New lipopeptide formulations

Abstract

Novel pharmaceutical liposomal formulations of lipopeptides and their uses and preparation are provided.

Description

[Background technology]

[0001] background Brevirtide is a novel drug candidate for the treatment of chronic hepatitis B and chronic delta hepatitis (also known as hepatitis D), and potentially a variety of inflammatory and metabolic diseases. Brevirtide is disclosed in WO 2009 / 092612. Brevirtide is a linear, 47 amino acid chemically synthesized peptide derived from the N-terminal domain of the large HBV surface protein (HBV=hepatitis B virus). The active substance is available as an acetate salt. Its antiviral mode of action relies on the specific binding and blocking of the hepatocyte surface protein NTCP. Brevirtide-mediated inhibition of NTCP prevents the entry of HBV and HDV (hepatitis D virus) into cells.

[0002] It has been recently discovered that hepatitis B virus and hepatitis delta virus enter hepatocytes via binding to NTCP with the HBV preS1 surface protein domain. As a mimetic of the preS1 domain, myrcudex B specifically blocks the corresponding NTCP binding site, thereby inhibiting viral entry into hepatocytes.

[0003] Blockade of NTCP is not only relevant for the development of antiviral drugs: the effects of this mechanism on lipid metabolism, particularly elevated bile acid levels, may have positive implications for the treatment of a variety of other diseases.

[0004] Hepatitis delta is the most severe form of viral hepatitis. It is caused by HDV, a small RNA virus that requires helper functions from HBV for virion assembly and propagation, and uses the HBV envelope for viral release and infection of new cells. Approximately 5% of chronically infected HBV patients are co-infected with HDV.

[0005] The presence of HDV is associated with a more severe and rapid progression of liver disease than HBV infection alone. During the course of HBV / HDV coinfection, cirrhosis and decompensation occur earlier and more frequently than in HBV infection alone.

[0006] Antiviral agents active against HBV are inactive against HDV, so treatment options for patients with HDV coinfection are extremely limited.

[0007] For the treatment of HDV infection, for example, Brevirtide, also known as Hepcludex®, was conditionally approved in July 2020 as one of the first specific agents against HDV. Brevirtide appears as a white or off-white hygroscopic powder. Brevirtide is practically insoluble in water and soluble at a concentration of 1 mg / ml in 50% acetic acid and about 7 mg / ml in carbonate buffer at pH 8.8, respectively. Hepcludex® is available as a powder that can be stored at -20°C or at a temperature of 2°C to 8°C for up to 3 months according to the instructions for Hepcludex® 2 mg by MYR Pharmaceuticals. At the time of use, Hepcludex® is reconstituted with water for injection, followed by a subcutaneous injection, which is required once a day for the course of treatment.

[0008] There are various methods to solubilize poorly soluble compounds for parenteral administration. Typical approaches are pH optimization or the use of cosolvents 15 (e.g., PEG300, PEG400, propylene glycol or ethanol). If these approaches are not feasible for some reason, the use of surfactants can be considered (e.g., Tween® 80 or Cremophor EL®). However, these types of surfactants are often associated with adverse effects.

[0009] Cyclodextrins are established as safe solubilizers, but are limited in that they are not effective solubilizers for all compounds, and compounds that are highly soluble in 20 natural oils (e.g., propofol) can be solubilized in parenteral fat emulsions.

[0010] Another possibility for solubilizing poorly soluble compounds is the use of phospholipids (van Hoogevest P., Xiangli L., and Alfred F. “Drug delivery strategies for poorly water-soluble drugs: the industrial perspective” Expert Opinion an Drug Delivery 2011, 8(11), 1481-1500 (Non-Patent Document 1)). However, the solubilization of a particular poorly soluble compound by phospholipids cannot be predicted, and the size of the liposomes must usually be adapted so that the liposomes are suitable for pharmaceutical use.

[0011] US 8,591,942 and US 9,655,846 disclosed a method for preparing liposomes containing docetaxel. In US 8,591,942, the method includes dispersing soybean phosphatidylcholine and sodium oleate in an aqueous medium to produce dispersed liposomes.

[0012] US 2008 / 0166403 (Patent Document 4) disclosed long-circulating liposomes comprising a phospholipid bilayer and a hydrophilic core containing a vitamin E derivative (D-alpha tocopheryl polyethylene glycol 1000 succinate, TPGS).

[0013] Liposomes are usually prepared by dissolving lipids in an organic solvent, lyophilizing, and subsequently hydrating (which usually results in multilamellar liposomes that are not suitable for most applications due to their large size and low encapsulation volume). Additional steps such as extrusion or complex homogenization must be used to reduce the size of large liposomes. However, the encapsulation efficiency of these processes is usually less than 70%. 50% is considered high, and generally, encapsulation efficiencies of 20% to 30% can be obtained (Dan Lasic, TibTech July 1998, Vol. 16, pages 307 to 321 (Non-Patent Document 2)).

[0014] In some cases, encapsulation efficiencies of about 90% have been observed, but these observations result in high and undesirable lipid-to-drug ratios for MLV (multilamellar vesicle) encapsulation, which typically have diameters of 5-50 μm (Liposome-Based Depot Injection Technologies Nandini V. Katre Am J Drug Deliv 2004;2(4):213-227 1175-9038 / 04 / 0004-0213 / $31.00 / 0 (Non-Patent Document 3)).

[0015] WO 2014 / 167435 (Patent Document 5) disclosed a surface-functionalized liposomal formulation comprising an anticancer drug as an active ingredient, liposomes surrounded by a functional coating of Da-tocopheryl polyethylene glycol 1000 succinate (TPGS), the anticancer drug being encapsulated within the liposome, and further the formulation having an encapsulation efficiency of >70%.

[0016] Muthu et al;Biomaterials.2012 Apr;33(12):3494-501 (Non-Patent Document 4) disclosed TPGS-coated liposomes for brain delivery of docetaxel, prepared by solvent injection method. The reported formulation resulted in an encapsulation efficiency of about 64.10±0.57%, which was significantly lower than the present invention (encapsulation efficiency of about 95%).

[0017] WO 2010 / 078045 A2 (Patent Document 6) discloses a method for preparing liposomes of a restricted particle size by substantially continuously mixing a substantially continuously flowing stream of water with a substantially continuously flowing stream of an organic solvent-containing lipid capable of forming liposomes and cooling the mixture so that liposomes are formed, the ratio of the flow rate of the water stream to the flow rate of the organic solvent stream and the cooling rate of the mixture being controlled to obtain a preparation of liposomes such that at least about 90% of the liposomes are less than about 200 nm in size.

[0018] CN 110339166 A (Patent Document 7) relates to liraglutide multivesicular liposomes and its preparation method and use. More specifically, the liraglutide multivesicular liposomes include liraglutide, a membrane material, an osmotic pressure regulator and a stabilizer.

[0019] Zhang et al. (Drug Delivery 23(9):3358-3363, 2016) discloses subcutaneous liraglutide-loaded multivesicular liposomes for treating diabetes by using a two-step water-in-oil-in-water double emulsification process.

[0020] CN 102688192 A (Patent Document 8) discloses a preparation of a polypeptide drug for treating diabetes, which is mainly composed of phosphatide, sesame oil, glycerin, liraglutide, salt and ethanol, and a method for producing the same.

[0021] Uhl et al. (European Journal of Pharmaceutics and biopharmaceutics, 103:159-166, 2016) (Non-Patent Document 6) disclose a liposomal formulation containing certain tetraether lipids for oral administration of the investigational hepatitis B peptide drug Myrcudex B.

[0022] Surprisingly, it has been observed that formulations of lipopeptides such as brevirtide or liraglutide can be prepared with high encapsulation efficiency by using the simple preparation method according to the present invention.The preparation method according to the present invention not only produces a surprisingly high loading of lipopeptide, such as brevirtide, but also a single-phase nanodispersion that can be lyophilized.Rehydration of such a lyophilized single-phase nanodispersion produces a liposome formulation with a liposome size suitable for subcutaneous injection and a favorable liposome / drug ratio.

[0023] In particular, it has been surprisingly found that a single-phase nanodispersion can be easily prepared using the method of the present invention by combining an organic phase with an aqueous phase, the organic phase comprising one or more phospholipids and one or more organic solvents, the one or more organic solvents forming a single-phase mixture with the aqueous phase that can be frozen and sublimed. The single-phase nanodispersion mixture is generated (spontaneously) by simply mixing the organic phase with the aqueous phase, without the need for mechanical means such as high-shear mixers, high-pressure homogenizers or ultrasound to reduce the size of vesicles. The single-phase nanodispersion contains micelles that can be sterile filtered and freeze-dried without any mechanical size reduction steps that are usually required in the current techniques listed above. This is particularly advantageous, since such freeze-dried single-phase nanodispersions are found to be stable and therefore can be stored and / or shipped without special temperature requirements and / or time constraints.

[0024] Also, lipopeptides such as brevirtide can be well preserved in such single-phase nanodispersions prepared according to the present invention, especially in lyophilized form, although the lipopeptide may not be stable in solution as in the case of brevirtide.When required for administration, the lyophilized preparation of single-phase nanodispersions can be easily reconstituted by adding an aqueous solution, for example, as typically used for the reconstitution of lyophilized drugs.By reconstituting the single-phase nanodispersions according to the present invention, preferably with an aqueous salt-containing solution such as saline or buffer, liposomes are prepared in situ.In particular, larger multi-layered liposomes can be prepared using the method of the present invention, compared to the current approach based on direct preparation and mechanical size reduction of liposomes, which are sterile-filtered using a pharmacy approved filter with a nominal pore size of 0.22 μm before lyophilization. The ability to generate multilamellar liposomes in situ with D90 between 1 μm and 4.5 μm according to the present invention allows the liposomes to be advantageously used for subcutaneous injections forming depots that allow for prolonged release of the encapsulated drug, and also allows the liposomes to be loaded with more lipopeptides, such as brevirtide, compared to liposomes generated using current methods. Of note, with respect to this paragraph, the same applies to each formulation according to the present invention.

[0025] The pharmaceutical liposomal formulation of the present invention is particularly advantageous for the administration of lipopeptides such as brevirtide.Compared to subcutaneous injection of lipopeptides such as brevirtide in solution, administration using the pharmaceutical multi-layered liposomal formulation of the present invention results in prolonged bioavailability due to slower release of lipopeptides from subcutaneous depot.As a result, the pharmaceutical liposomal formulation of the present invention may be required, for example, once a week instead of daily.Therefore, the pharmaceutical liposomal formulation of the present invention, preferably comprising brevirtide, has the potential to significantly improve the quality of life of patients.

[0026] The present invention therefore relates to an easy and highly efficient method for the preparation of lipopeptide-containing formulations, based on the preparation of nanoscale systems by combining organic and aqueous phases and adding lipopeptides, which are thermodynamically stable and can be easily formulated, sterile filtered, filled and lyophilized according to standard procedures of pharmaceutical manufacturing. The lyophilized preparations are characterized by high stability and shelf life and can be easily reconstituted in situ, for example by adding water for injection or saline, to generate lipopeptide-containing liposomes ready for parenteral administration, in particular for subcutaneous injection.

[0027] The actual scope of the invention is defined by the following claims. Aspects of the invention are the subject of the dependent claims and are disclosed throughout the specification and drawings. [Prior art documents] [Patent documents]

[0028] [Patent Document 1] WO 2009 / 092612 [Patent Document 2] US 8,591,942 [Patent Document 3] US 9,655,846 [Patent Document 4] US 2008 / 0166403 [Patent Document 5] WO 2014 / 167435 [Patent Document 6] WO 2010 / 078045 A2 [Patent Document 7] CN 110339166A [Patent Document 8] CN 102688192A [Non-patent literature]

[0029] [Non-Patent Document 1] van Hoogevest P., Xiangli L., and Alfred F. “Drug delivery strategies for poorly water-soluble drugs: the industrial perspective” Expert Opinion an Drug Delivery 2011, 8(11), 1481-1500 [Non-Patent Document 2] Dan Lasic,TibTech July 1998,Vol.16,pages 307 to 321 [Non-Patent Document 3] Liposome-Based Depot Injection Technologies Nandini V.Katre Am J Drug Deliv 2004;2(4):213-227 [Non-Patent Document 4] Muthu et al;Biomaterials.2012 Apr;33(12):3494-501 [Non-Patent Document 5] Zhang et al.(Drug Delivery 23(9):3358-3363,2016) [Non-Patent Document 6] Uhl et al.(European Journal of Pharmaceutics and biopharmaceutics,103:159-166,2016) Summary of the Invention

[0030] overview The first phase (phase 1) is (a) one or more phospholipids selected from the group consisting of phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), or derivatives of any of the foregoing, or mixtures thereof, in the range of 40% to 99.7%, preferably 40% to 97%, and more preferably 40% to 75%, based on the total weight of (a) to (e); (b) a lipopeptide, preferably brevirtide or liraglutide, in the range of 0.3% to 20% based on the total weight of (a) to (e); (c) cholesterol or a derivative thereof, preferably in the range of 0% to 14%, preferably 4% to 14%, based on the total weight of (a) to (e); (d) in the range of 0% to 35%, preferably 15% to 35%, based on the total weight of (a) to (e), a bulking agent, preferably glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, or 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, or mixtures thereof; (e) an isotonicity agent other than (d), in the range of 0% to 35%, preferably in the range of 0.1% to 10%, based on the total weight of (a) to (e); 1. A formulation which is a single-phase nanodispersion system comprising: The sum of (a), (b), (c), (d), and (e) always totals 100%, and the combined amount of (a)-(e) is 10%-100% based on the total weight of the formulation. Thus, a formulation according to aspect 1 relates to a single-phase nanodispersion system comprising micelles loaded with a lipopeptide. The lipopeptide is preferably brevirtide or liraglutide, and preferably brevirtide is in the range of 3%-13% based on the total weight of (a)-(e), or liraglutide is in the range of 0.3%-2% based on the total weight of (a)-(e).

[0031] Preferred embodiment 1 relates to the formulation of aspect 1, wherein the formulation is a lyophilized formulation. This is advantageous because the lyophilized formulation according to preferred embodiment 1 is stable and therefore can be stored and / or shipped without specific temperature and / or time constraints.

[0032] Further preferred embodiment 2 relates to a lyophilized formulation according to preferred embodiment 1, in which the amount of brevirtide is 12 mg to 24 mg.

[0033] Further preferred embodiment 2 relates to the lyophilized formulation according to preferred embodiment 1, wherein the amount of liraglutide is 0.6 mg to 1.8 mg.

[0034] A further aspect is a formulation, which is a pharmaceutical liposomal formulation obtained by mixing / reconstituting the formulation of any of aspect 1, preferred embodiment 1, and / or the two further preferred embodiments 2 described hereinbefore, (a) one or more phospholipids selected from the group consisting of phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), or derivatives of any of the foregoing, or mixtures thereof, in the range of 40% to 99.7%, preferably 40% to 97%, and more preferably 40% to 75%, based on the total weight of (a) to (e); (b) a lipopeptide, preferably brevirtide or liraglutide, in the range of 0.3% to 20% based on the total weight of (a) to (e); (c) cholesterol or a derivative thereof in the range of 0% to 14%, preferably in the range of 4% to 14%, based on the total weight of (a) to (e); (d) 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, saccharose, xylitol, xylose, dextran or a mixture thereof, in the range of 0% to 35%, preferably 15% to 35%, based on the total weight of (a) to (e); (e) an isotonicity agent other than (d), in the range of 0% to 35%, preferably in the range of 0.1% to 10%, based on the total weight of (a) to (e); Including, The sum of (a), (b), (c), (d) and (e) totals 100%, and the combined amount of (a)-(e) is 10%-50% based on the total weight of the formulation further including a solvent, preferably water. Preferably, the pharmaceutical liposomal formulation according to said further aspect is obtained by mixing or reconstituting the formulation of any of aspect 1, preferred aspect 1 and / or the two further preferred aspects 2 described herein above with an aqueous solution, preferably a salt-containing aqueous solution such as saline or a buffer (physiologically acceptable solution). With respect to the lipopeptide, said lipopeptide is preferably brevirtide or liraglutide, preferably brevirtide is in the range of 3%-13% based on the total weight of (a)-(e) or liraglutide is in the range of 0.3%-2% based on the total weight of (a)-(e).

[0035] A further preferred embodiment 4 relates to a liposomal pharmaceutical composition according to a further aspect, wherein the tonicity agent is NaCl.

[0036] A further preferred embodiment 5 relates to a liposomal pharmaceutical formulation according to the further aspect and / or preferred embodiment 4, further comprising a buffer system.

[0037] A further preferred embodiment 6 relates to a liposomal pharmaceutical formulation according to any one of the further aspects and / or preferred embodiments 4 to 5, wherein the pH of the liposomal pharmaceutical formulation is 5 to 8, preferably 5 to 7.6, and in a more preferred embodiment 6 to 7.6 (e.g. either slightly acidic to neutral, e.g. 6.5 to 7, or 7.2 to 7.6, e.g. 7.3 to 7.5).

[0038] A further preferred embodiment 7 relates to a formulation according to aspect 1, the further aspects, and / or any one of preferred embodiments 1 and 6, wherein (a) is PC or a mixture of PC and one or more phospholipids selected from the group consisting of phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidic acid (PA), preferably PC.

[0039] A further preferred embodiment 8 relates to a formulation according to aspect 1, the further aspects, and / or any one of the preferred embodiments 1-7, wherein (e) is trehalose.

[0040] A further preferred embodiment 9 relates to a formulation according to aspect 1, the further aspects, and / or any one of the preferred embodiments 1-8, wherein (a) is PC, and (a) is 50% to 70% relative to the sum of (a), (b), (c), (d), and (e) in the formulation, and (b) is 5% to 11% relative to the sum of (a), (b), (c), (d), and (e) in the formulation, and (d) is 6% to 12% relative to the sum of (a), (b), (c), (d), and (e) in the formulation, and (e) is trehalose, and (e) is 20% to 30% relative to the sum of (a), (b), (c), (d), and (e) in the formulation, and the sum of (a), (b), (c), (d), and (e) add up to 100%.

[0041] A further preferred embodiment 10 relates to a pharmaceutical composition according to any of the further aspects and / or preferred embodiments 4 to 9 for use as a medicament.

[0042] A further preferred embodiment 11 relates to a pharmaceutical composition for use according to preferred embodiment 10, wherein said use is in the treatment of chronic hepatitis B and / or chronic hepatitis D. This has the advantage that the frequency of administration of a lipopeptide such as brevirtide can be reduced compared to the administration of the respective lipopeptide in solution, thus improving the quality of life of the patient.

[0043] A further preferred embodiment 11 relates to a pharmaceutical composition for use according to preferred embodiment 10, wherein said use is in the treatment of inflammatory, preferably inflammatory diseases.

[0044] A further preferred embodiment is (a) one or more phospholipids selected from the group consisting of phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), or derivatives of any of the foregoing, or mixtures thereof, in the range of 40% to 70% based on the total weight of (a) to (e); (b) a lipopeptide, preferably brevirtide or liraglutide, in the range of 0.3% to 20% based on the total weight of (a) to (e); (c) cholesterol or a derivative thereof in the range of 4% to 14% based on the total weight of (a) to (e); (d) in the range of 15% to 35% based on the total weight of (a) to (e), a bulking agent, preferably glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, or 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, or mixtures thereof; (e) an isotonicity agent other than (d), in the range of 0% to 35% based on the total weight of (a) to (e); Including, The formulation according to aspect 1, further aspects, and / or any of preferred embodiments 1-10 thereof, wherein the sum of (a), (b), (c), (d), and (e) total 100%.

[0045] In a preferred embodiment, the formulation according to aspect 1, or any of preferred embodiments 1 or 2 thereof, wherein the formulation further comprises tert-butanol in the range of 0.01% to 2% based on the total weight of the formulation.

[0046] In yet another preferred embodiment, the formulation according to aspect 1, or any of preferred embodiments 1 or 2 thereof, wherein the formulation further comprises tert-butanol in the range of 0.01% to 2% based on the total weight of the formulation, and the combined amount of (a) through (e) is 98% to 99.99% based on the total weight of the formulation.

[0047] A further aspect 2 relates to the use of a formulation according to aspect 1, or any one of the preferred aspects 1 or 2, for the preparation of a medicament for treating chronic hepatitis B and / or chronic hepatitis D or inflammatory diseases, preferably for the preparation of a medicament for treating chronic hepatitis B and / or chronic hepatitis D or inflammatory diseases.

[0048] Further aspect 3 relates to a kit comprising a formulation according to aspect 1 or any one of preferred aspects 1 or 2 and separating an aqueous pharmaceutical solution. For example, a kit comprising a formulation according to aspect 1 or any one of preferred aspects 1 or 2 in a container and an aqueous pharmaceutical solution in a second container. Optionally, further comprising instructions for mixing the two components of the two containers to receive a pharmaceutical liposomal formulation, preferably ready to use. Thus, aspect 3 relates to a kit comprising a lyophilized single-phase nanodispersion preferably formulated with lipopeptide and an aqueous pharmaceutical solution that can be used to reconstitute the lyophilized single-phase nanodispersion and thus prepare liposomes formulated with said lipopeptide for subsequent use, preferably for subsequent subcutaneous injection.

[0049] Further Phase 4 is (i) one or more phospholipids selected from the group consisting of phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), or a derivative of any of the foregoing, or a combination of any of the foregoing; Optionally, cholesterol or a derivative of cholesterol, At least one organic solvent providing an organic phase comprising (ii) an aqueous medium; Optionally, a bulking agent selected from the group consisting of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, a sugar moiety selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, xylitol, xylose, dextran, or a mixture of any of the foregoing; optionally a pharma- ceutically acceptable buffer; Optionally, a pharma- ceutically acceptable tonicity agent that is not a bulking agent selected from the group consisting of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, a sugar moiety selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, saccharose, xylitol, xylose, dextran, or a mixture of any of the foregoing. providing an aqueous phase comprising the pH of the aqueous phase is between 3 and 9, for example between 5.5 and 7, for example between 5.8 and 6.7 (for example by using a sodium acetate buffer); (iii) combining an organic phase with an aqueous phase, wherein the mixing ratio of the organic phase to the aqueous phase is 10:1 (v / v) to 1:10 (v / v), preferably 2:1 (v / v) to 1:4 (v / v), more preferably 1.5:1 (v / v) to 1:4 (v / v), for example 1:1 (v / v) to 1:3 (v / v), about 1±0.5:1±0.5 (v / v), about 1±0.5:2±0.5 (v / v), or about 1±0.5:3±0.5 (v / v), for example 1:1 (v / v), 1:2 (v / v) or 1:3 (v / v), which results in a combined organic phase and aqueous phase, wherein the at least one organic solvent and the aqueous phase form a single-phase mixture, preferably a freezable and sublimable single-phase mixture, (iv) either adding the lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the organic phase of step (i) and then mixing the organic phase containing the lipopeptide with the aqueous phase as described in step (iii), or adding the lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the aqueous phase of step (ii) and then mixing the aqueous phase containing the lipopeptide with the organic phase as described in step (iii), or adding the lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the combined phases of step (iii), preferably to the combined phases of step (iii), which results in a lipopeptide formulation, the lipopeptide formulation so obtained being a single-phase nanodispersion. The present invention relates to a method for preparing a formulation comprising:

[0050] It is therefore envisaged that the single-phase mixture formed by combining the aqueous phase with at least one organic solvent contained in the organic phase is preferably freezeable and sublimable. This has the advantage that the organic phase can be substantially removed from the formulation by lyophilization. In this specification, the term "freezable" refers to the physicochemical property of the mixture forming a solid matrix below a temperature in the range of 0°C to -60°C. In particular, it is preferred that at a temperature in the range of 0°C to -60°C, the single-phase mixture prepared in step (iii) substantially forms a solid matrix. Furthermore, the term "sublimable" is intended to be understood as the physicochemical property of the mixture passing from a solid state to a gaseous state without any intermediate liquid state at a pressure in the range of 1 Pa to 100 Pa and at a temperature in the range of 0°C to -60°C.

[0051] Thus, a preferred single-phase mixture can be obtained by combining an aqueous phase, preferably water, with an organic phase comprising at least one organic solvent, preferably selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, alcohol, preferably selected from the group consisting of 1-butanol, 2-butanol, and tert-butanol, acetic acid, ethyl lactate (ethyl 2-hydroxypropanoate), acetonitrile, and any combination of the foregoing.

[0052] Alternatively, or optionally, the at least one organic solvent may be selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, 1-butanol, 2-butanol, and tert-butanol, and combinations of any of the foregoing.

[0053] Alternatively or optionally, it is particularly preferred that the at least one organic solvent is selected from the group consisting of alcohols, preferably tert-butanol, anisole (phenoxymethane), dimethylsulfoxide, 1,4-dioxane, and dimethylcarbonate, and combinations thereof. Preferably, the at least one organic solvent comprises or is tert-butanol.

[0054] Alternatively, or optionally, the at least one organic solvent may be selected from the group consisting of acetic acid, ethyl lactate (ethyl 2-hydroxypropanoate) and acetonitrile.

[0055] It should be understood that the listed examples of organic solvents are given for illustration and not limitation, in particular, those skilled in the art know how to identify suitable organic solvents, and thus organic solvents that can preferably form freezable and sublimable single-phase mixtures with aqueous solutions such as water.

[0056] Surprisingly, it has been found that preparing a single-phase mixture according to the method of the present invention has the advantageous effect that one or more phospholipids contained in the organic phase constitute a single-phase nanodispersion system that can be sterile filtered.

[0057] In a preferred embodiment, the method according to aspect 4 comprises: (i) one or more phospholipids selected from the group consisting of phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), or a derivative of any of the foregoing, or a combination of any of the foregoing; Optionally, cholesterol or a derivative of cholesterol, at least one organic solvent selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, alcohol, preferably an alcohol selected from the group consisting of 1-butanol, 2-butanol, and tert-butanol, acetic acid, ethyl lactate (ethyl 2-hydroxypropanoate), acetonitrile, or a combination of any of the foregoing; providing an organic phase comprising Preferably, the at least one organic solvent is selected from the group consisting of an alcohol, preferably tert-butanol, anisole (phenoxymethane), dimethylsulfoxide, 1,4-dioxane, and dimethylcarbonate, and combinations thereof; (ii) an aqueous medium; optionally a pharma- ceutically acceptable buffer; Optionally, a bulking agent selected from the group consisting of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, a sugar moiety selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, xylitol, xylose, dextran, or a mixture of any of the foregoing; Optionally, a pharma- ceutically acceptable tonicity agent that is not a bulking agent selected from the group consisting of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, a sugar moiety selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, saccharose, xylitol, xylose, dextran, or a mixture of any of the foregoing. providing an aqueous phase comprising the pH of the aqueous phase is between 3 and 9, for example between 5.5 and 7, for example between 5.8 and 6.7 (for example by using a sodium acetate buffer); (iii) combining an organic phase with an aqueous phase, wherein the mixing ratio of the organic phase to the aqueous phase is between 10:1 (v / v) and 1:10 (v / v), more preferably between 2:1 (v / v) and 1:4 (v / v), even more preferably between 1.5:1 (v / v) and 1:4 (v / v), for example between 1:1 (v / v) and 1:3 (v / v), about 1±0.5:1±0.5 (v / v), about 1±0.5:2±0.5 (v / v), or about 1±0.5:3±0.5 (v / v), for example 1:1 (v / v), 1:2 (v / v) or 1:3 (v / v), which results in a combined organic phase and aqueous phase, wherein the at least one organic solvent and the aqueous phase form a single-phase mixture, preferably a freezable and sublimable single-phase mixture, (iv) either adding the lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the organic phase of step (i) and then mixing the organic phase containing the lipopeptide with the aqueous phase as described in step (iii); or adding the lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the aqueous phase of step (ii) and then mixing the aqueous phase containing the lipopeptide with the organic phase as described in step (iii); or adding the lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the combined phases of step (iii), preferably to the combined phases of step (iii), which results in a lipopeptide formulation, the lipopeptide formulation so obtained being a single-phase nanodispersion.

[0058] A preferred embodiment 1 of aspect 4, and the preferred embodiment above, relates to a process wherein the organic solvent is tert-butanol.

[0059] A preferred embodiment 2 of aspect 4, and preferred embodiments thereof, relates to a method, wherein the lipopeptide is brevirtide or liraglutide.

[0060] A preferred embodiment 3 of aspect 4, and preferred embodiments thereof, relates to a method, wherein the phospholipid comprises PC.

[0061] A preferred embodiment of aspect 4, and preferred embodiments thereof, relates to a process, wherein the pH of the aqueous phase is 5-6.

[0062] A preferred embodiment 5 of aspect 4, and preferred embodiments thereof, relates to a method further comprising a step (v) of freeze-drying the formulation (single-phase nanodispersion) obtained from step (iv), resulting in a lyophilisate, which is understood to be a preferred but purely optional step, referred to as "step (v)" merely for clarity.

[0063] A preferred embodiment 6 of aspect 4 and preferred embodiment 5 further comprise a step of rehydrating the lyophilized product obtained in step (v) with an aqueous solution, which results in a liposomal preparation. Thus, in particular, preferred embodiment 6 relates to a rehydration step in which the lyophilized product obtained in step (v) is mixed with an aqueous solution to result in a liposomal preparation. The liposomal preparation is a reconstituted liposomal preparation. In this specification, the term "reconstitution" refers to the rehydration of the lyophilized product (single-phase nanodispersion system) by mixing the lyophilized product with an aqueous solution, preferably with saline or water, more preferably with saline.

[0064] A preferred embodiment 7 of aspect 4, as well as preferred embodiment 6, relates to a method, wherein the D90 of the liposomes of the liposome formulation obtained from the rehydration step (reconstitution) is between 1 μm and 4.5 μm, preferably less than 2.5 μm, more preferably between 0.025 μm and 2.5 μm, even more preferably between 0.1 μm and 2 μm. Thus, the liposome formulation may be prepared by mixing or reconstituting the lyophilized single-phase nanodispersion with an aqueous solution, preferably saline or water, more preferably saline.

[0065] A preferred embodiment 8 of aspect 4, as well as preferred embodiments 6 and 7 thereof, wherein the aqueous solution comprises at least 95% water.

[0066] A preferred embodiment 9 of aspect 4, and preferred embodiments 6 to 8 thereof, relate to a method in which the aqueous solution used for mixing / reconstitution is an aqueous NaCl solution with an amount of NaCl between 8 g / l and 10 g / l, preferably between 8.8 g / l and 9.2 g / l, more preferably 9 g / l.

[0067] A preferred embodiment 10 of aspect 4 relates to a method according to aspect 4 and preferred embodiments 2 to 4 or preferred embodiments 6 to 9 thereof, wherein the lipopeptide or liposomal preparation is a pharmaceutical preparation.

[0068] A preferred embodiment 11 of aspect 4 relates to a method according to aspect 4 and its preferred embodiments 2 to 4 or 10, wherein D90 is less than 60 nm, preferably less than 25 nm, more preferably 20 nm or less, even more preferably between 3 nm and 20 nm. In a more preferred embodiment, D90 is between 10 nm and 20 nm, and in another more preferred embodiment, D90 is between 3 nm and 5 nm.

[0069] Another aspect 5 relates to a formulation prepared according to the method according to aspect 4, and preferred embodiments thereof disclosed herein.

[0070] In a preferred embodiment of aspect 5, the formulation is a pharmaceutical formulation.

[0071] definition Unless otherwise stated, amounts in % refer to % (weight / weight) ((w / w)).

[0072] Unless expressly stated otherwise (e.g., by using a term such as "particular" to mean "one"), the term "a" is an indefinite article that encompasses "one" and "one or more" / "more than one" nouns following the term "a".

[0073] As used herein, a "buffer" or "buffer system" is used to prevent changes in the pH of a solution, and suitable examples will be well known to those skilled in the art.

[0074] As used herein, "bulking agents," as the name implies, form the bulk of the lyophilized product and provide proper structure to the lyophilized cake. Non-limiting examples of bulking agents include mannitol, glycine, arginine, proline, glucose, sucrose, lactose, trehalose, and dextran.

[0075] The term "cholesterol" refers to 3β-hydroxy-5-cholestene (CAS number: 57-88-5). Examples of cholesterol derivatives include cholesteryl sulfate and its salts (e.g., sodium salts), cholesteryl hemisuccinate, cholesteryl succinate, cholesteryl oleate, polyethylene glycol derivatives of cholesterol (cholesterol-PEG), coprostanol, cholestanol, cholestan, cholic acid, cortisol, corticosterone, hydrocortisone, and calciferol. Thus, the cholesterol derivative is preferably selected from the group consisting of cholesteryl sulfate, salts of cholesteryl sulfate, cholesteryl hemisuccinate, cholesteryl succinate, cholesteryl oleate, cholesterol-PEG, coprostanol, cholestanol, cholestan, cholic acid, cortisol, corticosterone, hydrocortisone, and calciferol.

[0076] The term "container" as used herein means an ampoule or vial with a rubber stopper and cap, a single- or double-chamber syringe, an infusion bag or bottle made of polymeric material or glass, suitable for holding a composition for parenteral administration. It also includes any vessel for holding a liquid.

[0077] The term "D90" is well known to those skilled in the art and refers to the amount of vesicles with a diameter equal to or less than a given value, with respect to size distribution, that have a weight of 90% of the weight of the components that form such particles in the formulation. D90 can be determined by multi-angle light scattering (MALS).

[0078] The term "in the range of X% to Y%" (where X and Y represent any number between 0 and 100, and X% is less than Y%) refers to any number in the range of X% to Y%, inclusive.

[0079] As used herein, "nano-dispersion" refers to an aqueous formulation containing vesicles with a D90 of 60 nm or less, where the vesicles are not liposomes. More specifically, the vesicles can be considered as precursors of liposomes, since liposomes can be produced by reconstituting / mixing a lyophilisate of the aqueous formulation containing the vesicles, preferably with saline or water, more preferably with saline. The vesicles are also referred to herein as micelles in relation to the single-phase nano-dispersion. It should be noted here that the inventors have found that the single-phase nano-dispersion according to the invention has liquid crystal properties and, in the case of lyotropic liquid crystals, (spontaneously) forms nanoscale self-assembled structures similar to micelles (see also the English Wikipedia entry on lyotropic liquid crystals, last edited on September 12, 2022). In this specification, the terms "nano-disperse system" and "nano-dispersed system" are used interchangeably herein. Further, as used herein, a nanodispersion is a single-phase nanodispersion.

[0080] "Lipopeptides" as peptides, in which lipid chains are covalently attached to the peptide. Such modifications of peptides, especially when the peptide has less than 50 amino acids, have been found to inhibit proteolytic attack due to the lipid chains non-covalently interacting with, for example, serum albumin to increase the molecular weight and thus, when administered to a patient, to reduce, for example, renal filtration. Typically, there are three types of lipid additions, which differ based on the bond formation method between lipids and peptides, namely, amidation, esterification (S- or O-), and S-bond (ether or disulfide) formation. Amidation, and O-esterification form strong, irreversible covalent bonds, whereas the other two methods are weak, reversible covalent bonds. The method used, as well as the lipid chain, the position of lipid attachment, and the spacer used, all have a significant impact on the physicochemical properties and biological activity (Zhang and Bulaj “Converting Peptides into Drug Leads by Lipidation”. Current Medicinal Chemistry, Vol 19, Issue 11, 2012).

[0081] A "therapeutic lipopeptide" is a peptide or polypeptide (oligomer) used to treat disease. Naturally occurring peptides can serve as hormones, growth factors, neurotransmitters, ion channel ligands and anti-infectives. Lipopeptide therapeutics mimic such functions. Because peptides can be metabolized by the body, lipopeptide therapeutics are usually relatively safe and well tolerated.

[0082] Liraglutide (CAS number 204656-20-2)(N 26 -(Hexadecanoyl-gamma-glutamyl)-[34-arginine]GLP-1-(7-37)-peptide (WHO) or NN 2211, also known as γ-L-glutamoyl(N-α-hexadecanoyl)-Lys 26 , Arg 34-GLP-1(7-37)) is a lipopeptide with 31 amino acids. Liraglutide is an antidiabetic drug used to treat type 2 diabetes, obesity and chronic weight control. Therefore, liraglutide is also a therapeutic lipopeptide.

[0083] Brevirtide (CAS number: 2012558-47-1) is a lipopeptide with a 47 amino acid peptide with a fatty acid, myristoyl residue at the N-terminus and an amidated C-terminus. The active substance is available as the acetate salt. The counterion acetate is bound in ionic form to the basic groups of the peptide molecule and exists in a non-stoichiometric ratio. The chemical name of brevirtide is (N-myristoyl-glycyl-L-threonyl-L-asparaginyl-L-leucyl-L-seryl-L-valyl-L-prolyl-L-asparaginyl-L-prolyl-L-leucyl-glycyl-L-phenylalanyl-L-phenylalanyl-L-prolyl-L-aspartyl-L-histidyl-L-glutaminyl-L-leucyl-L-aspartyl-L-prolyl-L-alanyl-L-phenylalanyl-glycyl-L-alanyl-L-asparaginyl-L-seryl-L-asparaginyl-L-asparaginyl-L-prolyl-L-aspartyl- Brevirtide is an antiviral drug for the treatment of chronic hepatitis D. Brevirtide is administered by subcutaneous injection. Thus, Brevirtide is also a therapeutic lipopeptide. Brevirtide is usually sold in the form of its acetate salt.

[0084] "Liposome" is a spherical vesicle with at least one lipid bilayer. Liposome can be used as a vehicle for the administration of pharmaceuticals. Liposome is most often composed of phospholipids, especially phosphatidylcholine, but can also contain other lipids, such as egg phosphatidylethanolamine, as long as it is compatible with the lipid bilayer structure.

[0085] As used herein, "liposome formulation" refers to a liquid containing liposomes, which contain phospholipids, suitable for solubilizing lipopeptides, such as brevirtide or liraglutide, in an aqueous environment.

[0086] The liposome size or vesicle size (such as micelle size) disclosed herein refers to the size determined by multi-angle light scattering (MALS) or dynamic light scattering (DLS), respectively. Typically, the size of liposomes is within the range of 0.025 μm to 2.5 μm (Akbarzadeh et al. Nanoscale Research Letters 2013, 8:102).

[0087] By "formulated" is meant incorporating brevirtide into or transferring brevirtide to / encapsulating brevirtide by a liposome.

[0088] "Pharmaceutically acceptable" means approved or approvable by a national regulatory agency, or listed in the European Pharmacopoeia or the United States Pharmacopoeia or other generally recognized pharmacopoeias, for use in animals, or more specifically, humans.

[0089] PEG means polyethylene glycol.

[0090] PEGylation is the process of covalent and non-covalent attachment or fusion of PEG polymer chains to molecules and macrostructures, such as phospholipids, resulting in vesicles that are described as PEGylated. PEGylated phospholipids are well known and commercially available.

[0091] The "pharmaceutical composition" described herein is particularly a pharmaceutical liposomal composition. By "pharmaceutical liposomal composition" is meant a composition comprising liposomes, suitable for pharmaceutical administration.

[0092] The phospholipids used in the formulation of the present invention can be selected from the group consisting of natural phospholipids, synthetic phospholipids and their combinations.Lecithin is one of the natural sources of phospholipids.Lecithin is a mixture found in egg yolk and soybean.Lecithin includes several phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) or the (pharmaceutical acceptable) salt of any of the above.

[0093] Generally, the structure of a phospholipid as used herein is a phospholipid of structure (I): TIFF2024535127000001.tif55128In formula, R1 is C 10 ~C 24 Represents acyl, R2 is C 10 ~C 24 acyl, or hydrogen; R3 is 2-trimethylamino-1-ethyl (resulting in PC), 2-amino-2-carboxy-1-ethyl (resulting in PS), inosityl group (CH 11 O5) (yielding PI), 2-amino-1-ethyl (yielding PE), or hydrogen (yielding PA).

[0094] The terms "phosphatidic acid" and "PA" are used interchangeably herein. PA or its (pharmaceutical acceptable) salt can be derived from natural and / or synthetic sources. Non-limiting examples of PA include DLPA, DMPA, DPPA, DSPA, POPA, POPA, DEPA, HSPA, HEPA or any of the (pharmaceutical acceptable) salts mentioned above.

[0095] The terms "phosphatidylcholine" and "PC" are used interchangeably herein. PC or its (pharmaceutically acceptable) salt can be derived from natural and / or synthetic sources. PC can be PEGylated. Non-limiting examples of PC include dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), palmitoyl-oleoyl-phosphatidylcholine (POPC), dierucoylglycerophosphocholine (DEPC), hydrogenated soybean phosphatidylcholine (HSPC), hydrogenated egg phosphatidylcholine (HEPC), or any of the above (pharmaceutically acceptable) salts.

[0096] The terms "phosphoethanolamine" and "PE" are used interchangeably herein. PE can be PEGylated. PE or its (pharmaceutically acceptable) salt can be derived from natural and / or synthetic sources. Non-limiting examples of PE include DLPE, DMPE, DPPE, DSPE, POPE, POPE, DEPE, HSPE, HEPE, or any (pharmaceutically acceptable) salt of the foregoing.

[0097] The terms "phosphatidylglycerol" and "PG" are used interchangeably herein. PG or its (pharmaceutical acceptable) salt can be derived from natural and / or synthetic sources. Non-limiting examples of PG include DLPG, DMPG, DPPG, DSPG, POPG, POPG, DEPG, HSPG, HEPG, or any of the aforementioned (pharmaceutical acceptable) salts.

[0098] The terms "phosphatidylinositol" and "PI" are used interchangeably herein. PI can be PEGylated. PI or its (pharmaceutically acceptable) salt can be derived from natural and / or synthetic sources. Non-limiting examples of PI include DLPI, DMPI, DPPI, DSPI, POPI, POPI, DEPI, HSPI, HEPI, or any (pharmaceutically acceptable) salt of the above.

[0099] The terms "phosphoserine" and "PS" are used interchangeably herein. PS can be PEGylated. PS or its (pharmaceutically acceptable) salt can be derived from natural and / or synthetic sources. Non-limiting examples of PS include DLPS, DMPS, DPPS, DSPS, POPS, POPS, DEPS, HSPS, HEPS, or any (pharmaceutically acceptable) salt of the above.

[0100] Non-limiting examples of pharma- ceutically acceptable salts of any of the phospholipids include sodium or ammonium salts, e.g., PG-Na PG-NH4, DSPG-Na, or DSPG-NH4.

[0101] Thus, any derivative of a phospholipid selected from the group consisting of phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA) and phosphatidylglycerol (PG) can be used as a phospholipid derivative, such as DLPA, DMPA, DPPA, DSPA, POPA, POPA, DEPA, HSPA, HEPA, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC, HSPC, HEP C, DLPE, DMPE, DPPE, DSPE, POPE, POPE, DEPE, HSPE, HEPE, DLPG, DMPG, DPPG, DSPG, POPG, POPG, DEPG, HSPG, HEPG, DLPI, DMPI, DPPI, DSPI, POPI, POPI, DEPI, HSPI, HEPI, DLPS, DMPS, DPPS, DSPS, POPS, POPS, DEPS, HSPS, HEPS, PEGylated forms of any of the foregoing, and salts of any of the foregoing.

[0102] By mixing "just prior to administration to a patient" is meant up to 3 days, particularly up to 24 hours, for example up to 6 hours, prior to administration to a patient.

[0103] "Isotonicity agent" means a pharma- ceutically acceptable compound that can be added to a formulation to make it isotonic with human plasma. Isotonicity agents include, for example, dextrose, glucose, mannitol, sucrose, lactose, trehalose, glycerin and NaCl, particularly sucrose or glycerin or NaCl, more particularly sucrose or NaCl. Tonicity is the "effective osmolality" and is equal to the sum of the concentrations of solutes that have the ability to exert an osmotic force across a membrane. Parenteral formulations should be isotonic with plasma. Isotonicity agents are well known to those skilled in the art.

[0104] As used herein, the terms "treat," "treating," or "treatment of" any disease or disorder, in one aspect, refer to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the onset of the disease, or at least one of its clinical symptoms). In another aspect, "treat," "treating," or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another aspect, "treat," "treating," or "treatment" refers to modulating the disease or disorder physically (e.g., stabilizing discernible symptoms), physiologically (e.g., stabilizing physical parameters), or both.

[0105] The term "(a), (b), (c), (d) and (e) add up to 100%" means that the percentage values ​​given for components (a)-(e) must always be selected such that the values ​​of (a)-(e) add up to 100%. These values ​​thus give a ratio between components (a)-(e). However, a person skilled in the art will understand that the formulation may contain additional components such as solvents. However, the amount of such additional components is not taken into account when calculating the ratio between (a)-(e) (the % values ​​must always add up to 100%).

[0106] It is understood that any embodiment of the invention may be combined, regardless of the fact that the embodiment is an embodiment, a preferred embodiment, a more preferred embodiment, a particularly preferred embodiment, or a most preferred embodiment, unless such combination violates any law of nature, and that such combinations of two or more embodiments of the invention are disclosed herein by disclosing two or more embodiments, even if such combinations of two or more embodiments are not explicitly mentioned. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0107] Detailed Description The present invention relates to a simple method for preparing formulations containing lipopeptides. The method of the present invention allows for the easy preparation of liquid formulations (lipopeptide formulations) in which the vesicles, preferably micelles, have a D90 of 60 nm or less, more preferably 25 nm or less, preferably 20 nm or less. This single-phase nanodispersion formed during the method according to the present invention allows for the preparation of liposomal formulations without the need for mechanical reduction of the liposome size for the final liposomal formulation.

[0108] Alternatively, the method of the present invention further allows for the easy preparation of liposomal formulations in which the D90 of the liposomes is between 1 μm and 4.5 μm. The present invention further provides a formulation comprising a lipopeptide prepared according to the method of the present invention, more specifically a formulation of a lipopeptide, preferably a therapeutic lipopeptide, more preferably brevirtide or liraglutide, suitable for parenteral administration to a patient. In particular, such administration is by intravenous injection or infusion. The present invention further provides two separate formulations that can be mixed together immediately prior to administration to a patient to provide a liposomal composition suitable for administration. One formulation can be a lyophilisate comprising the lipopeptide and the second formulation can be an aqueous formulation. When the two separate formulations are mixed together, brevirtide is incorporated into the formed liposomes, allowing solubilisation of brevirtide, resulting in a pharmaceutical liposomal composition suitable for clinical use.

[0109] Preferably, a formulation containing a therapeutic lipopeptide should allow for efficient and optimal incorporation of the therapeutic lipopeptide into liposomes prior to administration to a patient.

[0110] Preferably, the present invention provides a pharmaceutical liposomal formulation that allows for rapid release of brevirtide from the liposomes after administration (injection).

[0111] Overall, the invention described herein allows for the effective administration of lipopeptides, preferably therapeutic lipopeptides (e.g., brevirtide or liraglutide), to patients despite the challenging chemical characteristics of the drug.

[0112] The formulations described herein are in particular pharmaceutical formulations, such as pharmaceutical liposomal compositions.

[0113] Preferably, the D90 of the liposome of the liposome preparation is from 1 μm to 4.5 μm.

[0114] In particular, the phospholipids described herein are selected from egg lecithin, soy lecithin or synthetic phospholipids.

[0115] Several aspects and embodiments of the present invention are described below.

[0116] formulation The first aspect is (a) one or more phospholipids selected from the group consisting of phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), or derivatives of any of the foregoing, or mixtures thereof, in the range of 40% to 99.7%, preferably in the range of 40% to 97%, based on the total weight of (a) to (e), such as about 60%, (b) a lipopeptide, preferably a therapeutic lipopeptide, more preferably brevirtide or liraglutide, in the range of 0.3% to 20%, preferably in the range of 3% to 18%, more preferably in the range of 3% to 13%, based on the total weight of (a) to (e), such as about 8% when the lipopeptide is brevirtide, or in the range of 0.3% to 2% when the lipopeptide is liraglutide, for example, 0.37±0.05% (i.e., 0.365% to 0.375%), 0.6±0.05%, 0.7±0.05%, 1.1±0.06%, 1.2±0.06%, 1.8±0.06%, etc. (c) cholesterol or a derivative thereof in the range of 0% to 14%, preferably 4% to 14%, based on the total weight of (a) to (e), such as about 9%; (d) a bulking agent, preferably glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, or 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, saccharose, xylitol, xylose, dextran or mixtures thereof, more preferably in the range of 0% to 35%, such as about 25%, based on the total weight of (a) to (e). glycine, arginine, proline, or a sugar moiety selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, saccharose, xylitol, xylose, dextran or mixtures thereof, even more preferably glycine, arginine, proline, mannitol, glucose, sucrose, lactose, trehalose or dextran, even more preferably mannitol, glycine or trehalose, (e) an isotonicity agent other than (d), in the range of 0% to 35% based on the total weight of (a) to (e); 1. A formulation which is a single-phase nanodispersion system comprising: The sum of (a), (b), (c), (d), and (e) total 100%, and the combined amounts of (a)-(e) are between 10% and 100%, based on the total weight of the formulation.

[0117] With respect to (c) and (e), these ingredients are in some formulations optionally demonstrated by ranges with 0% as the lowest boundary.

[0118] A person skilled in the art will understand that when the formulation consists of (a), (b), (d), optionally (c) and optionally (e), trace amounts of solvents and additives (such as tert-butanol, or water, or buffer systems) used in the preparation process, preferably the preparation process of the present invention further described below, may still be present in an amount of up to 2%.

[0119] A preferred embodiment relates to a formulation according to the invention, which consists of residues of (a), (b), (c), (d) and (e) and a solvent, preferably tert-butanol, and the amount of tert-butanol is 0.01%-2% based on the total weight of such formulation (e.g., in the case of a lyophilisate, e.g., the combined amounts of (a)-(e) and tert-butanol). More preferably, the amount of tert-butanol is 1% or less, e.g., 0.001%-0.8%.

[0120] Another preferred embodiment relates to a formulation according to the invention, wherein the formulation consists of (a), (b), (c), (d), and (e), tert-butanol and a buffer system, preferably an acetate buffer system, wherein the combined amount of tert-butanol and the buffer system is 0.01% to 2% based on the total weight of such formulation (e.g., in the case of a lyophilisate, e.g., the combined amounts of (a)-(e) and tert-butanol).

[0121] In yet another preferred embodiment, the formulation according to the present invention comprises liposomes, proliposomes, lipid clathrates, lipid colloidal dispersions, micelles, reverse micelles, discotic structures or combinations thereof.

[0122] A further preferred embodiment relates to a preferably pharmaceutical single-phase nanodispersion system, wherein the D90 of the vesicles is equal to or less than 60 nm, preferably less than 25 nm, more preferably equal to or less than 20 nm, for example between 10 nm and 20 nm, for example about 15 nm, or between 3 nm and 10 nm, for example about 5 nm.

[0123] Another preferred embodiment relates to a lyophilized formulation.

[0124] Another preferred embodiment relates to a lyophilized formulation (lyophilisate) in which the amount of lipopeptide (b), preferably a therapeutic lipopeptide, more preferably brevirtide or liraglutide, is between 12 mg and 24 mg, more preferably between 10 mg and 20 mg.

[0125] Yet another preferred embodiment relates to a lyophilized formulation (lyophilisate) in which the amount of lipopeptide (b), preferably a therapeutic lipopeptide, more preferably liraglutide, is between 0.5 mg and 1.8 mg, e.g. 0.6 mg, 1.2 mg or 1.8 mg, respectively.

[0126] In a lyophilized formulation, the sum of (a), (b), (c), (d), and (e) total 100%, with the combined amount of (a)-(e) being 98%-100% based on the total weight of the formulation. Preferably, the combined amount of (a)-(e) is 99%-100%, even more preferably 99.9%-100%, and most preferably 100%.

[0127] A preferred embodiment relates to a pharmaceutical liposomal formulation according to the invention comprising (a), (b), (c), (d), and (e) and water and optionally a buffer.

[0128] Preferably, especially in the latter two preferred embodiments, the combined amount of (a)-(e) is 10%-15% based on the total weight of the formulation.

[0129] In a further preferred embodiment, the pharma- ceutical active substance in the formulation according to the invention is entrapped by lipid clathrates, 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 brevirtide is incorporated in the liposome or micelle.

[0130] In a preferred embodiment, the pharma- ceutical active substance in the formulation according to the invention is entrapped by micelles (nanodispersion formulation / single-phase nanodispersion system).Most preferably, the micelles have a D90 of 60 nm or less, e.g., less than 25 nm, more preferably 20 nm or less, e.g., between 10 nm and 20 nm, or between 3 nm and 10 nm.

[0131] In another preferred embodiment, the pharma- ceutical active substance in the formulation according to the invention is entrapped by liposomes (liposomal formulation).

[0132] Another preferred embodiment relates to a formulation, preferably a liposomal or nanodispersed formulation, which is a pharmaceutical formulation.

[0133] Liposomal formulation A preferred embodiment relates to a pharmaceutical liposomal formulation obtained by mixing or reconstituting a lyophilized formulation (single-phase nanodispersion) preferably with an aqueous solution, more preferably saline or water, even more preferably saline.

[0134] Such pharmaceutical liposomal formulations are preferably suitable for injection, hi certain embodiments, the liposomal formulations are administered to the patient intravenously or, preferably, subcutaneously.

[0135] Another preferred embodiment relates to a pharmaceutical liposomal formulation in which the lipopeptide is brevirtide and the amount of brevirtide in the liposomal formulation is from 12 mg / 1.5 ml to 24 mg / 1.5 ml, more preferably from 15 mg / 1.5 ml to 20 mg / 1.5 ml.

[0136] Another preferred embodiment relates to a pharmaceutical liposomal formulation wherein the lipopeptide is liraglutide and the amount of liraglutide in the liposomal formulation is 0.5 mg / ml to 18 mg / ml, for example, 0.5 mg / ml to 12 mg / ml, more preferably 4 mg / ml to 8 mg / ml, even more preferably 5.8 to 6.2 mg / ml, for example, 6 mg / ml.

[0137] According to another preferred embodiment, the pharmaceutical liposomal formulation has a pH of 5 to 7.5, such as 5 to 6, or 6 to 7.5, or 7.3 to 7.4.

[0138] (a) Phospholipids Another preferred embodiment relates to formulations in which the one or more phospholipids comprise PC or a mixture of PC and one or more phospholipids selected from the group consisting of phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and phosphatidic acid (PA) phosphatidylglycerol (PG), or a pharma- ceutically acceptable salt of any of the foregoing.

[0139] Another preferred embodiment relates to formulations in which 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 pharma- ceutically acceptable salt of any of the foregoing.

[0140] In a preferred embodiment, at least one phospholipid is a PEGylated phospholipid.

[0141] Another more preferred embodiment relates to formulations in which the one or more phospholipids comprise PC, preferably selected from the group consisting of DLPC, DMPC, DPPC, DSPC, POPC, POPC, DEPC, HSPC, HEPC, or preferably a pharma- ceutically acceptable salt thereof, of any of the foregoing.

[0142] Another further preferred embodiment relates to a formulation wherein the one or more phospholipids comprise PEGylated PC, preferably selected from the group consisting of PEGylated DLPC, PEGylated DMPC, PEGylated DPPC, PEGylated DSPC, PEGylated POPC, PEGylated POPC, PEGylated DEPC, PEGylated HSPC, PEGylated HEPC, or a PEGylated PC, preferably a pharma- ceutically acceptable salt thereof, of any of the foregoing.

[0143] Another more preferred embodiment relates to a formulation in which one or more phospholipids comprise PC or preferably a pharma- ceutically acceptable salt thereof. In an even more preferred embodiment, the PC or preferably a pharma- ceutically acceptable salt thereof is derived from soybean (e.g. Lipoid S100), egg or synthetic, and in a most preferred embodiment, the PC or preferably a pharma- ceutically acceptable salt thereof is derived from soybean.

[0144] Another preferred embodiment relates to a formulation in which the amount of (a) is 50% to 65% compared to the sum of (a), (b), (c), (d), and (e) in the formulation, e.g., about 57%.

[0145] Another preferred embodiment relates to formulations in which the PG, or preferably a pharma- ceutically acceptable salt thereof, is PEGylated PG, or a PEGylated, preferably a pharma- ceutically acceptable salt thereof.

[0146] Another more preferred embodiment relates to formulations in which PG, or preferably a pharma- ceutically acceptable salt thereof, is selected from the group consisting of DLPG, DMPG, DPPG, DSPG, POPG, POPG, DEPG, HSPG, HEPG.

[0147] Another more preferred embodiment relates to formulations wherein PG, or preferably a pharma- ceutically acceptable salt thereof, is PEGylated and is selected from the group consisting of PEGylated DLPG, PEGylated DMPG, PEGylated DPPG, PEGylated DSPG, PEGylated POPG, PEGylated POPG, PEGylated DEPG, PEGylated HSPG, PEGylated HEPG, or preferably a pharma- ceutically acceptable salt of any of the foregoing.

[0148] (b) Lipopeptide In a preferred embodiment, the lipopeptide is a lipopeptide having 50 or fewer amino acids.

[0149] In a further preferred embodiment, the lipopeptide has 5 to 50 amino acids, preferably independently selected from the group consisting of alanine (ala), arginine (arg), asparagine (asn), aspartic acid (asp), cysteine ​​(cys), glutamine (gln), glutamic acid (glu), glycine (gly), histidine (his), isoleucine (ile), leucine (leu), lysine (lys), methionine (met), phenylalanine (phe), proline (pro), serine (ser), threonine (thr), tryptophan (trp), tyrosine (tyr) and valine (val).

[0150] In another preferred embodiment, the lipophilic residue is attached to the amino acid via amidation, esterification (S- or O-), or S-bond (ether or disulfide) formation.

[0151] In another preferred embodiment, the lipopeptide has at least one lipophilic residue selected from the group consisting of a glycosylphosphatidylinositol-anchor, a palmitoyl (C16:0) residue, or a myristoyl (C14:0) residue covalently attached to one of the 5 to 50 amino acids, e.g., a palmitoyl or myristoyl residue is attached to the cis residue.

[0152] In a preferred embodiment, the lipopeptide has a glycosylphosphatidylinositol anchor, a lipophilic residue selected from the group consisting of a palmitoyl (C16:0) residue or a myristoyl (C14:0) residue covalently attached to one of the 5 to 50 amino acids.

[0153] In yet another preferred embodiment, the lipopeptide is one of the same type (eg, only brevirtide or only liraglutide is present).

[0154] In another preferred embodiment, the lipopeptide used in the formulation (or method) according to the invention is brevirtide.

[0155] In yet another preferred embodiment, the lipopeptide used in the formulation (or method) according to the invention is liraglutide.

[0156] In yet another preferred embodiment, the lipopeptide is brevirtide.

[0157] In yet another preferred embodiment, the lipopeptide is liraglutide.

[0158] Another preferred embodiment relates to a formulation according to the invention, wherein brevirtide is used as its acetate salt. In one embodiment, the formulation according to the invention preferably contains acetate in the equimolar range as brevirtide.

[0159] Another preferred embodiment relates to a formulation in which the amount of lipopeptide (b)) is between 5% and 11% compared to the sum of (a), (b), (c), (d) and (e) in the formulation, e.g., about 8%.

[0160] In another preferred embodiment, the amount of the lipopeptide, preferably brevirtide or liraglutide, is 5% to 11% compared to the sum of (a), (b), (c), (d), and (e) in the formulation, and the concentration of the lipopeptide (c)) in the formulation is 15 mg / 1.5 ml to 20 mg / 1.5 ml, for example, when the formulation is a liquid formulation such as a single-phase nanodispersion containing brevirtide, or a liposomal formulation, preferably a pharmaceutical liposomal formulation, containing the lipopeptide.

[0161] In another preferred embodiment, the amount of (c) is 5% to 11% relative to the sum of (a), (b), (c), (d), and (e) in the formulation, and the concentration of (c) in the formulation is, for example, 8 mg per 176.6 mg of the sum of components (a)-(e) to 19.4 mg per 176.6 mg of the sum of components (a)-(e) when the formulation is a lyophilized formulation.

[0162] (c) Cholesterol Cholesterol is known to affect liposome stability and drug release.

[0163] Thus, in one preferred embodiment, the formulation according to the invention preferably comprises cholesterol or a derivative thereof (component (c)).

[0164] Another preferred embodiment relates to formulations in which (c) is selected from the group consisting of cholesterol or sodium cholesteryl sulfate. More preferably, component (c) is cholesterol.

[0165] Another preferred embodiment relates to a formulation in which the amount of (c) is between 6% and 12% relative to the sum of (a), (b), (c), (d), and (e) in the formulation, e.g., about 9%, e.g., between 8% and 10%.

[0166] (d) Bulking agent The presence of a bulking agent is advantageous, especially if the formulation according to the invention is to be lyophilizable or if the preparation of the formulation according to the invention via the method according to the invention requires a lyophilization step. Examples of preferred bulking agents are glycine, arginine, proline or any other amino acid known to be suitable as a bulking agent, or a sugar component (component (e) in the formulation according to the invention). Preferred sugar components are selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, xylitol, xylose, dextran or mixtures thereof.

[0167] A more preferred embodiment relates to a formulation in which (e) is trehalose.

[0168] Another preferred embodiment relates to a formulation in which the amount of (e) is 20% to 30% compared to the sum of (a), (b), (c), (d), and (e) in the formulation, e.g., about 25%.

[0169] In a preferred embodiment, the formulation according to the invention comprises, in addition to components (a) to (e), tert-butanol in the range of 0.01% to 2% relative to the total weight of the formulation (e.g., lyophilisate).

[0170] (e) Tonicity agent Another preferred embodiment relates to a pharmaceutical liposomal formulation comprising (a), (b), (c), (d) and one or more tonicity agents which are not (d) (component (e) in the formulation according to the invention).

[0171] Such a tonicity agent, preferably a pharma- ceutically acceptable one, should be present in the pharmaceutical liposomal formulation. Such a tonicity agent can be added during the preparation of the liposomal formulation by mixing the lyophilisate according to the invention with an aqueous phase containing said tonicity agent, or can be provided during the rehydration step of the lyophilisate.

[0172] Alternatively, such a tonicity agent may already be present in the lyophilisate in that it is part of the aqueous phase used in the method according to the invention for preparing the lipopeptide according to the invention. The skilled person knows that the tonicity agent may be partly and partly part of such aqueous phase. The skilled person can easily calculate the total amount of the tonicity agent that is required to have an appropriate concentration in the final pharmaceutical liposomal formulation in order to have an isotonic effect with respect to the medicinal product provided to the patient.

[0173] One of ordinary skill in the art will recognize that a tonicity agent is also sometimes a bulking agent, for example, when the tonicity agent is a sugar moiety such as glucose or trehalose.

[0174] Other tonicity agents, such as NaCl, do not have a bulking function. Thus, when using such a "single-functional component", e.g., NaCl, the formulation may include a bulking agent (e) and a tonicity agent (e). The formulation may include a bulking agent (e.g., trehalose) and "two" tonicity agents (trehalose and e.g., NaCl).

[0175] Tonicity agents are well known to those skilled in the art. In a preferred embodiment, the tonicity agent is selected from the group consisting of dextrose, glucose, mannitol, sucrose, lactose, trehalose, glycine, arginine, proline and NaCl, in particular glycine, mannitol, trehalose, glucose and NaCl, even more particularly glycine and NaCl, most preferably NaCl.

[0176] A person skilled in the art knows how to select the appropriate concentration of a particular tonicity agent in a formulation, preferably for subcutaneous injection.

[0177] In a preferred embodiment, the tonicity agent is NaCl, and the concentration of NaCl in the pharmaceutical liposomal formulation is 0.8% to 1%, more preferably about 0.9%, e.g., 0.9%±0.1%, more preferably 0.9%, based on the total weight of the pharmaceutical liposomal formulation.

[0178] In a preferred embodiment, component (e) is present in the formulation according to the invention in an amount of 0.1% to 10%, more preferably 0.4% to 1.5%, e.g. when the tonicity agent is NaCl, a preferred amount of NaCl in the formulation, e.g. a pharma- ceutically acceptable formulation, is 0.8% to 1.0% (0.9%±0.1%, more preferably 0.9%±0.05%), i.e. at or near physiological concentrations, with respect to plasma.

[0179] Depending on the tonicity agent and the type of formulation (e.g., a pharma- ceutically acceptable liposomal formulation or a lyophilisate), the skilled artisan will be familiar with how to calculate the amount of one or more tonicity agents to be present in the (final) formulation for administration to a patient in physiological concentrations.

[0180] Solvents for aqueous solutions Another preferred embodiment relates to a pharmaceutical liposomal formulation comprising (a), (b), (c), (d), and (e) as defined herein and one or more solvents.

[0181] Another preferred embodiment relates to a pharmaceutical liposomal formulation, wherein one or more solvents are present in an amount of 10% to 90%, such as 80% to 90%, or even 85% to 90%, based on the total weight of the final formulation.

[0182] A suitable solvent can be selected by a person skilled in the art. In a preferred embodiment, the solvent or solvents are selected from the group consisting of water and, for example, an aqueous solution in which a salt is present. The aqueous solution in which a salt is present is preferably a solution, preferably water, containing at least one salt in a physiological concentration, preferably at least one salt in an isotonic concentration, such as, for example, saline, lactated Ringer's solution and / or Plasma Lyte. In this specification, saline may also be referred to as saline solution, and preferably relates to a mixture of sodium chloride and water with a sodium chloride concentration of 9 g salt per liter (0.9%) solution (see also Example 4, in which such a 0.9% (w / v) saline solution is used for the reconstitution and formation of liposomes). Lactated Ringer's solution refers to a sodium lactate solution as a mixture of sodium chloride, sodium lactate, potassium chloride and calcium chloride in water. Plasma Lyte may also be referred to as Plasma Lyte 148 (pH 7.4). Thus, the aqueous solution preferably has a salt concentration, osmolality and pH that reflects the physiological plasma electrolyte concentration, osmolality and pH of humans.However, in a more preferred embodiment, the solvent is water or a combination of water and at least one salt, preferably at an isotonic concentration, and the amount of water of the total amount of solvent is at least 80%, preferably at least 90%, more preferably at least 95%.Preferably, the solvent is water, saline, lactated Ringer's solution or Plasmalyte.

[0183] More preferably, the solvent or solvents are water or saline. Most preferably, the solvent or solvents are water.

[0184] buffer solution Another preferred embodiment relates to a formulation which is a pharmaceutical liposomal formulation comprising (a), (b), (c), (d), and (e) as defined herein, and a buffer.

[0185] Another preferred embodiment relates to a formulation which is a pharmaceutical liposomal formulation consisting of (a), (b), (c), (d), and (e) as defined herein and a solvent.

[0186] Another preferred embodiment relates to a formulation which is a pharmaceutical liposomal formulation comprising (a), (b), (c), (d), and (e) as defined herein, a solvent, and one or more isotonicity agents, and a buffer.

[0187] In addition to the components (a)-(e) in the pharmaceutical liposomal formulation according to the present invention, any pharma- ceutically acceptable components, such as one or more solvents, buffers, salts, other additives, etc., are summarized as a pharmaceutical aqueous solution. Thus, the pharmaceutical liposomal formulation according to the present invention consists of the components (a)-(e) and the pharmaceutical aqueous solution. Those skilled in the art will understand that the components forming the pharmaceutical aqueous solution can be combined sequentially with the formulation comprising (a)-(e) as defined herein, or can be mixed first and then added to the formulation comprising (a)-(e) as defined herein (i.e., the formulation can comprise only (a)-(e) and, for example, a buffer, or can be provided together / in the pharmaceutical aqueous solution or (f), and the aqueous solution is provided sequentially, resulting in a pharmaceutical liposomal formulation consisting of the components (a)-(e) and the pharmaceutical aqueous solution according to the present invention.

[0188] Vesicle size in single-phase nanodispersions In yet another embodiment, the lipopeptide formulation of step (iv) of the method according to the invention (see further below) is a monophasic nanodispersion, wherein the D90 of the vesicles is equal to or less than 60 nm, more preferably less than 25 nm, even more preferably equal to or less than 20 nm, such as between 10 nm and 20 nm, or between 3 nm and 10 nm.

[0189] Liposome size In yet another embodiment, the liposomes of the liposomal formulation according to the present invention have a D90 size distribution of 1 μm to 4.5 μm.

[0190] Treatment method / How to use One aspect relates to the use of a formulation according to the invention, wherein the lipopeptide is brevirtide, for the preparation of a medicament for the treatment of chronic hepatitis B and / or chronic hepatitis D.

[0191] Another aspect relates to the use of a formulation according to the invention, wherein the lipopeptide is brevirtide, for the preparation of a medicament for treating an inflammatory, preferably an inflammatory disease.

[0192] Another aspect relates to a method for treating chronic hepatitis B and / or chronic hepatitis D, comprising providing to a patient a pharmaceutical liposomal formulation according to the present invention, wherein the lipopeptide is brevirtide. Preferably, providing the pharmaceutical liposomal formulation according to the present invention is by injection.

[0193] One aspect relates to the use of a formulation according to the invention, wherein the lipopeptide is liraglutide, for the preparation of a medicament for treating type 2 diabetes.

[0194] Another aspect relates to a method of treating type 2 diabetes comprising providing to a patient a pharmaceutical liposomal formulation according to the invention, wherein the lipopeptide is liraglutide. Preferably, providing the pharmaceutical liposomal formulation according to the invention is by injection.

[0195] Preparation method Another aspect is (i) providing an organic phase comprising one or more phospholipids (component (a) in the formulation according to the invention), and optionally cholesterol or a derivative thereof (component (c) in the formulation according to the invention), and at least one organic solvent, preferably selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, an alcohol, preferably an alcohol selected from the group consisting of 1-butanol, 2-butanol and tert-butanol, more preferably tert-butanol, acetic acid, ethyl lactate (ethyl 2-hydroxypropanoate), acetonitrile and any combination of the foregoing, (ii) an aqueous medium; Optionally, a bulking agent selected from the group consisting of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, a sugar moiety selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, saccharose, xylitol, xylose, dextran, or a mixture of any of the foregoing; optionally a pharma- ceutically acceptable buffer; Optionally, a pharma- ceutically acceptable tonicity agent. providing an aqueous phase comprising the pH of the aqueous phase is between 3 and 9, for example between 5.5 and 7, for example between 5.8 and 6.7 (for example by using a sodium acetate buffer); (iii) combining an organic phase with an aqueous phase, the mixing ratio of the organic phase to the aqueous phase being between 10:1 (v / v) and 1:10 (v / v), resulting in a combined organic phase and aqueous phase, the at least one organic solvent and the aqueous phase forming a single-phase mixture, preferably a freezable and sublimable single-phase mixture; (iv) adding a lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the combined phase to receive a lipopeptide formulation according to the invention; or adding a lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the organic phase prior to mixing the organic phase with the aqueous phase in step (iii); or adding a lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the aqueous phase prior to mixing the aqueous phase with the organic phase in step (iii); providing a lipopeptide formulation, the lipopeptide formulation thus obtained being a single-phase nanodispersion; The present invention relates to a method for preparing a formulation according to the present invention, comprising:

[0196] Preferably, the aqueous phase comprises a buffer.

[0197] A preferred embodiment is (i) providing an organic phase comprising one or more phospholipids (component (a) in the formulation according to the invention), and optionally cholesterol or a derivative thereof (component (c) in the formulation according to the invention), and at least one organic solvent, preferably selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, an alcohol, preferably an alcohol selected from the group consisting of 1-butanol, 2-butanol and tert-butanol, more preferably tert-butanol, acetic acid, ethyl lactate (ethyl 2-hydroxypropanoate), acetonitrile and any combination of the foregoing, (ii) an aqueous medium; Optionally, a bulking agent selected from the group consisting of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, a sugar moiety selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, saccharose, xylitol, xylose, dextran, or a mixture of any of the foregoing; a pharma- ceutically acceptable buffer; Optionally, a pharma- ceutically acceptable tonicity agent. providing an aqueous phase comprising the pH of the aqueous phase is between 3 and 9, for example between 5.5 and 7, for example between 5.8 and 6.7 (for example by using a sodium acetate buffer); (iii) combining an organic phase with an aqueous phase, the mixing ratio of the organic phase to the aqueous phase being between 10:1 (v / v) and 1:10 (v / v), resulting in a combined organic phase and aqueous phase, the at least one organic solvent and the aqueous phase forming a single-phase mixture, preferably a freezable and sublimable single-phase mixture; (iv) adding a lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the combined phase to receive a lipopeptide formulation according to the invention; or adding a lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the organic phase prior to mixing the organic phase with the aqueous phase in step (iii); or adding a lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the aqueous phase prior to mixing the aqueous phase with the organic phase in step (iii); providing a lipopeptide formulation, the lipopeptide formulation thus obtained being a single-phase nanodispersion; The present invention relates to a method for preparing a formulation according to the present invention, comprising:

[0198] Therefore, another preferred embodiment is (i) providing an organic phase comprising one or more phospholipids (component (a) in the formulation according to the invention), and optionally cholesterol or a derivative thereof (component (c) in the formulation according to the invention), and at least one organic solvent, preferably selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, an alcohol, preferably an alcohol selected from the group consisting of 1-butanol, 2-butanol and tert-butanol, more preferably tert-butanol, acetic acid, ethyl lactate (ethyl 2-hydroxypropanoate), acetonitrile and any combination of the foregoing, (ii) an aqueous medium; Optionally, a bulking agent selected from the group consisting of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, a sugar moiety selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, saccharose, xylitol, xylose, dextran, or a mixture of any of the foregoing; optionally a pharma- ceutically acceptable buffer; Optionally, a pharma- ceutically acceptable tonicity agent. providing an aqueous phase comprising the pH of the aqueous phase is between 3 and 9, for example between 5.5 and 7, for example between 5.8 and 6.7 (for example by using a sodium acetate buffer); (iii) combining an organic phase with an aqueous phase, the mixing ratio of the organic phase to the aqueous phase being between 10:1 (v / v) and 1:10 (v / v), resulting in a combined organic phase and aqueous phase, the at least one organic solvent and the aqueous phase forming a single-phase mixture, preferably a freezable and sublimable single-phase mixture; (iv) adding a lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the combined phase to receive a lipopeptide formulation according to the invention, the lipopeptide formulation thus obtained being a single-phase nanodispersion system. The present invention relates to a method for preparing a formulation according to the present invention, comprising:

[0199] Preferably, the aqueous phase comprises a buffer.

[0200] Another aspect is (i) providing an organic phase comprising one or more phospholipids (component (a) in the formulation according to the invention), a lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide (component (b) in the formulation according to the invention), and optionally cholesterol or a derivative thereof (component (c) in the formulation according to the invention), and at least one organic solvent, preferably selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, an alcohol, preferably an alcohol selected from the group consisting of 1-butanol, 2-butanol and tert-butanol, more preferably tert-butanol, acetic acid, ethyl lactate (ethyl 2-hydroxypropanoate), acetonitrile, and any combination of the foregoing, (ii) an aqueous medium; Optionally, a bulking agent selected from the group consisting of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, a sugar moiety selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, saccharose, xylitol, xylose, dextran, or a mixture of any of the foregoing; optionally a pharma- ceutically acceptable buffer; Optionally, a pharma- ceutically acceptable tonicity agent. providing an aqueous phase comprising the pH of the aqueous phase is between 3 and 9, for example between 5.5 and 7, for example between 5.8 and 6.7 (for example by using a sodium acetate buffer); (iii) combining an organic phase with an aqueous phase, the mixing ratio of the organic phase to the aqueous phase being between 10:1 (v / v) and 1:10 (v / v), resulting in a combined organic phase and aqueous phase, the at least one organic solvent and the aqueous phase forming a single-phase mixture, preferably a freezable and sublimable single-phase mixture. The present invention relates to a method for preparing a formulation according to the present invention, comprising:

[0201] Preferably, the aqueous phase comprises a buffer.

[0202] Another aspect is (i) providing an organic phase comprising one or more phospholipids (component (a) in the formulation according to the invention), and optionally cholesterol or a derivative thereof (component (c) in the formulation according to the invention), and at least one organic solvent, preferably selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, an alcohol, preferably an alcohol selected from the group consisting of 1-butanol, 2-butanol and tert-butanol, more preferably tert-butanol, acetic acid, ethyl lactate (ethyl 2-hydroxypropanoate), acetonitrile and any combination of the foregoing, (ii) an aqueous medium; A lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide (component (b) in the formulation according to the invention), Optionally, a bulking agent selected from the group consisting of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, a sugar moiety selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, saccharose, xylitol, xylose, dextran, or a mixture of any of the foregoing; optionally a pharma- ceutically acceptable buffer; Optionally, a pharma- ceutically acceptable tonicity agent. providing an aqueous phase comprising the pH of the aqueous phase is between 3 and 9, for example between 5.5 and 7, for example between 5.8 and 6.7 (for example by using a sodium acetate buffer); (iii) combining an organic phase with an aqueous phase, the mixing ratio of the organic phase to the aqueous phase being between 10:1 (v / v) and 1:10 (v / v), resulting in a combined organic phase and aqueous phase, the at least one organic solvent and the aqueous phase forming a single-phase mixture, preferably a freezable and sublimable single-phase mixture. The present invention relates to a method for preparing a formulation according to the present invention, comprising:

[0203] Preferably, the aqueous phase comprises a buffer.

[0204] For the purpose of explanation, a mixing ratio of 10:1 (v / v) means that 10 parts by volume of organic phase and 1 part by volume of aqueous phase are combined (e.g., 10 ml and 1 ml).Preferably, the mixing ratio in the method according to the invention is 5:1 (v / v) to 1:5 (v / v), for example, 1:1, or about 1, for example, 2:1 to 1:2.

[0205] aqueous medium The aqueous medium is preferably water or, for example, an aqueous solution in which a salt is present. The aqueous solution in which a salt is present is preferably a solution, preferably water, containing at least one salt in physiological concentration, preferably at least one salt in isotonic concentration, such as, for example, saline, Ringer's lactate and / or Plasmalyte. Thus, the aqueous medium preferably has a salt concentration, osmolality and pH that reflects the physiological plasma electrolyte concentration, osmolality and pH of humans. The aqueous medium is preferably water or a combination of water and at least one salt in isotonic concentration, preferably saline, Ringer's lactate and / or Plasmalyte. The amount of water of the total amount of solvent of the aqueous medium is at least 80%, preferably at least 90%, more preferably at least 95%. More preferably, the aqueous medium is water or saline. Most preferably, the aqueous medium is water.

[0206] Preferred buffers, bulking agents and tonicity adjusting agents for use in the methods according to the invention are the same as the respective buffers, bulking agents and tonicity adjusting agents described for the formulations of the invention.

[0207] aqueous phase The aqueous phase of step (ii) comprises an aqueous medium, preferably the aqueous medium consists of water, ie water is the only solvent in the aqueous phase.

[0208] In a preferred embodiment, the pH of the aqueous phase is selected such that the lipopeptide is substantially not soluble in the aqueous phase. Substantially not soluble in this context means that at most 20% (w / w)(ii) of the lipopeptide (relative to the total amount mixed with the aqueous phase), more preferably at most 10% (w / w), even more preferably at most 5% (w / w), even more preferably at most 1% (w / w), and most preferably at most 0.1% (w / w) of the lipopeptide is soluble in the aqueous phase.

[0209] Preferred buffers, bulking agents and tonicity adjusting agents for use in the methods according to the invention are the same as the respective buffers, bulking agents and tonicity adjusting agents described for the formulations of the invention.

[0210] Aqueous phase buffer (step (i)) In a preferred embodiment, the aqueous phase comprises a buffer.

[0211] A person skilled in the art knows how to select a buffer for a particular pH value. Preferred buffers are acetate buffers (e.g., acetate / acetic acid) (preferably for a pH of 3.7-6.5), phosphate or citrate buffers (e.g., Na2HPO4 / citric acid, Na2HPO4 / NaH2PO4, or Na2HPO4 / NaOH) (preferably for a pH of 5.4-8.0), sodium citrate / citric acid (preferred pH of 3.0-6.2).

[0212] In a preferred embodiment, the aqueous phase comprises at least 70% (w / w), more preferably at least 90% (w / w), of an aqueous medium, preferably water, optionally a bulking agent (component (d) in the formulation according to the invention), the amount of bulking agent in the aqueous phase being preferably 1% (w / w) to 25% (w / w), and a buffer, the amount of buffer being preferably 0.001% (w / w) to 5% (w / w), more preferably 0.001% (w / w) to 1% (w / w), for example 0.01% (w / w) to 0.1% (w / w).

[0213] If a bulking agent is present in the aqueous phase, the skilled person knows how to select the total volume of the aqueous phase and the amount of bulking agent (and the amount of any further components such as buffers or tonicity agents different from the bulking agent) to arrive at a formulation according to the invention.

[0214] In another preferred embodiment, water is the only solvent in the aqueous medium and is therefore the aqueous phase.

[0215] Acetate buffer is most preferred, especially when brevirtide is the lipopeptide (b). Preferably, the pH of such aqueous phase is 5 to 6.8, for example 5 to 6.

[0216] In a further preferred embodiment, the aqueous phase consists of an aqueous medium, a bulking agent, a buffer, and optionally the lipopeptide. If the lipopeptide is present in the aqueous phase, the pH of the aqueous phase should be within the range in which the lipopeptide is soluble in the aqueous phase. If the lipopeptide is not soluble in the aqueous phase, it is advantageous to add the lipopeptide to the organic phase or to the combined organic and aqueous phases.

[0217] In an even more preferred embodiment, the aqueous phase consists of water, a bulking agent, a buffer, and optionally a lipopeptide.

[0218] In an even more preferred embodiment, the aqueous phase consists of an aqueous medium, preferably water, a buffer, and optionally a lipopeptide.

[0219] In highly preferred embodiments, all of the components, such as the aqueous medium, buffer and tonicity agent, are pharma- ceutically acceptable components.

[0220] Typically, the buffer is present at a concentration of from 0.05 mM to 100 mM, for example from 1 mM to 50 mM.

[0221] organic phase In addition to the definitions and illustrative examples of the at least one organic solvent provided herein, for the organic phase, at least one suitable lyophilizable, water-miscible organic solvent can be selected by one of skill in the art.

[0222] The organic solvent should be miscible with water under standard conditions (25° C. and 1.013 bar). The organic solvent should also be lyophilizable so that it can be removed by sublimation (the direct transition of a substance from a solid state to a gaseous state without passing through a liquid state). A person skilled in the art knows how to select a suitable organic solvent, as well as a suitable temperature and pressure for lyophilizing the organic solvent, for example, by using pressure-temperature (PT) diagrams of organic solvents known in the art.

[0223] Preferred organic solvents are selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol and alcohols, such as butanol, preferably selected from the group consisting of 1-butanol, 2-butanol, and tert-butanol, acetic acid, ethyl lactate (ethyl 2-hydroxypropanoate), acetonitrile, or combinations of any of the foregoing, preferably at least one organic solvent is selected from the group consisting of alcohols, preferably tert-butanol, anisole, dimethyl sulfoxide, 1,4-dioxane, and dimethyl carbonate, and combinations thereof.

[0224] In a preferred embodiment, the organic solvent is anisole (CAS 100-66-3).

[0225] In a preferred embodiment, the organic solvent is ethyl acetate (CAS 141-78-6).

[0226] In a preferred embodiment, the organic solvent is 1,4-dioxane (CAS 123-91-1).

[0227] In a preferred embodiment, the organic solvent is dimethyl carbonate (CAS 616-38-6).

[0228] In a preferred embodiment, the organic solvent is dimethylsulfoxide (CAS 67-68-5).

[0229] In a preferred embodiment, the organic solvent is glycofurol (CAS 31692-85-0).

[0230] In a preferred embodiment, the organic solvent is N,N-dimethylacetamide (CAS 127-19-5).

[0231] In a preferred embodiment, the organic solvent is N,N-dimethylformamide (CAS 68-12-2).

[0232] In a preferred embodiment, the organic solvent is N-methyl-2-pyrrolidone (CAS 872-50-4).

[0233] In a preferred embodiment, the organic solvent is isopropylideneglycerol (CAS 100-79-8).

[0234] In a preferred embodiment, the organic solvent is 1-butanol (CAS 71-36-3).

[0235] In a preferred embodiment, the organic solvent is 2-butanol (CAS 78-92-2).

[0236] In a preferred embodiment, the organic solvent is tert-butanol (CAS 75-65-0).

[0237] In a preferred embodiment, the organic solvent is acetic acid.

[0238] In a preferred embodiment, the organic solvent is ethyl lactate (ethyl 2-hydroxypropanoate).

[0239] In a preferred embodiment, the organic solvent is acetonitrile.

[0240] Surprisingly, it has been found that the use of butanol, preferably tert-butanol (TBA), results in a preferred single-phase nanodispersion when the organic phase from step (i) is combined with the aqueous phase from step (ii).

[0241] Thus, in a more preferred embodiment, the organic solvent is tert-butanol (CAS 75-65-0).

[0242] Although higher and lower concentrations of lipopeptide components in the organic phase suitable for the method according to the invention can be used, it is preferred that the total of the one or more lipopeptides in the organic phase is between 3% and 30%, such as between 5% and 25%, for example between 10% and 20%, based on the total weight of the organic phase.

[0243] The skilled person is able, without undue burden, to calculate the concentrations and amounts of the various components of the organic and aqueous phases to be combined in step (iii) in order to arrive at a formulation according to the invention having the requirements regarding the ratio between the various components (a) to (e).

[0244] Surprisingly, it has been found that the lipopeptide formulation of step (iv) is a single-phase nanodispersion, the D90 of the vesicles being equal to or less than 60 nm, preferably less than 25 nm, more preferably equal to or less than 20 nm, such as between 10 nm and 20 nm, e.g., about 15 nm, or between 3 nm and 10 nm, e.g., about 5 nm.

[0245] In other words, as bound by the description, by combining the organic phase from (i) with the aqueous phase from (ii), no liposomes are obtained, but a single-phase nanodispersion having a micelle (particle) D90 size of 60 nm or less, preferably less than 25 nm, which nanodispersion can be sterile filtered.

[0246] In a preferred embodiment, the ratio between the organic phase and the aqueous phase is 2:1 (v / v) to 1:4.5 (v / v), even more preferably 1.5:1 (v / v) to 1:4 (v / v), such as 1:1 (v / v) to 1:4 (v / v), such as 1:1 (v / v) to 1:3.5 (v / v), such as about 1:1 (v / v), about 1:2 (v / v) or about 1:3 (v / v). For the purposes of explanation, "(v / v)" refers to the volume ratio of the two phases, for example a ratio of 1:1 refers to 1 ml of organic phase and 1 ml of aqueous phase.

[0247] In a preferred embodiment, the D90 of the vesicles in the resulting single-phase nanodispersion is 15 nm or less, more preferably 10 nm or less, for example, 3 nm to 10 nm, when the ratio between the two phases is 1:1. Without being bound by the explanation, a high ratio of organic solvent may result in a predominantly molecular solution of the components in the solvent mixture.

[0248] In another preferred embodiment, the D90 of the vesicles in the single-phase nanodispersion is 60 nm or less, more preferably less than 25 nm, even more preferably 20 nm or less, for example, between 5 nm and 20 nm, when the ratio between the two phases is 1:3. Without being bound by the explanation, at this higher proportion of water, a micellar solution is formed that exhibits the typical size and homogeneity of micelles. The mixture exhibits a nearly transparent appearance with a slight Tyndall effect and is freely filterable through a sterile filter with a nominal pore size of 0.22 μm.

[0249] At a solvent mixing ratio of 1:5, the resulting preparation is turbid. Size distribution measurements by DLS reveal a broad heterogeneous spectrum of vesicles with an average size of approximately 1,000 μm (1000 nm). This dispersion can be filtered through a membrane sterilizing filter with a nominal pore size of 0.22 μm simply by applying high pressure.

[0250] In a preferred embodiment, the ratio between the organic phases is 1:1 to 1:4 and the size of the vesicles, preferably micelles, in the single-phase nanodispersion system is 5 nm to 60 nm.

[0251] The method according to the present invention allows high loading of lipopeptides into vesicles.After lyophilization, small amounts of organic solvent, preferably butanol, more preferably tert-butanol, may still be present in the lyophilisate.Therefore, the formulation according to the present invention, when prepared according to the method of the present invention, may still contain small amounts of organic solvent, preferably butanol, more preferably tert-butanol.

[0252] Those skilled in the art will understand that the order of step (i) and step (ii) can be interchanged.Similarly, those skilled in the art will understand that PG and phospholipid (a) can be dissolved separately in an amount of organic solvent, and then the two organic phases can be combined to produce the organic phase of step (i), or both compounds can be dissolved in the same amount of organic solvent in parallel or successively to produce the organic phase of step (i).

[0253] Preferred lipopeptides for the methods according to the invention are also described above for the formulations according to the invention.

[0254] Typically, steps (i)-(iv) may be carried out at or near room temperature (25° C.) and at or near standard pressure (101.325 kPa). Generally, steps (i), (ii), (iii) and (iv) may be carried out individually at temperatures preferably between 0° C. and 40° C., more preferably between 15° C. and 35° C., e.g., between 18° C. and 28° C. Any of the four steps may also be carried out individually at higher and lower pressures, but preferably any of the steps are carried out individually at a pressure between 90 kPa and 112 kPa, more preferably between 95 kPa and 116 kPa, and most preferably near standard pressure, e.g., 101.325 kPa±2%.

[0255] Optionally, the method comprises one or more further steps.

[0256] Preferably, one further step is the step "sterile filtration": · Sterile filtering the resulting formulation comprising (a), (b), (d), and optionally (e), and optionally (d).

[0257] Preferably, the sterile filtration is performed by using a membrane filter, such as a PVDF membrane filter, preferably having a nominal pore size of 200 nm or less.

[0258] The same preferred temperatures and pressures as in steps (i)-(iv) also apply to the sterile filtration step.

[0259] Optionally, the method further comprises the optional step of "lyophilizing" the nanodispersion formulation with the lipopeptide: lyophilizing the formulation resulting from step (iv) (or the sterile filtration step after step (iv)), comprising (a), (b), (c), (d), and (e) as defined herein, to result in a lyophilisate comprising (a), (b), (c), (d), and (e) as defined herein.

[0260] Those skilled in the art are familiar with the freeze drying (lyophilization) technique. Usually, the freeze drying of the preparation according to the invention is carried out at a temperature of +40°C to -40°C, preferably +30°C to -10°C. The freeze drying process may be repeated once or several times. Usually, the pressure is between 1000hPa and 0.001hPa (1hPa=1mbar). Preferably, the freeze drying process is carried out at a pressure between 1hPa and 0.01hPa, for example about 0.1hPa.

[0261] Optionally, the method includes a step of lyophilization, and the method further includes a step of rehydrating the lyophilizate comprising (a), (b), (d), and optionally (c), and optionally (e), preferably with an aqueous pharmaceutical solution.

[0262] Another preferred embodiment is (i) preparing an organic phase by dissolving phospholipids (a)) preferably comprising PC and PG (component (a)) and, optionally, cholesterol (component (c)) in butanol, preferably tert-butanol; (ii) preparing an aqueous phase by dissolving a sugar component (d), preferably trehalose, and optionally an isotonicity agent (component (e)) which is not component (d), in an aqueous medium, preferably the aqueous medium having a pH between 3 and 9, e.g. about 5.5 (e.g. by using a sodium acetate buffer); (iii) combining the organic phase and the aqueous phase; (iv) either adding the lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the organic phase of step (i) and then mixing the organic phase containing the lipopeptide with the aqueous phase as described in step (iii), or adding the lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the aqueous phase of step (ii) and then mixing the aqueous phase containing the lipopeptide with the organic phase as described in step (iii), or adding the lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the combined phases of step (iii), preferably to the combined phases of step (iii), which results in a lipopeptide formulation, the lipopeptide formulation so obtained being a single-phase nanodispersion, (v) lyophilizing the formulation of step (iv) to result in a lyophilizate comprising (a), (b), (d), and optionally (c), and optionally (e). The present invention relates to a method for preparing a formulation according to the present invention, comprising:

[0263] Optionally, the method comprises further steps, preferably before step (iv) or before step (v), such as a "sterile filtration" step.

[0264] Another preferred embodiment is (i) preparing an organic phase by dissolving phospholipids (a)), preferably comprising PC and PG (component (a)), and optionally cholesterol (component (c)), in butanol, preferably tert-butanol; (ii) preparing an aqueous phase by dissolving a sugar component (e), preferably trehalose, in an aqueous medium, preferably the aqueous medium having a pH of 3 to 9, e.g. about 5.5 (e.g. by using a sodium acetate buffer); (iii) combining the organic phase and the aqueous phase; (iv) adding a lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the combined phase to receive a lipopeptide formulation according to the invention; or adding a lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the organic phase prior to mixing the organic phase with the aqueous phase in step (iii); or adding a lipopeptide, preferably brevirtide or liraglutide, more preferably brevirtide, to the aqueous phase prior to mixing the aqueous phase with the organic phase in step (iii); The present invention relates to a method for preparing a liposomal formulation, preferably a pharmaceutical liposomal formulation according to the present invention, comprising: (v) lyophilizing the lipopeptide formulation of step (iv) to result in a lyophilizate comprising (a), (b), (d), and optionally (c), and optionally (e). (vi) rehydrating the lyophilisate of step (v) preferably with an aqueous pharmaceutical solution, preferably resulting in a pharmaceutical liposomal formulation.

[0265] Another preferred embodiment relates to a method for preparing a pharmaceutical liposomal formulation according to the invention, comprising a step of "rehydration" of the lyophilisate, preferably prepared according to steps (i) to (iv) above, and at least the optional step "lyophilisation" outlined above, comprising (a), (b), (c), optionally (d), (e) and optionally (f), rehydrating the lyophilisate with an aqueous pharmaceutical solution.

[0266] Typically, such steps may be carried out at the same temperatures and pressures as described for steps (i) to (iv).

[0267] Gentle swirling or vortexing of the resulting liposomal dispersion is sufficient for homogenization. There is no need to reduce the size of the liposomes by further complicated steps if the liposomal preparation is to be suitable for injection into a patient in need of a lipopeptide preparation.

[0268] In a preferred embodiment, the ratio between the lipopeptide and one or more phospholipids in the organic phase, the combined organic and aqueous phase, the lipopeptide formulation, the lyophilisate or the liposomal formulation, respectively, is 1:333 to 1:2, such as 1:333 to 1:49 (where the lipopeptide is, for example, liraglutide), or 1:33 to 1:6.7 (where the lipopeptide is, for example, brevirtide), based on the total amount of lipopeptide and phospholipid in the organic phase, the combined organic and aqueous phase, the lipopeptide formulation, the lyophilisate or the liposomal formulation, respectively.

[0269] aqueous solution As used herein, preferably, pharmaceutical aqueous solution comprises pharmaceutical aqueous medium, preferably water, or (preferably pharmaceutical) aqueous medium, for example, salt is present. The aqueous medium in which salt is present is preferably an aqueous medium, preferably water, containing at least one salt in physiological concentration, preferably at least one salt in isotonic concentration, such as saline, Ringer's lactate and / or Plasmalyte. Thus, (preferably pharmaceutical) aqueous medium preferably has salt concentration, osmolality and pH that reflects human physiological plasma electrolyte concentration, osmolality and pH. Preferably, pharmaceutical aqueous solution is water, or a combination of water and at least one salt, preferably in isotonic concentration, and the amount of water in the total amount of solvent is preferably at least 80%. More preferably, (preferably pharmaceutical) aqueous solution is water or saline.

[0270] In one embodiment, the pharmaceutical aqueous solution consists of water or a combination of water and a salt, such as saline, lactated Ringer's solution and / or Plasmalyte, and the amount of water in the total amount of solvent is at least 80%. More preferably, the (preferably pharmaceutical) aqueous solution is water or saline. In an even more preferred embodiment, the pharmaceutical aqueous solution consists of water.

[0271] A further preferred embodiment relates to an aqueous pharmaceutical solution comprising a solvent as described herein and an isotonicity agent. Preferred isotonicity agents for use in the aqueous solution have already been described for the formulations according to the invention.

[0272] A further preferred embodiment relates to an aqueous pharmaceutical solution comprising a solvent as described herein, and a tonicity agent and a buffer. Preferred tonicity agents and buffer systems for use in the aqueous solution have already been described for the formulations according to the invention.

[0273] Preferably, the pH of the aqueous pharmaceutical solution is selected so that the pH of the resulting liposomal formulation is 5-8, preferably 5-7.6, more preferably 6-7.6 (e.g. either slightly acidic to neutral, e.g. 6.5-7, or 7.2-7.6, e.g. 7.3-7.5). The skilled person knows that depending on the preparation method according to the invention, the lyophilisate can already contain a buffer system or the aqueous pharmaceutical solution is provided in such a buffer system. The skilled person is able to calculate the requirements for the aqueous pharmaceutical solution for preparing the final liposomal formulation without undue burden.

[0274] Preferred tonicity agents, buffers, solvents, phospholipids, phosphatidylglycerol, lipopeptides, cholesterol (and derivatives thereof), sucrose components, ratios and amounts of any of the foregoing that may be used in the methods of the present invention have already been described above for the formulations according to the invention.

[0275] Preferably, the ratios of the various components used in the method according to the invention are selected by a person skilled in the art to prepare a formulation having the requirements described herein. (a) a phospholipid selected from the group consisting of phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), or a derivative of any of the foregoing, or a mixture thereof, in the range of 40% to 93% based on the total weight of (a) to (e); (b) brevirtide in the range of 3% to 13% based on the total weight of (a) to (e), or liraglutide in the range of 0.3% to 2% based on the total weight of (a) to (e); (c) cholesterol or a derivative thereof in the range of 0% to 14% based on the total weight of (a) to (e); (d) in the range of 0% to 35% based on the total weight of (a) to (e), glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, or 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, saccharose, xylitol, xylose, dextran, or a mixture of any of the foregoing; (e) an isotonicity agent other than (d), in the range of 0% to 35% based on the total weight of (a) to (e); A formulation comprising: One can readily calculate the amounts and concentrations required to prepare a formulation in which the sum of (a), (b), (c), (d), and (e) total 100%, and the combined amounts of (a)-(e) are between 10% and 100%, based on the total weight of the formulation and any preferred formulation.

[0276] In a preferred embodiment, in the method according to the invention, a buffer must be present either in the aqueous phase or in the aqueous solution.

[0277] In another preferred embodiment, in the method according to the invention, the same or different buffers are present in the aqueous phase and in the aqueous solution, preferably the buffers are the same.

[0278] In yet another preferred embodiment, in the process according to the invention, a weighting agent is present in the aqueous phase.

[0279] In yet another preferred embodiment, in the method according to the invention the bulking agent and the buffer are present in the aqueous phase.

[0280] In yet another preferred embodiment, in the method according to the invention, a tonicity agent is present in the aqueous phase.

[0281] In yet another preferred embodiment, in the method according to the invention, the tonicity agent and the buffer are present in the aqueous phase.

[0282] In yet another preferred embodiment, in the method according to the invention, the tonicity agent, bulking agent and buffer are present in the aqueous phase.

[0283] In yet another preferred embodiment, in the method according to the invention, the tonicity agent is present in the aqueous solution.

[0284] In yet another preferred embodiment, in the method according to the invention, the tonicity agent and the buffer are present in an aqueous solution.

[0285] kit A further aspect of the invention relates to a kit comprising a formulation according to the invention and a separate aqueous pharmaceutical solution, for example where the formulation according to the invention is in one container and the aqueous pharmaceutical solution is in another container.

[0286] In a preferred embodiment, the formulation according to the invention is a lyophilized formulation.

[0287] In another preferred embodiment, the formulation according to the present invention is a lyophilized formulation, and a portion of the formulation in the container contains brevirtide (compound (c)) at a concentration of 8 mg per 176.6 mg of the total of components (a)-(e) to 19.4 mg per 176.6 mg of the total of components (a)-(e), the amount of brevirtide being 5%-11% compared to the total of (a), (b), (c), (d), and (e) in the formulation. In a preferred embodiment, the weight of the portion of the formulation according to the present invention in the container is 100 mg-250 mg.

[0288] In a preferred embodiment, after adding a portion of the aqueous pharmaceutical solution to the lyophilized formulation according to the present invention (or vice versa), the aqueous pharmaceutical solution is divided into separate containers such that the weight of the portion of the aqueous pharmaceutical solution is calculated such that the total amount of (a), (b), (c), (d), and (e) in the resulting pharmaceutical liposomal formulation according to the present invention is between 20% and 2%, more preferably between 15% and 7%, e.g., about 12%.

[0289] Mixing the two-component aqueous pharmaceutical solution and the lyophilized formulation allows the pharmaceutical liposomal formulation to be prepared immediately prior to administration to a patient.

[0290] While the described invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various modifications can be made and equivalents can be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the objective of the invention. All such modifications are intended to be within the scope of the appended claims. [Brief description of the drawings]

[0291] drawing [Figure 1] FIG. 1 shows a process chart of the freeze-drying process. [Diagram 2] FIG. 2 shows the observed size distribution of vesicles as a function of the organic to aqueous phase ratio (v / v). [Diagram 3]FIG. 3 shows the average size of 1.9 μm liposomes after reconstitution in 0.9% NaCl solution (D90=3.6 μm). [Figure 4] FIG. 4 shows the average size of 0.2 μm liposomes after reconstitution in purified water (D90=2 μm). [Diagram 5] FIG. 5 shows the RP-HPLC chromatogram of the excess liposome volume (unencapsulated brevirtide). [Figure 6] FIG. 6 shows the force-displacement curve of brevirtide acetate liposomal formulation (1.5 ml) from a single-use 3 mL syringe fitted with a 27G×1 inch cannula.

[0292] Other aspects and advantages of the present invention are described in the following examples, which are given by way of illustration and not by way of limitation.

[0293] Each publication, patent, patent application or other document cited herein is incorporated herein by reference in its entirety. EXAMPLES

[0294] Substances and Materials The following substances and materials were used (Table 1).

[0295] (Table 1) Substances and materials TIFF2024535127000002.tif217162

[0296] The following equipment and devices were used: HPLC equipment: Identification number: Sys 1 Manufacturer: Agilent Technologies (Santa Clara, California USA) Type:1260 Series Pilot Freeze Dryer (GT1): Manufacturer: Hof Sonderanlagenbau (Lohra, Germany) 0.5m2 shelf area Particle sizer: Manufacturer:Malvern(Malvern,GB) Type:Mastersizer 2000 DLS Plate Reader: Manufacturer: Wyatt Technology Corporation (Santa Barbara, CA, USA) Type: Dynapro Plate Reader II Additional Laboratory Equipment ·Magnetic stirrer (IKA Werke) Volumetric pipette (Gilson) Camera equipment (Canon EOS 600D with DGMacro 105mm 1:2.8) pH meter (Mettler Toledo, SevenMulti with InLab Micro electrode) ·Balance (Kern, EW6200-2NM, ABJ-NM ABS3204N ·Purified water supply (Siemens, Ultra Clean UV UF(trademark)) Vortex mixer (Scientific Industries Inc., Vortex Genie II) Centrifuge: ThermoScientific, Heraeus Pico 17 Orbital shaker: Wisd Laboratory Instruments, WiseShake SHO-1D

[0297] The following protocol was used for the RP-HPLC analysis (see Table 2).

[0298] Table 2: RP-HPLC chromatographic conditions TIFF2024535127000003.tif200164

[0299] Additional Laboratory Equipment ·Magnetic stirrer (IKA Werke) Volumetric pipette (Gilson) Camera equipment (Canon EOS 600D with DGMacro 105mm 1:2.8) pH meter (Mettler Toledo, SevenMulti with InLab Micro electrode) ·Balance (Kern, EW6200-2NM, ABJ-NM ABS3204N ·Purified water supply (Siemens, Ultra Clean UV UF(trademark)) Vortex mixer (Scientific Industries Inc., Vortex Genie II) Centrifuge: ThermoScientific, Heraeus Pico 17 Orbital shaker: Wisd Laboratory Instruments, WiseShake SHO-1D

[0300] Preparation of liposomal formulations using single-phase nanodispersion systems TIFF2024535127000004.tif151160

[0301] Example 1: Preparation of nanodispersion Dispersion formation using TBA involved the following steps.

[0302] The organic phase containing phosphatidylglycerol (PG), S100 (highly purified soy lecithin) and tert-butanol (TBA) was weighed, followed by dissolving PG and S100 in the TBA by heating (70 °C) and stirring for approximately 40-80 min.

[0303] Once the components were homogeneously dissolved, the organic phase was cooled to ambient temperature.

[0304] Aqueous phase: 10 mM sodium acetate buffer was adjusted to pH 5.5 with acetic acid. Trehalose (5%) was added and dissolved under stirring.

[0305] The aqueous solution was added to the organic phase in small portions under continuous stirring. The first part of the aqueous solution was dissolved into a transparent micellar system. After complete addition of the aqueous phase, a milky white single-phase nanodispersion with low viscosity was formed.

[0306] With gentle stirring, the brevirtide acetate was added and incorporated into the micellar system.

[0307] The final bulk solution was sterile filtered through a PVDF membrane filter with a nominal pore size of 200 nm. TIFF2024535127000005.tif166160

[0308] Example 2: Freeze-drying The formulation obtained from Example 1 was filled into sterile glass vials (1.5 g in a 2R vial).

[0309] The filled vials were stoppered in the lyophilization position and placed into the lyophilizer.

[0310] The following lyophilization cycles were performed (Table 3).

[0311] (Table 3) Freeze-drying process program TIFF2024535127000006.tif145160

[0312] The following equipment was used: Pilot freeze dryer (GT3) (Hof Sonderanlagenbau (Lohra, Germany)), shelf area 0.25 m 2 , Ice condenser capacity 5kg.

[0313] The process was monitored by online data acquisition. The process chart (see Figure 1) showed that the freeze-drying process was completed successfully.

[0314] Example 3: Preparation of liposomal formulations The lyophilisates were reconstituted with either 1.5 ml of purified water or 1.5 ml of 0.9% sodium chloride solution. Reconstitution of the lyophilisates was rapid and spontaneous within 10 seconds. The resulting dispersions were homogenised by gentle swirling or vortex mixing within 5 minutes.

[0315] In both cases, visually different liposome dispersions were obtained. The lyophilizates reconstituted with purified water showed higher transparency and milky white color, and showed submicron-sized particles, whereas those reconstituted with purified water showed higher turbidity and a more cloudy appearance. More specifically, the lyophilizates reconstituted with purified water showed higher transparency and milky white color, and showed submicron-sized particles, whereas those reconstituted with saline showed higher turbidity and a more cloudy appearance, and showed multi-lamellar liposomes with larger diameters. Therefore, it is advantageous to reconstitute the lyophilizates using saline, especially for subcutaneous depot formulations.

[0316] Both reconstituted mutants were analyzed using the MALS particle sizer.

[0317] Example 4: Vesicle / Liposome Size Distribution MALS (Multi-Angle Light Scattering): Liposome / vesicle size was determined using a Malvern, Mastersizer 2000 equipped with a hydro 2000μP sample cell instrument (MALS) with a liquid sample cell. The sample cell was filled with dispersant (approximately 18mL, 0.9% sodium chloride or purified water) and air bubbles were removed by increasing the circulation pump speed. The instrument was blanked. Sample was added until sufficient signal absorption was reached. Measurements were started and then taken in triplicate. Results were calculated from summarized data.

[0318] DLS (Dynamic Light Scattering): Liposome / vesicle size distribution was also determined using a Dynapro Plate Reader (Wyatt Technology) instrument. Samples (30 μL) were loaded into a 96-well plate with a clear bottom, the plate was transferred to the DLS plate reader, and 3 wells were loaded per sample. The measurement was started. The temperature was set to 25° C. Acquisition time: 5 s, 5 acquisitions per well were performed. The mass-weighted average radius of the observed particles was calculated.

[0319] 4.1 Vesicle (micelle) size in single-phase nanodispersion systems To determine the size of the formed vesicles, various ratios of organic to aqueous phase were analyzed by dynamic light scattering.

[0320] The ratios tested were 1:1, 1:3 and 1:5 organic:aqueous phase.

[0321] A 1:1 ratio resulted in a clear solution with no turbidity. A few very small particles could be detected. The average size was about 5 nm (3 nm to 10 nm, see Figure 2). Without being bound to this explanation, it appears that a high ratio of organic solvent results in a predominantly molecular solution of the components within the solvent mixture.

[0322] Within a 1:3 solvent mixing ratio, the number and size of the observed particles increased substantially to an average size of approximately 15 nm (10 nm to 20 nm, see Figure 2). A micellar solution was formed, showing the typical size and homogeneity of micelles. The mixture exhibited a nearly transparent appearance with a slight Tyndall effect and was freely filterable through sterile filters with a nominal pore size of 0.22 μm.

[0323] At a solvent mixing ratio of 1:5, the resulting preparation was turbid. Size distribution measurements by DLS revealed a broad heterogeneous spectrum of vesicles with an average size of about 1 μm, typical of multilamellar liposomes. This liposome dispersion can be filtered through a membrane sterilizing filter with a nominal pore size of 0.22 μm simply by applying high pressure, resulting in the deformation of the vesicles. Figure 2 shows the observed size distribution of vesicles as a function of the ratio of organic to aqueous phases (v / v).

[0324] 4.2 Liposome size in liposome formulation (reconstituted lyophilized product) The following size distribution was observed: When reconstituted in 0.9% NaCl solution, the average size of the liposomes is 1.9 μm (D90=3.6 μm, see FIG. 3). When reconstituted in purified water, the average size of the liposomes is 0.2 μm (D90=2 μm, see FIG. 4).

[0325] The measurements confirmed the visual impression of the dispersion: larger liposomes were formed when reconstituted in sodium chloride solution (major fraction of about 1.9 μm, D90 of 3.6 μm), whereas smaller liposome sizes were observed when reconstituted in purified water (major fraction of about 0.2 μm, D90 of 2 μm). As mentioned above, larger (multi-lamellar) liposomes are preferred due to the increased drug loading.

[0326] Example 5: Liposome encapsulation of brevirtide Determination of Brevirtide and formulation content and purity.

[0327] Liposome encapsulation of brevirtide was tested by determining the amount of free (unencapsulated) brevirtide.

[0328] Lyophilized samples from Example 2 were reconstituted with 1.5 ml of 0.9% (w / w) NaCl solution or water. Samples for determining liposomal encapsulation of brevirtide were mixed and left for 20 minutes to allow complete hydrazination and liposome formation. The reconstituted samples were then diluted 1:1 with 0.9% sodium chloride solution and mixed thoroughly. Samples were centrifuged at 21460 x g to separate the lipid phase from the solvent. The supernatant was collected and analyzed by RP-HPLC.

[0329] The RP-HPLC method was used as provided by the drug substance manufacturer, Chengdu Shengnuo Biopharm Co. Ltd. Chromatographic conditions were used for the analysis as outlined in Table 2.

[0330] The samples were reconstituted with 1.5 mL of 0.9% (w / w) sodium chloride solution. The samples were mixed thoroughly and left on the bench for 20 minutes to allow complete hydrazination and liposome formation. The reconstituted samples were then diluted 1:1 with 0.9% sodium chloride solution and mixed thoroughly. The samples were centrifuged at 21460 x g to separate the lipid phase from the solvent. The supernatant was collected and analyzed.

[0331] Results are reported as mg / mL using DS calibration standards and the peak area of ​​brevirtide from the chromatogram obtained by sample preparation. Figure 5 shows the RP-HPLC chromatogram of the excess liposome volume (unencapsulated brevirtide).

[0332] Small amounts of brevirtide could be detected in the supernatant. By comparison with calibration standards, the amount of free brevirtide acetate was calculated to be 847 μg (per vial), which corresponds to an encapsulation efficiency of 94%. In a second vial of the same preparation, an encapsulation efficiency of 96% (584 μg / vial) was determined.

[0333] Example 6: Extrusion force After reconstitution of the lyophilisate with water or 0.9% NaCl, respectively, the liposome dispersion was drawn into a standard single-use 3 mL PP syringe, fitted with a 27G x 1 inch cannula, and residual air was removed from the syringe. The syringe contents were dispensed at a defined traverse speed using a force / displacement measuring device (Thumler, Z3).

[0334] The results are shown in Figure 6, which shows the force-displacement curves of the brevirtide acetate liposomal formulation (1.5 ml) from a single-use 3 mL syringe fitted with a 27G x 1 inch cannula. Strain 1 in Figure 1: speed 200 mm / min; Strain 2 and 3 in Figure 5: speed 500 mm / min.

[0335] Extrusion force evaluation showed that a moderate force of 30-50 N was sufficient to dispense the liposomal brevirtide formulation.

Claims

1. A method for preparing a formulation, comprising the steps of: (i) one or more phospholipids selected from the group consisting of phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), or a derivative of any of the foregoing, or a combination of any of the foregoing; Optionally, cholesterol or a derivative thereof, at least one organic solvent; providing an organic phase comprising (ii) an aqueous medium; optionally a pharmaceutically acceptable buffer; and Optionally, a bulking agent 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, saccharose, xylitol, xylose, dextran, or a mixture of any of the foregoing; Optionally, a pharmaceutically acceptable tonicity agent that is not a bulking agent selected from the group consisting of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, a sugar moiety selected from the group consisting of sucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose, lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose, ribose, sorbitol, saccharose, xylitol, xylose, dextran, or a mixture of any of the foregoing. providing an aqueous phase comprising the aqueous phase has a pH of 3 to 9; (iii) combining the organic phase and the aqueous phase, wherein the mixing ratio of the organic phase to the aqueous phase is between 10:1 (v / v) and 1:10 (v / v), resulting in a combined organic and aqueous phase, wherein the at least one organic solvent and the aqueous phase form a single-phase mixture, preferably a freezable and sublimable single-phase mixture; (iv) adding the lipopeptide to the organic phase of step (i) and then mixing the organic phase containing the lipopeptide with the aqueous phase as described in step (iii); or adding the lipopeptide to the aqueous phase of step (ii) and then mixing the aqueous phase containing the lipopeptide with the organic phase as described in step (iii); or adding the lipopeptide to the combined phase of step (iii) Either of the following: This results in a lipopeptide formulation, wherein the lipopeptide formulation thus obtained is a single-phase nanodispersion system.

2. 2. The method of claim 1, wherein the at least one organic solvent is selected from the group consisting of anisole, ethyl acetate, 1,4-dioxane, dimethyl carbonate, dimethyl sulfoxide, glycofurol, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropylideneglycerol, alcohol, preferably selected from the group consisting of 1-butanol, 2-butanol, and tert-butanol, acetic acid, ethyl lactate (ethyl 2-hydroxypropanoate), acetonitrile, or a combination of any of the foregoing; preferably, the at least one organic solvent is selected from the group consisting of alcohol, preferably tert-butanol, anisole (phenoxymethane), dimethyl sulfoxide, 1,4-dioxane, and dimethyl carbonate, and a combination thereof.

3. 2. The method of claim 1, wherein the derivative of any of the foregoing is selected from the group consisting of DLPA, DMPA, DPPA, DSPA, POPA, POPA, DEPA, HSPA, HEPA, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC, HSPC, HEPC, DLPE, DMPE, DPPE, DSPE, POPE, POPE, DEPE, HSPE, HEPE, DLPG, DMPG, DPPG, DSPG, POPG, POPG, DEPG, HSPG, HEPG, DLPI, DMPI, DPPI, DSPI, POPI, POPI, DEPI, HSPI, HEPI, DLPS, DMPS, DPPS, DSPS, POPS, POPS, DEPS, HSPS, HEPS, PEGylated forms of any of the foregoing, and salts of any of the foregoing.

4. 3. The method of claim 2, wherein the organic solvent is tert-butanol.

5. 4. The method of claim 3, wherein the lipopeptide is brevirtide or liraglutide.

6. 2. The method of claim 1, wherein the phospholipid comprises PC.

7. 10. The method of claim 1, wherein the pH of the aqueous phase is between 5 and 7.

5.

8. 10. The method of claim 1, further comprising sterile filtration of the single-phase nanodispersion.

9. 10. The method of claim 9, further comprising a step (v) of freeze-drying the formulation (single-phase nanodispersion) obtained from step (iv), resulting in a lyophilisate.

10. 10. The method of claim 9, further comprising a rehydration step of mixing the lyophilized product obtained in step (v) with an aqueous solution, which results in a liposomal preparation.

11. 11. The method of claim 10, wherein the liposomes of the liposome formulation obtained from the rehydration step (reconstitution) have a D90 of 1 μm to 4.5 μm.

12. 11. The method of claim 10, wherein the aqueous solution used for mixing / reconstituting is an aqueous NaCl solution having an amount of NaCl of 8 g / l to 10 g / l, preferably 8.8 g / l to 9.2 g / l, more preferably 9 g / l.

13. 2. The method of claim 1, wherein the D90 of the micelles of the single-phase nanodispersion is 60 nm or less.

14. 11. The method of claim 10, wherein the lipopeptide formulation is a pharmaceutical formulation.

15. A pharmaceutical formulation prepared according to the method of any one of claims 1 to 14.

16. (a) a phospholipid selected from the group consisting of phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), or a derivative of any of the foregoing, or a mixture thereof, in the range of 40% to 97% based on the total weight of (a) to (e); (b) brevirtide in the range of 3% to 13% based on the total weight of (a) to (e), or liraglutide in the range of 0.3% to 2% based on the total weight of (a) to (e); (c) cholesterol or a derivative thereof in the range of 0% to 14% based on the total weight of (a) to (e); (d) in the range of 0% to 35%, preferably 15% to 35%, based on the total weight of (a) through (e), of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, or 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, saccharose, xylitol, xylose, dextran, or a mixture of any of the foregoing; (e) a tonicity agent other than (d), in the range of 0% to 35% based on the total weight of (a) to (e); 1. A formulation which is a single-phase nanodispersion comprising: The formulation, wherein the sum of (a), (b), (c), (d), and (e) always total 100%, and the combined amount of (a) through (e) is from 10% to 100% based on the total weight of the formulation.

17. 17. The formulation of claim 16, which is a lyophilized formulation.

18. 18. A pharmaceutical liposomal formulation obtained by mixing or reconstituting the formulation according to claim 17, preferably with an aqueous solution, more preferably with saline or water, (a) a phospholipid selected from the group consisting of phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), or a derivative of any of the foregoing, or a mixture thereof, in the range of 40% to 75% based on the total weight of (a) to (e); (b) brevirtide in the range of 3% to 13% based on the total weight of (a) to (e), or liraglutide in the range of 0.3% to 2% based on the total weight of (a) to (e); (c) cholesterol or a derivative thereof in the range of 4% to 14% based on the total weight of (a) to (e); (d) in the range of 15% to 35% based on the total weight of (a) through (e) of glycine, arginine, proline, or any other amino acid known to be suitable as a bulking agent, or 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, saccharose, xylitol, xylose, dextran, or mixtures thereof; (e) 0.1% to 10% by weight of an isotonic agent other than (d), based on the total weight of (a) to (e). Including, The formulation, wherein (a), (b), (c), (d), and (e) total 100% and the combined amount of (a) through (e) is 10% to 50% based on the total weight of the formulation, which further includes a solvent and a tonicity agent.

19. 18. A kit comprising the formulation of claim 16 or 17 in a container and an aqueous pharmaceutical solution in a second container.

20. 17. The formulation of claim 16, wherein the derivative of cholesterol is selected from the group consisting of cholesteryl sulfate, salts of cholesteryl sulfate, cholesteryl hemisuccinate, cholesteryl succinate, cholesteryl oleate, cholesterol-PEG, coprostanol, cholestanol, cholestane, cholic acid, cortisol, corticosterone, hydrocortisone, and calciferol.

21. 19. The formulation of claim 18, wherein the derivative of cholesterol is selected from the group consisting of cholesteryl sulfate, salts of cholesteryl sulfate, cholesteryl hemisuccinate, cholesteryl succinate, cholesteryl oleate, cholesterol-PEG, coprostanol, cholestanol, cholestane, cholic acid, cortisol, corticosterone, hydrocortisone, and calciferol.

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