Novel duogliptin preparation and preparation method thereof

Abstract

The invention provides a novel duogliptin liposome preparation medicine and a preparation method thereof.

Description

Background Art

[0001] Dulagliptin (dugliptin, CAS 852329-66-9) is designed as a regenerative drug to reduce and repair myocardial damage. As an inhibitor of the dipeptidyl peptidase-IV (DPP4) enzyme, it is involved in the degradation of stromal derived factor-1α (SDF-1α). In actual clinical trials, the drug was given to patients twice a day, 60-100 mg subcutaneous injection in combination with a subcutaneous dose of filgrastim.

[0002] Usually, dulagliptin is prepared into its tartrate. Dulagliptin was originally administered orally. However, for acute cardiac indications, parenteral administration is required to obtain optimal pharmacokinetic and pharmacodynamic activity. In order to achieve the desired activity with the current preparation, it is necessary to administer at least twice a day for 2 weeks, which leads to problems with patient compliance, acceptance and injection site. Therefore, it is desirable to reduce the frequency of parenteral administration. Therefore, the purpose of the present application is to meet this demand and reduce the number of parenteral administrations, and therefore the purpose of the present invention is to provide a preparation of (freeze-dried) multilamellar vesicles (MLV) containing encapsulated dulagliptin, and to provide a liposomal dulagliptin preparation. These are advantageously suitable for, for example, subcutaneous (sc) or intramuscular (im) applications. Summary of the invention

[0003] The first aspect of 1A relates to a method for preparing a liposomal dugliptin formulation, comprising the following steps:

[0004] (A)

[0005] A.1) providing an organic phase, wherein the organic phase comprises

[0006] (a) one or more phospholipids, wherein the amount of the 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);

[0007] (b) one or more organic solvents selected from anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, tetrahydrofuran glycol ether, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropyl glycerol, 1-butanol, 2-butanol and tert-butanol or any combination of the foregoing; wherein the amount of the one or more organic solvents is based on (a),

[0008] The sum of (b) and (c) is 60% (w / w) to 97% (w / w), preferably 74.43% (w / w) based on the sum of (a), (b) and (c);

[0009] (c) optionally one or more further organic components which are neither (a) nor (b), wherein the amount of the one or more further organic components is up to 10% (w / w), based on the sum of (a), (b) and (c);

[0010] wherein the sum of (a), (b) and (c) is preferably 100%; and

[0011] A.2) providing an aqueous phase, wherein the aqueous phase comprises

[0012] (d) an aqueous medium, wherein 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);

[0013] (e) optionally one or more further components selected from buffer systems, pH adjusting acids and / or bases (e.g. NaOH, HCl) and stabilizers, wherein the amount of all further components is at most 2% (w / w) based on the sum of (d), (e), (f) and (g);

[0014] (f) optionally a bulking agent, preferably trehalose, wherein the amount of the 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);

[0015] (g) dulagliptin, preferably wherein the dulagliptin is provided in the form of its tartrate salt or its free base form, more preferably in the form of its free base, wherein when dulagliptin is provided in the form of its tartrate salt, the amount of dulagliptin is 5% (w / w) to 22% (w / w) based on the sum of (d), (e), (f) and (g), preferably 11±1% (w / w) based on the sum of (d), (e), (f) and (g), or when dulagliptin is provided in the form of its free base, it is (6.2±0.7)% (w / w) based on the sum of (d), (e), (f) and (g);

[0016] wherein the sum of (d), (e), (f) and (g) is 100%; and

[0017] A.3) mixing the aqueous phase and the organic phase until the ratio of the aqueous phase to the organic phase reaches 3:1 to 1:3, preferably 1:1, to obtain a nano-dispersion system containing doxepin; and

[0018] A.4) freeze-drying the nanodispersion system comprising dulagliptin to obtain a lyophilisate; and

[0019] A.5) reconstituting the lyophilisate described in step A.4 with an aqueous solution to produce a liposomal dulagliptin formulation, wherein the aqueous solution comprises 80% (w / w) to 100% (w / w) of an aqueous medium based on the total weight of the aqueous solution, optionally further comprising a tonicity regulator, wherein the amount of the tonicity regulator is up to 20% (w / w) based on the total weight of the aqueous solution, optionally further comprising a buffer system, preferably, wherein the final concentration of dulagliptin in the liposomal dulagliptin formulation is 25 mg / ml to 60 mg / ml; or

[0020] (B)

[0021] B.1) providing an organic phase comprising

[0022] (a) one or more phospholipids, wherein the amount of the 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);

[0023] (b) one or more organic solvents selected from anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropyl glycerol, 1-butanol, 2-butanol and tert-butanol or any combination of the foregoing; wherein the amount of the one or more organic solvents is 60% (w / w) and 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);

[0024] (c) optionally one or more further organic components which are neither (a) nor (b), wherein the amount of the one or more further organic components is up to 10%, based on the sum of (a), (b) and (c);

[0025] wherein the sum of (a), (b) and (c) is 100%; and

[0026] B.2) providing an aqueous phase, comprising

[0027] (d) an aqueous medium, wherein the amount of the aqueous medium is 66% (w / w) and 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);

[0028] (e) optionally one or more further components selected from a buffer system, NaOH, HCl and a stabilizer, wherein the amount of all further components is at most 2% based on the sum of (d), (e) and (f)

[0029] (w / w);

[0030] (f) optionally a bulking agent, preferably trehalose, wherein 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);

[0031] wherein the sum of (d), (e) and (f) is 100%; and

[0032] B.3) mixing the aqueous phase and the organic phase until the ratio of the aqueous phase to the organic phase reaches 3:1-1:3, preferably 1:1, to obtain a nano-dispersed system;

[0033] B.4) freeze-drying the nanodispersion system to obtain a lyophilized product.

[0034] B.5) reconstitute the lyophilized product described in step B.4 with an aqueous solution to obtain a liposomal dulalagliptin preparation, preferably, wherein the final concentration of dulalagliptin in the liposomal dulalagliptin preparation is 25 mg / ml to 60 mg / ml, wherein the aqueous solution comprises

[0035] (i) from 67% (w / w) to 100% (w / w) of an aqueous medium, based on the total weight of the aqueous solution, optionally further comprising a tonicity adjuster, wherein the amount of the tonicity adjuster is at most 20% (w / w), based on the total weight of the aqueous solution, optionally further comprising a buffer system,

[0036] (ii) dulagliptin, preferably in the form of its tartrate salt or its free base form, wherein when dulagliptin is in the form of its tartrate salt or its free base form, the amount of dulagliptin is 5% (w / w) to 13% (w / w), more preferably 10±0.5% (w / w), based on the total weight of the aqueous solution; or when dulagliptin is in the form of its free base, the amount is (6±0.4)% (w / w), based on the total weight of the aqueous solution; or

[0037] (C)

[0038] C.1) providing an organic phase, wherein the organic phase comprises

[0039] (a) one or more phospholipids, wherein the amount of the one or more phospholipids is 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);

[0040] (b) one or more organic solvents selected from anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropyl glycerol, 1-butanol, 2-butanol and tert-butanol or any combination of the foregoing; 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),

[0041] (c) optionally one or more further organic components which are neither (a) nor (b), wherein the amount of the one or more further organic components is up to 10% (w / w), based on the sum of (a), (b) and (c);

[0042] wherein the sum of (a), (b) and (c) is 100%; and

[0043] C.2) providing an aqueous phase, comprising

[0044] (d) an aqueous medium, wherein the amount of the aqueous medium is 66% (w / w) and 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);

[0045] (e) optionally one or more further components selected from buffer systems, pH adjusting acids and / or bases (e.g. NaOH, HCl) and stabilizers, wherein the amounts of all further components are based on (d), (e),

[0046] The sum of (f) and (g) is at most 2% (w / w);

[0047] (f) optionally a bulking agent, preferably trehalose, wherein the amount of the bulking agent is up to 10% (w / w) based on the sum of (d), (e), (f) and (g), preferably 3% based on the sum of (d), (e), (f) and (g)

[0048] (w / w) to 7% (w / w);

[0049] (g) Dulagliptin, wherein the Dulagliptin is provided in the form of one of its fatty acid salts, wherein the amount of the fatty acid salt of Dulagliptin is 5% (w / w) and 22% (w / w) based on the sum of (d), (e), (f) and (g).

[0050] (w / w);

[0051] wherein the sum of (d), (e), (f) and (g) is 100%; and

[0052] C.3) mixing the aqueous phase and the organic phase until the ratio of the aqueous phase to the organic phase reaches 3:1-1:3, preferably 1:1, to obtain a nano-dispersion system containing doxepin;

[0053] C.4) lyophilizing in a nanodispersion system comprising dulagliflozin to obtain a lyophilisate;

[0054] C.5) reconstitute the lyophilisate of step C.4 with an aqueous solution to produce a liposomal dulalagliptin formulation, wherein the aqueous solution comprises 80% (w / w) to 100% (w / w) of an aqueous medium based on the total weight of the aqueous solution, optionally further comprising a tonicity regulator, wherein the amount of the tonicity regulator is up to 20% (w / w) based on the total weight of the aqueous solution, optionally further comprising a buffer system, preferably, wherein the final concentration of dulalagliptin in the liposomal dulalagliptin formulation is 25 mg / ml to 60 mg / ml.

[0055] One embodiment of aspect 1 or any dependent embodiment thereof relates to a process, wherein the organic solvent is tert-butyl alcohol (TBA).

[0056] Another embodiment of the 1st aspect or any dependent embodiment thereof relates to the method, wherein the organic phase further comprises cholesterol, and wherein the amount of cholesterol is 2.5% (w / w) to 4% (w / w), preferably 3.2% (w / w).

[0057] Another embodiment of aspect 1 or any dependent embodiment thereof relates to the method, wherein the organic phase comprises the phospholipid lecithin, and wherein the amount of lecithin is from 18.1% (w / w) to 26.2%

[0058] (w / w), more preferably 22.13% (w / w).

[0059] Another embodiment of aspect 1 or any dependent embodiment thereof relates to a method, wherein the organic phase further comprises PG as phospholipid, preferably DOPG-Na, wherein the total amount of PG is from 0.1% (w / w) to 0.4% (w / w), preferably 0.23% (w / w).

[0060] Another embodiment of aspect 1 or any dependent embodiment thereof relates to the process, wherein the pH of the aqueous phase is between 7 and 7.8, preferably 7.4.

[0061] Another embodiment of the first aspect or any dependent embodiment thereof relates to the method, wherein the D90 of the liposomes of the liposomal dulalagliptin formulation produced by the reconstitution step is from 1 μm to 4.5 μm.

[0062] Another embodiment of aspect 1 or any dependent embodiment thereof relates to the method, wherein the only solvent in the aqueous solution is water.

[0063] Another embodiment of aspect 1 or any dependent embodiment thereof relates to the method, wherein the aqueous solution is an aqueous NaCl solution, wherein 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.

[0064] Another embodiment of aspect 1 or any dependent embodiment thereof relates to the method, wherein said aqueous solution in step B.5 is a tartrate gliptin solution.

[0065] Another embodiment relates to the latter embodiment, wherein the aqueous solution further comprises 9 g / l NaCl.

[0066] Another embodiment of the first aspect or any dependent embodiment thereof relates to the method, wherein the total amount of dolagliflozin in said liposomal dolagliflozin formulation is between 60 mg and 100 mg.

[0067] Another aspect (Aspect 2) relates to a liposomal dugliptin formulation comprising

[0068] (i) an aqueous medium, preferably water, wherein the amount of the aqueous medium is 65% (w / w) and 90% (w / w) based on the total weight of the liposomal doxepin formulation;

[0069] (ii) optionally a lipid, preferably cholesterol, wherein the amount of the lipid, preferably cholesterol, is 1% (w / w) to 2.5% (w / w) based on the total weight of the liposomal dugliptin formulation;

[0070] (iii) one or more phospholipids, wherein the amount of the one or more phospholipids is 7% (w / w) and 15% (w / w), preferably (11.2±0.4) (w / w), based on the total weight of the liposomal dugliptin formulation, preferably, wherein the one or more phospholipids are PC, PG and DSPG;

[0071] (iv) optionally one or more agents selected from glycine, arginine, proline or any other amino acid known to be suitable as a bulking agent, a sugar component selected from sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, xylitol, xylose, dextran, glucose, and NaCl, wherein the total amount of the one or more agents is from 1.5% (w / w) to 5.5% (w / w);

[0072] (v) dulagliptin, wherein the amount of dulagliptin, preferably its tartrate form or 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 dulagliptin formulation, and the final concentration of dulagliptin in the liposomal dulagliptin formulation is 25 mg / ml and 60 mg / ml;

[0073] The sum of (i) to (v) is 100%).

[0074] Another aspect (the third aspect) relates to a kit comprising the lyophilisate described in the first aspect and embodiments thereof in a container and the aqueous pharmaceutical solution described in the first aspect and embodiments thereof in a second container.

[0075] One embodiment of the third aspect relates to a kit further comprising instructions on how to combine the contents of the first and second containers to contain the liposomal dulalagliptin formulation according to the last embodiment of the first aspect; or the liposomal dulalagliptin formulation of the second aspect.

[0076] definition

[0077] If not stated otherwise, amounts in % refer to % (weight / weight) ((w / w)).

[0078] If not expressly stated otherwise (for example, by use of a term such as "a particular" which means "one"), the term "a" is an indefinite article and includes both "one" and "one or more" / "more than one" of the noun following the term "a".

[0079] The term "amount of one or more" components, such as the amount of one or more organic solvents, refers to the sum of all these components. For example, if there are two organic solvents in the organic phase, such as 20% (w / w) tert-butyl alcohol based on the total weight of the organic phase and 10% (w / w) propanol based on the total weight of the organic phase, then the amount of the one or more organic solvents (the two organic solvents propanol and tert-butyl alcohol) is 30% (w / w) based on the total weight of the organic phase.

[0080] As used herein, a "buffer" or "buffer system" is used to prevent major changes in the pH of a solution, and suitable examples are well known to those skilled in the art.

[0081] As used herein, "bulking agent" and its name are meant to form the bulk of the lyophilized product and provide sufficient structure to the lyophilized cake. Non-limiting examples of bulking agents are mannitol, glycine, arginine, proline, glucose, sucrose, lactose, trehalose and dextran.

[0082] The term "cholesterol" refers to 3β-hydroxy-5-cholestene (CAS No. 57-88-5). Examples of cholesterol derivatives are cholesterol sulfate and its salts (e.g. sodium salt), cholesterol hemisuccinate, cholesterol succinate, cholesterol oleate, polyethylene glycol derivatives of cholesterol (cholesterol-PEG), coprolol, cholestanol, cholestane, bile acid, cortisol, corticosterone, hydrocortisone and calciferol.

[0083] The term "container" as used herein refers to an ampoule or vial made of polymeric material or glass with a rubber stopper and a cap, a single-chamber or double-chamber syringe, an infusion bag or a bottle, which is suitable for containing a composition for parenteral administration. It also includes any container for containing liquids.

[0084] The term "D90" is well known to the skilled person and is related to size distribution and refers to the number of vesicles with a diameter equal to or below a given value, which weight is 90% of the weight of the components forming such particles in the formulation. D90 can be determined by multi-angle light scattering (MALS).

[0085] "Dulagliptin" is an effective and selective dipeptidyl peptidase-4 (DPP4) inhibitor for the treatment of type 2 diabetes. It has the following chemical structure:

[0086]

[0087] The term dulalagliptin includes the molecule in free base form or any pharmaceutically acceptable salt thereof, such as a tartrate or fatty acid salt thereof.

[0088] When calculating the concentration of dulagliflozin (mg / ml), the concentration is based on the dulagliflozin free base molecule.

[0089] As used herein, "other organic compounds" refers to organic compounds that are non-solvents and miscible with water, such as lipids such as cholesterol, polysaccharides and polypeptides. The term "lipid" as used herein does not include phospholipids. The term lipid used in the present invention refers to a lipid selected from 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 (isopentenol lipids) and waxes.

[0090] As used herein, "miscible with water" means that the organic solvent can be mixed with water in all proportions, for example, ethanol, propanol, butanol can be mixed with water respectively.

[0091] The term "between X% and Y%" (wherein X and Y represent any number between 0 and 100, and X% is less than Y%) refers to any number within the range between X% and Y%, including X% and Y%.

[0092] As used herein, "nanodispersion system" refers to a combination of an organic phase and an aqueous phase, ie, an aqueous formulation comprising vesicles or micelles having a D90 of 60 nm or less, wherein the vesicles or micelles are not liposomes.

[0093] "Liposomes" are spherical vesicles having at least one lipid bilayer. Liposomes can be used as carriers for drug administration. Liposomes are most commonly composed of phospholipids, especially phosphatidylcholine, but may also include other lipids, such as egg phosphatidylethanolamine, as long as they are compatible with the lipid bilayer structure.

[0094] As used herein, "liposomal formulation" refers to a liquid containing liposomes, wherein the liposomes contain phospholipids. The liposomal formulation is suitable for encapsulating dulagliptin in the aqueous environment of the liposomes.

[0095] As disclosed herein, liposome size or lipophilic vesicle size (e.g., micelle size) refers to the size determined by multi-angle light scattering (MALS) or alternatively by dynamic light scattering (DLS), respectively. Typically, the size of liposomes is in the range of 0,025 μm to 2.5 μm (Akbarzadeh et al., Nacleic Research Letters 2013, 8: 102).

[0096] The liposomal formulation of the present invention may have the advantage of reducing the number of injections or ingestions of dulaliptin required per day for a patient. Thus, it contributes to patient convenience and / or patient compliance.

[0097] "Loading" refers to the incorporation or transfer of dulagliptin into / encapsulation of dulagliptin with liposomes.

[0098] As used herein, "organic component" refers to any molecule containing at least one -(CH2)- moiety.

[0099] "Pharmaceutically acceptable" means approved or approved by a regulatory agency of a country or listed in the European or US Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[0100] PEG refers to polyethylene glycol.

[0101] PEGylation is the process by which PEG polymer chains are covalently and non-covalently attached or fused to molecules and macrostructures such as phospholipids, which produces vesicles, which are then described as PEGylated. PEGylated phospholipids are well known and commercially available.

[0102] The "pharmaceutical composition" described herein is particularly a pharmaceutical liposome composition. A "pharmaceutical liposome composition" refers to a composition comprising liposomes suitable for pharmaceutical administration.

[0103] "Phospholipids" used in the preparations of the present invention include a hydrophilic phosphate portion and two hydrophobic carbohydrate portions. For the sake of clarification, the term lipid does not encompass phospholipids. Phospholipids can be selected from 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 contains a variety of phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) or a (pharmaceutically acceptable) salt of any of the above substances.

[0104] Generally, the structure of the phospholipids used herein is a phospholipid of structure (I)

[0105]

[0106] in

[0107] R1 represents C 10 -C 24 Acyl:

[0108] R2 represents C 10 -C 24 acyl or hydrogen;

[0109] R3 represents 2-trimethylamino-1-ethyl (yielding PC), 2-amino-2-carboxy-1-ethyl (yielding PS), inositol (C6H 11 O5) (yielding PI), 2-amino-1-ethyl (yielding PE) or hydrogen (yielding PA).

[0110] The terms "phosphatidic acid" and "PA" are used interchangeably herein. PA or its (pharmaceutically acceptable) salt may be derived from natural sources and / or synthetic sources. Non-limiting examples of PA are DLPA, DMPA, DPPA, DSPA, POPA, DEPA, HSPA, HEPA or a (pharmaceutically acceptable) salt of any of the foregoing.

[0111] The terms "phosphatidylcholine" and "PC" are used interchangeably herein. PC or its (pharmaceutically acceptable) salt may be derived from natural sources and / or synthetic sources. PC may be PEGylated. Non-limiting examples of PC are dilaurylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), palmitoyloleoylphosphatidylcholine (POPC), dierucylphosphatidylcholine (DEPC), hydrogenated soy phosphatidylcholine (HSPC), hydrogenated egg phosphatidylcholine (HEPC), or a (pharmaceutically acceptable) salt of any of the foregoing.

[0112] The terms "phosphoethanolamine" and "PE" are used interchangeably herein. PE may be pegylated. PE or a (pharmaceutically acceptable) salt thereof may be derived from a natural source and / or a synthetic source. Non-limiting examples of PE are DLPE, DMPE, DPPE, DSPE, POPE, DEPE, HSPE, HEPE or a (pharmaceutically acceptable) salt of any of the foregoing.

[0113] The terms "phosphatidylglycerol" and "PG" are used interchangeably herein. PG or a (pharmaceutically acceptable) salt thereof may be derived from natural sources and / or synthetic sources. Non-limiting examples of PG are DLPG, DMPG, DPPG, DSPG, POPG, DEPG, HSPG, HEPG or a (pharmaceutically acceptable) salt of any of the foregoing.

[0114] The terms "phosphatidylinositol" and "PI" are used interchangeably herein. The PI may be pegylated. The PI or its (pharmaceutically acceptable) salt may be derived from a natural source and / or a synthetic source. Non-limiting examples of PIs are DLPI, DMPI, DPPI, DSPI, POPI, DEPI, HSPI, HEPI or a (pharmaceutically acceptable) salt of any of the foregoing.

[0115] The terms "phosphoserine" and "PS" are used interchangeably herein. PS may be pegylated. PS or a (pharmaceutically acceptable) salt thereof may be derived from natural sources and / or synthetic sources. Non-limiting examples of PS are DLPS, DMPS, DPPS, DSPS, POPS, DEPS, HSPS, HEPS or a (pharmaceutically acceptable) salt of any of the foregoing.

[0116] Non-limiting examples of pharmaceutically acceptable salts of any phospholipid are sodium or ammonium salts, such as PG-Na, PG-NH4, DSPG-Na or DSPG-NH4.

[0117] When the terms PC, PG, PE, PA, PS and / or PI are used, if not otherwise stated, these terms also include pharmaceutically acceptable salt forms of the phospholipids or PEGylated modifications of the phospholipids.

[0118] The term "reconstitution of a lyophilisate" refers to mixing the lyophilisate with a solution.

[0119] Mixing "shortly before administration to a patient" means up to three days before administration to a patient, in particular up to 24 hours before administration to a patient, for example up to 6 hours before administration to a patient.

[0120] "Tonicity modifier" refers to a pharmaceutically acceptable compound that can be added to a formulation to make it isotonic with human plasma. Tensonicity modifiers include, for example, dextrose, glucose, mannitol, sucrose, lactose, trehalose, glycerol and NaCl, particularly sucrose or glycerol or NaCl, more particularly sucrose or NaCl. Tension is the "effective osmotic molar concentration", which is equal to the sum of the concentrations of solutes that have the ability to exert osmotic force across a membrane. Parenteral formulations should be isotonic with plasma. Tensonicity modifiers are well known to those skilled in the art. The skilled person knows that in some cases, tension modifiers may also have other functions, for example, trehalose is also a filler.

[0121] As used herein, the terms "treat," "treating," or "treatment" of any disease or condition refer, in one embodiment, to ameliorating the disease or condition (i.e., slowing or arresting or reducing the development of the disease or at least one clinical symptom thereof). In another embodiment, "treating" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In another embodiment, "treating" refers to modulating a disease or condition physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of a physical parameter), or both.

[0122] The term "wherein the sum of a), b), c) etc. (and further enumerations) adds up to 100%" means that the percentage values ​​given to components a) to e) always have to be chosen in such a way that the sum of the values ​​of a) to e) is 100. These values ​​thus give the ratios between components a) to e). However, the skilled person understands that the formulations may comprise further components, such as solvents. However, when calculating the ratio between a) and c), the amounts of such additional components are not taken into account (the values ​​in % always have to add up to 100%).

[0123] It should be understood that as long as the combination does not violate any natural laws, all embodiments of the present invention can be combined, regardless of whether the embodiment is an embodiment, a preferred embodiment, a more preferred embodiment, a particularly preferred embodiment or a most preferred embodiment, and even if the combination of two or more embodiments is not explicitly mentioned, such a combination of two or more embodiments of the present invention can be disclosed by disclosing two or more embodiments.

[0124] It will be understood by those skilled in the art that all embodiments described herein, whether or not an embodiment is an embodiment, a preferred embodiment, a more preferred embodiment, etc., can be combined, unless such combination contradicts the laws of nature. It will be understood by those skilled in the art that embodiments such as preferred embodiments of one aspect of the present invention can also be applied to another aspect. Therefore, for example, a preferred embodiment according to the method of the present invention is also a preferred embodiment, if applicable, for example, for a lyophilized product or liposomal dulagliptin formulation according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0126] The present invention relates to a liposome preparation of dulagliptin and a method for preparing the liposome preparation containing dulagliptin.

[0127] Therefore, one aspect of the present invention relates to a liposomal formulation comprising dulagliptin, comprising

[0128] (i) an aqueous medium, preferably water, wherein the amount of the aqueous medium is between 65% and 90% based on the total weight of the liposomal doxepin formulation;

[0129] (ii) optionally a lipid, preferably cholesterol, wherein the amount of the lipid, preferably cholesterol, is between 1% (w / w) and 2.5% (w / w) based on the total weight of the liposomal doxepin formulation;

[0130] (iii) one or more phospholipids, wherein the amount of the 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 dugliptin formulation;

[0131] (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);

[0132] (v) dulagliptin, wherein the amount of dulagliptin, preferably in its tartrate form or 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 dulagliptin formulation; and preferably, the final concentration of dulagliptin in the liposomal dulagliptin formulation is between 25 mg / ml and 60 mg / ml;

[0133] The sum of (i) to (v) is 100%.

[0134] Another aspect relates to a method for preparing a liposome formulation of dulagliptin, comprising the following steps:

[0135] (A)

[0136] A.1) providing an organic phase, wherein the organic phase comprises

[0137] (a) one or more phospholipids, wherein the amount of the one or more phospholipids 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);

[0138] (b) one or more organic solvents selected from anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropyl glycerol, 1-butanol, 2-butanol and tert-butanol, or any combination of the foregoing; wherein the amount of the one or more organic solvents is between 60% (w / w) and 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);

[0139] (c) optionally one or more further organic components which are neither (a) nor (b), wherein the amount of the one or more further organic components is up to 10% (w / w), based on the sum of (a), (b) and (c);

[0140] wherein the sum of (a), (b) and (c) is 100%; and

[0141] A.2) providing an aqueous phase, wherein the aqueous phase comprises

[0142] (d) an aqueous medium, wherein the amount of the aqueous medium is between 66% (w / w) and 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).

[0143] (w / w);

[0144] (e) optionally one or more further components selected from buffer systems and pH adjusting acids and / or bases (e.g. NaOH, HCl), wherein the amount of all further components is at most 2% (w / w) based on the sum of (d), (e), (f) and (g);

[0145] (f) optionally a bulking agent, preferably trehalose, wherein the amount of the 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);

[0146] (g) Dulagliptin, preferably wherein the dulagliptin is provided in the form of its tartrate salt or its free base form, more preferably in the form of its free base, wherein when dulagliptin is provided in the form of its tartrate salt, the amount of dulagliptin is between 5% (w / w) and 22% (w / w), based on the sum of (d), (e), (f) and (g), preferably 11±1% (w / w), based on the sum of (d), (e), (f) and (g); or when dulagliptin is provided in the form of its free base, the amount of dulagliptin is (6.2±0.7)% based on the sum of (d), (e), (f) and (g).

[0147] (w / w);

[0148] wherein the sum of (d), (e), (f) and (g) is 100%; and

[0149] A.3) mixing the aqueous phase and the organic phase until the ratio of the aqueous phase to the organic phase reaches 3:1 to 1:3, preferably 1:1, to obtain a nanodispersed system containing dulagliptin; and

[0150] A.4) freeze-drying the nanodispersion system comprising dulagliptin to obtain a lyophilisate; and optionally

[0151] A.5) reconstituting the lyophilisate of step A.4 with an aqueous solution to produce a liposomal dulagliptin formulation, wherein the aqueous solution comprises 80% (w / w) to 100% (w / w) of an aqueous medium based on the total weight of the aqueous solution, optionally further comprising a tonicity regulator, wherein the amount of the tonicity regulator is up to 20% (w / w) based on the total weight of the aqueous solution, optionally further comprising a buffer system, preferably, wherein the final concentration of dulagliptin in the liposomal dulagliptin formulation is 25 mg / ml to 60 mg / ml; or

[0152] (B)

[0153] B.1) providing an organic phase comprising

[0154] (a) one or more phospholipids, wherein the amount of the one or more phospholipids 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);

[0155] (b) one or more organic solvents selected from anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropyl glycerol, 1-butanol, 2-butanol and tert-butanol, or any combination of the foregoing; wherein the amount of the one or more organic solvents is between 60% (w / w) and 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);

[0156] (c) optionally one or more further organic components which are neither (a) nor (b), wherein the amount of the one or more further organic components is up to 10%, based on the sum of (a), (b) and (c);

[0157] wherein the sum of (a), (b) and (c) is 100%; and

[0158] B.2) providing an aqueous phase, comprising

[0159] (d) an aqueous medium, wherein the amount of the aqueous medium is between 66% (w / w) and 90% (w / w) based on the sum of (d), (e) and (f), preferably 84% based on the sum of (d), (e) and (f);

[0160] In one embodiment, in (d), the amount of the aqueous medium is 88% (w / w) to 100% (w / w) based on the sum of (d), (e) and (f);

[0161] In one embodiment, in (d), the amount of the aqueous medium is 88% (w / w) to 99.99% (w / w) based on the sum of (d), (e) and (f), wherein (f) is at most 10% (w / w) based on the sum of (d), (e) and (f).

[0162] In one embodiment, in (d), the amount of the aqueous medium is 91% (w / w) to 96.99% (w / w) based on the sum of (d), (e) and (f), wherein (f) is 3% (w / w) to 7% (w / w) based on the sum of (d), (e) and (f).

[0163] The "99.99" and "96.99" indicate that (e) and (f) may be present, but at very low concentrations.

[0164] In one embodiment, in (d), the amount of the aqueous medium is 91% (w / w) to 96.5% (w / w) based on the sum of (d), (e) and (f), wherein (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).

[0165] (e) optionally one or more further components selected from buffer systems and pH adjusting acids and / or bases (e.g. NaOH, HCl), wherein the amount of all further components is at most 2% (w / w) based on the sum of (d), (e) and (f);

[0166] In one embodiment, in (e), the amount of all other components is 0.5-2% (w / w) based on the sum of (d), (e) and (f),

[0167] (f) optionally a bulking agent, preferably trehalose, wherein 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);

[0168] wherein the sum of (d), (e) and (f) is 100%; and

[0169] B.3) mixing the aqueous phase and the organic phase until the ratio of the aqueous phase to the organic phase reaches 3:1-1:3, preferably 1:1, to obtain a nano-dispersed system;

[0170] B.4) freeze-drying in a nanodispersion system to obtain a freeze-dried product; optionally

[0171] B.5) reconstitute the lyophilisate of step B.4 with an aqueous solution to produce a liposomal dulagliptin formulation, preferably, wherein the final concentration of dulagliptin in the liposomal dulagliptin formulation is between 25 mg / ml and 60 mg / ml, wherein the aqueous solution comprises

[0172] (i) 67% (w / w) and 100% (w / w) of an aqueous medium based on the total weight of the aqueous solution, optionally further comprising a tonicity adjuster, wherein the amount of the tonicity adjuster is up to 20% (w / w), preferably 10% (w / w) to 20% (w / w), based on the total weight of the aqueous solution, optionally further comprising a buffer system,

[0173] (ii) dulagliptin, preferably in the form of its tartrate salt or its free base form, wherein when dulagliptin is in the form of its tartrate salt, the amount of dulagliptin is 5% (w / w) to 13% (w / w), more preferably 10±0.5% (w / w), based on the total weight of the aqueous solution; or when dulagliptin is in the form of its free base, the amount is (6±0.4)% (w / w), based on the total weight of the aqueous solution; or

[0174] (C)

[0175] C.1) providing an organic phase, wherein the organic phase comprises

[0176] (a) one or more phospholipids, wherein the amount of the one or more phospholipids 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);

[0177] (b) one or more organic solvents selected from anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropyl glycerol, 1-butanol, 2-butanol and tert-butanol, or any combination of the foregoing; 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),

[0178] (c) optionally one or more further organic components which are neither (a) nor (b), wherein the amount of the one or more further organic components is up to 10% (w / w), based on the sum of (a), (b) and (c);

[0179] wherein the sum of (a), (b) and (c) is 100%; and

[0180] C.2) providing an aqueous phase, comprising

[0181] (d) an aqueous medium, wherein the amount of the aqueous medium is between 66% (w / w) and 90% (w / w) based on the sum of (d), (e), (f) and (g), preferably 84% based on the sum of (d), (e), (f) and (g).

[0182] (w / w);

[0183] (e) optionally one or more further components selected from buffer systems and pH adjusting acids and / or bases (e.g. NaOH, HCl), wherein the amount of all further components is at most 2% (w / w), based on the sum of (d), (e), (f) and (g);

[0184] (f) optionally a bulking agent, preferably trehalose, wherein the amount of the bulking agent is up to 10% (w / w) based on the sum of (d), (e), (f) and (g), preferably 3% based on the sum of (d), (e), (f) and (g)

[0185] (w / w) to 7% (w / w);

[0186] (g) Dulagliptin, wherein the Dulagliptin is provided in the form of one of its fatty acid salts, wherein the amount of the fatty acid salt of Dulagliptin is between 5% (w / w) and 22% based on the sum of (d), (e), (f) and (g).

[0187] (w / w); and

[0188] wherein the sum of (d), (e), (f) and (g) is 100%; and

[0189] C.3) mixing the aqueous phase and the organic phase until the ratio of the aqueous phase to the organic phase reaches 3:1-1:3, preferably 1:1, to obtain a nano-dispersion system containing doxepin; and

[0190] C.4) freeze-drying the nanodispersed system comprising dulalagliptin to obtain a lyophilisate; optionally

[0191] C.5) reconstitute the lyophilisate of step C.4 with an aqueous solution to produce a liposomal dulalagliptin formulation, wherein the aqueous solution comprises 80% (w / w) to 100% (w / w) of an aqueous medium based on the total weight of the aqueous solution, optionally further comprises a tonicity regulator, wherein the amount of the tonicity regulator is up to 20% (w / w) based on the total weight of the aqueous solution, optionally further comprises a buffer system, preferably, wherein the final concentration of dulalagliptin in the liposomal dulalagliptin formulation is 25 mg / ml to 60 mg / ml.

[0192] Method C may optionally include a step before step C1, which involves preparing a fatty acid salt of duogaliptin by replacing the tartrate anion in duogaliptin tartrate with a fatty acid anion (preferably laurate, myristate, palmitate, stearate or bile salt hydrate, more preferably bile salt hydrate);

[0193] The method of the present invention allows the direct preparation of a liquid intermediate formulation nanodispersion system, wherein the D90 of the lipophilic vesicles or micelles is 60 nm or less, more preferably 25 nm or less, preferably 20 nm or less. Such a nanodispersion system formed in the method according to the present invention allows the preparation of liposomal formulations without the need to mechanically reduce the size of the liposomes (e.g., the doxepin formulation can be sterile filtered before filling into a sterile container and / or freeze drying).

[0194] Alternatively, the method of the present invention further allows for easy preparation of liposome preparations, wherein the D90 of the liposomes is 1 μm to 4.5 μm. The present invention also provides a preparation comprising dugliptin prepared according to the method of the present invention, more specifically, a preparation comprising dugliptin suitable for administration to a patient is provided. In particular, this administration is by subcutaneous injection or infusion, more preferably by subcutaneous injection. The present invention further provides two separate preparations, which can be mixed together shortly before administration to the patient to provide a liposome composition suitable for administration. In one embodiment, a preparation containing phospholipids can be a lyophilisate, and the second preparation can be an aqueous preparation, provided that at least one of the lyophilisate or the aqueous solution contains dugliptin. When the two separate preparations are mixed together, dugliptin is loaded into the formed liposomes, and a drug liposome dugliptin preparation suitable for clinical use is produced. Preferably, the final concentration of dugliptin in the liposome dugliptin preparation is between 25 mg / ml and 60 mg / ml.

[0195] Formulations comprising dulaliptin should be capable of efficient and optimal loading of dulaliptin into liposomes prior to administration to a patient.

[0196] In a preferred embodiment, at least 25% (w / w) of dulagliptin, more preferably at least 30% (w / w) of dulagliptin is liposome-encapsulated in the liposomal dulagliptin formulation.

[0197] Preferably, the present invention provides a pharmaceutical liposome formulation that enables prolonged release of dulaliptin from the liposomes after administration (injection).

[0198] The formulations described herein are particularly pharmaceutical formulations, such as pharmaceutical liposomal compositions.

[0199] Preferably, the D90 of the liposomes of the liposome formulation is from 1 μm to 4.5 μm.

[0200] In particular, the phospholipid or a pharmaceutically acceptable salt thereof as described herein is selected from egg lecithin, soybean lecithin or a synthetic lecithin.

[0201] Various aspects and embodiments of the invention are described below.

[0202] preparation

[0203] A first aspect relates to a liposomal dugliptin formulation as described herein.

[0204] The skilled person will understand that trace amounts of solvents and additives (e.g., tert-butyl alcohol, or water, or buffer systems) used in the preparation method, preferably in the preparation method of the present invention further described below, may still be present in an amount of up to 2%; or other additives may be present in the liposomal dulagliptin formulation. Without being limited to this explanation, other additives may be required for the purpose of stability or tolerance of such liposomal dulagliptin formulations by patients.

[0205] In a preferred embodiment, the total weight of (i) to (v) is at least 98% (w / w) based on the total weight of the liposomal dugliptin formulation.

[0206] In another preferred embodiment, the total weight of (i) to (v) is at least 99% (w / w) based on the total weight of the liposomal dugliptin formulation.

[0207] In a preferred embodiment, the total weight of (i) to (v) is at least 99.9% (w / w) based on the total weight of the liposomal dulagliflozin formulation.

[0208] A preferred embodiment relates to a liposomal dulalagliptin formulation, wherein the residual amount of organic solvent (e.g. tert-butyl alcohol) is less than 2% (w / w) based on the total weight of the formulation (e.g. the total amount of (i) to (v) and tert-butyl alcohol). More preferably, the amount of tert-butyl alcohol is 1% or less, such as less than 0.8% or even less than 0.01%.

[0209] A preferred embodiment relates to a preparation according to the invention, wherein the preparation consists of (i) to (v).

[0210] 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 total amount of tert-butanol and buffer system is 0.01% to 2%, based on the total weight of the formulation (e.g. a) to e) and tert-butanol (e.g. in the case of a lyophilisate).

[0211] A further preferred embodiment relates to a nanodispersion system (or a method comprising preparing a nanodispersion system) comprising a preferred drug of dulagliptin, wherein the D90 of the lipophilic vesicles is less than 25 nm, more preferably 20 nm or less, such as 10 nm to 20 nm, such as about 15 nm, or 3 nm to 10 nm, such as about 5 nm.

[0212] Another preferred embodiment relates to a lyophilized formulation comprising dulagliptin.

[0213] Another preferred embodiment relates to a freeze-dried formulation (lyophilisate), wherein the amount of dulagliptin is between 50 mg and 120 mg, more preferably between 60 mg and 100 mg.

[0214] In a further preferred embodiment, the pharmaceutically active substance in the formulation of the present invention is encapsulated by lipid inclusion complexes, proliposomes, micelles or liposomes, more preferably liposomes or micelles. In other words, the composition comprises, for example, at least one liposome or at least one micelle, respectively, and dulagliptin is loaded into the liposomes or micelles.

[0215] In a preferred embodiment, the dulalagliptin in the formulation of the present invention is encapsulated by micelles (nanodispersed formulation / nanodispersed system). Most preferably, the D90 of the micelle is 60 nm or less, such as less than 25 nm, more preferably 20 nm or less, such as 10 nm-20 nm, or 3 nm-10 nm.

[0216] In another preferred embodiment, dulagliptin in the formulation of the present invention is encapsulated by liposomes (liposomal formulation).

[0217] Another preferred embodiment relates to a formulation, wherein said formulation, preferably a liposomal formulation or a nanodisperse formulation, is a pharmaceutical formulation.

[0218] Liposome preparation

[0219] A preferred embodiment relates to liposomal formulations of the drug.

[0220] Such liposomal formulations of the drug are preferably suitable for injection, preferably subcutaneous injection.

[0221] Another preferred embodiment relates to the pharmaceutical liposomal formulation, wherein the concentration of doxagliptin in the liposomal formulation is between 25 mg / ml and 60 mg / ml, such as between 30 mg / ml and 50 mg / ml.

[0222] According to another preferred embodiment, the pH value of the pharmaceutical liposomal formulation is 6 to 8, preferably 7 to 8, more preferably 7 to 7.8, such as 7.4±2.

[0223] (i) Solvents for liposome preparations

[0224] Aqueous medium

[0225] The aqueous medium of the liposomal formulation is water or a combination of water and a solvent selected from 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, in particular propylene glycol and ethanol, wherein the amount of water accounts for at least 60% (w / w), preferably at least 90% (w / w), and more preferably at least 95% (w / w) of the total amount of solvent in the aqueous medium.

[0226] In a preferred embodiment, water is the only solvent in the aqueous medium and, therefore, the only solvent in the liposomal formulation.

[0227] Typically, the liposomal formulation comprises an aqueous medium, wherein the amount of aqueous medium is between 65% (w / w) and 90% (w / w) based on the sum of (i) to (v).

[0228] (ii) Lipids

[0229] In a preferred embodiment, the formulations of the invention comprise one or more lipids.

[0230] In a preferred embodiment, the lipid used in the present invention is a lipid selected from the following: fatty acids, sterols (such as cholesterol), fat-soluble vitamins (such as vitamins A, D, E and K), glycerolipids (such as monoglycerides, diglycerides, triglycerides), sphingolipids, glycolipids, polyketides, isoprenoids (isoprenol lipids) and waxes, more preferably selected from fatty acids, sterols (such as cholesterol), fat-soluble vitamins (such as vitamins A, D, E and K), glycerolipids (such as monoglycerides, diglycerides, triglycerides), sphingolipids and glycolipids.

[0231] Cholesterol is known to have an effect on liposome stability and drug release.

[0232] Therefore, in a more preferred embodiment, the formulation of the present invention preferably comprises cholesterol or a derivative thereof.

[0233] Another preferred embodiment relates to a formulation wherein the additional organic component is selected from cholesterol or sodium cholesterol sulfate. More preferably, the additional organic component is cholesterol.

[0234] Another preferred embodiment relates to a formulation wherein the amount of cholesterol is between 0.5% and 3% compared to the sum of all components of the formulation, for example about 1.6%, such as between 1.3% and 1.9%.

[0235] (iii) Phospholipids

[0236] Another preferred embodiment relates to a formulation wherein 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 pharmaceutically acceptable salt of any of the foregoing.

[0237] Another preferred embodiment relates to a formulation wherein the one or more phospholipids is 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 pharmaceutically acceptable salt of any of the foregoing.

[0238] In a preferred embodiment, the pharmaceutically acceptable salt of the phospholipid is the Na salt of the phospholipid.

[0239] In a preferred embodiment, at least one phospholipid is a PEGylated phospholipid (or a pharmaceutically acceptable salt thereof).

[0240] In another preferred embodiment, PC or a pharmaceutically acceptable salt thereof is pegylated.

[0241] In another preferred embodiment, the formulation comprises PC or a pharmaceutically acceptable salt thereof and PG or a pharmaceutically acceptable salt thereof. In a preferred embodiment, PG is DOPG-Na.

[0242] Another more preferred embodiment relates to a formulation wherein the one or more phospholipids comprise PC or preferably a pharmaceutically acceptable salt thereof, preferably selected from DLPC, DMPC, DPPC, DSPC, POPC, DEPC, HSPC, HEPC or preferably a pharmaceutically acceptable salt of any of the foregoing.

[0243] Another more preferred embodiment relates to a formulation wherein the one or more phospholipids comprise PEGylated PC or preferably a pharmaceutically acceptable salt thereof, preferably selected from PEGylated DLPC, PEGylated DMPC, PEGylated DPPC, PEGylated DSPC, PEGylated POPC, PEGylated DEPC, PEGylated HSPC, PEGylated HEPC or preferably a pharmaceutically acceptable salt of any of the foregoing.

[0244] Another preferred embodiment relates to a formulation wherein the amount of the one or more phospholipids relative to the sum of all components of the liposome formulation is 7% (w / w)-15% (w / w), such as about 11.2%, such as (11.2±0.5)%.

[0245] Another more preferred embodiment relates to a formulation wherein the one or more phospholipids include PC or a preferred pharmaceutically acceptable salt thereof. In an even more preferred embodiment, PC or a preferred pharmaceutically acceptable salt thereof is derived from soy (e.g. lipid S100), egg or synthetic, and in a most preferred embodiment, PC or a preferred pharmaceutically acceptable salt thereof is derived from soy.

[0246] Another more preferred embodiment relates to a formulation, wherein the PC or preferably pharmaceutically acceptable PC is selected from DLPC, DMPC, DPPC, DSPC, POPC, DEPC, HSPC, HEPC or a combination thereof.

[0247] Another more preferred embodiment relates to a formulation wherein the PG or preferably a pharmaceutically acceptable salt thereof is pegylated and is selected from pegylated DLPG, pegylated DMPG, pegylated DPPG, pegylated DSPG, pegylated POPG, pegylated POPG, pegylated DEPG, pegylated HSPG, pegylated HEPG or preferably a pharmaceutically acceptable salt of any of the foregoing.

[0248] Another preferred embodiment relates to a formulation, wherein the one or more phospholipids comprise PG or preferably a pharmaceutically acceptable salt thereof, and the PG or the salt thereof is PEGylated PG or a PEGylated salt thereof.

[0249] Another more preferred embodiment relates to a formulation, wherein the PG or preferably a pharmaceutically acceptable salt thereof is selected from DLPG, DMPG, DPPG, DSPG, POPG, POPG, DEPG, HSPG, HEPG.

[0250] Another more preferred embodiment relates to a formulation wherein the PG or preferably a pharmaceutically acceptable salt thereof is pegylated and is selected from pegylated DLPG, pegylated DMPG, pegylated DPPG, pegylated DSPG, pegylated POPG, pegylated POPG, pegylated DEPG, pegylated HSPG, pegylated HEPG or preferably a pharmaceutically acceptable salt of any of the foregoing.

[0251] (iv) Agents (bulking agents and / or tonicity adjusting agents)

[0252] In particular, when the components of the formulation of the present invention are to be lyophilizable, or when a lyophilization step is required for the preparation of the formulation of the present invention by the method of the present invention, the presence of a filler is advantageous. Examples of preferred fillers are glycine, arginine, proline or any other amino acid known to be suitable as a filler or sugar component (component (iv) or (f) respectively). Preferred sugar components are selected from sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, xylitol, xylose, dextran or a mixture thereof.

[0253] A more preferred embodiment relates to a formulation wherein (f) in the aqueous phase in the method according to the invention (and, therefore, (iv) in the liposome formulation according to the invention, respectively) is trehalose.

[0254] A preferred embodiment relates to a liposomal formulation, wherein one agent is trehalose, and wherein the amount of trehalose in the formulation is from 1.5% (w / w) to 3.5% based on the total weight of the liposomal formulation.

[0255] (w / w), for example (2.5±0.5)% (w / w).

[0256] Another preferred embodiment relates to a liposomal formulation of a drug comprising a tonicity modifier.

[0257] Such a tonicity regulator, preferably a pharmaceutically acceptable tonicity regulator, should be present in the pharmaceutical liposome formulation. It can be added during the preparation of the liposome formulation by mixing the lyophilisate of the present invention with an aqueous phase containing the tonicity regulator, or it can be provided during the rehydration step of the lyophilisate.

[0258] Alternatively, it may already be present in the lyophilisate, since the tonicity modifier is part of the aqueous phase used in the process of the invention to prepare the lipopeptide of the invention. The skilled person is aware that the tonicity modifier may also be partly part of such an aqueous phase and partly part of the aqueous phase. The skilled person can easily calculate the total amount of tonicity modifier that is required to have the correct concentration in the final pharmaceutical liposome formulation to have an isotonic effect with respect to the drug provided to the patient.

[0259] The skilled artisan will recognize that a tonicity adjuster is also a bulking agent in some cases, for example where the tonicity adjuster is a carbohydrate component such as glucose or trehalose.

[0260] Other tonicity modifiers such as NaCl do not have a swelling function. Therefore, if such a "single functional component" is used, such as NaCl, the formulation may contain filler e) and tonicity modifier e). It may even contain a filler (such as trehalose) and "two" tonicity modifiers (trehalose and, for example, NaCl).

[0261] Tonicity regulators are well known to those skilled in the art. In a preferred embodiment, the tonicity regulator is selected from dextrose, glucose, mannitol, sucrose, lactose, trehalose, glycine, arginine, proline and NaCl, particularly glycine, mannitol, trehalose, glucose and NaCl, even more particularly glycine and NaCl, most preferably NaCl.

[0262] The skilled artisan knows how to select the correct concentration of a particular tonicity adjusting agent in a formulation, preferably for subcutaneous injection.

[0263] In a preferred embodiment, the tonicity modifier is NaCl, and the concentration of NaCl in the liposome formulation is 0.8% to 1%, more preferably about 0.9%, such as 0.9%±0.1%, more preferably 0.9%, based on the total weight of the pharmaceutical liposome formulation.

[0264] Depending on the type of tonicity modifier and formulation (eg, pharmaceutically acceptable liposomal formulation or lyophilisate), one skilled in the art is well aware of how to calculate the amount of one or more tonicity modifiers present in the (final) formulation in order to administer to a patient at physiological concentrations.

[0265] (v) Dulagliptin

[0266] In a preferred embodiment, the concentration of dulagliflozin in the liposome formulation is 25 mg / ml-60 mg / ml, more preferably 30 mg / ml-50 mg / ml, for example, a 2 ml volume of injection contains 50 mg-120 mg, more preferably 60 mg-100 mg dulagliflozin.

[0267] Freeze-dried

[0268] Another aspect of the present invention relates to a lyophilisate comprising dulagliptin ((g)), (a), optionally (c), optionally (e) and optionally (f), as defined in the methods herein.

[0269] Preferably, the total amount of dulagliptin is between 50 mg and 120 mg, more preferably between 60 mg and 100 mg.

[0270] Preferably, wherein

[0271] The amount of (g) is between 12% (w / w) and 53% (w / w) based on the total amount of the lyophilisate;

[0272] The amount of (a) is between 7.2% (w / w) and 73% (w / w) based on the total amount of the lyophilisate;

[0273] The amount of (c) is at most 24.3% (w / w), preferably (c) comprises cholesterol, and the amount of cholesterol is 4.8% (w / w) to 9.7% (w / w) based on the total amount of the lyophilisate;

[0274] (e) in an amount of up to 4.9% (w / w) based on the total amount of the lyophilisate;

[0275] The amount of (f) is at most 24.3% (w / w), preferably (f) comprises trehalose, and the amount of trehalose is 12% (w / w) to 53% (w / w) based on the total amount of the lyophilisate.

[0276] In a preferred embodiment, (c) consists of cholesterol.

[0277] In another preferred embodiment, (f) consists of trehalose.

[0278] Another aspect relates to the use of a lyophilisate comprising dulagliptin as described herein for the preparation of a medicament, preferably a liposomal dulagliptin formulation.

[0279] Liposome size

[0280] In another embodiment, the liposomes of the liposome formulation of the present invention have a D90 size distribution of 1 μm to 4.5 μm.

[0281] Treatment / Use

[0282] One aspect relates to the use of the liposome preparation according to the present invention in the preparation of a medicament for treating diabetes, in particular type 2 diabetes.

[0283] Preparation method

[0284] Another aspect relates to a method (A) for preparing the liposome formulation of the present invention.

[0285] Another aspect relates to a method (B) for preparing the liposome formulation of the present invention.

[0286] Another aspect relates to a method (C) for preparing the liposome formulation of the present invention.

[0287] Organic phase

[0288] (a) Phospholipids

[0289] In a 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 pharmaceutically acceptable salt of any of the foregoing.

[0290] In a preferred embodiment, the one or more phospholipids in the organic phase comprise PC or a pharmaceutically acceptable salt thereof and PG or a pharmaceutically acceptable salt thereof.

[0291] In a preferred embodiment, the at least one phospholipid is a pegylated phospholipid.

[0292] Another more preferred embodiment relates to a formulation wherein the one or more phospholipids comprise PC or a preferred pharmaceutically acceptable salt thereof, preferably selected from DLPC, DMPC, DPPC, DSPC, POPC, POPC, DEPC, HSPC, HEPC or a preferred pharmaceutically acceptable salt of any of the foregoing.

[0293] Another more preferred embodiment relates to a formulation wherein the one or more phospholipids comprise PEGylated PC or preferably a pharmaceutically acceptable salt thereof, preferably selected from PEGylated DLPC, PEGylated DMPC, PEGylated DPPC, PEGylated DSPC, PEGylated POPC, PEGylated POPC, PEGylated DEPC, PEGylated HSPC, PEGylated HEPC or preferably a pharmaceutically acceptable salt of any of the foregoing.

[0294] Another more preferred embodiment relates to a formulation wherein the one or more phospholipids include PC or a preferred pharmaceutically acceptable salt thereof. In an even more preferred embodiment, PC or a preferred pharmaceutically acceptable salt thereof is derived from soy (e.g. lipid S100), egg or synthetic, and in a most preferred embodiment, PC or a preferred pharmaceutically acceptable salt thereof is derived from soy.

[0295] Therefore, a preferred embodiment relates to a method for preparing a liposomal formulation, wherein the organic phase comprises 3% (w / w) to 30% (w / w) based on the sum of (a), (b) and (c), preferably 15% (w / w) to 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).

[0296] Another preferred embodiment relates to an organic phase, wherein the PG or preferably a pharmaceutically acceptable salt thereof is PEGylated PG or preferably a pharmaceutically acceptable salt thereof.

[0297] Another more preferred embodiment relates to the organic phase, wherein PG or preferably a pharmaceutically acceptable salt thereof is selected from DLPG, DMPG, DPPG, DSPG, POPG, POPG, DEPG, HSPG, HEPG.

[0298] Another more preferred embodiment relates to an organic phase, wherein the PG or preferably a pharmaceutically acceptable salt thereof is pegylated and is selected from pegylated DLPG, pegylated DMPG, pegylated DPPG, pegylated DSPG, pegylated POPG, pegylated POPG, pegylated DEPG, pegylated HSPG, pegylated HEPG or preferably a pharmaceutically acceptable salt of any of the foregoing.

[0299] Another preferred embodiment relates to the organic phase, wherein the one or more phospholipids comprise or consist of PC in an amount of 3.2% (w / w) to 29.8% based on the sum of (a), (b) and (c).

[0300] (w / w), preferably 15.2% (w / w) to 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).

[0301] Another preferred embodiment relates to the organic phase, wherein the one or more phospholipids comprise or consist of PC (or a pharmaceutically acceptable salt thereof) and PG (or a pharmaceutically acceptable salt thereof), and the amount of PC (or a pharmaceutically 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 29.9% (w / w) based on the sum of (a), (b) and (c). 27.9% (w / w), for example, based on the sum of (a), (b) and (c), it is between (22.1±0.5)% (w / w), and the total amount 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), for example, based on the sum of (a), (b) and (c), it is (0.23±0.1)% (w / w).

[0302] (b) Organic solvents

[0303] For the organic phase, a skilled formulator can select a suitable water-miscible, lyophilizable organic solvent.

[0304] The organic solvent should be miscible with water under standard conditions (25°C and 1,013 bar). In addition, the organic solvent should be lyophilizable, i.e. the solvent can be removed by sublimation (transition of a substance directly from a solid to a gaseous state without passing through a liquid state). The skilled person knows how to select a suitable organic solvent and a suitable temperature and pressure to lyophilize the organic solvent by, for example, using a pressure-temperature (PT) diagram of the solvent known in the art.

[0305] Preferred organic solvents are selected from anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropyl glycerol and alcohols such as methanol, ethanol, propanol, butanol or pentanol.

[0306] In a preferred embodiment, the organic solvent is anisole (CAS 100-66-3).

[0307] In a preferred embodiment, the organic solvent is ethyl acetate (CAS 141-78-6).

[0308] In a preferred embodiment, the organic solvent is 1,4-dioxane (CAS 123-91-1).

[0309] In a preferred embodiment, the organic solvent is dimethyl carbonate (CAS 616-38-6).

[0310] In a preferred embodiment, the organic solvent is dimethyl sulfoxide (CAS 67-68-5).

[0311] In a preferred embodiment, the organic solvent is tetrahydrofuran polyglycol ether (CAS

[0312] 31692-85-0).

[0313] In a preferred embodiment, the organic solvent is N,N-dimethylacetamide (CAS

[0314] 127-19-5).

[0315] In a preferred embodiment, the organic solvent is N,N-dimethylformamide (CAS

[0316] 68-12-2).

[0317] In a preferred embodiment, the organic solvent is N-methyl-2-pyrrolidone (CAS

[0318] 872-50-4).

[0319] In a preferred embodiment, the organic solvent is isopropylglycerol (CAS 100-79-8).

[0320] In a preferred embodiment, the organic solvent is 1-butanol (CAS 71-36-3).

[0321] In a preferred embodiment, the organic solvent is 2-butanol (CAS 78-92-2).

[0322] In a preferred embodiment, the organic solvent is tert-butyl alcohol (CAS 75-65-0).

[0323] Surprisingly it was found that the use of butanol, preferably tert-butanol (TBA), leads to a preferred nanodisperse system when the organic phase from step A.1 / B.1 / C.1 is combined with the aqueous phase from step A.2 / B.2 / C.2, respectively.

[0324] Therefore, in a more preferred embodiment, the organic solvent is tert-butyl alcohol (CAS

[0325] 75-65-0).

[0326] In a preferred embodiment, the 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) based on the sum of (a), (b) and (c) in method A, for example (74, 5±2)% (w / w) based on the sum of (i), (ii) and (iii) in method B, or (1), (2) and (3) in method C.

[0327] (c) Further organic components

[0328] Generally, any organic component may be present. However, it is preferred that the organic component is pharmaceutically acceptable to humans.

[0329] 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.

[0330] Cholesterol is known to have an effect on liposome stability and drug release.

[0331] Therefore, in a preferred embodiment, the organic phase of the method of the present invention preferably further comprises cholesterol or a derivative thereof.

[0332] Another preferred embodiment relates to the organic phase, wherein the additional organic component is selected from cholesterol or sodium cholesterol sulfate. More preferably, the additional organic component is cholesterol.

[0333] Another preferred embodiment relates to a formulation wherein 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).

[0334] In a 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.

[0335] In a preferred embodiment, the organic phase consists of (a), (b) and (c).

[0336] In another preferred embodiment, (a) consists of PC and PG, preferably consists of PC and DOPG, (b) consists of TBA, and (c) consists of cholesterol.

[0337] In another preferred embodiment, the organic phase consists of (a), (b) and (c), and (a) comprises PC and PG, preferably PC and DOPG, (b) comprises TBA, and (c) comprises cholesterol.

[0338] In another preferred embodiment, the organic phase consists of (a), (b) and (c), and (a) consists of PC and PG, preferably PC and DOPG, (b) consists of TBA, and (c) consists of cholesterol.

[0339] Water Phase

[0340] (d) Aqueous medium

[0341] The aqueous medium of the aqueous phase is water or a combination of water and a solvent, wherein the solvent is selected from 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, in particular propylene glycol and ethanol, wherein the amount of water in the total amount of solvent in the aqueous medium is at least 60%, preferably at least 90%, more preferably at least 95%.

[0342] In a preferred embodiment, water is the only solvent in the aqueous medium and, therefore, the only solvent of the aqueous phase.

[0343] Typically, the aqueous phase comprises an aqueous medium, wherein the amount of the aqueous medium is 76% (w / w) to 92% (w / w) based on the sum of (d), (e), (f) and (g), more preferably 80% (w / w) to 88% (w / w) based on the sum of (d), (e), (f) and (g), for example (84.2±2)% (w / w).

[0344] (e) further components selected from buffer systems and pH adjusting acids and / or bases (e.g. NaOH, HCl)

[0345] Optionally, the aqueous phase may comprise a buffer system. Suitable pharmaceutically 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. The technician knows how to select a buffer for a specific pH value. For example, an acetate buffer (e.g., acetate / acetic acid) can be used to adjust the pH value to between 3.7 and 6.5, a phosphate buffer or a citrate buffer (e.g., Na2HPO4 / citric acid, Na2HPO4 / NaH2PO4 or Na2HPO4 / NaOH) can be used to adjust the pH value to between 5.4 and 8.0, and sodium citrate / citric acid can be used to adjust the pH value to between 3.0 and 6.2.

[0346] Optionally, the aqueous phase may contain HCl and / or NaOH. These two components may, for example, be used to adjust the pH of the aqueous phase optionally containing dulagliptin to a pH in the range of between 6 and 8, preferably between 7 and 8, more preferably between 7.0 and 7.8, for example 7.4 ± 0.2.

[0347] (f) Fillers

[0348] The aqueous phase may optionally contain fillers. Suitable fillers for use in liposomal dugliptin formulations, and therefore suitable fillers for use in the aqueous phase for preparing such formulations, have been described above.

[0349] In a preferred embodiment, the bulking agent is trehalose.

[0350] In another preferred embodiment, the amount of filler present in the aqueous phase is 1% (w / w) to 10% (w / w) based on the sum of (d), (e), (f) and (g), more preferably 3% (w / w) to 8% (w / w) based on the sum of (d), (e), (f) and (g), for example (5±1)% based on the sum of (d), (e), (f) and (g).

[0351] (w / w).

[0352] In yet another preferred embodiment, the filler is trehalose and the trehalose is present in the aqueous phase in an amount of 1% (w / w) to 10% (w / w) based on the sum of (d), (e), (f) and (g), more preferably 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).

[0353] (g) Dulagliptin

[0354] Optionally, the aqueous phase comprises dulagliptin. However, for the process of the present invention, it is a prerequisite that the aqueous phase or aqueous solution (used to reconstitute the lyophilisate) comprises dulagliptin.

[0355] In a preferred embodiment, the aqueous phase comprises duogaliptin, and duogaliptin is provided in its free base form.

[0356] In another preferred embodiment, the aqueous phase comprises dulagliptin, and dulagliptin is provided in the form of one of its fatty acid salts, preferably in the form of its sodium laurate, sodium myristate, sodium palmitate, sodium stearate or its sodium cholate hydrate salt.

[0357] In a preferred embodiment, when doxagliptin is provided in its free base form, the amount of doxagliptin in the aqueous phase is 5% (w / w) to 22% (w / w) based on the sum of (d), (e), (f) and (g), preferably 6% (w / w) to 14% (w / w) based on the sum of (d), (e), (f) and (g), for example (6.6±0.5)% (w / w) based on the sum of (d), (e), (f) and (g); or for example when doxagliptin is provided in one of its free fatty acid salt forms, it is (16.3±3)% (w / w) based on the sum of (d), (e), (f) and (g).

[0358] In a preferred embodiment, the method for preparing the liposome formulation is method (A), and the aqueous phase is prepared by providing (g) dulagliptin in the form of a tartrate salt.

[0359] In an even more preferred embodiment, the method for preparing the liposomal formulation is method (A) and the aqueous phase is prepared by providing (g) dulagliptin in free base form.

[0360] In another preferred embodiment, the method for preparing the liposome formulation is method (B), and the aqueous phase does not include (g) and the aqueous solution includes (g), preferably by using dulagliptin in the form of tartrate to prepare the aqueous solution.

[0361] In another preferred embodiment, the method for preparing the liposome formulation is method (B), and the aqueous phase does not contain (g), and the aqueous solution contains (g), preferably in the form of a tartrate salt.

[0362] In another preferred embodiment, the method for preparing the liposome formulation is method (B), and the aqueous phase does not contain (g), and the aqueous solution contains (g), preferably by using the free base form of dulagliptin to prepare the aqueous solution.

[0363] In another preferred embodiment, the method for preparing the liposome formulation is method (B), and the aqueous phase does not contain (g), and the aqueous solution contains (g), preferably in the form of its free base.

[0364] In a very preferred embodiment, all components such as aqueous media, buffers and tonicity adjusting agents are pharmaceutically acceptable components.

[0365] Typically, the concentration of the buffer is 0.05 mM-100 mM, such as 1 mM-50 mM.

[0366] In a preferred embodiment, the aqueous phase comprises (d) consists of water, (e) comprises NaOH and / or HCl (e.g., trace amounts of one or both components for adjusting the pH), (f) comprises, preferably consists of, trehalose, and (g) consists of dulagliptin free base (see, e.g., Method A).

[0367] In another preferred embodiment, the aqueous phase comprises (d) water, (e) NaOH and / or HCl (e.g., trace amounts of one or both components for adjusting pH), (f) trehalose, and (g) a fatty acid salt form of doxepin (see, e.g., Method C).

[0368] In another preferred embodiment, the aqueous phase comprises (d) water, (e) NaOH and / or HCl (e.g., trace amounts of one or both components for adjusting pH), (f) trehalose, and (g) doxagliptin free base (see, e.g., Method A). Preferably, the amount of (d) is between 80% (w / w) and 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 between 3% (w / w) and 8% (w / w) based on the sum of (d), (e), (f) and (g), and the amount of (g) is between 6% (w / w) and 14% (w / w) based on the sum of (d), (e), (f) and (g).

[0369] In another preferred embodiment, the aqueous phase comprises (d) water, (e) NaOH and / or HCl (e.g., trace amounts of one or both components for adjusting pH), (f) trehalose, and (g) a fatty acid salt form of doxagliptin (see, e.g., Method C). Preferably, the amount of (d) is between 80% (w / w) and 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), and the amount of (f) is between 3% (w / w) and 8% (w / w) based on the sum of (d), (e), (f) and (g).

[0370] The amount of (g) is between 6% (w / w) and 14% (w / w) based on the sum of (d), (e), (f) and (g).

[0371] 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.

[0372] In a preferred embodiment, the aqueous phase consists of (d), (e), (f) and (g).

[0373] Aqueous solution

[0374] The aqueous solution as used herein comprises a (pharmaceutical) aqueous medium, preferably water or a combination of water and any 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, in particular propylene glycol and ethanol or a combination thereof, preferably wherein the amount of water is at least 80% of the total amount of solvent. In an even more preferred embodiment, water is the only solvent in the aqueous solution.

[0375] In one embodiment, the aqueous solution consists of water or a combination of water and any 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, in particular propylene glycol and ethanol or any combination thereof, wherein the amount of water accounts for at least 80% of the total amount of solvent. In a more preferred embodiment, the aqueous pharmaceutical solution consists of water.

[0376] A further preferred embodiment relates to an aqueous solution comprising a solvent as described herein and a tonicity adjuster.Preferred tonicity adjusters for use in aqueous solutions have been described for the formulations of the present invention.

[0377] Further preferred embodiments relate to aqueous solutions comprising solvents and tonicity modifiers and buffers as described herein. For the liposome formulations of the present invention, preferred tonicity modifiers and buffer systems for aqueous solutions have been described. The preferred tonicity modifier is NaCl, preferably, the amount of NaCl in the aqueous solution is (0.9 ± 0.1)% (w / w) based on the total amount of the aqueous solution, most preferably 0.9% (w / w).

[0378] Preferably, the pH value of the aqueous drug solution is selected so that the pH value of the resulting liposome preparation is between 6 and 8, preferably between 7 and 8.

[0379] In one embodiment, the aqueous formulation further comprises dulagliptin, preferably in the form of its tartrate salt or its free base form, more preferably in the form of its tartrate salt. Preferably, when dulagliptin is present in the form of its tartrate salt, the amount of dulagliptin is 5% (w / w) to 13% (w / w), more preferably (10±0.5)% (w / w), based on the total weight of the aqueous solution; or when dulagliptin is present in the form of its free base, the total weight of the aqueous solution is (6±0.4)% (w / w).

[0380] In one embodiment, the method for preparing a liposomal formulation of dulagliptin is method (B), and the aqueous solution comprises dulagliptin, preferably in the form of its tartrate salt.

[0381] In another preferred embodiment, the method for preparing the liposomal formulation of dulagliptin is method (B), and the aqueous solution comprises dulagliptin, preferably in the free base form thereof.

[0382] Those skilled in the art know that according to the preparation method of the present invention, the lyophilisate may already contain a buffer system or such a buffer system may be provided together with the drug aqueous solution. The technician can calculate the requirements for the drug aqueous solution for preparing the final liposome formulation without excessive burden.

[0383] Preferred tonicity modifiers, buffers, solvents, phospholipids, phosphatidylglycerols, lipopeptides, cholesterol (and its derivatives), sucrose components, ratios and amounts of any of the foregoing that can be used in the methods of the present invention have been described above with respect to the formulations of the present invention.

[0384] Calculate the concentration / amount of organic, aqueous and water phases

[0385] The skilled person is able to calculate the concentrations and amounts of the various components of the organic phase and the aqueous phase combined in steps A.3 / B.3 / C.3 and the concentrations and amounts of the various components of the aqueous solutions of the various steps to obtain the preparation according to the invention as required regarding the ratios between the various components without undue burden.

[0386] Surprisingly, it was found that the preparation of step A.3 / B.3 / C.3 is a nanodispersion system, wherein the D90 of the lipophilic vesicles is 60nm or less, preferably less than 25nm, more preferably 20nm or less, for example 10nm to 20nm, for example about 15nm, or 3nm to 10nm, for example about 5nm.

[0387] In other words, and without limitation to explanation, by combining the organic phase from A.1 / B.1 / C.1 and the aqueous phase from A.2 / B.2 / C.2, no liposomes are produced, but rather a nanodispersion system, presumably micelles, having a D90 particle size of 60 nm or less, preferably less than 25 nm is obtained. The nanodispersion can be sterile filtered.

[0388] In a preferred embodiment, the ratio between the organic phase and the aqueous phase is 3:1 (v / v) to 1:3 (v / v), even more preferably 2:1 (v / v) to 1:2 (v / v), such as about 1:1 (v / v). For the purpose 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.

[0389] In a preferred embodiment, when the ratio between the two phases is 1:1, the D90 of the vesicles in the resulting nanodispersion system is 15 nm or less, more preferably 10 nm or less, such as 3 nm to 10 nm. Without being bound by explanation, a high proportion of organic solvent can produce a predominantly molecular solution of the components within the solvent mixture.

[0390] In another preferred embodiment, when the ratio between the two phases is 1:3, the D90 of the vesicles in the nanodispersion system 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. Without being bound by explanation, under such a higher ratio of water, a micellar solution is formed, showing the typical size and uniformity of micelles. The mixture shows an almost transparent appearance, has a slight Tyndall effect, and can be freely filtered through a sterile filter with a nominal pore size of 0.22 μm.

[0391] At a solvent mixing ratio of 1:3, the resulting preparation was turbid. The size distribution measured by DLS revealed a broad, heterogeneous range of vesicle spectra with an average size of about 1.000 μm (1000 nm). The dispersion could be filtered through a membrane sterile filter with a nominal pore size of 0.22 μm simply by applying high pressure.

[0392] Generally, all steps of methods (A), (B) and (C) can be carried out at about room temperature (25°C) and at room temperature and at about standard pressure (101.325 kPa) and at standard pressure. If not otherwise specified, for example, for the freeze-drying step, each step can be carried out individually at a temperature of preferably 0°C-40°C, more preferably 15°C-35°C, such as 18°C-28°C. Although any step can also be carried out individually at higher and lower pressures, it is preferred that any step be carried out individually at a pressure of 90 kPa-112 kPa, more preferably 95 kPa-116 kPa, most preferably near standard pressure, such as 101.325 kPa±2%.

[0393] The freeze drying (lyophilization) technique is well known to the skilled person. Typically, freeze drying of the preparation of the invention is carried out at a temperature between +40°C and -40°C, preferably between +30°C and -10°C. The freeze drying step can be repeated one or more times. Typically, the pressure is 1000 hPa-0.001 hPa (1 hPa=1 mbar). Preferably, the freeze drying step is carried out at a pressure between 1 hPa and 0.01 hPa, for example about 0.1 hPa.

[0394] Optionally, the method comprises one or more additional steps.

[0395] Preferably, a further step of the method of the invention is a step of "sterile filtering" the nanodispersed system or the reconstituted lyophilized solution.

[0396] Preferably, sterile filtration is performed by using a membrane filter, such as a PVDF membrane filter. Preferably, the nominal pore size of the membrane filter is 200 nm or less.

[0397] It should be noted that for homogenization purposes, gently swirling or vortexing the obtained liposomal dispersion is sufficient. When the liposomal preparation shall be suitable for injection into patients in need of lipopeptide preparations, it is not necessary to reduce the size of the liposomes by further complicated steps.

[0398] The preparation of liposomal dugliptin formulation can be carried out according to or similar to the following examples.

[0399] Preparation of liposomal dulagliptin formulation using dulagliptin fatty acid salt can be performed similarly to Example 1 by replacing dulagliptin tartrate with dulagliptin fatty acid salt.

[0400] Preparation of liposomal dulagliptin formulation using dulagliptin fatty acid salt can be performed similarly to Example 2 by replacing dulagliptin tartrate with dulagliptin fatty acid salt.

[0401] The preparation of liposomal dulagliptin formulation using dulagliptin fatty acid salt can be carried out by adding dulagliptin fatty acid salt to the organic phase and then following the route of Example 1, but omitting dulagliptin in the aqueous phase.

[0402] The preparation of liposomal dulagliptin formulation using dulagliptin free base form can be performed similarly to Example 1 by replacing dulagliptin tartrate with dulagliptin free base form.

[0403] The preparation of liposomal dulagliptin formulation using dulagliptin free base form can be performed similarly to Example 2 by replacing dulagliptin tartrate with dulagliptin free base form.

[0404] The preparation of liposomal dulagliptin formulation using dulagliptin free base form can be carried out by adding dulagliptin free base form to the organic phase and then following the route of Example 1, but omitting dulagliptin in the aqueous phase.

[0405] Reagent test kit

[0406] Another aspect of the present invention relates to a kit comprising a lyophilisate as described herein and an aqueous formulation as described herein separated therefrom to prepare a liposomal formulation of the present invention. For example, the formulation of the present invention is in one container and the aqueous drug solution is in another container.

[0407] In a preferred embodiment, the lyophilisate contains a total amount of 60 mg to 100 mg of doxagliptin, and the amount of doxagliptin in the lyophilisate is 12% (w / w) to 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; if present, the amount of (c) is at most 24.3% (w / w), preferably, (c) contains cholesterol, and the amount of cholesterol is 4.8% (w / w) to 9.7% (w / w), based on the total amount of the lyophilisate; if present, the amount of (e) is at most 4.9% (w / w), based on the total amount of the lyophilisate; if present, the amount of (f) is at most 24.3% (w / w), preferably, (f) contains trehalose, and the amount of trehalose is 12% (w / w) to 53% (w / w), based on the total amount of the lyophilisate.

[0408] In a preferred embodiment, the drug aqueous solution is dispensed into another container, and the weight of this portion of the drug aqueous solution is calculated so that the total amount and ratio of the components i), ii, iii), iv) and v) of the resulting liposome preparation is the total amount and ratio of the liposome preparation of the present invention after the portion of the drug aqueous solution is added to the lyophilized material (or vice versa).

[0409] In a preferred embodiment, the volume of the aqueous solution added to the lyophilisate is 1.8 ml to 2.2 ml, for example 2 ml. Preferably, the volume of the aqueous solution in the container refers to a volume for subcutaneous injection.

[0410] Mixing the two-component aqueous drug solution and the lyophilized formulation allows for the preparation of a liposomal formulation of the drug shortly before administration to a patient.

[0411] Although the described invention has been described with reference to specific embodiments of the invention, it will be appreciated by those skilled in the art that various changes 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 specific circumstances, materials, compositions of matter, process steps or steps to achieve the intended scope of the invention. All such modifications are within the scope of the appended claims.

[0412] It is noteworthy that all preferred components mentioned in any phase, solution, formulation, lyophilisate, kit or method also refer to any other phase, solution, formulation, kit or method disclosed herein, even if not explicitly mentioned thereby and if applicable. For example, when a preferred filler is present in the organic phase, the same preferred agent is of course also a preferred filler for the lyophilisate and the resulting liposomal formulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0413] Figure 1 The two-phase system of the aqueous phase (lower phase) of dulagliptin and the organic TBA phase (upper phase) is shown. The left figure shows the aqueous phase (lower phase) of dulagliptin + TBA phase, and the right figure shows the aqueous phase (lower phase) of dulagliptin + TBA / PC / PG phase (each 1:1 w / w).

[0414] Figure 2 An online process data graph is shown. The freeze drying process was completed successfully and the process performed matched the preset parameters described in Section E5. Figure 2 The following parameters are shown:

[0415]

[0416] Figure 3 A photograph of a 50 mg dulalagliptin formulation is shown: Method Magnification: 40x (λ / 4 polarized microscope). There are various types of liposomes: small unilamellar vesicles, large unilamellar vesicles, multilamellar vesicles and oligolamellar vesicles. Example

[0417] E1 Substances and materials

[0418] The following substances and materials were used (Table).

[0419] Table 1: Substances and materials

[0420]

[0421]

[0422] The following equipment and apparatus were used: The following equipment and apparatus were used: HPLC Equipment Sys4:

[0423]

[0424]

[0425] Pilot freeze dryer (GT2):

[0426] Manufacturer: Hof Sonderanlagenbau (Lohra, Germany)

[0427] 0.36m 2 Shelf area

[0428] Temperature recording in bottle

[0429] Differential pressure measurement

[0430] Vacuum regulation: MKS and Pirani

[0431] ELISA reader

[0432] Manufacturer: Tecan( Switzerland)

[0433] Type: Safire SN: 12901300247

[0434] Additional laboratory equipment

[0435] Magnetic stirrer (IKA Werke)

[0436] Volumetric pipette (Gilson)

[0437] Camera (Canon, EOS 600D with DGMacro 105mm 1:2.8)

[0438] pH meter (Mettler Toledo, Sevenmulti with InLab microelectrode)

[0439] Balance (Kern, EW6200-2NM, ABJ-NM ABS3204N

[0440] Purified water supply system (Siemens, ultra-clean UV UF TM)

[0441] Vortex mixer (Scientific Industries Inc., Vortex Genie II)

[0442] Centrifuge: ThermoScientific, Heraeus Pico 17

[0443] Orbital shaker: Wisd Laboratory Instruments, WiseShake SHO-1DRP-HPLC analysis was performed using the following method

[0444] E2 RP-HPLC method

[0445] The intended use of this method is to determine the assay and purity of dulagliflozin drug product.

[0446] The AM-C16031637-G-01.03 method was used as supplied. The following chromatographic conditions were used:

[0447]

[0448] 25 mM sodium dodecyl sulfate (SDS)

[0449] Weigh 7.3 g of SDS into 1000 mL of pure water, dissolve and mix thoroughly.

[0450] Mobile phase A: 25 mM SDS: ACN: TFA = 600: 400: 1 (v / v / v)

[0451] To prepare 1 L of mobile phase, mix 600 ml of 25 mM SDS solution with 400 ml ACN and 1.0 ml 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.

[0452] Mobile phase B: 25mMSDS:ACN:TFA=400:600:1 (v / v / v)

[0453] To prepare 1 L of mobile phase, mix 400 ml of 25 mM SDS solution with 600 ml ACN and 1.0 ml TFA in a suitable container and mix thoroughly. Filter through a 0.45 μm nylon membrane (or other suitable solvent resin filter) and degas before use.

[0454] Thinner

[0455] Mobile phase A

[0456] E3 Salt Precipitation / Anion Exchange

[0457] Assuming that the ionic lattice of tartrate doxagliptin (dissolved in water) is pulled apart so that individual doxagliptin and tartrate ions are present in solution, exchange should occur by adding excess fatty acid anions. Due to the reduced solubility, the resulting hydrophobic salt should form and precipitate.

[0458] To facilitate the exchange, the salt dissolved at room temperature was cooled to 2-8°C after the addition of tartrate gliptin and stirred overnight.

[0459] E4 liposome encapsulation efficiency / entrapment volume

[0460] Liposomes were prepared in a solution of the fluorescent dye calcein. The fraction of the total volume in the liposomes was obtained as the fraction of fluorescence remaining after the addition of cobalt (II) ions, which quench the fluorescence when activated by calcein. Multilamellar placebo liposome lyophilisates were prepared according to the method described in Example 1, but without tartrate calcein, and reconstituted in 1 mL of Mops (4-morpholinopropanesulfonic acid)-buffered saline (pH 7.2) containing 50 nmol calcein.

[0461] 6.25 μL of liposome suspension was diluted to 250 μL with Mops buffered saline, transferred to a 96-well plate, and the fluorescence of the suspension was measured before (Ftot) and after the addition of 1.25 μL 10 mM CoCl2(Fin). The fluorescence plate reader parameters are listed in Table 2. Subsequently, 12.5 μL 10% Triton X-100 was added and the fluorescence was measured again (Ftotq). The latter addition destroyed the integrity of the liposomes. The capture volume can be calculated using the following formula:

[0462]

[0463] Among them, F tot Indicates the fluorescence of all calcein present

[0464] F in The fluorescence from the inner compartment plus the unquenched portion from the outer compartment

[0465] F totq Fluorescence representing the equilibrium concentration of free calcein

[0466] r represents the dilution factor caused by the addition of Triton X-100 and cobalt, currently 1.06

[0467] Table 2 Tecan fluorescence plate reader parameters

[0468]

[0469] E5 freeze-dried

[0470] Lyo-solution (nanodispersion system) was filled into sterile glass vials (1.5 g in 6R vials). The filled vials were stoppered at the lyophilization position and loaded into the freeze dryer. The following lyophilization cycle was performed.

[0471] Table 3: Procedure for a conservative, general lyophilization method

[0472]

[0473] E6 Polarization Microscope

[0474] A drop of the well-mixed reconstituted liposome formulation was placed on a microscope slide and covered with a microscope cover slip. The samples were examined with and without a polarizer at 400x magnification to identify encapsulated dulalagliptin / liposomes. Photos were taken at 40X magnification.

[0475] The following equipment was used: microscope: Axio Lab., A1-HAL with polarizer, Zeiss; camera: EOS600D, Canon.

[0476] Example 1: Method using tartrate (A):

[0477] Preparation of Nanodispersed System of Tartaric Acid Gliptin in Aqueous Phase

[0478] For the formation of liposomes, two phases are required: an organic solution and an aqueous solution.

[0479] Organic phase

[0480] For the organic phase, phosphatidylglycerol (PG) and S100 (highly purified soybean lecithin) were dissolved in tert-butyl alcohol (TBA) by heating (70° C.) and stirring for 40-80 minutes.

[0481] Once the ingredients were homogeneously dissolved, the organic phase was cooled to ambient temperature.

[0482] Water Phase

[0483] For the aqueous phase, a tartrate solution of gliptin obtained from Recardio (adjusted to pH 7.4) and trehalose were added to the water and dissolved under stirring.

[0484] Under continuous stirring, the aqueous solution is added in small portions to the organic phase. Depending on the amount of aqueous phase added to the organic phase, different liquid crystal phases may appear. The first aqueous solution is dissolved into a clear micellar system. When further aqueous solutions are added, cubic phases, hexagonal phases and final lamellar phases with different viscosities and turbidities may appear. After the aqueous phase is completely added, a low-viscosity opalescent nanodispersion system is obtained.

[0485] The final bulk solution was sterile filtered through a PVDF membrane filter with a nominal pore size of 200 nm.

[0486]

[0487]

[0488] Direct Encapsulation of Tartaric Acid Gliptin

[0489] In this mixture, a generally stable, homogeneous nanodispersed mesophase is formed with lipids. However, in the case of tartrate gliptin dissolved in the aqueous phase, TBA containing organic and aqueous phases does not form a stable solution, and phase separation of the nanodispersed system occurs after a short time (≤ 5 minutes).

[0490] Comparison with the lipid-free TBA solution and the aqueous phase in which the tartrate-containing gliptin was dissolved showed that the development of a two-phase system was even more pronounced and spontaneous (see Figure 1 ).

[0491] To suppress phase separation, the emulsion was stirred constantly during filling. 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 method was performed as described in section E5.

[0492] The lyophilized cake was reconstituted by adding water to prepare a liposomal suspension. A stable liposomal suspension was formed rapidly.

[0493] The reconstituted lyophilisate was observed under a polarizing microscope directly after reconstitution / homogenization at 22°C. Photos were taken for documentation (see Figure 3 ).

[0494] Liposomal encapsulation of dulagliptin was investigated by determining the amount of free (unencapsulated) dulagliptin. Equal intra- and extra-liposomal volumes refer to a 1:1 equilibrium concentration of the hydrophilic active pharmaceutical ingredient (API). A reduction in dulagliptin tartrate in the extra-liposomal matrix will result in a positive liposomal encapsulation efficiency.

[0495] After reconstitution, the liposomal dispersion was drawn into a standard single-use 3 mL PP syringe. A 27G×1" cannula (Sterican, B. Braun) was connected and the remaining air was removed from the syringe. The contents of the syringe were dispensed manually. Evaluation of the push-out force showed that a force of up to 20 N, more preferably up to 15 N, was sufficient to dispense the liposomal tartrate gliptin formulation.

[0496] It is possible to separate liposomes and aqueous phase by centrifugation only when using MLV dispersion. Therefore, the amount of liposomes in the extraliposome volume is determined by centrifugation of the lyophilized product reconstituted with water. The lyophilized product is mixed with 1mL saline, shaken and stored for 20 minutes at room temperature and slight stirring to form liposomes. In subsequent centrifugation (with 17000xg, 5 minutes), the liposomes cannot be successfully separated. Only after further dilution with 1mL water (total water volume 2mL) can successful separation be achieved. In order to avoid possible dilution errors, a comparative variant directly mixed with 2mL saline is produced. The fat-free supernatant (Table 4) of these samples is analyzed by RP-HPLC.

[0497] Adjusting equal intra-liposomal and extra-liposomal volumes, a 1:1 equilibrium concentration of hydrophilic dulalagliptin is expected. A decrease in the concentration of dulalagliptin in the extra-liposomal matrix indicates a positive liposome encapsulation efficiency.

[0498] Table 4 RP-HPLC results: Concentration of dulagliptin in the extraliposome volume after liposome preparation

[0499]

[0500] *Reconstitute the lyophilizate with 2 mL and, after liposome development, further dilute with 2 mL WFI.

[0501] **Decreases the concentration of extralipidic dugliptin.

[0502] Example 2: Method (B) Reconstitution of (placebo) lipid lyophilisate with aqueous solution of doxagliptin and determination of free drug concentration

[0503] In large-scale production, it is difficult to fill unstable emulsions that tend to phase separate. Therefore, an alternative manufacturing method was tested:

[0504] First, a (placebo) liposome lyophilisate without tartaric acid dapoxetine was prepared according to the above method. Then 0.9% NaCl (saline) was added to the tartaric acid dapoxetine solution to increase the analogy and ζ-potential. The resulting solution (100 mg / mL, 0.9% NaCl) was added to the (placebo) lyophilisate. After the vial was gently swirled for a few minutes, a uniform liposome emulsion of tartaric acid dapoxetine was formed.

[0505] The reconstituted vials were centrifuged directly and after the addition of 1 mL of saline (0.9% NaCl solution) (Table 5). The concentration of metoprolol in the separated aqueous phase was determined by RP-HPLC.

[0506] Table 5 RP-HPLC results: Concentration of dapoxetine in the extraliposome volume after liposomes were prepared with tartrate dapoxetine solution (100 mg / mL, 0.9% NaCl)

[0507]

[0508] *Reconstitute frozen material with 1 mL and further dilute with 1 mL saline after liposome expansion

[0509] Phase separation can be performed by centrifugation. However, due to the extremely high g-forces during centrifugation, the liposomes may be disrupted and leak, leading to invalid results for free drug concentrations. However, the results are close to theoretical values. Since tartrate-gliptin is highly water-soluble, it can be expected that the drug concentration inside and outside the liposomes is equal and that no drug is associated with the lipid phase of the vesicles.

[0510] In order to determine the amount of drug encapsulated, the volume of aqueous phase entrapped by the liposomes must be quantified.

[0511] Capture volume and derived encapsulation efficiency

[0512] The literature method for determining the liposome inclusion volume described in 3.6. is subject to certain uncertainties. Evaluation of fluorescence measurements showed an increased fluorescence quenching of liposome solutions compared to free calcein solutions. These are not structurally determined but rather determined by the components of the liposome preparation, which makes it necessary to consider the results individually according to the liposome composition.

[0513] The experiments to determine the liposome inclusion volume were performed in triplicate. A series of experiments were performed with double diluted / reconstituted liposome solutions. An overview is provided in Table 6.

[0514] Table 6. Normalized fluorescence signals (arbitrary units)

[0515]

[0516] *F tot : represents the fluorescence of all calcein present; F in : fluorescence from the inner compartment plus the unquenched portion from the outer compartment; F totq : Fluorescence representing the equilibrium concentration of free calcein

[0517] In case the mass-dependent liposome proportion of the reconstituted lyophilisate was 12.8% (w / w), the inclusion volume was determined to be approximately 30% of the total volume.

[0518] The encapsulation efficiency of the reconstituted liposome formulation containing 12.8% lipid, 100 mg total drug, and 1.0 mL saline was calculated by the following formula:

[0519] m L =100mg–100mg / mL+V L *0.3mL*100mg / mL=30mg

[0520] m L =Mass of liposome-encapsulated DGT

[0521] V L = aqueous phase volume of liposomes

[0522] Conclusion: About 1 / 3 of the total drug was encapsulated in the vesicles.

[0523] Example 3: Method (C): Preparation of a liposome formulation of dulaliptin containing a fatty acid salt of dulaliptin

[0524] Five different physiological fatty acids (as sodium salts) were prepared by anion exchange of tartrate and double molar salts at reduced temperature (reduced tartaric acid gliptin solubility). The most promising fatty acids were selected for further preparation. The solubility of the fatty acid salts was tested at 2-8°C (see table).

[0525] Table 7 Fatty acid sodium salts used to prepare water-insoluble gliptin salts

[0526]

[0527] Testing of the solubility of the sodium salts showed that, among the salts used, only sodium cholate remained sufficiently soluble under the given conditions (cooling to 2-8° C.) Upon addition of 100 mg / ml tartrate solution, the solution became slightly yellow, but there was no visible sign of turbidity or salt precipitation.

[0528] Example 4: Method (A) Preparation of a liposome formulation of dulagliptin containing dulagliptin free base

[0529] The protocol of the Comparative Example can be used with the modification that the free base form of dulagliptin is substituted for dulagliptin tartrate.

Claims

1. A method for preparing a liposomal dugliptin preparation, comprising the following steps: (B) B.1) providing an organic phase comprising (a) one or more phospholipids, wherein the amount of the 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 anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropyl glycerol, 1-butanol, 2-butanol and tert-butanol, or any combination of the foregoing; 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 further organic components which are neither (a) nor (b), wherein the amount of the one or more further organic components is up to 10%, based on the sum of (a), (b) and (c); wherein the sum of (a), (b) and (c) is 100%; and B.2) providing an aqueous phase, comprising (d) an aqueous medium, wherein 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 further components selected from a buffer system, NaOH and HCl, wherein the amount of all further components is at most 2% (w / w) based on the sum of (d), (e) and (f); (f) optionally a bulking agent, preferably trehalose, wherein 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); wherein the sum of (d), (e) and (f) is 100%; and B.3) mixing the aqueous phase and the organic phase until the ratio of the aqueous phase to the organic phase reaches 3:1-1:3, preferably 1:1, to obtain a nano-dispersed system; B.4) freeze-drying the nano-dispersed system to obtain a frozen product; B.5) reconstitute the lyophilized product described in step B.4 with an aqueous solution to obtain a liposomal dulalagliptin preparation, preferably, wherein the final concentration of dulalagliptin in the liposomal dulalagliptin preparation is 25 mg / ml to 60 mg / ml, wherein the aqueous solution comprises (i) from 67% (w / w) to 100% (w / w) of an aqueous medium, based on the total weight of the aqueous solution, optionally further comprising a tonicity adjuster, wherein the amount of the tonicity adjuster is at most 20% (w / w), based on the total weight of the aqueous solution, optionally further comprising a buffer system, (ii) dulagliptin, preferably in the form of its tartrate salt or its free base form, wherein when dulagliptin is present in the form of its tartrate salt or its free base form, the amount of dulagliptin is 5% (w / w) to 13% (w / w), more preferably 10±0.5)% (w / w), based on the total weight of the aqueous solution; or when dulagliptin is present in the form of its free base, the amount is (6±0.4)% (w / w), based on the total weight of the aqueous solution.

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

3. The process according to any one of the preceding claims, wherein the organic phase comprises lecithin as phospholipid and wherein the amount of lecithin is from 18.1% (w / w) to 26.2% (w / w), more preferably 22.13% (w / w).

4. The method according to any one of the preceding claims, wherein the organic phase further comprises PG, preferably DOPG-Na, as phospholipid, wherein the total amount of PG is from 0.1% (w / w) to 0.4% (w / w), preferably 0.23% (w / w).

5. The process according to any one of the preceding claims, wherein the pH of the aqueous phase is between 7 and 7.8, preferably 7.

4.

6. A method according to any one of the preceding claims, wherein: The D90 of the liposomes of the liposomal dulalagliptin formulation produced by the reconstitution step is 1 μm to 4.5 μm.

7. A method according to any one of the preceding claims, wherein the only solvent in the aqueous solution is water.

8. The method according to any one of the preceding claims, wherein the aqueous solution is an aqueous NaCl solution, wherein the amount of NaCl is between 8 g / l and 10 g / l, preferably between 8.8 g / l and 9.2 g / l, more preferably 9 g / l.

9. The method according to any one of the preceding claims, wherein the aqueous solution in step B.5 is a tartrate solution of gliptin.

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

11. The method according to any one of the preceding claims, wherein the total amount of dolagliflozin in the liposomal dolagliflozin formulation is between 60 mg and 100 mg.

12. A liposomal doxepin formulation preparable according to the method of claims 1 to 11.

13. A liposomal dugliptin preparation comprising (vi) an aqueous medium, preferably water, wherein the amount of the aqueous medium is between 65% (w / w) and 90% (w / w) based on the total weight of the liposomal doxepin formulation; (vii) optionally a lipid, preferably cholesterol, wherein the amount of the lipid, preferably cholesterol, is 1% (w / w) to 2.5% (w / w) based on the total weight of the liposomal dugliptin formulation; (viii) one or more phospholipids, wherein the amount of the 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 dugliptin formulation, preferably, wherein 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, 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 from 1.5% (w / w) to 5.5% (w / w); (x) dulagliptin, wherein the amount of dulagliptin, preferably in the form of its tartrate salt or free base, is between 3% (w / w) and 12% (w / w), preferably (5.3±0.3) (w / w), based on the total weight of the liposomal dulagliptin formulation, and the final concentration of dulagliptin in the liposomal dulagliptin formulation is between 25 mg / ml and 60 mg / ml; The sum of (i) to (v) is 100%.

14. A kit comprising the lyophilisate of claims 1 to 5 in a container and the aqueous pharmaceutical solution of claim 1, 7, 8 or 10 in a second container.

15. The kit according to claim 14, further comprising instructions on how to combine the contents of the first container and the second container to contain the liposomal dugliptin formulation according to claim 12 or 13.

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