Hydrophobic phase composition comprising dehydrated liposomes with nutritional supplement

Abstract

The present invention relates to a composition for administration to a subject, comprising dehydrated liposomal vesicles in a hydrophobic phase, wherein the dehydrated liposomal vesicles comprise a nutritional supplement selected from the group consisting of vitamins, minerals, enzymes, proteins, amino acids, peptides, and / or herbal extracts and to a method for preparing the composition.

Description

[0001] Title: HYDROPHOBIC PHASE COMPOSITION COMPRISING DEHYDRATED LIPOSOMES WITH NUTRITIONAL SUPPLEMENT

[0002] Technical Field

[0003] The current disclosure relates to a hydrophobic phase composition for administration to a subject, comprising dehydrated liposomal vesicles, wherein the dehydrated liposomal vesicles comprise a nutritional supplement, and to a method for preparing the composition.

[0004] Background Art

[0005] A liposome is a spherical vesicle having at least one lipid bilayer. Liposomes are most often composed of phospholipids, but may also include other lipids. The lipid bilayer closely resembles human cell membranes, which is why the content of liposomes can be more readily absorbed after administration. Hydrophilic biologically active components may be dissolved in the core of a liposome, whereas lipophilic components associate with the bilayer. A liposome can hence be loaded with hydrophobic and / or hydrophilic molecules. To deliver the molecules to a site of action, the lipid bilayer can fuse with other bilayers such as the cell membrane. Liposomes are most commonly used in the drug / medicine industry. Compared to conventional (i.e. non-liposomal) oral drug delivery methods, use of drug-loaded liposomes leads to an increased bioavailability of the drugs, and therefore an increased efficacy of the treatment.

[0006] Use of liposomes for delivery of nutritional supplements is much less common. In the art, liposomes loaded with drug molecules are commonly prepared as aqueous dispersions. A problem with these dispersions is that they only have limited physical stability. The liposomes can aggregate and precipitate as sediment. Additionally, on storage the biologically active compounds may be lost into the external aqueous phase. Furthermore, depending upon the type of lipid and biologically active compound present in the liposome, there is the potential for chemical degradation of the lipid components and / or the biologically active components in the aqueous dispersion. Thereto, US 4830858 discloses a method for preparing a spray-dried mixture of liposomal components which may be stored dry and reconstituted to form a liposome. US 4830858 does not relate to nutritional supplements.

[0007] WO2019221598 discloses a composition comprising dehydrated liposomes with a nutritional supplement. Before administration to a subject, said dehydrated liposomes with nutritional supplement are resuspended in aqueous medium. However, upon resuspension, the problem remains that the liposomes can aggregate and quickly precipitate as sediment. Also, the absorption of the nutritional supplement by the subject leaves something to be desired.

[0008] Summary of disclosure

[0009] It is an objective of the present disclosure to solve one or more of the above- mentioned or other problems in the art. In particular, it is an objective to provide a formulation with good bioavailability for nutritional supplements. A further objective of the present disclosure is to provide a form that is stable during use.

[0010] Thereto, the present disclosure provides a composition, preferably for intranasal or oral administration to a (human) subject, comprising dehydrated liposomal vesicles in a hydrophobic phase, wherein the dehydrated liposomal vesicles comprise a nutritional supplement.

[0011] Surprisingly it has been found that administration of such a composition displays very satisfactory bioavailability. In addition, it was found that the composition is very stable, i.e. the dehydrated liposomal vesicles are homogenously distributed in the composition, and do not or hardly aggregate / precipitate. Also the oxidation of the lecithin is prevented.

[0012] The nutritional supplement, which can be selected from vitamins, minerals, enzymes, proteins, peptides, amino acids and / or herbal extracts, may be fat-soluble (preferred), water- soluble (more preferred), and / or both. Even such molecules with relatively low water and / or low fat solubility may be used. For example the (nutritional) supplement may be a peptide of between 1-500 kDa, or between 1-100, or 1-10 kDa. In addition or alternatively, the (nutritional) supplement may be a peptide of between 2-100 amino acids in length, preferably 2-75 or 2-50 amino acids in length. In particular, the peptide may be BPC-157, TB-500; Colostrinine; or Epitalon. The peptide may find use in therapy such as in longevity, recovery (from injury), and / or cognitive enhancement.

[0013] The dehydrated liposomal vesicles preferably comprise a bilayer of lecithin extracts, which lecithin extracts may comprise at least 10 w% phosphatidylcholine.

[0014] The disclosure also relates to a method for preparing the composition, the method comprising the steps of: a) dissolving lecithin extracts, preferably comprising at least 10 w% phosphatidylcholine, in an organic solvent to form a solution, b) simultaneously stirring and drying the solution in a vessel, preferably under reduced pressure (relative to atmospheric pressure), forming a dry film on the wall of the vessel, c) adding to the vessel an aqueous medium such as a physiological salt solution and agitating the solution, thereby removing the dry film from the wall of the vessel and creating vesicles, d) drying the vesicles, optionally in the presence of a pharmaceutically acceptable carrier, preferably by vacuum drying, belt drying and / or spray-drying, e) mixing the dried vesicles with a hydrophobic phase, wherein in step a) a fat-soluble nutritional supplement is added to the solution, and / or in step c) a water-soluble nutritional supplement is added to the solution.

[0015] The preferred lecithin extracts comprising at least 10 w% of phosphatidylcholine result in (spray-and or freeze)dryable liposomal vesicles comprising the nutritional supplement(s) after step c).

[0016] It is known from Ingvarsson et al., Expert Opinion on Drug Delivery 2011, 8 (3), 375- 388, that stabilization of liposomes during drying is an issue. Two main stress factors (heat and high shearing forces) are involved in e.g. the spray-drying process and may disrupt the liposomal bilayer structure and result in degradation of the lipid components during the process. In a preferred embodiment, when the lecithin extracts comprise at least 10 w% of phosphatidylcholine, the dehydrated liposomal vesicles are more stable during the dehydration / drying process.

[0017] The resulting composition is a dry (i.e. dehydrated) powder, which can be mixed with hydrophobic (continuous) phase, such as oil, fat, fatty acids, and / or wax for oral / intranasal / sublingual intake. It may for example be used as a food supplement or a medicament as described below.

[0018] Description of Embodiments

[0019] Preferably, the nutritional supplement is chosen from cannabidiol (CBD), vitamin C, folic acid, nicotinamide adenine dinucleotide (NAD), NMN (i.e. Nicotinamide Mononucleotide, a nucleotide derived from niacin (vitamin B3), Nicotinamide- riboside, Gamma-Aminobutyric Acid (GABA), melatonin, taurine and / or vitamin B12 etc. Mucosal uptake in the mouth or in the nose is improved as compared to conventional delivery methods such as tablets, powders and capsules (not comprising liposomes), as proven in the experimental section by increased blood plasma levels.

[0020] Cannabidiol (CBD) is one of the at least 113 cannabinoids identified in cannabis. It is a major phytocannabinoid, which accounts for 40% of the plant’s extract. CBD does not appear to have any psychoactive effects such as those caused by tetrahydrocannabinol (THC). It is believed to have a downregulating impact on disordered thinking and anxiety. Potential uses are the subject of ongoing research. Cannabidiol is insoluble in water but soluble in organic solvents such as pentane and edible oils.

[0021] Vitamin C is also known as ascorbic acid or L-ascorbic acid. Instead of the pure vitamin it may be used in the form of salts like sodium ascorbate, potassium ascorbate and / or any other pharmaceutically acceptable salt. These salts have lesser pH values and are therefore result in lesser gastro-intestinal problems. Vitamin C and it salts are water soluble and an essential nutrient involved in the repair of tissue and the enzymatic production of certain neurotransmitters. Vitamin C is required for the functioning of several enzymes and is important for immune system function. It also functions as an antioxidant. High-dose vitamin C has been studied as a treatment for patients with cancer since the 1970s. When taken by intravenous infusion, vitamin C can reach much higher levels in the blood than when the same amount is taken orally. As reported in Proc. Natl. Acad. Sci. USA 93 (1996) 3704-3709, Fig. 1 C, the maximal plasma vitamin C plateau level when taken orally is about 80 pM. This level is obtained by a dosage of 1 to 2.5 g per day, and higher plasma levels do not seem obtainable, even at higher dosages. When higher plasma levels are desired, intravenous intake is required. With the present disclosure, the required high plasma levels are obtainable by oral intake. This eliminates the need for intravenous infusion.

[0022] Folic acid is converted into folate by the body, is used as a dietary supplement and in food fortification as it is more stable during processing and storage. Folate is also known as vitamin B9 and folacin, and is one of the B vitamins.

[0023] Nicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism. [3] Found in all living cells, NAD is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine nucleobase and the other, nicotinamide. NAD exists in two forms: an oxidized and reduced form, abbreviated as NAD+ and NADH (H for hydrogen), respectively.

[0024] Nicotinamide Mononucleotide (NMN) is a nucleotide derived from niacin (vitamin B3) and is a precursor to Nicotinamide Adenine Dinucleotide (NAD+), a coenzyme that is crucial for energy metabolism and various cellular processes. Health benefits include anti-aging, improving insulin sensitivity and improving cardiovascular health.

[0025] Gamma-Aminobutyric Acid (GABA) is a naturally occurring amino acid that functions as the primary inhibitory neurotransmitter in the central nervous system (CNS) of mammals, including humans. It plays a crucial role in reducing neuronal excitability and maintaining a balance between excitation and inhibition in the brain. GABA Supplements are often marketed to promote relaxation, reduce anxiety, and improve sleep. However, their efficacy can vary, as GABA itself has limited ability to cross the blood-brain barrier.

[0026] Melatonin is a hormone primarily produced by the pineal gland in the brain. It plays a crucial role in regulating sleep-wake cycles, also known as the circadian rhythm. Melatonin supplements are commonly used to treat sleep disorders, such as insomnia and delayed sleep phase syndrome. They are also used to help with sleep disturbances caused by shift work or jet lag.

[0027] Taurine is an amino sulfonic acid that is important in several physiological processes. Health Benefits include improving cardiovascular health, improving insulin sensitivity and blood sugar control and maintenance of retinal health. More preferably, the nutritional supplement is vitamin B12. Vitamin B12 is a vitamin that plays a role in mammalian growth, haematopoiesis, production of epithelial cells, and maintenance of the nervous system. It is quite water-soluble and thus could be expected to be easily available to human subjects. However, the absorption from the gut of normal dietary amounts of vitamin B12 is believed to be dependent on gastric Intrinsic Factor (GIF), and the loss of Intrinsic Factor leads to vitamin B12 deficiency. The loss of ability to absorb vitamin B12 (B12) is the most common cause of adult B12 deficiency. Such a loss may, for example, be due to pernicious anaemia (with loss of Intrinsic Factor) or to a number of other conditions that decrease production of gastric acid, which also plays a part in absorption of B12 from foods. Deficiency is most significantly linked to inadequate absorption rather than low consumption, as those who consume high amounts of vitamin B12 may still experience deficiency as evidenced by a low blood concentration.

[0028] Vitamin B12 deficiency results in various undesirable conditions such as fatigue, depression, poor memory, etc. Other causes of vitamin B12 deficiency include atrophic gastritis (a thinning of the stomach lining), surgery in which part of the stomach and / or small intestine is removed, conditions affecting the small intestine (such as Crohn's disease, celiac disease, bacterial growth, or a parasite), excessive alcohol consumption, autoimmune disorders (such as Graves' disease or systemic lupus erythematosus) and drug abuse. Treatment of vitamin B12 deficiency is traditionally accomplished by highly dosed intramuscular injections due to low bioavailability of orally ingested (non-liposomal) vitamin B12. Such injection are usually given by a health physician. With the composition of the present disclosure, intramuscular injections are not necessary. Therefore, visiting a health professional in order to receive the treatment for vitamin B12 deficiency is also not necessary. The subject can administer the vitamin B12 himself. Moreover, as shown in the experiments, the vitamin B12 plasma concentration obtainable with vitamin B12 compositions according to the disclosure greatly exceeds values obtainable in the prior art.

[0029] In the present application, the term vitamin B12 includes cyano-cobalamin, hydroxycobalamin, methyl-cobalamin, 5’-deoxyadenosyl-cobalamin, aquacobalamin, glutathionyl- cobalamin and nitrilocobalamin, including the pharmaceutically acceptable salts thereof, and including mixtures thereof.

[0030] Preferably the vitamin B12 is methyl-cobalamin. Methyl-cobalamin is considered a powerful drug because it decomposes easily in water. Methyl-cobalamin is an active form of vitamin B12 in the central nervous system and is absorbed readily into the bloodstream. The value of using methyl-cobalamin has not been realized in compositions according to the prior art, i.e. liquid compositions, as methyl-cobalamin cannot be easily stored. Pharmaceutical compositions comprising methyl-cobalamin in aqueous solution have to be kept frozen, and therefore a liposomal composition with methyl-cobalamin is not self-evident. In fact, both commercially available solutions that supposedly contained methyl-cobalamin from well- known suppliers that were tested by the inventors (and that were stored by the inventors as prescribed by the respective suppliers) did in fact not contain any methyl-cobalamin, but only hydroxyl-cobalamin. This is likely due to degradation of methyl-cobalamin to hydroxylcobalamin during storage and transport. The composition of the present disclosure will benefit from improved stability, in particular for methyl-cobalamin.

[0031] According to a favourable embodiment, the hydrophobic (continuous) phase is chosen from at least one of oil, fat, fatty acids, and wax. Thus, a hydrophobic environment for the nutritional supplement is provided. In other words, the nutritional supplement is dispersed in the hydrophobic (continuous) phase. In general, the fatty acids have a length of the carbon chain of at least 6. According to a favourable embodiment, the hydrophobic continuous phase is (edible) oil / fat. The term “hydrophobic” is clear to the skilled person and means that mixing a liquid continuous phase with water forms an emulsion (with or without emulsifier). A hydrophobic phase typically has a water solubility of below 500, 250, 100, 50, 10, preferably below 5, or 1 mg / L pure water at room temperature, i.e. 20 degrees Celcius. If the hydrophobic continuous phase comprises more than one constituent, the average of their water solubility is considered, taking into account their relative wt.% with respect to the hydrophobic continuous phase as a whole.

[0032] The term fat-soluble (or oil-soluble) means that the respective compound, e.g. the nutritional supplement, has a solubility in sesame oil of more than 1, 5, 10, 50, 100, 250, 500 mg / L pure sesame oil and / or a solubility in water of less than 100, 50, 10, 5, 1 mg / L. The term water-soluble means that the respective compound, e.g. the nutritional supplement, has a solubility in water of more than 1, 5, 10, 50, 100, 250, 500 mg / L pure water and / or a solubility in sesame oil of less than 100, 50, 10, 5, 1 mg / L.

[0033] According to a favourable embodiment, the hydrophobic continuous phase is anhydrous. This promotes the release of vitamin B12 from the pharmaceutically acceptable carrier into the mucous membrane. Anhydrous, within the context of the present disclosure, means a water content of less than 5 wt.%, preferably less than 1 wt.% and more preferably with less than 0.2 wt.% with respect to the weight of the hydrophobic continuous phase.

[0034] It is possible to strengthen the composition (or the dehydrated liposomal vesicles) with a pharmaceutically acceptable and water soluble carrier. Suitable pharmaceutically acceptable carriers include microcrystalline cellulose, microfine cellulose, lactose, starch, pregelatinized starch, calcium carbonate, calcium sulfate, sugar, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, kaolin, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, polymethacrylate, potassium chloride, powdered cellulose, sodium chloride, sorbitol, talc, acacia, alginic acid, carbomer, carboxymethylcellulose sodium, dextrin, ethyl cellulose, gelatin, guar gum, hydrogenated vegetable oil, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, liquid glucose, magnesium aluminum silicate, maltodextrin, methylcellulose, polymethacrylates, povidone, pregelatinized starch, sodium alginate, starch, alginic acid, carboxymethyl cellulose calcium, colloidal silicon dioxide, croscarmellose sodium, crospovidone, guar gum, magnesium aluminum silicate, methyl cellulose, polacrilin potassium, powdered cellulose, pregelatinized starch, aerosil, sodium alginate and sodium starch glycolate.

[0035] Preferably, the pharmaceutically acceptable carrier is chosen from the group consisting of monosaccharides, polysaccharides, phospholipid(s), maltodextrin, cellulose, aerosil, and starches. More preferably the pharmaceutically acceptable carrier is chosen from the group consisting of maltodextrin, cellulose, aerosil, and starches. These carriers are readily soluble in / miscible with water and have a pleasant taste.

[0036] Preferably, the composition (or the dehydrated liposomal vesicles) comprises between 5-90 w% or between 10 and 75 w% of the carrier.

[0037] Preferably, the dehydrated liposomal vesicles comprise a bilayer of lecithin extracts, which lecithin extracts preferably comprise at least 5, 10, 20, 25 w% phosphatidylcholine. Preferably, the remainder of the lecithin extracts are phospholipids other than phosphatidylcholine. Surprisingly, vesicles with these compositions have been shown not to suffer from any disadvantageous effects of the drying step. The size of the liposomes before drying as well as after drying and resuspension in water is similar.

[0038] The term “dehydrated” means that the dehydrated liposomal vesicles are substantially free of water, i.e. “dehydrated liposomal vesicles” refers to liposomal vesicles preferably comprising at most 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 % water with respect to the total weight of the liposomal vesicles. In addition or alternatively, “dehydrated liposomal vesicles” refers to a (substantially dry) powder comprising liposomal vesicles. The term “vesicle” in the context of the present disclosure particularly refers to a (bi)layer-bound (or membrane-bound) sac which can comprise / store / transport substance(s) (e.g. nutritional supplement). The vesicle typically is spherical-shaped in water, but may have an alternate shape when dried or when in hydrophobic phase (e.g. oil). The term “nutritional supplement” can refer to any substance / compound which preferably can be added to a diet, therapy and / or is nutritious. The term “nutritional supplement” may be replaced by the term “supplement”’. The term “liposomal” preferably refers to the characteristic of having a bilayer of phospholipids (which are molecules with hydrophilic (water-attracting) heads and hydrophobic (water-repelling) tails). This bilayer structure forms a vesicle that can encapsulate substances / compounds.

[0039] More preferably, the lecithin extracts comprise between 10 - 75 w% phosphatidylcholine. Preferably, the dehydrated liposomal vesicles have size of between 50 - 500 nm for a good uptake of the vesicles. Preferably the dehydrated liposomal vesicles have size of between 50 - 250 nm, to provide for better uptake. Most preferably the dehydrated liposomal vesicles have size of between 50 - 200 nm. Uptake of such vesicles is optimal.

[0040] Preferably, the composition comprises between 1 - 50 w / w% of nutritional supplement.

[0041] The composition according to the disclosure may be used as a medicament. Notably, the composition wherein the dehydrated liposomal vesicles comprise vitamin B12 may be used as a medicament. Most notably, the composition wherein the dehydrated liposomal vesicles comprise methyl-cobalamin may be used as a medicament. Specifically, these compositions may be used in the treatment of vitamin B12 deficiency. The vitamin B12 deficiency is any condition where an increased level would be of benefit to the subject, which can be a human or an animal. It may be a condition chosen from pernicious anaemia, autism spectrum disorder, fatigue, memory deficiency, ALS, Alzheimer, deficiency caused by drug abuse, thinning of the stomach lining, vitamin B12 deficiency after surgery in which part of the stomach and / or small intestine is removed, Crohn's disease, celiac disease, Graves' disease, systemic lupus erythematosus and migraine. At present, treatment of vitamin B12 deficiency is conducted with vitamin B12 injections.

[0042] Oral or intranasal use as a medicament of a composition comprising vitamin B12 for treatment of vitamin B12 deficiency is not self-evident. Vitamin B12 is traditionally administered by intramuscular injections due to low bioavailability of orally ingested (non- liposomal) vitamin B12. Literature studies have indicated that with conventional orally ingested vitamin B12 compositions (non-liposomal), plasma levels higher than 400 pmol / L are not obtainable, even after prolonged treatment. The composition of the disclosure is particularly effective for oral / intranasal treatment of vitamin B12 deficiency, since a single dose was shown to be effective for reaching a plasma level much higher than 400 pmol / L.

[0043] The composition according to the present disclosure preferably does not comprise

[0044] - preservative(s) such as chosen from benzalkonium chloride, phenylmercuric nitrate, phenylmercuric acetate, chlorobutanol, thimerosal, potassium sorbate, and disodium EDTA; and / or

[0045] - silica such as chosen from amorphous silica or any other substance comprising SiC>2.

[0046] Where in the present disclosure reference is made to oral administration, rectal administration is a viable alternative. The preferred embodiments discussed above are equally applicable to this use, are included by reference for this use, and are not repeated for the sake of brevity only.

[0047] The method according to the disclosure optionally comprises a step c’) of sieving the vesicles to remove large vesicles and aggregates, after between step c) and d). This ensures a homogeneous particles size distribution. As required, detailed embodiments of the present disclosure are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the disclosure, which can be embodied in various forms.

[0048] The terms "a" / "an", as used herein, are defined as one or more than one. The terms including and / or having, as used herein, are defined as comprising (i.e. , open language, not excluding other elements or steps). The mere fact that certain measures are recited in mutually different dependent clauses does not indicate that a combination of these measures cannot be used to advantage. All reaction conditions are under atmospheric pressure, unless otherwise indicated.

[0049] Examples

[0050] General protocol

[0051] Lecithin extracts comprising at least 25 w% phosphatidylcholine and up to 75 w% other phospholipids (the total adding up to 100 % phospholipids) and any fat-soluble ingredients (e.g. CBD) are dissolved in a flask in an organic solvent such as ethanol, methanol, chloroform, ethyl acetate pentane and / or any other organic solvent which can easily be evaporated.

[0052] The organic solvent is removed under reduced pressure while agitating, thereby forming a dry lipid film with fat-soluble active ingredients on the internal wall of the flask. Subsequently the material is diluted with a physiological salt solution, comprising any water- soluble active ingredients (e.g. vitamin C and / or B12). All materials are mixed under firm agitation, thereby creating a slurry.

[0053] Nano sized particles of about 50 - 500 nm are obtained by sonification of the slurry and / or by high pressure extrusion. The particles may optionally be sieved through a 0.2 micron sieve in order to separate the particles with a size of 200 nm or less from the larger particles and aggregates.

[0054] After sieving, the particles are checked with light microscopy and laser diffraction by checking a liposomal suspension comprising the chosen nutritional supplement in water.

[0055] The suspended particles can also be dried in the presence of a carrier (e.g. maltodextrine, cellulose, etc.) by either vacuum drying, belt drying and / or spray drying to obtain a powder. This powder is mixed with a hydrophobic phase.

[0056] Reference example - liposomal vitamin B12 in water, intranasal intake

[0057] A volunteer was administered liposomal vitamin B12 (in the form of hydroxylcobalamin) at a dose of 1 mg. The powder was administered intranasally after first mixing the powder in water. Within 30 minutes the plasma concentration increased from 411 pmol / L to 1075 pmol / L. This is a significant improvement with respect to oral intake of non-liposomal vitamin B12 in tablets and capsules as found in literature, e.g. in Sharabi et al., J. Clin. Pharmacol., 56, 635-638. In the latter paper, the plasma concentration did not exceed 400 pmol / L, even after several weeks of treatment.

[0058] Reference example - liposomal vitamin B12 in water, oral intake

[0059] The same volunteer as in the previous example (but one week later) was administered liposomal vitamin B12 (in the form of hydroxyl-cobalamin) at a dose of 10 mg. The powder was administered orally after first mixing the powder in water. Within 4 hours the plasma concentration increased from 3761 pmol / L to 1051 pmol / L.

[0060] Example - liposomal vitamin B 12 in hydrophobic phase, intranasal intake

[0061] The same volunteer as in the reference example (but one week later) was administered liposomal vitamin B12 (in the form of hydroxyl-cobalamin) at a dose of 1 mg. The powder was administered intranasally after first mixing the powder in sesame oil. Within 30 minutes the plasma concentration increased from 387 pmol / L to >1476 pmol / L. This is a significant improvement with respect to intranasal intake of liposomal vitamin B12 in water.

[0062] Example - liposomal vitamin B 12 in hydrophobic phase, oral intake

[0063] Another volunteer was administered liposomal vitamin B12 (in the form of hydroxylcobalamin) at a dose of 10 mg. The powder was administered orally after first mixing the powder in sesame oil. Within 2 hours the plasma concentration increased from 259 pmol / L to >1476 pmol / L. This is a significant improvement with respect to oral intake of liposomal vitamin B12 in water.

[0064] Example - liposomal folic acid in hydrophobic phase, intranasal intake

[0065] The same volunteer as in the previous example (but one week later) was administered liposomal folic acid at a dose of 100 pg. The powder was administered intranasally after first mixing the powder in sesame oil. Within 30 minutes the plasma concentration increased from 8.4 nmol / l to >45.3 nmol / l. This is a significant improvement with respect to intranasal intake of liposomal folic acid in water.

[0066] Example - Examination of water and fat solubility of liposomes

[0067] Research Question:

[0068] In a nasal drop formulation based on oil (such as MCT oil) or another hydrophobic solvent, we have previously suspended a water-soluble compound. It was observed that this water- soluble substance is rapidly absorbed through the nasal mucosa. The reason is that a water- soluble molecule is more compatible with the aqueous environment of the nasal mucosa and can therefore be absorbed quickly. This process has also been demonstrated in the oral mucosa. As such, water-soluble molecules like vitamin B12 tend to be more efficiently absorbed when administered from a fatty base.

[0069] But what about lipophilic substances like cannabidiol (CBD) and coenzyme Q10, which are highly fat-soluble and poorly water-soluble — up to 3000 times less soluble in water? It can be expected that in such formulations, these compounds remain in the oil phase and do not migrate into the aqueous phase.

[0070] To demonstrate this, coenzyme Q10 was dissolved in MCT oil, resulting in a vivid red solution. In Figure 1 , Photo 1, this oily Q10 solution was mixed with water. As shown, the fatty Q10 floats on the surface of the water and does not mix. In Figure 1, Photo 2, oil was poured onto the mixture. The Q10 immediately dissolved into the oil, giving it a dark yellow to orange color, while the water layer changed only slightly in color. This demonstrates that the oily Q10 does not mix with water to any significant extent. The same result was observed with cannabidiol; photos are not shown here because CBD dissolves completely colorless in oil and is therefore not visually distinctive.

[0071] To increase the water solubility or dispersibility of coenzyme Q10, we incorporated it into liposomes. The same experiment was repeated, but now using liposomal Q10 in powder form. Figure 2, Photo 3 illustrates the result. The tube on the left contains the oil-dissolved form of Q10, which floats on the surface of the water layer, which is very lightly yellow- colored. In contrast, the tube on the right clearly shows that the liposomal Q10 has fully migrated into the lower aqueous phase and hardly at all into the upper oil phase. We have effectively rendered the lipophilic Q10 hydrophilic.

[0072] What is the implication of this in a nasal drop formulation?

[0073] From an aqueous nasal drop, a lipophilic compound such as Q10, CBD, or any other fatsoluble substance will not dissolve. Such substances only dissolve in an oil-based drop. An oil-based nasal drop offers the advantage that a hydrophilic compound can be absorbed through the nasal mucosa. This is not the case for a lipophilic substance — unless we make it hydrophilic. Liposomes offer a natural way to achieve this transformation.

[0074] Therefore, incorporating liposomes into an oil-based nasal drop is an excellent strategy to improve the stability of hydrophilic compounds in an oily medium and to convert hydrophobic compounds into hydrophilic ones, thereby increasing the likelihood that they will pass through the nasal mucosa. This makes the use of liposomes a valuable enhancement to oil-based nasal drops, allowing a broader range of active ingredients to be absorbed.

[0075] Comparative Example 1 - Dehydrated liposomes redispersed in water - instability without preservative Objective:

[0076] To show that dehydrated liposomes, when redispersed in water degrade or lose stability over time unless a preservative is added.

[0077] Materials and Methods:

[0078] Liposomal vitamin B12 powder was prepared by spray-drying liposomes made from lecithin containing 25 wt% phosphatidylcholine. The powder was redispersed in sterile water at a concentration of 10 mg / mL (hydroxocobalamin). Two formulations were prepared:

[0079] • 1.1 Without preservative.

[0080] • 1.2 With 0.1 % w / v benzyl alcohol as preservative.

[0081] Both formulations were stored at 25°C for 14 days in sealed glass vials. Samples were taken at day 0, day 7, and day 14. The physical stability was assessed visually (for sedimentation and color changes) and analytically using:

[0082] • Dynamic light scattering (DLS) for particle size.

[0083] • HPLC for vitamin B12 content.

[0084] Results:

[0085] • Formulation 1.1 (no preservative) showed visible aggregation and sedimentation after 7 days, with a significant loss of vitamin B12 (40% decrease) after 14 days.

[0086] • Formulation 1.2 (with preservative) remained physically stable over the same period with no aggregation and <5% loss of active.

[0087] Conclusion:

[0088] This comparative example confirms that redispersed liposomes in water are chemically and physically unstable over time without preservatives, which may necessitate sterile production or antimicrobial additives.

[0089] Comparative Example 2: Vitamin B 12 in oil - sedimentation and sprayability issues without silica

[0090] Objective:

[0091] To show that vitamin B12 dissolved or dispersed in oil is physically unstable and unsuitable for nasal spray administration unless silica is added.

[0092] Materials and Methods:

[0093] Vitamin B12 (hydroxocobalamin) was dispersed in MCT oil at 1 mg / mL. Two formulations were tested:

[0094] • 2.1 Without any excipient.

[0095] • 2.2 With 2% w / w colloidal silica (Aerosil 200) to increase viscosity and suspension stability.

[0096] Both formulations were filled into 10 mL nasal spray bottles and stored at 25°C.

[0097] Sedimentation was monitored visually and by UV-Vis spectrophotometry (absorbance at 361 nm) from the upper 1 mL layer. Sprayability was tested using a standard pump nasal spray device (0.1 mL per actuation).

[0098] Results:

[0099] • Formulation 2.1 (no silica) exhibited complete sedimentation of B12 within 24 hours. The top layer became clear, and the nasal spray pump failed to deliver an even suspension (clogging occurred).

[0100] • Formulation 2.2 (with silica) showed improved suspension stability, with no visible sedimentation after 7 days. Sprayability was acceptable.

[0101] Conclusion:

[0102] Oil-based B12 formulations are not sprayable without silica, but silica is not always desirable (e.g. for regulatory or safety reasons).

[0103] Example According to the Invention: Dehydrated liposomes in oil - stable and sprayable without silica or preservatives

[0104] Objective:

[0105] To demonstrate that dehydrated liposomes containing vitamin B12, dispersed in oil, form a stable and effective nasal spray formulation without the need for preservatives or silica. Materials and Methods:

[0106] Dehydrated liposomal powder containing methylcobalamin was prepared by dissolving lecithin (25% phosphatidylcholine) in ethanol, forming a lipid film, hydrating with aqueous B12, followed by spray-drying with 10% maltodextrin as carrier. The resulting powder was dispersed in MCT oil at 1 mg / mL B12 concentration.

[0107] This formulation was filled into nasal spray bottles and stored at 25°C. Physical stability was assessed by:

[0108] • Visual inspection (for sedimentation / phase separation),

[0109] • DLS (for particle size),

[0110] • UV-Vis spectroscopy (top-layer B12 quantification),

[0111] • Sprayability testing (uniformity and clogging).

[0112] Samples were tested on day 0, 7, 14, and 28.

[0113] Results:

[0114] • The formulation remained physically stable throughout the 28-day test, with no visible sedimentation or phase separation.

[0115] • Particle size remained consistent (-180 nm).

[0116] • Sprayability was excellent; no clogging or inconsistencies observed over 100 actuations.

[0117] • No preservatives or silica were needed to maintain formulation quality. Conclusion:

[0118] This example shows that dehydrated liposomes dispersed in oil form a stable, sprayable nasal formulation without the drawbacks of the comparative examples. It addresses the problems of instability without preservatives in water and instability and poor sprayability of B12 in oil without silica.

Claims

CLAIMS1. Composition comprising dehydrated liposomal vesicles in a hydrophobic phase, wherein the dehydrated liposomal vesicles comprise a nutritional supplement.

2. Composition according to any one of the preceding claims, wherein the nutritional supplement is water soluble.

3. Composition according to any one of the preceding claims, wherein the hydrophobic phase is an oil, fat or a wax.

4. Composition according to any one of the preceding claims, wherein the composition is for oral, sublingual or nasal administration to a subject.

5. Composition according to any one of the preceding clauses, wherein the nutritional supplement is chosen from vitamin, mineral, enzyme, protein, peptide, amino acid, and / or herbal extract.

6. Composition according to any one of the preceding claims, wherein the nutritional supplement is chosen from vitamin B12, vitamin C, folic acid and / or nicotinamide adenine dinucleotide.

7. Composition according to any one of the preceding claims, wherein the nutritional supplement is vitamin B12, preferably chosen from cyano-cobalamin, hydroxo-cobalamin, methyl-cobalamin, 5’-deoxyadenosyl-cobalamin, aquacobalamin, glutathionyl-cobalamin and nitrilocobalamin.

8. Composition according to any one of the preceding claims, wherein the composition does not comprise- preservative(s) such as chosen from benzalkonium chloride, phenylmercuric nitrate, phenylmercuric acetate, chlorobutanol, thimerosal, potassium sorbate, and disodium EDTA; and / or- silica such as chosen from amorphous silica or any other substance comprising SiC>2.

9. Composition according to any one of the preceding claims for use as a medicament.

10. Composition according to claim 7, for use in the treatment of vitamin B12 deficiency.

11. Method for preparing a composition according to any one of claims 1-8, comprising the steps of: a) dissolving lecithin extracts comprising at least 10 w% phosphatidylcholine in an organic solvent to form a solution, b) simultaneously stirring and drying the solution in a vessel under reduced pressure, forming a dry film on the wall of the vessel, c) adding to the vessel an aqueous medium and agitating the solution, thereby removing the dry film from the wall of the vessel and creating vesicles, d) drying the vesicles, preferably by vacuum drying, belt drying and / or spray-drying; e) mixing the dried vesicles with a hydrophobic phase, wherein in step a) a fat-soluble nutritional supplement is added to the solution, and / or in step c) a water-soluble nutritional supplement is added to the solution.

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