Novel dutogliptin formulations and their preparation

Abstract

Novel pharmaceutical liposomal formulations of dutogliptin and methods for their preparation are provided.

Description

[Background technology]

[0001] background Dutogliptin (CAS 852329-66-9) is designed as a regenerative medicine drug to reduce and repair myocardial injury. It acts as an inhibitor of the dipeptidyl peptidase IV (DPP4) enzyme, which is involved in the degradation of stromal-derived factor 1α (SDF-1α). The drug has been given to patients as subcutaneous injections of 60-100 mg twice daily in combination with a subcutaneous dose of filgrastim in actual clinical trials.

[0002] Generally, dutogliptin is prepared as a tartrate salt. Initially, dutogliptin was administered orally. However, for acute cardiac indications, it needs to be administered parenterally to achieve optimal pharmacokinetic and pharmacodynamic activity. To achieve the required activity with current formulations, it needs to be administered at least twice a day for two weeks, which leads to problems with patient compliance, acceptance, and injection sites. It is therefore desirable to reduce the frequency of parenteral administration. The goal of the present application is therefore to meet such a need and to reduce the number of parenteral administrations, and therefore the object of the present invention is to provide a preparation of (freeze-dried) multilamellar vesicles (MLVs) containing encapsulated dutogliptin, and to provide liposomal dutogliptin formulations. These are advantageously suitable for, for example, subcutaneous (sc) or intramuscular (im) application. Summary of the Invention

[0003] A first aspect relates to a method for the preparation of a liposomal dutogliptin formulation, comprising the steps of: (A) (A.1) providing an organic phase, (a) the one or more phospholipids, the amount of which is between 3% (w / w) and 30% (w / w) based on the sum of (a), (b), and (c), preferably 22.36% (w / w) based on the sum of (a), (b), and (c); (b) one or more organic solvents selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, 1-butanol, 2-butanol, and tert-butanol, or any combination thereof, wherein the amount of the one or more organic solvents is 60% (w / w) to 97% (w / w) based on the sum of (a), (b), and (c), preferably 74.43% (w / w) based on the sum of (a), (b), and (c); (c) optionally, one or more additional organic components other than (a) or (b), the amount of said one or more additional organic components being up to 10% (w / w) based on the sum of (a), (b), and (c); wherein preferably the sum of (a), (b), and (c) is 100%; and (A.2) providing an aqueous phase, (d) the amount of the aqueous medium is 66% (w / w) to 95% (w / w) based on the sum of (d), (e), (f), and (g), preferably 84% based on the sum of (d), (e), (f), and (g); (e) optionally, one or more additional components selected from the group consisting of a buffer system, acids and / or bases for adjusting pH (e.g., NaOH, HCl), and stabilizers, wherein the amount of all additional components is up to 2% (w / w) based on the sum of (d), (e), (f), and (g); (f) optionally a bulking agent, preferably trehalose, wherein the amount of bulking agent is up to 10% (w / w) based on the sum of (d), (e), (f), and (g), preferably 3% (w / w) to 7% (w / w) based on the sum of (d), (e), (f), and (g); (g) dutogliptin, preferably in its tartrate form or its free base form, more preferably dutogliptin provided in its free base form, wherein the amount of dutogliptin is 5% (w / w) to 22% (w / w) based on the sum of (d), (e), (f), and (g) when dutogliptin is provided in its tartrate form, preferably 11±1% (w / w) based on the sum of (d), (e), (f), and (g), or 6.2±0.7% (w / w) based on the sum of (d), (e), (f), and (g) when dutogliptin is provided in its free base form. wherein the sum of (d), (e), (f), and (g) is 100%; and (A.3) mixing the aqueous phase and the organic phase until a ratio of 3:1 to 1:3, preferably 1:1, of the aqueous phase to the organic phase is reached, resulting in a nanodispersion system comprising dutogliptin; and (A.4) lyophilizing the nanodispersion comprising dutogliptin to obtain a lyophilisate; and (A.5) reconstituting the lyophilized product of step (A.4) with an aqueous solution to obtain a liposomal dutogliptin formulation, the aqueous solution comprising an aqueous medium of 80% (w / w) to 100% (w / w) based on the total weight of the aqueous solution, optionally further comprising an osmotic agent in an amount of up to 20% (w / w) based on the total weight of the aqueous solution, and optionally further comprising a buffer system, preferably wherein the final concentration of dutogliptin in the liposomal dutogliptin formulation is between 25 mg / ml and 60 mg / ml; or (B) (B.1) Below: (a) the one or more phospholipids, the amount of which is between 3% (w / w) and 30% (w / w) based on the sum of (a), (b), and (c), preferably 22.36% (w / w) based on the sum of (a), (b), and (c); (b) one or more organic solvents selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, 1-butanol, 2-butanol, and tert-butanol, or any combination thereof, wherein the amount of the one or more organic solvents is 60% (w / w) to 97% (w / w) based on the sum of (a), (b), and (c), preferably 74.43% (w / w) based on the sum of (a), (b), and (c); (c) optionally, one or more additional organic components other than (a) or (b), the amount of the one or more additional organic components being up to 10% based on the sum of (a), (b), and (c); wherein the sum of (a), (b), and (c) equals 100%; and (B.2) Below: (d) the amount of the aqueous medium is 66% (w / w) to 90% (w / w) based on the sum of (d), (e), and (f), preferably 84% (w / w) based on the sum of (d), (e), and (f); (e) optionally, one or more additional components selected from the group consisting of a buffer system, NaOH, HCl, and stabilizers, wherein the amount of all additional components is up to 2% (w / w) based on the sum of (d), (e), and (f); (f) optionally, a bulking agent, preferably trehalose, in an amount of up to 10% (w / w) based on the sum of (d), (e), and (f), preferably 3% (w / w) to 7% (w / w) based on the sum of (d), (e), and (f); wherein the sum of (d), (e), and (f) equals 100%; and (B.3) mixing the aqueous phase and the organic phase until a ratio of 3:1 to 1:3, preferably 1:1, of the aqueous phase to the organic phase is reached, resulting in a nanodispersion; (B.4) freeze-drying the nanodispersion to yield a lyophilisate; (B.5) Reconstituting the lyophilized product of step (B.4) with an aqueous solution to obtain a liposomal dutogliptin formulation, preferably having a final concentration of dutogliptin in the liposomal dutogliptin formulation of 25 mg / ml to 60 mg / ml, and (i) between 67% (w / w) and 100% (w / w) of an aqueous medium based on the total weight of the aqueous solution, optionally further comprising an osmotic agent in an amount of up to 20% (w / w) based on the total weight of the aqueous solution, and optionally further comprising a buffer system; (ii) dutogliptin, preferably in the form of its tartrate salt or in its free base form, wherein the amount of dutogliptin is 5% (w / w) to 13% (w / w) based on the total weight of the aqueous solution when dutogliptin is present in its tartrate salt form or in its free base form, more preferably 10±0.5% (w / w); or 6±0.4% (w / w) based on the total weight of the aqueous solution when dutogliptin is present in its free base form. or (C) (C.1) providing an organic phase, the organic phase (a) the amount of one or more phospholipids is between 3% (w / w) and 30% (w / w) based on the sum of (a), (b), and (c), preferably between 22 and 36% (w / w) based on the sum of (a), (b), and (c); (b) one or more organic solvents selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, 1-butanol, 2-butanol, and tert-butanol, or any combination thereof, wherein the amount of the one or more organic solvents is 60% (w / w) to 97% (w / w) based on the total of (a), (b), and (c), preferably 74.43% (w / w) based on the total of (a), (b), and (c); (c) optionally, one or more additional organic components other than (a) or (b), the amount of said one or more additional organic components being up to 10% (w / w) based on the sum of (a), (b), and (c); wherein the sum of (a), (b), and (c) is 100%; and (C.2) Below: (d) the amount of the aqueous medium is 66% (w / w) to 90% (w / w) based on the sum of (d), (e), (f), and (g), preferably 84% (w / w) based on the sum of (d), (e), (f), and (g); (e) optionally, one or more additional components selected from the group consisting of a buffer system, acids and / or bases for adjusting pH (e.g., NaOH, HCl), and stabilizers, wherein the amount of all additional components is up to 2% (w / w) based on the sum of (d), (e), (f), and (g); (f) optionally a bulking agent, preferably trehalose, wherein the amount of bulking agent is up to 10% (w / w) based on the sum of (d), (e), (f), and (g), preferably 3% (w / w) to 7% (w / w) based on the sum of (d), (e), (f), and (g); (g) dutogliptin provided in one of its fatty acid salt forms, wherein the amount of dutogliptin fatty acid salt is 5% (w / w) to 22% (w / w) based on the sum of (d), (e), (f), and (g). wherein the sum of (d), (e), (f), and (g) equals 100%; and (C.3) mixing the aqueous phase and the organic phase until a ratio of 3:1 to 1:3, preferably 1:1, of the aqueous phase to the organic phase is reached, resulting in a nanodispersion system comprising dutogliptin; (C.4) lyophilizing the nanodispersion comprising dutogliptin to obtain a lyophilisate; (C.5) Reconstituting the lyophilized material of step (C.4) with an aqueous solution to obtain a liposomal dutogliptin formulation, the aqueous solution comprising an aqueous medium of 80% (w / w) to 100% (w / w) based on the total weight of the aqueous solution, optionally further comprising an osmotic adjuster in an amount of up to 20% (w / w) based on the total weight of the aqueous solution, and optionally further comprising a buffer system, preferably wherein the final concentration of dutogliptin in the liposomal dutogliptin formulation is 25 mg / ml to 60 mg / ml.

[0004] An embodiment of aspect 1, or any of its subembodiments, relates to a process wherein the organic solvent is tert-butanol (TBA).

[0005] Another embodiment of aspect 1, or any of its subembodiments, relates to the process, wherein the organic phase further comprises cholesterol, the amount of cholesterol being between 2.5% (w / w) and 4% (w / w), preferably 3.2% (w / w).

[0006] Another embodiment of aspect 1, or any of its subembodiments, relates to a process wherein the organic phase comprises lecithin as the phospholipid, and the amount of lecithin is between 18.1% (w / w) and 26.2% (w / w), more preferably 22.13% (w / w).

[0007] Another embodiment of aspect 1, or any of its subembodiments, relates to a method, wherein the organic phase further comprises PG as a phospholipid, preferably DOPG-Na, and the total amount of PG is 0.1% (w / w) to 0.4% (w / w), preferably 0.23% (w / w).

[0008] Another embodiment of aspect 1, or any of its subembodiments, relates to a process, wherein the pH of the aqueous phase is between 7 and 7.8, preferably 7.4.

[0009] Another embodiment of aspect 1, or any of its subembodiments, relates to a method, wherein the liposomal dutogliptin formulation resulting from the reconstitution step has a D90 of liposomes between 1 μm and 4.5 μm.

[0010] Another embodiment of Aspect 1, or any of its subembodiments, relates to a method, wherein the only solvent in the aqueous solution is water.

[0011] Another embodiment of aspect 1, or any of its subembodiments, relates to a method wherein the aqueous solution is an aqueous NaCl solution, and the amount of NaCl is between 8 g / l and 10 g / l, preferably between 8.8 g / l and 9.2 g / l, and more preferably 9 g / l.

[0012] Another embodiment of Aspect 1, or any of its subembodiments, relates to the method, wherein the aqueous solution of step (B.5) is a dutogliptin tartrate solution.

[0013] Another embodiment relates to the latter embodiment, wherein the aqueous solution further comprises 9 g / l NaCl.

[0014] Another embodiment of aspect 1, or any of its subembodiments, relates to a method, wherein the total amount of dutogliptin in the liposomal dutogliptin formulation is between 60 mg and 100 mg.

[0015] The other phase (phase 2) is (i) an aqueous medium, preferably water, wherein the amount of the aqueous medium is 65% (w / w) to 90% (w / w) based on the total weight of the liposomal dutogliptin formulation; (ii) optionally, a lipid, preferably cholesterol, wherein the amount of said lipid, preferably cholesterol, is between 1% (w / w) and 2.5% (w / w), based on the total weight of the liposomal dutogliptin formulation; (iii) the amount of one or more phospholipids is between 7% (w / w) and 15% (w / w), preferably 11.2±0.4 (w / w), based on the total weight of the liposomal dutogliptin formulation, preferably the one or more phospholipids are PC, PG, and DSPG; (iv) optionally, one or more agents selected from the group consisting of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, a sugar component selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, xylitol, xylose, dextran, dextrose, and NaCl, wherein the total amount of the one or more agents is between 1.5% (w / w) and 5.5% (w / w); (v) the amount of dutogliptin, preferably in its tartrate form or in its free base form, is 3% (w / w) to 12% (w / w), preferably 5.3±0.3 (w / w), based on the total weight of the liposomal dutogliptin formulation, and the final concentration of dutogliptin in the liposomal dutogliptin formulation is 25 mg / ml to 60 mg / ml; The present invention relates to a liposomal dutogliptin formulation comprising the above-mentioned formula (i) to (v), wherein the total of (i) to (v) is 100%.

[0016] Another aspect (Aspect 3) relates to a kit comprising in one container a lyophilisate as described in Aspect 1 and its embodiments, and in a second container an aqueous pharmaceutical solution as described in Aspect 1 and its embodiments.

[0017] One embodiment of Aspect 3 relates to a kit further comprising a liposomal dutogliptin formulation according to the last embodiment of Aspect 1; or instructions on how to combine the contents of the first and second containers to obtain the liposomal dutogliptin formulation of Aspect 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] definition Unless otherwise specified, % amounts refer to % (weight / weight) ((w / w)).

[0019] Unless explicitly stated otherwise (for example, by using a term such as "one" in the "specific" sense), the word "a" is an indefinite article that includes "one" and "one or more" / "more than one" of the noun following the word "a".

[0020] The term "amount of one or more components", such as the amount of one or more organic solvents, refers to the sum of all such components. For example, if two organic solvents, such as 20% (w / w) tert-butanol based on the total amount of the organic phase and 10% (w / w) propanol based on the total weight of the organic phase, are present in the organic phase, then the amount of the one or more organic solvents (the two organic solvents, propanol and tert-butanol) is 30% (w / w) based on the total weight of the organic phase.

[0021] A "buffer" or "buffer system", as used herein, is used to prevent large changes in the pH of a solution, and suitable examples will be well known to those skilled in the art.

[0022] "Bulking agents" are used herein, as the name suggests, to form the majority of the lyophilized product and provide proper structure to the lyophilized cake. Non-limiting examples of bulking agents are mannitol, glycine, arginine, proline, glucose, sucrose, lactose, trehalose, and dextran.

[0023] The term "cholesterol" refers to 3β-hydroxy-5-cholestene (CAS number: 57-88-5). Examples of derivatives of cholesterol are cholesteryl sulfate and its salts (e.g., sodium salt), cholesteryl hemisuccinate, cholesteryl succinate, cholesteryl oleate, polyethylene glycol derivatives of cholesterol (cholesterol-PEG), coprostanol, cholestanol, cholestane, cholic acid, cortisol, corticosterone, hydrocortisone, and calciferol.

[0024] The term "container" as used herein means an ampoule or vial with a rubber stopper and cap, a single- or double-chamber syringe, an infusion bag or bottle made of polymeric material or glass, suitable for containing a composition for parenteral administration, and also includes any container for holding a liquid.

[0025] The term "D90" is well known to those skilled in the art and refers to the number of vesicles with a diameter equal to or less than a given value in relation to a size distribution, the weight of which is 90% of the weight of the components forming such particles in the formulation. D90 can be determined via multi-angle light scattering (MALS).

[0026] "Dutogliptin" is a potent and selective dipeptidyl peptidase 4 (DPP4) inhibitor for the treatment of type 2 diabetes. It has the following chemical structure: TIFF2025515490000001.tif34128

[0027] The term dutogliptin encompasses the molecule as a free base or any of its pharma- ceutically acceptable salts, such as tartrate or fatty acid salts.

[0028] When calculating the concentration of dutogliptin (mg / ml), the concentration is based on the dutogliptin free base molecule.

[0029] "Additional organic compounds" as used herein refer to organic compounds that are not solvents and are miscible with water, such as lipids, e.g., cholesterol, polysaccharides, and polypeptides. The term "lipids" as used herein excludes phospholipids. The term lipids as used herein refers to lipids selected from the group consisting of fatty acids, sterols (e.g., cholesterol), fat-soluble vitamins (e.g., vitamins A, D, E, and K), glycerolipids (e.g., monoglycerides, diglycerides, triglycerides), sphingolipids, glycolipids, polyketides, isoprenoids (prenol lipids), and waxes.

[0030] "Miscible with water," as used herein, means that the organic solvent can be mixed with water in all proportions, for example, ethanol, propanol, butanol can each be mixed with water.

[0031] The term "X% to Y%" (where X and Y represent any number between 0 and 100, and X% is less than Y%) refers to any number in the range of X% to Y%, inclusive.

[0032] "Nanodispersion system", as used herein, refers to a combination of an organic phase and an aqueous phase that comprises vesicles or micelles with a D90 of 60 nm or less, i.e., an aqueous formulation, where the vesicles or micelles are not liposomes.

[0033] "Liposome" is a spherical vesicle having at least one lipid bilayer. Liposomes can be used as a vehicle for the administration of pharmaceutical drugs. Liposomes are most often composed of phospholipids, specifically phosphatidylcholine, but may also contain other lipids, such as egg phosphatidylethanolamine, as long as they are compatible with the lipid bilayer structure.

[0034] "Liposomal formulation," as used herein, refers to a liquid that contains liposomes that include phospholipids. The liposomal formulation is suitable for encapsulation of dutogliptin in the aqueous environment of the liposomes.

[0035] Liposome size or lipophilic vesicle size (e.g., micelle size) refers to the size determined by multi-angle light scattering (MALS) or dynamic light scattering (DLS), respectively, as disclosed herein. Generally, liposome sizes range from 0.025 μm to 2.5 μm (Akbarzadeh et al. Nanoscale Research Letters 2013, 8:102).

[0036] The liposomal formulations of the present invention may have the advantage of reducing the number of injections or ingestion of dutogliptin a patient needs to take each day, thus contributing to patient convenience and / or patient compliance.

[0037] By "loading" is meant incorporating or introducing dutogliptin into the liposome / encapsulating dutogliptin in the liposome.

[0038] "Organic moiety," as used herein, refers to any molecule that contains at least one -(CH2)- moiety.

[0039] "Pharmaceutically acceptable" means approved or approvable by a regulatory authority in a country, or listed in the European or American Pharmacopoeia or other generally recognized pharmacopoeias, for use in animals, or more specifically, in humans.

[0040] PEG means polyethylene glycol.

[0041] PEGylation is the process of both covalent and non-covalent attachment or fusion of PEG polymer chains to molecules and macrostructures, such as phospholipids, resulting in vesicles that are described as PEGylated. PEGylated phospholipids are well known and commercially available.

[0042] The "pharmaceutical composition" described herein is specifically a pharmaceutical liposomal composition. By "pharmaceutical liposomal composition" is meant a composition comprising liposomes suitable for pharmaceutical administration.

[0043] The "phospholipid" used in the formulation of the present invention comprises a hydrophilic phosphate moiety and two hydrophobic carbohydrate moieties. For clarity, phospholipids are not included in the term lipid. Phospholipids may be selected from the group consisting of natural phospholipids, synthetic phospholipids, and combinations thereof. Lecithin is one of the natural sources of phospholipids. Lecithin is a mixture found in egg yolk and soybeans. It comprises a number of phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI), or any of their (pharmaceutical acceptable) salts.

[0044] Generally, the structure of a phospholipid as used herein has the structure (I): TIFF2025515490000002.tif34128, wherein R1 is C 10 -C 24 Represents acyl; R2 is C 10 -C24 represents acyl or hydrogen; R3 is 2-trimethylamino-1-ethyl (resulting in PC), 2-amino-2-carboxy-1-ethyl (resulting in PS), inosityl group (CH) (resulting in PI) 11 O5), 2-amino-1-ethyl (resulting in PE), or hydrogen (resulting in PA).

[0045] The terms "phosphatidic acid" and "PA" are used interchangeably herein. PA or its (pharmaceutical acceptable) salt can be derived from natural and / or synthetic origin. Non-limiting examples of PA are DLPA, DMPA, DPPA, DSPA, POPA, POPA, DEPA, HSPA, HEPA, or any of their (pharmaceutical acceptable) salts.

[0046] The terms "phosphatidylcholine" and "PC" are used interchangeably herein. PC or its (pharmaceutically acceptable) salts can be derived from natural and / or synthetic sources. PC can be PEGylated. Non-limiting examples of PC are dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), palmitoyloleoylphosphatidylcholine (POPC), dierucoylglycerophosphocholine (DEPC), hydrogenated soybean phosphatidylcholine (HSPC), hydrogenated egg phosphatidylcholine (HEPC), or any of their (pharmaceutically acceptable) salts.

[0047] The terms "phosphoethanolamine" and "PE" are used interchangeably herein. PE may be PEGylated. PE or its (pharmaceutically acceptable) salt may be derived from natural and / or synthetic sources. Non-limiting examples of PE are DLPE, DMPE, DPPE, DSPE, POPE, POPE, DEPE, HSPE, HEPE, or any (pharmaceutically acceptable) salt thereof.

[0048] The terms "phosphatidylglycerol" and "PG" are used interchangeably herein. PG or its (pharmaceutical acceptable) salt can be derived from natural and / or synthetic origin. Non-limiting examples of PG are DLPG, DMPG, DPPG, DSPG, POPG, POPG, DEPG, HSPG, HEPG, or any (pharmaceutical acceptable) salt thereof.

[0049] The terms "phosphatidylinositol" and "PI" are used interchangeably herein. PI may be PEGylated. PI or its (pharmaceutically acceptable) salt may be derived from natural and / or synthetic origin. Non-limiting examples of PI are DLPI, DMPI, DPPI, DSPI, POPI, POPI, DEPI, HSPI, HEPI, or any (pharmaceutically acceptable) salt thereof.

[0050] The terms "phosphoserine" and "PS" are used interchangeably herein. PS may be PEGylated. PS or its (pharmaceutically acceptable) salt may be derived from natural and / or synthetic origin. Non-limiting examples of PS are DLPS, DMPS, DPPS, DSPS, POPS, POPS, DEPS, HSPS, HEPS, or any (pharmaceutically acceptable) salt thereof.

[0051] Non-limiting examples of pharma- ceutically acceptable salts of any of the phospholipids are sodium or ammonium salts, e.g., PG-Na, PG-NH4, DSPG-Na, or DSPG-NH4.

[0052] When the terms PC, PG, PE, PA, PS, and / or PI are used, these terms also encompass pharma- ceutically acceptable salt forms of that phospholipid or PEGylated modifications of that phospholipid, unless otherwise indicated.

[0053] The term "reconstituting a lyophilisate" means mixing the lyophilisate with a solution.

[0054] Admixture "immediately prior to administration to a patient" means within 3 days, specifically within 24 hours, for example within 6 hours, prior to administration to a patient.

[0055] "Osmotic modifier" means a pharma- ceutically acceptable compound that can be added to a formulation to make it isotonic with human plasma. Osmotic modifiers include, for example, dextrose, glucose, mannitol, sucrose, lactose, trehalose, glycerin, and NaCl, specifically sucrose or glycerin or NaCl, more specifically sucrose or NaCl. Osmolarity is the "effective osmolarity" and is equal to the sum of the concentrations of solutes that have the ability to exert osmotic force through a membrane. Parenteral formulations should be isotonic with plasma. Osmotic modifiers are well known to those skilled in the art. Those skilled in the art recognize that in some cases, osmotic modifiers can also have other functions, for example, trehalose is also a bulking agent.

[0056] As used herein, the terms "treat", "treating", or "treatment" of any disease or disorder refer, in one embodiment, to ameliorating the disease or disorder (i.e., delaying, arresting, or reducing the onset of the disease or at least one of its clinical symptoms). In another embodiment, "treat", "treating", or "treatment" refers to alleviating or ameliorating at least one physical parameter, including those that are not discernible by the patient. In yet another embodiment, "treat", "treating", or "treatment" refers to modulating the disease or disorder physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both.

[0057] The term "(a), (b), (c), etc. (and further enumerations) sum to 100%" means that the percentage values ​​given for components (a)-(e) must be selected such that the values ​​for (a)-(e) necessarily sum to 100. These values ​​thus give the ratios of components (a)-(e). However, one of skill in the art will appreciate that the formulation may contain additional components, e.g., solvents. However, the amounts of such additional components are not taken into account when calculating the ratios of (a)-(c) (which must necessarily sum to 100%).

[0058] All aspects of the present invention may be combined, regardless of whether the aspects are embodiments, preferred aspects, more preferred aspects, particularly preferred aspects, or most preferred aspects, unless such combination violates any law of nature, and it is understood that such combinations of two or more aspects of the present invention are disclosed herein by disclosing those two or more aspects, even if such combinations of two or more aspects are not explicitly mentioned.

[0059] Those skilled in the art will understand that all embodiments described herein, regardless of whether the embodiment is an embodiment, a preferred embodiment, a more preferred embodiment, etc., can be combined, as long as such combination is not inconsistent with the laws of nature. Those skilled in the art will understand that an embodiment, e.g., a preferred embodiment, of one aspect of the invention can also apply to another aspect. Thus, for example, a preferred embodiment of the method according to the invention is also a preferred embodiment, if applicable, of, e.g., a lyophilisate or liposomal dutogliptin formulation according to the invention.

[0060] Detailed Description The present invention relates to liposomal formulations of dutogliptin and methods for the preparation of liposomal formulations containing dutogliptin.

[0061] Thus, one aspect of the present invention is (i) an aqueous medium, preferably water, wherein the amount of the aqueous medium is 65%-90% based on the total weight of the liposomal dutogliptin formulation; (ii) optionally, a lipid, preferably cholesterol, wherein the amount of said lipid, preferably cholesterol, is between 1% (w / w) and 2.5% (w / w), based on the total weight of the liposomal dutogliptin formulation; (iii) the amount of one or more phospholipids is between 7% (w / w) and 15% (w / w), preferably 11.2±0.4 (w / w), based on the total weight of the liposomal dutogliptin formulation; (iv) optionally, one or more agents selected from the group consisting of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, xylitol, xylose, dextran, dextrose, NaCl, a buffer system, and a stabilizer, wherein the total amount of the one or more agents is between 1.5% (w / w) and 5.5% (w / w); (v) the amount of dutogliptin, preferably in its tartrate form or in its free base form, is between 3% (w / w) and 12% (w / w), preferably 5.3±0.3 (w / w), based on the total weight of the liposomal dutogliptin formulation; preferably, the final concentration of dutogliptin in the liposomal dutogliptin formulation is between 25 mg / ml and 60 mg / ml; The present invention relates to a liposome formulation of dutogliptin comprising the above-mentioned formula (i) to (v), wherein the total of (i) to (v) is 100%.

[0062] Another embodiment relates to a method for the preparation of a liposomal formulation of dutogliptin comprising the steps of: (A) (A.1) providing an organic phase, (a) the one or more phospholipids, the amount of which is between 3% (w / w) and 30% (w / w) based on the sum of (a), (b), and (c), preferably 22.36% (w / w) based on the sum of (a), (b), and (c); (b) one or more organic solvents selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, 1-butanol, 2-butanol, and tert-butanol, or any combination thereof, wherein the amount of the one or more organic solvents is 60% (w / w) to 97% (w / w) based on the sum of (a), (b), and (c), preferably 74.43% (w / w) based on the sum of (a), (b), and (c); (c) optionally, one or more additional organic components which are not (a) or (b), wherein the amount of said one or more additional organic components is up to 10% (w / w) based on the sum of (a), (b), and (c); wherein the sum of (a), (b), and (c) is 100%; and (A.2) providing an aqueous phase, (d) the amount of the aqueous medium is 66% (w / w) to 95% (w / w) based on the sum of (d), (e), (f), and (g), preferably 84% (w / w) based on the sum of (d), (e), (f), and (g); (e) optionally, one or more additional components selected from the group consisting of a buffer system and acids and / or bases for adjusting pH (e.g., NaOH, HCl), wherein the amount of all additional components is up to 2% (w / w) based on the sum of (d), (e), (f), and (g); (f) optionally a bulking agent, preferably trehalose, wherein the amount of bulking agent is up to 10% (w / w) based on the sum of (d), (e), (f), and (g), preferably 3% (w / w) to 7% (w / w) based on the sum of (d), (e), (f), and (g); (g) dutogliptin, preferably in its tartrate salt form or in its free base form, more preferably dutogliptin provided in its free base form, wherein the amount of dutogliptin is 5% (w / w) to 22% (w / w) based on the sum of (d), (e), (f), and (g) when dutogliptin is provided in its tartrate salt form, preferably 11±1% (w / w) based on the sum of (d), (e), (f), and (g); or 6.2±0.7% (w / w) based on the sum of (d), (e), (f), and (g) when dutogliptin is provided in its free base form. wherein the sum of (d), (e), (f), and (g) is 100%; and (A.3) mixing the aqueous phase and the organic phase until a ratio of 3:1 to 1:3, preferably 1:1, of the aqueous phase to the organic phase is reached, resulting in a nanodispersion system comprising dutogliptin; and (A.4) lyophilizing the nanodispersion comprising dutogliptin to obtain a lyophilisate; and optionally (A.5) reconstituting the lyophilized product of step (A.4) with an aqueous solution to obtain a liposomal dutogliptin formulation, the aqueous solution comprising an aqueous medium of 80% (w / w) to 100% (w / w) based on the total weight of the aqueous solution, optionally further comprising an osmotic agent in an amount of up to 20% (w / w) based on the total weight of the aqueous solution, and optionally further comprising a buffer system, preferably wherein the final concentration of dutogliptin in the liposomal dutogliptin formulation is between 25 mg / ml and 60 mg / ml; or (B) (B.1) Below: (a) the one or more phospholipids, the amount of which is between 3% (w / w) and 30% (w / w) based on the sum of (a), (b), and (c), preferably 22.36% (w / w) based on the sum of (a), (b), and (c); (b) one or more organic solvents selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, 1-butanol, 2-butanol, and tert-butanol, or any combination thereof, wherein the amount of the one or more organic solvents is 60% (w / w) to 97% (w / w) based on the sum of (a), (b), and (c), preferably 74.43% (w / w) based on the sum of (a), (b), and (c); (c) optionally, one or more additional organic components other than (a) or (b), the amount of the one or more additional organic components being up to 10% based on the sum of (a), (b), and (c); wherein the sum of (a), (b), and (c) equals 100%; and (B.2) Below: (d) the amount of the aqueous medium is 66% (w / w) to 90% (w / w) based on the sum of (d), (e), and (f), preferably 84% based on the sum of (d), (e), and (f); In one embodiment, in (d), the amount of the aqueous medium is 88% (w / w) to 100% (w / w) based on the total of (d), (e), and (f); In one embodiment, in (d), the amount of aqueous medium is 88% (w / w) to 99.99% (w / w) based on the total of (d), (e), and (f), and (f) is at most 10% (w / w) based on the total of (d), (e), and (f); In one embodiment, in (d), the amount of the aqueous medium is 91% (w / w) to 96.99% (w / w) based on the total of (d), (e), and (f), and (f) is 3% (w / w) to 7% (w / w) based on the total of (d), (e), and (f); where "99.99" and "96.99" represent the fact that (e) and (f) may be present, but in very low concentrations; In one embodiment, in (d), the amount of aqueous medium is 91% (w / w) to 96.5% (w / w) based on the sum of (d), (e), and (f), (f) is 3% (w / w) to 7% (w / w) based on the sum of (d), (e), and (f), and (e) is 0.5 to 2% (w / w) based on the sum of (d), (e), and (f); (e) optionally, one or more additional components selected from the group consisting of a buffer system, an acid and / or a base for adjusting pH (e.g., NaOH, HCl), wherein the amount of all additional components is up to 2% (w / w) based on the sum of (d), (e), and (f); In one embodiment, in (e), the amount of all additional ingredients is 0.5-2% (w / w) based on the total of (d), (e), and (f); (f) optionally, a bulking agent, preferably trehalose, in an amount of up to 10% (w / w) based on the sum of (d), (e), and (f), preferably 3% (w / w) to 7% (w / w) based on the sum of (d), (e), and (f); wherein the sum of (d), (e), and (f) equals 100%; and (B.3) mixing the aqueous phase and the organic phase until a ratio of 3:1 to 1:3, preferably 1:1, of the aqueous phase to the organic phase is reached, resulting in a nanodispersion; (B.4) freeze-drying the nanodispersion to yield a lyophilisate; optionally (B.5) Reconstituting the lyophilized product of step (B.4) with an aqueous solution to obtain a liposomal dutogliptin formulation, preferably having a final concentration of dutogliptin in the liposomal dutogliptin formulation of 25 mg / ml to 60 mg / ml, and (i) between 67% (w / w) and 100% (w / w) of an aqueous medium based on the total weight of the aqueous solution, optionally further comprising an osmolality adjusting agent in an amount of up to 20% (w / w), preferably between 10% (w / w) and 20% (w / w), based on the total weight of the aqueous solution, and optionally further comprising a buffer system; (ii) dutogliptin, preferably in the form of its tartrate salt or in its free base form, wherein the amount of dutogliptin is 5% (w / w) to 13% (w / w) based on the total weight of the aqueous solution when dutogliptin is present in its tartrate salt form, more preferably 10±0.5% (w / w); or 6±0.4% (w / w) based on the total weight of the aqueous solution when dutogliptin is present in its free base form. or (C) (C.1) providing an organic phase, the organic phase (a) the one or more phospholipids, the amount of which is between 3% (w / w) and 30% (w / w) based on the sum of (a), (b), and (c), preferably 22.36% (w / w) based on the sum of (a), (b), and (c); (b) one or more organic solvents selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, 1-butanol, 2-butanol, and tert-butanol, or any combination thereof, wherein the amount of the one or more organic solvents is 60% (w / w) to 97% (w / w) based on the total of (a), (b), and (c), preferably 74.43% (w / w) based on the total of (a), (b), and (c); (c) optionally, one or more additional organic components other than (a) or (b), the amount of said one or more additional organic components being up to 10% (w / w) based on the sum of (a), (b), and (c); wherein the sum of (a), (b), and (c) is 100%; and (C.2) Below: (d) the amount of the aqueous medium is 66% (w / w) to 90% (w / w) based on the sum of (d), (e), (f), and (g), preferably 84% (w / w) based on the sum of (d), (e), (f), and (g); (e) optionally, one or more additional components selected from the group consisting of a buffer system and acids and / or bases for adjusting pH (e.g., NaOH, HCl), wherein the amount of all additional components is up to 2% (w / w) based on the sum of (d), (e), (f), and (g); (f) optionally a bulking agent, preferably trehalose, wherein the amount of bulking agent is up to 10% (w / w) based on the sum of (d), (e), (f), and (g), preferably 3% (w / w) to 7% (w / w) based on the sum of (d), (e), (f), and (g); (g) dutogliptin provided in one of its fatty acid salt forms, wherein the amount of dutogliptin fatty acid salt is 5% (w / w) to 22% (w / w) based on the sum of (d), (e), (f), and (g). wherein the sum of (d), (e), (f), and (g) equals 100%; and (C.3) mixing the aqueous phase and the organic phase until a ratio of 3:1 to 1:3, preferably 1:1, of the aqueous phase to the organic phase is reached, resulting in a nanodispersion system comprising dutogliptin; (C.4) lyophilizing the nanodispersion comprising dutogliptin to obtain a lyophilisate; optionally (C.5) Reconstituting the lyophilized material of step (C.4) with an aqueous solution to obtain a liposomal dutogliptin formulation, the aqueous solution comprising an aqueous medium of 80% (w / w) to 100% (w / w) based on the total weight of the aqueous solution, optionally further comprising an osmotic adjuster in an amount of up to 20% (w / w) based on the total weight of the aqueous solution, and optionally further comprising a buffer system, preferably wherein the final concentration of dutogliptin in the liposomal dutogliptin formulation is 25 mg / ml to 60 mg / ml.

[0063] Method (C) may optionally comprise, prior to step (C1), a step involving preparing a fatty acid salt of dutogliptin by replacing the tartrate anion in dutogliptin tartrate with a fatty acid anion (preferably lauric acid, myristic acid, palmitic acid, stearic acid, or cholic acid hydrate, more preferably cholic acid hydrate).

[0064] The method of the invention allows for the simple preparation of nanodispersions, which are liquid intermediate formulations, in which the D90 of the lipophilic vesicles or micelles is 60 nm or less, more preferably 25 nm or less, preferably 20 nm or less. This nanodispersion formed in the method according to the invention allows for the preparation of liposomal formulations without the need for mechanical reduction in liposome size (e.g., to allow for sterile filtration of the dutogliptin formulation prior to filling into sterile containers and / or freeze-drying).

[0065] Alternatively, the method of the present invention further allows for the easy preparation of liposomal formulations in which the liposomes have a D90 of 1 μm to 4.5 μm. The present invention further provides a formulation comprising dutogliptin prepared according to the method of the present invention, more particularly a formulation comprising dutogliptin suitable for administration to a patient. Specifically, such administration is by subcutaneous injection or infusion, more preferably by subcutaneous injection. The present invention further provides two separate formulations that can be mixed together immediately prior to administration to a patient to provide a liposomal composition suitable for administration. In one embodiment, one formulation comprising phospholipids can be a lyophilisate and the second formulation can be an aqueous formulation, with the proviso that at least one of the lyophilisate or aqueous solution comprises dutogliptin. When the two separate formulations are mixed together, the formed liposomes are loaded with dutogliptin, resulting in a pharmaceutical liposomal dutogliptin formulation suitable for clinical use. Preferably, the final concentration of dutogliptin in the liposomal dutogliptin formulation is 25 mg / ml to 60 mg / ml.

[0066] A formulation containing dutogliptin should allow for efficient and optimal loading of dutogliptin into liposomes prior to administration to a patient.

[0067] In one preferred embodiment, at least 25% (w / w) of the dutogliptin, more preferably at least 30% (w / w) of the dutogliptin is encapsulated in liposomes in the liposomal dutogliptin formulation.

[0068] Preferably, the present invention provides pharmaceutical liposomal formulations that allow for an extended release of dutogliptin from the liposomes following administration (injection).

[0069] The formulations described herein are specifically pharmaceutical formulations, such as pharmaceutical liposomal compositions.

[0070] Preferably, the D90 of the liposome of the liposome preparation is from 1 μm to 4.5 μm.

[0071] Specifically, the phospholipids or pharma- ceutically acceptable salts thereof described herein are selected from egg lecithin, soybean lecithin, or synthetic phospholipids.

[0072] Numerous aspects and embodiments of the present invention are described below.

[0073] formulation The first aspect relates to a liposomal dutogliptin formulation described herein.

[0074] Those skilled in the art will understand that in the liposomal dutogliptin formulation, trace amounts, up to 2%, of the solvents and additives (e.g., tert-butanol or water or buffer system) used in the preparation method of the present invention, preferably further described below, may remain; or additional additives may be present. Without being bound by this explanation, additional additives may be required for the stabilization of such liposomal dutogliptin formulations or for patient tolerability.

[0075] In one preferred embodiment, the sum of the weights of (i)-(v) is at least 98% (w / w) based on the total weight of the liposomal dutogliptin formulation.

[0076] In another preferred embodiment, the sum of the weights of (i)-(v) is at least 99% (w / w) based on the total weight of the liposomal dutogliptin formulation.

[0077] In one preferred embodiment, the sum of the weights of (i)-(v) is at least 99.9% (w / w) based on the total weight of the liposomal dutogliptin formulation.

[0078] One preferred embodiment relates to liposomal dutogliptin formulations having residual organic solvents, such as tert-butanol, of less than 2% (w / w) based on the total weight of such formulation (e.g., the sum of the amounts of (i)-(v) and tert-butanol). More preferably, the amount of tert-butanol is 1% or less, such as less than 0.8% or even less than 0.01%.

[0079] One preferred embodiment relates to a formulation according to the present invention comprising (i) to (v).

[0080] Another preferred embodiment relates to a formulation according to the invention, wherein the formulation consists of (i) to (v), tert-butanol, and a buffer system, preferably an acetate buffer system, wherein the sum of the amounts of tert-butanol and the buffer system is 0.01% to 2% based on the total weight of such formulation (e.g. in the case of a lyophilisate, e.g. the sum of the amounts of (a) to (e) and tert-butanol).

[0081] A further preferred embodiment relates to a dutogliptin-containing, preferably pharmaceutical, nanodispersion (or a method comprising the preparation of a nanodispersion), in which the D90 of the lipophilic vesicles is less than 25 nm, more preferably 20 nm or less, such as between 10 nm and 20 nm, for example around 15 nm, or between 3 nm and 10 nm, for example around 5 nm.

[0082] Another preferred embodiment relates to a lyophilized formulation comprising dutogliptin.

[0083] Another preferred embodiment relates to a lyophilized preparation (lyophilisate) in which the amount of dutogliptin is 50 mg to 120 mg, more preferably 60 mg to 100 mg.

[0084] In a further preferred embodiment, the pharma- ceutical active substance in the formulation according to the invention is entrapped in a lipid inclusion complex, a proliposome, a micelle, or a liposome, more preferably in a liposome or a micelle, in other words, the composition comprises, for example, at least one liposome or at least one micelle, respectively, and dutogliptin is loaded into the liposome or micelle.

[0085] In one preferred embodiment, dutogliptin in the formulation according to the invention is entrapped in micelles (nanodispersion formulations / nanodispersion systems). Most preferably, the micelles have a D90 of 60 nm or less, e.g., less than 25 nm, more preferably, 20 nm or less, e.g., between 10 nm and 20 nm, or between 3 nm and 10 nm.

[0086] In another preferred embodiment, the dutogliptin in the formulation according to the invention is entrapped in liposomes (liposomal formulation).

[0087] Another preferred embodiment relates to a formulation, preferably a liposomal or nanodispersion formulation, which is a pharmaceutical formulation.

[0088] Liposomal formulation One preferred embodiment relates to pharmaceutical liposomal formulations.

[0089] Such pharmaceutical liposomal formulations are preferably suitable for injection, preferably subcutaneous injection.

[0090] Another preferred embodiment relates to a pharmaceutical liposomal formulation, wherein the concentration of dutogliptin in the liposomal formulation is from 25 mg / ml to 60 mg / ml, for example from 30 mg / ml to 50 mg / ml.

[0091] According to another preferred embodiment, the pharmaceutical liposomal formulation has a pH of 6 to 8, preferably 7 to 8, more preferably 7 to 7.8, for example 7.4±2.

[0092] (i) Solvents for liposome formulations aqueous medium The aqueous medium of the liposome formulation is water or a combination of water and a solvent selected from the group consisting of propylene glycol, ethanol, isopropanol, glycerol, glycol ether (e.g., monobutyl ether of ethylene glycol or propylene glycol, or monoethyl ether or monomethyl ether of diethylene glycol, or any combination thereof), polyethylene glycol (PEG) 300, PEG 400, specifically, propylene glycol and ethanol, and the amount of water relative to the total amount of solvent in the aqueous medium is at least 60% (w / w), preferably at least 90% (w / w), more preferably at least 95% (w / w).

[0093] In one preferred embodiment, water is the only solvent in the aqueous medium and, therefore, the only solvent of the liposomal formulation.

[0094] In general, the liposome formulation comprises an aqueous medium, and the amount of the aqueous medium is 65% (w / w) to 90% (w / w) based on the total of (i) to (v).

[0095] (ii) Lipids In one preferred embodiment, the formulation according to the invention comprises one or more lipids.

[0096] In one preferred embodiment, the lipid used in the present invention is a lipid selected from the group consisting of fatty acids, sterols (e.g., cholesterol), fat-soluble vitamins (e.g., vitamins A, D, E, and K), glycerolipids (e.g., monoglycerides, diglycerides, triglycerides), sphingolipids, glycolipids, polyketides, isoprenoids (prenol lipids), and waxes, more preferably from the group consisting of fatty acids, sterols (e.g., cholesterol), fat-soluble vitamins (e.g., vitamins A, D, E, and K), glycerolipids (e.g., monoglycerides, diglycerides, triglycerides), sphingolipids, and glycolipids.

[0097] Cholesterol is known to affect liposome stability and drug release.

[0098] Therefore, in one more preferred embodiment, the formulation according to the invention preferably comprises cholesterol or a derivative thereof.

[0099] Another preferred embodiment relates to formulations in which the additional organic component is selected from the group consisting of cholesterol or sodium cholesteryl sulfate. More preferably, the additional organic component is cholesterol.

[0100] Another preferred embodiment relates to a formulation in which the amount of cholesterol is between 0.5% and 3% compared to the sum of all components in the formulation, for example around 1.6%, for example between 1.3% and 1.9%.

[0101] (iii) Phospholipids Another preferred embodiment relates to formulations in which the one or more phospholipids comprise PC or a mixture of PC and one or more phospholipids selected from the group consisting of phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidic acid (PA), phosphatidylglycerol (PG), or a pharma- ceutically acceptable salt of any of them.

[0102] Another preferred embodiment relates to formulations in which the one or more phospholipids is PC or a mixture of PC with one or more phospholipids selected from the group consisting of phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidic acid (PA), phosphatidylglycerol (PG), or a pharma- ceutically acceptable salt of any of them.

[0103] In one preferred embodiment, the pharma- ceutically acceptable salt of the phospholipid is a Na salt of the phospholipid.

[0104] In one preferred embodiment, at least one phospholipid is a PEGylated phospholipid (or a pharma- ceutically acceptable salt thereof).

[0105] In another preferred embodiment, the PC, or a pharma- ceutically acceptable salt thereof, is PEGylated.

[0106] In yet another preferred embodiment, the formulation comprises PC or a pharma- ceutically acceptable salt thereof and PG or a pharma- ceutically acceptable salt thereof. In one preferred embodiment, the PG is DOPG-Na.

[0107] Another more preferred embodiment relates to a formulation wherein the one or more phospholipids comprise PC, or preferably a pharma- ceutically acceptable salt thereof, more preferably selected from the group consisting of DLPC, DMPC, DPPC, DSPC, POPC, POPC, DEPC, HSPC, HEPC, or preferably a pharma- ceutically acceptable salt thereof.

[0108] Another more preferred embodiment relates to a formulation wherein the one or more phospholipids comprise PEGylated PC, or PEGylated DLPC, PEGylated DMPC, PEGylated DPPC, PEGylated DSPC, PEGylated POPC, PEGylated POPC, PEGylated DEPC, PEGylated HSPC, PEGylated HEPC, or preferably a pharma- ceutically acceptable salt thereof, more preferably selected from the group consisting of PEGylated PC, or PEGylated DLPC, PEGylated DMPC, PEGylated DPPC, PEGylated DSPC, PEGylated POPC, PEGylated DEPC, PEGylated HSPC, PEGylated HEPC, or preferably a pharma- ceutically acceptable salt thereof.

[0109] Another preferred embodiment relates to a formulation in which the amount of one or more phospholipids is between 7% (w / w) and 15% (w / w) compared to the sum of all components of the liposomal formulation, for example around 11.2%, for example 11.2±0.5%.

[0110] Another more preferred embodiment relates to a formulation in which the one or more phospholipids comprises PC or, preferably, a pharma- ceutically acceptable salt thereof. In a further preferred embodiment, the PC or, preferably, a pharma- ceutically acceptable salt thereof is derived from soybean (e.g., Lipoid S100), egg, or synthetic, and in a most preferred embodiment, the PC or, preferably, a pharma- ceutically acceptable salt thereof is derived from soybean.

[0111] Another more preferred embodiment relates to a formulation in which PC, or preferably a pharma- ceutically acceptable, is selected from the group consisting of DLPC, DMPC, DPPC, DSPC, POPC, POPC, DEPC, HSPC, HEPC, or a combination thereof.

[0112] Another more preferred embodiment relates to a formulation wherein the PG, or preferably a pharma- ceutically acceptable salt thereof, is PEGylated and is selected from the group consisting of PEGylated DLPG, PEGylated DMPG, PEGylated DPPG, PEGylated DSPG, PEGylated POPG, PEGylated POPG, PEGylated DEPG, PEGylated HSPG, PEGylated HEPG, or preferably a pharma- ceutically acceptable salt of any thereof.

[0113] Another preferred embodiment relates to a formulation in which the one or more phospholipids comprise PG or, preferably, a pharma- ceutically acceptable salt thereof, and the PG or salt thereof is PEGylated PG or a PEGylated salt thereof.

[0114] Another more preferred embodiment relates to a formulation wherein PG, or preferably a pharma- ceutically acceptable salt thereof, is selected from the group consisting of DLPG, DMPG, DPPG, DSPG, POPG, POPG, DEPG, HSPG, HEPG.

[0115] Another more preferred embodiment relates to a formulation wherein the PG, or preferably a pharma- ceutically acceptable salt thereof, is PEGylated and is selected from the group consisting of PEGylated DLPG, PEGylated DMPG, PEGylated DPPG, PEGylated DSPG, PEGylated POPG, PEGylated POPG, PEGylated DEPG, PEGylated HSPG, PEGylated HEPG, or preferably a pharma- ceutically acceptable salt of any thereof.

[0116] (iv) Drugs (bulking agents and / or osmolality adjusters) The presence of a bulking agent is advantageous, especially when the components of the formulation according to the invention are to be lyophilizable or the preparation of the formulation according to the invention via the method according to the invention requires a lyophilization step. Examples of preferred bulking agents are glycine, arginine, proline or any other amino acid known to be suitable as a bulking agent, or a sugar component (component (iv) or (f), respectively). Preferred sugar components are selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, xylitol, xylose, dextran, or mixtures thereof.

[0117] One more preferred embodiment relates to a formulation in which (f) in the aqueous phase of the method according to the invention (and therefore (iv) in the liposomal formulation according to the invention, respectively) is trehalose.

[0118] One preferred embodiment relates to a liposomal formulation in which one drug is trehalose and the amount of trehalose in the formulation is between 1.5% (w / w) and 3.5% (w / w), for example 2.5±0.5% (w / w), based on the total weight of the liposomal formulation.

[0119] Another preferred embodiment relates to pharmaceutical liposomal formulations that include an osmotic agent.

[0120] Such an osmolality modifier, preferably a pharma- ceutically acceptable osmolality modifier, should be present in the pharmaceutical liposomal formulation, which may be added during the preparation of the liposomal formulation by mixing the lyophilisate according to the invention with an aqueous phase containing the osmolality modifier, or may be provided in the rehydration step of the lyophilisate.

[0121] Alternatively, the osmotic modifier may already be present in the lyophilisate in that it is part of the aqueous phase used in the method according to the invention for preparing the lipopeptide according to the invention. The skilled person will be aware that the osmotic modifier may be partly and partly part of such aqueous phase. The skilled person will be able to easily calculate the total amount of osmotic modifier required to have an appropriate concentration in the final pharmaceutical liposomal formulation, in order to have an isotonic effect with respect to the medicament provided to the patient.

[0122] One of ordinary skill in the art will recognize that in some cases, the osmotic modifier is also a bulking agent, for example, when the osmotic modifier is a sugar moiety, such as glucose or trehalose.

[0123] Some osmotic adjusters, such as NaCl, do not have a bulking function. Thus, when using such a "single-function component", e.g., NaCl, the formulation may include a bulking agent (e) and an osmotic adjuster (e). It may further include a bulking agent (e.g., trehalose) and a "two" osmotic adjuster (e.g., trehalose and, e.g., NaCl).

[0124] Osmotic modifiers are well known to those skilled in the art. In one preferred embodiment, the osmotic modifier is selected from the group consisting of dextrose, glucose, mannitol, sucrose, lactose, trehalose, glycine, arginine, proline, and NaCl, specifically, glycine, mannitol, trehalose, glucose, and NaCl, more specifically, glycine and NaCl, most preferably NaCl.

[0125] Those skilled in the art will know how to select the appropriate concentration of a particular osmotic agent in a formulation, preferably for subcutaneous injection.

[0126] In a preferred embodiment, the osmotic agent is NaCl, and the concentration of NaCl in the liposomal formulation is 0.8% to 1%, more preferably around 0.9%, for example 0.9% ± 0.1%, more preferably 0.9%, based on the total weight of the pharmaceutical liposomal formulation.

[0127] Depending on the osmotic agent and the type of formulation (e.g., a pharma- ceutically acceptable liposomal formulation or a lyophilisate), the skilled artisan is well aware how to calculate the amount of one or more osmotic agents to be present in the (final) formulation for administration to a patient in physiological concentrations.

[0128] (v) dutogliptin In one preferred embodiment, the concentration of dutogliptin in the liposomal formulation is 25 mg / ml to 60 mg / ml, more preferably 30 mg / ml to 50 mg / ml, for example, an injection in a volume of 2 ml contains 50 mg to 120 mg, more preferably 60 mg to 100 mg of dutogliptin.

[0129] Freeze-dried product A further aspect of the present invention relates to a lyophilisate comprising dutogliptin ((g)), (a), optionally (c), optionally (e), and optionally (f), as defined in the methods herein.

[0130] Preferably, the total amount of dutogliptin is 50 mg to 120 mg, more preferably 60 mg to 100 mg.

[0131] Preferably, The amount of (g) is 12% (w / w) to 53% (w / w) based on the total amount of the lyophilisate; The amount of (a) is 7.2% (w / w) to 73% (w / w) based on the total amount of the lyophilisate; the amount of (c) is up to 24.3% (w / w), preferably (c) comprises cholesterol, the amount of cholesterol being between 4.8% (w / w) and 9.7% (w / w), based on the total amount of the lyophilisate; the amount of (e) is at most 4.9% (w / w) based on the total amount of the lyophilisate; The amount of (f) is up to 24.3% (w / w), preferably (f) comprises trehalose, the amount of trehalose being 12% (w / w) to 53% (w / w) based on the total amount of the lyophilisate.

[0132] In one preferred embodiment, (c) consists of cholesterol.

[0133] In another preferred embodiment, (f) consists of trehalose.

[0134] Another aspect relates to the use of a lyophilisate comprising dutogliptin as described herein for the preparation of a medicament, preferably a pharmaceutical liposomal dutogliptin formulation.

[0135] Liposome size In yet another embodiment, the liposomes of the liposomal formulation according to the invention have a D90 size distribution of 1 μm to 4.5 μm.

[0136] Method of Treatment / Use One aspect relates to the use of a liposomal formulation according to the invention for the preparation of a medicament for treating diabetes, in particular type 2 diabetes.

[0137] Method of preparation Another aspect relates to a method (A) for the preparation of a liposomal formulation according to the invention.

[0138] Another aspect relates to a method (B) for the preparation of a liposomal formulation according to the invention.

[0139] Another aspect relates to a method (C) for the preparation of a liposomal formulation according to the invention.

[0140] organic phase (a) Phospholipids In one preferred embodiment, the one or more phospholipids in the organic phase comprise PC, or a mixture of PC and one or more phospholipids selected from the group consisting of phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidic acid (PA), phosphatidylglycerol (PG), or a pharma- ceutically acceptable salt of any of them.

[0141] In one preferred embodiment, the one or more phospholipids in the organic phase comprise PC, or a pharma- ceutically acceptable salt thereof, and PG, or a pharma- ceutically acceptable salt thereof.

[0142] In one preferred embodiment, at least one phospholipid is a PEGylated phospholipid.

[0143] Another more preferred embodiment relates to a formulation wherein the one or more phospholipids comprise PC, or preferably a pharma- ceutically acceptable salt thereof, more preferably selected from the group consisting of DLPC, DMPC, DPPC, DSPC, POPC, POPC, DEPC, HSPC, HEPC, or preferably a pharma- ceutically acceptable salt thereof.

[0144] Another more preferred embodiment relates to a formulation wherein the one or more phospholipids comprise PEGylated PC, or PEGylated DLPC, PEGylated DMPC, PEGylated DPPC, PEGylated DSPC, PEGylated POPC, PEGylated POPC, PEGylated DEPC, PEGylated HSPC, PEGylated HEPC, or preferably a pharma- ceutically acceptable salt thereof, more preferably selected from the group consisting of PEGylated PC, or PEGylated DLPC, PEGylated DMPC, PEGylated DPPC, PEGylated DSPC, PEGylated POPC, PEGylated DEPC, PEGylated HSPC, PEGylated HEPC, or preferably a pharma- ceutically acceptable salt thereof.

[0145] Another more preferred embodiment relates to a formulation in which the one or more phospholipids comprises PC or, preferably, a pharma- ceutically acceptable salt thereof. In an even more preferred embodiment, the PC or, preferably, a pharma- ceutically acceptable salt thereof is derived from soybean (e.g., Lipoid S100), egg, or synthetic, and in a most preferred embodiment, the PC or, preferably, a pharma- ceutically acceptable salt thereof is derived from soybean.

[0146] Thus, one preferred embodiment relates to a method for preparing a liposomal formulation, wherein the organic phase constitutes between 3% (w / w) and 30% (w / w) based on the sum of (a), (b) and (c), preferably between 15% (w / w) and 28% (w / w) based on the sum of (a), (b) and (c), for example 22.3±0.5% (w / w) based on the sum of (a), (b) and (c).

[0147] Another preferred embodiment relates to an organic phase in which PG, or preferably a pharma- ceutically acceptable salt thereof, is PEGylated PG, or a PEGylated, preferably a pharma- ceutically acceptable salt thereof.

[0148] Another more preferred embodiment relates to an organic phase in which PG, or preferably a pharma- ceutically acceptable salt thereof, is selected from the group consisting of DLPG, DMPG, DPPG, DSPG, POPG, POPG, DEPG, HSPG, HEPG.

[0149] Another more preferred embodiment relates to an organic phase wherein PG, or preferably a pharma- ceutically acceptable salt thereof, is PEGylated and selected from the group consisting of PEGylated DLPG, PEGylated DMPG, PEGylated DPPG, PEGylated DSPG, PEGylated POPG, PEGylated POPG, PEGylated DEPG, PEGylated HSPG, PEGylated HEPG, or preferably a pharma- ceutically acceptable salt of any thereof.

[0150] Another preferred embodiment relates to an organic phase in which the one or more phospholipids comprise or consist of PC, the amount of PC being between 3.2% (w / w) and 29.8% (w / w) based on the sum of (a), (b) and (c), preferably between 15.2% (w / w) and 27.8% (w / w) based on the sum of (a), (b) and (c), for example 22.1±0.5% (w / w) based on the sum of (a), (b) and (c).

[0151] Another preferred embodiment is one in which the one or more phospholipids comprise or consist of PC (or a pharma- ceutically acceptable salt thereof) and PG (or a pharma- ceutically acceptable salt thereof), and the amount of PC (or a pharma- ceutically acceptable salt thereof) is between 3.1% (w / w) and 29.9% (w / w) based on the sum of (a), (b) and (c), preferably between 15.1% (w / w) and 27.9% (w / w) based on the sum of (a), (b) and (c), For example, 22.1±0.5% (w / w) based on the sum of (a), (b) and (c), and the sum of the amounts of DOPG and PG is between 0.1% (w / w) and 0.3% (w / w) based on the sum of (a), (b) and (c), preferably between 0.15% (w / w) and 0.28% (w / w) based on the sum of (a), (b) and (c), for example 0.23±0.1% (w / w) based on the sum of (a), (b) and (c).

[0152] (b) Organic Solvent A suitable water-miscible, lyophilizable organic solvent for the organic phase can be selected by one of skill in the art.

[0153] The organic solvent should be miscible with water at standard conditions (25° C. and 1,013 bar). Furthermore, the organic solvent should be lyophilizable, i.e., the solvent can be removed by sublimation (direct transition of a substance from a solid state to a gaseous state without passing through a liquid state). Those skilled in the art know how to select a suitable organic solvent, as well as the appropriate temperatures and pressures for lyophilizing the organic solvent, for example, by using pressure-temperature (PT) phase diagrams of the solvents known in the art.

[0154] Preferred organic solvents are selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, and alcohols, such as methanol, ethanol, propanol, butanol, or pentanol.

[0155] In one preferred embodiment, the organic solvent is anisole (CAS 100-66-3).

[0156] In one preferred embodiment, the organic solvent is ethyl acetate (CAS 141-78-6).

[0157] In one preferred embodiment, the organic solvent is 1,4-dioxane (CAS 123-91-1).

[0158] In one preferred embodiment, the organic solvent is dimethyl carbonate (CAS 616-38-6).

[0159] In one preferred embodiment, the organic solvent is dimethylsulfoxide (CAS 67-68-5).

[0160] In one preferred embodiment, the organic solvent is glycofurol (CAS 31692-85-0).

[0161] In one preferred embodiment, the organic solvent is N,N-dimethylacetamide (CAS 127-19-5).

[0162] In one preferred embodiment, the organic solvent is N,N-dimethylformamide (CAS 68-12-2).

[0163] In one preferred embodiment, the organic solvent is N-methyl-2-pyrrolidone (CAS 872-50-4).

[0164] In one preferred embodiment, the organic solvent is isopropylidene glycerol (CAS 100-79-8).

[0165] In one preferred embodiment, the organic solvent is 1-butanol (CAS 71-36-3).

[0166] In one preferred embodiment, the organic solvent is 2-butanol (CAS 78-92-2).

[0167] In one preferred embodiment, the organic solvent is tert-butanol (CAS 75-65-0).

[0168] Surprisingly, it has been found that the use of butanol, preferably tert-butanol (TBA), results in a preferred nanodispersion when the organic phase from steps (A.1) / (B.1) / (C.1) is combined with the aqueous phase from steps (A.2) / (B.2) / (C.2), respectively.

[0169] Thus, in one more preferred embodiment, the organic solvent is tert-butanol (CAS 75-65-0).

[0170] In one preferred embodiment, one or more organic solvents are present in the organic phase in an amount of 60% (w / w) to 97% (w / w) based on the sum of (a), (b) and (c), preferably 65% ​​(w / w) to 85% (w / w), for example 74.5±2% (w / w), based on the sum of (a), (b) and (c) in method (A), based on the sum of (i), (ii) and (iii) in method (B), or based on the sum of (1), (2) and (3) in method (C), respectively.

[0171] (c) Additional organic components In general, any organic component may be present, however it is preferred if the organic component is pharma- ceutically acceptable to humans.

[0172] Preferably, the organic component is a sugar, such as trehalose, a polysaccharide, such as cellulose or sodium carboxymethylcellulose, a lipid, such as cholesterol, an amino acid, an amino acid chain, or the like.

[0173] Cholesterol is known to affect liposome stability and drug release.

[0174] Thus, in one preferred embodiment, the organic phase of the process according to the invention preferably further comprises cholesterol or a derivative thereof.

[0175] Another preferred embodiment relates to an organic phase in which the additional organic component is selected from the group consisting of cholesterol or sodium cholesteryl sulfate. More preferably, the additional organic component is cholesterol.

[0176] Another preferred embodiment relates to a formulation in which the amount of cholesterol is from 1% (w / w) based on the sum of (a), (b) and (c) to 8% (w / w) based on the sum of (a), (b) and (c), preferably from 2% (w / w) to 4.4% (w / w) based on the sum of (a), (b) and (c), for example 3.2±0.3% (w / w).

[0177] In one preferred embodiment, the sum of the weights of (a), (b), and (c) is 95% (w / w) or more based on the total weight of the organic phase, preferably 98% (w / w) or more based on the total weight of the organic phase.

[0178] In one preferred embodiment, the organic phase consists of (a), (b), and (c).

[0179] In another preferred embodiment, (a) consists of PC and PG, preferably PC and DOPG, (b) consists of TBA, and (c) consists of cholesterol.

[0180] In another preferred embodiment, the organic phase consists of (a), (b), and (c), where (a) comprises PC and PG, preferably PC and DOPG, (b) comprises TBA, and (c) comprises cholesterol.

[0181] In another preferred embodiment, the organic phase consists of (a), (b), and (c), where (a) consists of PC and PG, preferably PC and DOPG, (b) consists of TBA, and (c) consists of cholesterol.

[0182] aqueous phase (d) Aqueous medium The aqueous medium of the aqueous phase is water or a combination of water and a solvent selected from the group consisting of propylene glycol, ethanol, isopropanol, glycerol, glycol ethers (e.g., monobutyl ether of ethylene glycol or propylene glycol, or monoethyl ether or monomethyl ether of diethylene glycol, or any combination thereof), polyethylene glycol (PEG) 300, PEG 400, specifically, propylene glycol and ethanol, and the amount of water relative to the total amount of solvent in the aqueous medium is at least 60%, preferably at least 90%, more preferably at least 95%.

[0183] In one preferred embodiment, water is the only solvent in the aqueous medium and thus the only solvent of the aqueous phase.

[0184] Generally, the aqueous phase comprises an aqueous medium having an amount of aqueous medium ranging from 76% (w / w) to 92% (w / w) based on the sum of (d), (e), (f), and (g), more preferably from 80% (w / w) to 88% (w / w), for example, 84.2±2% (w / w), based on the sum of (d), (e), (f), and (g).

[0185] (e) additional components selected from the group consisting of buffer systems and acids and / or bases for adjusting pH (e.g., NaOH, HCl); Optionally, the aqueous phase may contain a buffer system. Suitable pharma- ceutically acceptable buffer systems are well known to those skilled in the art. Non-limiting examples are systems based on sodium phosphate, citric acid, acetic acid, tromethamine (TRIS), histidine, gluconic acid, lactic acid, tartaric acid, aspartic acid, glutamic acid, citric acid, fumaric acid, α-ketoglutaric acid, malic acid, and succinic acid. Those skilled in the art will know how to select a buffer for a particular pH value. For example, acetate buffers (e.g., acetate / acetic acid) may be used for pH adjustments of 3.7 to 6.5, phosphate or citrate buffers (e.g., Na2HPO4 / citric acid, Na2HPO4 / NaH2PO4, or Na2HPO4 / NaOH) may be used for pH adjustments of 5.4 to 8.0, and sodium citrate / citric acid may be used for pH adjustments of 3.0 to 6.2.

[0186] Optionally, the aqueous phase may contain HCl and / or NaOH. These two components may be used, for example, to adjust the pH of the aqueous phase, optionally containing dutogliptin, to be in the range of 6 to 8, preferably 7 to 8, more preferably 7.0 to 7.8, e.g., 7.4±0.2.

[0187] (f) Bulking agents The aqueous phase may optionally contain a bulking agent. Suitable bulking agents for liposomal dutogliptin formulations, and therefore for the aqueous phase for preparing such formulations, are as described above.

[0188] In one preferred embodiment, the bulking agent is trehalose.

[0189] In another preferred embodiment, the bulking agent is present in the aqueous phase in an amount of from 1% (w / w) to 10% (w / w) based on the sum of (d), (e), (f), and (g), more preferably from 3% (w / w) to 8% (w / w) based on the sum of (d), (e), (f), and (g), for example, 5±1% (w / w) based on the sum of (d), (e), (f), and (g).

[0190] In yet another preferred embodiment, the bulking agent is trehalose and the trehalose is present in the aqueous phase in an amount of from 1% (w / w) to 10% (w / w) based on the sum of (d), (e), (f), and (g), more preferably from 3% (w / w) to 8% (w / w) based on the sum of (d), (e), (f), and (g), for example, 5±1% (w / w) based on the sum of (d), (e), (f), and (g).

[0191] (g) dutogliptin Optionally, the aqueous phase contains dutogliptin. However, a condition for the process according to the invention is that either the aqueous phase or the aqueous solution (for reconstituting the lyophilisate) contains dutogliptin.

[0192] In one preferred embodiment, the aqueous phase comprises dutogliptin, wherein the dutogliptin is provided in its free base form.

[0193] In yet another preferred embodiment, the aqueous phase comprises dutogliptin, which is provided in the form of one of the fatty acid salts, preferably the sodium laurate, sodium myristic acid, sodium palmitate, sodium stearate, or sodium cholic acid hydrate.

[0194] In one preferred embodiment, the amount of dutogliptin in the aqueous phase is between 5% (w / w) and 22% (w / w) based on the sum of (d), (e), (f), and (g), preferably between 6% (w / w) and 14% (w / w) based on the sum of (d), (e), (f), and (g), when dutogliptin is provided in its free base form, e.g., 6.6±0.5% (w / w) based on the sum of (d), (e), (f), and (g); or, e.g., 16.3±3% (w / w) based on the sum of (d), (e), (f), and (g), when dutogliptin is provided in one of its free fatty acid salt forms.

[0195] In one preferred embodiment, the method for preparation of the liposomal formulation is method (A) and the aqueous phase is prepared by providing (g) dutogliptin in the tartrate salt form.

[0196] In an even more preferred embodiment, the method for preparation of the liposomal formulation is method (A) and the aqueous phase is prepared by providing (g) dutogliptin in free base form.

[0197] In another preferred embodiment, the method for preparation of the liposomal formulation is method (B), wherein the aqueous phase does not contain (g) and the aqueous solution contains (g), preferably, the aqueous solution is prepared by using the tartrate salt form of dutogliptin.

[0198] In another preferred embodiment, the process for the preparation of liposomal formulations is process (B), wherein the aqueous phase does not contain (g) and the aqueous solution contains (g), preferably in the tartrate salt form.

[0199] In yet another preferred embodiment, the method for preparation of the liposomal formulation is method (B), wherein the aqueous phase does not comprise (g) and the aqueous solution comprises (g), preferably, the aqueous solution is prepared by using the free base form of dutogliptin.

[0200] In another preferred embodiment, the process for preparation of the liposomal formulation is process (B), wherein the aqueous phase does not contain (g) and the aqueous solution contains (g), preferably in free base form.

[0201] In highly preferred embodiments, the components such as the aqueous medium, buffer, and osmolality adjusting agent are all pharma- ceutically acceptable components.

[0202] Generally, the buffering agent is present at a concentration of from 0.05 mM to 100 mM, for example from 1 mM to 50 mM.

[0203] In one preferred embodiment, the aqueous phase comprises (d) water, (e) NaOH and / or HCl (e.g., trace amounts of one or both pH adjusting ingredients), (f) trehalose, preferably trehalose, and (g) dutogliptin free base (see, e.g., method (A)).

[0204] In another preferred embodiment, the aqueous phase comprises (d) water, (e) NaOH and / or HCl (e.g., trace amounts of one or both pH adjusting ingredients), (f) trehalose, and (g) dutogliptin in one of its fatty acid salt forms (see, e.g., method (C)).

[0205] In yet another preferred embodiment, the aqueous phase comprises (d) water, (e) NaOH and / or HCl (e.g., trace amounts of one or both pH adjusting ingredients), (f) trehalose, and (g) dutogliptin free base (see, e.g., method (A)). Preferably, the amount of (d) is 80% (w / w) to 91% (w / w) based on the sum of (d), (e), (f), and (g), the amount of (e) is less than 0.1% (w / w) based on the sum of (d), (e), (f), and (g), the amount of (f) is 3% (w / w) to 8% (w / w) based on the sum of (d), (e), (f), and (g), and the amount of (g) is 6% (w / w) to 14% (w / w) based on the sum of (d), (e), (f), and (g).

[0206] In another preferred embodiment, the aqueous phase comprises (d) water, (e) NaOH and / or HCl (e.g., trace amounts of one or both pH adjusting ingredients), (f) trehalose, and (g) dutogliptin in one of its fatty acid salt forms (see, e.g., method (C)). Preferably, the amount of (d) is 80% (w / w) to 91% (w / w) based on the sum of (d), (e), (f), and (g), the amount of (e) is less than 0.1% (w / w) based on the sum of (d), (e), (f), and (g), the amount of (f) is 3% (w / w) to 8% (w / w) based on the sum of (d), (e), (f), and (g), and the amount of (g) is 6% (w / w) to 14% (w / w) based on the sum of (d), (e), (f), and (g).

[0207] In another preferred embodiment, the sum of the weights of (d), (e), (f), and (g) is 95% (w / w) or more based on the total weight of the aqueous phase, preferably 98% (w / w) or more based on the total weight of the aqueous phase.

[0208] In one preferred embodiment, the aqueous phase consists of (d), (e), (f), and (g).

[0209] aqueous solution The aqueous solution used herein comprises a (pharmaceutical) aqueous medium, preferably water, or a combination of water and any of the solvents selected from the group consisting of propylene glycol, ethanol, isopropanol, glycerol, glycol ether (e.g., monobutyl ether of ethylene glycol or propylene glycol, or monoethyl ether or monomethyl ether of diethylene glycol, or any combination thereof), polyethylene glycol (PEG) 300, PEG 400, specifically propylene glycol and ethanol, or any combination thereof, and preferably the amount of water relative to the total amount of solvent is at least 80%.In even more preferred embodiments, water is the only solvent of the aqueous solution.

[0210] In one embodiment, the aqueous solution consists of water or a combination of water and any of the solvents selected from the group consisting of propylene glycol, ethanol, isopropanol, glycerol, glycol ethers (e.g., monobutyl ether of ethylene glycol or propylene glycol, or monoethyl ether or monomethyl ether of diethylene glycol, or any combination thereof), polyethylene glycol (PEG) 300, PEG 400, specifically propylene glycol and ethanol, or combinations thereof, wherein the amount of water relative to the total amount of solvent is at least 80%. In an even more preferred embodiment, the aqueous pharmaceutical solution consists of water.

[0211] A further preferred embodiment relates to an aqueous solution comprising a solvent as described herein and an osmolality modifier. Preferred osmolality modifiers for use in the aqueous solution are as described above for the formulations according to the invention.

[0212] A further preferred embodiment relates to an aqueous solution comprising a solvent as described herein and an osmotic modifier and a buffer. Preferred osmotic modifiers and buffer systems for use in the aqueous solution are as described above for the liposomal formulation according to the invention. A preferred osmotic modifier is NaCl, preferably the amount of NaCl in the aqueous solution is 0.9±0.1% (w / w), most preferably 0.9% (w / w), based on the total volume of the aqueous solution.

[0213] Preferably, the pH of the aqueous pharmaceutical solution is selected so that the resulting liposomal formulation has a pH of 6-8, preferably 7-8.

[0214] In one embodiment, the aqueous formulation also comprises dutogliptin, preferably in the form of its tartrate salt or its free base form, more preferably in the form of its tartrate salt. Preferably, the amount of dutogliptin is 5% (w / w) to 13% (w / w) based on the total weight of the aqueous solution when dutogliptin is present in its tartrate salt form, more preferably 10±0.5% (w / w); or 6±0.4% (w / w) based on the total weight of the aqueous solution when dutogliptin is present in its free base form.

[0215] In one embodiment, the process for the preparation of a liposomal dutogliptin formulation is process (B), and the aqueous solution comprises dutogliptin, preferably in the tartrate salt form.

[0216] In another preferred embodiment, the process for preparation of a liposomal dutogliptin formulation is process (B), wherein the aqueous solution comprises dutogliptin, preferably dutogliptin in its free base form.

[0217] The skilled person is aware that depending on the preparation method according to the invention, the lyophilisate may already contain a buffer system or such a buffer system is provided together with the aqueous pharmaceutical solution. The skilled person is able to calculate, without undue burden, the requirement for the aqueous pharmaceutical solution for preparing the final liposomal formulation.

[0218] Preferred osmotic agents, buffers, solvents, phospholipids, phosphatidylglycerol, lipopeptides, cholesterol (and derivatives thereof), sucrose components, ratios and amounts of any of them that may be used in the methods of the present invention are as described above for the formulations according to the present invention.

[0219] Calculation of organic phase, aqueous phase, and aqueous solution concentrations / volumes The skilled person will be able, without undue burden, to calculate, depending on the requirements regarding the ratios between the various components, the concentrations and amounts of the various components for the organic and aqueous phases to be combined in steps (A.3) / (B.3) / (C.3), as well as the concentrations and amounts of the various components of the aqueous solutions of the steps to arrive at the formulations according to the invention.

[0220] Surprisingly, it has been found that the formulation of step (A.3) / (B.3) / (C.3) is a nanodispersion system in which the D90 of the lipophilic vesicles is equal to or less than 60 nm, preferably less than 25 nm, more preferably equal to or less than 20 nm, such as between 10 nm and 20 nm, for example around 15 nm, or between 3 nm and 10 nm, for example around 5 nm.

[0221] In other words, and being bound by the description, combining the organic phase from (A.1) / (B.1) / (C.1) with the aqueous phase from (A.2) / (B.2) / (C.2) produces a nanodispersion, possibly a micelle, rather than a liposome, with a particle size D90 of 60 nm or less, preferably less than 25 nm. This nanodispersion can be sterile filtered.

[0222] In one preferred embodiment, the ratio of organic phase to aqueous phase is between 3:1 (v / v) and 1:3 (v / v), more preferably between 2:1 (v / v) and 1:2 (v / v), for example around 1:1 (v / v). For purposes of explanation, "(v / v)" refers to the volume ratio of the two phases, for example a ratio of 1:1 refers to 1 ml of organic phase and 1 ml of aqueous phase.

[0223] In one preferred embodiment, the D90 of the vesicles in the resulting nanodispersion is 15 nm or less, more preferably 10 nm or less, for example, 3 nm to 10 nm, when the ratio between the two phases is 1:1. Without being bound by any explanation, a high proportion of organic solvent may result in a predominantly molecular solution of the components in the solvent mixture.

[0224] In another preferred embodiment, the D90 of the vesicles in the nanodispersion is 60 nm or less, more preferably less than 25 nm, even more preferably 20 nm or less, for example 5 nm to 20 nm, when the ratio between the two phases is 1:3. Without being bound by explanation, at this higher water ratio, a micellar solution is formed that shows the typical size and homogeneity of micelles. The mixture shows an almost transparent appearance with slight Tyndall phenomenon and is freely filterable through a sterile filter with a nominal pore size of 0.22 μm.

[0225] At a solvent mixing ratio of 1:3, the resulting preparation is turbid. Size distribution measurements by DLS reveal a broad, heterogeneous spectrum of vesicles with an average size of about 1.000 μm (1000 nm). This dispersion can be filtered through a membrane sterilizing filter with a nominal pore size of 0.22 μm only by applying high pressure.

[0226] Generally, the steps of methods (A), (B), and (C) may all be carried out at about room temperature (25° C.) and at about standard pressure (101.325 kPa). Unless otherwise specified, for example, for the freeze-drying step, the steps may be individually carried out at temperatures preferably between 0° C. and 40° C., more preferably between 15° C. and 35° C., e.g., between 18° C. and 28° C. Any of the steps may be individually carried out at higher and lower pressures, but preferably any of the steps are individually carried out at a pressure between 90 kPa and 112 kPa, more preferably between 95 kPa and 116 kPa, and most preferably near standard pressure, e.g., 101.325 kPa±2%.

[0227] The skilled artisan is well aware of the freeze-drying (lyophilization) technique. Generally, the freeze-drying of the formulation according to the invention is carried out at a temperature between +40°C and -40°C, preferably between +30°C and -10°C. The freeze-drying process can be repeated once or several times. Generally, the pressure is between 1000hPa and 0.001hPa (1hPa = 1mbar). Preferably, the freeze-drying process is carried out at a pressure between 1hPa and 0.01hPa, for example around 0.1hPa.

[0228] Optionally, the method includes one or more additional steps.

[0229] Preferably, one additional step in the method according to the invention is a step of "sterile filtering" the nanodispersion or the reconstituted lyophilisate solution.

[0230] Preferably, the sterile filtration is performed by using a membrane filter, such as a PVDF membrane filter, preferably having a nominal pore size of 200 nm or less.

[0231] It should be noted that gentle rotation or vortexing of the resulting liposome dispersion is sufficient for homogenization. When the liposome formulation is to be suitable for injection into a patient in need of a lipopeptide formulation, there is no need to reduce the size of the liposomes by additional complicated steps.

[0232] The preparation of liposomal dutogliptin formulations can be carried out according to or similar to the examples below.

[0233] The preparation of liposomal dutogliptin formulations using dutogliptin fatty acid salts can be carried out similarly to Example 1 by replacing dutogliptin tartrate with dutogliptin fatty acid salts.

[0234] The preparation of liposomal dutogliptin formulations using dutogliptin fatty acid salts can be carried out similarly to Example 2 by replacing dutogliptin tartrate with dutogliptin fatty acid salts.

[0235] Preparation of liposomal dutogliptin formulations using dutogliptin fatty acid salts can be carried out by following the route of Example 1, adding the dutogliptin fatty acid salt to the organic phase and then omitting the dutogliptin in the aqueous phase.

[0236] Preparation of liposomal dutogliptin formulations using dutogliptin free base form can be carried out similarly to Example 1 by substituting dutogliptin tartrate with dutogliptin free base form.

[0237] Preparation of liposomal dutogliptin formulations using dutogliptin free base form can be carried out similarly to Example 2 by substituting dutogliptin tartrate with dutogliptin free base form.

[0238] Preparation of liposomal dutogliptin formulations using the dutogliptin free base form can be carried out by adding the dutogliptin free base form to the organic phase and then following the route of Example 1, omitting the dutogliptin in the aqueous phase.

[0239] kit A further aspect of the present invention relates to a kit for preparing a liposomal formulation according to the present invention, comprising a lyophilisate as described herein and, separately therefrom, an aqueous formulation as described herein, e.g., the formulation according to the present invention in one container and an aqueous pharmaceutical solution in another container.

[0240] In one preferred embodiment, the lyophilisate comprises a total amount of dutogliptin between 60 mg and 100 mg, the amount of dutogliptin in the lyophilisate is between 12% (w / w) and 53% (w / w) based on the total amount of the lyophilisate; the amount of (a) is between 7.2% (w / w) and 73% (w / w) based on the total amount of the lyophilisate; the amount of (c), if present, is up to 24.3% (w / w), preferably (c) is a cholesterol-lowering agent. (c) is a glycerol-based lyophilisate having an amount of 0.01 to 0.1% (w / w) based on the total weight of the lyophilisate; (d) is a glycerol-based lyophilisate having an amount of 0.01 to 0.1% (w / w) based on the total weight of the lyophilisate; (e ...f) is a glycerol-based lyophilisate having an amount of 0.01 to 0.1% (w / w) based on the total weight of the lyophilisate;

[0241] In one preferred embodiment, the aqueous pharmaceutical solution is dispensed into another container and the weight of that portion of the aqueous pharmaceutical solution is calculated such that the amounts and ratios of the different components (i), (ii), (iii), (iv) and (v) of the liposomal formulation obtained after adding that portion of the aqueous pharmaceutical solution to the lyophilisate (or vice versa) are those of the liposomal formulation according to the invention.

[0242] In one preferred embodiment, the volume of the aqueous solution added to the lyophilized product is 1.8 ml to 2.2 ml, for example, 2 ml. Preferably, the volume of the aqueous solution in the container refers to the volume for subcutaneous injection.

[0243] Mixing the two components, the aqueous pharmaceutical solution and the lyophilized formulation, allows the pharmaceutical liposomal formulation to be prepared immediately prior to administration to a patient.

[0244] While the described invention has been described with respect to specific embodiments thereof, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step(s) to the objective scope of the invention. All such modifications are intended to be within the scope of the claims appended hereto.

[0245] It should be noted that, where applicable, even if not explicitly mentioned, all preferred ingredients mentioned in any of the phases, solutions, formulations, lyophilisates, kits, or methods also relate to any of the other phases, solutions, formulations, kits, or methods disclosed herein, For example, when a preferred bulking agent is present in the organic phase, the same preferred agent is naturally also a preferred bulking agent for the lyophilisate and the resulting liposomal formulation. [Brief description of the drawings]

[0246] (Figure 1) Figure 1 shows a two-phase system of a dutogliptin aqueous phase (bottom phase) and an organic TBA phase (top phase). The dutogliptin aqueous phase (bottom phase) + TBA phase is shown on the left and the dutogliptin aqueous phase (bottom phase) + TBA / PC / PG phase is shown on the right (1:1 w / w each). (FIG. 2) FIG. 2 shows the online process data chart. The freeze-drying process was completed successfully and the process performed was consistent with the preset parameters described in Section E5. The following parameters are shown in FIG. 2: TIFF2025515490000003.tif29128 (Figure 3) Figure 3 shows a photograph of a 50 mg dutogliptin formulation at 40x magnification (λ / 4 polarizing microscope). There are various types of liposomes: small unilamellar vesicles, large unilamellar vesicles, multilamellar vesicles, and oligolamellar vesicles. EXAMPLES

[0247] Working Example E1 Substances and equipment The following materials and equipment were used (Table):

[0248] Table 1. Materials and equipment TIFF2025515490000004.tif179165TIFF2025515490000005.tif153165

[0249] The following equipment and instruments were used:

[0250] The following equipment and instruments were used: TIFF2025515490000006.tif88137

[0251] Additional Experimental Equipment ·Magnetic stirrer (IKA Werke) Volumetric pipette (Gilson) Camera equipment (Canon EOS 600D with DGMacro 105mm 1:2.8) pH meter (Mettler Toledo, SevenMulti with InLab Micro electrode) ·Balance (Kern,EW6200-2NM,ABJ-NM ABS3204N ·Purified water supply (Siemens, Ultra Clean UV UF TM) Vortex mixer (Scientific Industries Inc., Vortex Genie II) Centrifuge: ThermoScientific, Heraeus Pico 17 Orbital shaker: Wisd Laboratory Instruments, WiseShake SHO-1D

[0252] For RP-HPLC analysis the following protocol was used.

[0253] E2 RP-HPLC method The intended use of this method is to determine the content and purity of dutogliptin drug product. Method AM-C16031637-G-01.03 was used as provided. The following chromatographic conditions were used: TIFF2025515490000007.tif181128

[0254] 25mM sodium dodecyl sulfate (SDS) 7. Weigh 3 g of SDS into 1000 mL of purified water, dissolve and mix thoroughly.

[0255] Mobile phase A: 25mM SDS:ACN:TFA = 600:400:1(v / v / v) To prepare 1 L of mobile phase, combine 600 mL of 25 mM SDS solution with 400 mL of ACN and 1.0 mL of TFA in a suitable container and mix thoroughly. Filter through a 0.45 μm nylon membrane (or other suitable solvent-resistant filter) and degas before use.

[0256] Mobile phase B: 25mM SDS:ACN:TFA = 400:600:1(v / v / v) To prepare 1 L of mobile phase, combine 400 mL of 25 mM SDS solution with 600 mL of ACN and 1.0 mL of TFA in a suitable container and mix thoroughly. Filter through a 0.45 μm nylon membrane (or other suitable solvent-resistant filter) and degas before use.

[0257] Diluent Mobile phase A

[0258] E3 Salt Precipitation / Anion Exchange Assuming that the ionic lattice of dutogliptin tartrate (dissolved in water) is pulled apart and the individual dutogliptin and tartrate ions are in solution, by adding excess fatty acid anions, an exchange should occur, resulting in the formation of a hydrophobic salt, which should precipitate due to reduced solubility.

[0259] To facilitate the exchange, the salts dissolved at room temperature were cooled to 2-8°C after addition of dutogliptin tartrate and stirred overnight.

[0260] E4 Liposome Encapsulation Efficiency / Trapped Volume Liposomes were prepared in a solution of calcein, a fluorescent dye. After addition of cobalt(II) ions, which quench the fluorescence of calcein when they form a chelate complex with it, the fraction of fluorescence remaining was taken as the fraction within the liposome relative to the total volume. Multilamellar placebo liposome lyophilisates were prepared according to the method described in Example 1 without dutogliptin tartrate and reconstituted in 1 mL of Mops (4-morpholinepropanesulfonic acid) buffered saline (pH 7.2) containing 50 nmol calcein.

[0261] 6.25 μL of the liposome suspension was diluted to 250 μL with Mops-buffered saline and transferred to a 96-well plate prior to the addition of 1.25 μL of 10 mM CoCl2 (F tot ) and after addition (F inThe fluorescence of the suspension was measured. The parameters of the fluorescence plate reader are shown in Table 2. Subsequently, 12.5 μL of 10% Triton X-100 was added and the fluorescence was measured again (F totq The addition of the latter destroyed the integrity of the liposomes. The trapped volume was: TIFF2025515490000008.tif11128, where: F tot represents the fluorescence of all calcein present, F in represents the fluorescence from the unquenched fractions of the inner and outer compartments, F totq represents the fluorescence of free calcein at equilibrium concentration, r represents the dilution factor due to the addition of Triton X-100 and cobalt (in this case 1.06).

[0262] Table 2: Tecan fluorescence plate reader parameters TIFF2025515490000009.tif34128

[0263] E5 Lyophilization Lyo-solution (nano dispersion) was filled into sterile glass vials (1.5 g in 6R vials). The filled vials were stoppered in the freeze-drying position loaded into the freeze-dryer. The following freeze-drying cycle was performed:

[0264] Table 3: A typical conservative freeze-drying process program TIFF2025515490000010.tif121159

[0265] E6 Polarizing Microscope A drop of the thoroughly mixed reconstituted liposome formulation was placed on a microscope slide and covered with a microscope cover slip. To identify encapsulated dutogliptin / liposomes, the samples were examined at 400x magnification with and without polarizers. Photographs were taken at 40x magnification.

[0266] The following equipment was used: Microscope: Axio Lab. A1-HAL with polarizer, Zeiss; Camera system: EOS 600D, Canon.

[0267] Example 1: Tartrate Method (A): Preparation of nanodispersions with aqueous phase containing dutogliptin tartrate For the formation of liposomes, two phases were required: an organic solution and an aqueous solution.

[0268] organic phase For the organic phase, phosphatidylglycerol (PG) and S100 (highly purified soy lecithin) were dissolved in tert-butanol (TBA) by heating (70°C) and stirring for 40-80 min.

[0269] Once the components were homogeneously dissolved, the organic phase was cooled to ambient temperature.

[0270] aqueous phase For the aqueous phase, dutogliptin tartrate solution from Recardio (adjusted to pH 7.4) and trehalose were added to the water with stirring and allowed to dissolve.

[0271] The aqueous solution is added to the organic phase in small portions under constant stirring. Depending on the amount of aqueous phase added to the organic phase, different liquid crystal phases can occur. The first part of the aqueous solution dissolves into a clear micellar system. With further addition of the aqueous solution, cubic, hexagonal and finally lamellar phases with different viscosities and turbidities can appear. After complete addition of the aqueous phase, a milky nanodispersion with low viscosity results.

[0272] The final bulk solution is sterile filtered through a PVDF membrane filter with a nominal pore size of 200 nm. TIFF2025515490000011.tif131139

[0273] Direct encapsulation of dutogliptin tartrate Typically, in this mixture with lipids, a stable homogeneous nanodisperse mesophase is formed. However, in this case with dutogliptin tartrate dissolved in the aqueous phase, the organic phase containing TBA and the aqueous phase do not form a stable solution, and after a short time (5 minutes or less), phase separation of the nanodisperse system occurs.

[0274] Comparison with the lipid-free TBA solution and the aqueous phase in which dutogliptin tartrate was dissolved showed that the occurrence of a two-phase system was more evident and formed spontaneously (see FIG. 1).

[0275] The emulsion was constantly stirred during filling to prevent phase separation. The emulsion was filled into 6R Type 1 glass vials at 1.5 g / vial. The filled vials were transferred to a freeze-dryer and the freeze-drying process was started. The freeze-drying process was carried out as described in Section E5.

[0276] Reconstitution of the lyophilized cake for preparation of the liposomal suspension was performed by adding water, and a stable liposomal suspension was rapidly formed.

[0277] The reconstituted lyophilisates were observed under a polarising microscope immediately after reconstitution / homogenisation at 22° C. Photographs were taken for documentation purposes (see FIG. 3).

[0278] Liposomal encapsulation of dutogliptin was studied by determining the amount of free (unencapsulated) dutogliptin. Equal intraliposomal and extraliposomal volumes refer to a 1:1 equilibrium concentration of the hydrophilic active pharmaceutical ingredient (API). The reduction of dutogliptin tartrate in the extraliposomal matrix results in a positive liposomal encapsulation efficiency.

[0279] After reconstitution, the liposomal dispersion was drawn into a standard disposable 3 mL PP syringe. A 27G x 1 inch cannula (Sterican, B. Braun) was attached and any remaining air was removed from the syringe. The contents of the syringe were manually expelled. Evaluation of the expulsion force indicated that a force of up to 20 N, more preferably up to 15 N, was sufficient to expel the liposomal dutogliptin tartrate formulation.

[0280] Centrifugation to separate liposomes and aqueous phase is only possible with MLV dispersions. Therefore, the amount of dutogliptin in the extraliposomal volume was determined by centrifugation of the lyophilisate reconstituted with water. The lyophilisate was mixed with 1 mL of saline, shaken and stored at room temperature for 20 min under gentle stirring to allow liposome formation. Subsequent centrifugation (17000×g for 5 min) did not allow successful separation of the liposomes. Successful separation was only achieved after further dilution with 1 mL of water (total water volume of 2 mL). To avoid possible dilution errors, a comparative variant was made that was directly mixed with 2 mL of saline. The lipid-free supernatants of these samples were analyzed by RP-HPLC (Table 4).

[0281] Adjusting for equal intraliposomal and extraliposomal volumes predicts a 1:1 equilibrium concentration of hydrophilic dutogliptin. A decrease in the concentration of dutogliptin in the extraliposomal matrix indicates a positive liposomal encapsulation efficiency.

[0282] Table 4. RP-HPLC results: Concentration of dutogliptin in the extraliposomal volume after liposome preparation TIFF2025515490000012.tif57159 * The lyophilisate was reconstituted in 2 mL and, after liposome generation, further diluted with 2 mL WFI. ** Decreased extraliposomal dutogliptin concentrations.

[0283] Example 2: Method (B) Reconstitution of (placebo) lipid lyophilisates with aqueous dutogliptin solutions and determination of free drug concentrations Packaging of unstable emulsions prone to phase separation is difficult in large scale manufacturing, therefore alternative manufacturing methods were examined.

[0284] First, a (placebo) liposomal lyophilizate without dutogliptin tartrate was prepared according to the method described above. Then, 0.9% NaCl (saline) was added to the dutogliptin tartrate solution to increase the imagery and zeta potential. The resulting solution (100 mg / mL, 0.9% NaCl) was added to the (placebo) lyophilizate. After gently rotating the vial for a few minutes, a homogenous dutogliptin liposomal emulsion was formed.

[0285] The reconstituted vials were centrifuged directly and after addition of 1 mL of saline (0.9% NaCl solution) (Table 5). The concentration of dutogliptin was determined in the separated aqueous phase by RP-HPLC.

[0286] Table 5. RP-HPLC results: Concentration of dutogliptin in the extraliposomal volume after liposome preparation with dutogliptin tartrate solution (100 mg / mL, 0.9% NaCl) TIFF2025515490000013.tif89134 * The lyophilisate was reconstituted in 1 mL and, after liposome generation, further diluted with 1 mL of saline.

[0287] Phase separation was possible by centrifugation. However, due to the very high G-forces in centrifugation, liposomes may be damaged and leak out, leading to uncertain results about the free drug concentration. However, the results are close to the theoretical values. Since dutogliptin tartrate is highly water soluble, it is expected that the drug concentrations inside and outside the liposomes are equal and that the drug is not associated with the lipid phase of the vesicles.

[0288] To determine the amount of encapsulated drug, the volume of the aqueous phase entrapped in the liposomes must be quantified.

[0289] Trapped volume and derived entrapment efficiency There are some uncertainties in the literature method for determining the intraliposomal volume described in 3.6. Evaluation of fluorescence measurements showed an increase in the fluorescence quenching of liposomal solutions compared to free calcein solutions. These are not structurally determined but rather by the components of the liposomal preparation, necessitating individual consideration of the results depending on the liposomal composition.

[0290] Experiments to determine intraliposomal volume loading were performed in triplicate. A series of tests with doubly diluted / reconstituted liposome solutions were performed. A summary is provided in Table 6.

[0291] Table 6: Normalized fluorescence signals (free units) TIFF2025515490000014.tif85159 * F tot represents the fluorescence of all calcein present; F in represents the fluorescence from the unquenched fractions of the inner and outer compartments; F totq represents the fluorescence of free calcein at equilibrium concentration.

[0292] A mass-dependent liposome fraction of 12.8% (w / w) of the reconstituted lyophilisate determined an internal volume of approximately 30% of the total volume.

[0293] The encapsulation efficacy for the reconstituted liposomal formulation with 12.8% lipid, 100 mg total drug, and 1.0 mL saline was calculated by the following formula: m L = 100mg - 100mg / mL + V L * 0.3mL * 100mg / mL = 30mg m L = Mass of DGT encapsulated in liposomes V L = Aqueous phase volume of liposomes Conclusion: Approximately one-third of the total drug is encapsulated within the vesicles.

[0294] Example 3: Method (C): Preparation of Liposomal Dutogliptin Formulations with Dutogliptin Fatty Acid Salts Five different physiological fatty acids (as sodium salts) were prepared by anion exchange of the tartrate salt with two-fold molar addition of the salt at low temperature (reduced solubility of dutogliptin tartrate). The most promising fatty acid was selected for further preparation. The solubility of the fatty acid salts at 2-8°C was investigated (see table).

[0295] Table 7. Sodium fatty acid salts for the preparation of water insoluble dutogliptin salts TIFF2025515490000015.tif94159

[0296] Tests on the solubility of the sodium salts showed that of the salts used, only sodium cholate remained sufficiently soluble at the given conditions (cooling to 2-8°C). After addition of a 100 mg / ml dutogliptin tartrate solution, the solution turned slightly yellow, but there was no visual evidence of turbidity or salt precipitation.

[0297] Example 4: Method (A) Preparation of Liposomal Dutogliptin Formulations with Dutogliptin Free Base The protocol of the Comparative Example can be used with the modification that dutogliptin tartrate is replaced with the free base form of dutogliptin.

Claims

1. A method for preparing a liposomal dutogliptin formulation, comprising the following steps: （B） (B.1) Below: (a) one or more phospholipids, wherein the amount of one or more phospholipids is 3% (w / w) to 30% (w / w) based on the sum of (a), (b), and (c), preferably 22.36% (w / w) based on the sum of (a), (b), and (c); (b) One or more organic solvents selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycoflor, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, 1-butanol, 2-butanol, and tert-butanol, or any combination thereof, wherein the amount of the one or more organic solvents is 60% (w / w) to 97% (w / w) based on the sum of (a), (b), and (c), preferably 74.43% (w / w) based on the sum of (a), (b), and (c); and (c) Optionally, one or more additional organic components that are neither (a) nor (b), the amount of which is up to 10% based on the sum of (a), (b), and (c). A step of providing an organic phase comprising (a), (b), and (c) such that the sum of (a), (b), and (c) is 100%; Furthermore (B.2) Below: (d) an aqueous medium in an amount of 66% (w / w) to 90% (w / w) based on the sum of (d), (e), and (f), preferably 84% (w / w) based on the sum of (d), (e), and (f); (e) Optionally, one or more additional components selected from the group consisting of buffer systems, NaOH, and HCl, wherein the amount of all additional components is at most 2% (w / w) based on the sum of (d), (e), and (f); and (f) Optionally, the amount of the bulking agent is up to 10% (w / w) based on the sum of (d), (e), and (f), preferably 3% (w / w) to 7% (w / w) based on the sum of (d), (e), and (f), preferably trehalose. A step of providing an aqueous phase containing (d), (e), and (f) such that the sum of (d), (e), and (f) is 100%; Furthermore (B.3) A step of mixing the aqueous phase and the organic phase until a ratio of aqueous phase to organic phase of 3:1 to 1:3, preferably 1:1, is reached, resulting in a nanodispersion; Furthermore (B.4) A step of freeze-drying the nanodispersion system to obtain a freeze-dried product; Furthermore (B.5) A step of reconstituting the lyophilized product from step (B.4) with an aqueous solution to obtain a liposome dutogliptin formulation, wherein the final concentration of dutogliptin in the liposome dutogliptin formulation is preferably 25 mg / ml to 60 mg / ml, and the aqueous solution is (i) an aqueous medium of 67% (w / w) and 100% (w / w) based on the total weight of the aqueous solution, optionally further comprising up to 20% (w / w) of an osmotic pressure regulator based on the total weight of the aqueous solution, and optionally further comprising a buffer system, and (ii) Dutogliptin, preferably in the form of its tartrate or free base, wherein the amount of dutogliptin is 5% (w / w) to 13% (w / w), more preferably 10 ± 0.5% (w / w), based on the total weight of the aqueous solution when the dutogliptin is present in the form of its tartrate or free base; or 6 ± 0.4% (w / w), based on the total weight of the aqueous solution when the dutogliptin is present in the form of its free base. The process, including the process.

2. (i) The organic solvent is tert-butanol (TBA), and / or (ii) The method according to claim 1, wherein the organic phase further contains cholesterol, the amount of which is 2.5% (w / w) to 4% (w / w), preferably 3.2% (w / w).

3. The method according to claim 1, wherein the organic phase contains lecithin as a phospholipid, and the amount of lecithin is 18.1% (w / w) to 26.2% (w / w), more preferably 22.13% (w / w).

4. The method according to claim 1, wherein the organic phase further contains PG, preferably DOPG-Na, as a phospholipid, and the total amount of PG is 0.1% (w / w) to 0.4% (w / w), preferably 0.23% (w / w).

5. The method according to claim 1, wherein the pH of the aqueous phase is 7 to 7.8, preferably 7.

4.

6. The method according to claim 1, wherein the D90 of the liposomes of the liposomal dutogliptin preparation obtained from the reconstitution step is 1 μm to 4.5 μm.

7. The method according to claim 1, wherein the sole solvent of the aqueous solution is water.

8. The method according to claim 1, wherein the aqueous solution is an aqueous solution of NaCl, and the amount of NaCl is 8 g / l to 10 g / l, preferably 8.8 g / l to 9.2 g / l, more preferably 9 g / l.

9. The method according to claim 1, wherein the aqueous solution in step (B.5) is a dutogliptin tartrate solution.

10. The method according to claim 9, wherein the aqueous solution further comprises 9 g / l NaCl.

11. The method according to claim 1, wherein the total amount of dutogliptin in the liposomal dutogliptin preparation is 60 mg to 100 mg.

12. A liposomal dutogliptin preparation that can be prepared by the method described in any one of claims 1 to 11.

13. (vi) an aqueous medium, preferably water, wherein the amount of the aqueous medium is 65% (w / w) to 90% (w / w) based on the total weight of the liposomal dutogliptin preparation; (vii) Optionally, the amount of lipids, preferably cholesterol, is 1% (w / w) to 2.5% (w / w) based on the total weight of the liposomal dutogliptin preparation; (viii) The amount of one or more phospholipids is 7% (w / w) to 15% (w / w), preferably 11.2 ± 0.4 (w / w), based on the total weight of the liposomal dutogliptin preparation, and preferably the one or more phospholipids are PC, PG, and DSPG; (ix) Optionally, one or more agents selected from the group consisting of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, xylitol, xylose, dextran, dextrose, and NaCl, wherein the total amount of the one or more agents is 1.5% (w / w) to 5.5% (w / w); and (x) The amount of dutogliptin, preferably in the form of its tartrate or free base, is 3% (w / w) to 12% (w / w), preferably 5.3 ± 0.3 (w / w), based on the total weight of the liposomal dutogliptin preparation, and the final concentration of dutogliptin in the liposomal dutogliptin preparation is 25 mg / ml to 60 mg / ml. A liposomal dutogliptin preparation comprising (i) to (v), wherein the sum of (i) to (v) is 100%.

14. A kit comprising a freeze-dried product according to any one of claims 1 to 5 in one container, and a pharmaceutical aqueous solution according to claim 1, 7, 8, or 10 in a second container.

15. The kit according to claim 14, further comprising a manual for a method of combining the contents of the first and second containers to obtain the liposomal dutogliptin formulation according to claim 12.

16. The kit according to claim 14, further comprising a manual for a method of combining the contents of the first and second containers to obtain the liposomal dutogliptin formulation according to claim 13.

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