EP4153143A1

EP4153143A1 - Process for preparing an active liposome, related compositions and uses

Info

Publication number: EP4153143A1
Application number: EP21732514.1A
Authority: EP
Inventors: Giulia Nannoni; Ruggero MARTIGNONI; Michele GIUNTINI
Original assignee: Labiotre Srl
Current assignee: Labiotre Srl
Priority date: 2020-05-18
Filing date: 2021-05-17
Publication date: 2023-03-29
Also published as: WO2021234538A1

Abstract

The present invention relates to a process for preparing an active liposome which comprises an active ingredient in a liposome. Furthermore, the present invention relates to said active liposome as such (per se), or obtained from such process. Furthermore, the present invention relates to a composition comprising said active liposome and, optionally, food-grade or pharmaceutical-grade additives and/or excipients.

Description

PROCESS FOR PREPARING AN ACTIVE LIPOSOME, RELATED COMPOSITIONS AND USES

The present invention relates to a process for preparing an active liposome which comprises an active ingredient in a liposome. Furthermore, the present invention relates to said active liposome as such (per se), or obtained from said process.

Furthermore, the present invention relates to a composition comprising said active liposome and, optionally, food-grade or pharmaceutical-grade additives and/or excipients. Finally, the present invention relates to a composition comprising said active liposome and, optionally, food-grade or pharmaceutical-grade additives and/or excipients, for use in a method for:

(a) treating and/or improving and/or regulating the digestive function, the hepatic function, intestinal transit, the functionality of the digestive system, or the functionality of the cardiovascular apparatus;

(b) treating and/or improving and/or regulating the drainage of bodily liquids (heaviness in the legs), the micro-circulation functionality, or to combat the blemishes of cellulite;

(c) treating chronic venous insufficiency (CVI), hemorrhoids and varicose veins.

The presence on the market of a vast range of finished products is known, for example food products, or products for food supplements, or products for medical devices under Reg. (EU) 2017/745, all the foregoing in various pharmaceutical forms of administration, such as for example tablets, capsules, granules or solutions or dispersions. The finished products present on the market can contain, among other things, various types of active ingredients such as for example botanical extracts, alkaloids extracted from various plants, molecules of plant origin such as for example bioflavonoids, or enzymes, such as for example the enzymes present in the juice of some fruits. However, the nature and the chemical and physical characteristics of these active ingredients (such as for example the hydrophilic or hydrophobic nature of an active ingredient) very often limit their practical use and their workability in a process for preparing finished products. In particular, the chemical and physical nature of these active ingredients makes it difficult to obtain repeatable and standardizable concentrations of active ingredients with values equal to those that the market requires in order to be capable of preparing and making them available in increasingly efficacious solid pharmaceutical forms.

Therefore, in recent years research and development in the sector of nutraceuticals and supplements (for example, products for food supplements or products for medical devices) has striven to develop increasingly high- performing working processes, such as for example particular extraction techniques, in order to be capable of preparing active ingredients and, therefore, finished products, that have a very high concentration, in a manner which is repeatable and standardizable.

However, it has been observed that a very high concentration of active ingredient does not always correspond to an adequate level of stability over time. Furthermore, it has been observed that a very high concentration of active ingredient does not always correspond to its efficacy and/or bioavailability in the organism when that active ingredient is administered, for example orally via the administration of a finished product, to a consumer. This is because the chemical form with which the active ingredient is prepared plays a very important, and sometimes essential, role.

Among the chemical forms known and used in the sector of food supplements and medical devices there are also liposomes.

In pharmaceutical technology, liposomes are microscopic vesicles which are obtained from molecules of phospholipids in an aqueous environment. Liposomes can be used to store substances of various types (antiblastic and immuno stimulant drugs, in particular). If injected into an organism intravenously, they are assimilated by the cells of the reticuloendothelial system which degrade them, allowing the substances they contain to be freed. Liposomes find application in the field of experimental pharmacology, as vehicles for the administration of drugs of which liposomes reduce the toxicity.

There are numerous processes for preparing liposomes. By way of example, the prior art document EP0736299A1 discloses a method of preparing a solid liposomal formulation by way of using an organic solvent. The prior art document W098/36735A1 discloses an aqueous pharmaceutical composition that comprises a component that is highly insoluble in water, wherein a dispersion containing said component is subjected to a series of freeze-drying steps. The prior art document EP0488142A1 discloses a process for encapsulating solid or liquid lipophilic active ingredients in liposomes with an average size distribution of less than 200 nm.

However, the known processes for preparing liposomes are not satisfactory, since they need to be conducted in an organic environment and in the presence of solvents.

Furthermore, the known liposomal forms and liposomes do not always make it possible to make the active ingredients encapsulated or contained therein to be bioavailable within a short length of time (in particular: within two hours, preferably within approximately one hour) after administration, and at a concentration that is useful and effective.

The Applicant, after lengthy and intensive research and development activity, has devised a process for preparing an active liposome that is capable of providing an adequate solution to the existing limitations, drawbacks and problems.

An object of the present invention is a process for preparing an active liposome which comprises an active ingredient in a liposome, having the features according to the appended claims. An object of the present invention is said active liposome as such (per se), i.e. obtained from said process, having the features according to the appended claims.

An object of the present invention is a composition comprising said active liposome and, optionally, food-grade or pharmaceutical-grade additives and/or excipients, which has the features according to the appended claims.

An object of the present invention is a composition comprising said active liposome and, optionally, food-grade or pharmaceutical-grade additives and/or excipients, for use in a treatment method according to the appended claims.

An object of the present invention is an active liposome comprising berberine, as an active ingredient, in a lecithin-based liposome, for use in a method for (a) treating and/or improving and/or regulating the digestive function, the hepatic function, intestinal transit, the functionality of the digestive system, or the functionality of the cardiovascular apparatus, which has the features according to the appended claims.

An object of the present invention is an active liposome comprising bromelain, as an active ingredient, in a liposome, for example based on lecithin, for use in a method for (b) treating and/or improving and/or regulating the digestive function, drainage of bodily liquids (heaviness in the legs), the micro-circulation functionality, or to combat the blemishes of cellulite, which has the features according to the appended claims.

An object of the present invention is an active liposome comprising diosmin, as an active ingredient, in a liposome, for example based on lecithin, for use in a method for (c) treating chronic venous insufficiency (CVI), hemorrhoids and varicose veins, which has the features according to the appended claims.

In the context of the present invention the term “composition(s)” means a pharmaceutical composition, a food supplement composition, a food product composition, or a composition for medical devices under Reg. (EU) 2017/745.

The present invention will now be described by reference to the accompanying drawings, which are provided purely as examples and, therefore, are non-limiting, wherein:

- Figure 1 is a flowchart of a process according to the present invention, according to a possible embodiment;

- Figure 2 is an exemplary chromatogram of a sample at 120 minutes and a standard sample, showing the two traces for each sample corresponding to berberine (transition 336>291) and benzanilide (198>105);

- Figure 3 is a diagram of the absorption speed of an active liposome according to the present invention, containing berberine as an active ingredient, with respect to the absorption speed of a prior art 97% berberine solution in water;

- Figures 4 and 5 are, respectively, two 63 Ox enlargements of the active ingredient in the form of a prior art 97% berberine solution in water and of the active liposome according to the present invention dispersed in water, containing berberine as an active ingredient, taken in an optical microscope using a Moticam instrument (Moticam, Hong Kong);

- Figures 6 and 7 show two SEM images, respectively at 500x and 1500x, of a sample in powder form of crystalline berberine, deposited on a stub and then metalized; the samples in powder form were deposited on a stub, metalized and then observed using a scanning electron microscope. The instrument used is a Leica Stereoscan 410. These are respectively samples of a dry extract of Berberis aristata bark, titrated to 97% berberine, and of a granular liposome containing the same extract and having a titer of 20% berberine;

- Figures 8 and 9 are two SEM images, respectively at lOOx and 500x, of a sample in powder form of an active liposome according to the present invention dispersed in water containing berberine, deposited on a stub and then metalized; the samples in powder form were deposited on a stub, metalized and then observed using a scanning electron microscope. The instrument used is a Leica Stereoscan 410. These are respectively samples of a dry extract of Berberis aristata bark, titrated to 97% berberine, and of a granular liposome containing the same extract and having a titer of 20% berberine.

The Applicant has devised a process for preparing an active liposome, which comprises an active ingredient in a liposome, wherein said process comprises the following steps of:

(i) preparing a granulation mixture or a granulating solution comprising or, alternatively, consisting of:

(a) at least one active ingredient selected from the group comprising or, alternatively, consisting of: berberine, diosmin, bromelain, or salts thereof, or mixtures thereof;

(b) at least one coating agent selected from the group comprising or, alternatively, consisting of: phospholipids, phosphoglycerides, sphingophospholipids, lecithin, phosphatidylcholines, phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol, or mixtures thereof;

(c) water, and

(d) optionally technological additives and/or excipients;

(ii) subjecting said granulation mixture or granulating solution, obtained from step (i), to a step of granulation carried out in a fluid bed granulator, preferably by way of nebulization or spraying, bringing said granulation mixture or granulating solution into contact with said coating agent (step ii(a)) or, alternatively, with a granulation aid in solid form so as to obtain a wet granulate;

(iii) subjecting said wet granulate, obtained from step (ii), to a step of drying, (or desiccation) by bringing said wet granulate into contact with a heat source so as to obtain an active liposome in granular form. The active ingredient (a) is preferably selected from the group comprising or, alternatively, consisting of: berberine, diosmin, bromelain, and/or salts thereof and/or mixtures thereof.

Berberine (CAS N. 633-66-9) is a quaternary ammonium salt belonging to the group of benzylisoquinoline alkaloids, which has antimicrobial effects on bacteria, viruses, fungi, protozoa, helminths, and chlamydia. Preferably, in the mixture of step (i) of the present invention, the active ingredient (a) is a dry extract of berberine obtained from a plant of the Berberis genus, more preferably an extract of the root, rhizome, stalk and/or bark of a plant of the Berberis genus, even more preferably a dry extract of the bark of Berberis aristata DC. For example, Berberis Aristata ( root bark ) in dry extract form can be an extract of 97% berberine (analysis method by titration) in the form of a fine, yellow-colored powder. When sieved, 90% of the powder passes through a mesh size of from 40 to 60 mesh and can have a humidity content lower than 12% by weight, preferably lower than 8% by weight, and an ash residue lower than 8% by weight, preferably lower than 4% by weight.

Bromelain is a generic term used to refer to two proteolytic enzymes (i.e. capable of degrading the proteins in amino acids) that are extracted from Ananas comosus , a plant of the Bromeliaceae family originating from South America. In addition to their proteolytic activity, several other effects have been attributed to these enzymes (anti-inflammatory and anti thrombotic). The first form of bromelain is found in the pineapple fruit, while the second is found in the stem; since in the stem the amounts of the enzyme are larger, for commercial purposes it is preferred to use this latter source. Preferably, in the mixture of step (i) of the present invention, the bromelain is an extract of the fruit and/or of the stem of Ananas comosus , preferably a dry extract. For example, a bromelain with 2500 GDU/g of proteolytic activity can be obtained from the stem of Ananas comosus by extraction with water solvent and can have a bulk density of 0.2-0.8 and a particle size of 100% (40 mesh). It takes the form of a hygroscopic powder, white in color.

Diosmin (CAS N. 520-27-4) is obtained from plant sources, or as a semi-synthetic molecule, modified starting from the hesperidin molecule, which belongs to the flavonoid family. Diosmin is a phlebotropic medicine used for vascular protection. Diosmin is considered a vascular protection agent, used to help improve chronic venous insufficiency (CVI), hemorrhoids, lymphedema and varicose veins. For example, a diosmin can be in the form of a light yellow hygroscopic powder, practically insoluble in water, soluble in dimethyl sulfoxide DMSO, insoluble in 96% ethanol, and can be dissolved in diluted solutions of alkali hydroxides.

The active liposome, prepared according to the process of the present invention (steps (i) to (iii)), is in solid granulate, flake, pellet or powder form, preferably the active liposome is in granular form. In a preferred embodiment, the active liposome, prepared according to the present invention, has a phospholipid bilayer.

The granulation mixture prepared in step (i) can be obtained, in a first embodiment, by mixing the components (a), (b) and (c) and, optionally, (d) all together, in any order and proportion, in a container provided with heating and agitation means, such as for example a mixer with rotating blades, so as to obtain a granulation mixture [(a)+(b)+(c) and optionally (d)]. The components (a), (b), (c) and, optionally, (d) can all be, or only some of them can be, in liquid form, for example aqueous liquid; liquid extract, for example aqueous extract; suspension, for example aqueous suspension; dispersion, for example aqueous dispersion; or in solid or semi solid form, for example powder, granules, flakes or pellets. The granulation mixture [(a)+(b)+(c) and optionally (d)] can be in liquid or suspension or gel or solid or semi-solid form, preferably it is in solid or semi-solid form.

In the granulation mixture (step (i)) the components (a), (b) and (c) are present in a quantity by weight (a):(b):(c) comprised from 1:1:1 to 1:1:15, preferably comprised from 1:1:15 to 1:1:10, for example 1:1:8, for example 200 g of extract of 97% Berberis aristata (see above), 200 g of lecithin in powder form, for example a sunflower lecithin in powder form, and 1600 g of water.

In the wet granulate (step (ii)) the components (a), (b) and (c) are present in a quantity by weight (a):(b):(c) comprised from 1:2:4 to 1:6:12, for example 1:4:8; for example 200 g of extract of 97% Berberis aristata , 200 g of lecithin in powder form, for example a sunflower lecithin in powder form (step (i)) and 600 g of lecithin in powder form, for example of a sunflower lecithin in powder form (step (ii)) and 1600 g of water.

The granulation mixture prepared in step (i) can be obtained, in a second embodiment, by mixing the components (a) and (b) together, in a container provided with heating and agitation means, such as for example a mixer with rotating blades, so as to obtain a mixture [(a)+(b)], for example in liquid or solid or semi-solid form. Then, subsequently, the water component (c) is added to this mixture [(a)+(b)], and, optionally, also the component (d) so as to obtain a granulation mixture [(a)+(b)+(c)+ optionally

The components (a), (b), (c) and, optionally, (d) can all be, or only some of them can be, in liquid form, for example aqueous liquid; liquid extract, for example aqueous extract; suspension, for example aqueous suspension; dispersion, for example aqueous dispersion; or in solid or semi solid form, for example powder, granules, flakes or pellets. The granulation mixture [(a)+(b)+(c) and optionally (d)] can be in liquid or suspension or gel or solid or semi-solid form, preferably it is in solid or semi-solid form.

In the granulation mixture (step (i)) the components (a) and (b) are present in a quantity by weight (a):(b) comprised from 1:3 to 3:1, preferably 1:2, or 1:1; for example 200 g of extract of 97% Berberis aristata and 200 g of lecithin in powder form, for example a sunflower lecithin in powder form. Then, subsequently, the water component (c) is added so as to obtain [(a)+(b)+(c)] where the components (a), (b) and (c) are present in a quantity by weight comprised (a):(b):(c) of 1:1:8; for example 200 g of extract of 97% Berberis aristata and 200 g of lecithin in powder form, for example a sunflower lecithin in powder form and 1600 g of water.

In the wet granulate (step (ii)) the components (a), (b) and (c) are present in a quantity by weight comprised (a):(b):(c) of 1:4:8; for example 200 g of extract of 97% Berberis aristata , 200g of lecithin in powder form, for example a sunflower lecithin in powder form (step (i)) and 600 g of lecithin in powder form, for example a sunflower lecithin (step (ii)) and 1600 g of water.

In the first and second embodiments of step (i), the granulation mixture is prepared preferably at an external pressure of 1 Atm, using water, preferably at a temperature from 10°C to 40°C, even more preferably from 15°C to 30°C.

In the first and second embodiments of step (i), the granulation mixture is prepared preferably at an external pressure of 1 Atm and maintaining a temperature, preferably at a temperature from 10°C to 40°C, even more preferably from 15°C to 30°C, for a mixing time preferably from 5 minutes to 240 minutes, even more preferably from 30 minutes to 180 minutes, for example from 60 minutes to 120 minutes, so as to obtain a granulation mixture with a water content from 55% to 85% by weight, preferably from 65% to 75% by weight, of the total weight of the granulation solution.

Subsequently this granulation mixture, obtained from step (i), is subjected to a step of granulation (step (ii)) which can be carried out in two ways: a step ii(a) or a step ii(b).

In step ii(a), the granulation mixture, obtained from step (i), is brought into contact with a granulation aid which is represented by the same coating agent used in step (i), such as for example a lecithin, preferably in powder form, so as to obtain a wet granulate. In step ii(b), the granulation mixture, obtained from step (i), is brought into contact with a granulation aid in solid form so as to obtain a wet granulate. The granulation aid in solid form is selected from the group comprising or, alternatively, consisting of an inorganic aid like silica (Si0₂); plant fibers selected from: inulin, maltodextrin, FOS, GOS, XOS; a cellulose-derived polymer selected from: carboxymethylcellulose, carboxyethylcellulose, carboxypropylcellulose; a natural rubber selected from: gum Arabic, guar gum, tara gum or Xanthan gum; polyalcohols; lecithin, such as for example soya, maize or sunflower lecithin, or mixtures thereof.

The wet granulate prepared in step (ii) can be obtained by addition of this granulation mixture, preferably in the form of aqueous solution (granulating solution), to a granulation aid. The granulation aid is selected from the group comprising or, alternatively, consisting of an inorganic aid like silica (Si0₂); plant fibers selected from: inulin, maltodextrin, FOS, GOS, XOS; a cellulose-derived polymer selected from: carboxymethylcellulose, carboxyethylcellulose, carboxypropylcellulose; a natural rubber selected from: gum Arabic, guar gum, tara gum or Xanthan gum; polyalcohols; lecithin, such as for example soya, maize or sunflower lecithin, or mixtures thereof.

The quantity by weight [(a)+(b)+(c)+ optionally (d)] : [(granulation aid)] is from 12:1 to 1:1, preferably from 6:1 to 1:1, even more preferably from 3:1 to 1:1.

In an embodiment, the step of granulation (step (ii)) is preferably carried out using a fluid bed granulator, in the presence of said granulation aid which is represented by the same coating agent (step (ii(a))) used in step (i), such as for example a lecithin, preferably in powder form, so as to obtain a wet granulate to be subjected to step (iii).

In another embodiment, the step of granulation (step (ii)) is preferably carried out using a fluid bed granulator, in the presence of said granulation aid (step (ii(b))) in solid form so as to obtain a wet granulate to be subjected to step (iii).

Preferably, both in step (ii(a)) and in step (ii(b)) the fluid bed granulator can use hot air, at a temperature from 25°C to 50°C, preferably from 30°C to 45°C, even more preferably from 35°C to 40°C in order to facilitate the evaporation of the aqueous solvent (c) contained in the granulation mixture (i) and the formation of the granules in the wet granulate to be subjected to the step of drying (iii).

The wet granulate prepared in step (ii) contains a quantity of water comprised between 10% and 30% by weight, preferably between 15% and 25% by weight, with respect to the weight [(a)+(b)+(c)+ optionally (d)]: [(granulation aid)] .

Subsequently, said wet granulate, obtained from the step of granulation (ii), is subjected to a step of drying (step (iii)) which brings said wet granulate into contact with a source of heat in order to eliminate the excess water or humidity, so as to obtain an active liposome in granular form.

In a preferred embodiment, the step of dehydration (step (iii)) is represented by a step of drying which is preferably carried out using a fluid bed granulator, using hot air at a temperature from 25°C to 60°C, preferably from 30°C to 55°C, even more preferably from 35°C to 50°C in order to eliminate the residual water present in the wet granulate, so as to obtain an active liposome in granular form.

The active liposome in granular form contains water in a quantity by weight from 1% to 15%; preferably from 3% to 12%; even more preferably from 5% to 10%; of the total weight of said active liposome.

The active liposome in granular form contains a quantity of said at least one active ingredient, for example berberine, from 5% to 45%; preferably from 10% to 35%; even more preferably from 15% to 25%, of the total weight of said active liposome. The final titer, or the final quantity of liposomal granulate, obtained according to the process of the present invention, is determined using qualitative/quantitative techniques of high performance liquid chromatography (HPLC).

The step of granulation (step (ii) and the step of dehydration (step (iii)), preferably step of drying, can also be carried out together in the fluid bed granulator, as a function of the operating conditions adopted.

An embodiment of the present invention is shown schematically in Figure 1. In step (i), Figure 1, of the process according to the present invention, a mixture is provided comprising or, alternatively, consisting of: (a) at least one active ingredient (also named hydrophobic active ingredient), (b) at least one coating agent (also named encapsulation agent), preferably a phospholipid agent; and (c) optional technological additives/excipients. Preferably, in said mixture of step (i), both (a) and (b) are independently in solid form, or in liquid form or in semi-liquid form (meaning in an intermediate physical state between a solid and a fluid); more preferably, (a) and (b) are both in solid form.

In said mixture of step (i), Figure 1, a weight ratio (a):(b) is preferably comprised between 1.5:1 and 1:10, preferably comprised between 1.1:1 and 1:8, even more preferably comprised between 1 : 1 and 1 :4.

Preferably, a quantity of active ingredient (a) in the mixture of step (i) is from 5% to 20%, by weight, preferably from 10% to 15% by weight, of the total weight of the mixture.

Said at least one coating agent (b) is preferably a phospholipid. Phospholipids are complex lipids in which one or more fatty acid molecules are linked to a phosphoric group or to a nucleobase. Based on their chemical structure, phospholipids can be divided into two classes: phosphoglycerides (also phosphoglycerols or glycerophospholipids) and sphingophospholipids (also sphingolipids or sphingophosphatids). In phosphoglycerides, unlike triglycerides, only two hydroxyls of glycerol are esterified with corresponding glycerol molecules, while the third hydroxyl is esterified with orthophosphoric acid (or phosphoric acid).

Of the various phospholipids, the most structurally simple is phosphatidic acid. Lecithin belongs to the class of phosphoglycerides. In lecithin, the phosphoric group is linked to choline, an amino alcohol choline, and it is for this reason that sometimes they are also called phosphatidylcholines. Depending on the hydroxyl to which the phosphoric group is linked, the lecithin is alpha-lecithin (more common) or beta- lecithin. Other phospholipids belonging to the class of phosphoglycerides which have interesting characteristics are phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol.

In an embodiment, said coating agent (b) comprises or, alternatively, consists of lecithin, even more preferably soya lecithin, sunflower lecithin, maize lecithin, or mixtures thereof, in an aqueous solution or in powder form.

The optional technical additives (c) are preferably present in the mixture of step (i) in a quantity from 1% to 50%, preferably from 5% to 40%, more preferably from 10% to 30%, by weight, of the total weight of such mixture.

Preferably, the technical additives (c) that can be used in step (i) are selected from the group comprising or, alternatively, consisting of: emulsifiers, suspending agents, coating agents for modified release.

The active liposome obtained from the preparation process described above contains a quantity of active ingredient (a) - determined by high performance liquid chromatography or HPLC/DAD - preferably comprised between 5% and 45% by weight, preferably comprised between 10% and 35% by weight, even more preferably comprised between 15% and 25% by weight.

Said active liposome has an average size distribution - determined by laser light scattering analysis - comprised between 150 nm and 1800 nm, preferably comprised between 250 nm and 1500 nm, even more preferably comprised between 550 nm and 660 nm. More preferably, a polydispersity index of said active liposome is comprised between 0.3 and 0.4, even more preferably comprised between 0.320 and 0.350.

By way of example, said average size distribution may be determined by laser light scattering analysis (correlation spectroscopy) using a 90 Plus Instrument (Brookhaven, USA), in aqueous suspension at a temperature of 25°C, using a laser beam at the wavelength of 638 nm at 90°. Preferably, a percentage average size distribution per size class may be as indicated in Table 1 below.

Table 1

A further object of the present invention is an active liposome obtained by said method of preparation in the aqueous phase.

Embodiments En of the present invention are given below.

El. A process for preparing an active liposome, which comprises an active ingredient in a liposome, wherein said process comprises the following steps of:

(c) water, and

(d) optionally technological additives and/or excipients;

(ii) subjecting said granulation mixture or granulating solution, obtained from step (i), to a step of granulation carried out in a fluid bed granulator, preferably by way of nebulization or spraying, bringing said granulation mixture or granulating solution into contact with said coating agent (step ii(a)) or, alternatively, with a granulation aid in solid form (step ii(b)) so as to obtain a wet granulate;

(iii) subjecting said wet granulate, obtained from step (ii), to a step of drying (or desiccation), by bringing said wet granulate into contact with a heat source so as to obtain an active liposome in granular form.

E2. The process according to El, wherein in the granulation mixture or granulating solution, obtained from step (i), the components (a), (b) and (c) are present in a quantity by weight comprised between 1:1:1 and 1:1:15, preferably comprised between 1:1:15 and 1:1:10, even more preferably in a quantity by weight of 1 : 1 : 8.

E3. The process according to El or E2, wherein said at least one active ingredient (a) is berberine, preferably an extract of Berberis aristata 97%, said at least one coating agent is lecithin, preferably a lecithin of soya, maize, sunflower, or mixtures thereof, and said (c) is water; preferably in a quantity by weight of (a):(b):(c)=l : 1 :8.

E4. The process according to any one of El -3, wherein said granulation mixture, obtained from step (i), is subjected to a step of granulation (step (ii)) so as to obtain a wet granulate, said step (ii) being preferably carried out in a step ii(a) wherein the granulation mixture, obtained from step (i), is brought into contact with a granulation aid which is represented by the same coating agent used in step (i), preferably a lecithin, preferably a lecithin in powder form, even more preferably a lecithin of soya, maize or sunflower, or mixtures thereof, so as to obtain a wet granulate.

E5. The process according to E4, wherein in the wet granulate, obtained from step (ii), the components (a), (b) and (c) are present in a quantity by weight (a):(b):(c) comprised between 1:2:4 and 1:6:12, preferably in a quantity by weight of 1 :4: 8.

E6. The process according to E5, wherein said at least one active ingredient (a) is berberine, preferably an extract of Berberis aristata 97%, said at least one coating agent is lecithin, preferably a lecithin of soya, maize, sunflower, or mixtures thereof, and said (c) is water; preferably in a quantity by weight of (a) : (b) : (c)= 1:4:8.

E7. The process according to E6, wherein said wet granulate prepared in step (ii) contains a quantity of water comprised between 10% and 30% by weight, preferably between 15% and 25% by weight, with respect to the weight [(a)+(b)+(c)+ optional (d)] : [(coating agent)] . E8. The process according to any one of El -7, wherein said wet granulate, obtained from the step of granulation (ii), is subjected to a step of drying (step (iii)) which is preferably conducted using a fluid bed granulator, using hot air at a temperature comprised between 25°C and 60°C, preferably between 30°C and 55°C, even more preferably between 35°C and 50°C in order to eliminate the residual water present in the wet granulate so as to obtain an active liposome in granular form that has a quantity by weight of water comprised between 1% and 15%; preferably comprised between 3% and 12%; even more preferably comprised between 5% and 10%; with respect to the total weight of said active liposome. E9. An active liposome obtainable from a process according to any one of El -8; preferably containing berberine as the active ingredient (a), preferably an extract of Berberis aristata 91% in a quantity by weight comprised between 5% and 45%, of the total weight of said active liposome, and a lecithin as the coating agent, preferably a lecithin of soya, maize, sunflower, or mixtures thereof, and water. E10. A composition comprising a mixture comprising or, alternatively, consisting of an active liposome according to E9, and optionally a food- grade or pharmaceutical-grade additive and/or excipient; said composition being for use in a method for (a) treating and/or improving and/or regulating the digestive function, the hepatic function, intestinal transit, the functionality of the digestive system, or the functionality of the cardiovascular apparatus.

Below are some examples, provided by way of non-limiting examples, of the present invention.

EXAMPLES.

Example 1: Preparation of a liposome with a phospholipid bilayer encapsulating berberine (active ingredient) which comprises a dry extract of 97% Berberis aristata in berberine; lecithin of soya and water. Example 2: Pharmacokinetics of an active liposome encapsulating berberine.

The project entails the pharmacokinetic analysis of the berberine in rat blood samples taken at 10, 30, 60, 120 and 480 minutes from oral administration via stomach tube of a 20% formulation of berberine (Berberina Phytobilayer™, batch 2017030048) prepared as per Figure 1 and 97% berberine hydrochloride (batch DNBAJ20161205) used as a control.

The two raw materials were dissolved in methanol, sonicated for approximately ten minutes and the titer of berberine was determined by HPLC-DAD using a C8 4.6x150 column for the stationary phase and the eluting gradient shown in Table 2. Table 2

A flow of 1 ml/minute was used and the reading was taken at 350 nm. A standard solution of berberine was used as a control and was prepared under the same conditions as the samples and a calibration curve was drawn in the range of 10-100 pg/ml. The measured titer of the two raw materials is shown in Table 3, and complies with the declared values.

Table 3

The pharmacokinetics were measured after administration via stomach tube of 50 mg/kg of 97% berberine and Berberine Phytobilayer™ (the subject matter of the present invention) with equidose of berberine. Both were suspended in water at a concentration of 12.5 mg/ml of berberine equivalents and used for oral administration. The blood samples were taken after 10, 30, 60, 120 and 480 minutes from administration. For the analysis, 500 microliters of heparinized blood are added to 500 microliters of benzanilide solution in methanol (900 ng/ml), 3 ml of water are added, and mixed. 5 ml of ethyl acetate are added and extraction is carried out. The organic phase is separated and dried under vacuum, then the residue is recovered with 300 microliters of methanol and used for the chromatography. For calibration, solutions of berberine and benzanilide were prepared in the range of 1-0.01 ng/ml. Fixed amounts of benzanilide (900 ng) and variable amounts of berberine in a range of 4-100 ng/ml were added to untreated blood samples. The calibration samples were prepared as described previously. For the analysis, the column used was an Agilent Polaris 3 C18A 150 x 3 mm. As mobile phases, 1% formic acid in water and acetonitrile. A flow of 0.3 ml/minute was used and the eluting gradient is given in Table 4.

Table 4

Figure 2 shows an exemplary chromatogram of a sample at 120 minutes and a standard sample, showing the two traces for each sample corresponding to berberine (transition 336>291) and benzanilide (198>105). For the analysis, an Agilent 1260 system was used fitted with a 320 triple quadrupole detector (TQDMS). For the berberine, the 336>291 transition was used; for benzanilide 198>105. The LOD and LOQ are 0.3 ng/ml and 0.9 ng/ml respectively. Table 5 below gives the results of the pharmacokinetic analyses expressed in ng/ml, shown in graphic form in the accompanying Figure 3. Table 5

From the analyses, at 60 minutes after administration of Berberine Phytobilayer™ (according to the present invention), a clear increase of the blood peak of 5.6 times with respect to the control was observed. Furthermore, maintenance of higher blood levels (approximately double with respect to the control) was also observed at 120 minutes and 480 minutes. Considering the dose of 50 mg/kg and the areas under the curve, and considering the amount administered, it was observed that the amount of berberine absorbed is 1.3% for the oral suspension, but a full 2.5% for the active liposome.

Example 3: Stability tests of an active liposome encapsulating berberine.

The active liposome encapsulating berberine was subjected to an evaluation of physical stability over time when stored for one year, at two different temperatures (4°C and 22°C). A dry extract of 97% berberine was used for comparison. From these tests it becomes clear that the dry extract of 97% berberine exhibits some changes that could be correlated to the hygroscopicity of the dry extract and to the tendency of berberine to crystallize in hydrated form.

By contrast, for the active liposome encapsulating berberine it can be seen that the storage at different temperatures has substantially no effects on the sample.

Figures from 4 to 9 show optical microscope images and SEM analyses conducted on the various samples.

Advantageously, the active liposome according to the present invention allows to increase the bioavailability of the active ingredients encapsulated therein, and to obtain a longer release of such active ingredients over time.

Advantageously, the active liposome obtained with the process according to the present invention allows to obtain a greater solubility in water of active ingredients that otherwise have poor solubility or are even insoluble.

Advantageously, the active liposome obtained with the process according to the present invention allows to mask the flavors of the encapsulated active ingredients. Advantageously, the active liposome obtained with the process according to the present invention has a high stability over time.

The disclosures in Italian Patent Application No. 102020000011395 from which this application claims priority are incorporated herein by reference.

Claims

1. A process for preparing an active liposome, which comprises an active ingredient in a liposome, wherein said process comprises the following steps of:

(c) water, and

(d) optionally technological additives and/or excipients;

(ii) subjecting said granulation mixture or granulating solution, obtained from step (i), to a step of granulation carried out in a fluid bed granulator, preferably by nebulization or spraying, bringing said granulation mixture or granulating solution into contact with said coating agent (step ii(a)) or, alternatively, with a granulation aid in solid form (step ii(b)) so as to obtain a wet granulate;

2. The process according to claim 1, wherein in the granulation mixture or granulating solution, obtained from step (i), the components (a), (b) and (c) are present in a quantity by weight comprised between 1:1:1 and 1:1:15, preferably comprised between 1:1:15 and 1:1:10, even more preferably in a quantity by weight of 1 : 1 : 8.

3. The process according to claim 1 or 2, wherein said at least one active ingredient (a) is berberine, preferably an extract of Berberis aristata 97%, said at least one coating agent is lecithin, preferably a lecithin of soya, maize, sunflower, or mixtures thereof, and said (c) is water; preferably in a quantity by weight of (a):(b):(c)=l : 1 :8.

4. The process according to any one of claims 1-3, wherein said granulation mixture, obtained from step (i), is subjected to a step of granulation (step (ii)) so as to obtain a wet granulate, said step (ii) being preferably carried out in a step ii(a) wherein the granulation mixture, obtained from step (i), is brought into contact with a granulation aid which is represented by the same coating agent used in step (i), preferably a lecithin, preferably a lecithin in powder form, even more preferably a lecithin of soya, maize or sunflower, or mixtures thereof, so as to obtain a wet granulate.

5. The process according to claim 4, wherein in the wet granulate, obtained from step (ii), the components (a), (b) and (c) are present in a quantity by weight (a):(b):(c) from 1:2:4 to 1:6:12, preferably in a quantity by weight of 1:4:8.

6. The process according to claim 5, wherein said at least one active ingredient (a) is berberine, preferably an extract of Berberis aristata 97%, said at least one coating agent is lecithin, preferably a lecithin of soya, maize, sunflower, or mixtures thereof, and said (c) is water; preferably in a quantity by weight of (a):(b):(c)=l:4:8.

7. The process according to claim 6, wherein said wet granulate prepared in step (ii) contains a quantity of water comprised between 10% and 30% by weight, preferably between 15% and 25% by weight, with respect to the weight [(a)+(b)+(c)+ optionally (d)] : [(coating agent)] .

8. The process according to any one of the preceding claims, wherein said wet granulate, obtained from the step of granulation (ii), is subjected to a step of drying (step (iii)) which is preferably conducted using a fluid bed granulator, using hot air at a temperature comprised between 25°C and 60°C, preferably between 30°C and 55°C, even more preferably between 35°C and 50°C in order to eliminate the residual water present in the wet granulate so as to obtain an active liposome in granular form that has a quantity by weight of water comprised between 1% and 15%; preferably comprised between 3% and 12%; even more preferably comprised between 5% and 10%; with respect to the total weight of said active liposome.

9. An active liposome obtainable from a process according to any one of the preceding claims 1-8; preferably containing berberine as the active ingredient (a), preferably an extract of Berberis aristata 91% in a quantity by weight comprised between 5% and 45%, of the total weight of said active liposome, and a lecithin as the coating agent, preferably a lecithin of soya, maize, sunflower, or mixtures thereof, and water.

10. A composition comprising a mixture that comprises or, alternatively, consists of an active liposome according to claim 9, and optionally a food-grade or pharmaceutical-grade additive and/or excipient; said composition being for use in a method for (a) treating and/or improving and/or regulating the digestive function, the hepatic function, intestinal transit, the functionality of the digestive system, or the functionality of the cardiovascular apparatus.