NL2035231B1

NL2035231B1 - New method comprising an aqueous nanoparticle composition comprising a hydrophobic compound

Info

Publication number: NL2035231B1
Application number: NL2035231A
Authority: NL
Inventors: Pieter Beintema Rients; Peter Joubert George
Original assignee: Biogntx R&D B V
Priority date: 2023-06-30
Filing date: 2023-06-30
Publication date: 2025-01-09
Also published as: WO2025005799A1; AR133128A1

Abstract

Described is a method for the preparation of an aqueous nanoparticle composition comprising a hydrophobic compound, comprising the steps of: a. Providing an emulsifier or a blend of emulsifiers in powder form; 10 b. Mixing one or more oils at a temperature above 40°C where all oils have become liquid, wherein said oils differ in melting temperature and which mixture comprises at least a sufficient amount of medium chain triglycerides to enable the composition formed in step g to be liquid at temperatures around about 4°C; 15 c. Adding the hydrophobic compound in any hydrophobic solvent to the oil mixture; d. Optionally letting the mixture cool down to room temperature; e. Adding the emulsifier powder and water to the oil mixture and letting the mixture emulsify, under optional agitation and heating to 30-40°C; 20 f. Subjecting the emulsified mixture to a sonication treatment comprising optionally mixing or fluidisation, until the average particle size of the mixture remains stable; g. Cooling down the sonicated mixture. and compositions produced by the above method. 25

Description

New method comprising an aqueous nanoparticle composition comprising a hydrophobic compound

BACKGROUND

Nanoparticles (NPs) and nanocarrier (NC) formulations of highly lipophilic drugs enable the delivery of compounds that previously could not be administered at therapeutic levels by conventional formulations. Complex NC constructs, such as liposomes, nanocapsules, polymeric NPs, micelles and polymersomes can improve the observed therapeutic effect of drug compounds by increasing solubility, improving pharmacokinetics or altering biodistribution. Metallic, organic, inorganic and polymeric nanostructures, including dendrimers, micelles, and liposomes are frequently considered in designing the target-specific drug delivery systems. In particular, those drugs having poor solubility with less absorption ability are tagged with these nanoparticles. However, the efficacy of these nanostructures as drug delivery vehicles varies depending on the size, shape, and other inherent biophysical/chemical characteristics. For these reasons, there is still need for a new, stable nanoparticle composition comprising a hydrophobic compound enabling long- term storage and easy application for producing a pharmaceutical composition comprising the hydrophobic compound.

In practice, (oral) cannabinoid compositions are usually provided in the form of a solution in an oily solvent wherein the cannabinoids such as cannabidiol (CBD) and tetrahydrocannabinol (THC) dissolve, allowing a rather concentrated cannabinoid content. Most of the known compositions are oil-based, i.e. an oily solution wherein the cannabinoids are dissolved, or a water-in oil dispersion, wherein the cannabinoids are in the oily phase. For oral administration, the oily solvent needs to be food grade and acceptable for oral administration. The composition is defined as oily or oil-based when more than half of the volume of the composition is an oil, and in case of a dispersion, the oily phase should be the continuous phase. Ingestion of oil is however cumbersome and since the ingestion volume is limited, the cannabinoid compositions known in the art are highly concentrated, e.g. in concentrations of 5 w/w% to 60 w/w%.

Such high concentrated composition are however difficult to dose properly and often, undesired side effects are observed. Furthermore, the bioavailability of cannabinoids from oily preparations is low, which means that much of the ingested active compound is not utilized.

In more recent times many approaches have been published to provide water- based compositions comprising cannabinoids. The present invention, which provides an aqueous nanoparticle composition, is advantageously suited for comprising a plant extract, such as a cannabinoid composition and thereby overcomes many of the disadvantages of the presently available aqueous dosage forms of cannabinoids.

Nanoparticles have been a big step forward in getting lipophilic drugs, like some components of cannabis, into the body. They have helped drugs work better by making them dissolve more easily, getting them to the right places in the body, and changing how they spread out once they are there. However, there are still some problems to solve. One big issue is finding a way to create nanoparticles that are stable for a long time and are easy to apply, especially for drugs that do not like water.

Right now, most methods for delivering cannabis components use an oily solution.

This is far from ideal. It is not easy to ingest, and the body does not absorb much of the drug. It also takes a long time (about 3.5 hours) for the drug to start working, and the dose (and thus the effects) can be unpredictable. Moreover, these methods usually have a high concentration of the drug, which can make dosing difficult and cause unwanted side effects. They also go through a process in the body called first pass metabolism, which can increase the drug's interaction with other substances in the body, complicating things further

SUMMARY OF THE INVENTION

The current invention provides a new method that uses an aqueous composition to deliver hydrophobic or amphiphilic components, such as cannabinoids. It is quick, customizable, and consistent. People taking the composition can feel the effects within seconds, offering immediate and consistent relief. It gets around many of the issues with oil-based methods, like the unpredictable absorption of oils, leading to results that are more reliable. In addition, it reduces first pass metabolism, so it does not interact as much with other substances in the body.

Even better, the present method entails a drug delivery system that may be programmed to release the drug in a specific way. This means specific tissues can be targeted, which can make the drug more effective and which can reduce side effects.

This invention addresses many of the problems with current drug delivery methods, and it could transform the way cannabinoids and other drugs that do not like water are administered.

The present invention relates to a method for the preparation of an aqueous nanoparticle composition comprising a hydrophobic compound, comprising the steps of: a. Providing an emulsifier or a blend of emulsifiers in powder form; b. Mixing one or more oils at a temperature above 40°C where all oils have become liquid, wherein said oils differ in melting temperature and which mixture comprises at least a sufficient amount of medium chain triglycerides to enable the composition formed in step g to be liquid at temperatures around about 4°C; c. Adding the hydrophobic compound in any hydrophobic solvent to the oil mixture; d. Optionally letting the mixture cool down to room temperature; e. Adding the emulsifier powder and water to the oil mixture and letting the mixture emulsify, under optional agitation and heating to 30-40°C; f. Subjecting the emulsified mixture to a sonication treatment comprising optionally mixing or fluidisation until the average particle size of the mixture remains stable; g. Cooling down the sonicated mixture.

Preferably in such a method the emulsifier is a blend of emulsifiers, preferably wherein said emulsifiers are non-toxic emulsifiers, more preferably wherein said blend comprises sugar-based emulsifiers, such as sucrose ester and/or cyclodextrin.

Especially preferred is an emulsifier blend comprising sucrose ester, cyclodextrin and lecithin, preferably sunflower lecithin, more preferably wherein the amount of lecithin is such, that in the final sonicated mixture from step g. the concentration of lecithin is less than 5%, preferably less than 2%, more preferably less than 1%. In this embodiment it is further preferred that the amount of sugar-based emulsifiers is at least two times the amount of lecithin, preferably at least four times. It is also preferred that the ratio between sucrose ester, cyclodextrin and lecithinis 2 : 2 : 1.

In another embodiment, the method preferably comprises oils or fats that are non toxic.

It is also preferred that the oil mixture comprises at least one oil with a melting point above 50°C, preferably above 60°C. In another preferred embodiment, the oil mixture comprises an oil with a melting point in between room temperature and body temperature.

In order to achieve the above conditions, the oil mixture preferably comprises stearic acid, coconut oil and medium chain triglycerides. Then preferably the oil mixture, when mixed with the hydrophobic compound comprises the components in a ratio of stearic acid : coconut oil : medium chain triglycerides : solvent with hydrophobic compound of 1:2:9:5.

In a further preferred embodiment a non-toxic antioxidant is added to the oil mixture, preferably wherein said antioxidant is a blend of antioxidants, more preferably wherein said antioxidant or blend of antioxidants in total does not exceed the amount of 10% of the oil mixture, preferably not exceed the amount of 5% of the oil mixture, more specifically wherein said blend of antioxidants comprises linseed oil, hempseed oil, tocopherol and/or rosemary extract; preferably where it comprises linseed oil, hempseed oil, tocopherol and rosemary extract, preferably inaratioof2:2:2: 1.

It is further preferred in the method of the invention that the water that is used to prepare the composition is food-grade water.

In a further preferred embodiment, the nanoparticles in the sonicated mixture will have a mean particle size of 10 — 600 nm, preferably of 50 — 150 nm and more preferably of 80 — 130 nm, most preferably about 110 nm.

Also preferred is a method as detailed herein wherein glycerol is added to the sonicated mixture, more preferably wherein the concentration of glycerol is more than 20%, preferably more than 25%.

The hydrophobic compound used in the method of the invention preferably is a plant- based extract in oil, more preferably the plant-based extract is an extract of Cannabis sativa, preferably, said extract comprises a cannabinoid, more preferably, said extract comprises a cannabinoid chosen from the group consisting of A9-tetrahydrocannabinol (THC), A9-tetrahydrocannabinolic acid (A9-THCA or THCA), A9-tetrahydrocannabio- rolic acid (A9-THCA-C1 or THCA-C1), A9-tetra-hydrocannabiorcol (A9-THCO-C1 or

THCO-C1), A9-tetrahydrocanna-biorcolic acid (A9-THCOA or THCOA), A9-tetra- hydrocannabivarin (A9-THCV or THCV), A9-tetrahydrocannabivarinic acid (A9-

THCVA or THCVA), trihydroxy-A9-tetrahydro-cannabinol (TRIOH-THC), A10- tetrahydro-cannabinol (A10-THC), tetrahydro-cannabiphorol (THCP), THC-O acetate

(THCO), hexa-hydrocannabinol (HHC), 10-oxo- A6a-tetrahydrocannabinol (OTHC),

A8-tetra-hydrocannabinol (A8-THC), AS8-tetrahydrocannabinolic acid (A8-THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidiorcol (CBDC1), cannabidiol-

C4 (CBDC4), cannabidiol dimethyl ether (CBDD), cannabidiol monomethyl ether 5 (CBDM), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), nabilone, nabiximol, anandamide, cannabigerol (CBG), cannabigerolic acid (CBGA), cannabigerolic acid A monomethykether (CBGAM), canna-bigerovarin (CBGV), cannabigerovarinic acid (CBGVA), cannabigerol mono-methylether (CBGM), cannabinol (CBN), cannabinolic acid (CBNA), cannabdiorcol (CBN-C1), cannabinol-C2 (CBN-C2), cannabivarin (CBN-

C3), cannabinol-C4 (CBN-C4), cannabinodivarin (CBNDC3), cannabinol methylether (CBNM-C5), cannabichromene (CBC), cannabichromenc acid (CBCA), cannabichromanon (CBCN-C5), cannabicoumaronone (CBCON-C5), cannabi- chromanone-C3 (CBCN-C3), cannabichromevarin (CBCV), cannabichromevarinic acid (CBCVA), cannabielsoin (CBE-C5), cannabigelndol-C3 (OH-iso-HHCVC3), C3- canna-bielsoicacid B (CBEA-C3 B), cannabifuran (CBF), dehydrocannabifuran (DCBF-C5), cannabifuran (CBF-C5), dehydrocannabifuran (DCBF or CBFD), cannabicyclol (CBL-C5), cannabicyclovarin (CBLV-C3), cannabitriol (CBT), cannabitriolvarin (CBTV), cannabiripsol (CBR), cannabinodivarin (CBV or CBVD), 2- arachidonoylglycerol (2-AG), 2-arachidonoylglycerol ether (2-AGE), isotetra- hydrocannabinol, isotetrahydrocannabivarin, palmitoylethanolamide, epigallo- catechin (EGC), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG), most preferably said extract comprises THC or a blend with THC.

In another embodiment the present invention relates to a method as described above, wherein an additional step of diluting the sonicated mixture obtained in step g. is performed to obtain a diluted composition, preferably, wherein the mixture is diluted with water, more preferably wherein the mixture is diluted in such a way that the dilution comprises between 0.001% and 5% of the hydrophobic compound, preferably between 0.005% and 1%, more preferably between 0.01% and 0.5%, more preferably between 0,02 % and 0.1%. Preferably the water used for the dilution is food grade.

Further it is preferred to add a stabiliser to the composition, preferably wherein said stabiliser is a food grade stabiliser, more preferably wherein said stabiliser is a gum, more preferably wherein said stabiliser comprises guar gum and/or xanthan gum, more preferably wherein the concentration of guar gum and/or xanthan gum in the diluted composition is between 0.01 and 0.05%, more preferably about 0.02%. In another preferred embodiment a preservative is added to the composition, preferably wherein said preservative is a food grade preservative, more preferably, wherein said preservative is chosen from the group consisting of ascorbic acid, sodium ascorbate, isoascorbic acid, sodium isoascorbicate, citric acid, sorbic acid, calcium sorbate, benzoic acid, potassium benzoate, acetic acid, erythorbic acid, sodium erythorbate, ethyl lauroyl arginate, long-chain glycolipids from Dacryopinax spathularia MUCL 53181, methyl-p-hydroxybenzoate, nisin, sulphurous acid, dimethyl dicarbonate, ascorbyl palmitate and blends thereof, more preferably .wherein said preservative comprises ascorbic acid, citric acid or sorbic acid or a blend thereof, preferably wherein the ascorbic acid, if present, is present at a concentration between 0.01% and 0,1%, preferably about 0.05%, and wherein the citric acid, if present, is present at a concentration between 0.005% and 0.05%, preferably at about 0.01%, and wherein the sorbic acid, if present, is present at a concentration between 0.05% and 0.5%, preferably about 0,1%. Further, it is preferred to add a flavouring compound, preferably a food grade flavouring compound.

In another preferred embodiment, panthenol is added to the oil mixture, preferably wherein panthenol is added to an amount between 0.5 and 5% of the oil mixture, more preferably an amount between 1 and 3% of the oil mixture.

The present invention also relates to an aqueous nanoparticle composition provided by a method as described above. Further, the invention also relates to a pharmaceutical composition comprising such an aqueous nanoparticle composition.

DETAILED DESCRIPTION

In the present invention, a nanoparticle composition is made by emulsifying an oil composition comprising a hydrophobic compound of interest with water by adding an emulsifier, after which the emulsion is sonicated to produce an aqueous nanoparticle solution.

A first step in the present invention for preparing the aqueous nanoparticle composition of the present invention is to provide an emulsifier or, preferably, a blend of emulsifiers. The goal of these emulsifiers is to provide a system from which nanoparticles may be produced and for this purpose, the emulsifier should be able to provide a sufficient stability. Further, since it is highly likely that eventually the composition is taken orally, it should also have a sufficient safety profile and it should provide for an acceptable taste. One preferred emulsifier is lecithin, this in itself already being a blend of glycerophospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and phosphatidic acid. The lecithin may be derived from various sources, such as soybean, rapeseed, cottonseed or sunflower. Lecithin is a food-grade product, has GRAS status and is admitted in Europe as food additive E322. However, due to its off-flavour, it is desirable that the amount of lecithin in the final composition is relatively low. Therefore, care should be taken that the concentration of lecithin is less than 5%, preferably less than 2%, more preferably less than 1% of the final sonified mixture as obtained in the process according to the present claims. Next to this low amount, it is also deemed advantageous to include further emulsifiers that would be able to mask the bad taste of lecithin. For this purpose, sugar-based emulsifiers are preferred, since they provide a sweet taste. Any sugar-based emulsifier that is non-toxic and which has a sweet taste can be used, such as sucrose esters, cyclodextrin, sucralose esters, sophorolipids, and the like. For an optimal taste masking effect the concentration of these sugar-based emulsifiers is at least two times the concentration of lecithin and preferably at least four times. The most preferable combination of emulsifiers is a blend of sunflower lecithin, sucrose ester (e.g. Ryoto™ sugar ester P-1670 obtainable from Mitsubishi Chemical Corporation) and B-cyclodextrin, most preferably in a ratio of 1:2:2. This blend also shows a lower toxicity profile than traditional emulsifiers or emulsifier blends with a lower formation of harmful free radicals and degradation products.

In a second step of the method, a mixture of oils is provided. Similar to the blend of emulsifiers, also for the oil mixture only oils should be included which are non-toxic and food-grade. Further, in order to be able to regulate the viscosity and stability of the nanoparticle solution oils of different melting temperatures should be used. For obtaining a good stability of the nanoparticles at least one oil with a melting temperature of more than 50°C, preferably more than 60°C should be used, such as myristic acid, palmitic acid, stearic acid or arachidic acid. Preferably, stearic acid (E570), that has a melting point of nearly 70°C, is used. Stearic acid is one of the most common saturated fatty acids found in nature and in the food supply and it is often used in (nonalcoholic) beverages. Stearic acid is preferably used since it advantageously stabilises the nanoparticles that will be formed in the process.

Further preferred in the oil mix is an oil that has a melting point that lies between room temperature and body temperature. Such an oil may for example be chosen from coconut oil, cocoa butter, palm kernel oil, peanut oil and babassu oil. Preferable is coconut oil since this is cheap and easily commercially obtainable. Lastly, the oil mixture should also contain a component that would provide for a low melting point, such that the mixture of oil, water and emulsifier still is liquid at about 4°C. For this ingredient an oily substance with a very low melting point should be taken, such as olive oil, rapeseed oil, sunflower oil, soybean oil, castor oil, tung oil, cottonseed oil, or medium chain triglycerides (MCTs). MCTs are triglycerides with two or three fatty acids having an aliphatic tail of 8 — 12 carbon atoms, ie. medium-chain fatty acids (MCFAs). Preferably, medium chain triglycerides are used since these are completely saturated, which means that they are unlikely to react during sonication or mixing. Further MCTs are stable over a wide temperature range through all processing conditions. Further, it is a cheap source of oil and safely, rapidly metabolized by the body into known, safe metabolites with an extremely favourable safety profile. Also, they produce a small particle size that easily sonicates and MCTs can easily be obtained in high purity. In a further preferred embodiment, C8 MCT (caprylic acid MCT) is used. The oil mixture is prepared by adding all components at a temperature at which all the oils/fats are liquid (and which is below the boiling temperature of any of the present components). To this oil mixture the hydrophobic compound of interest is added which may or may not be present in a hydrophobic solvent. If such a hydrophobic solvent is used, care should be taken that the solvent is nontoxic and acceptable in food applications, at least at a concentration at which it will be available in the final product. Preferably the hydrophobic compound in a solvent is a plant extract in oil, preferably an extract from hemp (Cannabis sativa) comprising one or more cannabinoids, more preferably comprising at least THC (A9-tetrahydrocannabinol).

However, any cannabinoid compound may be included, such as selected from the group of A9-tetrahydrocannabinol (THC), A9-tetrahydrocannabinolic acid (A9-THCA or THCA), A9-tetrahydrocannabiorolic acid (A9-THCA-C1 or THCA-C1), A9-tetra- hydrocannabiorcol (A9-THCO-C1 or THCO-C1), A9-tetrahydrocanna-biorcolic acid (A9-THCOA or THCOA), A9-tetra-hydrocannabivarin (A9-THCV or THCV), A9-tetra- hydrocannabivarinic acid (A9-THCVA or THCVA), trihydroxy-A9-tetrahydro- cannabinol (TRIOH-THC), A10-tetrahydro-cannabinol (A10-THC), tetrahydrocanna- biphorol (THCP), THC-O acetate (THCO), hexa-hydrocannabinol (HHC), 10-0x0-A6a- tetrahydrocannabinol (OTHC), A8-tetra-hydrocannabinol (A8-THC), A8-tetrahydro-

cannabinolic acid (A8-THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidiorcol (CBDC1), cannabidiol-C4 (CBDC4), cannabidiol dimethyl ether (CBDD), cannabidiol monomethyl ether (CBDM), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), nabilone, nabiximol, anandamide, cannabigerol (CBG), cannabigerolic acid (CBGA), cannabigerolic acid A monomethyk lether (CBGAM), cannabigerovarin (CBGV), cannabigerovarinic acid (CBGVA), cannabigerol mono-methylether (CBGM), cannabinol (CBN), cannabinolic acid (CBNA), cannabdiorcol (CBN-C1), cannabinol-C2 (CBN-C2), cannabivarin (CBN-C3), cannabinol-C4 (CBN-C4), cannabinodivarin (CBNDC3), cannabinol methylether (CBNM-C5), cannabichromene (CBC), cannabichromenc acid (CBCA), cannabichromanon (CBCN-C5), cannabicoumaronone (CBCON-C5), cannabi- chromanone-C3 (CBCN-C3), cannabichromevarin (CBCV), cannabichromevarinic acid (CBCVA), cannabielsoin (CBE-C5), cannabiglendol-C3 (OH-iso-HHCVC3), C3- canna-bielsoicacid B (CBEA-C3 B}, cannabifuran (CBF), dehydrocannabifuran (DCBF-C5), cannabifuran (CBF-C5), dehydrocannabifuran (DCBF or CBFD), cannabicyclol (CBL-C5), cannabicyclovarin (CBLV-C3), cannabitriol (CBT), cannabitriolvarin (CBTV), cannabiripsol (CBR), cannabinodivarin (CBV or CBVD), 2- arachidonoylglycerol (2-AG), 2-arachidonoylglycerol ether (2-AGE), isotetra- hydrocannabinol, isotetrahydrocannabivarin, palmitoylethanolamide, epigallo- catechin (EGC), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG). The hydrophobic compound may be added to the oil mixture in the form as is, i.e. in the hydrophobic solvent, but, if available, it may also be added in dry or semi- dry form. Preferably, the hydrophobic solvent is non-toxic and food-grade, such as an oil.

It has been found that a mixture with stearic acid, coconut oil and medium chain triglycerides is yielding excellent results with respect to the production of stable nanoparticles. The ratio between the oils/fats with different melting points enables control of the melting behaviour of the nanoparticles and thereby release of the hydrophobic compound associated with these nanoparticles after entering the human body. An increase in the amount of oils with a low melting point provides a more rapid release, while an increase in the amount of oils with a high melting point provides for a retarded release of the hydrophobic compound. The ratio of components that provides a stable nanoparticle composition with the desired release properties may be achieved by mixing stearic acid, coconut oil, medium chain triglycerides and solvent with hydrophobic compound in a ratio of approximately 1 : 2 : 9 : 5. However, depending on the nature and melting points of the individual components other ratios may be equally applicable. The skilled person will know how to vary the parameters involved in preparing the oil mixture to obtain the desired release characteristics.

Optionally one or more antioxidants may be added to the oil mixture. However, in order to maintain the antioxidant activity of such compounds, these should only be added when the oil mixture is cooled down to about RT. Antioxidants prevent free radical induced cell and biological targets damage by preventing the formation of radicals, scavenging them, or by promoting their decomposition. Moreover, antioxidants prevents the oxidative reaction, which is responsible for rancid odors and flavors within fats and oils which reduces nutritional quality of foods. Thus, antioxidants play an important role to enable a long-term storage of compositions comprising oils and fats and act advantageously in the body. Luckily, there are sufficient hydrophobic compounds that may be added to the oil mixture that can function as antioxidant (see e.g. Papas AM, Oil-Soluble Antioxidants in Foods, Toxicology and industrial Health, 1683,8(1-23 123-148; Fan L and Micheal Eskim NA, The Use of Antioxidants in the

Praservation of Edible cis, In: Handbook of Antioxidants for Food Preservation,

Woodhead Publishing Series in Food Science, Technology and Nutrition, 2015, 373- 388.). Many plant oils, such as olive oil, rapeseed oil, linseed oil, peanut oil, sunflower oil, carrot seed oil, palm oil, corn oil, hempseed oil or cottonseed oil can be used, but also other plant derived components, such as vitamin E (tocopherol), extracts from rosemary, sage, thyme, and the like. The main purpose of adding these antioxidants to the oil mixture is to protect the active hydrophobic ingredient during sonication without presenting a toxicity threat to the user of the composition. Although all of the mentioned antioxidants as a single component or as a blend may be added in such an amount to achieve the desired protection, we found that a mixture of linseed oil, hempseed oil, tocopherol and rosemary extract provided sufficient antioxidant protection in the process of the invention. Tocopherol also enhances tissue absorption of the hydrophobic compound.

A further component that may be added to the oil mixture is panthenol, which is a provitamin of vitamin B5. It is a moisturizer and humectant that is often found in shampoos and skin care products. In the present invention it enhances the binding of the nanoparticles to water, i.e. it increases the Zeta potential of the nanoparticle solution. Because of this, it enhances tissue absorption rates of the hydrophobic compound of interest. Other additions may be pyridoxal 5’-phosphate or pyridoxine hydrochloride (vitamin B6) or melatonin.

The oil mixture is mixed with the emulsifier (blend) and water at a slightly elevated temperature (about 30 - 40°C). Normally on 1 liter of the oil mixture a total of 25 grams of the emulsifier (blend) is used and 500 ml water. The water preferably is food-grade water.

The addition of these three components results in an emulsion with discontinuous oil droplets containing a load of the hydrophobic component dispersed in the continuous aqueous medium. The mixture is preferably homogenised to obtain an emulsion in which the oil droplets are uniformly dispersed in the continuous phase.

Such homogenisation can be performed with any type of mixing apparatus, such as a high-speed blender, a homogenizer, an immersion blender, an overhead stirrer, a magnetic stirrer or even a kitchen mixer or whisk (for small batches). It is also possible to obtain homogenisation through fluidisation. After this optional step of homogenisation the process of sonication is started. By this process, the oil droplets in the emulsion will fall apart into smaller droplets, finally resulting into nanoparticles.

The result is an aqueous medium in which nano-size oil droplets loaded with the hydrophobic component are available, i.e. the droplets are a mixed solid and liquid composition. The sonication process may be performed with any commercially available sonicator and should be continued until the moment that the average particle size of the nanoparticles no longer decreases, i.e. until the mean particle size of the nanoparticles is stable. Care should be taken not to overheat the sonicated mixture.

The sonication process itself produces heat, which may jeopardize the formation and stability of the nanodroplets formed. Cooling can be performed by external cooling of the container in which the sonication process takes place, but a better way is to immediately cool down the formed nanoparticle solution at the moment that the sonication process is (nearly) completed. This can be achieved by putting the solution on ice, which can be done already during the sonication process, but a more preferred cooling method is adding glycerol in an amount up to 25% of the nanoparticle solution.

The addition of glycerol has the additional advantage that it crystallizes the particles, thereby increasing the shelf life of the product. Depending on the sonication equipment used and the components that were used in the oil mixture and emulsifier blend the nanoparticles will have a mean particle size of 10 — 600 nm, preferably of 50 — 150 nm and more preferably of 80 — 130 nm, most preferably about 110 nm. Calculation of the mean particle size may be exprsssed as D50 determined in accordance with ISO 9276- 2 (14th Edition, September 4, 2019) or with tunable resistive pulse sensing (TRPS) such as obtainable by using an Izon Exoid™ apparatus. Other methods of measuring the droplet size in a nanoemulsion may be used, such as dynamic light scattering, nanoparticle tracking analysis, transmission electron microscopy, scanning electron microscopy or laser diffraction. Monitoring the particle size during the sonication is preferably achieved by TRPS on samples taken during sonication.

After sonication, but before glycerol is added, the mixture is preferably filtered to remove larger particles and microorganisms, such as bacteria. For such a filtration, a filter with a cut-off at e.g 200 nm is used. Several filter types may be used, such as polyetherculfone (PES) filter, polyvinylidene fluoride (PVDF) filters, polytetrafluoroethylene (PTFE) filters, mixed cellulose ester (MCE) filters, polypropylene (PP) filters or nylon filters. All such filters may be pre-sterilized or can be sterilized by the user and these are readily commercially available. .

This invention uniquely employs nano-sized oil droplets to execute a regulated release of hydrophobic components. The modulated release mechanism is triggered when the aqueous solution is exposed to varying temperatures. These temperature variations catalyse a transformative effect on the nanostructure conformation, creating a programmable delivery system that responds to body temperature, thus initiating decomposition.

Upon the initiation of crystallization in the nanoparticles, a displacement of the hydrophobic components from the core to the surrounding medium occurs. This unique characteristic distinguishes the invention from existing drug delivery nanoparticle systems. Previous systems have relied on a solid base, known as Solid Lipid

Nanoparticles {SLNs), which struggle with encapsulation inefficiencies and less than optimal drug release kinetics. Alternatively, they may use a liquid base, or nanoemulsion, which suffers from poor shelf-life stability as the nanoparticles tend to merge and lose their unique nanoscale attributes.

The present method takes advantage of partial crystallization, the degree of which is dictated by the melting temperature of the oils in the mixture. This approach bestows the nanoparticles with a unique stability that aids in resisting flocculation, all while preserving the ability to hold substantial amounts of hydrophilic or amphiphilic compounds. The result is a maintained encapsulation efficiency, which ultimately enhances the overall efficacy and potential of our novel nanoparticle system.

For shelfing the product obtained according to the above-described process, the product should be packaged in a sterile packaging, which can be of any inert material, such as glass or vacuum packaging materials, which are normally used for airtight packaging of food products. When packaged in such a way the shelf life of the product is extremely long.

The dispersion that is obtained through the above described method is a robust solution teeming with nanostructured lipid carriers, or nanoparticles, loaded with the desired hydrophobic compound. This formulation guarantees an impressively long shelf life spanning several years, with minimal perceivable alterations in its consistency. Furthermore, it serves as a rich, concentrated source of the specified hydrophobic compound.

Despite the potential susceptibility of the dispersion to Ostwald ripening—a process that could trigger flocculation and result in a reduction in efficacy over time—the meticulous formulation and preparation of the product are designed to effectively counter this. It is important to clarify that the product's packaging does not inhibit

Ostwald ripening. Instead, it is the precise composition and processing of the product that forestalls this phenomenon. Nonetheless, the product's packaging plays an instrumental role in preserving its viability. As long as the integrity of the sterile packaging is maintained, it protects the product from microbial contamination, air- induced degradation and, depending on the packaging and storage conditions, possible radiation damage. Even in instances where potency may be slightly diminished, the viability of the product remains intact, thanks to the packaging.

Should flocculation occur, the product is designed to be reprocessable to reclaim its initial efficacy. This measure may be undertaken if continuous testing, conducted after the retest period, identifies such a need. This characteristic emphasizes the resilient and flexible nature of the innovative nanoparticle composition of the invention, further accentuating its value and potential.

If needed the product can be diluted to decrease the amount of active ingredient in order to obtain a suitable dose form. Dilution normally will take place by adding (food- grade) water. When diluting, additionally stabilizers, colourants, preservatives and/or flavourants may be added. For stabilizers preferably a food grade stabiliser is chosen.

Preferably the stabiliser is a gum, such as guar gum (E412), arabic gum (E414), xanthan gum (E415), alginic acid (E400), carrageenan (E407), ghatti gum, tragacanth gum (E413), karaya gum (E418), locust bean gum (E410), dammar gum, glucomannan (E425), tara gum (E417), gellan gum or beta-glucan. We have found that addition of a combination of guar gum and xanthan gum works well as a stabilizer when it is applied in a concentration in the diluted composition of between 0.01 and 0.05%, more preferably about 0.02%.

Preservatives may be chosen from the group consisting of ascorbic acid, sodium ascorbate, isoascorbic acid, sodium isoascorbicate, citric acid, sorbic acid, calcium sorbate, benzoic acid, potassium benzoate, acetic acid, erythorbic acid, sodium erythorbate, ethyl lauroyl arginate, long-chain glycolipids from Dacryopinax spathularia MUCL 53181, methyl-p-hydroxybenzoate, nisin, sulphurous acid, dimethyl dicarbonate, ascorbyl palmitate and blends thereof. In the present invention it was found that a combination of ascorbic acid, citric acid and sorbic acid provides the desired result when the ascorbic acid, if present, is present at a concentration between 0.01% and 0,1%, preferably about 0.05%, and when the citric acid, if present, is present at a concentration between 0.005% and 0.05%, preferably at about 0.01%, and when the sorbic acid, if present, is present at a concentration between 0.05% and 0.5%, preferably about 0,1%

Although the composition as produced according to the above described method already has an acceptable, if not pleasant taste, a further flavouring compound may be added, if desired. Any flavouring compound that can be used in food, including drinks, may be added, such as flavouring essences. These are readily available in any taste and the skilled person will know how to apply these and which concentration is needed for the flavouring to provide a pleasant taste.

In the products that were obtained in which the hydrophobic compound of interest was a cannabinoid a 50 times dilution was made, which resulted in a product that comprised approximately 8 mg of the cannabinoid in 25 ml of product (used as a dosage form), which is a concentration of about 0,05% (meaning that the undiluted product had a concentration of about 2.5%). However, it has appeared that in the current system that concentrations up to 5% of hydrophobic compound of interest may be reached. This level, of course, would depend on the nature of the hydrophobic compound, the concentration of the hydrophobic compound in the solvent in which it is added to the oil mixture, the amount of oil mixture and the amount of water that is used in the emulsifying reaction, etc.

With the specific compositions as exemplified herein it was found that a superior system was made that surpasses the stability, flavor, and safety profiles of existing counterparts currently in the market. The chosen co-emulsifiers (sunflower lecithin, beta-cyclodextrin, and sucrose ester) interact synergistically to enhance nanoparticle stability, offering a robust formulation capable of maintaining product quality under various storage conditions. The combination also enhances the flavor profile of the composition, ensuring an enjoyable consumption experience. Further, the blend exhibits a lower toxicity profile compared to traditional emulsifiers, thus preventing the formation of harmful free radicals and degradation products during high-intensity processing. The thermal process, as exemplified herein, involving a hot emulsion phase followed by cooling before homogenization, results in a more efficient procedure with minimized energy requirements and side reactions, especially since a natural antioxidant blend is employed to shield the active ingredients, mitigating potential oxidative damage to consumers. Further, some molecules (tocopherol, vitamin B5 precursor) may be incorporated to enhance tissue absorption rates and facilitate efficient drug release kinetics.

By adjusting the solid-to-liquid lipid ratios, the formulation allows precise manipulation of drug release kinetics, offering a customizable delivery experience, which is unknown for at least cannabinoids. The unique temperature-responsive release mechanism in the nanoparticles ensures a stable product at room temperature that allows controlled release upon ingestion. The formulation, however, is versatile and allows for ultra-stable nanoparticles capable of being loaded with a variety of hydrophobic drugs, expanding potential applications beyond cannabinoid delivery. The optimized nanoparticle size in the formulation supports efficient tissue penetration and helps in overcoming drug resistance mechanisms. The unique combination of emulsifiers, lipid vehicles, and natural antioxidants in the formulation not only provides a safe and stable delivery system, especially for cannabinoids, but also enhances bioavailability and release kinetics. The tested formulation offered enhanced bioavailability of cannabinoids, being up to 10 times more bioavailable than normal cannabinoid oil and showing effects in minutes rather than hours. Nevertheless, the release kinetics of compounds from the nanoparticles can be programmed to suit the needs of different consumers, ranging from rapid to delayed release. The technology also supports the targeted delivery of cannabinoids or other therapeutic agents specifically inside tumors, providing a valuable tool for personalized medicine. The nanoparticle design supports potential sequential release of multiple therapeutic agents, facilitating a coordinated treatment approach. The technology can be extended to controlled-release drug delivery systems, improving patient compliance and therapeutic outcomes.

Further, the nanoparticles are transdermally bioavailable, expanding the delivery routes for therapeutic agents. The technology can be applied to improve the efficacy of cosmetic formulations, potentially enabling better skin penetration and longer- lasting effects. In addition, the nanoparticle design allows for encapsulation and preservation of volatile or sensitive substances, extending shelf-life and maintaining compound efficacy. The formulation enables the loading and delivery of hydrophilic, amphiphilic, or charged bioactive compounds, expanding potential applications. For instance, the technology can be used to improve oral delivery of drugs with low bioavailability due to first-pass metabolism. Incorporation of bicessential compounds can potentially improve the stability and bioavailability of probiotics or other beneficial gut microflora and thus co-administered with these. The formulation can also potentially protect and enhance the delivery of probiotics, supporting their survival during transit through the harsh stomach environment.

Next to the transdermal delivery, the nanoparticles also allow for penetration through the blood-brain barrier, providing potential solutions for neurological conditions.

The nanoparticle design might further allow for cell-specific targeting of therapeutics by incorporating specific ligands or antibodies on the surface of the nanoparticles. The nanoparticle design could potentially enable the delivery of genes or RNA therapies, extending the potential applications to the burgeoning field of gene therapy. The formulation can further potentially improve the delivery and efficacy of vaccines by protecting the antigen and providing adjuvant effects.

As is indicated above, the nanoparticle composition is designed to accommodate various release triggers within the nanostructures. These triggers ensure a responsive and customizable release mechanism under a range of conditions. As already has been disclosed, antibodies can serve as trigger to initiate release at the presence of an antigen to which the antibody may bind, thereby enabling targeted drug delivery in response to specific (pathological) markers. For targeting of tumors pH-sensitev compounds may be applied that are able to release the drug load in an area of low pH. This can of course also be used to effect release in the stomach. The release may also be made responsive to enzymes by including certain peptides or proteins in the nanoparticles. Another possibility is to make use of photoresponsive material by which a release can be established through exposure to specific wavelengths of light. In the same way magnetic materials may be used in which case a magnetic field may trigger the release.

The potential applications of the technology extend to animal health, potentially improving the delivery and absorption of veterinary therapeutics.

Example 1

Preparation of an aqueous nanoparticle cannabinoid composition

A mixture of emulsifiers was prepared by combining 10g beta cyclodextrin (Landor

Trading Comp.), 10g sucrose ester (Ryoto TM P-1670 from Mitsubishi Chemical

Company), and 5g sunflower lecithin (buXtrade). This mixture was then diluted to 800ml with purified water at 25°C.

Separately, an oily mixture was prepared by melting 10g stearic acid, 50g natural cannabis sativa extract, 20g coconut oil (Ekoplaza), 30g C8 MCT (Lus Health

Ingredients), 2g hempseed oil (Holland and Barrett), 2g linseed oil (Holland and

Barrett), 1g natural tocopherols concentrate (soapqueen.nlj, and 1g rosemary extract in order of descending melting point, using a heated stirrer. The temperature during this process did not exceed 75°C and the total process lasted for approximately 30 minutes.

The oily phase and emulsifier solution were then combined using a mixer until the mixture was visibly homogeneous. This mixture was then further processed with 600W of sonication power (U.S. SOLID sonicator) in a 1L beaker, with a cycle of 10 seconds on and 2 seconds off for 7 minutes and 30 seconds.

The mixture was cooled to room temperature (approximately 20°C) using a water and ice bath before being sonicated for a further 2 minutes with the same conditions.

The final product was filtered using a 200nm filter to remove any larger particles. The resulting filtrate had a mean particle size of 114.8 nm (measured using a Izon Science

Apparatus (using TPRS) and a THC concentration of 50 mg/ml.

The filtrate could be preserved using 25% glycerol if not intended for immediate use and sterile packaged.

Example 2

Preparation of a dosage form of the composition from Example 1

A strawberry-flavored preparation was made by adding 20ml of the THC filtrate from

Example 1, 200ml of strawberry syrup BP, 1g of potassium sorbate, 0.5g Guar Gum (buXtrade), 150mg Ascorbic Acid (buXtrade), and 50mg Citric Acid (buXtrade) to 1L of water. After thorough mixing, it was divided into dosage forms containing 25 ml of the preparation. The preparation should be consumed within 2 days of preparation or sterile packaged.

Claims

CONCLUSIONS

1. A method for preparing an aqueous nanoparticle composition comprising a hydrophobic compound, comprising the steps of:

a. Providing an emulsifier or a mixture of emulsifiers in powder form;

b. Mixing one or more oils at a temperature above 40°C at which all the oils have become liquid, said oils differing in melting temperature and which mixture comprises at least a sufficient amount of medium chain triglycerides to enable the composition formed in step g to be liquid at temperatures around about 4°C;

c. Add to the oil mixture the hydrophobic compound in any hydrophobic solvent;

d. Optionally, allow the mixture to cool to room temperature;

e. Adding the emulsifier powder and water to the oil mixture and allowing the mixture to emulsify while optionally agitating and heating to 30-40°C;

f. Subjecting the emulsified mixture to a sonication treatment with optional mixing or fluidization until the average particle size of the mixture remains stable;

g. Allowing the sonicated mixture to cool.

2. A method according to claim 1, wherein the emulsifier is a mixture of emulsifiers, preferably wherein said emulsifiers are non-toxic emulsifiers, more preferably wherein said mixture comprises sugar-based emulsifiers such as sucrose ester and/or cyclodextrin.

3. A method according to claim 2, wherein the emulsifier is a mixture comprising sucrose ester, cyclodextrin and lecithin, preferably sunflower lecithin.

4. A method according to claim 3, wherein the amount of lecithin is such that in the final sonicated mixture of step g the concentration of lecithin is less than 5%, preferably less than 2%, more preferably less than 1%.

5. A method according to claim 3 or 4, wherein the amount of sugar-based emulsifiers is at least twice the amount of lecithin, preferably at least four times.

6. A method according to any one of claims 3 to 6, wherein the ratio of sucrose ester, cyclodextrin and lecithin is 2:2:1.

7. A method according to any preceding claim, wherein the oil mixture comprises oils or fats which are non-toxic,

8. A method according to any one of the preceding claims, wherein the oil mixture comprises at least one oil having a melting point above 50°C, preferably above 60°C.

9. A method according to any preceding claim, wherein the oil mixture comprises an oil having a melting point between room temperature and body temperature.

10. A method according to any preceding claim, wherein the oil mixture comprises stearic acid, coconut oil and medium chain triglycerides.

11. A method according to any preceding claim, wherein the oil mixture, when mixed with the hydrophobic compound, comprises the component in a ratio of stearic acid:

coconut oil: medium chain triglycerides: solvent with hydrophobic compound of 1:2:9:5.

12. A method according to any preceding claim, wherein a non-toxic antioxidant is added to the oil mixture, preferably wherein said antioxidant is a mixture of antioxidants, more preferably wherein the total amount of said antioxidant or mixture of antioxidants does not exceed 10% of the oil mixture, preferably not exceeding 5% of the oil mixture.

13. A method according to claim 12, wherein said mixture of antioxidants comprises linseed oil, hemp seed oil, tocopherol and/or rosemary extract, preferably comprising linseed oil, hemp seed oil, tocopherol and rosemary extract, preferably in a ratio of 2:2 12:1.

14. A method according to any preceding claim, wherein the water is of food grade quality.

15. A method according to any preceding claim, wherein the nanoparticles in the sonicated mixture have an average particle size of 10 - 600 nm, preferably 50 - 150 nm, and more preferably 80 - 130 nm and most preferably about 110 nm.

16. A method according to any preceding claim wherein glycerol is added to the sonicated mixture, preferably wherein the concentration of glycerol is more than 20%, preferably more than 25%.

17. A method according to any preceding claim, wherein the hydrophobic compound is a plant extract in oil.

18. The method of claim 17, wherein the plant extract is an extract of Cannabis sativa, preferably wherein said extract comprises a cannabinoid, more preferably wherein said extract comprises a cannabinoid selected from the group consisting of Δ9-tetrahydrocannabinol (THC), Δ9-tetrahydrocannabinolic acid (Δ9-THCA or THCA), Δ9-tetrahydrocannabiorolic acid (Δ9-THCA-C1 or THCA-C1), Δ9-tetrahydrocannabiorcol (Δ9-THCO-C1 or THCO-C1), Δ9-tetrahydrocannabiorcolic acid (Δ9-THCOA or THCOA), Δ9-tetrahydrocannabivarin (Δ9-THCV or THCV), Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA or THCVA), trihydroxy-Δ9-tetrahydrocannabinol (TRIOH-THC), A10-tetrahydrocannabinol (A10-THC), tetrahydrocannabiforol (THCP), THC-O acetate (THCO}, hexahydrocannabinol (HHC), 10-oxo-A6a-tetrahydrocannabinol (OTHC), A8-tetra-hydrocannabinol (A8-THC), A8-tetrahydrocannabinolic acid (A8-THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidiorcol (CBDC1), cannabidiol-C4 (CBDC4), cannabidiol dimethyl ether (CBDD), cannabidiol monomethyl ether (CBDM), cannabidivarin (CBDV), cannabidivaric acid (CBDVA), nabilone, nabiximol, anandamide, cannabigerol (CBG), cannabigerolic acid (CBGA), cannabigerolic acid A monomethyl ether (CBGAM), cannabigerovarin (CBGV), cannabigerovaric acid (CBGVA), cannabigerol monomethyl ether {CBGM), cannabinol (CBN), cannabinolic acid (CBNA}, cannabdiorcol (CBN-C1), cannabinol-C2 {CBN-C2), cannabivarin (CBN-C3}, cannabinol-C4 (CBN-C4), cannabinodivarin (CBNDC3), cannabinol methyl ether (CBNM-C5), cannabichromene (CBC), cannabichromenic acid (CBCA}, cannabichromanone (CBCN-C5), cannabicoumaronone (CBCON-C5), cannabi-chromanone-C3 (CBCN-C3}, cannabichromevarine (CBCV), cannabichromevaric acid (CBCVA), cannabielsoine (CBE-C5), cannabiglendol-C3 (OH-iso-HHCVC3]}, C3-canna-bielsoic acid B (CBEA-C3 B}, cannabifuran (CBF), dehydrocannabifuran (DCBF-C5), cannabifuran (CBF-C5), dehydrocannabifuran (DCBF or CBFD), cannabicyclol (CBL-

C5), cannabicyclovarin (CBLV-C3), cannabitriol (CBT), cannabitriolvarin (CBTV), cannabiripsol (CBR), cannabinodivarin (CBV or CBVD), 2-arachidonoylglycerol (2-AG), 2-arachidonoyiglycerol ether (2-AGE), isotetrahydrocannabinol, isotetrahydrocannabivarin, palmitoylethanolamide, epigallocatechin (EGC), (-}-epicatechin gallate (ECG) and (-}-epigallocatechin gallate (EGCG).

19. A method according to any preceding claim, wherein said extract comprises THC or a mixture containing THC.

A method according to any preceding claim, wherein an additional step of diluting the sonicated mixture obtained in step g is carried out to obtain a diluted composition, preferably wherein the mixture is diluted with water, more preferably wherein the mixture is diluted such that the dilution comprises between 0.001% and 5% of the hydrophobic compound, preferably between 0.005% and 1%, more preferably between 0.01% and 0.5%, more preferably between 0.02% and 0.1%.

21. The method of claim 20, wherein the water is of food grade.

22. A method according to claim 20 or 21, wherein a stabilizer is further added to the composition, preferably wherein said stabilizer is a food grade stabilizer, more preferably wherein said stabilizer is a gum, more preferably wherein said stabilizer comprises guar gum and/or xanthan gum, more preferably wherein the concentration of guar gum and/or xanthan gum in the diluted composition is between 0.01 and 0.05%, more preferably about 0.02%.

23. A method according to any one of claims 20 to 22, wherein a preservative is further added to the composition, preferably wherein said preservative is a food grade preservative, more preferably wherein said preservative is selected from the group consisting of ascorbic acid, sodium ascorbate, isoascorbic acid, sodium isoascorbate, citric acid, sorbic acid, calcium sorbate, benzoic acid, potassium benzoate, acetic acid, erythorbic acid, sodium erythorbate, ethyl lauroyl arginate, long chain glycolipids from Dacropinax sphatularia MUCL 53181, methyl p-hydroxybenzoate, nisin, sulfurous acid, dimethyl dicarbonate, ascorbyl palmitate and mixtures thereof.

24. A method according to claim 23, wherein said preservative comprises ascorbic acid, citric acid or sorbic acid or a mixture thereof, preferably wherein the ascorbic acid, if present, is present in a concentration of between 0.01 and 0.1%, preferably about 0.05%, and wherein the citric acid, if present, is present in a concentration of between 0.005% and 0.05%, preferably about 0.01% and wherein the sorbic acid, if present, is present in a concentration of between 0.05% and 0.5%, preferably about 0.1%.

25. A method according to any one of claims 20 to 24, further comprising adding a flavouring agent, preferably a food grade flavouring agent.

26. A method according to any preceding claim, wherein panthenol is added to the oil mixture, preferably wherein panthenol is added to an amount between 0.5 and 5% of the oil mixture, more preferably an amount between 1 and 3% of the oil mixture.

27. An aqueous nanoparticle composition provided by a method according to any one of claims 1 to 26.

28. A pharmaceutical composition comprising the aqueous nanoparticle composition of claim 27.