Bhb liposome and method for preparing the same

Abstract

The present invention provides a liposome comprising an active ingredient; and one membrane, or one inner membrane and one outer membrane. Each membrane comprises a plurality of lipid molecules, and the active ingredient is entrapped within an interior space bounded by the membrane or inner membrane, or is embedded in the membrane and forms part of the membrane.

Description

[0001] Cross-reference to related applications

[0002] This application is the national phase application of PCT / CN2022 / 075372 filed on February 7, 2022. It claims priority to international application PCT / CN2021 / 074773 filed on February 2, 2021, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0003] BHB stands for β-hydroxybutyrate (also known as β-hydroxybutyrate or 3-hydroxybutyric acid), and is one of the three main ketone bodies (acetoacetic acid, acetone, and BHB). It provides a clean energy source for the brain and muscles. While the body normally relies on glucose for energy, when glucose supply is too low for the body's energy needs, such as during prolonged exercise, starvation, or a lack of dietary carbohydrates, the body turns to fat as fuel. Since the brain and central nervous system cannot directly use fat as energy, the liver produces ketone bodies (also known as ketones) from fatty acids as an alternative fuel source, which are then released into the blood / plasma. Ketones not only fuel the brain but are also used by bones and heart muscle. Numerous studies on exogenous ketones have shown that elevated blood ketone levels contribute to improved athletic performance, recovery, appetite control, and mental clarity.

[0004] Recognized research data indicates that BHB not only increases blood ketone levels but also helps the body enter ketosis more quickly. Simultaneously, BHB can help improve endurance performance, support appetite control, and provide powerful energy to the brain, bones, and heart muscle tissue. Currently, BHB is widely commercialized as a dietary supplement. Due to the high hygroscopicity and low melting point of its free acid form, BHB is typically sold as mineral salts, such as sodium, calcium, and magnesium BHB.

[0005] Liposomes are small, spherical artificial vesicles composed of one or more lipid layers (usually bilayers). Studies have shown that liposomes can serve as useful drug carriers for encapsulating active ingredients, making them more effective. For example, vitamin C liposomes can reduce the degradation of vitamin C in the gastrointestinal tract, slow its release, and enhance absorption, thereby improving bioavailability. Liposomes can also alleviate potential disturbances in the gastrointestinal tract, allowing high doses of active ingredients to exert their effects for extended periods (Maciej et al. J. Liposome Res., (2019) 30(3), 227-234).

[0006] Typically, liposomes have a common vesicle structure containing an aqueous lumen surrounded by a lipid bilayer membrane. Hydrophilic or water-soluble molecules can be enclosed in the aqueous core and theoretically completely embedded, while hydrophobic or oil-soluble molecules can be embedded in the hydrophobic regions of the phospholipid bilayer, forming part of the membrane. Alternatively, reverse vesicles are spherical containers in organic liquids (oils), consisting of an oily core surrounded by a reverse bilayer. They can carry oil-soluble substances, while hydrophilic substances can be embedded in the hydrophilic regions of the bilayer (Tung et al. J. Am. Chem. Soc. (2008) Vol. 130 (27), 8813-8817).

[0007] Conventional methods for preparing liposome drugs include thin-film dispersion, injection, reverse-phase evaporation, double emulsion, freeze-drying, ultrasonication, and high-pressure homogenization. The choice of method depends on: (1) the physical and chemical properties of the liposomes and the encapsulated components; (2) the concentration of the encapsulated components; (3) the particle size of the liposomes; and (4) the cost, reproducibility, and applicability of industrial production. For example, Chinese patent application CN111920702A discloses a method for preparing liposomes by thin-film dispersion; CN110279590A discloses a method for preparing liposomes by injection; CN102488656A discloses a method for preparing liposomes by supercritical reverse-phase evaporation; and CN102935068A discloses a method for preparing liposomes by freeze-drying.

[0008] However, existing liposome formulations and preparation methods still face various technical challenges, such as low effective components in liposomes, making large-scale production difficult. Summary of the Invention

[0009] The summary is provided to introduce, in a simplified form, some concepts that will be further described in the following detailed description. The summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to limit the scope of the claimed subject matter.

[0010] This invention generally relates to formulations of β-hydroxybutyrate (BHB) liposomes and methods for preparing such BHB liposomes. Specifically, the BHB liposomes of this invention comprise lipids (e.g., phospholipids) and active ingredients (e.g., BHB free acid, its physiologically acceptable salts, esters, or mixtures, and optionally one or more additional hydrophilic components) and are capable of efficiently delivering BHB. These BHB liposomes can be prepared using the improved preparation method of this invention and used to increase or maintain blood ketone levels in subjects.

[0011] One aspect of the invention provides a liposome comprising an active ingredient; and a membrane, or an inner membrane and an outer membrane, wherein each of the membrane or the inner and outer membranes defines an internal space within one membrane or the inner membrane and / or between the inner and outer membranes, each membrane comprising a plurality of lipid molecules, and the active ingredient being retained within the internal space or embedded in the membrane. The lipids comprise phospholipids, and the active ingredient comprises β-hydroxybutyric acid (BHB), a physiologically acceptable salt, ester, or mixture thereof.

[0012] In some implementations, the active ingredient is the free acid form of BHB.

[0013] In some embodiments, the BHB is of type R, type S, or a mixture of types R and S. Preferably, the BHB is of type R.

[0014] In some implementations, depending on the proportions of the components, BHB liposomes have a common vesicle structure in which BHB is trapped within an internal space defined by a membrane or inner membrane.

[0015] In some implementations, each membrane is a double membrane.

[0016] In some implementations, liposomes comprise a membrane.

[0017] In some other embodiments, depending on the proportions of the components, BHB liposomes have an inverted vesicle structure in which BHB is embedded in a membrane and forms part of the membrane.

[0018] In some embodiments, the active ingredient comprises BHB and / or one or more additional hydrophilic components, comprising 1% to 95% by mass of the total liposome mass. Examples of hydrophilic components include, but are not limited to, vitamin C, vitamin B1, vitamin B6, folic acid, and other water-soluble vitamins.

[0019] In some other embodiments, the active ingredient is 60% to 85% of the total mass of the liposomes.

[0020] In some embodiments, phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, dilinoleoylphosphatidylcholine, distearateoylphosphatidylcholine, phosphatidylcholine, or combinations thereof (e.g., a mixture of any two or any three in any ratio).

[0021] In some implementations, the phospholipid is phosphatidylcholine.

[0022] In some embodiments, the mass percentage of lipids is 1% to 40% of the total mass of the liposomes. Preferably, the mass percentage of lipids is 5% to 10% of the total mass of the liposomes.

[0023] In some embodiments, BHB liposomes further comprise a co-emulsifier. Examples of suitable co-emulsifiers include polyethylene glycol 200-2000, glycerol, sorbitol, or combinations thereof (e.g., a mixture of any two or three in any ratio).

[0024] In some implementations, the mass percentage of the co-emulsifier is 1% to 40% (e.g., 5% to 10%) of the total mass of the liposomes.

[0025] In some embodiments, BHB liposomes further comprise stabilizers. Examples of stabilizers include, but are not limited to, medium-chain triglycerides, soybean oil, sunflower oil, or combinations thereof (e.g., a mixture of any two or three in any ratio).

[0026] In some embodiments, the stabilizer is 1% to 40% of the total mass of the liposomes, or preferably 5% to 10%.

[0027] In some embodiments, BHB liposomes further comprise a solvent (e.g., water). The mass percentage of the solvent ranges from 0.01% to 10% of the total mass of the liposomes, or preferably from 0.01% to 1%.

[0028] In some embodiments, the BHB liposomes of the present invention comprise: (a) an active ingredient, including BHB or a salt or ester thereof; (b) a phospholipid; (c) a co-emulsifier; (d) a stabilizer; and (e) water.

[0029] In some embodiments, BHB liposomes comprise (a) 1% to 95% by weight of an active ingredient; (b) 1% to 40% by weight of phospholipids; (c) 1% to 40% by weight of a co-emulsifier; (d) 1% to 40% by weight of a stabilizer; and (e) 0.01% to 10% by weight of water of the total mass of the liposomes.

[0030] In some other embodiments, BHB liposomes comprise (a) 60% to 85% by weight of the active ingredient; (b) 5% to 10% by weight of phospholipids; (c) 5% to 10% by weight of an emulsifier; (d) 5% to 10% by weight of a stabilizer; and (e) 0.01% to 1% by weight of water of the total mass of the liposomes.

[0031] In some embodiments, the active ingredient comprises BHB and optionally one or more other hydrophilic components. Examples of hydrophilic components include, but are not limited to, water-soluble vitamins such as vitamin C, vitamin B1, vitamin B6, folic acid, or any mixture thereof.

[0032] In some embodiments, phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, dilinoleoylphosphatidylcholine, distearateoylphosphatidylcholine, phosphatidylcholine, or combinations thereof.

[0033] In some implementations, the co-emulsifier includes polyethylene glycol 200-2000, glycerin, sorbitol, or combinations thereof.

[0034] In some implementations, stabilizers include medium-chain triglycerides, soybean oil, sunflower oil, or combinations thereof.

[0035] Another aspect of the present invention provides a method for preparing BHB liposomes. Specifically, the method includes preparing a crude BHB suspension.

[0036] In some embodiments, the method for preparing BHB liposomes includes the following steps: melting crystalline BHB; dissolving phospholipids; preparing a crude BHB suspension; and preparing BHB liposomes.

[0037] In some embodiments, melting crystalline BHB involves heating the crystalline BHB at a desired temperature (e.g., 50-80°C) for a period of time (e.g., 10-60 minutes) until it melts into a clear liquid.

[0038] In some embodiments, phospholipid dissolution involves mixing phospholipids and a stabilizer, heating at a desired temperature (e.g., 50-80°C) for a period of time (e.g., 10-60 minutes) to obtain a yellow liquid.

[0039] In some embodiments, the preparation of a crude BHB suspension includes mixing water, a co-emulsifier, and molten BHB liquid, adding a solution of phospholipids and stabilizers from a previous step to the mixture, and mixing it for a period of time (e.g., 5-30 minutes) using a mixing device to obtain a crude BHB suspension.

[0040] In some implementations, the preparation of BHB liposomes includes homogenizing a crude BHB suspension using a liposome preparation device to obtain BHB liposomes.

[0041] In some other embodiments, the method for preparing BHB liposomes includes: melting crystallized BHB; preparing a crude BHB suspension; and preparing BHB liposomes.

[0042] In some embodiments, melting crystalline BHB involves heating the crystalline BHB at a desired temperature (e.g., 50-80°C) for a period of time (e.g., 10-60 minutes) until it melts into a clear liquid.

[0043] In some embodiments, the preparation of a crude BHB suspension involves dissolving molten BHB liquid and phospholipids in an organic solvent, removing the organic solvent by evaporation to form a uniformly spread lipid film, then adding water, a co-emulsifier, and a stabilizer to the lipid film, and dispersing it for a period of time (e.g., 5-30 minutes) using a mixing device to obtain a crude BHB suspension.

[0044] In some implementations, the preparation of BHB liposomes includes homogenizing a crude BHB suspension using a liposome preparation device to obtain BHB liposomes.

[0045] Examples of mixing equipment include, but are not limited to, high-shear emulsifiers and / or high-speed dispersers.

[0046] Examples of liposome preparation equipment include, but are not limited to, nanoliposome extruders, high-pressure homogenizers, high-pressure microfluidic systems, and / or ultrasonic cell disruption.

[0047] In another aspect, the present invention provides a method for increasing or maintaining blood ketone levels in a subject, comprising administering the BHB liposome of the present invention according to the present invention.

[0048] In some implementations, the subjects are humans.

[0049] As used herein, the term “or” is intended to include both “and” and “or”. In other words, the term “or” can also be replaced with “and / or”.

[0050] As used herein, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” are intended to include the plural forms as well. Attached Figure Description

[0051] The following figures are illustrative by way of example and not limitation. For the sake of brevity and clarity, each feature of a given structure is not always labeled in every figure in which the structure appears. The same reference numerals do not necessarily represent the same structure. Conversely, the same reference numerals can be used to represent similar features or features with similar functions, and different reference numerals may also be used.

[0052] Figure 1 The invention illustrates a β-hydroxybutyric acid (BHB) liposome in a conventional vesicle form according to one embodiment of the invention.

[0053] Figure 2 BHB liposomes in reverse vesicle form according to another embodiment of the present invention are shown.

[0054] Figure 3A-3GThe stability of BHB liposomes after long-term storage under refrigerated conditions (5°C) was demonstrated. Peaks labeled with BHB represent the dry content of BHB, while peaks labeled with BHB dimer or BHB trimer represent degradation products and impurities of BHB.

[0055] Figure 4A-4G The stability of BHB liposomes after long-term storage under normal conditions (20°C and 60% relative humidity) was demonstrated. Peaks labeled with BHB represent the dry content of BHB, while peaks labeled with BHB dimer or BHB trimer represent degradation products and impurities of BHB.

[0056] Figure 5 The study showed blood ketone levels at different time points after oral administration of BHB acid or BHB liposomes. Detailed Implementation

[0057] The present invention will now be further described with reference to preferred embodiments thereof. While the invention will be described in conjunction with preferred embodiments, it should be understood that they are not intended to limit the invention to these embodiments. Rather, the invention is intended to cover alternatives, modifications, and equivalents that may be included within the spirit and scope of the invention as defined in the claims. Furthermore, numerous specific details are set forth in the detailed description of the invention to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention may be practiced without these specific details. In other instances, well-known methods, procedures, components, and other features have not been described in detail so as not to unnecessarily obscure aspects of the invention.

[0058] definition

[0059] As used herein, the terms “subject” and “patient” are used interchangeably and, as used herein, refer to any mammal, including but not limited to humans, including human patients or subjects to whom the compositions of the present invention may be administered. The term “mammal” includes human patients and non-human primates, as well as laboratory animals such as rabbits, rats, and mice, and other animals.

[0060] The chemical structure of β-hydroxybutyric acid (also known as 3-hydroxybutyric acid, β-HB, BHB, or beta-hydroxybutyric acid) is shown below.

[0061]

[0062] BHB is a chiral molecule with a 3′ hydroxyl group and has two enantiomers, R / D and S / L.

[0063] The term "amphiphilic" refers to a molecule composed of a water-soluble (hydrophilic) portion and an organic solvent-soluble (lipophilic) portion. Amphiphilic lipids are often the main components of lipid vesicles.

[0064] General

[0065] Generally, various embodiments of the present invention provide formulations of BHB liposomes comprising lipids (e.g., phospholipids), an active ingredient (e.g., β-hydroxybutyric acid (BHB), its physiologically acceptable salts, esters, or mixtures, and optionally one or more additional hydrophilic active ingredients), a co-emulsifier, a stabilizer, and a solvent (e.g., water). In some embodiments, the BHB liposomes of the present invention are made from the following raw materials in parts by weight: (a) active ingredient: 1–95%; (b) phospholipids: 1–40%; (c) co-emulsifier: 1–40%; (d) stabilizer: 1–40%; and (e) water: 0.01–10%.

[0066] In some preferred embodiments, the BHB liposomes of the present invention are preferably made from the following raw materials in parts by weight: (a) active ingredient: 60-85%; (b) phospholipid: 5-10%; (c) co-emulsifier: 5-10%; (d) stabilizer: 5-10%; and (e) water: 0.01-1%.

[0067] The BHB liposomes of the present invention can be in the form of a conventional vesicle structure or an inverted vesicle structure, depending on the proportions of the components. For example... Figure 1 As shown, for a typical vesicle, the membrane consists of one or more phospholipid bilayers with hydrophilic head groups arranged on the periphery. Therefore, the hydrophilic BHB resides within an aqueous core surrounded by a lipid bilayer membrane. Alternatively, as... Figure 2 As shown, BHB liposomes can form reverse vesicles, consisting of a nonpolar core surrounded by a bilayer with hydrophilic head groups arranged internally. Therefore, hydrophilic BHB is embedded in the lipid bilayer membrane and forms part of the membrane.

[0068] The present invention also provides a method for preparing such BHB liposomes, including the preparation of a crude BHB suspension.

[0069] More specifically, an exemplary preparation method according to the present invention includes: (1) melting BHB crystals: heating crystalline BHB free acid at 50-80°C for 10-60 minutes until it melts into a clear liquid; (2) dissolving phospholipids: mixing phospholipids and a stabilizer, heating at 50-80°C for 10-60 minutes to obtain a yellow liquid; (3) preparing a crude BHB suspension: mixing water, a co-emulsifier, and the molten BHB liquid from step 1, adding a solution of phospholipids and a stabilizer from step 2 to the mixture, and continuing to stir with a mixing device for 5-30 minutes to obtain a crude BHB suspension; and (4) preparing BHB liposomes: homogenizing the crude BHB suspension using conventional liposome preparation equipment to obtain BHB liposomes.

[0070] Another exemplary preparation method according to the present invention includes: (1) melting BHB crystals: heating crystalline BHB at 50-80°C for 10-60 minutes until it melts into a clear liquid state; (2) preparation of BHB crude suspension: dissolving the molten BHB liquid from step 1 and phospholipids in an organic solvent, evaporating to remove the organic solvent, forming a uniformly spread lipid film, then adding water, co-emulsifier and stabilizer to the lipid film, dispersing it with a mixing device for 5-30 minutes to obtain BHB crude suspension; (3) preparation of BHB liposomes: homogenizing the BHB crude suspension with conventional liposome preparation equipment to obtain BHB liposomes.

[0071] The following examples illustrate selected embodiments of the invention, but are not intended to limit the scope of the invention.

[0072] Example 1

[0073] The BHB liposome formulation in Example 1 was made from the raw materials in the mass percentages shown in Table 1 below.

[0074] Table 1

[0075]

[0076]

[0077] The above-mentioned BHB liposome formulation was prepared as follows:

[0078] (1) Melting of BHB crystals: Heat 1 kg of crystallized BHB in a 50°C water bath for 60 minutes until it melts into a clear liquid.

[0079] (2) Phospholipid dissolution: Mix 2 kg of phosphatidylcholine and 4 kg of medium-chain fatty acid glycerides, and heat in a 50°C water bath for 60 minutes to obtain a yellow liquid;

[0080] (3) Preparation of BHB crude suspension: Mix 1 kg of water, 2 kg of glycerol and 1 kg of BHB molten liquid, add 2 kg of phosphatidylcholine and 4 kg of medium chain fatty acid glycerides from step 2 as yellow liquid, and stir the mixture for 30 minutes using a high shear emulsifier to obtain BHB crude suspension.

[0081] (4) Preparation of BHB liposomes: Using a liposome extruder system, the crude BHB suspension was passed through a filter with a specified pore size under high pressure (6000 psi) to obtain uniform BHB liposomes, wherein the solid content of BHB was 10%.

[0082] Example 2

[0083] The BHB liposome formulation in Example 2 was made from the raw materials in the mass percentages shown in Table 2 below.

[0084] Table 2

[0085] Formula ingredients Mass (kg) Quality percentage (%) BHB free acid 1.00 10.00 Phosphatidylcholine 2.00 20.00 glycerin 2.00 20.00 Medium-chain fatty acid glycerides 4.00 40.00 water 1.00 10.00

[0086] The above-mentioned BHB liposome formulation was prepared as follows:

[0087] (1) Melting of BHB crystals: Heat 1 kg of crystallized BHB in a 50°C water bath for 60 minutes until it melts into a clear liquid.

[0088] (2) Preparation of crude BHB suspension: 1 kg of molten BHB liquid and 2 kg of phosphatidylcholine were mixed and dissolved in 10 kg of ethanol. The ethanol was evaporated to remove the ethanol and a uniformly spread lipid film was formed. Then, 1 kg of water, 2 kg of glycerol and 4 kg of medium-chain fatty acid glycerides were added to the lipid film and dispersed in a high-shear emulsifier for 30 minutes to obtain crude BHB suspension.

[0089] (3) Preparation of BHB liposomes: BHB crude suspension was passed through a filter with a specified pore size under high pressure (6000 psi) using a liposome extruder system to obtain uniform BHB liposomes, wherein the solid content of BHB was 10%.

[0090] Example 3

[0091] The BHB liposome formulation in Example 3 was made from the raw materials in the mass percentages shown in Table 3 below.

[0092] Table 3

[0093] Formula ingredients Mass (kg) Quality percentage (%) BHB free acid 3.00 30.00 Phosphatidylethanolamine 1.00 10.00 Polyethylene glycol 400 1.00 10.00 soybean oil 4.00 40.00 water 1.00 10.00

[0094] The above-mentioned BHB liposome formulation was prepared as follows:

[0095] (1) BHB crystal melting: Heat 3 kg of crystallized BHB in a 50°C water bath for 60 minutes until it melts into a clear liquid.

[0096] (2) Phospholipid dissolution: Mix 1 kg of phosphatidylethanolamine and 4 kg of soybean oil, and heat in a 50°C water bath for 60 minutes to obtain a yellow liquid;

[0097] (3) Preparation of BHB crude suspension: Mix 1 kg of water, 1 kg of polyethylene glycol 400 and 3 kg of BHB molten liquid, add 1 kg of phosphatidylethanolamine and 4 kg of soybean oil yellow liquid from step 2 to the mixture, disperse the mixture for 30 minutes using a high-speed disperser to obtain BHB crude suspension.

[0098] (4) Preparation of BHB liposomes: The crude BHB suspension was pressurized (6000 psi) using a high-pressure microfluidic system to obtain homogeneous BHB liposomes, in which the solid content of BHB was 30%.

[0099] Example 4

[0100] The BHB liposome formulation in Example 4 was made from the raw materials in the mass percentages shown in Table 4 below.

[0101] Table 4

[0102] Formula ingredients Mass (kg) Quality percentage (%) BHB free acid 5.00 50.00 Phosphatidylserine 1.00 10.00 Sorbitol 1.00 10.00 Sunflower seed oil 2.90 29.00 water 0.10 1.00

[0103] The above-mentioned BHB liposome formulation was prepared as follows:

[0104] (1) BHB crystal melting: Heat 5 kg of crystallized BHB in a 50°C water bath for 60 minutes until it melts into a clear liquid.

[0105] (2) Phospholipid dissolution: Mix 1 kg of phosphatidylserine and 2.9 kg of sunflower seed oil, and heat in a 50°C water bath for 60 minutes to obtain a yellow liquid;

[0106] (3) Preparation of BHB crude suspension: Mix 0.1 kg water, 1 kg sorbitol and 5 kg BHB molten liquid, add 1 kg phosphatidylserine and 2.9 kg sunflower seed oil yellow liquid from step 2 to the mixture, stir the mixture for 30 minutes with a high shear emulsifier to obtain BHB crude suspension;

[0107] (4) Preparation of BHB liposomes: The crude BHB suspension was homogenized twice using a high-pressure homogenizer. The first time the pressure was 35-45 MPa and the second time the pressure was 60-70 MPa to obtain uniform BHB liposomes, in which the solid content of BHB was 50%.

[0108] Example 5

[0109] The BHB liposome formulation in Example 5 was made from the raw materials in the mass percentages shown in Table 5 below.

[0110] Table 5

[0111] Formula ingredients Mass (kg) Quality percentage (%) BHB free acid 5.00 50.00 Phosphatidylserine 1.00 10.00 Sorbitol 1.00 10.00 Sunflower seed oil 2.90 29.00 water 0.10 1.00

[0112] The above-mentioned BHB liposome formulation was prepared as follows:

[0113] (1) BHB crystal melting: Heat 5 kg of crystallized BHB in a 50°C water bath for 60 minutes until it melts into a clear liquid.

[0114] (2) Preparation of crude BHB suspension: 5 kg of molten BHB liquid and 1 kg of phosphatidylcholine were mixed and dissolved in 10 kg of ethanol. The ethanol was evaporated to remove the ethanol and a uniformly spread lipid film was formed. Then, 0.1 kg of water, 1 kg of sorbitol and 2.9 kg of sunflower seed oil were added to the lipid film and dispersed in a high-shear emulsifier for 30 minutes to obtain crude BHB suspension.

[0115] (3) Preparation of BHB liposomes: The crude BHB suspension was homogenized twice using a high-pressure homogenizer. The first time the pressure was 35-45 MPa and the second time the pressure was 60-70 MPa to obtain uniform BHB liposomes, in which the solid content of BHB was 50%.

[0116] Example 6

[0117] The BHB liposome formulation in Example 6 was made from the raw materials in the mass percentages shown in Table 6 below.

[0118] Table 6

[0119] Formula ingredients Mass (kg) Quality percentage (%) BHB free acid 7.00 70.00 Phosphatidylcholine 0.60 6.00 Polyethylene glycol 400 0.60 6.00 Medium-chain fatty acid glycerides 1.74 17.40 water 0.06 0.60

[0120] The above-mentioned BHB liposome formulation was prepared as follows:

[0121] (1) BHB crystal melting: Heat 7 kg of crystallized BHB in an 80°C water bath for 20 minutes until it melts into a clear liquid.

[0122] (2) Phospholipid dissolution: Mix 0.6 kg of phosphatidylcholine and 1.74 kg of medium-chain fatty acid glycerides and heat in an 80°C water bath for 20 minutes to obtain a yellow liquid;

[0123] (3) Preparation of BHB crude suspension: Mix 0.06 kg water, 0.6 kg polyethylene glycol 400 and 7 kg BHB molten liquid, add 0.6 kg phosphatidylcholine and 1.74 kg medium chain fatty acid glycerides from step 2 to the mixture, stir for 30 minutes with a high shear emulsifier to obtain BHB crude suspension;

[0124] (4) Preparation of BHB liposomes: Using a liposome extruder system, the crude BHB suspension was passed through a filter with a specified pore size under high pressure (6000 psi) to obtain uniform BHB liposomes, wherein the solid content of BHB was 70%.

[0125] Example 7

[0126] The BHB liposome formulation in Example 7 was made from the raw materials in the mass percentages shown in Table 7 below.

[0127] Table 7

[0128]

[0129]

[0130] The above-mentioned BHB liposome formulation was prepared as follows:

[0131] (1) Melting of BHB crystals: Heat 8 kg of BHB free acid crystals in an 80°C water bath for 20 minutes until they melt into a clear liquid.

[0132] (2) Preparation of crude BHB suspension: 8 kg of molten BHB liquid and 0.6 kg of phosphatidylinositol were mixed and dissolved in 10 kg of ethanol. The ethanol was evaporated to remove the ethanol and a uniformly spread lipid film was formed. Then, 0.06 kg of water, 0.6 kg of polyethylene glycol 400 and 0.74 kg of soybean oil were added to the lipid film and stirred for 30 minutes with a high-shear emulsifier to obtain crude BHB suspension.

[0133] (3) Preparation of BHB liposomes: BHB crude suspension was passed through a filter with a specified pore size under high pressure (6000 psi) using a liposome extruder system to obtain uniform BHB liposomes with a BHB solid content of 80%.

[0134] Example 8

[0135] The BHB liposome formulation in Example 8 was made from the raw materials in the mass percentages shown in Table 8 below.

[0136] Table 8

[0137] Formula ingredients Mass (kg) Quality percentage (%) BHB free acid 8.00 80.00 Phosphatidylinositol 0.60 6.00 Polyethylene glycol 400 0.60 6.00 soybean oil 0.74 7.40 water 0.06 0.60

[0138] The above-mentioned BHB liposome formulation was prepared as follows:

[0139] (1) Melting of BHB crystals: Heat 8 kg of BHB free acid crystals in an 80°C water bath for 20 minutes until they melt into a clear liquid.

[0140] (2) Phospholipid dissolution: Mix 0.6 kg of phosphatidylinositol with 0.74 kg of soybean oil and heat in an 80°C water bath for 20 minutes to obtain a yellow liquid;

[0141] (3) Preparation of BHB crude suspension: Mix 0.06 kg water, 0.6 kg polyethylene glycol 400 and 8 kg BHB molten liquid, then add 0.6 kg phosphatidylinositol and 0.74 kg soybean oil yellow liquid from step 2 to the mixture, stir for 30 minutes with a high shear emulsifier to obtain BHB crude suspension;

[0142] (4) Preparation of BHB liposomes: Using a liposome extruder system, the crude BHB suspension was passed through a filter with a specified pore size under high pressure (6000 psi) to obtain uniform BHB liposomes, wherein the solid content of BHB was 80%.

[0143] Example 9: Stability analysis of BHB liposomes

[0144] We conducted this study to evaluate the stability of BHB liposomes after long-term storage under normal conditions (20°C and 60% relative humidity) and refrigerated conditions (5°C). This study was performed by measuring BHB liposome samples using HPLC at the initial stage and at weeks 1, 2, 3, and 4. Figures 3 and 4 show the results of the stability analysis. As shown in the figures, peaks labeled BHB represent the dry content of BHB, while peaks labeled BHB dimers or BHB trimers represent degradation products and impurities of BHB.

[0145] Figure 3A-3G The stability of BHB liposomes after long-term storage under refrigerated conditions (5°C) was demonstrated. More specifically, Figure 3A The initial HPLC analysis results of BHB liposomes (week 0) are shown. Figure 3B The HPLC analysis results of BHB liposomes after storage under refrigerated conditions (5°C) for 1 week are shown. Figure 3C The HPLC analysis results of BHB liposomes after storage under refrigerated conditions (5°C) for 2 weeks are shown. Figure 3D The HPLC analysis results of BHB liposomes after 3 weeks of storage under refrigerated conditions (5°C) are shown. Figure 3E The HPLC analysis results of BHB liposomes after 4 weeks of storage under refrigerated conditions (5°C) are shown. Figure 3F The BHB dry content (%) of BHB liposomes stored under refrigeration conditions is shown. Figure 3G The BHB dimer content (%) of BHB liposomes stored under refrigeration conditions is shown (BHB dimer represents the degradation products of BHB).

[0146] Figure 4A-4G The stability of BHB liposomes after long-term storage under normal conditions of 20°C and 60% relative humidity was demonstrated. More specifically, Figure 4A The initial HPLC analysis of BHB liposomes is shown (week 0). Figure 4B The HPLC analysis results of BHB liposomes after 1 week of storage (20℃ / 60%) are shown. Figure 4C The HPLC analysis results of BHB liposomes after 2 weeks of storage (20℃ / 60%) are shown. Figure 4D The HPLC analysis results of BHB liposomes after 3 weeks of storage (20℃ / 60%) are shown. Figure 4EThe HPLC analysis results of BHB liposomes after 4 weeks of storage (20℃ / 60%) are shown. Figure 4F The BHB dry content (%) of BHB liposomes stored under normal conditions is shown. Figure 4G The BHB dimer content (%) of BHB liposomes stored under normal conditions is shown (BHB dimer represents the degradation products of BHB).

[0147] like Figure 3A-3G As shown, the content of BHB liposomes did not change significantly after long-term storage under refrigerated conditions (5℃). Figure 4A-4G As shown, after long-term storage at 20°C and 60% relative humidity, the dry BHB content decreased slightly from 70.1% to 68.8%, and the impurities (expressed as BHB dimers) increased slightly from 3.0% to 4.7%, both within acceptable ranges. Therefore, stability analysis indicates that the BHB liposomes of the present invention exhibit good stability.

[0148] Example 10: Effects of BHB acid and BHB liposomes on blood ketone levels

[0149] The ketogenic potential of BHB acid and BHB liposomes was analyzed using a slightly modified Csilla method (Nutrients. Oct 2019; 11(10):2330).

[0150] Specific pathogen-free (SPF) Investigative Cancer Research Institute (ICR) mice were purchased from Qinglongshan Animal Husbandry Center. All mice were fed a standard diet and kept individually in steel cages in a 24°C room under a standard light-dark cycle (12:12 hour light-dark cycle), with tap water readily available. The mice were fasted for 16 hours before treatment.

[0151] In this treatment, 30 mice were randomly divided into three groups: a control group, a BHB group, and a BHB liposome group. As shown in Table 9, mice were treated orally via tube feeding at a dose of 0.1 ml / 10 g body weight. Mice in the BHB group were treated with a free acid form of R-BHB aqueous solution (once daily) at 9.92 mmol / kg, mice in the BHB liposome group were treated with a liposome form of R-BHB free acid aqueous solution (once daily) at 9.92 mmol / kg, and mice in the control group were treated with water. The pH of both the treatment solution and the water was adjusted to 7 before administration. Blood ketone levels were measured using a ketone meter at 0, 10, 20, 30, 45, 60, 90, 120, and 240 minutes after administration.

[0152] Table 9. Treatment Scheme

[0153]

[0154] Figure 5Table 9 shows the serum ketone levels at different time points after oral administration of BHB acid or BHB liposomes. The highest serum ketone levels in the BHB acid treatment group and the BHB liposome treatment group were 2.8 mmol / L and 4.1 mmol / L, respectively. The highest serum ketone level of R-BHB liposomes was 1.41 times that of R-BHB free acid. The area under the curve for R-BHB liposomes was 1.25 times that of R-BHB free acid; therefore, the bioavailability of BHB liposomes was significantly higher than that of R-BHB. In conclusion, compared with BHB free acid, BHB liposomes showed higher ketogenic potential, better bioavailability, and achieved better nutritional ketosis.

[0155] While specific embodiments and examples of the invention have been described herein, those skilled in the art will understand that any modifications and variations can be made without departing from the principles of the invention. The above embodiments and descriptions do not limit the scope of the invention. Any combination of embodiments of the invention, as well as any obvious extensions or analogies thereof, are within the scope of the invention. Furthermore, the invention covers any arrangement intended to achieve the same purpose, and all such variations and modifications falling within the scope of the appended claims.

[0156] Unless otherwise expressly stated, all features disclosed in this specification (including any appended claims, abstract, and drawings) may be replaced by alternative features for the same, equivalent, or similar purposes. Therefore, unless otherwise expressly stated, each disclosed feature is an example of a series of equivalent or similar features.

Claims

1. A liposome comprising an active ingredient; and a membrane, or an inner membrane and an outer membrane, characterized in that, Each of the membranes, or the inner and outer membranes, defines an internal space within the membrane or the inner membrane and / or between the inner and outer membranes. Each membrane contains a plurality of lipid molecules, and the active ingredient is trapped within the internal space or embedded in the membrane. The active ingredient comprises β-hydroxybutyric acid (BHB) and a physiologically acceptable salt thereof, and the active ingredient constitutes 10% to 80% of the total mass of the liposomes by mass. The lipid is a phospholipid, and the phospholipid constitutes 6% to 20% of the total mass of the liposomes by mass, and the phospholipid is phosphatidylcholine. The liposomes contain phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol; the liposomes further contain a co-emulsifier, the co-emulsifier being 6% to 20% of the total mass of the liposomes by mass, and the co-emulsifier being polyethylene glycol 400, glycerol, or sorbitol; the liposomes further contain a stabilizer, the stabilizer being 7.4% to 40% of the total mass of the liposomes by mass, and the stabilizer being medium-chain triglycerides, soybean oil, or sunflower oil; the liposomes further contain water, the water being 0.6% to 10% of the total mass of the liposomes by mass.

2. The liposomes according to claim 1, characterized in that, The active ingredient is BHB in the form of a free acid.

3. The liposomes according to claim 1, characterized in that, The BHB is in R-form, S-form, or a mixture of R- and S-forms.

4. The liposomes according to claim 1, characterized in that, The liposomes have a typical vesicle structure, and the active ingredient is trapped within the internal space defined by the membrane or the inner membrane.

5. The liposomes according to claim 1, characterized in that, Each membrane is a double membrane.

6. The liposomes according to claim 1, characterized in that, Each liposome contains a membrane.

7. The liposomes according to claim 6, characterized in that, The liposomes have an inverted vesicle structure, and the active ingredient is embedded in the membrane and forms part of the membrane.

8. The liposomes according to claim 1, characterized in that, The active ingredient further comprises one or more additional hydrophilic components.

9. The liposomes according to claim 8, characterized in that, The other hydrophilic component is one or more water-soluble vitamins.

10. The liposomes according to claim 9, characterized in that, The water-soluble vitamins include vitamin C, vitamin B1, vitamin B6, folic acid, or any mixture thereof.

11. A method for preparing liposomes as described in any one of claims 1 to 10, characterized in that, The method includes preparing a crude suspension of BHB.

12. The method according to claim 11, characterized in that, The method includes the following steps: melting crystalline BHB; dissolving phospholipids; preparing a crude BHB suspension; and preparing BHB liposomes.

13. The method according to claim 12, characterized in that, The step of melting crystalline BHB includes heating the crystalline BHB at 50-80°C for 10-60 minutes until the BHB melts into a clear liquid.

14. The method according to claim 12, characterized in that, The phospholipid dissolution step includes mixing the phospholipid and the stabilizer together, and heating the mixture of phospholipid and stabilizer at 50-80°C for 10-60 minutes to obtain a yellow liquid.

15. The method according to claim 12, characterized in that, The BHB coarse suspension preparation step includes mixing water, a co-emulsifier, and molten BHB from claim 13 together, then adding the yellow liquid obtained from claim 14 to the mixture, and stirring the mixture for 5-30 minutes using a mixing device to obtain the BHB coarse suspension.

16. The method according to claim 11, characterized in that, BHB liposome preparation involves homogenizing the crude BHB suspension using a liposome preparation device to obtain BHB liposomes.

17. The method according to claim 11, characterized in that, The method includes the following steps: melting crystalline BHB; preparing a crude BHB suspension; and preparing BHB liposomes.

18. The method according to claim 17, characterized in that, The step of melting crystalline BHB includes heating the crystalline BHB at 50-80°C for 10-60 minutes until the BHB melts into a clear liquid.

19. The method according to claim 17, characterized in that, The preparation of the crude BHB suspension includes dissolving molten BHB and phospholipids in an organic solvent, evaporating the organic solvent to remove it, forming a uniformly spread lipid film, then adding water, a co-emulsifier, and a stabilizer to the lipid film, and dispersing it for 5-30 minutes using a mixing device to obtain the crude BHB suspension.

20. The method according to claim 17, characterized in that, BHB liposome preparation involves homogenizing the crude BHB suspension using a liposome preparation device to obtain BHB liposomes.

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