RU2499610C2 - General method for air, fluid and surface cleansing with biocompatible microporous silicon for decontamination of viruses in household use, medicine and industries - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to air, fluid and surface cleansing and disinfection, and may be used in household use, medicine and industries. A method for air, fluid and surface cleansing consists in the decontamination of viruses from the same with a microporous silicon solution.

EFFECT: invention provides the effective air, fluid and surface decontamination of viruses with the low concentration of the working solution.

4 dwg, 3 tbl

Description

The invention relates to the field of medicine, virology, biotechnology and nanotechnology.

From the level of medicine, technical solutions of a similar nature are well known.

So, from the level of medicine known are "Methods and products having high antiviral and antibacterial effectiveness", disclosed in the application for invention No. 2008152442/15, A01N 25/08, 06/04/2007. In particular, there is known a method of killing viruses and bacteria on an inanimate surface, comprising treating the surface with a composition containing a compound capable of lowering the pH of the inanimate surface to less than about 4 for at least about 0.5 hours and up to eight hours, where a compound capable of lowering the pH of an inanimate surface is selected from the group consisting of (a) an organic acid, (b) an inorganic acid, (c) an inorganic salt comprising a cation having a valency of 2, 3 or 4, and a counterion capable of which can reduce the pH of the skin to less than about 4, (d) aluminum, zirconium or an aluminum-zirconium complex and (e) mixtures thereof, and a compound capable of lowering the pH of an inanimate surface is present in the composition in an amount of from about 0.05 to about 15 % by weight of the composition, and the composition has a pH of from about 2 to less than about 5, where the compound forms a substantially continuous barrier layer on an inanimate surface.

Also known from the level of medicine are "Compositions with a strong antiviral effect", disclosed in the description of the application 2008152402/15, A01N 37/36, 05.29.2007. In particular, a method for reducing a virus population on a surface is provided, comprising treating the surface with a solid composition comprising: (a) a powdered solid substrate selected from the group consisting of silicon oxide, clay, a superabsorbent polymer, and mixtures thereof; and (b) an effective virucidal effect of an organic acid comprising one or more of polycarboxylic acids comprising two to four carboxyl groups, optionally containing one or more hydroxyl groups, amino groups or both, and a polymeric acid containing a plurality of carboxylic acids, phosphate, sulfonate and / or sulfate fragments, in which the composition forms a substantially continuous barrier layer comprising an organic acid on the surface.

In addition, the “Method for improving the fight against viruses on the skin”, disclosed in the description of the application 2008151426/14, A61K 31/78, 05/23/2007, is known. In particular, a method for reducing a viral population on a living surface is described, comprising: (a) contacting the surface with a pretreatment composition comprising an anionic surfactant, a nonionic surfactant or mixture thereof and having a pH of from about 5 to about 7, then (b) contacting the surface with an antiviral composition capable of achieving a logarithmic decrease of at least 4 orders of magnitude against non-enveloped viruses after 30 seconds of contact, where the antiviral composition comprises: (i) from about 25% to about 75%, by weight, of a disinfecting alcohol 1-6 or mixtures thereof; (ii) a virucidally effective amount of an organic acid comprising (i) two or more polycarboxylic acids containing from two to four carboxyl groups, each optionally containing one or more hydroxyl groups, amino groups, or both, and (ii) a polymeric an acid having many carboxyl, phosphate, sulfonate and / or sulfate moieties; and (iii) from 0 to about 5%, by weight, of a gelling agent selected from the group consisting of cellulose, cellulose derivative, guar, guar derivative, algin, algin derivative, water insoluble C8-C20 alcohol, carrageenan, smectite clay polyquaternium compounds and mixtures thereof, and has a pH of from about 2 to about 5, where the antiviral composition forms on the living surface a substantially continuous barrier layer containing organic acid.

Also known is the "Means for the treatment and prevention of acute respiratory viral and bacterial diseases and a method for its preparation", disclosed in the description of the patent of the Russian Federation No. 2407738 C07D 235/14, C07D 401/06, C07D 403/06, C07D 403/12, C07D 405/06, A61K 31/454, A61K 31/4178, A61K 31/496, A61K 31/5355, A61P 31/12, 03/03/2009. The invention relates to new antiviral active components, which are substituted 2-aminomethyl-1-benzyl-1H-benzo [d] imidazole of general formula 1 or their pharmaceutically acceptable salts, pharmaceutical composition, antiviral drugs, a method for the prevention and treatment of viral diseases, especially caused by hepatitis C viruses (HCV). In the general formula 1, R1 represents one or two substituents of the cyclic system, including a hydrogen atom, a C1-C3 alkyl, a C1-C3 alkyloxy or a halogen atom; R2 represents a hydrogen atom or C1-C3 alkyl; R3 and R4 are unequal amino substituents, including hydrogen, C1-C2 alkyl, C1-C3 alkanoyl, optionally substituted furanoyl; optionally substituted or optionally annelated with 5-6 membered azaheterocycle phenyl; or R3 and R4, together with the nitrogen atom to which they are attached, form morpholin-4-yl, 4- (C1-C3 alkyl substituted) piperazin-1-yl, and R1 and R2 have the above meaning, or piperidin-1-yl, with the proviso that R1 has the above meaning, a R2 is C1-C3 alkyl, or with the proviso that R1 is hydrogen or o-fluoro, a R2 has the above meaning.

In addition, technologies of a similar purpose are disclosed from the medical level as disclosed in the descriptions of foreign security documents, for example, US 7910595, US 7910587, US 7910523, US 7910313, US 7906550, WO 2010000207, WO 2010047295, EA 201070053, etc.

The nearest analogues were not found.

The problem to which the invention is directed, is to create a universal method for cleaning air, liquid media and surfaces using biocompatible microporous silicon for antiviral treatment in everyday life, in medicine and in industry.

When implementing this invention, several results are achieved, one of which is the possibility of unification of the starting materials to create effective antiviral drugs, and the other is the versatility of its use.

There are three main methods of disinfection: physical, biological and chemical.

The physical method includes: exposure to high temperature, for example, in the form of steam, boiling, sterilization with hot air, calcination, burning; UV or ultrasonic treatment.

Biological disinfection methods are implemented using biological filters, biothermal chambers and composting.

Chemical disinfection agents include exposure to chemical compounds, for example, chlorine-containing reagents, bromine, iodine and their derivatives, phenols and cresols, guanidine, aldehydes, alcohols, oxides, acids, alkalis, etc.

The traditional chemical disinfectants are chlorative preparations of an organic (chloramine, chlorine derivatives of cyanuric acid and hydantoin) and inorganic (hypochlorites) nature.

A number of chloroactive substances are cheap, but their sharp odor, corrosive effect are related to their shortcomings; for some, poor solubility in water and instability during storage are characteristic.

Of the iodine compounds, the most widely used are iodophors - substances representing a complex of iodine and a carrier, which are used as high-molecular substances and surfactants.

A pronounced bactericidal, tuberculocidal, fungicidal and virucidal effect, as well as the sporocidal effect of iodophors, determine the use of these substances as antiseptics and are very limited for the disinfection of individual objects.

It is widely used in healthcare for disinfection, sterilization and pre-sterilization treatment of hydrogen peroxide. It has such valuable qualities as the absence of smell, the rapid decomposition in the environment into non-toxic products, and the absence of allergenic effects.

However, hydrogen peroxide is unstable, produces a pronounced local irritant effect and, in comparison with other disinfectants, has a low bactericidal activity. The liquid form of hydrogen peroxide-based products is inconvenient for use, which prompts researchers to search for peroxide compounds in solid form.

In the last decade, due to its properties, disinfectants from the surfactant group have become very widespread. By their ability to ionize in aqueous solutions, they are divided into cationic, anionic, ampholytic and nonionic surfactants. As independent disinfectants, cationic and ampholytic surfactants are used, compounds of all groups are used as potentiating additives in the composition of composite disinfectants.

High antimicrobial activity and a wide spectrum of action are distinguished by drugs from the group of peracids. Such disinfectants are deoxone-1, deoxone-4. The last drug is characterized by the presence of a stabilizer, allowing its solutions to be used at a temperature of 500C. The active principle of these drugs is peracetic acid (5-6%). Deoxoxones have high bactericidal, virucidal, tuberculocidal, fungicidal and sporocidal properties. The presence of such a wide spectrum of action allows the use of drugs to disinfect various objects in infections of various etiologies. However, a sufficiently strong smell of vinegar, inherent in deoxones, complicates their widespread use. Deoxon-1 is one of the few drugs that can be used to sterilize flexible endoscopes.

From the group of aldehydes, formaldehyde, glutaraldehyde and disinfectants based on them are used in the practice of disinfection. Formaldehyde is used in the form of solutions (water or alcohol) and in the form of a gas in combination with steam. Despite the high antimicrobial activity, the use of formaldehyde is limited due to the strong irritating effect and the presence of carcinogenic properties. Glutaraldehyde is more widely used as a disinfectant, but for its effective action, an alkaline environment is required, obtained by adding activators.

Thus, a brief analysis shows that much attention is paid to the creation of new, more advanced disinfectants, the study of their properties, antimicrobial activity, resistance, and the study of toxicity.

Recently, active research is being carried out on the biomedical properties of various nanomaterials, in particular, based on silver.

There are reports that silver ions have a pronounced ability to inactivate vaccinia viruses, influenza strains A-1, B, Mitrs strain, some entero and adenoviruses, as well as inhibit the AIDS virus and have a good therapeutic effect in the treatment of Marburg virus disease, viral enteritis and plague in dogs.

In this case, a great advantage of colloidal silver therapy was revealed compared to standard therapy.

However, it should be noted that the pharmacological preparations of aqueous colloidal silver are not stable, silver particles are aggregated randomly into large aggregates during storage, which sharply reduces the antibacterial properties of silver.

Back in 2009, Professor Tymoshenko V.Yu. assumptions were made about the virucidal properties of crystalline silicon (c-Si) and mesoporous silicon (meso-PC) nanoparticles with sizes of 100-200 nm.

Such assumptions were based on the proportionality of nanocrystals and viruses, which in principle did not exclude their interaction.

The results of some studies of this interaction when using provided by Professor Timoshenko V.Yu. c-Si and meso-PC nanoparticles confirmed the presence of virucidal properties of nanocrystalline silicon solutions. However, a pronounced virucidal effect was obtained only at sufficiently high concentrations of more than 2 mg / ml [1].

Further studies showed a significant dependence of the virucidal properties of silicon nanocrystals on their size.

This allowed us to suggest that there are much more complex processes of physico-biological interaction of silicon nanoparticles and viral biological substances than previously thought.

The aqueous solutions of microporous silicon (micro-PC, size less than 10 nm) that we studied showed more than an order of magnitude greater Vilulicidal efficacy at a significantly lower concentration.

The following is a brief description of experimental studies of the virucidal effect of micro-PCs, the characteristics of the materials and samples used, which in no way limit the possible options for experimental studies.

The initial solutions were microporous silicon particles. Based on the initial solutions of preparations with a concentration of 2 mg / ml were prepared. Moreover, PBS with a pH of 7.0 was used as a solvent.

Next, using the same solvent, 2-fold dilutions from 1/2 to 1/128 in a volume of 200 μl were prepared.

To each dilution, 20 μl of virus-containing fluid (HIV-1 / Bru strain, 1.5 * 10 ³ TCID50) was added and mixed using Wortex equipment for 20 minutes.

Then, the samples were centrifuged at 1600 rpm for 10 min at room temperature. The resulting supernatant in a volume of 20 μl was used to infect CEM SS cells. The final multiplicity was 75 TCID50.

The results of experimental studies are presented in table 1. The table shows the results of infection control in percent depending on the concentration of suspensions.

Table 1 Breeding 1/1 1/2 1/4 1/8 1/6 1/32 1/64 1/128 Concentration, mg / ml 2 one 0.5 0.25 0.12 0.06 0,03 0.015 Infection rate,% 0.7 2.2 1.9 10.9 3.6 67.8 98.8 93,4

The data obtained indicate that preparations based on microporous silicon exhibit a significant virucidal effect even at a concentration of their aqueous solution of 0.12 mg / ml.

The most well-known and common diseases in humans are associated with respiratory infections. One of the causative agents of such infections is the respiratory syncytial virus (RSV). RSV is rapidly transmitted from person to person by airborne droplets and causes upper and lower respiratory tract infections. RSV is the most common cause of disease in people, and especially children.

The cultivation of the respiratory syncytial virus (RSV) of strain Long was carried out in MA-104 cells (cells of the embryonic kidney of rhesus macaque). For this, the cells were cultured in complete DMEM medium (10% ETS, 300 mg / L glutamine, 80 mg / L Gentamicin) for a day until a monolayer of 75% confluency was formed, then the medium was changed to serum-free DMEM (300 mg / L glutamine, 80 mg / l Gentamicin) and infected cells with the RSV virus at MOI = 0.01, followed by incubation for 4 hours at 37 ° C, in a 5% CO ₂ atmosphere. Then the virion-containing medium was removed, the cells were washed with serum-free DMEM from unbound viral particles, and fresh serum-free DMEM medium was added, followed by incubation for 4 days until approximately 90% of the cytopathic effect was observed, which was observed by light microscopy.

The medium obtained from the cultivation of infected cells, which contained newly formed RSV virions, was centrifuged at 2000 rpm for 5 minutes to precipitate cell debris. The viral particle supernatant was aliquoted and stored at -70 ° C until used in experiments.

The resulting viral material was mixed in equal volumes with particles of microporous silicon of various concentrations dissolved in sterile phosphate-buffered saline (PBS). FSB without micro-PC was used as a positive control. FSB without RSV was used as a negative control. The resulting mixture of RSV and micro-PC (particle sizes less than 10 nm) was stirred on a rocking chair for 20 minutes at 1200 rpm. The mixture was then centrifuged at 2000 rpm for 10 minutes to deposit silicon particles. In the obtained supernatant, the number of viable virions was determined by titration on the prepared cell monolayer.

For this, MA-104 cells were seeded on a 96-well plate in an amount of 104 cells / moon in complete DMEM medium and incubated for one day until a monolayer was formed. Then the cells were washed with serum-free DMEM and infected with the obtained supernatant, performing binary titration. Infection was carried out in four repetitions, in a final volume of 100 μl / moon. Cells with supernatant added were incubated for 8 days and the formation of syncytium caused by the action of RSV on the monolayer by light microscopy was evaluated. Table 2 shows the results of 5 independent experiments with the respiratory virus (RS virus Long). The numbers given in Table 2 indicate the level of cell infection in% relative to the control (0 mg / ml = 100%).

table 2 Concentration, mg / ml 0 0.002 0.2 2 Infection rate,% 100 ± 10 60 ± 15 17 ± 10 0.02 ± 0.1

Figure 1 presents graphs of the dependence of the level of infection on the concentration of the aqueous solution of micro-PC, constructed using the data of table 1 and table 2. Similar data were obtained in the study of the virucidal effect on the common enveloped herpes virus.

In order to visualize the interaction of micro-PCs and viruses, images of the various stages of this process were obtained using an electron microscope.

Figure 2-4 presents photographs obtained using an electron microscope that explain the mechanism of the virucidal effect.

As can be seen in figure 2, the investigated viruses are a group of spherical substances. After the effects of the proposed drugs on viruses, the latter turn out to be micro-PK particles blocked on all sides, which makes their further interaction with the cells of living organisms impossible (see figure 3). With a larger magnification of the electron microscope, you can see how a single virus is blocked by particles of microporous silicon (see figure 4).

Based on the results of basic experiments, recommendations for the use of the considered virucidal effect for cleaning air, liquids, and the surface of objects have been developed and practically verified, which in no way limit the possible concentrations of micro-PC solutions and the specific consumption of solutions for various applications (see table 3) .

Table 3 Air Water Surface Initial virus concentration, TCID50 one hundred 1500 1500 The concentration of the solution, mg / ml 0.01 0.1 0.2 Specific consumption 140 ml / m ^{3 *)} 0.1 mg / ml 100 ml / m ² Note:
*) - the specific consumption of micro-PC solutions for air purification depends on the cleaning method used. The specific consumption shown in Table 3 characterizes a method based on bubbling of air flow through a solution layer with a linear velocity of 4-6 cm / s with a time interval of 2 hours between changes of cartridges with a solution of 12.5 liters.

Thus, on the basis of experimentally obtained data, it can be concluded that the studied microporous silicon preparations have a universal virucidal effect, reducing the infectious activity of the virus by 80-100% at solution concentrations of 0.1-0.2 mg / ml and can be widely used in everyday life, in medicine and in industry.

Used sources

1. Gilyazova Alla Vladimirovna. Microbicides with anti-HIV activity. Abstract of dissertation for the degree of candidate of biological sciences. Moscow 2010.

Claims (1)

A method of cleaning air, liquid media and surfaces, including antiviral treatment, for which a solution of microporous silicon particles is used.

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