RU2189231C1 - Emulsion preparation for prophylaxis of respiratory viral infections - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: invention relates to preparations in aerosol form based on natural polyisoprenoids. An emulsion preparation for prophylaxis of respiratory viral infections comprises polyisoprenoids, nonionogenic surface-active substance and an aqueous base. Natural polyprenols, in part, Siberian fir wood greens (Abies sibirica) are used as polyisoprenoids. The ready emulsion form consists of an aqueous microemulsion of turbidimetric parameter P, mainly, 2.0, not less, in the following wt.% ratio of components, wt.%: Siberian fir polyisoprenoids, 0.01-5.0; nonionogenic surface-active substance, 0.05-25; aqueous base, the balance, up to 100%. EFFECT: enhanced prophylactic protection against respiratory viral infections. 3 cl, 3 tbl, 15 ex

Description

 The invention relates to medicine, namely to preparations in aerosol form based on natural polyisoprenoids for the prevention of respiratory viral infections.

 Polyisoprenoids represent a large group of regularly built linear 1,5 - polyenes with various configuration of double bonds, widely distributed in nature. An important place among these compounds belongs to polyprenols, the molecular structure of which is expressed by the general formula (I), and their derivatives, dolichols (II), which have a wide spectrum of biological activity [1], including immunomodulating and protective in bacterial and viral infections (colibacteriosis and Sendai- infection) [1-3].

 Polyprenols are more common in plants, and polyprenols with m = 2 (betulaprenols) are characteristic of gymnosperms, and compounds with m = 3 (phycaprenols) are more characteristic of angiosperms. Dihydro derivatives of polyprenols - dolichols are distributed mainly in animal organisms, where they perform important regulatory functions. Currently, these groups of substances are considered as a new class of low molecular weight bioregulators (especially important in the biosynthesis of polysaccharides and glycoproteins).

Долихолы обладают более высокой биологической активностью и предпочтительны для разработок новых лечебно-профилактических препаратов. Однако концентрация долихолов в животных тканях очень низка (например, из 10 кг свиной печени можно выделить около 0,6 г долихолов). Более того, в настоящее время имеются большие трудности в препаративном получении долихолов полностью синтетическим путем. Эти обстоятельства сильно затрудняют как исследования биологической активности долихолов, так и разработки новых препаратов.

Dolichols have higher biological activity and are preferred for the development of new therapeutic and prophylactic drugs. However, the concentration of dolicholes in animal tissues is very low (for example, about 10 grams of dolicholes can be isolated from 10 kg of pork liver). Moreover, at present, there are great difficulties in the preparative preparation of dolichols in a completely synthetic way. These circumstances greatly complicate both the study of the biological activity of dolicholes and the development of new drugs.

 At the same time, mixtures of natural polyprenols can be extracted from the green parts of many plants (for example, Ginkgo biloba leaves / raw materials for the medicinal product BILOBIL / in late autumn contain up to 2% polyprenols). Other conifers (Pinaceae family) also contain significant amounts of polyprenols, and the composition of the polyprenols (m = 2, n = 12 - 19, mainly n = 13, 14) corresponds to the composition of natural dolichols (m = 2, n = 12 - 19 , mainly n = 13-15) [1,4].

 In the scientific and patent literature, the ability of various polyisoprenoids (including various polyprenols, dolichols, and their ethers) to normalize the body's immune function and increase its resistance to infections has been repeatedly noted [1–3, 6, 8–11].

 The protective mechanism of polyisoprenoids and their derivatives is usually associated with their ability to stimulate the generation of neutrophils and activate macrophages of RES [1, 2, 8, 10].

 Known pharmaceutical preparations based on various polyisoprenoids (tablets, capsules, powders), external (creams and lazinesses) for injection, intranasal or oral use. In these pharmaceutical compositions to increase the resistance of animal and human organisms, for the prevention and treatment of diseases caused by immunodeficiency, and also in combination with antibiotics - for the treatment of infectious diseases, the use of various polyisoprenoids and their derivatives (including dihydropolyprene and their simple and complex esters) is noted 1-3, 6, 8 - 11].

 However, in known sources of information there is no information on the use of these compositions and forms of drugs for the prevention of respiratory viral infections. At the same time, it is unknown from publications that immunostimulating and antiviral activity (including Siberian fir polyprenols) when inhaled in aerosol form can be significantly increased due to the use of emulsifiers and solubilizers and ensuring the required characteristics of emulsions (mainly microemulsions).

 The closest analogue (prototype) to the proposed technical solution is an emulsion composition for injection (intramuscular) use containing synthetic polyprenylalcohol and lecithin as an emulsifier. The drug has activity as a prophylactic for E. coli infection in white mice [11].

 The disadvantages of this method include the need for high doses of the active substance (about 100 mg / kg) for intramuscular use. In addition, there is evidence of low prophylactic efficacy of the injectable use of polyisoprenoids for respiratory infections [3].

 The objective of the invention is the creation of such a drug that would enhance its prophylactic effect by increasing the efficiency of delivery and disposal of polyisoprenoids in vivo by designing emulsion compositions of polyisoprenoids with the required physicochemical characteristics suitable for their use in aerosol form.

This problem is solved in that in an emulsion preparation for the prevention of respiratory viral infections, including polyisoprenoids, a nonionic surfactant and an aqueous base, according to the invention, natural polyprenols are used as polyisoprenoids, in particular Siberian fir polyprenols (Abies sibirica), and the finished emulsion form the preparation is an aqueous microemulsion with a turbidimetric parameter P, preferably not less than 2.0, with the following percentage of components, weight. %:
Siberian fir polyprenols - 0.01-5.0
Nonionic surfactant - 0.05-25.0
Water base - The rest is up to 100%
Fatty acid esters with polyethylene glycol, or fatty acid esters with polyhydric alcohols (sorbitol), or block copolymers of ethylene and propylene, or ethoxylated castor oil, or higher fatty alcohols (C ₁₆ -C ₁₈ ) are used as a nonionic surfactant.

 As the aqueous base, water-salt or water-alcohol compositions are used.

 In the development of pharmaceutical preparations (including those based on various polyisoprenoids), it is known to use emulsifiers to improve the biological activity of drugs. However, they are used for the preparation of injectable forms, creams and liniment [6, 8, 10, 11].

 A feature of this invention is the inhalation form of the use of polyisoprenoids (in aerosol form). The advantage of the proposed solution is the ability to significantly reduce the applied dose of the drug, as well as to provide selective activation of the respiratory tract immunity system in order to provide a therapeutic and preventive effect against respiratory infections (especially anti-influenza activity).

 Polyprenols of Siberian fir (Abies sibirica) were selected on the basis of raw material availability and for economic reasons (focusing on the previously developed inexpensive method for their isolation from needles [5]).

 General information about the technology used to prepare the inventive preparations in the form of microemulsions.

 The inventive preparations are prepared on the basis of polyprenols isolated from Abies sibirica, according to the method described in [5]. Polyprenol (PPR) is a chromatographically purified natural mixture of isoprenologists described by the general formula (1), where m = 2; n = from 6 to 16 (all members of the series are present, mainly with n = 12 and 13).

 To obtain medicinal compositions of lipophilic polyprenols of Siberian fir (obtained by extraction of needles), the following methods were used: emulsification and solubilization.

 The quality of emulsions for aerosol inhalation is determined (with all other conditions being equal) by their dispersion, i.e. micelle size of the dispersed phase, and stability, i.e. the ability to exist for a long time without stratification. The aggregation stability of emulsions is most affected by the nature of the emulsifier and its content in the system. As emulsifiers, the most diverse substances can be used: ionic and nonionic surfactants and high molecular weight compounds.

 The solubility of drugs in water is an important condition for their preventive effect. In pharmaceutical technology, the solubility of hydrophobic drugs is achieved by the use of complexing agents, solubilizers and various cosolvents. Solubilization promotes the production of drugs with high dispersion, which leads to their rapid and complete absorption. In addition, some solubilizers have the ability to enhance the pharmacological effect of drugs, which allows them to reduce their dose. In pharmaceutical practice, non-ionic surfactants are often used as solubilizers, since they have several advantages: absolute resistance in hard water, selective solubilizing ability, relatively low toxicity, etc.

To obtain medicinal compositions of polyprenols, nonionic surfactants were used as emulsifiers and solubilizers: Fatty acid esters with polyethylene glycol, fatty acid esters with polyhydric alcohols (sorbitol), or block copolymers of ethylene and propylene, or ethoxylated higher alcohols (C ₁₆ -C ₁₈ ), or castor oil.

The dispersion characteristics of emulsions (capable of influencing the distribution of particles over aerosol fractions) were controlled by the turbidimetric method, recording the turbidity of the emulsions and / or the turbidimetric parameter P [7], determined by the turbidity spectra A (λ) (in the wavelength range λ from 400 to 650 nm) formula interpolation
LogA (λ) = CP • Log (λ),
where C is a constant.

The turbidimetric parameter P is defined as the linear regression coefficient of the dependence of A (λ) in the coordinates Log (A) on Log (λ).
With particle sizes less than 300 nm, the size (turbidimetric radius) can be quantified by the theoretical calibration curve. In the case of emulsions of particles having particles with sizes greater than 500 nm, or polydisperse emulsions, the indicator P allows a comparative analysis of the emulsions, control the reproducibility of the preparation methods and study the changes in the properties of preparations during storage. Smaller P values correspond to larger emulsion micelles.

The dispersibility of emulsions (used for inhalation) and the properties of the aerosol were characterized by the following parameters:
the effectiveness of the conversion of the drug to aerosol
fractionally dispersed aerosol composition and
loading the target fraction under dispersion conditions simulating inhaled use.

Emulsions were dispersed on a Vulkan-1 ultrasonic aerosol inhaler with an average emitter power, a sample volume of 20 ml and a temperature of 37 ^o C. To determine the mass concentration of aerosol and fractionally dispersed composition, isokinetic sampling of the aerosol flow was carried out directly at the exit of the inhaler to a four-stage cascade impactor and filter. Air flow 25 l / min was controlled by a rotameter.

 To record the amount of aerosol, a water-soluble fluorescent label - sodium fluorescein (uranin) was used. To determine the content of polyprenols in the aerosol (loading of the target fraction), a lipid-soluble fluorescent label, perylene, was used (taking into account the spectral characteristics).

 The selected aerosol was washed off the impactor chippers with an alkaline solution (NaOH in water, pH 11). The fluorescence intensity was measured on a PERKIN-ELMER-1000 fluorimeter.

 The following is a specific technology for producing microemulsions and examples of the proposed compositions in emulsion form.

 To ensure effective delivery of the active substance to the respiratory tract and disposal of polyisoprenoids in vivo (to enhance immunomodulating and protective properties), drugs were prepared in one way or another, providing the formation of microemulsions with a turbidimetric parameter P, mainly not less than 2.0.

Example 1. The emulsion form of polyprenols is prepared using a surfactant - Tween 80 (Serva). 1 g of polyprenols needles are mixed with 5.0 g of Tween 80 and the components are thoroughly mixed until a visually homogeneous mixture is obtained (to accelerate the process, the mixture was heated in a water bath at a temperature of 60 ^o C). Then, with stirring, distilled water (heated to 60 ^{° C.} ) is added to a total volume of 20 ml. The resulting emulsion has P = 2.2 (particle size of about 0.5 microns).

The percentage of components: weight. %:
Siberian fir polyprenols - 5.0
Surfactant Tween 80 - 25.0
Water base (water) - Up to 100%
Example 2. The emulsion form of polyprenols is prepared on the basis of a nonionic surfactant - Span 20 (Serva). 2 ml of a solution of PPR needles in pentane (100 mg / ml) is mixed with 1 g of Span 20 (diluted with 2 ml of ethanol), the mixture is dried on a rotary evaporator, diluted with 2 ml of ethanol, and then quickly introduced into 50 ml of Hanks solution (heated up to 50 ^o C) with vigorous stirring. The resulting stable emulsion has P = 2.1.

The percentage of components: weight. %:
Siberian fir polyprenols - 0.4
Nonionic Surfactant Span 20 - 2.0
Water base (Hanks solution with 4% ethanol) - Up to 100%
Example 3. The emulsion form of polyprenols is prepared on the basis of a nonionic surfactant - Span 20 (Serva). 2 ml of a solution of PPR needles in pentane (100 mg / ml) is mixed with 1 g of Span 20 (diluted with 2 ml of ethanol). Next, the resulting mixture is quickly injected into 100 ml of Hanks solution (heated to 40 - 50 ^o C) with vigorous stirring. The resulting stable emulsion has P = 2.4.

The percentage of components: weight. %:
Siberian fir polyprenols - 0.2
Nonionic Surfactant Span 20 - 1.0
Water base (Hanks solution with 2% ethanol) - Up to 100%
Example 4. The emulsion form of polyprenols is prepared on the basis of nonionic PLV - Tween 80 (Serva). 2 ml of a solution of polyprenols of needles in pentane (100 mg / ml) are mixed with 1 g of Tween 80 (diluted with 2 ml of ethanol), the mixture is dried on a rotary evaporator, diluted with 2 ml of ethanol, and then quickly introduced into 18 ml of distilled water under intense stirring. The resulting stable emulsion has P = 2.0 and A ₄₀₀ = 0.45.

The percentage of components: weight. %:
Siberian fir polyprenols - 1.0
Non-ionic Surfactant Tween 80 - 5.0
Water base (10% aqueous ethanol solution) - Up to 100%
Example 5. The emulsion form of polyprenols is prepared on the basis of a nonionic surfactant - Tween 80 (Serva). 2 ml of a solution of polyprenols of needles in pentane (100 mg / ml) are mixed with 1 g of Tween 80 (diluted with 2 ml of ethanol). The resulting mixture was quickly injected into 20 ml of Hanks solution (heated to 40 ^o C) with vigorous stirring. The resulting stable emulsion has P = 2.44 and A ₄₀₀ = 0.17 (turbidimetric micelle diameter of about 0.2 μm).

The percentage of components: weight. %:
Siberian fir polyprenols - 1.0
Non-ionic Surfactant Tween 80 - 5.0
Water base (Hanks solution with 10% ethanol) - Up to 100%
Example 6. The emulsion form of polyprenols is prepared on the basis of a nonionic surfactant - Tween 80 (Serva). 2 ml of a solution of polyprenols of needles in pentane (100 mg / ml) are mixed with 1 g of Tween 80 (diluted with 2 ml of ethanol). The resulting mixture was slowly (dropwise) introduced into 18 ml of Hanks solution (heated to 50 ^{° C.} ) with vigorous stirring. The obtained stable emulsion has P = 3.5 and A ₄₀₀ = 0.1 (the turbidimetric diameter of the micelles is less than 0.1 μm).

The percentage of components: weight. %:
Siberian fir polyprenols - 1.0
Non-ionic surfactant Tween 80 - 5.0
Water base (Hanks solution with 10% ethanol) - Up to 100%
Example 7. The emulsion form of polyprenols is prepared on the basis of a nonionic surfactant - Tween 80 (Serva) and Span 20. 2 ml of a solution of pine needles polyprenols in pentane (100 mg / ml) are mixed with 1 g Tween 80 and 0.2 g Span 20 (diluted in 2 ml of ethanol). The resulting mixture (solution of PPR and surfactant in ethanol) is quickly injected into 18 ml of Hanks solution, heated to 50 ^o C, with vigorous stirring. The resulting stable emulsion has P = 2.29.

The percentage of components: weight. %:
Siberian fir polyprenols - 1.0
Surfactants: * Tween 80 - 5.0
Span 20 - 1.0
Water base (Hanks solution with 10% ethanol) - Up to 100%
Example 8. The emulsion form of polyprenols is prepared on the basis of a nonionic surfactant - emulsifier RO-35 (ethoxylated castor oil).

2 ml of a solution of polyprenols of needles in pentane (100 mg / ml) is mixed with 1 g of emulsifier RO-35 (ethoxylated castor oil) diluted with 2 ml of ethanol). A solution of PPR and RO-35 in ethanol is slowly (dropwise) introduced into 18 ml of Hanks solution (heated to 50 ^{° C.} ) with vigorous stirring. The resulting clear microemulsion (with P = 3.3) has a percentage of components: weight. %:
Siberian fir polyprenols - 1.0
Nonionic surfactant - emulsifier RO-35 - 5.0
Water base (Hanks solution with 10% ethanol) - Up to 100%
Example 9. The emulsion form of polyprenols is prepared on the basis of a nonionic surfactant - emulsifier "Emulgin B2" (based on ethoxylated higher alcohols C ₁₆ -C ₁₈ ). 2 ml of a solution of polyprenols of needles in pentane (100 mg / ml) is mixed with 1 g of Emulsin B2 emulsifier diluted with 2 ml of ethanol. The resulting mixture was slowly (dropwise) introduced into 18 ml of Hanks solution (heated to 50 ^{° C.} ) with vigorous stirring. The resulting transparent ultraemulsion (with P = 3.5) has a percentage of components: weight. %:
Siberian fir polyprenols - 1.0
Nonionic surfactant Emulgin B2 - 5.0
Water base (Hanks solution with 10% ethanol) - Up to 100%
Example 10. The emulsion form of polyprenols is prepared on the basis of a nonionic surfactant - emulsifier "Lutrol F68" (a poloximer based on block copolymers of ethylene and propylene). 2 ml of a solution of pine needles polyprenols in pentane (100 mg / ml) are mixed with 1 g of Lutrol F68 emulsifier (diluted with 2 ml of ethanol). A mixture of a solution of PPR and an emulsifier in ethanol is slowly (dropwise) introduced into 18 ml of a Hanks solution (heated to 50 ^{° C.} ) with vigorous stirring. The resulting clear microemulsion has a percentage of components: weight. %:
Siberian fir polyprenols - 1.0
Nonionic surfactant "Lutrol F68" - 5.0
Water base (Hanks solution with 10% ethanol) - Up to 100%
For comparison, liposomal and emulsion forms of the preparation are prepared that are close to analogues [8 or 11] and are used for injection.

 Example 11. The liposomal form of polyprenols is prepared on the basis of egg lecithin / company Rosma (Russia) /. 2 ml of a solution of polyprenols of pine needles in pentane (100 mg / ml) are mixed with 20 ml of lecithin in chloroform (100 mg / ml), dried on a rotary evaporator in the form of a thin film, thoroughly emulsified in 20 ml of Hanks solution and dispersed by ultrasound treatment in USN- 1.

The percentage of components: weight. %:
Siberian fir polyprenols - 1.0
Nonionic surfactant lecithin - 10.0
Water base (Hanks solution) - Up to 100%
The resulting emulsion has A ₄₀₀ = 16.4; P = 0.59.

 The selected method for the preparation of lecithin preparations provides stable emulsions (not decomposing during storage). Within a few days, a small precipitate forms in the emulsion (easily dispersible with stirring).

 Example 12 (control). The emulsion form of polyprenols is prepared on the basis of a nonionic surfactant - Span 20 (Serva). 2 ml of a solution of polyprenols of needles in pentane (100 mg / ml) are mixed with 1 g of Span 20 (diluted with 2 ml of ethanol), the mixture is dried on a rotary evaporator, diluted with 2 ml of ethanol, and then mixed with 18 ml of Hanks solution and dispersed by treatment ultrasound at UZDN-1.

 A feature of emulsions of this type (prototype preparation) is their low stability, which leads to the formation of a fraction of large particles (more than 10 μm) when visible under a light microscope (the turbidimetric parameter P of the emulsion changed during storage for several days from 0, 56 to 0.24, which indicates an increase in emulsion particles. During long-term storage, the formation of large droplets of the oil phase is observed).

The percentage of components: weight. %:
Siberian fir polyprenols - 1.0
Non-ionic Surfactant Span 20 - 5.0
Water base (Hanks solution with 10% ethanol) - Up to 100%
Example 13. Evaluation of the aerophysical characteristics of preparations of polyisoprenoids
The preparations prepared in example 5 and examples 11 and 12 (controls) were used as samples for research.

 Analysis by methods of light and electron microscopy, as well as turbidimetry showed that the preparation prepared in accordance with example 11, is a stable polydisperse emulsion, consisting mainly of submicron particles. The preparation prepared in accordance with example 12, consists of fairly large particles (reaching 5 μm) and is unstable. The preparation prepared in accordance with example 5 is a microemulsion, that is, a transparent (opalescent) preparation with a micelle size of about 0.2 μm (turbidimetric diameter).

Thus, for aerosol experiments, 3 different emulsion options were selected:
polydisperse unstable emulsion P = 0.43 (example 12);
stable emulsion with submicron micelles P = 0.59 (example 11);
stable emulsion with micelles of about 0.2 μm P = 2.44 (example 5).

 As the most adequate characteristics of the dispersibility of the preparations, the following were measured: the efficiency of transferring the preparation to an aerosol, the fractionally dispersed composition of the aerosol, and the loading of the target fraction under dispersion conditions corresponding to inhalation use.

The experiments were carried out on a Vulcan-1 ultrasonic aerosol inhaler with an average emitter power, sample volume of 20 ml and a temperature of 37 ^o C. To determine the mass concentration of aerosol and fractionally dispersed composition, isokinetic sampling of the aerosol stream was carried out directly at the exit of the inhaler to a four-stage cascade impactor and filter. Air flow 25 l / min was controlled by a rotameter.

 To record the amount of aerosol, a water-soluble fluorescent label - sodium fluorescein (uranin) was used. To determine the content of polyprenols in an aerosol, a lipid-soluble fluorescent label is perylene.

 The selected aerosol was washed off the impactor chippers with an alkaline solution (NaOH in water, pH 11). The fluorescence intensity was measured on a PERKIN-ELMER-1000 fluorimeter.

 Table 1 shows the experimental results.

Thus, the preparation prepared in accordance with Example 5 is most effectively converted to aerosol. Judging by the results, a further decrease in the micelle size, as obtained for the preparation in Examples 6, 8-10, can only slightly increase the efficiency of conversion to aerosol. The use of emulsions with P less than 2.0 is undesirable due to a decrease in the efficiency of conversion to aerosol
Example 14. Evaluation of the prophylactic effect of polyisoprenoid preparations when injected into animals aerogenically infected with influenza virus.

 The preparations prepared in accordance with examples 5, 11 and 12 were used as samples for a comparative study of their prophylactic activity in experimental influenza infection in mice.

 In order to compare the biological activity of the preparations with the prototype (for injection use) as a control, tube-dispersed preparations were prepared according to examples 11 and 2 (by analogy with the prototype).

Used the influenza virus strain A / Aichi / 2/68 (H3N2) obtained from the Institute of Virology. DI. Ivanovo RAMS, past 12 passages in mice and two in developing chicken embryos (RCE). In the experiments, a virus-allantoic fluid was used, obtained by culturing the influenza virus in 9-11-day RCE, with a biological concentration of the virus of 10 ⁸ - 10 ⁹ 50% of embryonic infectious doses (EID ₅₀ ) in 1 ml. Prior to research, viral preparations were stored at a temperature of minus 12 ^o C.

 The experiments were carried out on outbred mice of both sexes weighing 15-17 g (without antibodies to influenza A virus).

Infection of animals with a virus-containing aerosol was carried out at a temperature of 20-24 ^o C and a relative humidity of 50-70% in the installation, which included:
A sprayer for virus-containing suspensions of the Collison type [l2], operating at an overpressure of 0.8 - 1.0 atm with air flow rates of 16 l / min and a suspension of 0.1 ml / min and creating an aerosol with a median-mass aerodynamic particle diameter of 1.1 μm ((σ _g = 2.1).
Aerosol dispenser that allows you to smoothly reduce the concentration of aerosol from 1: 1 to 1: 100.

 A small dynamic chamber for 20 mice [13] with air flow rates of up to 2 m / s with an uneven concentration of aerosol in the chamber volume of not more than 10%.

Sampling devices:
impinger with an air sampling rate of 10 l / min and a volume of sorbing liquid of 10 ml [12] for determining the concentration of the virus in an aerosol (composition of the sorbing liquid: Hanks solution with 2% by volume of cattle serum, 100 units / ml of penicillin and 100 μg streptomycin);
four-stage impactor of a design similar to Andersen’s device for biological sampling [14] with a volume flow of 25 l / min for analysis of the dispersed composition of aerosols of preparations and virus-containing aerosols.

When studying the aerosol activity of the virus for animals, the exposure dose (DE) was determined by the formula
DE = Cb • T • W,
where Sat is the biological concentration of the virus in the aerosol (in logEID ₅₀ / l).

 The specificity of the death of the experimental animals was determined by the hemagglutinating activity of the allantoic fluid taken from RKE inoculated with a suspension of homogenate from the lungs of the dead animals.

50% aerogenic lethal doses (ALD ₅₀ ) were calculated by the modified Van der Waerden method [15] using the values of the exposure doses.

 The preparations were administered intramuscularly to animals by 0.1 ml of each preparation (1000 μg per head or approximately 60 mg / kg of animal weight) or Hanks solution 2 days before infection with the virus. Infection of mice was carried out by the aerogenous method using 5 animals per dose.

 Table 2 shows the results of the evaluation of the prophylactic anti-influenza activity of preparations based on Siberian fir polyprenols for injection use in order to compare the protective activity of the preparations with the prototype.

 Thus, only a preparation of the type of coarse emulsion with large sizes of micelles had a reliable protective anti-influenza effect for injectable use (example 12). The highly dispersed preparation of Example 5 (microemulsion) did not show any noticeable prophylactic activity. At the same time, the preparation according to example 11, which occupied an intermediate position in the size of micelles, did not have a reliable preventive effect in case of influenza infection.

 Comparison with the prototype.

In the prototype, the protective effect of the preparations based on polyprenylalkagol during intramuscular administration of the drugs with the infection caused by E. coli was analyzed (with the subcutaneous method of infection of animals). It was shown that the best drugs at doses of 50 mg / kg of weight protected up to 50-60% of animals from a dose of about 6 LD _{50 of} this infection.

 In case of influenza infection from the tested preparations, the controls of examples 11 and 12 imitated a prototype (coarse emulsion) in their physicochemical characteristics. Of these, the preparation of Example 12 protected 40% of the animals from about the same aerosol dose of the influenza virus when the animals were administered in an amount of about 60 mg / kg of their weight. At the same time, the preparation 11 (stabilized by lecithin by analogy with the prototype), which took an intermediate position in the size of micelles, did not have a reliable preventive effect in case of influenza infection.

 Thus, as in the prototype, the preparation of the type of coarse emulsion with large sizes of micelles had a reliable protective anti-influenza effect when injected, (example 12). PPR preparations prepared using nonionic surfactants in the form of a thin emulsion (Example 5) do not exhibit a protective effect upon intramuscular administration. This is also fully consistent with the examples and results shown in the prototype, where it is noted that the non-ionic solubilizer NSO-60 (providing microemulsions) used as one of the options does not have a protective effect when administered intramuscularly.

 The preparations prepared in accordance with examples 1-10, and the prototype show general patterns (depending on the characteristics of the emulsion) and a similar protective effect when administered intramuscularly (despite the significant difference in the diseases caused by the influenza virus and E. coli, and some differences in the composition of emulsifiers). This indicates close values of the biological activity of Siberian fir polyprenols and synthetic polyprenylalcagols of the prototype, as well as the possibility of a quantitative comparison of the biological activity of the developed formulations and prototype.

 Example 15. Evaluation of the prophylactic effect of PPR preparations during aerosol administration in animals aerogenically infected with influenza virus.

 The preparations prepared in accordance with examples 5, 6 and 11, 12 were used as samples for a comparative study of their prophylactic activity in aerosol administration against experimental influenza infection in mice.

 The preparation according to example 11 is a stable emulsion with submicron micelles and an admixture of large particles.

 The preparation according to example 12 is a polydisperse unstable emulsion with particle sizes up to 10 microns.

 The preparation of example 5 is a stable ultra-emulsion with micelles with a diameter of about 0.2 microns.

 The preparation according to example 6 is a stable ultra-emulsion with micelles with a diameter of less than 0.1 microns.

 Used influenza virus strain A / Aichi / 2/68 (H3N2), described in detail above.

 The experiments were carried out on outbred mice of both sexes weighing 15-17 g (without antibodies to influenza A virus).

The treatment of mice with aerosols of preparations and influenza virus was carried out at a temperature of 20-24 ^o C and a relative humidity of 50-70% on the installation, which included, in addition to the equipment previously described, also an ultrasonic inhaler "Volcan-1" for dispersing the preparations, providing flow aerosol up to 30 l / min and the spray rate of the drug up to 3 ml / min. Using such an inhaler, an aerosol was created, 85% by weight of which was composed of particles with MMA from 0.8 to 2 μm.

To determine the weight concentration of the aerosol, a water-soluble fluorescent label, sodium fluorescein (uranin), was used. Fluorescence intensity was measured at λ uraninite _pn = 520 nm (excitation at λ _exc = 472 nm).

The aerosol dose of preparations based on Siberian fir polyprenols obtained by mice (DEp) was estimated by the formula
DEp = Cp • T • W,
where Cn is the concentration of the aerosol of the preparations in the chamber (in μg / l),
T - time of exposure of animals (in min),
W is the minute respiratory volume of the mouse (l / min) - 15 ml / min, calculated according to the data presented in Guyton AC [16].

Evaluation of 50% aerogenic lethal doses (ALD ₅₀ ) and the specificity of animal death was carried out as described above.

 The effectiveness of the preventive effect of preparations based on Siberian fir polyprenols for influenza infection was determined by the proportion of surviving animals.

 The mice were treated with aerosol preparations at various doses 3 and / or 1 day before influenza virus aerosol infection. Data on the doses of the drug obtained by animals and their survival after infection with the influenza virus are summarized in table 3. The aerosols of the preparation prepared in the form of a microemulsion (drug 5) have the greatest protective effect: in 4 out of 8 studied patterns of use of the drug, animal survival exceeded 75% (in this case, in one case, it reached 100%).

 Aerosol treatment of animals with preparation 6 was carried out by varying the dose values and patterns of drug use (first treatment 3–9 and / or second treatment 1–6 days before infection). At high doses of double aerosol administration of the drug 6 (the first - 4.56-9.6 μg / mouse and the second 6 μg / mouse), a reliable protective effect in case of influenza infection is achieved with an increase in the time interval between the second treatment with the drug and infection (up to 3 days) .

 At the same time, when using small doses of the drug (1.4 and 1.4 μg / mouse in the first and second treatment), the prophylactic effect is detected when animals are infected 1 day after the second treatment.

Table 3 describes the protective effect of SPR preparations in aerosol form during aerosol infection of mice with the influenza virus A / Aichi / 2/68 (H3N2) in doses of 1-5 LD ₅₀ .

 An analysis of Table 3 shows that samples 5 and 6, which are finely dispersed preparations — a microemulsion with micelles of about 0.2 and less than 0.1 microns, respectively (compared with preparations 11 and 12 having a larger micelle size) have a fundamental advantage. The most pronounced protective effect when using drug 5 was observed when administered twice with an interval of 48 hours, 5.4 and 3.6 μg / mouse 1 day before infection, and drug 6 under the same conditions of use of 1.4 and 1.4 μg /mouse.

 The use of coarse emulsions (preparations 11 and 12) is also impractical due to the lower efficiency of aerosol transfer and the increased consumption of the drug to obtain the required therapeutic dose in the lungs.

 Thus, preparations based on Siberian fir polyprenols provide high protective activity in the case of aerosol delivery to animals when administered in doses approximately 100 times lower than when administered intramuscularly, providing reliable preventive protection against respiratory viral infections.

Sources of information
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 3. Iida J., Ishihara C., Mizukoshi N. et al. Prophylactic activity of dihydroheptaprenol, a synthetic polyprenol derivative, against Sendai virus infection in mice // Vaccine. - 1990. - Vol. 8, N4. - P.376-380.

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 5. Raldugin V.A. Polyprenols of Siberian fir wood greens: use of liquefied carbon dioxide in a new method for their isolation // Chemistry in the interests of sustainable development. 1997. N 5. S. 75-77.

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Claims (2)

1. An emulsion preparation for the prevention of respiratory viral infections, including polyisoprenoids, a nonionic surfactant and an aqueous base, characterized in that natural polyprenols, in particular polyprenols of Siberian fir wood (Abies sibirica), are used as the polyisoprenoids, and the finished emulsion form of the preparation represents an aqueous microemulsion with a turbidimetric parameter P, preferably not less than 2.0, with the following percentage of components, weight. %:
Siberian fir polyisoprenoids - 0.01-5.0
Nonionic surfactant - 0.05-25
Water base - The rest is up to 100%
2. The emulsion preparation according to claim 1, characterized in that fatty acid esters with polyethylene glycol, or fatty acid esters with polyhydric alcohols (sorbitol), or a block of ethylene-propylene copolymers, or ethoxylated castor oil are used as a nonionic surfactant, or higher alcohols (C ₁₆ -C ₁₈ ).  3. The emulsion preparation according to claim 1, characterized in that water-salt or water-alcohol compositions are used as the aqueous base.

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