RU2536208C1 - Composition of dihydroquercetine enclosed in phospholipid nanoparticles - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: therapeutic composition of dihydroquercetine in the form of nanoparticles, containing herbal phosphatidylcholine, maltose and dihydroquercetin in certain propotions.

EFFECT: composition possesses the high pharmacological activity, low toxicity; it is long-storable.

3 dwg, 1 tbl, 4 ex

Description

The invention relates to preventive medicine and relates to a therapeutic and prophylactic composition based on dihydroquercetin included in phospholipid nanoparticles, consisting of nanoparticles with a diameter of 8-30 nm based on plant phospholipids, with the antioxidant dihydroquercetin included in them.

Currently, it is known that with the help of therapeutic and prophylactic drugs it is possible (1) to organize a balanced diet and maintain the necessary vitality of the body (nutraceuticals) and (2) to prevent the development of various pathologies in the body, gently correct existing disorders (parapharmaceuticals). The use of therapeutic and prophylactic drugs not only contributes to the improvement of society and prolongs human life, but also plays an important role in the prevention of a number of diseases, such as cardiovascular, gastrointestinal, metabolic (obesity), and oncological ones. Treatment and prophylactic drugs can significantly reduce the intake of drugs, especially expensive ones.

From the perspective of the pathology of cellular and subcellular processes, the development of many diseases has common pathogenetic links, even in cases where their clinical manifestations are not the same. It is known that violations in the structure of cytoplasmic and intracellular biomembranes can be common pathogenetic elements of many painful processes [1]. All this determines the universality and effectiveness of drugs aimed at the repair of damaged cell membranes, primarily drugs based on phospholipids - universal components of the membranes of all types of cells. Currently, such preparations based on "essential" phospholipids are known and widely used (Phosphogliv, Essentiale, Lipostabil, Moslecithin).

The use of phospholipids to obtain medicinal and therapeutic compositions is an important and urgent task. Given the unique structure of phospholipid molecules and their most important role in the functioning of the cell and the body as a whole, phospholipids are an “essential” substance with pharmacological properties, based on which highly effective natural therapeutic and prophylactic drugs can be developed and introduced into medical practice [2, 3 ].

Due to the pharmacological properties of dihydroquercetin, preparations based on it are able to normalize the functioning of different cells of the body, prevent blood clots in the vessels, reduce blood cholesterol, serve as a regulator of fat metabolism in the body, have anti-allergic, anti-inflammatory, radioprotective and vasodilating effects; strengthen the walls of blood vessels, prevent the development of heart and liver diseases. In the top 20 dietary supplements in terms of pharmacy sales in Russia in 2011, Kapilar of the manufacturer DIOD OJSC containing dihydroquercitin is in 8th place.

The choice of dihydroquercetin for the development of the composition is due, on the one hand, to a wide spectrum of biological action and effectiveness, and, on the other hand, to poor solubility in water and low bioavailability.

The way out of this situation is the use of systems for the transport of biologically active compounds in the body. The use of nanosystems for the transport of such compounds opens up opportunities not only to increase the bioavailability of the latter, but also to ensure prolonged circulation of the biologically active compound in the bloodstream, thereby increasing the flow of the drug into organs, tissues and cells.

The most common nanocarriers are currently phospholipid-based liposomes. Dozens of new liposomal preparations are presented on the pharmaceutical market, even more of them are at different stages of clinical trials. The main advantage of the liposomal transport nanosystem is that liposomes, due to their structural organization, are capable of transporting both hydrophilic and hydrophobic drug substances. The particle diameter of phospholipid liposomal preparations, as a rule, is 100-400 nm, and the release form is a nanoemulsion, less commonly a lyophilized powder. It should be noted that, despite the listed number of advantages, the particle size of most of the liposomal drugs on the market contributes to their opsonization (interaction with opsonin proteins that accelerate the absorption of phagocytes) and the removal of the reticulo-endothelial system from circulation. So, it was shown that a decrease in the size of liposomes by 8 times prolongs their circulation in the bloodstream by almost 40 times [4].

Phospholipid nanoparticles, due to their chemical structure, are able to serve as carriers for both soluble and insoluble (hydrophobic) drugs in biological fluids. The incorporation of drug compounds into the lipid matrix of nanoparticles allows one to obtain new nanoforms of drugs with high efficiency, bioavailability and reduced side effects.

The high total surface area, in combination with nanoscale, creates optimal conditions for the interaction of such particles with the cell. In addition, nanoscale creates a unique opportunity for the introduction of particles in the region of gap intercellular junctions, the width of which in some areas can be 30-50 nm. Close sizes, along with the general nature of the surface, phospholipid nanoparticles and lipoproteins also create optimal conditions for their interaction with each other. In this case, phospholipid particles carrying a biologically active substance are included in the system of lipoproteins of blood plasma, with the participation of lipid transport proteins, as a result of which the molecules of the lipophilic drug together with phospholipids can be transported to the particles of lipoproteins.

Dispersible phospholipid-stabilized microparticles are known from the state of the art, which are a fast-dispersible solid dosage form consisting of a water-insoluble compound in the form of nanoscale or micromeric solid particles, the surface of which is stabilized by surface modifiers, for example a phospholipid, and the particles are dispersed in a volume-creating matrix (patent RF 2233654). The size of the resulting particles is 0.66-10.6 microns (660-10000 nm).

There is also known a method for producing submicron particles of a water-insoluble or poorly soluble organic pharmaceutically active compound, comprising the steps of dissolving this compound in a water-miscible first solvent, mixing this solution with a second solvent into which phosphatidylcholine can be added, and homogenizing, or homogenizing, in countercurrent to the obtained pre-suspension or exposure to it by ultrasound (RF patent 2272616). The size of the resulting particles is 0.1-2.46 microns (100-2500 nm). EP 0 556 394 A1 describes a method for preparing a lyophilized preparation for the delivery of drug substances based on refined soybean oil and refined egg phosphatidylcholine. The drug is easily soluble in water with the formation of particles with a size of 10-100 nm and is a fat emulsion.

The objective of the present invention is to develop a technology for producing a water-soluble composition based on dihydroquercetin incorporated into phospholipid nanoparticles with an average diameter of liposomal micellar particles up to 30 nm, which has increased biological activity, low toxicity and is able to withstand long-term storage.

The necessary technical result of the invention is achieved by increasing the solubility of dihydroquercetin embedded in phospholipid particles in comparison with free dihydroquercetin.

In accordance with the invention describes a composition in the form of phospholipid nanoparticles up to 30 nm in size, including phosphatidylcholine, maltose and dihydroquercetin in the following ratio, wt.%:

Phosphatidylcholine 20-43 Maltose 55-78 Dihydroquercetin 2-8

Also described is a method of obtaining a composition in the form of nanoparticles up to 30 nm in size, including 20-43% wt. phosphatidylcholine, 55-78% by weight, maltose, 2-8% by weight, of a biologically active compound, namely that the phospholipid and dihydroquercetin (hydrophobic drug) are dissolved in ethanol, ethanol is distilled off, water is added, suspended, maltose is added and the resulting suspension is subjected to several homogenization cycles under high pressure of 800-1500 bar at a temperature of 40-50 ° C, followed by lyophilization.

The number of homogenization cycles is usually 7-10 cycles, and the pH of the solution is in the range of 6.0-7.5.

Used phosphatidylcholine is the main component of highly purified vegetable soybean phospholipid, the content of phosphatidylcholine, in which not less than 78-95% of the mass. Other phospholipid components may be contained in amounts not exceeding permissible (lysophosphatidylcholine up to 4% by weight, trace amounts of other phospholipids).

As auxiliary pharmacologically acceptable substances, the composition contains a cryoprotectant, maltose, for the possibility of obtaining a lyophilisate capable of completely recovering its structure (in particular, particle size) after dissolution in physiological solution or water.

Materials and methods

The following materials were used in the work:

1. Soya phospholipid brand Lipoid S 100 company Lipoid, Germany.

2. Maltose monohydrate company MERCK, Germany.

3. Dihydroquercetin ("Lavitol (dihydroquercetin)")

4. Water for injection (according to FS No. 42-4587-95).

The invention is illustrated by the following examples.

Example 1. Technological stages of obtaining

Getting the primary (rough) emulsion. Samples of soybean phospholipid (phosphatidylcholine) and dihydroquercetin are added to an aqueous solution of maltose, homogenized by rotor-stator homogenization for 5-10 minutes at a temperature of 40-55 ° C and rotor speeds of 20,000-30000 rpm to obtain a uniform primary dispersion.

Microfluidization of concentrated dispersion. Homogenization is carried out using a microfluidizer Microfluidizer Processor, MHO EN-30K (USA). The primary dispersion obtained in the previous step is passed through a microfluidizer at a pressure of 1500-2000 bar ± 10%. The temperature of the dispersion is maintained between 40-65 ° C. The microfluidization process is carried out for 3-7 cycles until the transmittance of the working solution reaches> 40% (control by transmittance at 660 nm in a cuvette with an optical path length of 1 cm).

Filtration of a phospholipid emulsion. The filtering process is carried out in a tangential flow. Filtered liquid during filtration in a tangential flow from the storage tank with the help of a pump is fed into the filter housing and moves with high speed along the membranes. Part of the fluid passes through the pores into the membranes. All clean and filtered fluid is collected from all membranes and pumped out. The pump pumping out the filtered liquid periodically switches for a few seconds in the reverse direction, creating a water hammer in the membrane. Due to this, particles deposited on the surface of the membrane fly off it and are carried away by the flow of contaminated liquid.

Filling in pallets. The drug is bottled manually under aseptic conditions.

Freeze drying. The product is frozen to -40 ° C, then the shelves are heated to + 10 ° C. Under vacuum of 150 milliTor, the product is sublimated for 30 hours. Then the temperature of the shelves is increased to + 50 ° C and the preparation is dried under a vacuum of 50 milliTor for 8-10 hours.

As a package for finished medical and prophylactic products, sachets were selected.

The technological scheme is shown in figure 1.

Example 2. Particle Size

To determine the size of phospholipid nanoparticles, we used the method of photon correlation spectroscopy on a Beckman N5 Submicron Particle Size Analyzer (Beckman Coulter, USA) using Contin programs.

Particle Size Protocol

The contents of the sachet were dissolved in 10 ml of distilled water; 20 μl were taken from the resulting solution and transferred to a cuvette containing 3 ml of distilled water. A cuvette was installed in the device and the light scattering intensity was measured at a scattering angle of 90 °. The temperature of the thermostatic cell is 25 ° C, the time for balancing the temperature in the cell is 3 minutes.

The obtained particle size distribution data for both compositions are presented in FIG. 2.

Example 3. Comparison of the antioxidant activity of the therapeutic composition

Comparison of the antioxidant activity of the developed composition was determined spectrophotometrically by measuring the oxidation rate of the dianisidine reagent with peroxidase by increasing absorbance at 460 nm (ε = 30 mM-1 m-1). The reaction was carried out in the presence of riboflavin to initiate oxidation along the radical path.

Dihydroquercetin and its phospholipid analogue were added to the incubation medium before the addition of hydrogen peroxide, which started the oxidation process. The oxidation rate in the absence of antioxidants was taken as 100%. The introduction of antioxidants into the incubation medium reduces the oxidation rate of the dianisidine reagent. The results are presented in figure 3.

Example 4. Antihypoxic effect of dihydroquercetin

The antihypoxic effect of antioxidants was studied on the model of acute hypoxia [5, 6]: the animal absorbs oxygen in a confined space, reducing its content in the chamber. Due to the resulting oxygen deficiency, the so-called hypoxic hypoxia. At the same time, resistance to oxygen deficiency (life expectancy in conditions of hypoxia) is determined while taking the studied compositions.

The test was carried out as follows: the test composition and free substance were administered per os to white non-linear mice daily for 15 days (20 mg / kg dihydroquercetin). Then the animals were placed in a tightly closed jar. Using a stopwatch recorded life expectancy. The controls were intact animals. Catalase activity, the level of diene conjugates and lipid hydroperoxides in brain homogenates after death in an animal were determined as parameters characterizing the antihypoxic effect [7, 8, 9]. The results of the antihypoxic action of dihydroquercetin (DHA) and its phospholipid composition (NF-DHA) are presented in table 1.

Table 1 The effectiveness of dihydroquercetin and its phospholipid composition in the test "can hypoxia" Groups of animals Life span, min Malonic dialdehyde, nmol / mg protein Catalase, μmol H ₂ O ₂ / mg min Hydroperoxides of lipids, units opt.pl. at 480 nm Intact - 2.77 ± 0.32 9.59 ± 0.63 0.11 ± 0.01 The control 35.6 ± 2.2 4.58 ± 0.62 3.36 ± 0.52 0.45 ± 0.02 DHA substance 49.7 ± 1.8 * 3.26 ± 0.52 * 5.20 ± 0.81 * 0.31 ± 0.03 * NF-DGK 62.3 ± 2.0 ** 2.95 ± 0.40 ** 6.88 ± 0.38 ** 0.23 ± 0.02 ** * - significant differences from control, P <0.05
** - significant differences from the group receiving the free substance, P <0.05

As can be seen from the data, the use of the studied therapeutic and prophylactic composition significantly increased the life time of mice (almost 2 times), while the increase in the levels of malondialdehyde and lipid hydroperoxides in the brain tissue, as well as the decrease in the catalase activity of brain tissue, were significantly less than in control group. This indicates an increase in reserve antioxidant activity of the brain.

The list of drawings used in the description

Figure 1. Technological scheme for the production of dihydroquercetin incorporated into phospholipid nanoparticles.

Figure 2. The polydisperse distribution of phospholipid particles in volume for a composition containing dihydroquercetin.

Figure 3. The decrease in the oxidation rate of dianisidine reagent in the presence of antioxidant compositions and free substances.

Literature

1. Platova O.M. Phosphogliv: The mechanism of action and application in the clinic // State Research Institute of Biomedical Chemistry RAMS, 2005-318 p.

2. Kochetkova, A.A. and others. The modern theory of positive nutrition and functional products // Food industry. - 1999. - No. 4. - S.7-10.

3. Kolesnov A.Yu. Biochemical systems in assessing the quality of food. M .: Food industry, - 2000.

4. Drummond DD, Meyer Oh, Hong K, Kirpotin DB, Papahadjopoulos D: Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors. Pharmacol Rev. 51, 691-743 (1999).

5. Guide to physiology. Ecological physiology of man. Human adaptation to extreme environmental conditions. Edited by O.G. Gazenko. M., "Science", 1979, S. 333-336.

6. Guide to practical exercises by pathological physiologists. Ed. O.M. Pavlenko. M., "Medicine", 1974, p. 174-175.

7. Tyson S. A., Luman KD, Stephens RJ Age-related differences in G-SH-shuttle enzymes in NO ₂ - or O ₃ -exposed rat lungs - Arch. O ₃ . Env. Health, 1982, Vol. 37., No. 3, p. 167-176.

8. Research methods in occupational pathology. Ed. O.G. Arkhipova. M., 1988, p. 156-158.

9. Modern methods in biochemistry. Ed. V.N. Orekhovich. M., "Medicine", 1977, S. 62-64.

Claims (1)

A therapeutic and prophylactic composition based on dihydroquercetin in the form of nanoparticles with a size of 8-30 nm, including plant phosphatidylcholine 78-95%, maltose and dihydroquercetin in the following ratio, wt.%:
phosphatidylcholine 20-43 maltose 57-78 dihydroquercetin 2-8

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