Antiviral agent

Abstract

The invention relates to antiviral agents and specifically to synthetic biologically-active derivatives, and can be used in the pharmaceutical industry, in medicine, in plant husbandry and in biotechnology. The antiviral agent is based on poly-N1-hydrazino(imino)methyl-1,6-hexanediamine-poly-N1-amino(imino)methyl-1,6-hexane diamine of general formula (1), ##STR00001##
where: HX is an acid, n=3-20, and m=4-20, and which is active against viruses of humans, animals, plants, bacteria and fungi, said viruses being non-enveloped and enveloped and containing RNA or DNA. The antiviral agent exhibits a broad range of effectiveness, and is active against viruses both in intracellular and extracellular locations.

Claims

1. A method of treating a viral infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound comprising the structure ##STR00006## or a pharmaceutically acceptable salt thereof, wherein HX is an acid; n is 3-20; and m is 4-20.

2. The method of claim 1, wherein n is 5 and m is 6.

3. The method of claim 1, wherein n is 10 and m is 10.

4. The method of claim 1, wherein the infection is caused by a virus selected from the group consisting of: poliovirus; adenovirus; herpes virus; hepatitis C; immunodeficiency virus; influenza A virus; bacterial virus; and potato virus X.

5. The method of claim 1, wherein the infection is an infection of the respiratory organs, intestine, eye or lymphoid tissue.

6. The method of claim 1, wherein said viral infection is an infection of the respiratory organs.

7. The method of claim 1, wherein the compound is administered by topical administration.

8. A method of inhibiting a virus, comprising contacting the virus with an effective amount of a compound comprising the structure ##STR00007## or a pharmaceutically acceptable salt thereof, wherein HX is an acid; n is 3-20; and m is 4-20.

9. The method of claim 7, wherein n is 5 and m is 6.

10. The method of claim 7, wherein n is 10 and m is 10.

11. The method of claim 7, wherein the virus selected from the group consisting of: poliovirus; adenovirus; herpes virus; hepatitis C; immunodeficiency virus; influenza A virus; bacterial virus; and potato virus X.

12. The method of claim 7, wherein the virus is present on the surface of a structure.

13. A method of treating a viral infection in a subject in need thereof, the method comprising administering to the subject an effective amount of an antiviral agent, the antiviral agent consisting essentially of the compound ##STR00008## or a pharmaceutically acceptable salt thereof, wherein HX is an acid; n is 3-20; and m is 4-20.

14. The method of claim 13, wherein the antiviral agent consists of the compound ##STR00009## or a pharmaceutically acceptable salt thereof, wherein HX is an acid; n is 3-20; and m is 4-20.

15. The method of claim 13, wherein n is 5 and m is 6.

16. The method of claim 13, wherein n is 10 and m is 10.

17. The method of claim 13, wherein the infection is caused by a virus selected from the group consisting of: poliovirus; adenovirus; herpes virus; hepatitis C; immunodeficiency virus; influenza A virus; bacterial virus; and potato virus X.

18. The method of claim 13, wherein the infection is an infection of the respiratory organs, intestine, eye or lymphoid tissue.

19. The method of claim 13, wherein said viral infection is an infection of the respiratory organs.

20. The method of claim 13, wherein the compound is administered by topical administration.

Description

PREFERRED EMBODIMENT

Examples of Preparation of the Antiviral Agent

Example 1

(1) n=5, m=6.

(2) A three-necked 1 liter flask equipped with an inert gas supply tube, a thermometer and a gas outlet tube, were charged with 95.5 g (1 mole) of iminourea hydrochloride (also known as guanidine hydrochloride) (46.8 wt. %), 103.4 g (0.89 mol) of 1.6-diaminohexane (DH) (50.7 wt. %), and 5 g (0.1 mole) of hydrazine hydrate, after which the flask was purged with nitrogen. The contents of the flask were stirred and placed into an air bath, while the gas outlet tube was connected to the ammonia catchment receiver. Then, while purging with nitrogen at a rate of 30-40 ml/min, the reaction mixture was heated with gradual removal of water and ammonia over 1 hour adjusting the temperature of the mass to the constant reaction temperature of 190 C. The reaction was aged for 30 minutes, while the system was purged with nitrogen. At this point, the system was cooled to 160 C. and the hot syrupy mixture was poured onto a metal pan and cooled to obtain 169.9 of the product as a solid, almost colorless transparent vitrifaction.

Example 2

(3) The antiviral agent was prepared analogously to Example 1, with n=10, m=10, DH=0.50 mole, HH=0.1 mole. The reaction temperature and the holding time values are given in Table 1.

Example 3

(4) The substance was prepared analogously to Example 1, with n=28, m=20, DH=1.8 mole, HH=0.1 mole, the reaction temperature and the holding time values are given in Table 1.

(5) The resulting polymer is non-stereoregular, i.e. the mutual arrangement of alternating components of hydrazine and 1.6-diamine-hexane in the polymer chain may be arbitrary. But the average proportion of these components determined by the proportion of the initial reagents has a constant value in each example.

(6) The antiviral agent substance has a nanostructure.

(7) The nanostructure is determined by the methods of dynamic light scattering using a Malvern Instruments Nanosizer Nano-ZS particle size analyzer and a FEI Tecnai G212Cryo12 transmission electron microscope configured to cool the samples to the boiling point of liquid nitrogen.

(8) Using the dynamic light scattering method it was found that the solution contains globules having the size of 10-15 nm at the preparation concentration of 0.05 mg/ml. An electron-microscopic examination also revealed globules having the size of 10-15 nm.

(9) The following are examples of application of the present antiviral agent.

Example 1

(10) The study of antiviral action on a non-enveloped RNA containing poliovirus that causes poliomyelitis belonging to the Picornaviridae family causing human diseases.

(11) The antiviral agent was used in the form of a 1.0% aqueous solution. The virus dwell time with the solution was 0.5-2.0 minutes at the temperature of 202 C.

(12) Human viruses were grown in cell culture. Antiviral activity was determined by the method of inactivation on the surface of artificial leather. A neutralizer (bovine serum) was used in the experiments. Virus replication in the cells was evaluated by virus-induced cytopathic effect by the degree of inhibition of the infectious virus titer, measured in lg TCID.sub.50 (50% tissue cytopathic infectious dose). To work with the poliomyelitis virus a passaged culture of kidney cells of green monkey Vero was used.

(13) The findings on virucidal activity of the solution during treatment of the test objects infected with poliomyelitis virus with a 1.0% solution of the antiviral agent, are presented in Table 2.

(14) Thus, the antiviral agent exhibits virucidal activity against non-enveloped virus containing RNA.

Example 2

(15) The study of antiviral action of the antiviral agent on non-enveloped DNA containing adenoviruses. Adenoviruses of various serotypes cause infectious diseases in humans and animals (cattle, birds, sheep, dogs). The infectious diseases in animals caused by adenoviruses are characterized by lesions of the mucous membranes of the respiratory organs, intestines, eyes and lymphoid tissue.

(16) To work with the adenovirus the transplantable human cell line HeLa was used. The reproduction of viruses in cells was evaluated by virus-induced cytopathic effect by the degree of inhibition of the infectious virus titer, measured in lg TCID.sub.50.

(17) The antiviral agent was used in the form of a 0.5% aqueous solution. The virus dwell time with the solution was 0.5-2.0 minutes at the temperature of 202 C.

(18) The findings on virucidal activity of the antiviral solution during treatment of the test objects infected with adenoviruses with a 0.5% solution, are presented in Table 3.

(19) Thus, the tested antiviral solution exhibits virucidal activity against non-enveloped DNA-containing adenoviruses of humans and animals.

Example 3

(20) The study of antiviral action of the antiviral agent on enveloped DNA containing herpes simplex viruses. Herpes viruses of various serotypes cause infectious diseases in humans and animals. Infections of these viruses manifest in lesions of mucosae, skin, malignant transformation of cells.

(21) To work with the herpes virus a passaged culture of kidney cells of green monkey Vero was used. The virus reproduction in cells was evaluated by virus-induced cytopathic effect by the degree of inhibition of the infectious virus titer, measured in lg TCID.sub.50.

(22) The antiviral agent was used in the form of a 1.0% aqueous solution. The virus dwell time with the solution was 0.5-4.0 minutes at the temperature of 202 C.

(23) The findings on virucidal activity of the antiviral solution during treatment of the test objects infected with herpes simplex virus with a 1.0% solution, are presented in Table 4.

(24) Thus, the tested antiviral solution exhibits virucidal activity against enveloped DNA-containing viruses.

Example 4

(25) The study of antiviral action on an enveloped RNA-containing virus of hepatitis C, belonging to the Flaviviridae family and causing diseases in humans, dogs and primates.

(26) The antiviral agent was used in the form of a 0.5% aqueous solution. The virus dwell time with the solution was 0.5-4.0 minutes at the temperature of 202 C.

(27) The viruses of hepatitis C were grown in cell culture. Antiviral activity was determined by the method of inactivation on the surface of artificial leather. A neutralizer (bovine serum) was used in the experiments. The virus reproduction in cells was evaluated by virus-induced cytopathic effect by the degree of inhibition of the infectious virus titer, measured in lg TCID.sub.50. To work with the hepatitis C virus the pig embryo kidney cell culture (PEK) was used.

(28) The findings on virucidal activity of the antiviral solution during treatment of the test objects infected with hepatitis C virus with a 0.5% solution, are presented in Table 5.

(29) Thus, the tested antiviral solution exhibits virucidal activity against enveloped RNA-containing virus of hepatitis C.

Example 5

(30) The study of antiviral action of the antiviral agent on enveloped RNA-containing human immunodeficiency viruses. Human immunodeficiency viruses of various serotypes cause infectious diseases in humans and animals (monkeys).

(31) To work with the human immunodeficiency virus human lymphoblastoid MT-4 cells were used. The virus reproduction in cells was evaluated by virus-induced cytopathic effect by the degree of inhibition of the infectious virus titer, measured in lg TCID.sub.50.

(32) The antiviral agent was used in the form of a 1.0% aqueous solution. The virus dwell time with the solution was 0.5-4.0 minutes at the temperature of 202 C.

(33) The findings on virucidal activity of the antiviral solution during treatment of the test objects infected with the human immunodeficiency virus with a 1.0% solution, are presented in Table 6.

(34) Thus, the tested antiviral solution exhibits virucidal activity against enveloped RNA-containing virus of human and animal immunodeficiency.

Example 6

(35) The study of antiviral action of the antiviral agent on an enveloped RNA-containing influenza A virus causing diseases in humans and animals (birds, pigs, horses).

(36) The antiviral agent was used in the form of a 0.5% aqueous solution. The virus dwell time with the solution was 0.5-4.0 minutes at the temperature of 202 C.

(37) The viruses were grown in cell culture. Antiviral activity was determined by the method of inactivation on the surface of artificial leather. A neutralizer (bovine serum) was used in the experiments. The virus reproduction in cells was evaluated by virus-induced cytopathic effect by the degree of inhibition of the infectious virus titer, measured in lg TCID.sub.50. To work with the influenza A virus canine kidney cells (MDCK) were used.

(38) The findings on virucidal activity of the antiviral solution during treatment of the test objects infected with influenza A virus with a 0.5% solution, are presented in Table 7.

(39) Thus, the tested antiviral solution exhibits virucidal activity against enveloped RNA-containing influenza virus causing diseases in humans and animals.

Example 7

(40) The Antiviral Agent's Activity Against Bacterial Viruses (Bacteriophages)

(41) A commercial complex of bacterial viruses used for treating intestinal infections caused by Shigella, Escherichia, Salmonella Proteus, Pseudomonas Staphylococcus bacteria genera was used for the study.

(42) The testing was performed on Shigella flexneri 2a VT-13-678. P. aeruginosa VT-900, P. vulgaris VT-12-445 S. aureus VT-209 strain. Bacteriophages were placed into a 0.5% solution of the antiviral agent for 60 seconds, deposited on Millipore filters, washed with isotonic sodium chloride solution, after which the bacteriophages were washed away and their titer was determined by the agar layers method.

(43) Thus, a complete inactivation of the used mixture of viruses-bacteriophages takes place in one minute.

(44) The results of the bacterial viruses treatment with the antiviral agent are shown in Table 8.

Example 8

(45) The vast majority of plant viruses are non-enveloped RNA-containing viruses. So is the Potato Virus X used in the study (Potato virus X, PVX).

(46) The potato viruses were placed into a 1.0% solution of the antiviral agent for 60 seconds, deposited on Millipore filters, washed with isotonic sodium chloride solution, after which the viruses were washed away, the cells were prepared and infected under the conditions of a microclonal propagation in vitro.

(47) During the first cutting, an analysis for incidence of virus by the enzyme immunodetection method with fixation of the analyze results by photometer was carried out. The results showed that treatment of the Potato Virus X with the antiviral agent completely inhibits viral infection.

(48) The test results showed that the claimed solution possesses a virucidal activity against various, including unrelated non-enveloped and enveloped, DNA and RNA-containing viruses in human, animals, plants and bacteria.

(49) The results on incidence of the virus are shown in Table 9.

(50) The results showed that treatment of the Potato Virus X with the antiviral agent completely inhibits viral infection.

(51) The test results showed the claimed solution possesses a virucidal activity against various, including unrelated non-enveloped and enveloped, DNA and RNA-containing viruses in human, animals, plants and bacteria.

INDUSTRIAL APPLICABILITY

(52) The invention is implemented using common materials and equipment, resulting, according to the applicant's opinion, in compliance of the invention with the Industrial Applicability (IA) criterion for patentability.

(53) TABLE-US-00001 TABLE 1 Characteristics of the substance preparation according to Example 3. Reaction Average molecular temperature, Reaction weight of the product, Elemental analysis data, % Ex. No. C. time, h (formula) C H N C 1 190 0.5 2544 44.71 8.83 25.48 20.98 (C.sub.95H.sub.224Cl.sub.15N.sub.46) 2 210 1 13445 45.0 8.79 25.17 21.08 (C.sub.505H.sub.1171Cl.sub.80N.sub.241) 3 210 4.5 26769 45.03 8.77 25.08 20.09 (C.sub.1010H.sub.2339Cl.sub.160N.sub.481)

(54) TABLE-US-00002 TABLE 2 The findings on virucidal activity of the solution during treatment of the test objects infected with the poliomyelitis virus with a 1.0% solution of the antiviral agent. Disinfection Degree of inhibition, Method of Test time, min lg TCID.sub.50 treatment Artificial leather 0.5 3.2 Wiping 1.0 4.0 2.0 4.5 2.5 2 times 4.5

(55) TABLE-US-00003 TABLE 3 The findings on virucidal activity of the antiviral solution during treatment of the test objects infected with the adenoviruses with a 0.5% solution. Disinfection Degree of inhibition, Method of Test time, min lg TCID.sub.50 treatment Artificial leather 0.5 3.4 Wiping 1.0 4.0 2.0 4.0 1.5 2 times 4.0

(56) TABLE-US-00004 TABLE 4 The findings on virucidal activity of the antiviral solution during treatment of the test objects infected with herpes simplex virus with a 1.0% solution. Disinfection Degree of inhibition, Method of Test time, min lg TCID.sub.50 treatment Suspension test 1.0 1.0 Mixing, virus:solution (1:9) Artificial leather 0.5 2.0 Wiping 1.0 2.5 2.0 3.0 1.5 2 times 4.0

(57) TABLE-US-00005 TABLE 5 The study of virucidal activity of the solution in the course of treatment of the test objects infected with the hepatitis C virus with a 0.5% solution. Disinfection Degree of inhibition, Method of Test time, min lg TCID.sub.50 treatment Suspension test 0.5 3.5 Mixing, 1.0 4.5 virus:solution (1:9) Artificial leather 1.0 4.2 Wiping

(58) TABLE-US-00006 TABLE 6 The study of virucidal activity of the solution in the course of treatment of the test objects infected with the human immunodeficiency virus with a 1.0% solution. Disinfection Degree of inhibition, Method of Test time, min lg TCID.sub.50 treatment Suspension test 1.0 3.5 Mixing, virus:solution (1:9) Artificial leather 0.5 4.0 Wiping 1.0 4.0 2.0 4.5 1.5 2 times 5.0

(59) TABLE-US-00007 TABLE 7 The study of virucidal activity of the solution in the course of treatment of the test objects infected with the influenza A virus with a 0.5% solution. Disinfection Degree of inhibition, Method of Test time, min lg TCID.sub.50 treatment Suspension test 1.0 4.0 Mixing, virus:solution (1:9) Artificial leather 1.0 4.2 Wiping 2.0 4.7 1.5 2 times 5.0

(60) TABLE-US-00008 TABLE 8 Treatment of bacterial viruses with the antiviral agent. Virus titer Virus titer Virus titer before in 30 seconds in 60 seconds Test microbe treatment of exposure of exposure Shigella flexneri 2a 10.sup.5/ml 10 0 P. aeruginosa VT-900 10.sup.5/ml 10 0 P. vulgaris VT-12-445 10.sup.4/ml 0.5 0 S. aureus VT-209 10.sup.6 ml 40 0

(61) TABLE-US-00009 TABLE 9 Incidence of the virus. Absorbance at 490 nm Test plant 7 days 14 days Positive control 0.09 0.850 Negative control 0.07 0.09 Antiviral agent 0.07 0.08