RU2557987C1 - Method for producing agent for intracellular delivery of biologically active low-molecular nanoparticle-based compounds - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method is implemented as follows: preparing a mixture 1 by adding 0.5M aqueous solution of selenious acid 250 mcl in PEG 400 8 ml, mixing thoroughly in a magnetic mixture at min. 750 rpm with pH of the given mixture 7.55; that is followed by preparing a mixture 2 by adding 0.5M aqueous solution of hydrazine hydrochloride 250 mcl in PEG 400 8 ml, mixing thoroughly in a magnetic mixture at min. 750 rpm with pH of the given mixture 0.68. The mixture 1 is added to the mixture 2 by mixing thoroughly drop by drop. The prepared solution is dialysed against distilled water with removing PEG 400 and hydrazine hydrochloride; the surplus water is distilled off in a rotary evaporator at 60 rpm and 70°C. The prepared solution is added with a low-molecular compound specified in a group of: gentamicin, hexamethylene tetramine, methionine, cephalexin, indole-3-carbinol; pH is reduced to 7.2-7.4. The components are mixed in an amount to provide their content in the agent, wt %: biologically active low-molecular compound 0.001-5.0; selenium 0.0001-1.0; water up to 100.

EFFECT: simplifying the technology.

2 tbl, 3 ex, 1 dwg

Description

The invention relates to the field of pharmacology and medicine, including veterinary medicine, and can be used as an effective means of intracellular delivery of low molecular weight drugs and biologically active molecules in the treatment of diseases of various origins.

Intracellular delivery of drugs and biologically active molecules is an urgent problem in biology, medicine and veterinary medicine. Direct supply of drugs and biomolecules to target organs is ineffective, which is associated with enzymatic degradation of active substances, their neutralization by the liver and excretion from the body by the kidneys. Therefore, the search for effective and safe carriers to genes, cells, and target organs is an important area of research.

Structures such as fullerenes, dendrimers, liposomes, polyacrylate particles, micelles, colloidal gold and others are used as corpuscular carriers for biologically active substances.

The most promising developments in this area are lipid-containing (liposomal) nanosystems, polymer nanoparticles, colloidal particles, as well as porous silicon nanocarriers.

These transport nanosystems are imperfect and have a number of drawbacks (in particular, insufficient stability and high complexity of the production technology and, consequently, extremely high cost), which explains the absence of “targeted” drugs in practical medicine.

In particular, liposomal nanosystems are in most cases offered in the form of solutions containing particles constituting more than 100 nm. Liposomes are used as a “container” for delivering drugs to target organs.

Particles of this size are usually unstable and, accordingly, it is not possible to control the particle size during the formation of the system. In addition, when introduced into the body, liposomes are rapidly excreted from the bloodstream by the reticuloendothelial system, which leads to a decrease in the effectiveness of drugs.

Similar disadvantages are inherent in the following development - “Immunoliposomal nanosystem targeted delivery to connexin-43 positive tumor cells” (RF patent 2422154 according to class IPC A61K 39/00, published on 02.27.2011). The system includes biotinylated monoclonal antibodies to the extracellular fragment of connexin-43 (the first component) and pegylated liposome containers with a diameter of 70-100 nm, covalently linked to streptavidin (second component). The sequential introduction of the first and second components of the binary system into the living system leads to the selective delivery of liposomes to Cx-43-positive cells by specific binding of biotinylated antibodies to the Cx-43 extracellular fragment on glioma cell cytolemma and the subsequent formation of the streptavidin-biotin complex.

However, this nanosystem has a narrowly targeted effect - it is intended only for targeted delivery of liposomes to tumor cells. In addition, a significant disadvantage of this system is the low stability of liposomes during storage.

Also known is the dosage form for the delivery of water-insoluble and poorly soluble drugs in the form of nanoparticles and a method for producing a delivery system for the dosage form (see application No. 2006123043 according to class IPC A61K 31/00, publ. 01.20.2008). The method provides for the lyophilization of water-insoluble and poorly soluble biologically active substances with the formation of particles in the form of spherical nanoscale structures. Lyophilization is carried out by liposomes, consisting of lipids, which, upon association, form a vector environment around the nucleus - the liposome membrane. Nanoparticles have sizes from 5 to 1500 nm. As poorly soluble and water-insoluble biologically active substances, the system contains hypnotics, sedatives, anticonvulsants, tranquilizers, analgesic, anti-inflammatory, cardiovascular, hormonal drugs, vitamins, enzyme and metabolic stimulating agents, antifungal, antitumor, biologically active peptides and proteins. However, a significant disadvantage of this system is the low storage stability and the inability to control the particle size during the formation of the system.

The most stable is the highly dispersed nanosystem proposed in 2011 by the V.N. Scientific Research Institute of Biomedical Chemistry Orekhovich (IBMH RAMS). This system is based on soybean phospholipid and is a system for the transport of both fat-soluble (hydrophobic) and water-soluble (hydrophilic) biologically active compounds. The authors of the development, based on the screening of medicinal substances of different spectrum of action, analyzed their ability to incorporate into the phospholipid transport nanosystem, as well as conducted preliminary tests in vitro and in vivo. According to the results of these tests, drugs containing low molecular weight compounds of drugs Doxorubicin (Doxolip drug) and indomethacin (Indolip drug) equipped with a phospholipid transport nanosystem were selected as the most promising for industrial production. Preclinical studies of these nano-drugs were carried out, a complete set of regulatory and technical documentation necessary for their production was developed, documents were prepared for submission to the Ministry of Health of the Russian Federation for obtaining permission to conduct clinical trials.

The proposed technology allows to obtain nanomedicines in the form of lyophilized powder, which increases their shelf life up to 5 years, protects phospholipids from aggregation. However, a technological process that requires unique equipment is very complex and time-consuming. It includes the preparation of a stable nano-emulsion of phospholipid particles with an incorporated drug substance (high pressure homogenization) and subsequent freeze-drying of the resulting emulsions. This factor negatively affects the cost of finished products.

Porous silicon nanocarriers are represented by the following developments:

- “A method for producing a nanoscale drug delivery system based on silicon dioxide” (RF patent No. 2372890, class IPC A61J 3/00, A61K 47/04, A61K 38/33, publ. 20.11.2009),

- “Pharmaceutical preparations with nanocarriers. Production of pharmaceuticals with nanocarriers made of porous silicon ”(developers - Nanolek Holding Limited (Cyprus) and LLC Nanolek Russia).

The closest of them to the claimed method is a method of obtaining a nanoscale drug delivery system based on silicon dioxide (see RF patent No. 2372890, class IPC A61J 3/00, A61K 47/04, A61K 38/33, publ. 11/20/2009 ), namely, met-enkephalin on the hydrosol of SiO ₂ nanoparticles. The method includes mixing distilled water, hydrochloric acid and tetraethoxysilane, adding the prepared solution to NaOH, evaporating and filtering to obtain the SiO ₂ hydrosol, ultrasonically treating the obtained SiO ₂ hydrosol, adding met-enkephalin and a surfactant solution in an amount of 0.5-2% of the total the volume of the resulting system. The met-enkephalin delivery system is able to cross the blood-brain barrier and deliver the drug to brain cells. The method allows to create nanoparticles with a diameter of 6-10 nm. This drug and its delivery system have a narrowly targeted effect - it is intended only to overcome the blood-brain barrier.

As follows from the description of the project “Pharmaceutical preparations with nanocarriers. Production of pharmaceuticals with porous silicon nanocarriers ”(developers - Nanolek Holding Limited (Cyprus) and Nanolek Russia LLC) - technology for targeted delivery of porous silicon nanocarriers involves the prolongation of the biologically active substance by sorbing it on porous silicon nanoparticles that transport active substance, providing gradual release from pores of about 10 nm in size. On this principle, it is planned to develop a group of solid dosage forms, including drugs for the treatment of cardiovascular diseases, anticancer and antiviral drugs. The action of the already known active substances in these preparations is prolonged by their sorption on nanoparticles of porous silicon.

As follows from the description, this nanosystem does not imply targeted delivery, but only the prolongation of the action of active substances. The question of the rate of release of these substances from the pores of nanoparticles remains unclear. The probability of further utilization (biodegradation) of a sufficiently large silicon particle (more than 150 nm) located in the bloodstream is also doubtful. It is possible that these nanoparticles can be irritating to the immune system, which can subsequently lead to allergic and autoimmune diseases.

It should be noted that the main drawback of the currently used nanoparticles is their high toxicity compared to macroparticles. They are able to penetrate unchanged through cell barriers, through the blood-brain barrier into the central nervous system, circulate and accumulate in organs and tissues, causing more pronounced pathomorphological lesions of internal organs (for example, the formation of granulomas in the lungs, cirrhosis of the liver, glomerulonephrosis), as well as possessing long half-life.

The toxicity of nanoparticles is determined by their shape and size, while the smallest spindle-shaped nanoparticles cause more damaging effects in the body than similar spherical particles. Under the influence of nanoparticles on the body, the dependence “dose-effect” is clearly traced. Clinical manifestations are determined by the content of a chemical element in the composition of each particular nanoparticle, however, a significant increase in the toxic effect is observed.

In addition, nanoparticles can be irritating to the immune system. Since the removal of nanoparticles from the body takes a long time, the constant migration of nanostructures throughout the body in small quantities can lead to destabilization of the immune system and, in the future, to allergic and autoimmune diseases.

The invention is aimed at solving the problem of creating a non-toxic and effective means of intracellular delivery of biologically active low molecular weight compounds based on selenium nanoparticles, namely the development of a method for its production.

To solve the problem, the method is as follows

First, mix 1 is prepared by adding 250 μl of a 0.5 M aqueous solution of selenic acid in 8 ml of PEG 400, vigorously stirring on a magnetic stirrer at at least 750 rpm, the pH of this mixture is 7.55, then mix 2 is prepared by adding 250 μl 0.5 M aqueous solution of hydrazine hydrochloride in 8 ml of PEG 400, vigorous stirring on a magnetic stirrer at at least 750 rpm, the pH of this mixture is 0.68, with vigorous stirring, 1 mixture 2 is added dropwise to the mixture, the resulting solution is put on dialysis against distilled water removing PEG 400 and hydrochloric acid zine, excess water is distilled off on a rotary evaporator, a low molecular weight compound selected from the group of gentamicin, hexamethylenetetramine, methionine, cephalexin, indole-3-carbinol is introduced into the resulting solution. The pH was adjusted to 7.2-7.4.

As a result of the claimed method receive a tool of the following composition, in mass. %:

Biologically active low molecular weight compound 0.001-5.0 Selenium 0.0001-1.0 Water rest

As a low molecular weight biologically active substance, the compound may contain gentamicin, hexamethylenetetramine, methionine, cephalexin, indole-3-carbinol.

As selenium-containing substances, selenic acid is used.

Known to the authors, the sources of patent and scientific and technical information do not describe a non-toxic and effective means of intracellular delivery of low molecular weight biologically active substances and methods for its preparation, in which colloidal selenium is used as a carrier (nanoplatform), on which a number of biologically active molecules are conjugated.

It is known to use selenium as a therapeutic agent, i.e. a drug capable of participating in redox processes of body cells (see, for example, RF patents No. 2394583, No. 2426444, sites http://fitoapteka.com/read/ru: http://www.naturoprof.ru, http: //bezvreda.com/selen-v-pitanii/). Selenium normalizes the exchange of proteins and nucleic acids; regulates specific and nonspecific immunity by activating the function of neutrophils, proliferation of T-, B-lymphocytes, generating production of antibodies, lymphokines; improves body adaptation; reduces the toxic effect of substances, salts of heavy metals, drugs, other various antibiotics; prevents the development of the oxidative process, free radical diseases, including atherosclerosis and its complications, liver necrosis, pancreatitis, multiple sclerosis, parkinsonism, and other diseases.

It is generally recognized that the trace element selenium (Se) is a necessary nutrient for the normal functioning of the human body and animals, as it is part of most hormones and enzymes, actively participating in the metabolism. It performs catalytic, structural and regulatory functions in the body; interacts with vitamins, enzymes and biological membranes; participates in redox processes, cellular respiration, metabolism of fats, proteins and carbohydrates. The role of selenium in the body is largely determined by its inclusion in the composition of one of the most important enzymes - glutathione peroxidase, which protects cells from peroxidation products. Thus, selenium and its compounds exhibit significant antioxidant activity. This element is also a part of other enzymes, participates in the detoxification of xenobiotics, regulates the functions of the thyroid and pancreas, exhibits a hepatoprotective effect, stimulates the body's antitoxic defense, positively affects the reproductive system, and has a radioprotective effect.

The use of colloidal selenium as a nanocarrier for the delivery of low molecular weight biologically active substances is still unknown.

The advantage of the proposed method for the delivery of low molecular weight biologically active substances using nanoparticles based on colloidal selenium is the possibility of intracellular penetration of a nanoparticle of colloidal selenium with drug substances conjugated thereon - low molecular weight compounds. In this case, the biodynamics of drugs and biologically active molecules connected with colloidal selenium occurs by the lymphogenous route and to a lesser extent undergoes enzymatic degradation and neutralization by the liver.

Also, a certain advantage is the fact that colloidal selenium itself is part of the metabolic chain of the body and is able to be absorbed in the intracellular space. This eliminates the undesirable consequences associated with the "disposal" of the carrier itself.

The foregoing allows us to conclude that there is a criterion of "inventive step" in the claimed invention.

The agent obtained by the proposed method is a colloidal solution of selenium nanoparticles with particles of low molecular weight compounds adsorbed on its surface. It is a clear solution, the color of which varies from brick red to orange. The product is stored at a temperature of +2 to + 20 ° C in a dark place. Shelf life is 1 year.

To obtain a means of intracellular delivery of a biologically active low molecular weight compound, mixture 1 is prepared by adding 250 μl of a 0.5 M aqueous solution of selenic acid in 8 ml of PEG 400, intensive stirring on a magnetic stirrer at at least 750 rpm, the pH of this mixture is 7.55, then mix 2 is prepared by adding 250 μl of a 0.5 M aqueous solution of hydrazine hydrochloride in 8 ml of PEG 400, stirring vigorously with a magnetic stirrer at at least 750 rpm, the pH of this mixture is 0.68, with vigorous stirring, 1 mixture is added to the mixture 2 drop by drop, gender the scientific solution is put on dialysis against distilled water, removing PEG 400 and hydrazine hydrochloride, the excess water is distilled off on a rotary evaporator at 60 rpm and 70 ° C, a low molecular weight compound selected from the group of gentamicin, hexamethylenetetramine, methionine, cefalexin is introduced into the resulting solution indole-3-carbinol, at a concentration of 0.001-5.0% by weight, adjust the pH to 7.2-7.4.

Bringing the pH to 7.2-7.4 is due to the need to create a stable system, since acidity values above or below the specified limits will lead to the destruction of the colloidal solution.

The choice of the quantitative content of selenium in the agent is 0.0001-1.0 wt.%, As well as of the biologically active low molecular weight compound 0.001-5 wt.%, Due to the need to meet the task at hand. The values of the concentrations of the components ensures uniformity and stability of the obtained funds.

The preferred route of administration of an agent for intracellular delivery is parenteral. The dose is calculated depending on the biologically active substance used in the system, at the rate of 0.1-7 mg per kilogram of body weight of the biological organism.

Example 1

Mixture 1 is prepared by adding 250 μl of a 0.5 M aqueous solution of selenic acid in 8 ml of PEG 400, vigorous stirring with a magnetic stirrer at at least 750 rpm, the pH of this mixture is 7.55, then mixture 2 is prepared by adding 250 μl of 0 , 5M aqueous solution of hydrazine hydrochloride in 8 ml of PEG 400, vigorous stirring on a magnetic stirrer at at least 750 rpm, the pH of this mixture is 0.68, with vigorous stirring, 1 mixture of 2 is added dropwise to the mixture, the resulting solution is put on dialysis against distilled water, removing PEG 400 and hydrazine hydrochloride, A test of water is distilled off on a rotary evaporator at 60 rpm at 70 °, methionine is added to the resulting solution to a final concentration of 1.7 mass. %, adjust the pH to 7.2-7.4.

Total we get:

Low molecular weight biologically active substance methionine 0.017 g / ml Selenium 0,00062 g / ml Water rest

As a percentage of the total volume, the composition of the product will be as follows, in mass. %:

Low molecular weight biologically active substance methionine 1.7 Selenium 0,062 Water up to 100

Example 2

Mixture 1 is prepared by adding 250 μl of a 0.5 M aqueous solution of selenic acid in 8 ml of PEG 400, vigorous stirring with a magnetic stirrer at at least 750 rpm, the pH of this mixture is 7.55, then mixture 2 is prepared by adding 250 μl of 0 , 5M aqueous solution of hydrazine hydrochloride in 8 ml of PEG 400, vigorous stirring on a magnetic stirrer at at least 750 rpm, the pH of this mixture is 0.68, with vigorous stirring, 1 mixture of 2 is added dropwise to the mixture, the resulting solution is put on dialysis against distilled water, removing PEG 400 and hydrazine hydrochloride, A test of water is distilled off on a rotary evaporator at 60 rpm at 70 °, gentamicin or cephalexin is introduced into the resulting solution to a final concentration of 1 mass. %, adjust the pH to 7.2-7.4.

Total we get:

Low molecular weight biologically active substance gentamicin or cephalexin 0.095 g Selenium 0.0098 g

As a percentage of the total volume, the composition of the product will be as follows, in mass. %:

Low molecular weight biologically active substance gentamicin or cephalexin one Colloidal selenium 0.2 Water up to 100

Example 3

Mixture 1 is prepared by adding 250 μl of a 0.5 M aqueous solution of selenic acid in 8 ml of PEG 400, vigorous stirring with a magnetic stirrer at at least 750 rpm, the pH of this mixture is 7.55, then mixture 2 is prepared by adding 250 μl of 0 , 5M aqueous solution of hydrazine hydrochloride in 8 ml of PEG 400, vigorous stirring on a magnetic stirrer at at least 750 rpm, the pH of this mixture is 0.68, with vigorous stirring, 1 mixture of 2 is added dropwise to the mixture, the resulting solution is put on dialysis against distilled water, removing PEG 400 and hydrazine hydrochloride, A test of water is distilled off on a rotary evaporator at 60 rpm at 70 °, hexamethylenetetramine is introduced into the resulting solution to a final concentration of 5 wt. %, adjust the pH to 7.2-7.4.

Total we get:

Low molecular weight biologically active substance hexamethylenetetramine 0.05 g / ml Selenium 0,00062 g / ml

As a percentage of the total volume, the composition of the product will be as follows, in mass. %:

Low molecular weight biologically active substance hexamethylenetetramine 5 Selenium 0,062 Water up to 100

The particle size of the obtained agent was studied using electron microscopy, the results of which showed that the agent obtained by the proposed method contains selenium particles from 40 to 100 nm (see Fig. 1). The figure shows nanoparticles with sizes from 40 to 100 nm.

The effect of colloidal selenium on the intracellular penetration of methionine was determined by its accumulation in the intracellular space. The methionine measurement was determined chromatographically by the following procedure:

Determination of methionine concentration was carried out by high performance liquid chromatography. The concentration of methionine in the cells was determined using a Stayer liquid chromatograph, the analysis was carried out at a wavelength of 288 nm, a flow rate of 0.9 cm ³ / min, a sample volume of 20 μl, and an analysis temperature of -30-35 ° C , eluent - acetonitrile "for liquid chromatography" - 1% solution of acetic acid in a ratio of 2: 3 by volume. Under these conditions, standard solutions are chromatographed twice each. The peak area is measured at a retention time of 3.9-4.3 minutes (depending on column efficiency). Using the average of parallel measurements, the difference between them should not exceed 5% (rel.), Build a calibration graph in the coordinates: peak area - concentration mg / ml. Chromatography of the analyzed samples is carried out similarly. Using the obtained average value of two parallel measurements (the relative standard deviation of which should not exceed 5%), the concentration of methionine in the analyzed solution is found from the calibration graph.

Methionine accumulation was observed in rat peritoneal cells and lymphocytes.

Obtaining peritoneal macrophages

Peritoneal macrophages were obtained from the abdominal cavity of rats. 10 ml of medium was added intraperitoneally to the euthanized chloroform rats to isolate cells from the abdominal cavity (medium 199 + 2% horse serum + 100 units of heparin + 100 units of penicillin) and kept for 1 min, massaging the abdomen. Then the rat was opened and collected the introduced medium with washed macrophages. After the cells were besieged at 2500 rpm for 30 min and washed 3 times with medium for washing cells (medium 199 + 2% horse serum + 100 units of penicillin). Next, the cells were resuspended in culture medium (medium 199 + 10% horse serum + 100 units, penicillin). The content and leukogram of the isolated cells were determined on an Arkus hemanalyzer (Austria).

Receiving lymphocytes

Lymphoid cells were obtained from the peripheral blood of calves. Peripheral blood was centrifuged at 1500 rpm for 30 minutes, after which plasma was taken. The resulting plasma was diluted with Hanks 3: 1 solution. The diluted plasma was layered on ficol-verographin without violating the plasma / ficol interface and centrifuged at 1500 rpm for 30 minutes.

After centrifugation, the resulting mononuclear cell ring was collected in a separate tube, diluted with Hanks' solution 1: 1, and the cell fraction was precipitated at 3000 rpm for 10 min. The precipitate was washed with Hanks' solution 1: 7 from ficol-verographin, precipitating the cell fraction in the same way. The precipitate with the supernatant residue in a total volume of 1 ml was diluted with medium for receiving lymphocytes (medium 199 + 5% horse serum + 0.1% gelatin) 1: 1, precipitated at 2000 rpm for 10 min and the resulting precipitate was resuspended in the same environment. The content and leukogram of the isolated cells were determined on an Arkus hemanalyzer (Austria).

Incubation of particles of colloidal selenium conjugated with methionine with peritoneal macrophages and lymphocytes

The studied colloidal particles were incubated with peritoneal macrophages and lymphocytes for 3 hours in an incubator at 37 ° C.

After incubation, the cells were washed three times with cold saline to remove unabsorbed particles. After washing, the cells were suspended in 200 μl of physiological saline. In the obtained cell suspension, the amount of methionine accumulated in the cells was measured by HPLC, and smears were also prepared.

As a result of the experiments, the following data were obtained. During the cultivation of methionine conjugated with colloidal selenium as a carrier, the highest concentration of the drug was determined using rat peritoneal macrophages as an object of study. When using calf whole blood lymphocytes as an object of penetration, we observed a 2-fold decrease in the amount of methionine in the cell population (Table 1). The data obtained indicate the ability of colloidal selenium to transport methionine into the intracellular space.

Table 1 The concentration of methionine inside the cells when using colloidal selenium as a carrier Type of conjugate / type of cells Contributed methionine, mcg Found methionine, mcg × 10 ⁸ / Li methionine conjugate / selenium / peritoneal macrophages 30.45 29.13 methionine conjugate / selenium / whole blood lymphocytes 30.45 15.63 whole blood methionine / lymphocytes 30,4 0.06 methionine / peritoneal macrophages 30,4 2,4

Similar results were obtained using other low molecular weight substances - gentamicin, hexamethylenetetramine, cephalexin, indole-3 carbinol.

We studied the biodynamics of the means of intracellular delivery of biologically active substances by evaluating the penetrating ability of colloidal particles of selenium into the cell.

As the means of intracellular delivery used the tool obtained in accordance with example 1 or 2.

The drug was labeled with a fluorescent dye (FITC) according to the method described, for example, in the book: Immunological methods / Edited by G. Frimel, Translated from German by A.P. Tarasova. - M.: Medicine, 1987, p. 130.

The object of research was white mice weighing 20 grams.

All animals were divided into groups: the first group of mice (n = 3) a drug labeled with FITC was administered intramuscularly at a dose of 0.1 ml; the second - intraperitoneally at a dose of 0.2 ml, the third - orally at a dose of 0.2 ml. One mouse was a control.

After 2, 6, and 24 hours postmortal imprints of the liver, kidneys, spleen, bone marrow, and whole blood smear were obtained.

The research results are presented in table 2.

table 2 No. Organs Intracellular fluorescence 2 hours 6 o'clock 24 hours ORAL INTRODUCTION one. Blood + 2. Liver + + 3. Bud + four. Spleen + + + 5. Bone marrow + + Intraperitoneal Introduction one. Blood + + 2. Liver + + 3. Bud + four. Spleen + + 5. Bone marrow + + INTRAMUSCULAR MANAGEMENT one. Blood + + 2. Liver + + 3. Bud + four. Spleen + + 5. Bone marrow + +

As can be seen from table 2, the complex based on colloidal selenium with methionine or gentamicin in the animal body is located inside structural elements that have a shell, i.e. inside immunocompetent cells. In this case, the agent most often penetrates into the cells of the spleen. The drug is rapidly absorbed in the gastrointestinal tract.

However, with the oral administration of the drug into the cellular elements of the blood, it enters much later than with parenteral administration. At the same time, with parenteral administration, the drug reaches the liver and spleen later than with oral administration.

The data presented indicate the penetration of the drug into the cellular elements of immunocompetent organs, where its metabolism occurs. The absence of the drug in the intercellular space indicates its complete biodegradation by cellular elements.

Thus, the agent is not subjected to enzymatic degradation and neutralization by the liver, i.e. it completely penetrates into the intracellular space.

Colloidal selenium itself, being part of the metabolic chain of the body, is absorbed in the intracellular space, i.e. in this invention, the problem of "disposal" in the body of the carrier itself is solved.

The acute toxicity of the preparation was determined by the Kerber method.

For this, white outbred mice were used in an amount of 10 animals with a live weight of 20-25 g.

The drug based on methionine or gentamicin was administered intraperitoneally to mice at the maximum possible dose of 0.5 ml. The drug was administered during the day every 3-4 hours.

During the first day, the mice were closely monitored, noting the behavior, consumption of food, water, as well as the general condition.

All observations were carried out for 2 weeks, the death of mice was not observed. Thus, it can be stated that the drug belongs to the group of non-toxic compounds.

Similar results were obtained using other low molecular weight substances - hexamethylenetetramine, cephalexin, indole-3-carbinol.

The above experiments prove that the agent obtained by the developed method has low toxicity and high bioavailability.

In addition, the biodynamics of biologically active low molecular weight compounds conjugated to colloidal selenium occurs via the lymphogenous route and undergoes enzymatic degradation and neutralization of the liver to a lesser extent, while biologically active low molecular weight substances exert their effect directly in the pathological focus.

Claims (1)

A method of obtaining a means of intracellular delivery of a biologically active low molecular weight compound, which consists in preparing mixture 1 by adding 250 μl of a 0.5 M aqueous solution of selenic acid in 8 ml of PEG 400, intensive stirring on a magnetic stirrer at at least 750 rpm, pH of this the mixture is 7.55, then mixture 2 is prepared by adding 250 μl of a 0.5M aqueous solution of hydrazine hydrochloride in 8 ml of PEG 400, intensive stirring on a magnetic stirrer at at least 750 rpm, the pH of this mixture is 0.68, with intensive stirring is introduced into mixture 1 mixture 2 dropwise, the resulting solution is put on dialysis against distilled water, removing PEG 400 and hydrazine hydrochloride, the excess water is distilled off on a rotary evaporator at 60 rpm and 70 ° C, a low molecular weight compound selected from the gentamicin group is introduced into the resulting solution , hexamethylenetetramine, methionine, cephalexin, indole-3-carbinol, the pH is adjusted to 7.2-7.4, while the components are mixed in an amount ensuring their content in the product, in mass. %:
Biologically active low molecular weight compound 0.001-5.0 Selenium 0.0001-1.0 Water up to 100

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