EA044150B1 - AQUEOUS SOLUTION OF CHOLECALCIFEROL CLATHRATE WITH β-CYCLODEXTRIN AND METHODS OF PREPARATION - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to the fields of pharmaceuticals and nutraceuticals. In particular, a new composition and preparation of vitamin D3, as well as methods for their production and use in human and animal nutrition to compensate for vitamin D3 deficiency and as a medicine for the treatment of conditions and diseases associated with vitamin D3 deficiency, or conditions and diseases, are proposed. which can be treated with vitamin D3.

Information about the prior art

Vitamin D is a group of fat-soluble compounds responsible for various biological effects in humans and animals. Vitamin D is commonly found in the forms vitamin D2 (ergocalciferol) and D3 (cholecalciferol), and the latter is more effective than ergocalciferol in increasing serum levels of the total active form of the vitamin. The best known role of vitamin D is to maintain the homeostasis of calcium and phosphorus in the body, which is achieved by promoting their absorption in the small intestine, and therefore vitamin D, by promoting bone resorption, is an important component for bone development and maintenance of bone health. Vitamin D deficiency in children leads to rickets, and in the elderly it leads to decreased bone density and the risk of osteoporosis. In addition, vitamin D is involved in the functioning of the immune system. In general, the occurrence and/or severity of the following conditions and diseases are associated with vitamin D deficiency: rickets, osteomalacia, osteoporosis, bone fractures, muscle weakness, schizophrenia, depression, multiple sclerosis, rheumatoid arthritis, Parkinson's disease, tuberculosis, periodontal disease, respiratory infections and COVID-19, preeclampsia, cardiovascular, cancer, autoimmune diseases, diabetes, metabolic syndrome, etc.

Vitamin D3 deficiency can be assessed by the blood concentration of its active metabolite, calcidiol (25(OH)D), which is normally 30-50 ng/ml, and with acute deficiency drops below 12 ng/ml. According to some estimates, about a billion people around the world may suffer from vitamin D deficiency; Children and the elderly are at risk.

Approximately 1 billion people worldwide suffer from vitamin D deficiency, and about 50% of the world's population is vitamin D deficient, a problem affecting both developing and developed countries. In Russia, according to 2020 estimates, the deficit is observed in 65% of the population.

The main routes of vitamin D3 supply are synthesis in the skin under the influence of UV radiation and nutritional intake. The supply of vitamin D3 by synthesis in the skin is actually limited not only by the fact that most of Europe and North America, as well as parts of Asia, are located above the 37th parallel, where it is believed that people cannot get enough vitamin D3 this way, except in summer; but also by the need of people to protect themselves from the carcinogenic effects of UV radiation by using sunscreens or clothing. Dietary intake of vitamin D3 is also limited because few natural foods contain sufficient amounts of vitamin D3. In this regard, a promising way to eliminate vitamin D3 deficiency is the use of vitamin D preparations in the form of dietary supplements or medications. According to the methodological recommendations of Rospotrebnadzor (RF), the norm for vitamin D intake in the diet is 400-600 IU/day, which corresponds to the recommendations of the US National Academies (NASEM). The Russian Association of Endocrinologists recommends large amounts - 600-1000 IU/day; and to achieve the level of (25(OH)D) in the blood of 30 ng/ml for most people, it is suggested to take 1500-2000 IU/day; To achieve higher levels in case of established vitamin D3 deficiency or in case of aggravating conditions (malabsorption or obesity), larger dosages are recommended: 5000, 10000 IU/day and even more.

The main problem in obtaining effective vitamin D3 preparations is related to its lipophilicity, which limits absorption and, therefore, bioavailability. When taken orally, vitamin D3 appears to be soluble in dietary fat, emulsified by bile salts, and taken up by intestinal enterocytes. Vitamin D3 preparations found on the market include solid forms: tablets, capsules, powders; liquid: oil solutions and emulsions. They all differ in their bioavailability profile, with solid forms being the least preferred.

Sublingual and buccal routes of administration are known, which ensure entry into the systemic circulation by absorption of vitamin D3 through the oral mucosa, thereby bypassing the enteral route. When administered sublingually/buccally, bioavailability has been reported to be at or above the level of existing successful oral formulations. This method is especially suitable for people suffering from various absorption disorders (intestinal failure syndrome, consequences of cholecystectomy), but it is generally favorable for a wide range of people due to its ease of use.

Emulsification of vitamin D3 by encapsulation in micelles, microcapsules or liposomes seems to be one of the promising directions for the development of preparations of this vitamin, since it not only allows modifying the bioavailability profile, but also solving other related problems: tendency to aggregation/separation, appearance, stability, etc. .

One known approach involves obtaining a vitamin D3:e inclusion complex

- 1 044150 cyclodextrin (Szejtli J., Tardy-Lengyel M., Vitamin-D3 β-cyclodextrin inclusion complex. Drugs of the Future. V. 9, N. 9, 1984).

β-Cyclodextrin has a cyclic structure formed by seven glucose residues, while the outer part of the molecule contains hydroxyl groups and is hydrophilic, and the inner part contains carbon atoms, hydrogen and ether bonds, creating a hydrophobic region of the molecule that can accommodate molecules of other substances, and with the help of weak Van der Waals forces, hydrophobic interactions and other bonds form clathrates or inclusion compounds. Inclusion thus allows modification of the physicochemical properties of the product.

β-cyclodextrin itself has the advantage that it is absolutely safe when used in nutraceuticals and medicinal substances, has high biocompatibility, and is easily decomposed and excreted by the body.

Capelezzo, A. P. et al., 2018, β-cyclodextrins as Encapsulating Agents of Essential Oils, in P. Arora, N. Dhingra (eds.), Cyclodextrin - A Versatile Ingredient, IntechOpen, London, describes methods for encapsulating essential oils in β-Cyclodextrin: Spray drying, coacervation, solvent evaporation, extrusion, freeze drying, supercritical fluid.

In Bakirova et al., Obtaining and Investigation of the β-cyclodextrin Inclusion Complex with Vitamin D3 Oil Solution. Hindawi Scientifica, Volume 2020, Article ID 6148939, 8 p., describes the preparation of the complex by adding β-cyclodextrin and vitamin D3 to an ethanol:water solution, microwave, remove the solvent, wash with acetone and dry to obtain a white powder, which can then be dissolved in water.

The above documents do not mention methods based on the use of ultrasound or mixing with dry β-cyclodextrin.

There is a known method of encapsulating β-cyclodextrin using ultrasonic treatment, but of another substance - taxifolin (Kalinina et al., Sonochemical encapsulation of taxifolin into cyclodextrine for improving its bioavailability and bioactivity for food. Encapsulation of Active Molecules and Their Delivery System, Elsevier, 2020 , pp. 85-102).

A well-known liquid oral preparation of vitamin D3 is Aquadetrim from Polpharma, which is an almost transparent emulsion obtained with macrogol glyceryl ricinoleate. Although this preparation has a satisfactory and stable appearance, preparations with such an emulsifier are known to have a bioavailability profile that is even worse than that of oil formulations of vitamin D3 (see Egidijus Simoliunas, Ieva Rinkunait, Zivile Bukelskien, Virginija Bukelskiene. Bioavailability of Different Vitamin D Oral Supplements in Laboratory Animal Model Medicina 2019, 55, 265, see Fig. 3).

A commercially available preparation is known in the form of a spray for sublingual/buccal use SunD3 from LYL (lyl.eu), containing: purified water, sweetener xylitol, thickener xanthan gum, natural vitamin D3 (derived from lanolin), natural orange flavor, citric acid, preservative potassium sorbate. This drug has an unstable emulsion and must be shaken before each use to obtain the required dose of vitamin D3; The appearance of this emulsion is not entirely attractive, and therefore, apparently, it is produced in an opaque bottle.

Thus, there is still a need for improved aqueous formulations of vitamin D3 that have good bioavailability, stability, appearance, taste, and are suitable for sublingual and/or buccal administration, for example in the form of a spray or aerosol.

The essence of the invention

The present invention aims to solve the above problem by encapsulating vitamin D3 molecules in β-cyclodextrin using new methods.

In the first embodiment, a method for obtaining a vitamin D3 preparation is proposed, which includes the following stages:

a) applying vitamin D3 in the form of an oil solution to β-cyclodextrin powder or its derivative;

b) dissolving the resulting product in water or an aqueous solution with stirring to obtain an emulsion.

In particular embodiments of this method, in step (a) after application of vitamin D3, the β-cyclodextrin or derivative thereof is optionally maintained; the holding time can be 60-120 minutes, and the temperature - 10-25°C.

In step (b), an optional aqueous solution is used for dissolution, which may include one or more additional auxiliary ingredients, and mixing is preferably carried out at 1000-3000 rpm.

In the second embodiment, a method for obtaining a vitamin D3 preparation is proposed, which includes the following stages:

a) dissolving β-cyclodextrin or its derivative in water;

- 2 044150

b) adding vitamin D3 in the form of an oil solution to the solution obtained at stage (a) and subjecting the resulting mixture to ultrasonic treatment to form an emulsion.

The concentration of β-cyclodextrin in the solution obtained in step (a) is preferably 1.85 wt%.

The dissolution in step (a) is preferably carried out by stirring at 800-1000 rpm, preferably at 10-25°C.

The specific power of ultrasonic treatment in stage (b) is preferably 600-1000 W/l; preferably it is carried out at 30-50°C.

Both of the above-described embodiments of the method for producing a vitamin D3 preparation may optionally include the additional step of:

c) adding one or more auxiliary ingredients (nutraceutically and/or pharmaceutically acceptable) to the emulsion obtained in step (b).

The vitamin D3 oil solution used in the methods of the invention preferably additionally contains a mixture of tocopherols. The concentration of vitamin D3 in the oil solution is preferably 100,000-1,500,000 IU/ml. The oil in the vitamin D3 oil solution is selected from: refined or unrefined sunflower oil, refined or unrefined coconut oil, olive oil, soybean oil, peppermint oil, milk thistle oil, amaranth oil, cedar oil, flaxseed oil, fish oil, or any mixture thereof.

Excipients, if used, are selected from the group consisting of thickeners, acidity regulators, preservatives, carriers, sweeteners, colors and flavorings.

In yet another aspect, the invention relates to a vitamin D3 preparation prepared by the methods of the invention. The drug is distinguished by the fact that it maintains the stability of the emulsion for at least 14 months. In a preferred embodiment, the preparation according to the invention is in the form of a spray or aerosol and is therefore suitable for sublingual and/or buccal administration, without limitation.

In other aspects, the invention relates to the use of a vitamin D3 preparation for replenishing vitamin D3 deficiency and/or for treating conditions and diseases associated with vitamin D3 deficiency. Accordingly, the drug according to the invention can be used in the form of a nutraceutical (dietary supplement) or in the form of a drug. The preparation according to the invention can be used for the manufacture of drugs or nutraceuticals or for the fortification of food products with vitamin D3. In general, the preparation according to the invention is suitable for use for those purposes for which vitamin D3 is usually used.

The inventors have unexpectedly discovered that the vitamin D3 preparation with β-cyclodextrin obtained according to the invention has an excellent balance of properties. In particular, the preparation according to the invention is characterized by the following effects, individually or in any combination:

excellent physical stability of the emulsion, absence of separation within the shelf life of the drug; in particular, stability can be assessed by the absence of a significant change in optical density for at least 14 months, in particular more than 24 months or more;

excellent bioavailability, comparable to or even superior to commercially available vitamin D3 preparations;

excellent organoleptic properties, absence of unpleasant tastes, stability of organoleptic properties over time;

the drug has the particular advantage that it can be used by sublingual and/or buccal administration;

the drug has an attractive appearance: homogeneous light gray opalescent liquid; can be packaged in a transparent bottle;

a shelf life of up to 24 months is ensured without adding preservatives, even if the product base contains natural berry/fruit juices;

stability and organoleptic properties are maintained for a period longer than the shelf life of the ingredients that make up the drug.

In addition, we have unexpectedly discovered that vitamin D3 complex with β-cyclodextrin can be prepared by simple and low cost methods that provide substantially the same results. A separate stage of obtaining such a complex allows you to optimize the method of obtaining the drug as a whole and mix (mix) additional ingredients separately, providing individual conditions for different groups of ingredients (for example, temperature, humidity, duration, etc.); the production process is accelerated without negative consequences for bottling, emulsion stability, while dosage deviation is reduced and oxidation of active components is prevented. Finally, obtaining the drug does not involve the use of solvents such as acetone, ethanol and others, which reduces risks for consumers.

List of graphic materials

Fig. 1 - diagram of the dependence of optical density (vertical axis, OD ₆₀₀ ) on time (burn

- 3 044150 zontal axis, monthly measurement); experiment - a sample obtained according to the method according to the invention; control - a composition of the same composition, but obtained by ordinary mixing in solution.

Fig. 2 is a diagram showing the bioavailability of vitamin D3 in the blood of mice when applied to the oral mucosa (ng/ml); designations: 1 - aqueous emulsion of vitamin D3 10,000 IU/ml (60 IU/dose) with vitamin E (1%) and xylitol (25%); 2 - solution of vitamin D3 10000 IU/ml 60IU/dose) in oil; 3 - aqueous emulsion of vitamin D3 10,000 IU/ml (60 IU/dose) with xylitol (25%); 4 - aqueous emulsion of vitamin D3 10,000 IU/ml (60 IU/dose) with β-cyclodextrin (1.85%) and xylitol (25%); 5 - negative control (oil); 6 - positive control - drug SunD3 (LYL).

Detailed Description of the Invention

Terminology

All terms used have the meanings consistent with those accepted in the art, unless a more specific or different interpretation is defined herein or is clear from the context.

When using terms in the singular, it is implied, where the context allows, that the plural is also possible, and vice versa.

Terms including, containing, having, etc. mean that in addition to the specified elements, other unspecified (non-essential) elements may be contained, for example, the methods of the invention may include additional optional stages before, between or after those specifically specified, and the drug may include additional optional ingredients in addition to those specifically specified. In each case, these terms also include the meaning consisting of, i.e. in one of the embodiments, it is implied that the object being described is characterized by the presence of only the specified elements.

The numerical values mentioned mean both exactly the specified value and approximately corresponding to it (the terms about, approximately, approximately are also used for this), as determined by an increase and/or decrease deviation of 10, 5, 2 or 1% or, in the case of parameters that can be measured with a certain accuracy, within the limits of measurement error characteristic of the means typically used to measure a particular parameter.

The default intervals given imply that both the boundary values of the intervals and all the values contained in the interval are directly disclosed. Any disclosed numerical values can act as interval boundaries.

The terms biologically active additive (BAA), nutraceutical, biologically active substance, food additive, food ingredient are used interchangeably and designate a product, a biologically active substance and its compositions - suitable for consumption in food or with food. Dietary supplements are an additional source of biologically active substances intended to compensate for their deficiency or balance the diet. Dietary supplements can be given a form corresponding to a dosage form.

The nutraceutical according to the invention is suitable for administration not only to humans, but also to animals.

The term pharmaceutical composition refers to a medicinal composition, drug or drug that is suitable for the treatment and/or prevention of certain diseases. The pharmaceutical composition contains one or more active substances together with other components, in particular physiologically/pharmaceutically acceptable excipients, and can be optimized for the desired route of administration. The pharmaceutical composition includes an active substance in a therapeutically effective dose or a therapeutically effective amount, which means the dose or amount at which the therapeutic effects associated with the active substance occur.

Pharmaceutical or pharmaceutically acceptable refers to chemical compounds and compositions that do not result in an adverse, allergic or other undesirable reaction when administered to a mammal, particularly a human, or such reactions are minimized.

In the present invention, pharmaceutical compositions and preparations of vitamin D3 are used according to known uses and indications specific to vitamin D3.

M E stands for international unit; for vitamin D3 1 IU = 0.025 mcg.

Methods

In the first variant of the production method, an oil solution of vitamin D3 is applied to β-cyclodextrin powder or its derivative. In some embodiments, said powder may be further dried. The application method is not particularly limited and may be carried out by uniformly dispensing said solution onto the powder manually or in an automated manner (batch or continuous) using a suitable apparatus or apparatus capable of applying the solution to the powder. A non-limiting example is a granulator or other type of device that provides effective liquid-solid contact. Optionally and preferably, the resulting mixture of solution and powder is further stirred, even more preferably the stirring is carried out during application of the solution to the powder and/or immediately after such application.

- 4 044150

After the application step, the mixture is preferably kept for an unlimited time, for example from 0 to 24 hours, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or the specified time falls within any range formed by the specified values. In preferred embodiments, the holding time is 0-120 minutes, in particular 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 , 100, 105, 110, 115, 120 min or within any range formed by the specified values. The holding temperature is preferably 5-40°C, for example 5-35, 5-30, 10-35, 10-30, 10-25, 15-35, 15-30, 15-25, 15-20, 20-25 °C, preferably 1025°C, most preferably room temperature (about 25°C).

At the second stage of the first variant of the method, the resulting product (mixture) is dissolved in water or an aqueous solution with stirring. The dissolution time is not particularly limited and may be 024 hours, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours or within any range formed by the specified values. In preferred embodiments, the dissolution time is 0-120 minutes, in particular 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 , 100, 105, 110, 115, 120 min or within any range formed by the specified values. The stirrer speed is preferably 1000-3000 rpm, for example 1000, 1500, 2000, 2500, 3000 rpm, or selected from any range within these values. The dissolution temperature is preferably 20-40, 20-35, 20-30 or 25-30°C, most preferably about 25°C. The resulting solution is filtered, if necessary, and/or the excess oil phase is separated. Instead of water, you can use an aqueous solution that includes one or more additional auxiliary ingredients, such as those mentioned below. If water is used, then additional auxiliary ingredients can be added to the solution at the end of step (b).

In the second variant of the method, a solution of β-cyclodextrin or its derivative in water is first obtained. The dissolution temperature is preferably 10-40°C, for example 10-35, 10-30, 10-25, 1540, 15-35, 15-30, 15-25, 20-35, 20-30, 20-25, 25 -30°C, most preferably about 25°C. The stirrer speed is preferably 800-3000 rpm, for example 1000, 1500, 2000, 2500, 3000 rpm, or selected from any range formed by these values; preferably the stirrer speed is 900 rpm. The resulting solution is filtered if necessary.

At the next stage of the second variant of the method, an oil solution of vitamin D3 is added to the solution of β-cyclodextrin or its derivative and the resulting mixture is subjected to ultrasonic treatment to form an emulsion. Ultrasonic treatment can be carried out using an ultrasonic homogenizer (disintegrator), the parameters of which are set according to approaches known to specialists, depending, in particular, on the volume or flow of the liquid to be processed (see, for example, Peshkovsky A. S., Peshkovsky S. L., Bystryak S., Scalable high-power ultrasonic technology for the production of translucent nanoemulsions, Chemical Engineering and Processing: Process Intensification, V. 69, 2013, pp. 77-82; Taha A., Ahmed E., Ismaiel A. et al., Ultrasonic emulsification : An overview on the preparation of different emulsifiers-stabilized emulsions, Trends in Food Science & Technology, V. 105, 2020, pp. 363-377). Processing can be carried out in continuous, semi-continuous and batch mode. The processing frequency is typically about 19-24 kHz, for example 19, 20, 21, 22, 23, 24 (including any ranges formed by these values), preferably about 20 kHz. The power is selected in the range of 50-3000 W, and is not particularly limited, i.e. it can be above 3000 W. The choice of power is determined, as a rule, by production needs, as is known to specialists in this field, and can be expressed in terms of the volume of liquid processed. In non-limiting embodiments, the power density is 500-1500 W/l, in particular 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670 , 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920 , 930, 940, 950, 960, 970, 980, 990, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500 W/l, or within the range formed by any of the specified values . Preferably, the specific power of ultrasonic treatment is 600-1000 W/L.

In non-limiting embodiments, the processing time is 0-60 minutes, for example from 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 min, up to 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50 , 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25 , 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 min or selected from any range formed by the specified values. Preferably the processing time is 2-8 minutes. In non-limiting embodiments, the probe amplitude is 70 μm or greater, preferably 75 μm or greater, 80 μm or greater, 85 μm or greater, 90 μm or greater, 95 μm or greater, 97 μm or greater, 100 μm or greater, 110 μm or higher, 112 µm or higher, 115 µm or higher. The processing temperature is optionally from 10 to 60°C, for example from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 min to 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20 minutes, or selected from the range resulting

- 5 044150 can be any of the specified values.

In both variants of the method, an additional step of adding one or more auxiliary ingredients is possible. The addition can be carried out in one stage or in several, and in the latter case the auxiliary ingredients are added individually or several at a time. Depending on the specific ingredient, it may be added in liquid or solid form. In particular, in some embodiments it may be advisable to prepare a separate solution of the ingredient and add it to the emulsion obtained in step (b).

The finished preparation is packaged in a suitable container, preferably poured into containers suitable for direct use by the consumer.

The preparation of the invention has excellent physical stability, which can be assessed by the absence of significant change in optical density for at least 14 months. In particular, this period can reach at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or more, or this period may be included in the range formed by any of the specified values. Thus, the drug is characterized by the absence of a significant change in optical density during the period of use and/or shelf life of the drug, which may be 0.5, 1, 1.5, 2, 2.5, 3, or 3 or more years.

A drug

Vitamin D3 and β-cyclodextrin can be obtained from any source without limitation as known to those skilled in the art. For example, vitamin D3 can be obtained by extracting 7-dehydrocholesterol from lanolin and exposing it to UV irradiation, or by extracting vitamin D3 from lichen. β-Cyclodextrin is usually obtained by enzymatic treatment of starch.

The oil for preparing the vitamin D3 solution can be selected from essentially any edible oil of vegetable or animal origin. Examples of plant oils include peach, almond, castor, olive, sunflower, safflower, soybean, coconut, palm, peppermint, amaranth, flaxseed, milk thistle, peanut, corn, canola, hemp, cottonseed, apricot, grapeseed, walnut, pine nuts, wheat germ, sesame, hazelnut, macadamia, poppy, raspberry seeds, almond, passion fruit, sea buckthorn, rice, mustard, camelina, pumpkin, pistachio, their mixtures, fractions and mixtures of fractions. Oils can be refined or unrefined. Examples of animal oils include fish oils, amphibian, reptile, aquatic and terrestrial mammal liquid oils, mixtures, fractions and mixtures of fractions thereof. It is clear that oil (fat) should be liquid at room conditions.

Preferably, the vitamin D3 solution contains one or more substances from the vitamin E group (tocopherols (α, β, γ, δ) and tocotrienols (α, β, γ, δ)). The presence of vitamin E, which has natural antioxidant properties, helps stabilize vitamin D3.

The ratio of vitamin D3 and β-cyclodextrin is not specifically limited. The theoretical molar ratio to obtain the complex is 2:1 (β-cyclodextrin:vitamin D3), but in practical terms it is preferable to use an excess of β-cyclodextrin; for example, the molar ratio of β-cyclodextrin:vitamin D3 to obtain the drug can be from 2:1 to 5:1, for example 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2 ,4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.1:1, 3.2 :1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4 ,1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9 :1, 5:1 (including any subranges formed by any two of these values). In the embodiment of the production method in which a solution of β-cyclodextrin is used, it can be obtained in a maximum concentration, which at room temperature (25°C) in water is approximately 18.5 g/l (1.85 wt.%). Thus, any β-cyclodextrin concentration corresponding to 18.5 g/L or less can be used in the invention. For example, β-cyclodextrin can be used at a concentration of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 , 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15 , 15.5, 16, 16.5, 17, 17.5, 18, 18.5 g/l (including any subranges formed by any two of these values).

Instead of β-cyclodextrin as such, its derivatives can be used. It should be noted that the solubility of derivatives in water differs depending on the nature of the modification, which should be taken into account when calculating the composition of the drug. It is most advisable to use β-cyclodextrin derivatives modified with hydrophilic groups, since the solubility in water of such a derivative will be higher than the solubility of β-cyclodextrin. For some derivatives, the solubility can be 10-20 times higher than that of β-cyclodextrin. The most common β-cyclodextrin derivatives acceptable in the art include alkylated (e.g., methylated), hydroxyalkylated (e.g., hydroxypropylated), and sulfoalkylated (e.g., sulfopropylated or sulfobutylated), sulfated derivatives (Khalid, S. H. et al., 2019, 'Effect of Cyclodextrin Derivatization on Solubility and Efficacy of Drugs', in S. Karaku$. (ed.), Colloid Science in Pharmaceutical Nanotechnology, IntechOpen, London. 10.5772/intechopen.90364). Preferred examples of suitable derivatives include, but are not limited to, methyl-β

- 6 044150 cyclodextrin (MCD), hydroxypropyl-β-cyclodextrin (HPCD) and carboxymethyl-β-cyclodextrin (CMCD).

The content of vitamin D3 in the preparation is not particularly limited and is calculated, first of all, in such a way that when dosing, one dose contains a certain amount of ME vitamin D3. In non-limiting embodiments, the concentration of vitamin D3 in the preparation is from 0.025 to 10 g/L. For example, the lower limit of vitamin D3 content in the preparation (from) is (g/l): 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0 ,1, 0.105, 0.11, 0.115, 0.12, 0.125, 0.13, 0.135, 0.14, 0.145, 0.15, 0.155, 0.16, 0.165, 0.17, 0.175, 0.18 , 0.185, 0.19, 0.195, 0.2, 0.205, 0.21, 0.215, 0.22, 0.225, 0.23, 0.235, 0.24, 0.245, 0.25,

0.255, 0.26, 0.265, 0.27, 0.275, 0.28, 0.285, 0.29, 0.295, 0.3, 0.305, 0.31, 0.315, 0.32, 0.325, 0.33, 0.335, 0.34,

0.345, 0.35, 0.355, 0.36, 0.365, 0.37, 0.375, 0.38, 0.385, 0.39, 0.395, 0.4, 0.405, 0.41, 0.415, 0.42, 0.425, 0.43,

0.435, 0.44, 0.445, 0.45, 0.455, 0.46, 0.465, 0.47, 0.475, 0.48, 0.485, 0.49, 0.495, 0.5, 0.505, 0.51, 0.515, 0.52,

0.525, 0.53, 0.535, 0.54, 0.545, 0.55, 0.555, 0.56, 0.565, 0.57, 0.575, 0.58, 0.585, 0.59, 0.595, 0.6, 0.605, 0.61,

0.615, 0.62, 0.625, 0.63, 0.635, 0.64, 0.645, 0.65, 0.655, 0.66, 0.665, 0.67, 0.675, 0.68, 0.685, 0.69, 0.695, 0.7,

0.705, 0.71, 0.715, 0.72, 0.725, 0.73, 0.735, 0.74, 0.745, 0.75, 0.755, 0.76, 0.765, 0.77, 0.775, 0.78, 0.785, 0.79

0.795, 0.8, 0.805, 0.81, 0.815, 0.82, 0.825, 0.83, 0.835, 0.84, 0.845, 0.85, 0.855, 0.86, 0.865, 0.87, 0.875, 0.88

0.885, 0.89, 0.895, 0.9, 0.905, 0.91, 0.915, 0.92, 0.925, 0.93, 0.935, 0.94, 0.945, 0.95, 0.955, 0.96, 0.965, 0.97

0.975, 0.98, 0.985, 0.99, 0.995, 1; the upper limit of vitamin D3 content in the preparation (up to) is (g/l): 10, 9.9, 9.8, 9.7, 9.6, 9.5, 9.4, 9.3, 9.2 , 9.1, 9, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8, 7.9, 7 ,8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7, 6.9, 6.8, 6.7, 6.6, 6 ,5, 6.4, 6.3, 6.2, 6.1, 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5 ,2, 5.1, 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4, 3.9 , 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3, 2.9, 2.8, 2.7, 2.6 , 2.5, 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3 , 1.2, 1.1, 1, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5 , 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15.

In a preferred embodiment, one or more sweeteners may be included in the formulation, the choice of which is not substantially limited. By adding a sweetener it is possible to further improve the bioavailability of vitamin D3. In non-limiting embodiments, the sweetener is selected from sucrose, glucose, fructose, dextrose, galactose, inulin, acesulfame potassium, alitame, aspartame, isomalt, maltitol, mannitol, xylitol, sorbitol, sucralose, erythritol, stevioside, cyclamate, neohesperidin, thaumatin, glycyrrhizin, lac Toulose , lactose, saccharin or mixtures thereof. Particularly preferred are non-caloric substitutes, as well as those that are beneficial to the teeth. Preferably the sweetener is selected from sucralose, xylitol, erythritol, stevioside or mixtures thereof. The amount of sweetener is selected depending on the relative sweetness and can be determined by one skilled in the art.

To increase viscosity, the composition of the preparation according to the invention may optionally include one or more thickeners or viscosity modifiers, which are selected, without limitation, from gums (locust bean, arabic, xanthan, guar, tragacanth, alginates, carrageenan), gelatin, polyvinylpyrrolidone, polyvinyl alcohol, poloxamer, carbomers, cellulose derivatives (methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc.).

The preparation according to the invention optionally contains an acidity regulator, which is selected from fruit acids, for example, citric, malic, tartaric, lactic or mixtures thereof, but not limited to.

One or more preservatives selected from, but not limited to, sodium benzoate, sodium propionate, potassium sorbate, benzalkonium chloride, benzyl alcohol, sorbic acid may be added to the preparation. At the same time, the proposed composition of the drug provides a bacteriostatic effect, which allows you to do without a preservative with a shelf life of up to 24 months. Accordingly, in one preferred embodiment, the preparation of the invention does not contain a preservative.

The composition of the drug may optionally include flavoring agents of natural or synthetic origin, including flavorings based on berry or fruit juices.

In addition to the above, the preparation according to the invention may contain one or more other ingredients acceptable in the relevant field of application, including pharmaceutically and/or nutraceutically acceptable excipients, in particular surfactants or emulsifiers, buffers, antioxidants, chelating agents, dyes.

Application and dosage

The preparation according to the invention is suitable for enteral use according to any purposes and indications characteristic of vitamin D3, as known to one skilled in the field of nutraceuticals and pharmacology. Although the benefits of the drug are greatest when administered sublingually and buccally, the drug can also be administered orally.

In the case of pharmaceutical use, the drug can be used for the prevention and treatment of (but not limited to): vitamin D deficiency, for example in people who receive insufficient amounts of vitamin D from their diet, pregnant and lactating women, children; rickets and rickets-like diseases; hypocalcemic tetany; osteomalacia; metabolic osteopathies, for example

- 7 044150 measures hypoparathyroidism and pseudohypoparathyroidism; familial hypophosphatemia; for the treatment of osteoporosis, in particular postmenopausal (as part of complex therapy); for the prevention of depression, type 2 diabetes, metabolic syndrome, autoimmune diseases, proteinuria, hypertension, colon cancer, breast cancer, cardiovascular diseases, neuropsychiatric disorders, multiple sclerosis, rheumatoid arthritis, Parkinson's disease, tuberculosis, erectile dysfunction and etc.

The drug can be used as a raw material for the manufacture of medicines containing vitamin D3.

In the case of nutraceutical use, the drug can be used individually in the form of a dietary supplement to compensate for vitamin D deficiency and/or prevent and/or accompany the treatment of diseases or conditions for which vitamin D3 is indicated. Alternatively, the drug may be a human or animal feed additive.

One dose of the drug may contain 100-20,000 IU of vitamin D3. For example, the lower dose limit of vitamin D3 in the drug (from) may be (ME): 100, 200, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000; the upper dose limit of vitamin D3 in the preparation (from) can be (ME): 20000, 19000, 18000, 17000, 16000, 15000, 14000, 13000, 12000, 11000, 10000, 9500, 9000, 8500, 8000, 7 500, 7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500, 3000, 2950, 2900, 2850, 2800, 2750, 2700, 2650, 2600, 2550, 2500, 2450, 2400, 2 350, 2300, 2250, 2200, 2150, 2100, 2050, 2000, 1950, 1900, 1850, 1800, 1750, 1700, 1650, 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1200, 1150, 1 100, 1050, 1000, 950, 900, 850, 800, 750, 700, 650, 600.

In the case of pharmaceutical use, the specific dose is selected by the attending physician taking into account accepted recommendations and standards, the patient’s condition (specific disease/condition, metabolic characteristics, contraindications, etc.), as well as the dynamics of treatment. Preferably, blood 25(OH)D levels are monitored and the dose adjusted if necessary.

In the case of nutraceutical use, the preferred daily dose may be 400600 IU for children under 6 months of age, 600-2000 IU for children over 6 months and adults up to 75 years of age, 2000-4000 IU for the elderly over 75 years of age; therapeutic doses are usually higher and can be 1000-2000 IU for children under 1 month to one year of age, 2000-6000 IU for children over one year of age and adults. A daily dose of 10,000 IU is considered safe for most patients.

Details on how to prescribe doses and regimens of treatment and prevention with vitamin D3 are known to specialists (Bleizgys A. Vitamin D Dosing: Basic Principles and a Brief Algorithm (2021 Update). Nutrients. 2021 Dec 10;13(12):4415 ).

The drug may be packaged in a container suitable for dosing a specific or random dose. In particular, the container may be suitable for dispensing one dose as defined above in one use.

The container may have a pipette or dropper to dispense a specific amount of the drug. The container may have a spray nozzle (i.e. the drug is in the form of a spray), accordingly, one press can be adapted to dispense one dose. The container can be made in the form of a cylinder in which the vitamin D3 composition is under pressure (i.e. the drug is in the form of an aerosol), and the design of the spray nozzle can be made in such a way as to dispense a certain dose of the drug per press or dispense an indefinite dose drug. In particular, the aerosol canister may be suitable for dispensing one dose as defined above in one application. The design of suitable containers is known to those skilled in the art.

An additional advantage of the preparation according to the invention when used in the form of a spray or aerosol is that, due to the stability of the preparation, the spray nozzle is less likely to become clogged during its service life. This ensures less variation in the dispensed dose of vitamin D3 during the period of use of the drug and no need to rinse the nebulizer.

Information confirming the possibility of implementing the invention

Example 1.

Option 1:

The required amount of vitamin D3 oil solution (refined sunflower oil, mixture of tocopherols, vitamin D3 - 1,000,000 IU/ml) was applied to the required amount of dry β-cyclodextrin and left at 25°C for 60 minutes. After this, the resulting product was dissolved in the required amount of distilled water on a magnetic stirrer (1000 rpm) to obtain an emulsion in the form of a homogeneous light gray opalescent liquid.

Option 2:

β-Cyclodextrin was dissolved in water at a concentration of 1.85% (g/100 ml) on a magnetic stirrer (900 rpm) at 25°C. After this, an oil solution of vitamin D3 (refined sunflower oil, a mixture of tocopherols, vitamin D3 - 1,000,000 IU/ml) was added to the resulting solution. Further received

- 8 044150 the mixture was treated with an ultrasonic emulsifier (60 W) at 30°C until an emulsion was formed that looked like a homogeneous light gray opalescent liquid.

Example 2.

The stability of the emulsion obtained according to example 1 (option 1) was determined using photometry, determining the optical density in comparison with the control emulsion containing the same components and in the same quantities, except that the control emulsion was obtained in a way different from the method according to invention (mixing in a solution of β-cyclodextrin and vitamin D3 on a laboratory shaker). Optical density measurements were carried out once a month at a radiation wavelength of 600 nm. Before measurement, samples were diluted 4 times with sterile water. The measurement results are presented in Fig. 1.

In the case of the control sample, bleaching was observed (a decrease in optical density at 600 nm), which indicates stratification of the solution and destruction of the emulsion. In the case of an experimental emulsion, this does not happen; the appearance is preserved.

Similar results were obtained for the emulsion obtained according to option 2 from example 1.

Example 3.

The preservation of the organoleptic properties of the emulsion according to example 1 (option 1) was assessed by a group of volunteers (10 people - experimental emulsion and 10 people - control emulsion (contains the same components and in the same quantities, except that the control emulsion was obtained in a way different from the method according to the invention (mixing in a solution of β-cyclodextrin and vitamin D3 on a laboratory shaker)) on a 10-point scale. The assessment was carried out a week after receiving the emulsions and again a year later. The taste qualities of the experimental and control emulsions a week after mixing were assessed on average by 7.4 and 7.0 points, respectively. When re-evaluated a year later, the experimental emulsion was rated an average of 6.9 points, while the control emulsion was rated an average of 5.4 points. Similar results were obtained for the emulsion obtained according to option 2 from example 1. The results obtained indicate an improvement in the preservation of the organoleptic properties of the preparation according to the invention over time.

Example 4.

A study was carried out on the bioavailability of the drug according to the invention. The following compositions were studied:

Negative control without vitamin D3 (oil) (0 IU/dose)

Positive control (SunD3 (LYL)) 10000 IU/ml (60 IU/dose)

Vitamin D3 water emulsion (solution in oil) 10,000 IU/ml with 25% xylitol (60 IU/dose)

Vitamin D3 water emulsion (oil solution) 10,000 IU/ml (60 IU/dose) with 1% vitamin E and 25% xylitol

Aqueous emulsion of vitamin D3 (solution in oil) 10,000 IU/ml (60 IU/dose) with 1.85% β-cyclodextrin and 25% xylitol (emulsion prepared in a manner similar to example 1, option 1)

Vitamin D3 solution in oil 10,000 IU/ml (60 IU/dose)

Introduction

Over the course of 7 days, 6 groups of mice (5 individuals each) were injected with test samples (6 μl) into the oral cavity using a mechanical dispenser, ensuring contact with the oral mucosa (dose 60 IU/day). Blood sampling from mice was carried out an hour after the last administration, since at this time the _Cmax for 25(OH)D3 is observed.

The experiment was carried out according to a double-blind protocol: during the experiment, the person conducting the feeding and the person conducting the ELISA analysis did not know which of the analytes a specific group of mice was receiving. All samples and groups were anonymized and numbered.

Preparation of mouse sera

The selected blood samples were placed in a solid-state thermostat for 1.5 hours at 37°C, then centrifuged twice (15 minutes at 3500xg), followed by collection of blood serum (blood plasma without fibrinogen).

ELISA analysis and results

An enzyme immunoassay was performed using the 25-OH Vitamin D (total) EIA-5396 kit from DRG Instruments GmbH (Germany) according to the manufacturer's instructions. The 25(OH)D level in ng/ml was calculated.

Results and conclusions obtained

The results are presented in Fig. 2. From the diagram it follows that the experimental groups differ significantly in the content of vitamin D3 in the blood, and the studied drugs show different degrees of effectiveness in comparison with the control and depending on the composition. Compared to the commercially available drug SunD3, the drug according to the invention provides much greater bioavailability. Despite the fact that a composition that includes vitamin D3 and xylitol, but does not contain β-cyclodextrin, has even greater bioavailability than the drug according to the invention, it is unacceptable from the point of view of emulsion stability (subject to rapid separation) and this composition will require further

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Claims (12)

adding stabilizers. Example 5. The compositions of the preparations according to the invention, suitable for use in the form of sprays, are given in the table below Components Quantity, Drug 600 ME Quantity, Drug 2000 ME Role Water, ml (g) 24.5335 24.0372 solvent Xanthan gum (E415), g 0.24178 0.24178 thickener Citric acid (ECZO), g 0.11932 0.11932 regulator acidity Potassium sorbate (E202), g 0.0314 0.0314 preservative β-cyclodextrin (E459), g 0.0314 0.0314 carrier Sodium benzoate (E211), g Sucralose (E955), g Vitamin D3 (refined sunflower oil 96.5%, cholecalciferol 2.5%, tocopherol mixture concentrate (E306) as an antioxidant 1%), g 0.0314 0.007536 0.15 (vitamin content D3 - 3750 mcg = 150600 ME) 0.0314 0.007536 0.5 (vitamin content D3 - 12500 µg = 500000 ME) preservative sweetener active substance Flavor, ml 0.095 0.095 flavoring Total, ml 25.241336 25.095036 Total, g 31.4 31.4 The density of the resulting drug is 1.256 g/ml; one dose (injection) - 0.1 ml, 600 ME/2000 ME The examples presented above are intended to illustrate the invention only, but should not be construed as limiting the scope of the rights of the patent owner, which is determined solely by the claims. CLAIM 1. A method for producing a vitamin D3 preparation, including the following steps: a) applying vitamin D3 in the form of an oil solution to β-cyclodextrin powder or its derivative; b) dissolving the resulting product in water or an aqueous solution with stirring to obtain an emulsion. 2. The method according to claim 1, where in step (a) after applying vitamin D3, β-cyclodextrin or its derivative is kept at 10-25°C. 3. Method according to claim 2, where the holding time is 60-120 minutes. 4. The method according to any one of claims 1-3, where in step (b) the aqueous solution includes one or more additional auxiliary ingredients. 5. Method according to any one of claims 1-4, where in stage (b) mixing is carried out at 1000-3000 rpm. 6. A method for producing a vitamin D3 preparation, including the following steps: a) dissolving β-cyclodextrin or its derivative in water; b) adding vitamin D3 in the form of an oil solution to the solution obtained at stage (a) and subjecting the resulting mixture to ultrasonic treatment to form an emulsion. 7. The method according to claim 6, wherein the concentration of β-cyclodextrin in the solution obtained in step (a) is 1.85 wt%. 8. The method according to claim 6 or 7, where the dissolution in stage (a) is carried out by stirring at 800-1000 rpm. 9. Method according to any one of claims 6-8, where the dissolution in step (a) is carried out at 10-25°C. 10. Method according to any one of claims 6-9, where the specific power of ultrasonic treatment at stage (b) is 600-1000 W/l. 11. Method according to any one of claims 6-10, where ultrasonic treatment at stage (b) is carried out at 30-50°C. 12. The method according to any one of claims 1-11, further comprising the following step: c) adding one or more auxiliary ingredients to the emulsion obtained at stage (b) -