WO2021177829A1

WO2021177829A1 - Cartridges containing lipid formulations for health ingredient-containing hot drinks

Info

Publication number: WO2021177829A1
Application number: PCT/NL2021/050149
Authority: WO
Inventors: Josbert Maarten Metselaar; Grzegorz PAWLIK; Martino AMBROSINI
Original assignee: Liposoma Health B.V.
Priority date: 2020-03-04
Filing date: 2021-03-04
Publication date: 2021-09-10

Abstract

The invention relates to sealed cartridges for preparation of a phospholipid dispersion, wherein the cartridge is formed of a material which is water-impermeable or has at least one water-impermeable layer, and the cartridge forms a sealed chamber which encapsulates a mixture comprising one or more phospholipids, one or more surfactants, one or more lipophilic biologically active ingredients, and one or more liquid nontoxic polyols, monohydric alcohols and/or polyhydric alcohols, the cartridge further comprising an inlet port configured to allow a heated and pressurized liquid to be injected into the sealed chamber to form the phospholipid dispersion and an outlet port configured to allow the prepared phospholipid dispersion to exit the sealed chamber; to kit of parts comprising such cartridges and to methods for preparing liposome formulations using such cartridges.

Description

Title: CARTRIDGES CONTAINING LIPID FORMULATIONS FOR HEALTH INGREDIENT-CONTAINING HOT DRINKS

Field of the invention

The invention relates to formulations comprising mixtures of poorly water- soluble, lipophilic biological active substances and liposome forming phospholipids, suitable for encapsulation into a small cartridge that is compatible with - and can be extracted under pressure with warm water by - several existing coffee extraction systems so as to prepare a high quality uniformly reproducible fluid comestible product that contains a phospholipid dispersion of the biological active, which can improve the intestinal uptake after oral administration.

Background of the invention

Many poorly water soluble lipophilic biologically active compounds suffer from limited absorption in the gastrointestinal tract after oral administration, which leads to low bioavailability and suboptimal efficacy in the body. Liposomes are phospholipid bilayer vesicles that have proven ability to improve intestinal absorption by enhancing interaction with the enterocytes or the M-cells located in the intestinal wall, by helping the active compounds to remain dispersed in the gastrointestinal fluid, or by protecting the actives from premature degradation.

Successfully formulating a stable liposomal product of a poorly water soluble lipophilic biologically active substance depends on the way the active interacts with the phospholipid bilayer. Most lipophilic actives significantly disturb the way the phospholipids are arranged and cause the liposomes to destabilize, aggregate or disintegrate lipophilic substances can successfully incorporated in liposomes up to 5 to 10% of the total molar lipid quantity.

However, the required dose of a range of lipophilic actives is much higher than a feasible marketable liposomal product can accommodate for. Substances that need to be given in a daily dose range of around 0.5 to 1 gram would need around 10 gram of phospholipid for stable incorporation, which then is to be sold as 100 mL liquid in bottles for daily use or 20 grams freeze-dried powder in daily sachets. These products will be too bulky to handle and too costly to be marketed. When lower amounts of phospholipid are used to formulate the required daily dose of these agents, liposomes may still form but they are unstable and the formulation falls apart within hours after preparation.

Formulations are known in the art - referred to as ‘prodiposomes’ which comprise phospholipid mixtures in alcohol or other water-miscible solvents and upon exposure to an external excess of water or aqueous buffer form liposomal products. The basic composition of pro-liposomes are outlined in US patent US5004611A. This patent describes a composition, which spontaneously forms vesicles or liposomes in the presence of excess water, the composition comprising a uniform mixture of:

(a) at least one membrane lipid,

(b) at least one water-miscible organic liquid which is a solvent for the lipid, and up to 40% by weight of water, the proportion by weight of (a) to (b) being from 40: 1 to 1:20.

US5004611A also includes a method of forming an aqueous dispersion of liposomes, which method comprises mixing the dilutable pro-liposome composition with excess water. The resulting aqueous liposome dispersion comprise liposomes formed of membrane lipid which have diameters in the range of about 0.1 to 2.5 microns and contain at least 2 ml of entrapped aqueous fluid per gram of the lipid.

The limitation of this technology is, however, that the production of a liposomal dispersion out of these mixtures is achieved by gradual addition of water and vigorous agitation combined with prolonged incubation as shown by the examples in this patent. Very often small amounts of water need to be present in the pro liposome mixture beforehand to facilitate the formation of liposomes later upon exposure to water. Such a preparation method is not easy, quick and practical enough for daily use by a patient.

EP 3 216 443 A1 describes a beverage preparation capsule containing a solubilisate of a poorly water-soluble pharmaceutically active agent or dietary supplement. The solubilisate is defined as the composition of the substance to be solubilized, a solvent and a solubilizing agent and is characterized by the substantially complete solubilization of the substance. Hence, a solubilisate as described in EP 3 216 443 A1 is a thermodynamically stable mixture. The solubilisate of EP 3 216 443 remains the same during and after extraction, meaning that the extracted beverage contains the same solubilisate, and the extraction process is essentially a dilution of the formulation. E.g. in case of a liposomal solubilisate such as in examples 7 and 8, the liposomes already need to be present in the capsule. The limitation of this technology is, however, that having a liposomal solubilisate already in the beverage preparation capsule strongly limits the capacity to hold active ingredients. As the examples on liposomal solubilisates listed in D1 show, the active ingredients are roughly in the rather low range of 0.01 - 0.1% weight % of the total solubilisate in the capsule, meaning that the concentration in the beverage will be even lower.

Thus, there is a need for phospholipid mixtures accommodating high percentages and thus high doses of lipophilic actives that are packed and processed in a way that in one quick and easy step a homogenous liposomal dispersion is formed that can be orally administered right after preparation.

Summary of the invention

It is an object of the present invention to provide a phospholipid mixture with one or more lipophilic bioactive substances that can be prepared into a liposome dispersion for oral intake by simple preparation without laboratory equipment.

The invention therefore provides a sealed cartridge for preparation of a phospholipid dispersion, wherein the cartridge is formed of a material which is water-impermeable or has at least one water-impermeable layer, and the cartridge forms a sealed chamber which encapsules a mixture comprising one or more phospholipids, one or more surfactants, one or more lipophilic biologically active ingredients, and one or more liquid nontoxic monohydric or polyhydric alcohols, the cartridge further comprising an inlet port configured to allow a heated and pressurized liquid to be injected into the sealed chamber to form the phospholipid dispersion and an outlet port configured to allow the prepared phospholipid dispersion to exit the sealed chamber.

Such a cartridge allows simple preparation of a phospholipid dispersion using for example common coffee extractor household apparatuses that provide the heating and pressurized liquid. In a preferred embodiment, the one or more phospholipids are dispersed in said one or more liquid nontoxic monohydric or polyhydric alcohols, the dispersion being a suspension, a colloidal mixture and/or an emulsion

The phospholipid dispersion may be formed by heated and pressurized water in a pressure range of from 2 bar to 20 bar and with a temperature between 25 and 100 degrees Celsius.

The phospholipid dispersion that is formed may preferably be homogeneous with a particle size of below 1 pm, more preferably below 500 nm, more preferably between 50 and 500 nm, and a polydispersity index of below 0.5.

The cartridge may comprise a cup forming a chamber with an opening and a cup cover sealed to a lip of the cup wherein the cover extends across the opening to the lip and is sealed to the lip so that mixture is contained within the cup by the cover free from leaking, wherein the cover has a thickness and a breaking stress so that upon placement of the sealed cartridge in an apparatus for brewing a beverage under pressure. The sealed cartridge may be positioned in a holder of the apparatus in a way that the apparatus can pierce openings in the inlet port at a back of the cup and in the outlet port at the cover, which allows the flow of heated and pressurized liquid into the cup through the inlet port and the flow of prepared phospholipid dispersion through the outlet port.

The cartridge may be made of plastic or aluminium or any other airtight material.

Said one or more phospholipids, one or more surfactants and one or more lipophilic active ingredients may be dissolved and/or dispersed in said one or more liquid nontoxic monohydric or polyhydric alcohols.

Preferably, the mixture is a liquid, viscous liquid or paste.

In a further aspect, the invention provides a kit of part comprising a plurality of the above described cartridges.

In a further aspect, the invention provides a method for preparing a liposome formulation comprising adding an aqueous liquid having a temperature of at least 25 °C under pressure of at least 2 bar to a cartridge described above.

Said aqueous liquid may preferably be water. Further features and advantages of the invention are described in the following description and figures on which it is based. The figures show:

Figure 1 shows a cartridge according to an embodiment of the invention;

Figure 2 shows the cartridge of figure 1 in a section view; and

Figure 3 shows the cartridge of figure 1 in a corresponding apparatus in a section view;

Figure 4 shows examples of cartridges filled with formulations with different active ingredients.

Detailed description

The invention pertains to a sealed cartridge, which contains a formulation comprising phospholipids and a full dose unit of one or more lipophilic health ingredients dissolved in alcoholic solvent as a paste, which - upon extraction with warm water under pressure - forms a homogeneously dispersed phospholipid emulsion of the lipophilic active ingredient(s) that can be served as a drink in a cup or a glass. This emulsion remains stable without precipitation or phase separation until at least one hour after preparation. Within this timeframe the drink should be fully consumed to ensure the bioactive ingredient(s) remain molecularly dispersed or dissolved in the lipid matrix while passing through the gastrointestinal tract, so that maximal bioavailability and uptake are achieved.

The invention is directed to formulations that are encapsulated in a cartridge and that are mixtures of phospholipids and lipophilic biological active ingredients dissolved or dispersed in one or more liquid nontoxic polyhydric alcohols, in particular in the form of a suspension, a colloidal mixture or an emulsion that is not yet a liposomal solubilisate, and that can easily be filled into these cartridges. The formulations are suitable for hot water extraction in coffee extraction devices as present on the market. Upon extraction a stable homogenous fine phospholipid dispersion is formed of the formulated biologically active ingredient. The dispersion can be consumed as a warm beverage to ensure full stability of the dispersion also in the gastrointestinal tract. As shown in de examples the formulations to be incorporated in the cartridges can successfully be made with bioactive substances or mixtures thereof that are known to be lipophilic and poorly absorbable by the human body, including bioactive fatty acid esters and amides, cannabinoids, vitamins and provitamins, polyphenols and flavonoids, and essential oil components. When processed according to the methods as described in the art these formulations do not lead to a homogeneous liposome dispersion, whereas encapsulation in the cartridge and subsequent hot-water preparation in a coffee machine generates a smooth liposome dispersion without clearly visible aggregates, precipitates or other large scale inhomogeneities for at least a period of one hour after preparation.

The mixture preferably is a paste, liquid or viscous liquid. The mixture is preferably in the form of a solution, or dispersion, or a combination thereof. In one embodiment it is comprised of a fully dissolved mixture of the one or more lipophilic bioactive ingredients, the one or more phospholipids and the one or more surfactants. Preferably, at least part of said one or more phospholipids are dispersed in said one or more liquid nontoxic monohydric or polyhydric alcohols, the dispersion being a suspension, a colloidal mixture and/or an emulsion. The mixture present in the cartridge can also be a mixture of a solution and a suspension, a colloidal mixture and/or an emulsion. Hence, in a preferred embodiment, the mixture present in the cartridge is a suspension, a colloidal mixture and/or an emulsion or a combination of a solution and a suspension, a colloidal mixture and/or an emulsion. It is further preferred that less than 25% of the phospholipids are present in the mixture in the form of liposomes with a mean particle diameter of less than 1 pm, preferably less than 20%, more preferably less than 15%, more preferably less than 10%, more preferably less than 5%. I.e. preferably 75% or more of the one or more phospholipids is present in the form of a suspension, colloidal mixture and/or emulsion, preferably a suspension or colloidal mixture, preferably 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more of the one or more phospholipids is present in the form of a suspension, colloidal mixture or emulsion, preferably a suspension or colloidal mixture. The mixture is not a solubilisate. Preferably, the mixture comprises at least two different surfactants, one with a relatively high HLB and one with a relatively low HLB in a mixture of one or more polyol, monohydric or polyhydric nontoxic alcohols or a mixture thereof. The mixture in the cartridge may further comprise further ingredients such as agents that enhance taste, colour, viscosity, smell and/or chemical stability of the main ingredients.

The singular forms “a”, “an”, and “the” as used herein include plural referents unless the context clearly dictates otherwise . Thus, for example, reference to “a lipophilic compound” includes reference to one or more of such compounds.

“Liposomes” as used herein refers to vesicular structures that are comprised of one or more lipid bilayer membranes that enclose an aqueous compartment. The bilayer membranes are composed of two layers of lipids; an inner layer and an outer layer. The outer layer of the lipid molecules are oriented with their hydrophilic head portions towards the aqueous environment and their hydrophobic tails pointed downward toward the interior of the bilayer. Unilamellar liposomes feature one lipid bilayer membrane whereas multilamellar liposomes feature more than one lipid bilayer membrane, which membranes can enclose one or more than one closed aqueous compartment. The membranes are concentrically arranged so that the different membranes are separated by aqueous compartments, much like the structure of an onion. Mean diameter of liposomes, such as in the mixture in the cartridge of the present invention or in the prepared beverage, can be measured by electron microscopy, as is known to a person skilled in the art. Hence, sizes of liposomes as used herein refer to the mean diameter as determined using electron microscopy.

The term “cartridge” as used herein refers to a container suitable for storing the mixture as well as for preparation of the phospholipid dispersion by injecting heated and pressurized liquid into the cartridge containing the mixture. The cartridge may be compatible for use in available hot beverage preparation machines, in particular machines for single-serve coffee capsules.

The term “dispersion” as used herein refers to a mixture of two or more substances that are not soluble in each other and wherein one is present as dispersed droplets, particles, aggregates, colloids, or vesicles in the other substance that is then present in the form of a liquid. Before preparation the dispersion is present in the cartridge as a mixture of a solution and a suspension, a colloidal suspension and/or an emulsion with no majority of liposomes already being present. As used herein, “suspension” refers to a mixture of solid particles in a liquid or viscous phase. A “colloidal mixture” is distinguished from a suspension in that the particle size is generally smaller in the colloidal mixture, typically less than 10 pm. “Emulsion” as used herein refers to a mixture of fat particles in a liquid or viscous phase, which is thermodynamically unstable. As is well known to a person skilled in the art, suspensions, colloidal mixtures and emulsion are thermodynamically unstable in the short or long term. Preferably less than 25% of the phospholipids are present in the mixture in the form of liposomes with a mean particle diameter of less than 1 pm, preferably less than 20%, more preferably less than 15%, more preferably less than 10%, more preferably less than 5%.

After preparation with liquid under pressure and at elevated temperature the formulation is present in the form of a phospholipid dispersion, preferably a liposomal phospholipid dispersion. In this liposomal phospholipid dispersion is further preferred more than 50% of the one or more phospholipids are in the form of liposomes with a mean particle diameter of less than 1 pm, preferably more than 60%, more preferably more than 70%, preferably more than 80%. The phospholipid dispersion, preferably a liposomal phospholipid dispersion, that is formed is preferably homogeneous. It is further preferred that the liposomes have a mean particle diameter of below 1 pm, 500 nm, more preferably between 50 and 500 nm and a polydispersity index of below 0.5.

The term “stable” as used herein refers to the beverage remaining fully dispersed without visible flocculation, aggregation, precipitation, frothing or phase separation when kept, without agitation, for at least one hour after preparation.

The term “viscous” is used herein according to its art-recognized meaning and can be read as having a viscosity higher than that of water at the same temperature.

The terms “biologically active” and “bioactive” are used interchangeably herein. The terms refer to any natural or synthetic material that causes a biological response in living tissue or cell.

The term “paste” as used herein refers to a material that behaves as a soft solid with a firm consistency and without the clear properties of a liquid. The term “ HLB ” as used herein refers to a hydrophilic - lipophilic balance of a surface - active material ( e.g. , surfactant, emulsifier ) . An HLB value of 0 corresponds to a completely hydrophobic (i.e. , lipophilic) molecule , and a value of 20 corresponds to a completely hydrophilic (i.e., lipophobic ) molecule . The surface- active material having an HLB value of less than 10 is insoluble in water; whereas the surface -active material having an HLB value of more than 10 is soluble in water. A “high HLB” refers to an HLB of at least 10. A “low HLB” refers to a HLB of at most 10.

The mixture is preferably a paste, liquid or viscous liquid that is comprised of a mixture of the lipophilic bioactive ingredient(s), one or more phospholipids and one or more polyols, and/or monohydric or polyhydric nontoxic alcohols. More preferably, the mixture is a viscous liquid or paste. The one or more phospholipids, one or more surfactants and one or more lipophilic active ingredients are preferably dissolved and/or dispersed in said one or more nontoxic polyols, monohydric and/or polyhydric alcohols.

In the cartridge a total quantity between 1 gram and 20 gram can preferably be incorporated also depending in particular on the type of cartridge, e.g. if it is to be compatible with particular single-serve coffee capsule systems. The cartridge preferably has a content of between 5 and 20 ml, more preferably between 5 and 20 ml or between 10 and 20 ml.

The formulation may further comprise ingredients that enhance taste, color, viscosity, smell or chemical stability of the ingredients.

The bioactive ingredient is a lipophilic substance that is normally poorly soluble in water at room temperature, while it is well soluble in the polyol, monohydric or polyhydric alcohol or a mixture thereof, or in the matrix that comprises the polyol, monohydric or polyhydric alcohol or a mixture thereof and the phospholipids and surfactants. The bioactive ingredient may thus be a mixture of several lipophilic substances. With the term “lipophilic” is meant a log octanol/water value (log P value) of at least 0. The mixture may comprise more than one biologically active ingredient. The total amount of lipophilic bioactive ingredients present in the mixture is preferably at least 0.5 weight % in the mixture and preferably below 20 weight % of the total mixture in the cartridge. Preferably, the total amount of lipophilic bioactive ingredients present in the mixture is 1-20 weight% of the total mixture in the cartridge, more preferably 2-20 weight%, more preferably 2-10 weight%, more preferably 2-7 weight%, such as 3-15 weight%, 3-10 weight% or 4-10 weight% and 2-6 weight%. For instance, about 1%, about 2% or about 5% of the lipophilic bioactive ingredients present in the mixture.

The bioactive ingredients are preferably present in an amount sufficient to exert their bioactive effect.

In preferred embodiments the bioactive substance is a lipophilic vitamin or provitamin, a polyphenol or flavonoid substance, a endocannabinoid structure, a cannabinoid compound, a fatty acid neurotransmitter, an etheric or essential oil, a terpene derivative, or a saturated or polyunsaturated fatty acid or its chemical derivative, such as an ester or salt thereof, or combinations thereof.

In specific embodiments the active ingredient is a endocannabinoid substance from the N-acyl ethanolamine (NAEs) class, or the monoacylglycerol class or mixtures thereof.

In specific embodiments the active ingredient is an aliphatic (poly) alcohol ester or amide of a polyunsaturated fatty acid (omega 3, 6 or 9), or mixtures thereof.

In specific embodiments the active ingredient is a cannabinoid substance from the group of THC (tetrahydrocannabinol), THCA (tetrahydrocannabinolic acid), CBD (cannabidiol), CBDA (cannabidiolic acid), CBN (cannabinol), CBG (cannabigerol), CBC (cannabichromene), CBL (cannabicyclol)

CBV (cannabivarin), THCV (tetrahydrocannabivarin), CBDV (cannabidivarin)

CBCV (cannabichromevarin). CBGV (cannabigerovarin), CBGM (cannabigerol monomethyl ether), CBE (cannabielsoin), CBT (cannabicitran).

In specific embodiments the active ingredient is a hydrophobic vitamin, a hydrophobic provitamin, or a hydrophobic derivative of a hydrophilic vitamin.

In specific embodiments the active ingredient is a polyphenol substance from the group of plant flavonoids, stilbenoids, phenylethyl acids or their derivatives, or mixtures thereof. In specific embodiments the active ingredient is an essential oil from with a chemical structure based on plant terpenes or terpenoids.

Examples of cannabinoids: THC (tetrahydrocannabinol), THCA (tetrahydrocannabinolic acid), CBD (cannabidiol), CBDA (cannabidiolic acid), CBN (cannabinol), CBG (cannabigerol), CBC (cannabichromene), CBL (cannabicyclol)

CBV (cannabivarin), THCV (tetrahydrocannabivarin), CBDV (cannabidivarin)

Examples of vitamins and provitamins: vitamin A variants, derivatives and provitamins (including beta-carotene), the different vitamins B and derivatives, vitamin C and derivatives, vitamin D variants, vitamin E variants, vitamin K1 and K2 and derivatives, coenzyme Q10 and related quinones, NADH and NADP and variants.

Examples of plant polyphenol classes:

Flavonoids including their glycoside derivatives: Flavones (Apigenin, Luteolin), Flavonols (Isorhamnetin, Kaempferol, Myricetin), Quercetin, Flavanones (Hesperetin, Naringenin) Flavanols and their polymers (Catechin, Gallocatechin and their corresponding gallate esters Epicatechin, Epigallocatechin and their corresponding gallate esters, Theaflavin its gallate esters), Isoflavones (Genistein, Glycitein), Stilbenoids (Resveratrol, Pterostilbene), Anthocyanins (Cyanidin, Malvidin), Phenolic acids and their esters (Caffeic acid and esters, Cinnamic acid and esters, Ellagic acid and esters, Ferulic acid and esters, Gallic acid and esters), Salicylic acid and esters, Curcumin and related compounds, Flavonolignans (silymarin), Eugenol, Capsaicin, Alpha-lipoic acid.

Examples of carotenoid terpenoids: Alpha-carotene, Astaxanthin, Beta- carotene, Canthaxanthin, Cryptoxanthin, Lutein, Lycopene, Zeaxanthin

Examples of essential oil components from plants with monoterpene, diterpene or sequiterpene chemical structures: including but not limited to carvacrol, eugenol, nerolidol, thymol, beta-caryophyllene, humulene, linalool, menthol, geraniol, borneol, sabine ne. The polyol, monohydric or polyhydric alcohol phase constitutes between 20 % and 60% of the total mixture by weight. The polyol, monohydric or polyhydric alcohols or mixtures thereof have an aliphatic structure with a melting point of the mixture below 80 degrees Celsius. A preferred example of a polyol is polyethylene glycol), preferably of a molecular weight between 100 and 1000 dalton. Preferred examples of monohydric alcohols are ethanol, 2-propanol and tertiary butanol. Preferred examples of polyhydric alcohols are propylene glycol, 1,3 propane diol, glycerol, the several isomers that are available of butylene glycol, erythritol and other polyol sugar alcohols. An even more preferred example of the polyol, monohydric or polyhydric alcohol phase is a mixture of polyethylene glycol) and propylene glycol in a ratio of 1:1 and 1:5.

The weight ratio of the phospholipid fraction to the polyol, monohydric or polyhydric alcohol or the mixture thereof is preferably between 1:1 and 1:3. The phospholipid fraction can be a single phospholipid or a mixture of different phospholipids. Phospholipids that can be used in this invention are phospholipids known in the art for the production of liposomes, in particular amphipathic negatively charged or non-charged vesicle -forming phospholipids. The phospholipids can comprise saturated or unsaturated fatty acid side chains and feature a polar headgroup based on choline, glycerol, ethanolamine or phosphatidic acid. Alternative phospholipids of similar amphipathic vesicle-forming nature can also be included in the formulation. Non-limiting examples of suitable phospholipids are fat-free soy lecithin, sunflower lecithin, egg yolk lecithin, rapeseed lecithin, the (partially) hydrogenated versions thereof and synthetic phospholipids such as dimyristoyl phosphatidyl choline, dipalmitoyl phosphatidyl choline and distearoyl phosphatidyl choline.

The one or more surfactants preferably comprises an ionic amphiphilic molecule or a nonionic surfactant. In one preferred embodiment, the one or more surfactants preferably have a relatively high HLB, which is required to further improve consistency, texture or homogeneity, and are preferably present in a weight ratio to the one or more phospholipids together of between 1:20 and 1:1, preferably between 1:10 and 1:1.5. In another embodiment, the one or more surfactants are preferably present in a weight ratio to the one or more phospholipids of between 1:20 and 1:5. This surfactant preferably has a hydrophilicdipophilic balance (HLB) value of more than 10. This surfactant can be chosen from the well-known polysorbate emulsifiers in the art, which are PEG sorbitan fatty acid esters that include, but not are limited to, PEG-20 sorbitan monolaurate having an HLB value of 17 (e.g. TWEEN 20 from Croda , Inc.) PEG-20 sorbitan monopalmitate having an HLB value of 16 (e.g. TWEEN 40 from Croda , Inc.) ; PEG-20 sorbitan monostearate having an HLB value of 15 (e.g. TWEEN 60 from Croda , Inc.); and PEG-20 sorbitan monooleate having an HLB value of 15 (e.g. TWEEN 80 from Croda, Inc.), or combinations thereof. Alternatively, the one or more surfactant is selected from the group consisting of polyol- derivatized fat- soluble substances, which include PEGylated fatty acids, PEGylated lipids and tocopherol polyethylene glycol succinate (TPGS). In certain embodiments the surfactant is a chemical construct of a polyol derivative with fatty acids and polyethylene glycol or a block copolymer of polyethylene glycol and a hydrophobic polymer building block such as polypropylene glycol.

In another preferred embodiment the mixture comprises at least two surfactants, at least one surfactant with a relatively low HLB (herein referred to as “first surfactant”) and at least one surfactant with a relatively high HLB (herein referred to as “second surfactant”). The first surfactant serves to enable the stable phospholipid dispersion to form upon heated water extraction and the second surfactant serves to further improve consistency, texture or homogeneity of the formulation.

The at least one first surfactant, with a relatively low HLB and which enables the stable phospholipid dispersion to form upon heated water extraction, is preferably present in a weight ratio to the one or more phospholipids in the mixture of between 1:3 and 1:10, such as 1:4 and 1:8. This surfactant can be an ionic amphiphilic molecule or a nonionic surfactant and must have a hydrophilic- lipophilic balance value (HLB value) below 15. It is preferably made of a fatty acid ester of a hydrophilic sugar, polyol, amino acid or vitamin. Fatty acids in these esters can be saturated such as stearic acid, palmitic acid, lauric acid or unsaturated such as oleic acid. Also polyunsaturated fatty acids are a possibility. The hydrophilic moiety can for instance be sorbitan, glycerol, or vitamin C (ascorbic acid). Preferred examples of suitable surfactants are ascorbyl palmitate, sorbitan oleate, other sorbitan fatty acid derivatives and fatty acid derivatives of sugars such as the sucrose fatty acid esters, of which sucrose mono- or di-laurate, - palmitate and -stearate are examples.

In certain embodiments the first surfactant is a fatty acid ester of a hydrophilic substance that has reported bioactivity or can stimulate the therapeutic efficacy of active ingredient(s) in the formulation. A prime example is ascorbyl palmitate, which is converted in the body into vitamin C with well-known biologic activity.

The first surfactant can be a single surfactant or a combination of surfactants as described herein above.

The at least one second surfactant, with a relatively high HLB and which is required to further improve consistency, texture or homogeneity, is preferably present in a weight ratio to the one or more phospholipids together of between 1:20 and 1:1, preferably between 1:10 and 1:1.5. In another embodiment, the one or more second surfactants are preferably present in a weight ratio to the one or more phospholipids of between 1:20 and 1:5. This surfactant preferably has a hydrophilic-lipophilic balance (HLB) value of more than 10. This surfactant can be chosen from the well-known polysorbate emulsifiers in the art, which are PEG sorbitan fatty acid esters that include, but not are limited to, PEG-20 sorbitan monolaurate having an HLB value of 17 (e.g. TWEEN 20 from Croda , Inc.) PEG-20 sorbitan monopalmitate having an HLB value of 16 (e.g. TWEEN 40 from Croda , Inc.) ; PEG-20 sorbitan monostearate having an HLB value of 15 (e.g. TWEEN 60 from Croda , Inc.); and PEG-20 sorbitan monooleate having an HLB value of 15 (e.g. TWEEN 80 from Croda, Inc.), or combinations thereof. Alternatively, the second surfactant is selected from the group consisting of polyol-derivatized fat- soluble substances, which include PEGylated fatty acids, PEGylated lipids and tocopherol polyethylene glycol succinate (TPGS).

In certain embodiments the second surfactant is a lysophospholipid derived from hydrolyzed lecithin or synthetically synthesized.

In certain embodiments the second surfactant is a chemical construct of a polyol derivative with fatty acids and polyethylene glycol or a block copolymer of polyethylene glycol and a hydrophobic polymer building block such as polypropylene glycol.

Optionally additional surfactants can be added having an HLB value of less than 10.

Optionally, the mixture in the cartridge may contain one or more weak acids such as ascorbic acid and citric acid, or salts thereof. These agents appear to slightly further improve the consistency and texture of the liposome dispersion after preparation.

The formulation may further comprise triglycerides. Some triglycerides have bioactivity and thus complement the health benefit profile of the formulation. Examples of triglycerides are vegetable oils from soy, sunflower, hempseed, rosehip seed, pomegranate seed, linseed, sea buckthorn seed, raspberry seed and other berry seeds that are known to have health promoting effects.

The formulation may further comprise ingredients that enhance taste, color, viscosity, smell or chemical stability of the ingredients. Examples of additional ingredients are sweeteners including sugars or polyols, salts, minerals including carbonates or bicarbonates, coloring agents, etheric oils or other scent and taste enhancers, including coffee, tea, ginger, etc. Also substances can be added to the formulation that upon exposure to water form carbon dioxide gas, so as to impart the sensation of effervescence to the beverage. Examples are citric acid and sodium bicarbonate, which may be added separate from each other to avoid a premature reaction.

Optionally the mixtures are placed in the cartridges under a protective atmosphere of inert gases, examples of which are nitrogen and argon. To further improve stability the mixture is optionally pretreated with inert gases or mixtures thereof during mixing, optionally to the extent the resulting mixture gets a foamy texture. A cartridge according to an embodiment of the invention and a corresn on ding- apparatus are described in Figures 1 to 3.

Figure 1 shows a side view of the cartridge 10. This particular embodiment of the cartridge 10 has a rigid capsule part formed as a cup 12 with an opening disposed on the bottom side of the cup 12 as shown in Figure 1. The opening of the cup 12 is sealed by a cover 14 which extends across the opening of the cup and is sealed to a lip 16 of the cup 12 which extends around the circumference of the opening of the cup 12. The cup 12 and the cover 14 therefore form a chamber 22 which contains the formulation 24, which is shown in the section view of the cartridge 10 in Figure 2.

The cartridge 10 is formed of a material which is water-impermeable or has at least one water-impermeable layer. For example, the cup 12 and cover 14 may be formed of aluminum foil of different thicknesses or may be formed of plastic materials. The cartridge may also be formed of a material that is also air- or oxygen-impermeable or have at least one air- or oxygen-impermeable material. Examples of suitable materials are aluminum and plastic. The term “plastic” as used herein refers to any synthetic or semi-synthetic solid, suitable to be in contact with product intended for consummation.

The cartridge 10 therefore provides an encapsulation for the formulation 24 which shields the formulation 24 situated in the sealed chamber 22 in the cartridge 10 from the surroundings. Preferably, the formulation may be stored in the cartridge for at least one month, preferably a plurality of months.

The cartridge 10 may have a rigid capsule formed of a first material and at least one cover formed of a second material which seals an opening of the capsule. The first and second material are both water-impermeable or have at least one water-impermeable layer. The first and second material may further also be air- or oxygen-impermeable or have at least one air- or oxygen-impermeable material. The first material and second material can be the same or different. Alternatively, the cartridge may also be formed of the same uniform material.

The cartridge 10 has an inlet port 18 and an outlet port 20. In the present embodiment shown in the figures, the inlet port 18 is formed by a truncated conical section at the upper end of the cup 12. The outlet port 20 is formed by the cover 20 on the lower end of the cartridge 10.

When the cartridge 10 is inserted in to the apparatus26 as shown in figure 3, an inlet connector 28 of the apparatus 26 is connected to the inlet port 18 so that a heated and pressurized liquid may enter the cartridge 10 in order to mix with the formulation 24. In the present embodiment the inlet connector 28 consists of several pointed tubes which can pierce the wall of the cup 12 in the truncated conical section that forms the inlet port 18. The heated and pressurized liquid, preferably hot water under pressure, can then be supplied through the tubes of the inlet connector 28 as indicated by the arrows in figure 3.

The inlet port may be formed by a wall of the cartridge though which a water injector may pierce in order to inject water into the cartridge. Alternatively, the inlet port may be formed as an opening in a rigid capsule which can form a sealed connection with the inlet connector of the apparatus wherein the opening may be sealed with a cover that can be pierced by the inlet connector or removed by the user before inserting the cartridge into the apparatus.

The outlet port 20 of the present embodiment is formed by the cover 14 of the cartridge 10. The apparatus 26 comprises an outlet unit with a relief element 30 which is situated adjacent to the cover 14 when the cartridge is inserted into the apparatus 26. When the pressurized liquid is supplied to the chamber 22, the pressure inside the chamber 22 biases the cover 14 against the relief element 30 so as to tear open the cover 14. In the present embodiment, the relief element 30 is arranged to tear open the cover 14 at a plurality of locations so as to form a plurality of openings.

The outlet port of the cartridge is preferably formed of a plurality of openings through which the mixture of aqueous liquid and formulation is pressed due to the pressure of the supplied aqueous liquid. Alternatively, it is also possible that the outlet port only forms a single opening.

The plurality of openings may be formed by a sieve element such as disc comprising through holes or a filter element, such as a plastic woven fabric, which are situated between the chamber containing the formulation and the outlet port of the cartridge. Alternatively, the plurality of openings may be formed by the outlet port of the cartridge during the preparation of the liposome dispersion. The material of the outlet port may for example be pressed against a relief element of the outlet unit of the apparatus so as to tear open on the relief element at predetermined locations in order to provide the plurality of openings through which the mixture of mixture of aqueous liquid and formulation is then pressed due to the pressure of the supplied aqueous liquid.

The apparatus 26 for preparation of the liposome dispersion further comprises a heating unit which is able to provide a heated and pressurized aqueous liquid, preferably water. The apparatus further comprises a cartridge chamber which is able to house a cartridge 10 which is to be used for preparation of a liposome dispersion. The cartridge chamber comprises the inlet connector 28 for supplying the heated and pressurized aqueous liquid from the heating unit to the cartridge 10 and an outlet unit which delivers the prepared liposome dispersion from the cartridge to a beverage container. Alternatively, the cartridge 10 may form an outlet unit which delivers the prepared liposome dispersion directly to a beverage container.

Also provided is a kit of parts comprising a plurality of cartridges according to the invention. A “plurality” as used herein means at least two. For example, the cartridge comprises at least 4 cartridges, at least 5 cartridges, at least 6 cartridges, at least 7 cartridges, at least 8 cartridges, at least 9 cartridges, at least 10 cartridges preferably the kit of parts comprises the plurality of cartridges in an outer packaging.

The formulation can be easily included in a cartridge 10 that is compatible with the several coffee extraction machines that are present on the market. Examples of suitable cartridges are described in, and demonstrated in the drawings of US 5897899 A, US 7490542 B2, WO 2004/064584 Al, EP 2 611 713 Bl, WO 2012/037409 Al, US 6740345 B2 and WO 2005/092160 Al, which are incorporated herein by reference. There are no particular requirements for filling the cartridge or capsule with the mixture or components thereof. For instance, first, a mixture of all ingredients can be made, which is subsequently added to the cartridge. Alternatively, the components are separately added to the cartridge. Combinations are also possible, separate components and/or mixtures of two or more components are added separately to the cartridge. The mixtures of all or part of the components can be prepared at room temperature or at elevated temperature, such as at a temperature of between 20 and 80 °C.

These cartridges can be cylindrically shaped or flat spheroids as is needed to fit in their respective machines. The cartridges are airtight and pierced during processing to force hot water through and form the required stable liposome containing beverage upon extraction, which is collected in e.g. a cup to be orally administered as a beverage.

The invention therefore also provides a method for preparing a liposome formulation comprising adding an aqueous liquid having a temperature of at least 25 °C under pressure to a cartridge according to the invention. Said method preferably comprises extracting said mixture from said cartridge with an aqueous liquid having a temperature of at least 25 °C. The temperature is preferably between 25 °C and 100 °C, more preferably between 30 °C and 100 °C, more preferably between 40 °C and 100 °C, more preferably between 50 °C and 90 °C, such as between 70 °C and 90 °C or between 80 °C and 90 °C. The pressure applied during extraction is preferably at least 2 bar, more preferably between 2 and 20 bar, such as between 2 and 19 bar. Extraction can be performed with any apparatus that is commercially available and intended for prepared warm or hot beverage by extracting ingredients, e.g. coffee, from cartridges. Examples are the coffee extraction machines from Nespresso, Lavazza, Illy, Dolce Gusto, Keurig and Philips, Said aqueous liquid is preferably water. One full extraction is preferably achieved with a water volume of between 50 and 200 mL of water.

A person skilled in the art will understand that the cartridge and apparatus should be compatible, i.e. an apparatus should be used for extraction of the liposome dispersion that is compatible with the specific cartridge that contains the formulation and that allows extraction of the liposome dispersion from the specific cartridge. The liposome dispersion of the bioactive ingredient(s) that is formed upon extraction and collected for consumption is physically stable during at least one hour after preparation. With physical stability is meant that the formulation is opalescent and completely homogeneous, essentially without visual inhomogeneities such as precipitates, aggregates, clumps or phase separation. Opalescence is an indication of a fine particle dispersion in the nanosize range. This degree of fine dispersion is needed to ensure maximal bio avail ability of the encapsulated bioactive ingredient(s) after oral administration.

Features maybe described herein as part of the same or separate aspects or embodiments of the present invention for the purpose of clarity and a concise description. It will be appreciated by the skilled person that the scope of the invention may include embodiments having combinations of all or some of the features described herein as part of the same or separate embodiments.

The invention will be explained in more detail in the following, nondimiting examples.

Examples

Example 1

Failed preparation of a pro-liposome formulation according to patent US5004611A.

The aim of this experiment was to perform an attempt to produce a homogeneous and fine liposome dispersion using the methods as described in patent US5004611A (Prodiposomes).

The following cartridge with mixture was prepared:

Aluminum coffee cartridge 10 mL (iCafilas)

With a mixture of o 3.12 gram propylene glycol (Carl Roth GmbH, Germany) o 1.13 gram PEG400 (Carl Roth GmbH, Germany) o 2.4 gram soy phosphatidyl choline P45 (Lipoid, Germany) o 0.6 gram palmitoylethanolamide (lipophilic bioactive ingredient) (Ergomax, Netherlands) o 1.08 gram sorbitan oleate (first surfactant) (Carl Roth GmbH, Germany) o 0.26 gram polysorbate 80 (second surfactant) (Carl Roth GmbH, Germany) o 0.20 gram Citric acid (Natural Heroes BV, Netherlands)

All ingredients except for the citric acid were weighed, heated and mixed at about 80° C until fully dissolved. The resulting mixture was carefully poured into the coffee cartridge. It was a dark brown dense liquid at this temperature but solidified to a light brown paste at room temperature. At this stage citric acid was added to the capsule.

The cartridge with the mixture was then placed into a Nespresso coffee extractor machine and extracted with 100 niL warm (70° C) water. After preparation the resulting liposome dispersion very quickly formed off-white inhomogeneities or precipitates in the whole volume of formulation.

Example 2

Failed preparation of a pro-liposome preparation with PEA (active ingredient) according to patent US5004611A.

The following mixture was prepared: o 3.12 gram propylene glycol (Carl Roth GmbH, Germany) o 1.13 gram PEG400 (Carl Roth GmbH, Germany) o 2.4 gram soy phosphatidyl choline P45 (Lipoid, Ludwigshafen DE) o 0.6 gram palmitoylethanolamide (lipophilic bioactive ingredient) (Ergomax BV, Netherlands) o 0.54 gram ascorbyl palmitate (Dr Behr GmbH, Germany) o 0.26 gram polysorbate 80 (second surfactant) (Carl Roth GmbH, Germany) o 0.20 gram Citric acid (Natural Heroes BV, Netherlands)

All ingredients were weighed, heated and mixed at about 80° C until fully dissolved in line with patent US5004611A (Prodiposomes). Next, small quantities of warm water (70° C) were added to the pro-liposomal mixture. No homogeneous dispersion could be formed. The resulting mixture was equilibrated for lhl5 min and hand agitated for 1 min. with 15 min intervals.

The preparation failed because homogenous dispersion could only be obtained only after addition of water (equilibration) and two periods of hand agitation.

Example 3

Capsule containing 0.6 gram PEA in a lecithin/ascorbyl palmitate/polysorbate in glycerol/PEG400 formulation

The following cartridge with mixture was prepared:

Aluminum coffee cartridge 10 mL (iCafilas)

With a mixture of o 6.5 gram glycerin (Carl Roth GmbH, Germany) o 0.5 gram ethanol (Carl Roth GmbH, Germany) o 1.0 gram soy phosphatidyl choline (Lipoid, Germany) o 1.0 gram sunflower lecithin (Sternchemie, Germany) o 0.6 gram palmitoylethanolamide (lipophilic bioactive ingredient) (Ergomax BV, Netherlands) o 0.50 gram ascorbyl palmitate (first surfactant) (Dr Behr GmbH, Germany) o 1.3 gram polysorbate 80 (second surfactant) (Carl Roth GmbH, Germany) o 0.27 gram tocopherol (Nutrilo GmbH, Germany) o 10 mg steviol glycosides (sweetener) (Hhoya BV, Netherlands) o 0.79 flavouring agent in carrier (Pomona) All ingredients except for citric acid were weighed, heated and mixed at about 80° C until fully dissolved. The resulting mixture was carefully poured into the coffee cartridge. It formed a dark brown dense liquid at this temperature but solidified to a light brown paste at room temperature.

The cartridge with the mixture was then placed into a Nespresso coffee extractor machine and extracted with 100 mL warm water (about 80° C). The resulting liposome dispersion initially produced a white foam layer but quickly settled with a light amber color and a clear opalescent shine. It tasted neutral, slightly acidic with citrus aroma. Upon measurement with a dynamic light scattering measurement apparatus (Horiba, SZ-100V2) it showed a liposome particle diameter of around 350 nm and a polydispersity index of 0.4. It remained stable as a fine and clear dispersion for 1 hour after which it formed visible inhomogeneities.

Example 4

Capsule containing 0.6 gram PEA, 100 mg cannabidiol in a lecithin/ascorbyl palmitate/polysorbate in propylene glycol/PEG400 formulation with added taste enhancers.

The following cartridge with mixture was prepared:

Nespresso coffee cartridge 10 mL (iCafilas)

With a mixture of o 3.12 gram propylene glycol (Carl Roth GmbH, Germany) o 1.13 gram PEG400 (Carl Roth GmbH, Germany) o 2.4 gram soy phosphatidyl choline (Lipoid GmbH, DE) o 0.6 gram palmitoylethanolamide (lipophilic bioactive ingredient) (Ergomax BV, Netherlands) o 100 mg cannabidiol (CBD) (Endoca BV, Netherlands) o 0.54 gram ascorbyl palmitate (first surfactant) (Dr Behr GmbH, Germany) o 0.26 gram polysorbate 80 (second surfactant) (Carl Roth GmbH, Germany) o 0.20 gram sodium bicarbonate (Carl Roth GmbH, Germany) o 1 gram erythritol (sweetener) (Hhoya BV, Netherlands)

All ingredients except for sodium bicarbonate and erythritol were weighed, heated and mixed at about 80° C until fully dissolved. The resulting mixture was carefully poured into the coffee cartridge. It formed a dark brown dense liquid at this temperature but solidified to a light brown paste at room temperature. At this stage sodium bicarbonate and erythritol were added to the cartridge.

The cartridge with the mixture was then placed into a Nespresso coffee extractor machine and extracted with 100 mL warm water. The resulting liposome dispersion initially produced a white foam layer but quickly settled with a light amber color and a clear opalescent shine. It tasted neutral with a slight pungent aftertaste. Upon measurement with a dynamic light scattering measurement apparatus (Horiba, SZ-100V2) ) it showed a particle diameter of 270 nm and a polydispersity index of 0.4. It remained stable as a fine and clear dispersion for 1 hour after which it formed visible inhomogeneities

Example 5

Capsule containing 100 mg cannabidiol and 30 mg beta-caryophyllene in a lecithin/ascorbyl palmitate/polysorbate in propylene glycol/PEG400 formulation with added taste enhancers.

The following cartridge with mixture was prepared:

Nespresso coffee cartridge 10 mL (iCafilas)

With a mixture of o 3.12 gram propylene glycol (Carl Roth GmbH, Germany) o 1.13 gram PEG400 (Carl Roth GmbH, Germany) o 2.4 gram soy phosphatidyl choline (Lipoid GmbH, DE) o 100 mg cannabidiol (CBD) (Endoca BV, Netherlands) o 0.54 gram ascorbyl palmitate (first surfactant) (Dr Behr GmbH, Germany) o 0.26 gram polysorbate 80 (second surfactant) (Carl Roth GmbH, Germany) o 0.20 gram sodium bicarbonate (Carl Roth GmbH, Germany) o 1 gram erythritol (sweetener) (Hhoya BV, Netherlands)

All ingredients except for sodium bicarbonate and erythritol were weighed, heated and mixed at about 80° C until fully dissolved. The resulting mixture was carefully poured into the coffee cartridge. It formed a dark brown dense liquid at this temperature but solidified to a light brown paste at room temperature.

The cartridge with the mixture was then placed into a Nespresso coffee extractor machine and extracted with lOOmL warm water. The resulting liposome dispersion initially produced a white foam layer but quickly settled with a light amber color and a clear opalescent shine. It tasted neutral with a slight pungent aftertaste. Upon measurement with a dynamic light scattering measurement apparatus (Horiba, SZ-100V2) it showed a particle diameter of 380 nm and a polydispersity index of 0.5. It remained stable as a fine and clear dispersion for 1 hour after which it formed visible inhomogeneities.

Example 6

Capsule containing 500 mg vitamin C, 50 meg vitamin D, 267 mg vitamin E and 25 mg zinc in a lecithin/ascorbyl palmitate/polysorbate in glycerol/PEG400 formulation with added taste enhancers.

The following cartridge with mixture was prepared:

Nespresso coffee cartridge 10 mL (iCafilas)

With a mixture of o 5.0 gram glycerin (Carl Roth GmbH, Germany) o 0.56 gram PEG400 (Carl Roth GmbH, Germany) o 1.5 gram soy phosphatidyl choline (Lipoid GmbH, Germany) o 0.5 gram sunflower lecithin (Sternchemie GmbH, Germany) o 230 mg ascorbic acid (Health Sources Nutrition, China) o 160 mg zinc ascorbate (PureBulk Inc. USA) o 50 meg cholecalciferol (Brenntag BV, NL) o 0.40 gram ascorbyl palmitate (first surfactant) (Dr Behr GmbH, Germany) o 1.0 gram polysorbate 80 (second surfactant) (Carl Roth GmbH, Germany) o 0.27 gram tocopherol (Nutrilo GmbH, Germany) o 10 mg steviol glycosides (sweetener) (Hhoya BV, Netherlands) o 0.79 flavouring agent in carrier (Pomona)

All ingredients were weighed, heated and mixed at about 80° C until fully dissolved. The resulting mixture was carefully poured into the coffee cartridge. It formed a dark brown dense liquid at this temperature but solidified to a light brown paste at room temperature.

The cartridge with the mixture was then placed into a Nespresso coffee extractor machine and extracted with 100 mL warm water. The resulting liposome dispersion initially produced a white foam layer but quickly settled with a light amber color and a clear opalescent shine. It tasted neutral. Upon measurement with a dynamic light scattering measurement apparatus (Horiba, SZ-100V2) it showed a particle diameter of 450 nm and a polydispersity index of 0.5. It remained stable as a fine and clear dispersion for 1 hour after which it formed visible inhomogeneities.

Example 7

Capsule containing 300 mg resveratrol in a lecithin/ascorbyl palmitate/polysorbate in glycerol/PEG400 formulation with added taste enhancers.

The following cartridge with mixture was prepared:

Nespresso coffee cartridge 10 mL (iCafilas)

With a mixture of o 5.0 gram glycerin (Carl Roth GmbH, Germany) o 0.23 gram PEG400 (Carl Roth GmbH, Germany) o 2.0 gram soy phosphatidyl choline (Lipoid GmbH, DE) o 150 mg resveratrol (Making Cosmetics Inc, USA) o 0.40 gram ascorbyl palmitate (first surfactant) (Dr Behr GmbH, Germany) o 1.0 gram polysorbate 80 (second surfactant) (Carl Roth GmbH, Germany) o 0.27 gram tocopherol (Nutrilo GmbH, Germany) o 10 mg steviol glycosides (sweetener) (Hhoya BV, Netherlands) o 0.79 flavouring agent in carrier (Pomona)

The cartridge with the mixture was then placed into a Nespresso coffee extractor machine and extracted with 100 mL warm water. The resulting liposome dispersion initially produced a white foam layer but quickly settled with a light amber color and a clear opalescent shine. It tasted neutral. Upon measurement with a dynamic light scattering measurement apparatus (Horiba, SZ-100V2) it showed a particle diameter of around 250 nm and a polydispersity index of 0.4. It remained stable as a fine and clear dispersion for 1 hour after which it formed visible inhomogeneities.

Example 8

Capsule containing 300 mg epigallocatechin gallate (EGCG) in a lecithin/ascorbyl palmitate/polysorbate in a glycerol/PEG400 formulation with added taste enhancers.

The following cartridge with mixture was prepared:

Nespresso coffee cartridge 10 mL (iCafilas)

With a mixture of o 5.0 gram glycerin (Carl Roth GmbH, Germany) o 1,0 gram soy phosphatidyl choline (Lipoid GmbH, DE) o 1.0 gram sunflower lecithin (Sternchemie GmbH, DE) o 300 mg epigallocatechin gallate (Sunphenon, Taiyo International, USA) o 0.40 gram ascorbyl palmitate (first surfactant) (Dr Behr GmbH, Germany) o 1.0 gram polysorbate 80 (second surfactant) (Carl Roth GmbH, Germany) o 0.27 gram tocopherol (Nutrilo GmbH, Germany) o 0.50 gram PEG400 (Carl Roth GmbH, Germany) o 10 mg steviol glycosides (sweetener) (Hhoya BV, Netherlands) o 0.30 gram Matcha green tea powder (Marukyu Koyamaen Co Ltd Japan)

All ingredients were weighed, heated and mixed at about 80° C until fully dissolved. The resulting mixture was carefully poured into the coffee cartridge. It formed a brown-green dense liquid at this temperature but solidified to a light brown paste at room temperature.

The cartridge with the mixture was then placed into a Nespresso coffee extractor machine and extracted with lOOmL warm water. The resulting liposome dispersion initially produced a white foam layer but quickly settled with a light amber color and a clear opalescent shine. It tasted neutral. Upon measurement with a dynamic light scattering measurement apparatus (Horiba, SZ-100V2) it showed a particle diameter of 400 nm and a polydispersity index of 0.5. It remained stable as a fine and clear dispersion for 1 hour after which it formed visible inhomogeneities.

Claims

1. A sealed cartridge for preparation of a phospholipid dispersion, wherein the cartridge is formed of a material which is water-impermeable or has at least one water-impermeable layer, and the cartridge forms a sealed chamber which encapsulates a mixture comprising one or more phospholipids, one or more surfactants, one or more lipophilic biologically active ingredients, and one or more liquid nontoxic monohydric or polyhydric alcohols, the cartridge further comprising an inlet port configured to allow a heated and pressurized liquid to be injected into the sealed chamber to form the phospholipid dispersion and an outlet port configured to allow the prepared phospholipid dispersion to exit the sealed chamber.

2. A sealed cartridge according to claim 1, wherein at least part of said one or more phospholipids are dispersed in said one or more liquid nontoxic monohydric or polyhydric alcohols, the dispersion being a suspension, a colloidal mixture and/or an emulsion.

3. A sealed cartridge according to any of the preceding claims, wherein the phospholipid dispersion is formed by heated and pressurized water in a pressure range of from 2 bar to 20 bar and with a temperature between 25 and 100 degrees Celsius.

4. A sealed cartridge according to any of the preceding claims, wherein the cartridge comprises a cup forming a chamber with an opening and a cup cover sealed to a lip of the cup wherein the cover extends across the opening to the lip and is sealed to the lip so that mixture is contained within the cup by the cover free from leaking; wherein the cover has a thickness and a breaking stress so that upon placement of the sealed cartridge in an apparatus for brewing a beverage under pressure, wherein the sealed cartridge is positioned in a holder of the apparatus in a way that the apparatus can pierce openings in the inlet port at a back of the cup and in the outlet port at the cover, which allows the flow of heated and pressurized liquid into the cup through the inlet port and the flow of prepared phospholipid dispersion through the outlet port.

5. A sealed cartridge according to any of the preceding claims, wherein the cartridge being made of plastic or aluminium or any other airtight material.

6. A sealed cartridge according to any of the preceding claims wherein said one or more phospholipids, one or more surfactants and one or more lipophilic active ingredients are dissolved and/or dispersed in said one or more liquid nontoxic polyols, monohydric alcohols and/or polyhydric alcohols or a mixture thereof.

7. A sealed cartridge according to any of the preceding claims wherein the mixture in the cartridge is a liquid, viscous liquid or paste.

8. A sealed cartridge according to any of the preceding claims wherein the one or more phospholipids are selected from the group consisting of amphipathic negatively charged and non-charged vesicle-forming phospholipids.

9. A sealed cartridge according to any of the preceding claims wherein the one or more phospholipids comprise saturated or unsaturated fatty acid side chains and a polar headgroup based on choline, glycerol, ethanolamine or phosphatidic acid, preferably wherein the one or more phospholipids are selected from the group consisting of fat-free soy lecithin, sunflower lecithin, egg yolk lecithin, the (partially) hydrogenated versions thereof and synthetic phospholipids such as dimyristoyl phosphatidyl choline, dipalmitoyl phosphatidyl choline and distearoyl phosphatidyl choline.

10. A sealed cartridge according to any of the preceding claims comprising a surfactant with a relatively low HLB value selected from the group consisting of fatty acid esters of sugar, polyol, amino acid and vitamins, wherein the fatty acids are selected from the group consisting of stearic acid, palmitic acid, lauric acid, unsaturated fatty acids such as oleic acid and polyunsaturated fatty acids.

11. A sealed cartridge according to claim 10 wherein the surfactant with a relatively low HLB value is selected from the group of ascorbyl palmitate, sorbitan fatty acid derivatives and fatty acid derivatives of sugars, such as the sucrose fatty acid esters, of which sucrose mono- or di-laurate, -palmitate and -stearate are examples, preferably wherein the surfactant with a relatively low HLB value is ascorbyl palmitate.

12. A sealed cartridge according to any of the preceding claims comprising a surfactant with a relatively high HLB selected from the group of polysorbate emulsifiers, including PEG sorbitan fatty acid esters such as PEG-20 sorbitan monolaurate, PEG-20 sorbitan monop almitate, PEG-20 sorbitan monostearate, PEG-20 sorbitan monooleate, polyol-derivatized fat-soluble substances, which include PEGylated fatty acids, PEGylated lipids and tocopherol polyethylene glycol succinate (TPGS) and mixtures thereof, or selected from the group of hydrolyzed phospholipids (lysolipids), including hydrolyzed and purified sunflower lecithin or hydrolyzed and purified soy lecithin.

13. A sealed cartridge according to any of the preceding claims wherein said one or more biologically active ingredients have a logP value of 0 or higher, preferably a logP value of 1 or higher.

14. A sealed cartridge according to any of the preceding claims wherein said one or more biologically active ingredients are selected from the group of bioactive fatty acid esters and amides, endocannabinoids, vitamins and provitamins, polyphenols and flavonoids, and essential oil components.

15. A sealed cartridge according to any of the preceding claims wherein the total amount of lipophilic bioactive ingredients present in the mixture is 0.5-20 weight% of the total mixture in the cartridge.

16. A kit of part comprising a plurality of cartridges according to any of the preceding claims.

17. A method for preparing a liposome formulation comprising adding an aqueous liquid having a temperature of at least 25 °C under pressure of at least 2 bar to a cartridge according to any one of claims 1 to 15.