Estrosphere preparation, method of production thereof and use

Abstract

An estrosphere preparation comprising an ester phase and an aqueous phase characterised in that the ester phase comprises α-linolenic acid ethyl ester, or a mixture of α-linolenic acid ethyl ester with one or more components selected from the group comprising polyunsaturated fatty acids ethyl esters (PUFA ethyl esters) or triglycerides, ethanol, and emulsifier, in a weight ratio of between 2:0.5:1 and 8:2:1, respectively, and the aqueous phase comprises water with glycerol at a final concentration of 2.25% by weight, wherein the weight ratio of α-linolenic acid ethyl ester, or of the mixture of α-linolenic acid ethyl ester with one or more components to ethanol, emulsifier and water is between 2:0.5:1:45 and 8:2:1:45, respectively. A method for producing such an estrosphere preparation. Said preparation for use in the treatment of cancer and viral diseases.

Description

[0001] Estrosphere preparation, method of production thereof and use

[0002] The subject of the invention is an estrosphere preparation, a method of production and the use of the estrosphere preparation in the prevention and treatment of cancer and viral diseases. Drugs administered to patients are often rapidly metabolised, which depends on the route of administration. In the case of oral administration, metabolism may occur already in the gastrointestinal tract and then in the liver. Substances administered intravenously are mainly metabolised in the liver. Various strategies are used to counteract rapid metabolism and increase drug bioavailability.

[0003] For orally administered drugs, capsules are often used to delay the release of the drugs in the gastrointestinal tract. These capsules enable increased bioavailability of active substances and reduce their metabolism in the liver. However, in the case of substances administered intravenously, various types of drug carriers are used to increase stability and control the release of active substances.

[0004] The most commonly used drug carriers include liposomes, polymeric particles, polymeric micelles and various types of lipid emulsions. Among these, liposomes are particularly valued because they can encapsulate both hydrophilic (water-soluble) substances and lipophilic and hydrophobic substances. Lipophilic substances possessing ionizable moieties can be encapsulated inside liposomes using active encapsulation techniques. However, in the case of hydrophobic or lipophilic substances without ionized groups, their encapsulation in the lipid bilayer of liposomes is limited due to their low solubility.

[0005] It is a practical problem to encapsulate simultaneously hydrophilic substances in liposomes and hydrophobic substances in the lipid bilayer. The escape rate of hydrophobic substances from the lipid bilayer is much faster than that of hydrophilic substances, leading to increased permeability and destabilisation of the liposome structure. As a result, encapsulation of both hydrophilic and hydrophobic substances in one type of liposome may be difficult to achieve. The use of lipid emulsions solves this problem, but only for certain hydrophobic substances. However, lipid emulsions have their limitations related to their composition, as the main constituent forming a lipid emulsion is triglycerides, in which most lipophilic and hydrophobic drugs have limited solubility. This results in low affinity of drugs for the lipid phase, rapid escape of drugs during intravenous administration and low storage stability of the formulation. When medicinal substances and dietary supplements are administered orally, substances administered in tablet or capsule form have limited bioavailability due to the difficulty of dissolving in body fluids. A number of procedures are used here, such as micronisation or application to different types of carrier, for example microcrystalline cellulose. However, this approach has limitations as there is no controlled release of these substances, leading to the appearance of a high pharmacokinetic peak in the bloodstream.

[0006] One of the solutions to limit too rapid release of the substance and an unfavorable pharmacokinetic profile is the use of controlled release methods. The most common method is to coat the granule particles of the medicinal substance with various types of polymers, whose solubility is greater in certain parts of the digestive tract. It is also possible to mix two or three types of pellets with different coatings, which allows a gradual and controlled release of the contents in the digestive tract. However, the process is complex and requires specialised instrumentation.

[0007] Unexpectedly, the solution to the above problems is the use of a mixture of fatty acid ethyl esters with phospholipids, especially soy, sunflower, rapeseed or egg lecithin in various proportions, allowing to obtain structures called estrospheres by the inventors. Estrospheres have intermediate properties between liposomes and lipid emulsions.

[0008] Esterospheres have a hybrid structure, containing an aqueous solution in which hydrophilic substances can be encapsulated, and a lipid envelope that is much thicker than that of classical liposomes. This envelope better protects the aqueous solution encapsulated in the estrosphere structure. In addition, the lipid envelope is thick enough to encapsulate hydrophobic substances, which will then be slowly released. Thanks to the fact that the esters are composed of a mixture of ethyl esters of omega-3 fatty acids and phospholipids, the solubility of most hydrophobic and lipophilic drugs is much better than in classical emulsions composed of triglycerides. This significantly reduces the too rapid discharge of drugs during intravenous administration.

[0009] As estrospheres are made up of a mixture of fatty acid ethyl esters, which have medium HLB values, and phospholipids, which also have high HLB values, the formation of a lipid matrix follows the hydration of such a mixture. In this matrix, water-containing compartments and hydrophobic compartments are formed, resulting in such a sponge-like structure containing water, which has the ability to encapsulate both hydrophobic and hydrophilic substances. In addition, the process of drug release from such a structure is slowed down due to the high hydrophobicity of this lipid matrix and the inability to easily suspend as droplets. The breakdown process of the lipid matrix made up of fatty acid ethyl esters and phospholipids in the gastrointestinal tract is limited to the surface area digested by esterases, lipases and phospholipases. When drugs are encapsulated in such a structure, their release process will be gradual.

[0010] Appropriate homogenization of this lipid matrix leads to the formation of nanoparticles of approximately 100 nm in size, the properties of which have been described above.

[0011] The invention provides increased drug bioavailability and reduced drug metabolism through the use of unique drug carriers such as estrospheres, which combine the advantages of liposomes and lipid emulsions. These innovative approaches can significantly improve the therapeutic efficacy of administered drugs and dietary supplements.

[0012] Fatty acid ethyl esters are derivatives of triglycerides, however, in the process of transesterification of triglycerides, the released free fatty acids are subjected to an esterification reaction with ethanol in the presence of a catalyst to obtain glycerol-free derivatives. The fatty acid ethyl esters obtained have different physicochemical properties compared to triglycerides and a different effective molecular shape. Unlike triglycerides, there is one molecule of fatty acid for every molecule of alcohol. In the case of triglycerides, there are three molecules of fatty acids per molecule of glycerol, which originally has three hydroxyl groups.

[0013] The resulting fatty acid ethyl ester molecules have a different molecular shape, show solubility in ethanol (unlike triglycerides) and a different lipophilicity. Fatty acid ethyl esters have organoleptic properties similar to those of vegetable oils, but their properties differ from those of triglycerides. Due to the ability of fatty acid ethyl esters to dissolve in ethanol, it is possible to prepare a mixture of fatty acid ethyl esters and phospholipids, such as lecithin, in any weight proportions and then hydrate such a mixture.

[0014] The release process somewhat resembles that of the phospholipids themselves - there is an absorption of water and the production of lipid bilayer-like structures. Due to the presence of fatty acid ethyl esters in the mixture, typical liposomes are not formed, but a three-dimensional structure is created consisting of a mixture of fatty acid ethyl esters and phospholipids, containing water. The exact structure of this structure is not fully defined, as it contains both fatty acid ethyl esters and phospholipids in its composition and resembles hydrated phospholipid mixtures.

[0015] Due to the fact that fatty acid ethyl esters have a significantly higher HLB (Hydrophile-Lipophile Balance) than triglycerides, but lower than phospholipids, they form neither water-suspended emulsion droplets nor classical lipid bilayers. Macroscopically, the hydrated mixtures of fatty acid ethyl esters and phospholipids resemble a type of grease; however, due to the phospholipid content, these structures contain water. They adhere strongly to tissues, for example the palate or skin, and are very difficult to remove even with detergents.

[0016] The lipid matrix, which is formed by hydrating a mixture of fatty acids and phospholipids, such as soy lecithin, after being broken down into small particles using a high-pressure homogenizer, does not exhibit the characteristics of either a lipid emulsion or liposomes, but is an intermediate structure between a lipid emulsion and liposomes. These particles contain empty space, as in liposomes, but are surrounded not by a lipid bilayer, but by a layer of fatty acid ethyl esters surrounded by a lipid bilayer. This structure results in the resulting structures acquiring hitherto unknown properties. They are something intermediate between lipid emulsion and liposomes, being able to encapsulate both aqueous solutions and hydrophobic substances embedded in the lipid envelope of the estrosphere.

[0017] In the case of nanostructures, this structure allows for hitherto unknown properties compared to other drug carriers. Firstly, the release of hydrophilic substances encapsulated in the aqueous space of the estrosphere is significantly slowed down compared to classical liposomes, due to the considerable thickness of the envelope surrounding the aqueous solution of the estrosphere. Hydrophilic substances have not only a lipid bilayer barrier to overcome, which can be more easily destabilised under blood conditions, but also a layer made up of fatty acid ethyl esters and phospholipids. The cryo-electron microscope images provided show that the thickness of the lipid envelope is many times that of the classical lipid bilayer. Multilayer liposomes also have the ability to limit the diffusion of hydrophilic substances encapsulated in their structure, however, their size prevents practical application. This structure of the estrospheres allows a much better control of the release of hydrophilic substances.

[0018] In addition, the preparation of a lipid matrix from a mixture of fatty acid ethyl esters and phospholipids, such as lecithin, and placing them in capsules together with a suspended drug solution or its crystals, results in a controlled release of both hydrophilic and hydrophobic substances. One of the reasons why preparations encapsulated in such a lipid matrix and swallowed by the patient or consumer have the capacity for controlled release is that the mixture of fatty acid ethyl esters and phospholipids is not easily emulsified, but gradually digested from the surface by lipases, phospholipases and esterases present in the gastrointestinal tract.

[0019] Secondly, the resulting nanostructures can simultaneously be a carrier for hydrophobic substances such as anticancer drugs and others. Ethyl esters of fatty acids and phospholipids have much better solubility for most hydrophobic and lipophilic drugs compared to classical emulsions composed of triglycerides. In addition, as with liposomes, the estrosphere structures are surrounded by a lipid bilayer, which further prevents lipophilic and hydrophobic substances from easily escaping.

[0020] An important feature of estrospheres is that they can be constructed from any mixture of any proportion containing both soy or other phospholipids and fatty acid ethyl esters. Thus, the aqueous volume of the estrospheres and the thickness of the lipid envelope can be modified depending on whether one wants to use hydrophilic substances, hydrophobic substances or both at the same time, achieving different properties.

[0021] Various compositions containing ethyl esters of alpha-linolenic acid are known in the state of the art. CN101978950A discloses a nanoemulsion containing 200 g of a-linolenic acid ethyl ester, 12 g of lecithin, 21 g of glycerol and its method of manufacture and use.

[0022] CN108553419A concerns a nanoemulsion, with a particle size of 1-100 nm, of a-linolenic acid ethyl ester. The nanoemulsion of a-linolenic acid ethyl ester is prepared from 4-70 parts by weight of a-linolenic acid ethyl ester or an oil phase containing a-linolenic acid ethyl ester, 6-40 parts of a surfactant and co-surfactant (salicylic acid), 0-1.5 parts of an inorganic salt, 0.1-1.5 parts of an antioxidant and the balance water.

[0023] However, very importantly, such compositions known in the state of the art indicate that a system with nanoemulsion characteristics has been obtained, as indicated by electron microscopy images (if available) or by the way prepared according to the definition of nanoemulsion. There are no reports in the literature that precisely describe the development of the technology and the derivation of the ester-phospholipid-like spheres presented in this application. Unexpectedly, ethyl esters of alpha-linolenic acid (et-ALA) in the form of estrospheres have shown anticancer activity (cytotoxic as well as cytostatic) against several human cancers, and more importantly, no toxicity of such a system against normal cells has been demonstrated, which may mean that the proposed form of therapy meets the criteria for therapy. According to the present invention, the estrosphere technology developed on the basis of alpha-linolenic acid with the addition of natural, pharmaceutically pure soy lecithin used as an emulsifier exhibits a number of biological activities, including anticancer activity. The use of ethyl esters of fatty acids of animal (fish) origin, such as eicosapentaenoic acid (EP A) and docosahexaenoic acid (DHA), is known, but their use may be limited due to the potential toxicity of such acids when repeatedly administering high doses of these acids, which is necessary to achieve an anticancer effect. The administration of high doses of alpha linolenic acid ethyl esters (et-ALA) is safe, as they cannot be used directly by the body to form membrane lipids, as ALA acid is not a natural component of human cell membranes. In addition, ET-ALA can be taken as a dietary supplement and can be used to strengthen patients during cancer therapy. In the examples below, the anticancer effect of ET-ALA-based estrosphere technology was demonstrated for the first time on cell lines: LoVo, LoVo Dx and CaCo2 (cancer cell lines derived from the colon), A549, MCF7, Ca derived from the patient (various adenocarcinomas), CCRF / CEM (T lymphocytic leukemia) at the same time low toxicity was achieved towards normal cell lines - NHDF (human fibroblasts), L929 (mouse fibroblasts).

[0024] The course of the CO VID 19 pandemic clearly demonstrates that the epidemiological situation with regard to human infections and mortality is not a closed matter and, in all likelihood, the SARS-CoV2 virus will continue to pose a threat to public health for a long time to come. The high mutagenicity of the coronavirus means that it escapes the body's defence mechanisms and further research is necessary to develop effective methods and strategies to prevent and combat the disease. According to virologists' predictions, the gradual extinction of pandemics in the future will involve the simultaneous alternation of new viruses or the reappearance of long-defunct viruses with high pathogenic potential. Viruses constantly present in the human population and in the ecosystem, such as the influenza virus, herpes simplex or HIV, are also not insignificant. Most often, they do not cause mass deaths, but in individual cases they can lead to serious health damage or even death. Any form of inactivation or reduction of pathogens can provide invaluable assistance in preventing infectious diseases, both for individual patients and for the population. Body protection related to viral infections, should not be limited to the preventive sphere alone. The improvement of the patient's state of health during infection and the neutralisation of the sequelae of past infectious diseases should also be taken into account. For this purpose, it will be important to inhibit the cytokine storm and suppress inflammation resulting from infection. Unexpectedly, estrosphere technology based on ethyl esters of alphalinolenic acid (et-ALA) shows potent antiviral activity against a wide panel of human and selected animal viruses, and both attacking the respiratory tract and causing diseases such as herpes or obesity.

[0025] The studies shown in the examples below were carried out on viruses with respiratory affinity: human coronavirus OC43 (HcoV - OC 43) and adenovirus type 5 (HadV 5). In addition, its efficacy against the human infectious obesity virus adenovirus 36 (HadV 36) and herpes simplex virus (HSV) was tested. In addition, the antiviral properties were tested with equine herpes virus (EHV-1) causing abortions. The panel of infectious agents included both non-embryonic and enveloped viruses. In all cases, very high efficacy was obtained in eliminating the above pathogens at low concentrations of the estrosphere preparation, demonstrating its potential for use in therapies for diseases caused by these viruses.

[0026] The subject of the invention is an estrosphere preparation comprising an ester phase and an aqueous phase characterised in that

[0027] the ester phase comprises

[0028] a-linolenic acid ethyl ester, or a mixture of a-linolenic acid ethyl ester with one or more components selected from the group comprising polyunsaturated fatty acids ethyl esters (PUFA ethyl esters) or triglycerides,

[0029] emulsifier and

[0030] ethanol,

[0031] in a weight ratio of between 2:0.5: 1 and 8:2:1, respectively, and the aqueous phase comprises

[0032] water with glycerol at a final concentration of 2.25% by weight,

[0033] wherein the weight ratio of a-linolenic acid ethyl ester, or of the mixture of a-linolenic acid ethyl ester with one or more components to ethanol, emulsifier and water is between 2:0.5:1:45 and 8:2:1:45, respectively.

[0034] Preferably, the average particle size of the estrosphere in the preparation is between 20 and 200 nm.

[0035] Preferably, the the PUFA ethyl esters are selected from the group comprising a-linolenic acid ethyl ester, y-linolenic acid ethyl ester, eicosapentaenoic acid ethyl ester and docosahexaenoic acid ethyl ester, conjugated linoleic acid ethyl ester, linoleic acid ethyl ester, oleic acid ethyl ester, stearic acid ethyl ester, palmitic acid ethyl ester.

[0036] Preferably, the triglycerides are selected from the group of triglycerides comprising acids having 8 to 24 carbon atoms, in particular a-linolenic acid, y-linolenic acid (GLA), eicosapentaenoic acid (EP A), docosahexaenoic acid (DHA), conjugated linoleic acid (CLA), linoleic acid (LA) and oleic acid.

[0037] Preferably, the emulsifier is selected from the group comprising soy lecithin, pharmaceutical grade egg lecithin, fatty acid salts, detergents, lysolecithins and mono- and diglycerides of fatty acids.

[0038] Preferably, the fatty acid salt is the sodium salt of oleic acid.

[0039] Preferably, the detergent is selected from the group comprising Polysorbate 80, Polysorbate 20 and Span 80.

[0040] Preferably, the weight ratio of a-linolenic acid ethyl ester, or a mixture of a-linolenic acid ethyl ester with one or more components to ethanol, emulsifier and water is 4:0.5: 1 :45, respectively. The subject of the invention is also a method of production the estrosphere preparation characterised in that it comprises the following steps:

[0041] a. obtaining the ester phase

[0042] i. a-linolenic acid ethyl ester, or a mixture of a-linolenic acid ethyl ester with one or more components selected from the group comprising PUFA ethyl esters or triglycerides, is mixed with ethanol and emulsifier in the weight ratio of between 2:0.5: 1 and 8:2:1, respectively, with stirring at 30 to 40 rpm;

[0043] b. obtaining the aqueous phase

[0044] i. an aqueous glycerol solution of 2.25% by weight is prepared and mixed at 80 to 100 rpm;

[0045] c. phase mixing

[0046] i. the ester phase obtained in step a(i.) is added to the aqueous phase obtained in step b(i.) maintaining a weight ratio of either a-linolenic acid ethyl ester, or of the mixture of a-linolenic acid ethyl ester with one or more components to ethanol, emulsifier and water of between 2:0.5:1:45 and 8:2:1:45, respectively, and is mixed at 80 to 400 rpm;

[0047] ii. the obtained mixture is stirred further at 2000 to 2500 rpm for at least 10 min. iii. the mixture obtained in step c(ii.) is subjected to high-pressure homogenization by passing the mixture 6 times through the homogenization head at a pressure of between 300 and 600 bar.

[0048] Preferably, the mixture obtained in step a(i.) is heated at a temperature between 10 and 45 °C until the emulsifier is dissolved.

[0049] Preferably, the method is carried out under anaerobic conditions.

[0050] Preferably, in step c(i.) the weight ratio of a-linolenic acid ethyl ester, or a-linolenic acid ethyl ester mixture with one or more components to ethanol, emulsifier and water is 4:0.5:1:45, respectively.

[0051] In addition, the subject of the invention is the use of an estrosphere preparation in the prevention and treatment of cancer and viral diseases.

[0052] The cancer is preferably selected from a group comprising colorectal cancer, lung cancer, breast cancer, lymphocytic leukaemia, intestinal cancer, Burkitt's lymphoma, gastric cancer, glioma, melanoma and ovarian cancer.

[0053] Preferably, the viral disease is caused by equine herpesvirus type 1.

[0054] The subject of the invention is depicted by means of the figures of the drawing, in which: Fig. 1 shows a Cryo-EM microscopy image of an estrosphere preparation when an emulsifieresters weight ratio of 4: 1 is used.

[0055] Fig. 2 shows a Cryo-EM microscopy image of an estrosphere preparation when an emulsifieresters weight ratio of 2: 1 is used.

[0056] Fig. 3 shows a Cryo-EM microscopy image of an estrosphere preparation when an emulsifieresters weight ratio of 1 : 1 is used.

[0057] Fig. 4 shows a Cryo-EM microscopy image of an estrosphere preparation when an emulsifieresters weight ratio of 1 :2 is used.

[0058] Fig. 5 shows a Cryo-EM microscopy image of an estrosphere preparation when an emulsifieresters weight ratio of 1 :4 is used.

[0059] Fig. 6 shows photographs prepared using the cryo-electron microscopy (Cryo-EM) technique describing the visual character of estrosphere particles prepared under anaerobic conditions. Fig. 7 presents an analysis of the size distribution and poly dispersity of estrosphere particles with a weight ratio of ethyl esters:phospholipids (emulsifier) of 1:4, carried out using the Dynamic Light Scattering technique.

[0060] Fig. 8 presents an analysis of the size distribution and poly dispersity of estrosphere particles with a weight ratio of ethyl esters:phospholipids (emulsifier) of 1:2, carried out using the Dynamic Light Scattering technique.

[0061] Fig. 9 presents an analysis of the size distribution and poly dispersity of estrosphere particles with a weight ratio of ethyl esters:phospholipids (emulsifier) of 1:1, carried out using the Dynamic Light Scattering technique. Fig. 10 presents an analysis of the size distribution and poly dispersity of estrosphere particles with a weight ratio of ethyl esters:phospholipids (emulsifier) of 2:1, carried out using the Dynamic Light Scattering technique.

[0062] Fig. 11 presents an analysis of the size distribution and poly dispersity of estrosphere particles with a weight ratio of ethyl esters:phospholipids (emulsifier) of 4:1, carried out using the Dynamic Light Scattering technique.

[0063] Fig 12 shows an analysis of the size distribution and poly dispersity of the estrosphere particles made with the Dynamic Light Scattering technique for a preparation made under anaerobic conditions.

[0064] Fig. 13 shows the cytotoxic effect of an estrosphere preparation according to the invention containing a-linolenic acid ethyl ester on human colon-derived tumour line cells LoVo, LoVoDx, and CaCo2.

[0065] Fig. 14 shows the cytotoxic effect of an estrosphere preparation according to the invention containing a-linolenic acid ethyl ester on human lung cancer cells (A549,) breast cancer (MCF7), and cancer taken from a patient (Ca).

[0066] Fig. 15 shows the cytotoxic effect of an estrosphere preparation according to the invention containing a-linolenic acid ethyl ester on human CCRF / CEM lymphocytic leukaemia cells. Fig. 16 shows the cytotoxic effect of an estrosphere preparation according to the invention containing a-linolenic acid ethyl ester on normal human fibroblasts (NHDF) and normal mouse fibroblasts (L929).

[0067] Fig. 17 shows the kinetics of 4T1 tumour growth inhibition in response to intravenous (nanoemulsion) and oral (oil) administration of alpha linolenic acid ethyl esters.

[0068] Fig. 18 shows the kinetics of growth inhibition of mouse intestinal carcinoma CT26 in response to intravenous (estrosphere preparation) and oral (oil) administration of alpha linolenic acid ethyl esters.

[0069] The invention is illustrated in the following examples, , which are not intended to limit the scope of the invention but serve solely to illustrate its implementation.

[0070] Examples

[0071] General procedure for obtaining an estrospheres preparation

[0072] a. Obtaining the ester phase

[0073] i. a-linolenic acid ethyl ester, or a mixture of a-linolenic acid ethyl ester with one or more components selected from the group comprising other polyunsaturated fatty acids ethyl esters (PUFA ethyl esters) or triglycerides, is mixed with ethanol and an emulsifier in the weight ratio of between 2:0.5: 1 and 8:2:1, respectively, with stirring at 30 to 40 rpm; ii. optionally, the mixture obtained in step a(i.) is heated between 10 and 45°C until the emulsifier dissolves;

[0074] b. Obtaining the aqueous phase i. an aqueous glycerol solution of 2.25% by weight is prepared and mixed at 80 to 100 rpm;

[0075] c. Phase mixing

[0076] i. The ester phase obtained in step a(i.) or a(ii.) is added to the aqueous phase obtained in step b(i.), maintaining a weight ratio of either a- linolenic acid ethyl ester, or of the mixture of a-linolenic acid ethyl ester with one or more components to ethanol, emulsifier and water of between 2:0.5:1:45 and 8:2:1:45, respectively, and is mixed at 80 to 400 rpm;

[0077] ii. the obtained mixture is stirred further at 2000-2500 rpm for at least 10 min and

[0078] iii. the mixture obtained in step c(ii.) is subjected to high-pressure homogenization by passing the mixture 6 times through the homogenization head at a pressure of between 300 and 600 bar, Optionally, the method is carried out under anaerobic conditions.

[0079] Homogenization is carried out using an ultrasonic microprocessor with a power output of up to 150W. In particular, homogenization is carried out between 25 and 50% of the maximum power of the ultrasonic microprocessor.

[0080] The ethyl ester of a higher fatty acid is selected from the group comprising a-linolenic acid ethyl ester and other bioactive esters such as y-linolenic acid ethyl ester, eicosapentaenoic acid ethyl ester and docosahexaenoic acid ethyl ester, conjugated linolenic acid ethyl ester, linoleic acid ethyl ester, oleic acid ethyl ester, stearic acid ethyl ester, palmitic acid ethyl ester.

[0081] The fatty acids are selected from the group comprising acids with 8 to 24 carbon atoms, in particular those with beneficial biological activity such as a-linolenic acid and other bioactive fatty acids such as y-linolenic acid (GLA), eicosapentaenoic acid (EP A) and docosahexaenoic acid (DHA), conjugated linoleic acid (CLA), linoleic acid (LA), oleic acid.

[0082] The emulsifier is selected from a group including soy lecithin, pharmaceutically pure egg lecithin, fatty acid salts such as oleic acid sodium, pharmaceutically accepted detergents such as Polysorbate 80, Polysorbate 20, Span 80, lysolecithins and mono- and diglycerides of fatty acids.

[0083] In particular, soy or egg lecithin is the emulsifier.

[0084] In one variant, the emulsifier is in powder form.

[0085] Anaerobic conditions are achieved by passing a stream of nitrogen through at each stage. In one embodiment, the ethanol and water are previously deoxygenated by passing a stream of nitrogen.

[0086] In one embodiment, the weight ratio of the ethyl ester of the higher fatty acid to the ethanol, emulsifier and water is 4:0.5:1:45.

[0087] Example 1

[0088] Method of producing an estrosphere preparation obtained from pure fatty acid ethyl esters Liquid as well as solid lipid raw materials must be kept in the dark in a refrigerator below 10°C throughout their storage. Into a 150-mL glass ground-glass triple flask fitted with a high-speed mechanical stirrer, a dropper with gas outlet and a gas barb, 10 g of lecithin is introduced as an emulsifier with a purity of not less than 70%. Then, 5 g of ethanol (previously deoxygenated) is poured into the flask via a dropper. The two ingredients are mixed at 30 rpm with a mechanical stirrer until the emulsifier is dissolved, and then, once dissolved, 40 g of a mixture of PUFA ethyl esters obtained from the oil of plants such as flax, hemp or perilla is slowly added dropwise. Gaseous nitrogen is passed through the solution throughout the entire process. Glycerol (to a 2.25 % concentration) is then introduced into a second 1 000 mL flask containing 450 g of pharmaceutically pure water from which the air has previously been removed by a stream of nitrogen, while stirring at 80 rpm until the ingredients are mixed. The contents of the first flask are then introduced into the second flask while stirring with a mechanical stirrer at 80 rpm for 5 min, all the while passing a stream of nitrogen through the flask. In the next step, the stirrer speed is increased to at least 2000 rpm and stirred for a further 10 min while passing a stream of nitrogen through the flask to obtain the initial preparation of estrospheres. A ground glass tube is then inserted in place of the dropper and shaped to enable the transfer of the resulting preliminary estrosphere preparation, by generating an overpressure of gaseous nitrogen in the flask, into the buffer glass container pre-filled with this gas. In the next step, the preparation is subjected to a high-pressure homogenization process by passing the preparation 6 times through the homogenization head at 450 bar. Once the final estrosphere preparation is obtained, it is subjected to pre-sterilisation by being filtered through nylon or polycarbonate filters with a pore size of 0.6 micrometres.

[0089] In the next stage, the product is packaged using a dosing pump into final dark glass containers. When sealing, a stream of nitrogen is passed through the containers and fed over the liquid at the time of sealing and subjected to thermal sterilisation. Containers shall be stored at a temperature below 6°C, but not below 1°C.

[0090] The estrosphere preparation obtained is characterised by a mono-modal particle size distribution and an average particle size of 120 nm.

[0091] Example 2

[0092] Method of producing an estrosphere preparation obtained from ethyl esters of fatty acids and free fatty acids

[0093] Liquid as well as solid lipid raw materials must be kept in the dark in a refrigerator below 10°C throughout their storage. Into a 150-mL glass ground-glass triple flask fitted with a high-speed mechanical stirrer, a dropper with gas outlet and a gas barb, 10 g of lecithin is introduced as an emulsifier with a purity of not less than 70%. Then, 5 g of ethanol (previously deoxygenated) is poured into the flask via a dropper. The two ingredients are mixed at 30 rpm with a mechanical stirrer until the emulsifier is dissolved and then, once dissolved, 30g of a mixture of ethyl esters of PF As obtained from the oil of plants such as flax, hemp or pachyderm and 10g of a-linolenic acid is slowly added dropwise. Gaseous nitrogen is passed through the solution throughout the entire process. Glycerol (to a 2.25 % concentration) is then introduced into a second 1 000 mL flask containing 450 g of pharmaceutically pure water from which the air has previously been removed by a stream of nitrogen, while stirring at 80 rpm until the ingredients are mixed. The contents of the first flask are then introduced into the second flask while stirring with a mechanical stirrer at 80 rpm for 5 min, all the while passing a stream of nitrogen through the flask. In the next step, the stirrer speed is increased to at least 2 000 rpm and stirred for a further 10 min while passing a stream of nitrogen through the flask to obtain the initial preparation of estrospheres. A ground glass tube is then inserted in place of the dropper and shaped to enable the transfer of the resulting preliminary estrosphere preparation, by generating an overpressure of gaseous nitrogen in the flask, into the buffer glass container pre-filled with this gas. In the next step, the preparation is subjected to a high-pressure homogenization process by passing the preparation 6 times through the homogenization head at 300 bar. Once the final estrosphere preparation is obtained, it is subjected to pre-sterilisation by being filtered through nylon or polycarbonate filters with a pore size of 0.6 micrometres. In the next stage, the product is packaged using a dosing pump into final dark glass containers. When sealing, a stream of nitrogen is passed through the containers and fed over the liquid at the time of sealing and subjected to thermal sterilisation. Containers shall be stored at a temperature below 6°C, but not below 1°C.

[0094] The estrosphere preparation obtained is characterised by a mono-modal particle size distribution and an average particle size of 90 nm.

[0095] Example 3

[0096] Method of producing an estrosphere preparation containing ethyl esters of fatty acids and triglycerides

[0097] Liquid as well as solid lipid raw materials must be kept in the dark in a refrigerator below 10°C throughout their storage. Into a 150-mL glass ground-glass triple flask fitted with a high-speed mechanical stirrer, a dropper with gas outlet and a gas barb, 10 g of lecithin is introduced as an emulsifier with a purity of not less than 70%. Then, 5 g of ethanol (previously deoxygenated) is poured into the flask via a dropper. The two ingredients are mixed at 30 rpm with a mechanical stirrer until the emulsifier is dissolved and then, once dissolved, 20 g of a mixture of ethyl esters of PF As derived from the oil of plants such as flax, hemp or perilla and 20 g of triglycerides derived from fish oil are slowly added dropwise.

[0098] Gaseous nitrogen is passed through the solution throughout the entire process. Glycerol (to a 2.25 % concentration) is then introduced into a second 1 000 mL flask containing 450 g of pharmaceutically pure water from which the air has previously been removed by a stream of nitrogen, while stirring at 80 rpm until the ingredients are mixed. The contents of the first flask are then introduced into the second flask while stirring with a mechanical stirrer at 80 rpm for 5 min, all the while passing a stream of nitrogen through the flask. In the next step, the stirrer speed is increased to at least 2000 rpm and stirred for a further 10 min while passing a stream of nitrogen through the flask to obtain the preliminary estrosphere preparation. A ground glass tube is then inserted in place of the dropper and shaped to enable the transfer of the resulting final estrosphere preparation, by generating an overpressure of gaseous nitrogen in the flask, into the buffer glass container pre-filled with this gas. In the next step, the preparation is subjected to a high-pressure homogenization process by passing the preparation 6 times through the homogenization head at 600 bar. Once the final estrosphere preparation is obtained, it is subjected to pre-sterilisation by being filtered through nylon or polycarbonate filters with a pore size of 0.6 micrometres.

[0099] In the next stage, the product is packaged using a dosing pump into final dark glass containers. When sealing, a stream of nitrogen is passed through the containers and fed over the liquid at the time of sealing and subjected to thermal sterilisation. Containers shall be stored at a temperature below 6°C, but not below 1°C.

[0100] The estrosphere preparation obtained is characterised by a mono-modal particle size distribution and a size of 145 nm.

[0101] Example 4

[0102] Cytotoxic effects of the estrosphere preparation obtained in example 1 on human tumour cells of the colon-derived cancer lines LoVo, LoVoDx, and CaCo2.

[0103] The estrosphere preparation according to the invention obtained in example 1 was administered in increasing amounts of 0.01% to 5% by volume of the preparation relative to the culture medium to cells of the colon-derived cancer lines LoVo, LoVoDx and CaCo2.

[0104] The results for the preparation of esters and ethyl esters of a-linolenic acid, are shown in Fig.

[0105] 7.

[0106] A cytostatic effect was obtained for the CaCo and LoVo cell lines for concentrations of estrosphere preparation ranging from 0.5% to 50% and a cytotoxic effect for the LoVoDX line for concentrations of estrosphere preparation ranging from 0.25% to 5%.

[0107] Example 5

[0108] Cytotoxic effects of the estrosphere preparation obtained in example 2 containing a-linolenic acid ethyl ester on human lung cancer (A549,) breast cancer (MCF7), and patient-derived cancer (Ca) cells.

[0109] The estrosphere preparation according to the invention obtained in example 2 was administered in increasing amounts of 0.01% to 5% by volume of the preparation relative to the culture medium to lung cancer cells (A549,) breast cancer cells (MCF7), and patient-derived cancer (Ca). The results for the preparation of a-linolenic acid ethyl ester estrospheres are presented in Fig. 8.

[0110] For all lines, a cytotoxic effect was obtained for line A549 for an estrosphere formulation concentration of 0.25%, and for MCF7 and Patient Ca for an estrosphere formulation concentration of 0.01%.

[0111] Example 6

[0112] Cytotoxic effect of the estrosphere preparation obtained in example 2 containing a-linolenic acid ethyl ester on human CCRF / CEM lymphocytic leukaemia cells.

[0113] Estrosphere preparation obtained according to the invention in example 2 was administered in increasing amounts of 0.01% to 5% by volume of the preparation in relation to the culture medium to human CCRF / CEM lymphocytic leukaemia cells. The results for the estrosphere preparation are shown in Fig. 9.

[0114] The experiments demonstrated a strong growth-inhibitory effect on leukaemia cells. The activity is concentration dependent and is already visible at the lowest tested concentration of 0.01%.

[0115] Example 7

[0116] Cytotoxic effect of the estrosphere preparation obtained in example 2 containing a-linolenic acid ethyl ester on normal human fibroblasts (NHDF) and normal mouse fibroblasts (L929).

[0117] The estrosphere preparation obtained according to the invention in example 2 was administered in increasing amounts of 0.01% to 5% by volume of the preparation to the culture medium of normal human fibroblasts (NHDF) and normal mouse fibroblasts (L929). The results for the estrosphere preparation are shown in Fig. 10.

[0118] The results presented here indicate low toxicity of the estrosphere preparation to normal cells. In the case of the human line, cytotoxicity appears at a concentration of 5%, while in the case of mouse cells there is no such effect. Particularly noteworthy is the stimulation of cells to proliferate in the range of 0.05% to 1%. This makes it possible to use the estrosphere preparation in tissue regeneration processes, e.g. for preparations that will stimulate the healing process.

[0119] Example 8

[0120] Data from the above in vitro experiments were used to calculate the 50% inhibitory concentration (IC50).

[0121] The data obtained are presented in Table 1 showing the IC50 results obtained for the different cell lines treated with increasing volumes of the estrosphere preparation.

[0122] Table 1

[0123] IC50 (% of estrosphere

[0124] Line SD±

[0125] preparation in medium)

[0126] NHDF 3.550 0.170

[0127] L929 12.080 2.077

[0128] LOVO 0.215 0.078 LOVODX 0.152 0.009

[0129] A549 7.011 2.126

[0130] MCF7 0.498 0.039 CCRF / CEM 0.013 0.001

[0131] CaCo 0.096 0.021

[0132]

[0133] Patient Ca 0.183 0.016 High IC50 values were obtained for normal cell lines, while low and very low IC50 concentrations were obtained for most cancer cell lines, demonstrating the specificity of the estrosphere preparation towards cancer cells and its non-toxicity towards normal cells.

[0134] Example 9

[0135] Effect of intravenously administered estrosphere preparation and orally administered pure ET-ALA ethyl esters on the growth kinetics of mouse mammary tumour 4T1.

[0136] BalBc mice were orthotopically injected with 2xl05mouse mammary carcinoma cells - 4T1. The experiment started on day 4 after cell administration. The mice were administered the test preparations daily. First, ET-ALA ethyl esters were administered orally to the mice in a volume of 400 pL, and then the estrosphere preparation was administered intravenously in a volume of 200 pL. The compound was administered seven times. On day 8 after cell administration, tumours were harvested, weighed, and photographs of the tumours were taken. The results are shown in Fig. 11.

[0137] A statistically significant effect of the preparations was visible on days 6-8 and 10.

[0138] Example 10

[0139] Effect of intravenously administered estrosphere preparation and orally administered pure ET-ALA ethyl esters on the growth kinetics of mouse carcinoma CT26.

[0140] BalBc mice were orthotopically injected with 2xl05cells of mouse CT26 colon cancer. The experiment started on day 4 after cell administration. The mice were administered the test preparations daily. First, ET-ALA ethyl esters were administered orally to the mice in a volume of 400 pL, and then the tested estrosphere preparation was administered intravenously in a volume of 200 pL. The compound was administered seven times. On day 8 after cell administration, tumours were harvested, weighed, and photographs of the tumours were taken. The results are shown in Fig. 12.

[0141] A statistically significant effect of the preparations was visible on days 6-10.

[0142] Example 11

[0143] Determination of the virucidal activity of the estrosphere preparation against equine herpesvirus type 1 (EHV1)

[0144] Preparation of the test virus suspension

[0145] To prepare the virus suspension according to PN-EN 14476 + A2:2019-08 (EN 5.4), the RK-13 cell line was propagated in 175 cm3culture dishes (NEST SCIENTIFIC Biotechnology, New Jersey, USA) in the presence of Minimum Essential Medium and 10% foetal bovine serum (FBS). Equine herpesvirus type 1 (suspension at baseline concentration) was added to the monolayer for Ih at 37°C and gently shaken every 15 min. The culture was observed daily until the cytopathic effect appeared, after which it was frozen three times at -80°C and thawed. The suspension was centrifuged (10 min, 1500 x g) and the virus-containing supernatant was used for the test or stored at -80°C.

[0146] Infectivity testing Infectivity was determined by virus titration according to the standard (EN 5.5.7). In this experiment, each of the logarithmic dilutions tested was applied in a volume of 0.1 mL to four wells on a microplate containing 0.1 mL of freshly strypsinised RK-13 cells (10-15 xlO3cells per well). Microplates were incubated at 37°C with 5% CO2. The plate was observed daily until the appearance of a cytopathic effect at the highest dilution (7 days) under an inverted microscope (Axio Observer, Carl Zeiss MicroImaging GmbH). The infectious dose of TCIDso / mL was calculated using the Spearman and Karber method according to the following formula:

[0147] - logioTCIDso = xo - 0.5 + S r / n

[0148] where:

[0149] xo= log 10 of the highest dilution showing 100% positive reactions

[0150] r = number of positive determinations in the highest dilution showing 100% positive reactions and all positive results in the following dilutions

[0151] n = number of determinations for each dilution

[0152] Inactivation test

[0153] Virucidal efficacy of the estrosphere preparation at 5%, 2,5%, 1%, 0,5% and 0,025% concentration was tested according to standard PN-EN 14476 + A2:2019-08 (EN 5.5.2). Contact time was 60 minutes. For greater convenience of testing, a suspension of equal volumes (0.1 mL) of virus and interfering agent (PBS) and 0.8 mL of test agent was prepared. Immediately, after a defined contact time, the activity of the disinfectant was inhibited by making a series of dilutions to 10'12. Virus titration was performed after contact times of 0 min and 60 min.

[0154] Determination of cytotoxicity

[0155] The cytotoxicity of the preparation was determined according to the standard (EN 5.5.4.1) using 0.2 mL of hard water and 0.8 mL of the test agent.

[0156] Cell sensitivity

[0157] To control cell sensitivity (PN-EN 5.5.4.2), two volumes of doubly distilled water were mixed with eight volumes of the lowest, apparently non -toxic dilution of the product in PBS. The mixture was added to a volume of double-concentrated cell suspension. After one hour of incubation at 37°C, the cells were centrifuged and resuspended in culture fluid. The final step involved comparing virus suspension titres in cells treated with and untreated (PBS) with the preparation.

[0158] Checking the effectiveness of product inhibition

[0159] The preparation mixture (EN 5.5.2) with chilled DMEM + 2% FCS was incubated in an ice bath for 30 minutes, and a series of dilutions up to 10'8were made. Virus titres were determined and compared with test titre.

[0160] Reference virus inactivation test As a reference point for the determination of virucidal activity, according to the standard (EN 5.5.6.2iifh Ciifi Eh T Vtt ttttsrospereoc oeooc orsxyyxyu), 1.4% formaldehyde solution. The cytotoxicity of the formaldehyde suspension was d Ptrocu

[0161] determinid fldhdlttt preparaon procormaee conrouyed at a range of dilutions up to 10'5.

[0162] Verification of the method

[0163] The following cr Cittoncenraoiteria listed in section 5.7 PN-EN have been met:

[0164] n

[0165] a. Virus suspension titres allowed the determination of virus reduction by > 4 logio.

[0166] b. Cytotoxicity of the product at the use concentration allowed detection of a reduction in fi Itnererng

[0167] titre of 4 logio. bt ssancesu

[0168] c. The use concentration of the product showed a reduction in virus titres of > 41ogio within 60 min. Citt tonacme

[0169] d. Comparative virus titres ini ()mn RK-13 cells incubated in the presence of the product (1:10000 dilution) and not incubated showed acceptable differences (<logio) in virus titres.

[0170] e. The control for inhibitory efficacy of the product was < 0.5 log.

[0171] As all criteria given in the standard (EN 5.7) are fulfilled, the test with herpesvirus type 1 according to EN 14476 + A2:2019-08 was considered valid.

[0172] The results are shown in Table 2 - testing the virucidal properties of the estrosphere preparation against equine herpesvirus type 1.

[0173] Table 2.

[0174] Dilutions

[0175] 1 2 3 4 5 6 7 8 9 10 11 12 5%

[0176] 2.5%

[0177] Clean 60

[0178] 1%

[0179] conditions

[0180] 0.5%

[0181] 0.025%

[0182] 5%

[0183] 2.5%

[0184] Clean

[0185] 1% N.A.

[0186] conditions

[0187] 0.5%

[0188] 0.025%

[0189] 1.4% PBS N.A.

[0190] 0

[0191] Clean

[0192] N.A.

[0193] conditions

[0194] 60

[0195]

[0196] N. A. = not applicable

[0197] 0 = no virus present t =cytotoxicity

[0198] 1-4 = virus presence (degree of cytopathic effect intensity in 4 microplate wells) According to the premise of the standard, a disinfectant is considered virucidal if, after the recommended exposure time, the virus titre is reduced by at least 4 iog10 (inactivation > 99.99%). The estrosphere preparation was tested for equine herpesvirus type 1, at concentrations of 5%, 2.5%, 1%, 0.5% and 0.025%, under clean conditions. The exposure time was 60 minutes. After this time, the reduction in titre was determined:

[0199] > 4 loglO for concentrations of 2.5% and 1% (inactivation > 99.99%);

[0200] 3.5 loglO for concentrations of 5% and 0.5% (99.95% inactivation);

[0201] 2 loglO for concentrations of 0.025% (99% inactivation).

[0202] Therefore, the preparation of estrospheres at concentrations of 2.5% and 1% can be considered as a disinfectant against equine herpesvirus type 1.

[0203] Example 12

[0204] Determination of the virucidal activity of the estrosphere preparation against adenovirus type 5 (AdV 5)

[0205] Preparation of the test virus suspension

[0206] To prepare the virus suspension according to PN-EN 14476 + A2:2019-08 (EN 5.4), the MDCK cell line was propagated in 175 cm3culture dishes (NEST SCIENTIFIC Biotechnology, New Jersey, USA) in the presence of Dulbeco’s Minimum Essential Medium and 10% foetal bovine serum (FBS). Adenovirus type 5 (suspension at baseline concentration) was added to the monolayer for Ih at 37°C and gently shaken every 15 min. The culture was observed daily until the cytopathic effect appeared, after which it was frozen three times at -80°C and thawed. The suspension was centrifuged (10 min, 1500 x g) and the virus-containing supernatant was used for the test or stored at -80°C.

[0207] Infectivity testing

[0208] Infectivity was determined by virus titration according to the standard (EN 5.5.7). In this experiment, each of the logarithmic dilutions tested was applied in a volume of 0.1 mL to four wells on a microplate containing 0.1 mL of freshly strypsinised MDCK cells (10-15 xlO3cells per well). Microplates were incubated at 37°C with 5% CO2. The plate was observed daily until the appearance of a cytopathic effect at the highest dilution (7 days) under an inverted microscope (Axio Observer, Carl Zeiss MicroImaging GmbH). The infectious dose of TCIDso / mL was calculated using the Spearman and Karber method according to the following formula:

[0209] - logioTCIDso = xo - 0.5 + S r / n

[0210] where:

[0211] xo= log 10 of the highest dilution showing 100% positive reactions

[0212] r = number of positive determinations in the highest dilution showing 100% positive reactions and all positive results in the following dilutions n = number of determinations for each dilution

[0213] Inactivation test

[0214] Virucidal efficacy of the estrosphere preparation at concentrations: 5%, 2.5%, 1%, 0.5% and 0.025% were tested according to standard PN-EN 14476 + A2:2019-08 (EN 5.5.2). Contact time was 60 minutes. For greater convenience of testing, a suspension of equal volumes (0.1 mL) of virus and interfering agent (PBS) and 0.8 mL of test agent was prepared. Immediately, after a defined contact time, the activity of the disinfectant was inhibited by making a series of dilutions to 10'12.

[0215] Virus titration was performed after contact times of 0 min and 60 min.

[0216] Determination of cytotoxicity

[0217] The cytotoxicity of the preparation was determined according to the standard (EN 5.5.4.1) using 0.2 mL of hard water and 0.8 mL of the test agent.

[0218] Cell sensitivity

[0219] To control cell sensitivity (PN-EN 5.5.4.2), two volumes of doubly distilled water were mixed with eight volumes of the lowest, apparently non -toxic dilution of the product in PBS. The mixture was added to a volume of double-concentrated cell suspension. After one hour of incubation at 37°C, the cells were centrifuged and resuspended in culture fluid. The final step involved comparing virus suspension titres in cells treated with and untreated (PBS) with the preparation.

[0220] Checking the effectiveness of product inhibition

[0221] The preparation mixture (EN 5.5.2) with chilled DMEM + 2% FCS was incubated in an ice bath for 30 minutes, and a series of dilutions up to 10'8were made. Virus titres were determined and compared with test titre.

[0222] Reference virus inactivation test

[0223] As a reference point for the determination of virucidal activity, according to the standard (EN 5.5.6.2), 1.4% formaldehyde solution. The cytotoxicity of the formaldehyde suspension was determined at a range of dilutions up to 10'5.

[0224] Verification of the method

[0225] The following criteria listed in section 5.7 PN-EN have been met:

[0226] f. Virus suspension titres allowed the determination of virus reduction by > 4 logio. g. Cytotoxicity of the product at the use concentration allowed detection of a reduction in titre of 4 logio.

[0227] h. The use concentration of the product showed a reduction in virus titres of > 41ogio within 60 min.

[0228] i. Comparative virus titres in MDCK cells incubated in the presence of the product (1:10000 dilution) and not incubated showed acceptable differences (<logw) in virus titres. j. The control for inhibitory efficacy of the product was < 0.5 log. As all criteria given in the standard (EN 5.7) are fulfilled, the test with herpesvirus type 1 iifh Ciif Eh Ttt ttttsrospereoc oeooc oxyyxy

[0229] accordil P Vttrocrs conrouudid fldhdtt preparaon procormaeeuying to EN 14476 + A2:2019-08 is valid.

[0230] The results are shown in Table 3 - testing the virucidal properties of the nanoemulsion of the estrosphere preparation against adenovirus type 5.

[0231] Cittoncenraon

[0232] Table 3.

[0233] Dilutions

[0234] fi Itnererng

[0235] bt ssancesu

[0236] Citt tonacme

[0237] i ()mn

[0238] Clean

[0239] conditions

[0240] Clean

[0241] conditions

[0242] PBS

[0243] Clean

[0244] conditions

[0245]

[0246] N. A. = not applicable 0 = no virus present

[0247] t =cytotoxicity

[0248] 1-4 = virus presence (degree of cytopathic effect intensity in 4 microplate wells).

[0249] According to the premise of the standard, a disinfectant is considered virucidal if, after the recommended exposure time, the virus titre is reduced by at least 4 iog10 (inactivation > 99.99%). The estrosphere preparation was tested for adenovirus type 5, at concentrations of 5%, 2.5%, 1%, 0.5% and 0.025%, under pure conditions. The exposure time was 60 minutes. After this time, the reduction in titre was determined:

[0250] > 4 loglO for concentrations of 5%, 2.5%, 1% and 0.5% (inactivation > 99.99%);

[0251] 3 loglO for a concentration of 0.025% (99.9% inactivation). Therefore, the preparation in concentrations of 5%, 2.5%, 1% and 0.5% can be considered as a disinfectant against adenovirus type 5.

[0252] The estrosphere preparations according to the invention have applications as pharmaceutical preparations especially , especially for the treatment of cancer, and have antiviral effects, as well as have applications as dietary supplements, nutritional and cosmetic preparations.

Claims

Claims1. An estrosphere preparation comprising an ester phase and an aqueous phase characterised in thatthe ester phase comprisesa-linolenic acid ethyl ester, or a mixture of a-linolenic acid ethyl ester with one or more components selected from the group comprising polyunsaturated fatty acids ethyl esters (PUFA ethyl esters) or triglycerides,emulsifier andethanol,in a weight ratio of between 2:0.5: 1 and 8:2:1, respectively,and the aqueous phase compriseswater with glycerol at a final concentration of 2.25% by weight,wherein the weight ratio of a-linolenic acid ethyl ester, or of the mixture of a-linolenic acid ethyl ester with one or more components to ethanol, emulsifier and water is between 2:0.5:1:45 and 8:2:1:45, respectively.

2. Estrosphere preparation according to claim 1, wherein the average particle size of the estrospheres is between 20 and 200 nm.

3. Estrosphere preparation according to any of the preceding claims, wherein the PUFA ethyl esters are selected from the group comprising a-linolenic acid ethyl ester, y- linolenic acid ethyl ester, eicosapentaenoic acid ethyl ester and docosahexaenoic acid ethyl ester, conjugated linoleic acid ethyl ester, linoleic acid ethyl ester, oleic acid ethyl ester, stearic acid ethyl ester, palmitic acid ethyl ester.

4. Estrosphere preparation according to any of the preceding claims, wherein the triglycerides are selected from the group of triglycerides comprising acids having 8 to 24 carbon atoms, in particular a-linolenic acid, y-linolenic acid (GLA), eicosapentaenoic acid (EP A), docosahexaenoic acid (DHA), conjugated linoleic acid (CLA), linoleic acid (LA) and oleic acid.

5. Estrosphere preparation according to any of the preceding claims, wherein the emulsifier is selected from the group comprising soy lecithin, pharmaceutical grade egg lecithin, fatty acid salts, detergents, lysolecithins and mono- and diglycerides of fatty acids.

6. Estrosphere preparation according to claim 5, wherein the fatty acid salt is the sodium salt of oleic acid.

7. Estrosphere preparation according to claim 5, wherein the detergent is selected from the group comprising Polysorbate 80, Polysorbate 20 and Span 80.

8. Estrosphere preparation according to any of the preceding claims, wherein the weight ratio of a-linolenic acid ethyl ester, or a mixture of a-linolenic acid ethyl ester with one or more components to ethanol, emulsifier and water is 4:0.5:1:45, respectively.

9. Method of production an estrosphere preparation as defined in claim 1 to 8 characterized in that it includes the following steps:a. obtaining the ester phasei. a-linolenic acid ethyl ester, or a mixture of a-linolenic acid ethyl ester with one or more components selected from the group comprising PUFA ethyl esters or triglycerides, is mixed with ethanol and emulsifier in the weight ratio of between 2:0.5: 1 and 8:2:1, respectively, with stirring at 30 to 40 rpm;b. obtaining the aqueous phasei. an aqueous glycerol solution of 2.25% by weight is prepared and mixed at 80 to 100 rpm;c. phase mixingi. the ester phase obtained in step a(i.) is added to the aqueous phase obtained in step b(i.) maintaining a weight ratio of either a-linolenic acid ethyl ester, or of the mixture of a-linolenic acid ethyl ester with one or more components to ethanol, emulsifier and water of between 2:0.5: 1 :45 and 8:2: 1 :45, respectively, and is mixed at 80 to 400 rpm;ii. the obtained mixture is stirred further at 2000 to 2500 rpm for at least 10 min.iii. the mixture obtained in step c(ii.) is subjected to high-pressure homogenization by passing the mixture 6 times through the homogenization head at a pressure of between 300 and 600 bar.

10. Method according to claim 9, wherein the mixture obtained in step a(i.) is heated at a temperature between 10 and 45 °C until the emulsifier is dissolved.

11. Method according to claim 9, wherein the method is carried out under anaerobic conditions.

12. Method according to claims 9 to 11, wherein in step c(i.) the weight ratio of a-linolenic acid ethyl ester, or a-linolenic acid ethyl ester mixture with one or more components to ethanol, emulsifier and water is 4:0.5: 1 :45, respectively.

13. Estrosphere preparation as defined in claim 1 to 8 for use in the prevention and treatment of cancer and viral diseases.

14. Estrosphere preparation for use according to claim 13, wherein the cancer is selected from a group comprising colorectal cancer, lung cancer, breast cancer, lymphocytic leukaemia, intestinal cancer, Burkitt's lymphoma, gastric cancer, glioma, melanoma and ovarian cancer.

15. Estrosphere preparation for use according to claim 13, wherein the viral disease is caused by equine herpesvirus type 1.

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