The subject matter of the invention concerns stable self-emulsifying compositions containing naturally fat-soluble vitamins and provitamin derivatives thereof as well as molecular entities constituting their chemical modifications, including vitamin derivatives non-fat-soluble in their native forms and whose chemical analogs are useful as pharmaceutical and food ingredients, as well as active substances of natural origin with proven biological and/or therapeutic activity being lipophilic and/or hydrophobic compounds, including various extracts of hemp-derived compounds and standardized plant extracts. The present invention concerns novel compositions of single compounds or various combinations thereof which, according to the subject matter of the invention, form aqueous dispersions of substances with low solubility in water, having lipophilic properties in aqueous solutions. The proposed compositions may be readily diluted in aqueous solutions. Therefore, they are useful for manufacturing oral products based on aqueous solutions, in particular pharmaceutical compositions or foodstuffs which can be easily delivered with simple dosing of adequate amounts of active ingredients. In addition, the proposed compositions significantly increase the bioavailability of active substances which show poor gastrointestinal absorption in animals due to their hydrophobic and/or lipophilic properties.

Due to economic growth, the lifestyle and dietary habits of people have changed significantly these days. Consumption of processed foods having lower nutritional quality has recently increased. Poor dietary habits, excessive consumption of sweet and salty snacks as well as an irrational lifestyle, stress and sleep deprivation have resulted in a lower quality of life, which generates a number of health-related problems. However, research has proved that humans need approx. 90 nutrients every day, including 60 minerals, 16 vitamins, 21 amino acids or fatty acids, in particular of the omega-3 and omega-6 type. Therefore, dietary support by placing on the market dietary supplements, functional foods, foods for special purposes and nutraceutics in highly developed countries, such as the United States and Japan, is currently a very important aspect of nutrition and preventive health-related measures. Supplying adequate amounts of nutrients to the body in an appropriate form is one of the primary preventive and prophylactic measures against lifestyle diseases (cardiovascular conditions, obesity, hypertension, diabetes or nervous system diseases). Therefore, a number of authors are right in their beliefs that dietary supplements, nutraceutics and herbal medicines will become an integral health care system in the third millennium. Supplying nutrients and biologically active substances in a form that enables their health-related potential to be utilized as much as possible by increasing stability, absorption or quantities in products is considered most important in this respect, and this may be achieved for example by using adequate lipid nanoformulations (Rautiainen, S., Manson, J., Lichtenstein, A. et al., Dietary supplements and disease prevention — a global overview, Nature Reviews Endocrinology, 12; 407-420; 2016, Dwyer, J.T., Coates, P.M., Smith M.J., Dietary Supplements: Regulatory Challenges and Research Resources, Nutrients, 10 (1) 41; 2018)

Currently, one of the primary objectives when exploring novel oral compositions, mixtures and formulations is direct or indirect functionalization of active ingredients to enable their biological effects to be better used. Research shows that approx. 1/3 of all active substances with health-promoting or therapeutic properties have low solubility in water (hydrophobic and/or lipophilic); therefore, these substances show low bioavailability and/or efficacy. The availability of biological compounds in aqueous systems is one of the key factors for their efficacy when administered enterally. For oral intake is the primary route of delivering nutrients and active substances for the whole body. Therefore, one of the principal objectives for innovation in the development of novel compositions and formulations for the oral intake of various active chemical compounds is to increase their bioavailability and/or modification of the physicochemical parameters of the substances. (Helal, N.A., Eassa, H.A., Amer, A.M., Eltokhy, M.A., Edafiogho, I., Nounou, M.I., Nutraceuticals' Novel Formulations: The Good, the Bad, the Unknown and Patents Involved, Recent Patents on Drug Delivery & Formulation, 13 (2) 105-156; 2019, Rambran, T.F., A patent review of polyphenol nano-formulations and their commercialization, Trends in Food Science & Technology, 120; 111-122; 2020)

Research shows that as many as 40% of adult Americans have problems with swallowing tablets, even though most of them have no problems with solid or liquid foods. 80% admitted to feeling great discomfort when tablets pass through their throats, (https://www.pharmtech.com/view/your-tablet-hard- swallow-guidance-addresses-d rug-tablet-design) Therefore, many consumers seek comfort and prefer forms of administration more similar to foods, and liquid forms or alternative administration methods, such as fluid shots or gel sachets, are most convenient. Unfortunately, as much as 70% of a compound may be degraded during gastrointestinal passage following oral intake in fluid systems. The value may be particularly high for products delivered in aqueous systems in a non-modified form, in particular for hydrophobic compounds, wherein the substances precipitate and their bioavailability is low (typically less than 10%) (Rein, M.J., Renouf, M., Cruz-Hernandez, C., Actis-Goretta, L, Thakkar, S.K., da Silva Pinto, M. Bioavailability of bioactive food compounds: a challenging journey to bioefficacy, British journal of clinical pharmacology, 75 (3) 588- 602; 2013, Dima, C., Assadpour, E., Dima, S., Jafari, S.M., Bioavailability and bioaccessibility of food bioactive compounds; overview and assessment by in vitro methods, Comprehensive Reviews In Food Science And Food Safety, 19; 2862- 2884; 2020). In addition, they lack homogeneity, so that the products do not perform their functions as they fail to deliver the active substance in declared amounts or cause non-uniform delivery. As well as being delivered in aqueous systems, a number of other forms of foodstuffs are water-based or their manufacture and manufacturing processes require water. Therefore, many active substances with lipophilic properties need to be dissolved to be usable and to obtain uniform dosing. Hence, for the delivery of non-water-soluble active substances in liquid products (aqueous solutions), an adequate composition needs to be developed to form stable suspension for further processing.

A system of nanostructural lipid carriers is one of the most interesting colloid systems that provide an extremely attractive strategy to solve the problem of the delivery of active compo unds in liquid formulations for oral intake. Among the various forms that provide active delivery systems (micelles, liposomes, lipid micro- and nanoparticles), self-emulsifying systems (SELFs) in aqueous solutions have raised great interest. Self-emulsifying systems are lipid compositions made from blends of oils, surface active agents, solvents and co-solvents/surfactants (WO 2015/155703). When blended in adequate proportions, they may be used for designing formulations to improve oral absorption of highly lipophilic active compounds. The effectiveness of oral absorption depends on a number of formulation-related parameters, such as surface active agent concentration, oil-to-surface active agent ratio, polarity of emulsion, droplet size and charge, all of which essentially determine the self-emulsifying potential. Therefore, only very specific combinations of the aforementioned substances may facilitate the formation of effective self-emulsifying systems. Even though a number of studies have been performed, including those related to the pharmaceutical market, few products based on SELF technologies are available, which confirms the difficulties in developing stable hydrophobic compositions of active compounds in such formulations.

The efficacy of SELF systems depends on selecting ingredients of a composition and their ratios to achieve colloid dispersion during dilution in aqueous systems. When being formed, micelles (with typical sizes of up to 250 nm, and frequently below 100 nm) consist of a lipid nucleus (core) principally made of glycerides (essentially triglycerides, di- and monoglycerides) in which the active lipophilic substance is dissolved (molecular dispersion in the lipid core) and a layer of molecules of surfactant(s), which serve as emulsifiers, adsorbed at the oil/water interface and arranged so that the non-polar part of their molecules is immersed in the oil phase and polar parts are in the aqueous phase (WO 2002007712A2).

Vitamins are one of the most important among the multiple chemical compounds necessary for the normal function and course of a number of metabolic processes. Vitamins are low-molecular organic compounds whose presence in low amounts in the body frequently ensures appropriate activity of many enzymes. These are exogenous compounds for a number of organisms, including animals and humans, and they need to be supplied with food (WO 2015/169816). Certain vitamins may be synthesized from precursors known as provitamins, such as vitamin A from carotenoids, vitamin D3 from 7-dehydrocholesterol. Vitamins are classified first of all based on their physical properties, and they are divided into fat-soluble vitamins: vitamin A (retinol and derivatives), vitamin D (cholecalciferol), vitamin E (tocopherol), vitamin K (phylloquinone) and water-soluble vitamins: vitamin C (ascorbic acid), vitamin Bl (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B7 (biotin, vitamin H), vitamin B12 (cyanocobalamin) and folic acid.

The fat-soluble vitamins (A, D, E and K; ADEK in short) are non-polar hydrophobic molecules, being isoprene derivatives which should be supplied in everyday diet and theiravailability depends on the adequate absorption of fats. ADEK vitamins are involved in processes, such as regulation of calcium and phosphate metabolism, regulation of blood coagulation processes, act as antioxidants and they are necessary for the normal function of the epithelial tissue and processes of vision (US 6361800 Bl, US 9439913 Bl).

In other words, vitamin A is retinol and its derivatives. Carotenoids, containing at least one P-ionone ring, are direct precursors of vitamin A and they are very common in plants. In animal bodies, vitamin A is formed through the enzymatic oxidative breakdown of a provitamin. The vitamin has a prominent role as it is instrumental in healthy vision, adequate skin condition and healthy mucosae. In addition, vitamin A is beneficial for immunity.

Vitamin D (cholecalciferol and derivatives) is a group of steroid compounds. About 10 provitamins are known from which compounds having vitamin D activity form. In terms of human nutrition, vitamins D2 and D3, known as ergocalciferol and cholecalciferol, respectively, are most important. Vitamin D shows an effect on immunity, healthy structure of the skeletal system as well as on the nervous system, and it even regulates insulin production. Natural vitamin D forms in human skin exposed to sunlight. Unfortunately, irradiation is not sufficient at all latitudes to ensure adequate supply to the body. In addition, due to lifestyle changes, the vitamin is currently commonly supplemented not only in fall and winter but also in spring and summer. When deficiency of the vitamin occurs, diabetes, rickets in children and osteomalacia in adults as well as cardiovascular diseases may develop (Glowka, E., Stasiak J., Lulek, J., Drug Delivery Systems for Vitamin D Suppementation and Therapy, Pharmaceutics ,11 (347) 1-21; 2019).

Vitamin E (tocopherol) is an antioxidant whose function is to protect cells against oxidation and thus death by removing free radicals. What is also important, it has anticoagulant and anticancer properties. In addition, it has an effect on the function of the reproductive system. Vitamin E deficiency may result in keratosis, skin ageing, problems with concentration, loss of vision, anemia or even infertility (EP 0165352).

Vitamin K (phylloquinone, menadione) is necessary for normal blood coagulation. In the liver, it is required for the synthesis of prothrombin, a protein from which fibrin (blood clot) forms. It is also involved in bone tissue formation and has antibacterial, antifungal, analgesic and anti-inflammatory properties.

A number of chemical compounds, including vitamins, are chemically modified to affect their physicochemical parameters. For vitamin E, tocopherol acetate (US 7169943 B2, EP 0882036 Al) is an example of such a compound. However, derivatives are known which dramatically alter the physicochemical properties of chemical compounds. Aspartic acid palmitate ester (CN 102558115 A, CN 102260231 B) is a relevant example. Readily soluble in water, vitamin C (ascorbic acid) has high water solubility and provides a major oxidoreductive system active in acidic conditions. Ascorbyl palmitate, its derivative, yields a fat-soluble form of the vitamin and it is also used as a food-grade antioxidant (E304) in this form, approved in the European Union, the U.S., Australia and New Zealand. Furthermore, ascorbyl stearate is used as a source of vitamin C and as an antioxidative additive to margarine.

A number of bioactive lipophilic compounds are known which may be used in foods based on their health-promoting properties, such as carotenoids, omega-3 and omega-6 fatty acids (US 6284268), polyphenols, flavonoids, phytosterols and other standardized plant extracts. However, oral de livery of highly hydrophobic compounds, in particular in aqueous systems, is a considerable challenge not only due to their low water solubility, rapid oxidation and a low sensory threshold, but also based on their low bioavailability after gastrointestinal digestion. Therefore, within the present invention it is also important to develop selfemulsifying compositions with improved bioavailability useful for compounds of the aforementioned groups, in particular astaxanthin, zeaxanthin, lutein, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), conjugated linoleic acid (CLA), but also alpha-lipoic acid, naringenin, genistein, silymarin, curcumin, resveratrol, cannabidiol, cannabinoids, ubiquinone (coenzyme Q10) or various plant extracts.

Examples of oils from omega-3 fatty acids include for example long-chain polyunsaturated omega-3 fatty acids, such as EPA, DHA, a-linolenic acid (ALA), omega-3 fatty acid glycerol esters, such as mono-, di- and triglycerides; and omega-3 fatty acid esters with primary, secondary and/or tertiary alcohols, such as fatty acid ethyl esters. Omega-3 fatty acids, esters, triglycerides, derivatives, conjugates, precursors, salts and/or mixtures thereof of the present disclosure may be used in their pure form and/or as an ingredient of oil, e.g. as marine oil (fish oil, purified fish oil concentrates), algal oils, microbiological oils and other plant oils or mixtures thereof.

In addition, cannabinoids are an interesting group as natural chemical substances found in hemp (Cannabis sativa L.) - both wild (Cannabis sativa ruderalis J.) and cultivated (Cannabis sativa sativa L.) and Indian (Cannabis sativa indica). Tetrahydrocannabinol (THC) and cannabidiol (CBD) currently attract most attention. However, cannabinoids are a broad group of compounds which, as single compounds or mixtures in the form of cannabinoid extracts, are insoluble in water. Formulations have been reported, related to a self-dispersing emulsion using cannabinoids as a hydrophobic compound (US 20200037638). Even though the aforementioned application discloses compositions that increase the solubility of cannabinoids themselves, the emulsions do not form homogenous systems, and the present application concerns combinations of cannabinoids and/or cannabinoid extracts in self-emulsifying compositions in combination with various active compounds having health-promoting, active or therapeutic properties (US 2020/022386 Al, WO 2019/135224 Al).

The problem in this invention is the poor solubility of the active substances having lipophilic and/or hydrophobic properties in aqueous solutions, which results in their low gastrointestinal absorption in animals (low bioavailability). Another problem to be addressed by the present invention is the formation of homogenous suspensions. Currently, even though various lipid-based delivery forms for active substances having these parameters are used, no such solutions are available. These are typically so-called "milky" suspensions, unstable over time and showing a tendency of breaking, without proven active substance bioavailability. For most of these suspensions, vigorous stirring or expensive, complex or poorly effective devices, such as ultrasonic or high-pressure homogenizers, frequently need to be used to form a homogenous dispersed system in a short time. Another problem for the commonly used solutions is also that the compositions are not tolerant to dilution, and have low content of substances or inferior stability at low pH values (below 3), applicable to most of the commonly used drinks, which significantly limits the usability of such products. Another but very important factor is the ease of dose dispensing for the active substance, because excessive amounts of active substance (such as ADEK vitamins) are even disadvantageous.

The objective of the present invention is to disclose an improved composition with increased bioavailability which contains fat-soluble vitamins, and derivatives thereof and/or active substances of natural origin from groups of carotenoids, fatty acids and their derivatives, polyphenols, flavonoids, phytosterols, standardized plant extracts, etc. being lipophilic and/or hydrophobic active compounds with health-promoting and therapeutic properties, as a result of which a stable self-emulsifying emulsion is obtained which shows high transparency, short time to forming dispersion and yields a homogenous system, easily forming dispersion and providing nanoparticles with small hydrodynamic parameters, monodispersity of the nanosystem and high load (active substance content), wherein the quantity of the active substance may be easily dosed and delivered to the body. In addition, the invention provides high stability over time and a wide spectrum of action in terms of tolerance of dilution in aqueous solutions and tolerance of low pH values in the system.

For the purposes of this application, high transparency of a nanoemulsion after being added to an aqueous solution means transparency above 85%, most preferably above 90%. Transparency is synonymous to optic transmittance in the context of the invention. Transmittance of a nanoemulsion obtained according to the invention may be measured as a percentage value (%T) at wavelength X=600 nm (optionally in a range of ±80 nm, with no interference from excipient ingredients) using any spectrometer in this range. A Merck® Pharo 300 spectrometer was used for the measurements described herein.

For the purposes of this application, the short time to forming homogenous dispersion of active substances means time less than 60 sec from the addition of the last drop of the blend of ingredients of the composition to water until complete dissolution. Dispersion time may be measured using a spectrometric procedure with any spectrometer in the 520-680 nm range, wherein dispersion time means that a constant transmittance or absorbance value has been achieved which does not change on longer stirring of the dispersion in constant stirring conditions. Dispersion time may also be measured using automated analyzers of stability and ageing of emulsions, dispersions or suspensions, such as Turbiscan instruments. For the purposes of this application, the easy dispersibility means that vigorous stirring is not needed to form dispersion, i.e. high-speed stirring above 2500 rpm, homogenizers, high-pressure homogenization or use of sonication methods. It has been proved in the application that stirring below 150 rpm ensures adequate product parameters for the present compositions. Higher stirring speed (a range of up to 2500 rpm was tested) reduces the time needed and improves the quality of dispersions formed for the present compositions with active substances. Stirring speed is a relative term in this respect, because the quality of stirring is affected by parameters, such as the type and design of the mechanical stirrer, and design of the mixer, reactor or tank mixers.

For the purposes of this application, nanoparticles with small hydrodynamic d iameter mean nanoparticles with a hydrodynamic diameter of below 220 nm. Values of hydrodynamic diameter are expressed in nanometers (D _H , nm), and they may be measured using analyzers for determining particle size distribution based on dynamic light scattering, such as Zetasizer Nano-ZS from Malvern.

For the purposes of this application, monodispersity of a nanosystem means a polydispersity value of molecules dispersed in water of below 0.300. Polydispersity of molecules dispersed in water (Polydispersity Index) expressed as a Pdl value may be measured using a Zetasizer Nano-ZS instrument from Malvern.

For the purposes of this application, stability of a composition means stability above 12 months.

For the purposes of this application, the ease of dose dispensing for the composition and the active substance means that it may be dosed with minimal portioning by the consumer, which means for the composition that commercial pipettes/droppers with a pipette are used, wherein a validated drop weight for the product corresponds to a specific dose of the active substance. Alternatively, the composition may be used for manufacturing products with a different form for easy dosing or as a unit container.

For the purposes of this application, the high tolerance of dilution in aqueous solutions means high stability of a nanoemulsion with very high dilution, such as dilutions as high as 10,000x. Nanoemulsion stability with a dilution of 10,000x may be measured using a spectrometric procedure with any spectrometer in the 520-680 nm range, wherein a constant transmittance and/or absorbance value indicates stability during storage. Emulsion stability may also be measured using analyzers of stability and ageing of emulsions, such as Turbiscan. In addition, a constant value of hydrodynamic diameter and nanoparticle polydispersity in the system confirms that the emulsion is stable and no aggregation, sedimentation or flocculation occurs. Hydrodynamic diameter and nanoparticle polydispersity may be measured using analyzers for determining particle size distribution based on dynamic light scattering, such as Zetasizer Nano-ZS from Malvern.

For the purposes of this application, bioavailability of the active substance contained in a preferred composition is increased by at least 200% compared to a commercially available formulation containing an identical active substance dissolved only in plant oil or a mixture, calculated based on parameters determined from in vivo pharmacokinetic tests. Biological testing is preferably conducted in rats using healthy adult individuals. The testing is preferably conducted using all the required good laboratory practice. The active substance is assayed in blood samples collected over time using UPLC/MS/MS, having first completed full validation of the analytical procedure.

The technical purpose defined above has been achieved through the invention illustrated and described hereinbelow.

The subject matter of the invention is a self-emulsifying composition containing a surfactant, two auxiliary agents constituting distinct lipid fractions and one or more active substances with hydrophobic and/or lipophilic properties from the group of fat-soluble vitamins (A, D, E and K) and derivatives thereof, including provitamins, carotenoids (astaxanthin, zeaxanthin, lutein), fatty acids (such as eicosapentaenoic acid, docosahexaenoic acid, conjugated linoleic acid, alpha-linoleic acid) in the acid form, fatty acid glycerol esters (such as mono-, di- and triglycerides), fatty acid esters with alcohols (primary, secondary and/or tertiary alcohols), derivatives, polyphenols, flavonoids (naringenin, genistein, silymarin, curcumin, resveratrol, alpha-lipoic acid, ubiquinone), standardized plant extracts and other of the group of lipophilic and/or hydrophobic active compounds with health-promoting and therapeutic properties as well as excipients that affect the sensory attributes of the composition.

In a preferred example, the composition has very high stability, i.e. more than 12, preferably more than 24 months in standard storage conditions.

The composition of the invention preferably consists of at least one active compound with hydrophobic and/or lipophilic properties, a surfactant and two distinct lipid fractions and excipients that affect the sensory attributes of the composition.

In a preferred embodiment, the composition is characterized in that the active substance (or mixtures thereof) with lipophilic and/or hydrophobic properties and health-promoting and therapeutic properties comprises as much as up to 35% by weight of the composition, the surfactant comprises 20-50% by weight of the composition, the first lipophilic fraction comprises 15-35% by weight of the composition and the second lipophilic fraction comprises 15-35% by weight of the composition. The composition may optionally contain additional ingredients that make up the sensory texture (flavoring, aromatic and coloring substances with lipophilic and/or hydrophobic properties) whose content may be as high as up to 2.0% of the total weight.

In a preferred embodiment, the composition is characterized in that the surfactant is polysorbate 80, the first lipophilic fraction is a C8-C12 medium-chain triacylglyceride and the second lipophilic fraction is a C8-C12 medium-chain mono-diacylglyceride.

In a preferred embodiment, the composition is characterized in that it forms a clear monodisperse self-emulsifying system in an aqueous solution. In a preferred example, the composition enables the active substance to be easily dosed and portioned as a single dose (mg).

In a preferred embodiment, the composition is characterized in that the monodisperse system forms particles smaller than 220 nm.

In a preferred embodiment, the composition is characterized in that the polydispersity of particles dispersed in water is lower than 0.300.

In a preferred embodiment, the composition is characterized in that the monodisperse system forms within less than 60 s and no vigorous stirring is needed.

In a preferred embodiment, the composition is characterized in that the monodisperse system forms a stable nanoemulsion tolerant to dilution.

In a preferred embodiment, the composition is characterized in that it may contain flavoring and aromatic additives and colorants.

The composition of the invention is useful for manufacturing water-based oral products. The oral product is preferably a pharmaceutical composition or a foodstuff.

The composition of the invention may be used for manufacturing other products as an intermediate to finally obtain tablets, pills, film-coated tablets, capsules, suppositories, chewing gums, gels, powders, granules, syrups and dispersions, wherein the composition itself aids in the manufacturing process of these products.

Another subject matter of the invention is a manufacturing process for a stable monodisperse system characterized in that water or an aqueous solution is mixed with the composition of the invention as illustrated below: mixing is preferably conducted for not less than 120 seconds, preferably for not less than 60 seconds, with mixing speed not greater than 2500 rpm, preferably with mixing speed not greater than 150 rpm.

Another subject matter of the invention is a monodisperse system characterized in that it consists of a dispersion medium being water or an aqueous solution and a dispersed phase formed by particles obtained from the composition of the invention as defined above.

The monodisperse system of the invention preferably has transparency above 90%, monodispersity below 0.300, and the system maintains nanoemulsion stability when diluted with water or an aqueous solution up to 10,000x and/or maintains nanoemulsion stability with a pH value below 3.

When mixed in an aqueous environment, the ingredients of the composition of the invention spontaneously form essentially spherical nanoparticles in which the surfactant is the outer layer and the core (inner layer) is a mixture of lipids in which the active substance(s) or derivative(s) is dissolved. The surfactant that forms the outer layer is anchored in the inner core lipid layer with its lipophilic part (a fragment of the surfactant molecule that is the fatty acid residue). In addition, lipid 2 which has a key role in the structure of the nanoparticle of the invention, is involved in interactions between its hydrophilic outer layer and the inner hydrophobic core to furnish preferable properties of the nanoparticles illustrated in the application. When mixed in an aqueous environment, the ingredients of the composition spontaneously form nanoparticles of the invention whose arrangement changes as follows when going toward the center of the nanoparticle: the environment, that is, the aqueous solution, subsequently the surfactant which dominates in the outer nanoparticle layer, subsequently the mixture of lipids with the active substance (Lipid 1 + Lipid 2 + API), which form the nanoparticle core, wherein the Lipid 2 ingredient highly likely dominates in the outer core layer thus supporting the surfactant. Any possible sensory additives (OTHER: flavoring, aromatic and coloring) should be fat-soluble and form the nanoparticle core ((Lipid 1 + Lipid 2 + API + OTHER).

The principal technical effect of the developed composition that enables the active substance (or multiple such substances or mixtures thereof) with lipophilic and/or hydrophobic properties and healthpromoting and therapeutic properties to be dissolvedin an aqueous solution is:

• high loaded amount of active substances of as much as 35% by weight in the product,

• high stability of the composition above 12 months in standard storage conditions,

• high stability of the composition with very high dilution; dilution of up to 10,000x,

• nanoemulsion stability with sensory additives (flavoring, aromatic and coloring substances),

• high transparency of the nanoemulsion when added to an aqueous solution of more than 85%,

• very short time to forming homogenous dispersion when added to an aqueous solution of below 60 s,

• no need for vigorous stirring; stirring below 2500 rpm or even below 150 rpm,

• obtaining small-sized nanoparticles; size: hydrodynamic diameter below 220 nm,

• obtaining a monodisperse nanosystem; monodispersity below 0.300,

• high tolerance of low pH values (below 3),

• improved API bioavailability.

The following terms have been used throughout the specification:

• T80 - mixture of polyoxyethylated sorbitan and oleic acid derivatives, polysorbate 80, Tween® 80 surfactant,

• MCT - medium-chain triacylglycerides, C8-C12

• MCM - medium-chain mono-diglycerides, mainly of capric (C8) and caprylic (decanoic, CIO) acids,

• API - active ingredient, wherein API is first of all used in pharmacy and medicine and API means an active pharmaceutical ingredient. However, the term is very commonly used for active substances of natural origin. In the invention, it refers to one or more substances representing: fat-soluble vitamins (A, D, E and K) and derivatives thereof, including provitamins, carotenoids (astaxanthin, zeaxanthin, lutein), fatty acids (such as eicosapentaenoic acid, docosahexaenoic acid, conjugated linoleic acid, alpha-linoleic acid) in the acid form, fatty acid glycerol esters (such as mono-, di- and triglycerides), fatty acid esters with alcohols (primary, secondary and/or tertiary alcohols) and their derivatives, polyphenols, flavonoids (naringenin, genistein, silymarin, curcumin, resveratrol, alpha-lipoic acid, ubiquinone), cannabinoids, standardized plant extracts and other of the group of lipophilic and/or hydrophobic active compounds with health-promoting and therapeutic properties fo r oral delivery,

• OTHER - substances that affect sensory attributes of the product, that is aromatic compounds, colorants, flavoring substances with lipophilic and/or hydrophobic properties.

General preparation procedure for the composition of the invention

All ingredients of the composition are weighed out in a single tank protected from light. The contents of the vessel are stirred for 30 min at room temperature; alternatively, the whole mixture is heated to facilitate dissolution of the ingredients below 60°C and subsequently stirred to homogenize the mixture.

Preparation procedure for the emulsion from the composition of the invention

200 mL demineralized water (class II) with temperature of approx. 20°C is poured into a 600 mL beaker. A magnetic bar with a length of 4.5 cm is placed on the bottom of the beaker. The beaker is placed on a magnetic stirrer. Stirring is started (250 rpm) and an adequate quantity of the composition is added after 20 seconds.

Development of a self-emulsifying composition of the invention

The composition of the invention contains four principal ingredients and optionally the fifth ingredient. Optimal fractions of the ingredients are listed in Table 1 below, wherein the percentage values mean weight percentage.

Table 1. Fractions of respective ingredients in a preferred composition of the invention.

No. 1 : 2 : 3 : 4 : 5 Lipid Lipid

_ . .. _c , . . Active Sensory

Description Surfactant : . _ : fraction fraction , . substance ₁ additives

Preferred T80 API MCT MCM OTHER mass % 20-50 0 -35 15-38 15-38 0-2 The surfactant in the composition may be:

• monoacylated polyoxyethylated sorbitol with a trade name of Tween™, and Tween 20, 40, 60, 80 and 85 are available depending on the residue and substituent length. In the application, as well as Tween™ 80, polyoxyethylene sorbitol monolaurate (Polysorbate 20, Polyoxyethylene 20 sorbitan monolaurate), commercially available and offered by many manufacturers, was also evaluated as Tween 20. In the experiment, the following were used: Tween™ 20 from CRODA and polyoxyethylene sorbitol monostearate (Polysorbate 20, Polyoxyethylene 20 sorbitan monostearate), commercially available and offered by many manufacturers, as Tween 60,

• ethoxylated oil derivatives, a surfactant based on polyoxyethylene(35) castor oil (PEG-35 castor oil, Polyoxyl 35 Castor Oil), as Etocas, commercially available and offered by many manufacturers,

• hydrogenated ethoxylated oil derivatives, a surfactant based on hydrogenated castor oil(35) (Polyoxyl 40 hydrogenated castor oil, PEG-40 hydrogenated castor oil), as Croduret, commercially available and offered by many manufacturers,

• acyl polyglyceryl derivatives as Plurol (Polyglyceryl-3 dioleate) , commercially available surfactants offered by many manufacturers.

The fraction labelled as Lipid 1 may be medium-chain triacylglycerides with a mixture of C8-C12 fatty acids. The medium-chain triacylglycerides are primarily esters with caprylic (octanoic, C8:0) and capric (decanoic, C10:0) acids. MCT are commercially available and offered by many manufacturers under various trade names, such as: Crodamol GTCC triacylglyceride from CRODA, Labrafac lipophile from GATEFOSSE, Miglyol 808 from IOI OLEOCHEMICAL, Miglyol 812 N from IOI OLEOCHEMICAL, Imwitor® 928 from IOI OLEOCHEMICAL, Captex® 300 from ABITEC or Captex® 355 from ABITEC.

The fraction labelled as Lipid 2 may be medium-chain mono-diacylglycerides (MCM) with a mixture of C8- C12 fatty acids. Medium-chain mono-diacylglycerides are commercially available and offered by many manufacturers under various trade names, such as: Imwitor® 988 (Type I) from IOI OLEOCHEMICAL, Imwitor® 308 (Type II) from IOI OLEOCHEMICAL, Imwitor® 742 (Type I) from IOI OLEOCHEMICAL, Imwitor® 928 from IOI OLEOCHEMICAL, Campul MCM EP/NF (Type I) from ABITEC, Campul 808G EP/NF (Type II) from ABITEC, Campul MCM-8 (Type I) from ABITEC or Campul MCM NF (Type I) from ABITEC. In addition, MCM derivatives may be used as Lipid 2 in the composition, that is:

• medium-chain acid monoesters with propylene glycol, among which the following are most widespread:

• caprylic acid (C8:0) monoester with propylene glycol, type I in pharmaceutical classification, abbreviated as PGMC-I (propylene glycol monocaprylate, PG monocaprylate C8, Type I, NF); trade name: Capryol PGMC from GATEFOSSE, • caprylic acid (C8:0) monoester with propylene glycol, type II in pharmaceutica I classification, abbreviated as PGMC-II (propylene glycol monocaprylate, PG monocaprylate C8, Type II); trade name: Capryol 90 from GATEFOSSE,

• lauric acid (C12:0) monoester with propylene glycol, type II in pharmaceutica I classification, abbreviated as PGML (propylene glycol monolaurate, Type II); trade name: Capmul PG-12 NEP/NF from ABITEC,

• long-chain acid monoesters with glycerol, among which the following are most widespread:

• glycerol monoester with oleic acid (C18:l), abbreviated as GMO (glyceryl monooleates, Type 40); trade name: Peceol from GATEFOSSE,

• glycerol monoester with linoleic acid (C18:2), abbreviated as GML (glyceryl monolinoleate); trade name: Maisine CC from GATEFOSSE,

• glycerol monoester with ricinoleic acid (C18:1,-OH), abbreviated as GMR (glyceryl monoricinoleate/; trade name: Softigen 701 from 101 OLEOCHEMICAL.

APIs may be active substances with lipophilic and/or hydrophobic properties, among which the following are listed:

• fat-soluble vitamins (A, D, E and K) and derivatives thereof (such as K-2), including provitamins,

• carotenoids (astaxanthin, zeaxanthin, lutein),

• fatty acids (such as eicosapentaenoic acid, docosahexaenoic acid, conjugated linoleic acid, alphalinoleic acid) in the acid form, fatty acid glycerol esters (such as mono-, di- and triglycerides), fatty acid esters with alcohols (primary, secondary and/or tertiary alcohols) and their derivatives,

• polyphenols, flavonoids, cannabinoids, standardized plant extracts and other natural active substances, such as naringenin, genistein, silymarin, curcumin, resveratrol, alpha-lipoic acid or ubiquinone.

All the compounds considered OTHER are a group of compounds that affect the sensory texture of the composition and have lipophilic and/or hydrophobic properties. These may be flavonoids (anthocyanins), carotenoids or curcumin, but also ether compounds, that is, natural plant extracts or synthetic products that reproduce aromatic compositions through a mixture of chemical compounds, thus mimicking a profile and chemical fingerprint of natural plant extracts. Testing methodology

• dispersion time was measured in seconds (t, sec) from the addition of the last drop until complete dissolution,

• nanoemulsion transmittance was measured as a percentage value (%T) at wavelength X=600 nm using a Merck® Pharo 300 spectrometer,

• nanoparticle hydrodynamic diameter was expressed in nanometers (D _H , nm) and was measured using a Zetasizer Nano-ZS instrument from Malvern.

• particle polydispersity when dispersed in water (polydispersity index) was expressed as a Pdl value and measured using a Zetasizer Nano-ZS instrument from Malvern.

Biological testing of the composition in an animal model (in vivo pharmacokinetics).

The improved bioavailability of the active substance (API) in the composition of the invention was confirmed by in vivo pharmacokinetic tests according to the procedure discussed below.

The animal model involved rats (males) divided into three groups: the preferable composition with vitamin D3 was administered to the first group, a commercially available vitamin D3 solution in MCT oil used for vitamin supplementation to the second group and the third control group did not receive the active substance in any form. Each group consisted of 4 adult individuals except for the control of 3 individuals. The animals were maintained in controlled environmental conditions for at least five days before the testing started. Vitamin D3 at a dose of 0.5 mg/kg body weight was administered to the first and second groups. After administration, blood was collected from the rats after 15 min, 30 min, 1, 2, 4, 6, 8, 12, 24 and 48 hours into test tubes with an anticoagulant and centrifuged within 30 minutes; subsequently, plasma was harvested for testing. Plasma samples were stored at -20°C until UPLC/MS/MS analysis. Parameters, such as bioavailability, C _max , T _max , CL, d, AUC and T1/2, were determined based on vitamin D3 contents in the blood samples. It was found following analysis that the preferred composition significantly improved the bioavailability and peak serum concentration (C _max ) of the active substance compared to commercially available products based on plant oils, especially MCT.

The preferred composition comprises ingredients:

• Surfactant (T80) means a polyoxyethylated derivative of sorbitan and oleic acid (Polysorbate 80, polyoxyethylated sorbitol monooleate, Tween™ 80), commercially available and offered by many manufacturers. In the experiment, Tween™ 80 from CRODA was used,

• Lipid 1 (MCT) means medium-chain triacylglyceride with a mixture of C8-C12 acids, commercially available and offered by many manufacturers. In the experiment, Crodamol™ from GTCC was used, • Lipid 2 (MCM) means medium-chain mono-diglycerides, mainly of capric (C8) and caprylic (decanoic, CIO) acids, commercially available and offered by many manufacturers. In the experiment, Imwitor® 988 from 101 OLEOCHEMICAL was used,

• API (vitamin D3), as a typical example of a fat-soluble vitamin and an active substance with lipophilic and/or hydrophobic properties. Vitamin D from Fermenta Biotech Limited wa s used in the application.

• OTHER (ether compounds) as a typical example of compounds that affect sensory attributes of the composition, with lipophilic and/or hydrophobic properties; ether compounds were used. Terpenes from Eybna Technologies Ltd. were used in the application.

Exemplary preparation procedure for the composition containing vitamin D as an active substance:

The composition was prepared by weighing out 91.84 g surfactant (T80) making up 30.00% by mass of the composition, 212.40 mg of the active substance (vitamin D) making up 0.069% by mass of the composition, 106.43 g Lipid 1 fraction (MCT) making up 34.77% by mass of the composition, 106.64 g Lipid 2 fraction (MCM) making up 34.84% by mass of the composition and 0.99 g OTHER (ether compounds) making up 0.32% by mass of the composition into one vessel. This was stirred for 30 min using a magnetic bar at 300 rpm until homogenous at room temperature.

Exemplary dosing of the active substance in self-emulsifying nanoemulsions

The composition may be used in aqueous systems and other forms with defined unit containers. In the application, the composition was tested by precise addition of portions of the composition to aqueous systems. An adequate dosing system ensures precise delivery of the active substance in a specific and constant portion of the composition. Quantitative amounts of the active substance (here, vitamin D) in international units, wherein 1 pg = 40 IU) facilitated measurement of various nanoemulsion parameters based on the active substance dose. Within the application, a dosing system was tested using pipettes/droppers and appropriate droplets (droplet mass was validated by measuring the mass of ten droplets 100 times and mean droplets mass was determined for which the active substance dose was calculated), wherein one droplet was equivalent to 1000 IU. The results are shown in Table 2.

Table 2. Values of nanoemulsion parameters important for the application, obtained from self-emulsifying compositions based on an exemplary composition with vitamin D as the active substance according to the application.

In addition, compositions with various contents of vitamin D as the active substance were also prepared according to the example above. The results are shown in Table 3.

Table 3. Exemplary compositions with various contents of vitamin D3

Within the application, compositions were prepared with other active substances and ingredients of the composition which confirmed the action and unique character of the disclosed compositions. The results are shown in Table 4.

Table 4. Embodiments of the composition of the invention

In addition, the composition can be used for preparing mixtures that consist of at least two active substances (APIs) according to the procedure below.

Exemplary preparation procedure for the composition containing two active substances (APIs) The composition was prepared by weighing out 612.0 g surfactant (T80) making up 30.0% by mass of the composition, 632.0 mg of the Lipid 1 (MCT) fraction making up 31.0% by mass of the composition, 712.3 g of the Lipid 2 (MCM) fraction making up 34.92% by mass of the composition, 41.9 g vitamin D3 in the form of MCT solution with a concentration of 1 MIU/G making up 2.06% by mass of the composition, 38.5 g of 5% vitamin K2 MK7 solution (all-trans menaquinone-7) in MCT oil making up 1.9% by mass of the composition and OTHER (ether compounds) making up 0.17% by mass of the composition into one vessel. This was stirred for 30 min using a magnetic stirrer at 300 rpm until homogenous at room temperature.

The resulting mixture in a quantity of 100 mg was dispersed in 200 mL of class II water and the following nanoemulsion parameters were obtained:

Exemplary preparation procedure for the composition containing at least three active substances (APIs)

The composition was prepared by weighing out 604.7 g surfactant (T80) making up 29.4% by mass of the composition, 284.5 mg of the Lipid 1 (MCT) fraction making up 13.8% by mass of the composition, 716 g of the Lipid 2 (MCM) fraction making up 34.8% by mass of the composition, 50.4 g vitamin D3 in the form of MCT solution with a concentration of 1 MIU/G making up 2.5% by mass of the composition, 46.7 g of 5% vitamin K2 MK7 solution (all-trans menaquinone-7) in MCT oil making up 2.3% by mass of the composition, 47.4 g retinyl palmitate in the form of MCT solution with a concentration of 1 MIU/G making up 2.3% by mass of the composition, 305.6 g a-tocopherol acetate making up 14.9% by mass of the composition into one vessel. This was stirred for 30 min using a magnetic stirrer at 300 rpm until homogenous at room temperature.

The resulting mixture in a quantity of 100 mg was dispersed in 200 mL of class II water and the following nanoemulsion parameters were obtained: