JPS63183502A - Virus inactivating agent - Google Patents

Abstract

PURPOSE:To obtain an inactivating agent for causative viruses of diseases in livestock animals, etc., containing specific polyhexamethylenebiguanidines as an active ingredient and capable of effectively inactivating viruses without affecting humans, animals as well as plants and effectively preventing viral diseases. CONSTITUTION:An inactivating agent containing polyhexamethylenebiguandine having repeating units expressed by the formula, about 700-1,300, preferably 900-1,100 average molecular weight and antimicrobial activity against bacteria or salt thereof, e.g. hydrochloride, etc., as an active ingredient. A carrier or adjuvant is added to the above-mentioned active ingredient to prepare a solution agent or dust, which is used by directly spraying or applying to livestock animals, barns thereof, tools used therefor, instruments, materials, clothes, hands fingers, etc., ingredient is normally 0.01-30wt.%. The agent is useful for herpesviruses, coronatiruses, reoviruses, poxviruses, etc., as the above-mentioned viruses. Furthermore, cytotoxicity to human or animal dermal cells is low.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to an agent for inactivating disease-causing viruses that is harmless to humans, animals and plants.

Problems to be solved by conventional techniques and inventions Various human and animal diseases caused by viruses are known, but once they occur, there is no effective treatment. . Therefore, in many cases, humans and animals are vaccinated to prevent viral diseases.

However, vaccination alone is not necessarily sufficient prevention. The reality is that most people wash and disinfect their clothes, hands, and fingers, and no inactivating agent has been found to be effective against viruses that cause viral diseases in humans and animals.

In view of these circumstances, the present invention utilizes a compound known per se (U.S. Pat. No. 2,643,232) that has antibacterial activity against general bacteria (see Japanese Patent Publication No. 55-42611 and Japanese Patent Application Laid-Open No. 59-101425). and same No. 34285
This invention was developed based on the knowledge that polyhexamethylene biganadine or its salts (see specifications of No. 76) have an excellent inactivating effect on viruses that cause viral diseases in humans and animals. be.

Means for solving the problem, that is, the present invention, has a repeating unit represented by the formula (1) (CHJe NHCNHCNH (1) NHNH
The present invention relates to a virus inactivating agent containing polyhexamethylene biganadine No. 1300 or a salt thereof as an active ingredient.

The average molecular weight of polyhexamethylene biganadine is approximately 70
0-1300, preferably 900-1100.

Salts of polyhexamethylene biganadine include inorganic acid salts such as hydrohalides, sulfates, and nitrates, or lactates, tartrates, succinates, maleates, malonates, and gluconates. An example is an organic acid salt of

The preparation form of the virus inactivating agent containing polyhexamedylene biganadine or a salt thereof according to the present invention as an active ingredient is not particularly limited, but it is usually prepared using water or an organic solvent such as an aliphatic alcohol, glycol, or glycol ether. A solution or fine powder using a solvent such as kaolin,
Diatomaceous earth, talc? It is prepared as a powder using bentonite, calcium carbonate, powdered magnesium, etc. as a diluent.

The concentration of polyhexamethylene biganadine or its salt is usually about 0.01 to 30% by weight, preferably 0.03 to 30% by weight.
It is 20% by weight.

The virus inactivating agent according to the present invention may contain conventional additives such as wetting agents, dispersing agents, emulsifying agents, suspending agents, etc., if desired. Alternatively, it is possible to use it in combination with other antifungal agents, antibacterial agents, and antiviral agents.

Such solutions or powders are generally used by spraying or scattering them directly onto livestock animals, livestock stands, tools and equipment used for these, or the clothes and hands of workers.
Furthermore, in the case of a solution, it may be used as a cleaning liquid or the like. Such treatment can inactivate viruses existing in humans, animals, livestock stands, tools, equipment, etc., and can prevent virus infection from the outer ring.

The amount of the virus inactivating agent according to the present invention is not particularly limited, but it is usually about 100 to 50,000 times more polyhexamethylene biganadine or its salt, preferably 5 times more.
It may be diluted 00 to 20,000 times and used as appropriate. For example, in the case of an aqueous solution, an inactivating agent of IQ or higher is usually sprayed or washed 1 to 10 times per livestock stand L1 every day during the breeding period of livestock animals.

Viruses that can be targeted by the inactivating agent of the present invention include avian infectious laryngotracheitis virus, Marek's disease virus, avian infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus,
Viruses that cause diseases in chickens such as chicken encephalomyelitis virus, viruses that cause diseases in pigs such as swine infectious gastroenteritis virus, Aujeszky's disease virus, swine parvovirus, and influenza virus, bovine infectious rhinotracheitis virus, and newborns. Viruses that cause diseases in cattle include bovine diarrhea coronavirus, parainfluenza virus, and Akabane virus. In particular, the inactivating agent of the present invention has an excellent inactivating effect on herpesvirus, coronavirus, reovirus, poxvirus, or paramyxovirus. Infectious bronchitis virus, porcine infectious gastroenteritis virus, Aujeski disease virus, bovine infectious rhinotracheitis virus, neonatal calf diarrhea coronavirus, infectious bursal disease virus, fowlpox virus, Newcastle disease virus, cattle It is particularly effective against parainfluenza viruses.

Hereinafter, the present invention will be explained by examples.

Example 1 Hydrochloride of polyhexamethylene biganadine (average molecular weight 900-1100) was diluted 10,000 times with phosphate buffer and 2
Diluted 0,000 times and 40,000 times, mixed 0.21 dilution of ILT virus CE strain into this solution 2xQ, and incubated at 20°C.
5 minutes, 15 minutes, 30 minutes after contact.
After 60 and 120 minutes, the solutions 0 and IrnQ were inoculated into tissue-cultured chicken kidney cells. After culturing at 37°C for 4 days, cytopathic effects were observed and 50% infectious dose (T Cr
D5o) was determined by the R66d-Muench method. The results are shown in Table 1 and Figure 1. In the figure, (+),
(2) and (3) respectively show a 10,000-fold diluted area, a 20,000-fold diluted area, and a 40,000-fold diluted area of polyhexamethylene biganadine hydrochloride, and (4) shows a control area.

Table 1 *Dilution ratio of polyhexamethylene biganadine hydrochloride Example 2 HVT virus O1 strain was diluted with loM (l of phosphate buffer (+, 2x 1O6PFU/12)L, and this solution was used as the stock solution. , l01100,1000.1 in phosphate buffer
It was diluted 0,000 times.

0.2 drops of this virus solution was mixed with polyhexamethylene biganadine hydrochloride (average molecular weight 900-11).

00) diluted solution for 15 minutes or 60 minutes -8
= Sensitized. The dilution factor of polyhexamethylene biganadine hydrochloride is 1250.2500.5000.1000
0 times, and a phosphate buffer solution of 21 was used as a control.

After the action, dilute 100 times with phosphate buffer and tissue culture medium,
0 and 1 yttQ were inoculated into cultured chicken fetal fibroblast cells, the cytopathic effect was observed, and the 50% infectious dose (TC
ID5o) was determined by the Reed-Muench method.

The results are shown in Table-2.

*Dilution ratio of polyhexamethylene biganadine hydrochloride Example 3 Polyhexamethylene biganadine hydrochloride (average molecular weight 900-1100) was diluted 10,000 times with phosphate buffer, and ILT was added to 2 mQ of this solution. Dilution of virus SPL strain (103,6T CI Dsolo.

111112) 0,2 Add yQ, 20°C, 15
sensitized for minutes. 0.2 frogs of this sensitization solution were taken and added to 1.8 degrees of phosphate buffer to obtain a 10-fold dilution of the sensitization solution. Further, 0.2 mQ of this 10-fold dilution was taken and added to 1.8 mQ of phosphate buffer to obtain a 100-fold dilution of the sensitizing solution.

0°1if2 from the sensitizing solution and its diluted solution, respectively.
was taken and inoculated into tissue-cultured chicken kidney cells.

The cells were cultured at 37°C, and the cytopathic effect due to virus infection was observed for 10 days. In addition, phosphate buffer 2i (!I L
Diluted solution of T virus SPL strain (lo3゜8TCID5o
10. IJ! 12) Add 0.2JI12 and heat at 20℃, ■5
After sensitization for a minute, the same procedure was performed as a control.

The results are shown in Table-3.

Table 3 * Dilution ratio of polyhexamethylene biganadine hydrochloride 1; No cell degeneration due to virus infection +; Cell degeneration due to virus infection observed (The number in parentheses on the upper right is the number of culture days in which cell degeneration was observed. ) From these results, it was proved that the virus inactivating effect of polyhexamethylene biganadine hydrochloride is irreversible and that this compound has a virucidal effect.

Example 4 A 20% by weight solution of polyhexamethylene biganadine hydrochloride (average molecular weight 900-1100) was diluted with redistilled water to 1000% by weight.
．． Diluted 2000, 4000 and 8000 times.

Add herpes virus type II (S) to this solution 2H.
Mix 0 and 2 mQ of virus solution from A■) and let stand at 20°C. In addition, 2 mQ of double-distilled water was treated in the same manner as a control.

Samples were taken from each sensitization solution after 15, 30, 45 and 60 minutes, and a break assay (p1aque
assay), the amount of viable virus was quantified and expressed as the amount of virus reduction (%). The results are shown in Table 4 and Figure 2.

*20% by weight of polyhexaphthylene biganadine hydrochloride
Solution dilution factor: 11 - Example 5 A 20% by weight solution of polyhexamethylene biganadine hydrochloride (average molecular weight 900-1100) was diluted with phosphate buffer at 50% by weight.
Dilute 0x and 1000x, mix 0 and 2 mQ of TGE virus KB strain virus solution with this solution, and add 20 mQ of virus solution of TGE virus KB strain.
The cells were placed in a constant temperature bath at 0.degree. C. and sensitized for 3 hours. In addition, 2yttQ was taken as a phosphate buffer and operated in the same manner as a control.

After the action, a 100-fold dilution with phosphate buffer and tissue culture medium was inoculated into porcine kidney cell line LLC-PK, cultured for 4 days, and the presence or absence of cytopathic effect was observed. Table 5 shows the results.
Shown below.

Table-5 *20% by weight of polyhexamethylene biganadine hydrochloride
Liquid dilution factor +: Cytopathic effect -: No cytopathic effect Example 6 A 20% by weight solution of polyhexamethylene biganadine hydrochloride (average molecular weight 900-1100) was diluted with double-distilled water,
When 0.5 Mff of the virus solution is added to 1.5 mC of the drug dilution solution, the drug is diluted 500 times.

Virus solutions 0 and 5 of IBD virus PK-78 strain were mixed with 15 mQ of the drug dilution solution, and the mixture was allowed to stand at 20°C for 15 minutes of sensitization. In addition, double distilled water was taken for 1.5 hours and operated in the same manner as a control. After the action, a 100-fold dilution with phosphate buffer and tissue culture medium was inoculated into primary chicken embryo fibroblast cells, cultured for 7 days, and the presence or absence of cytopathic effect was observed. The results are shown in Table-6.

Table-6 *Polyhexamethylene biganadine hydrochloride 20% by weight
Diluted solution 12 Cytopathic effect: No cytopathic effect Example 7 Effect on fowlpox virus 20% by weight solution of polyhexamethylene biganadine hydrochloride (average molecular weight 900-1100) Dilute 500 times with double-distilled water, add fowlpox virus Nakan to 2 mQ of this solution.
o K ■ Mix strain virus solution 0, 2 x Q20
The cells were left standing at ℃ and sensitized for 15 minutes. Also, add double distilled water
rnQ was taken and operated in the same manner as a control.

After the action, a 100-fold dilution with phosphate buffer and tissue culture medium was inoculated into primary chicken embryo fibroblast cells, cultured for 5 days, and the presence or absence of cytopathic effect was observed. The results are shown in Table-7.

Table-7 *Polyhexamethylene biganadine hydrochloride 20% by weight
Diluted solution +: Cytopathic effect was observed ~: Cytopathic effect was not observed Example 8 A 20% by weight solution of polyhexamethylene biganadine hydrochloride (average molecular weight 900-1100) was added to a 15% aqueous ethanol solution. It was diluted 500 times. This solution 2111Q1.
: Virus fluid of ND virus TCND strain 0, 211 (
1 was mixed, left to stand at 20°C, and sensitized for 15 minutes, 30 minutes, and 1 hour.

After the action, a 100-fold dilution with phosphate buffer and tissue culture medium was inoculated into vero cells (African green monkey kidney cell line), cultured for 4 days, and the presence or absence of cytopathic effects was observed. When sensitized for 1 hour, no cytopathic effect occurred.

Example 9 The cytotoxicity of polyhexamethylene biganadine hydrochloride used in the present invention on skin cells was compared with that of a commercially available virus inactivator.

A 20% by weight aqueous solution of polyhexamethylene biganadine hydrochloride was diluted 1000 times and 2000 times with phosphate buffer and cell culture medium.
Test solutions were prepared by diluting the solution 1:4,000 and 8,000 times.

In addition, as a typical commercially available virus inactivator, a commercially available virus containing methyldodecylbenzyltrimedylammonium chloride, methyldodecylxylylene bistrimethylammonium chloride (50% liquid) 20g, and polyoxyethylene octylphenyl ether 5g in 100xQ is used. Add inactivating agent too much using phosphate buffer and cell culture medium.

Comparative test solutions were prepared by diluting the solution 2000 times, 4000 times, and 8000 times. Both wave detection was inoculated into human skin cell line NCTC2544.

After inoculation, culture was continued at 37°C, and after 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, and 6 hours, the culture medium in each incubator was removed and the cells were stained with trypan blue. was performed and subjected to microscopic examination. The total number of cells and the number of dead cells within a certain range were counted, and the cell survival rate (%) was calculated using the following formula.

A similar test for total cell count was conducted using a test solution (control) that did not contain a virus inactivator. The results are shown in Table-8.

Further, the results of 1000-fold dilution and 2000-fold dilution are shown in FIGS. 3 and 4, respectively.

Reference photo 1 (control) and reference photo 2 (polyhexamethylene biganadine hydrochloride) are micrographs of human skin cell lines stained with trypan blue after being in contact with a test solution 1000 times stronger than the virus inactivating agent for 1 hour. salt) and Reference Photo 3 (commercially available virus inactivator).

Dead cells are stained blue, live cells are unstained.

Example IO In the same manner as in Example 9, 10,000 ml of virus inactivating agent was prepared.
A doubly diluted test solution was prepared, human skin cell lines were brought into contact with it, and 24 hours later, the cultured cells were fixed with methanol and Giemsa staining was performed using a conventional method.

The micrographs are shown in Reference Photo 4 (control), Reference Photo 5 (polyhexamethylene biganadine), and Reference Photo 6 (commercially available virus inactivating agent).

Reference photos 4 to 6 show that polyhexamethylene biganadine has almost no skin toxicity.

Effects of the Invention The virus inactivating agent according to the present invention effectively inactivates viruses without adversely affecting humans, animals and plants, and effectively prevents viral diseases. In particular, it has low cytotoxicity to human or animal skin cells.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between the contact time at 20° C. (horizontal axis) and TCID6° (vertical axis) between the ILT virus and polyhexamethylene biganadine hydrochloride in Example 1. FIG. 2 is a graph showing the effect of polyhexamethylene biganadine hydrochloride on herpesvirus type II. Figures 3 and 4 are graphs showing the cytotoxicity of polyhexamethylene biganadine hydrochloride on skin cells.
000 times dilution area, (2) is the same <20000 times dilution area,
(3) is the same <40,000 times diluted area, (4) is the control area,
(5) is 1000 times diluted area, (6) is 2000 times diluted area, (7) is 4000 times diluted area, (8) is 8000 times diluted area, (9) is polyhexaphthylene biganadine hydrochloride,
(lO) indicates a commercially available virus inactivating agent. Patent jaw person Ueno Pharmaceutical Co., Ltd. Applied Research Agent Attorney Hiro Aoyama and 2 other patent attorneys Figure 1 ■ (

Claims (1)

[Claims] 1. Formula (I): ▲There are mathematical formulas, chemical formulas, tables, etc.▼An average molecular weight of approximately 700 to 700 having a repeating unit represented by (I)
A virus inactivating agent containing 1300 polyhexamethylene biganadine or a salt thereof as an active ingredient. 2. The average molecular weight of polyhexamethylene biganadine is 9
00-1100, the inactivating agent according to item 1. 3. The inactivating agent according to item 1, wherein the virus is a virus that causes disease in livestock animals. 4. The inactivating agent according to item 1, wherein the virus is a herpesvirus, coronavirus, reovirus, poxvirus or paramyxovirus.