JPH0977670A - Antiviral agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antiviral agent capable of inhibiting the proliferation of pathogenic virus and preventing non-infected cells from infection. SOLUTION: This antiviral agent contains oligodeoxynucleotide or polydeoxynucleotide equal to the partial structure of a DNA sequence hybridizable with a messenger RNA (m-RNA) to be developed in the proliferation of pathogenic virus. The oligodeoxynucleotide or polydeoxynucleotide is prepared, for example, by cloning a viral gene and then by fragmentation of the single strand of the resultant DNA. Besides, in order to reproduce a large amount of the cloned DNA, Escherichia coil possessing a phase or plasmid integrated with a viral gene is cultured by any conventional method in an appropriate volume of a culture fluid added, at need, with antibiotics, followed by collecting the cultured bacteria and then separating a DNA containing the cloned viral gene.

Description

Detailed Description of the Invention

The present invention relates to antiviral agents. More specifically, the present invention is characterized by containing an oligodeoxynucleotide or polydeoxynucleotide equivalent to a partial structure of a DNA sequence capable of hybridizing to messenger RNA generated during the growth of a pathogenic virus. It is related to agents. The present inventor has previously found that when an oligodeoxynucleotide or polydeoxynucleotide equivalent to a partial structure of a DNA sequence capable of hybridizing to a messenger RNA generated during the growth of a virus is supplied to the location of the messenger RNA, the virus grows. Based on this finding, a virus growth inhibition method was provided (Japanese Patent Application No. 58-19235).
No. 0), and as a result of repeated experiments in which the above-mentioned oligonucleotide or polydeoxynucleotide was applied to a virus-infected animal, the antiviral agent according to the present invention was provided. That is, as a means for carrying out the above-described method for inhibiting virus growth provided by the present inventor, the present invention provides oligos having a partial structure of a DNA sequence capable of hybridizing to messenger RNA generated during virus growth. The present invention provides an antiviral agent characterized by containing deoxynucleotides or polydeoxynucleotides.

The present invention is described in detail below. The present inventor previously used this RNA to artificially synthesize this RN.
Discovered the fact that by adding an oligodeoxynucleotide equivalent to a partial structure of a DNA sequence capable of hybridizing to A directly to a cell-free protein synthesis system, a protein that should be synthesized based on this RNA is not synthesized. (See Experimental Example 1 below). In addition, in this case, it was confirmed that when a similar experiment was performed using an oligodeoxynucleotide having a DNA sequence that could not hybridize to this RNA, a protein based on this RNA was synthesized. Then, the inventor
Α-globin messenger RNA existing in eukaryotic cells
Experiments were carried out to confirm whether or not they inhibit the production of α-globin and β-globin using various DNAs under physiological conditions using β and β-globin messenger RNA (see Experimental Example 2 described below).

As a result, α-globin messenger RN
Direct addition of a DNA capable of hybridizing to A to a cell-free protein synthesis system inhibits production of α-globin, and β-globin messenger RN
It was found that the addition of DNA capable of hybridizing to A directly to the cell-free protein synthesis system inhibits β-globin production. In addition, DN that cannot hybridize to α-globin messenger RNA
When A is used, the production of α globin is not inhibited,
It was also confirmed that the use of DNA that cannot hybridize to β-globin messenger RNA does not inhibit β-globin production.

Furthermore, in this experimental system, it was found that when the DNA used was an oligodeoxynucleotide, a high protein production inhibition rate was exhibited. Based on the results of these experiments, the messenger RN formed in the early stage of herpes simplex virus infection
When oligodeoxynucleotides that form a double chain with A were administered to herpes simplex virus-infected cells, syncytium of cells due to herpes simplex virus (plaque holes were formed in the cells on the culture Petri dish due to cell fusion) ) Was significantly inhibited.

In the above experiment, calcium chloride was used to enhance the uptake of oligodeoxynucleotide into cells. However, even when calcium chloride is not used, the oligodeoxynucleotide is taken up into cells and the cell culture system is used. It was confirmed that the growth of herpes simplex virus was inhibited (see Experimental Example 3 below). These experiments demonstrate that D capable of hybridizing to the messenger RNA of herpes simplex virus.
Although it was revealed that the direct addition of NA to herpes simplex virus-infected cells effectively inhibited the growth of herpes simplex virus, the present inventor further based on this fact, the messenger RNA of herpes simplex virus. On the other hand, when a hybridizable DNA was applied to a virus-infected mouse and a therapeutic test for a viral disease was conducted, it was confirmed that it could be used as an antiviral agent. The present invention has been made based on such findings.

The antiviral agent of the present invention is a DNA corresponding to the nucleotide sequence of messenger RNA derived from a virus.
In order to use an oligodeoxynucleotide or polydeoxynucleotide equivalent to the partial structure of the sequence, it is
It does not hybridize with messenger RNAs derived from other genes and thus the use of the antiviral agents of the invention does not affect protein synthesis by other genes.

Viruses include DNA virus (herpes virus, adenovirus, vaccinia virus, etc.) and R virus.
It is classified into NA virus (influenza virus, rhinovirus, poliovirus, etc.). RNA viruses include double-strand viruses and single-strand viruses, and single-strand viruses further include positive-strand and negative-strand viruses. In any case, viral protein synthesis is based on positive-strand RNA as messenger RNA.
Without the existence of. That is, in the double-stranded DNA virus, positive-strand RNA is produced from negative-strand DNA to become messenger RNA, and in the double-stranded RNA virus, positive-strand RNA is produced from negative-strand RNA. Then, it becomes messenger RNA. Among the single-stranded RNA viruses, the minus-strand RNA virus produces plus-strand RNA based on the minus-strand RNA, which results in messenger RNA.
Therefore, the positive-strand RNA virus itself acts as a messenger RNA. In the present invention, by using oligodeoxynucleotides or polydeoxynucleotides having the same partial structure of DNA as any messenger RNA of each virus, which is capable of forming a hybrid with a plus-strand RNA as messenger RNA, Anti-viral agents against the virus are provided.

In the present specification, the double-stranded DN is as follows.
The herpes simplex virus, which is A virus, will be described as an example. The antiviral agent of the present invention includes influenza virus, adenovirus, leukemia virus, dengue virus, rabies virus, hepatitis virus, measles virus, encephalitis virus, and parainfluenza. Applicable to pathogenic viruses such as viruses, Newcastle virus, poliovirus, rhinovirus, yellow fever virus, and EB virus. Therefore, the present invention can be widely used for treatment and prevention of various diseases derived from animal and plant viruses, and is thus extremely useful in various fields.

By using the antiviral agent of the present invention, DN
It is possible to stop the growth of the virus and prevent the infection of non-infected cells, regardless of A virus or RNA virus, without affecting the non-infected cells of the living body in which the virus is present. Regarding the effect of oligodeoxynucleotides or polydeoxynucleotides used in the antiviral agent of the present invention on normal cells, the present inventor has proposed a single-stranded DNA capable of hybridizing with the messenger RNA of the immediate early gene of herpes simplex virus.
The effect on hamster embryonic kidney cells was examined using a deoxynucleotide having the nucleotide sequence of 1., and the deoxynucleotide did not affect the survival of normal cells and did not affect the proliferative capacity thereof. It's known. Furthermore, during the therapeutic experiment on herpes simplex virus-infected mice, no change was observed in the behavior and appearance of the deoxynucleotide-administered group having a single-stranded DNA base sequence capable of hybridizing with the messenger RNA of the immediate early gene.

The oligodeoxynucleotide or polydeoxynucleotide used in the antiviral agent of the present invention is
As long as it has a desired DNA sequence partial structure such as one obtained by decomposing a single DNA strand of a virus by enzymatic treatment or the like, or one obtained by synthesizing a partial structure of a required DNA sequence, The origin may be any.
Examples of the method for preparing the oligodeoxynucleotide or polydeoxynucleotide used in the antiviral agent of the present invention include the following methods. Thus, one method is to clone the viral gene and fragment the single strand of its DNA.

When cloning a viral gene, in the case of RNA virus, D
It is based on the viral DNA that changes NA or is present in the virus-infected cells. When cloning a viral gene, for example, a method of obtaining a double-stranded DNA using a lambda phage or a plasmid vector such as pBR322, or a method of directly obtaining a single-stranded DNA using M13 phage is used. . The gene to be cloned may be any part of the nucleic acid chain of the gene contained in the virus, but the early (ea) gene that is expressed early during growth is used.
rly) gene or immediate early (immedia
te early) gene is preferably cloned.

In order to replicate a large amount of cloned DNA, for example, a phage into which a viral gene has been incorporated, or Escherichia coli harboring a plasmid, is added in an appropriate volume of a culture medium containing an antibiotic if necessary. The cells are cultivated by a conventional method, the cells are collected, and then the DNA containing the cloned viral gene is isolated. At the time of this separation, DNA can be extracted by treatment with an organic solvent such as phenol or phenol-chloroform.

To remove viral DNA from the separated DNA, it is digested with a restriction enzyme and agarose gel electrophoresis, column chromatography or the like is used. The obtained viral DNA is fragmented if necessary. For this purpose, treatment with a restriction enzyme such as endonuclease or ultrasonic treatment is performed. Fragment chain lengths are typically considered to range from 100 nucleotides to 9 nucleotides. When it is prepared using M13 phage, a direct single-chain fragment is obtained, but in consideration of the properties of the antiviral agent of the present invention, a messenger RNA of a viral gene is previously prepared.
A single-stranded DNA capable of forming a double-stranded DNA must be selected and cloned.

The viral DNA fragment obtained by using the pBR322, λ phage is heated to 100 ° C. for 10 minutes and then rapidly cooled to form a single chain. Since a single strand of the desired DNA fragment is present in the product, it can be used as it is for the antiviral agent of the present invention. However, in this product, a messenger RNA of a viral gene is also included. Since there are non-complementary single chain fragments, it is preferable to remove them by using affinity chromatography or the like.

The oligodeoxynucleotide or polydeoxynucleotide used in the antiviral agent of the present invention can also be produced by organic synthesis. In this case, the base sequence of the oligodeoxynucleotide or polydeoxynucleotide must be a base sequence capable of hybridizing with the base sequence of the messenger RNA of the viral gene. For example, in herpes simplex virus, one of the immediate early DNAs is plus D.
NA (5'-ATG / GCG / TCG / GAG ...) and negative DNA
(3'-TAC / CGC / AGC / CTC ...) forms a double strand. Of these, the DNA sequence on the TAC side (minus strand DNA) that forms a double strand with messenger RNA is selected. To synthesize.

[0016] For example, in the case of influenza virus, there are hemagglutin gene and NS (Non Structual) gene as influenza virus genes, but there are variations in the nucleotide sequence of DNA in order to inhibit virus growth. It is preferable to stop the expression of the NS gene rather than the hemagglutin gene, and it is preferable to select a nucleotide sequence of a single-stranded DNA that can hybridize with the messenger RNA of the NS gene.
For example, NS 1 gene (Lamb et al., Cell 21 475 (198
Taking 0)) as an example, a single-stranded DNA base sequence capable of hybridizing to the messenger RNA of the NS 1 gene, for example, 5 ′ -... ACT / TGA / CAC / AG
T, GTT, GGA, ATC, CAT ...- 3 'is selected to synthesize a nucleotide sequence of an appropriate length.

Furthermore, in the case of viruses related to animal husbandry, for example, Rous avian sarcoma virus, gag.po
l. Inhibits the expression of any gene of env. Here, in the case of inhibiting the expression of the gag gene, for example, considering the nucleotide sequence of P19 which is a part of the gag gene, 5 ′ -... AAT
-CAC / CTT / TAT / GAC / GGC / TTC ...- Select the base sequence of suitable length from the 3'base sequence and synthesize it.

Thus, in the case of any other virus, a single-chain D capable of hybridizing to the messenger RNA of the virus whose growth is to be inhibited
It is possible to select a base sequence having an appropriate length in the base sequence of NA, synthesize an oligodeoxynucleotide or a polydeoxynucleotide having the base sequence, and use the oligo (or poly) deoxynucleotide. Next, the preparation of oligodeoxynucleotides or polydeoxynucleotides used in the antiviral agent of the present invention will be described.

[0019]

[Specific Example] Specific Example 1 DNA obtained by treating herpes simplex virus with an organic solvent of chloroform-phenol was cut with a restriction enzyme, BamHI, and vector TBR phage ligase was added to vector pBR322 which had been cut with the restriction enzyme, BamHI in advance. It was incorporated by using. The obtained plasmid was given to Escherichia coli, and colonies were formed on an agar medium containing ampicillin. From these colonies, those hybridizing to the ³² P-labeled DNA of herpes simplex virus were selected by the filter hybridization method, and 2 liters of TSB (Tryptose Soy Broth) medium containing ampicillin at a concentration of 50 μg / ml was selected. It was cultured in. 10 ml of E. coli containing the above plasmid was added to 1 ml of this medium.
⁶ to 10 ⁷ cells were added, and the cells were cultivated with shaking in a 20 ml test tube at 37 ° C. for 15 hours. When the culture solution was cultured until the absorbance at 540 nm reached 0.5, 10 μg / ml chloramphenicol was added, and the culture was continued for further 15 hours.

The Escherichia coli culture broth thus obtained was centrifuged, 50 mM Tris-hydrochloric acid (pH = 8.0) containing 25% sucrose was added to the pellet, and the mixture was allowed to stand at 0 ° C. or lower for 5 minutes. The viscous liquid produced by adding 4 ml of Triton-X 100 was centrifuged at 30,000 G for 30 minutes at 0 ° C. To 8 ml of this supernatant was added 8 g of CsCl and Ethidium.
Add 1 ml of Bromide (5 μg / ml) and add 100,000.
The crude plasmid fraction produced by centrifugation at G for 48 hours was collected, mixed with 2 volumes of isopropyl alcohol, and the supernatant was removed.
It was dialyzed overnight against EDTA (pH = 8.0). The dialysis solution was concentrated, 200 μg of DNA equivalent was taken, the excess restriction enzyme (BamHI) was added and reacted, and then the herpes simplex virus DN was detected by agarose electrophoresis.
A was separated. This DNA is then partially digested with DNAase, heated at 100 ° C. for 10 minutes, and then rapidly cooled to give a chain length of 9
To obtain oligodeoxynucleotides or polydeoxynucleotides at position 100.

Example 2 A cloned M13 phage replicative double-stranded DNA was cloned into Ba
Cut with mHI. Next, the herpes simplex virus DNA isolated in Example 1 and the cleaved M13
Replicated double-stranded DNA of phage was ligated using T4 phage ligase. This bound DNA was put into 1ac (-) E. coli in the presence of CaCl ₂ by a conventional method,
Phage plaques were made in the presence of X-gal (indicator). Herpes simplex virus DN
M13 phage containing A formed phage plaques that showed no blue color after 24 hours. A plaque that hybridizes with the positive strand of the herpes simplex virus DNA labeled with ³² P (chain length 20 deoxynucleotides or more) was collected. This is because the phage plaques that do not show blue color contain only one of the double-stranded DNAs of the herpes simplex virus on one side. This M13 phage was treated with phenol-chloroform to separate DNA. The DNA obtained above contains herpes simplex virus single-stranded DNA, but may be further purified to remove the DNA of M13 phage contained therein, or the DNAase may be partially decomposed to give a chain length of 9 ~ 100
Position oligodeoxynucleotide or polydeoxynucleotide.

Example 3 Using a DNA synthesizer (manufactured by Applied Biosystem), among the negative DNA of immediate early DNA of herpes simplex virus,
Oligodeoxynucleotides that form double strands with plus DNA (3 '-... TAC / CGC / AGC / CTC / TTG / TTC / GTC /
20 deoxynucleotides (3'-C in GCG ...- 5 ')
GC / AGC / CTC / TTG / TTC / GTC / GC-5 ') were synthesized and purified. Hereafter, this 20 deoxynucleotide
Abbreviated as r. For this 20mer sequence,
It was confirmed by the Maxam-Kilbert method.

Example 4 Using a DNA synthesizer (manufactured by Applied Biosystem), of the oligodeoxynucleotides forming a double strand with the plus DNA among the minus DNA of the immediate early DNA of herpes simplex virus 20 deoxynucleotides (3'-GCA / CCC
-GGG-ACC-TTT-ACC-GC-5 ') was synthesized and purified. Hereinafter, this 20 deoxynucleotide is abbreviated as 20B. This 20B sequence was confirmed by the Maxam-Gilbert method.

The antiviral agent of the present invention is prepared by using the oligodeoxynucleotide or polydeoxynucleotide described above, if appropriate, using appropriate solvents, excipients, adjuvants and the like which are usually used in pharmaceutical preparations. It is prepared as a liquid preparation, an injection preparation, a suppository preparation, etc. according to a conventional manufacturing method. In the formulation, desired oligodeoxynucleotides or polydeoxynucleotides can be used alone or in appropriate combination, and other pharmaceutically active ingredients may be blended.

In these combinations and blends, the components to be combined or blended must not inhibit the antiviral action of the above oligodeoxynucleotides or polydeoxynucleotides. The oligodeoxynucleotides or polydeoxynucleotides described above can be mixed with a suitable base material inactive to them to prepare external preparations such as creams, ointments and poultices.

When prepared as a liquid preparation or an injection, distilled water for injection, physiological saline, an aqueous dextrose solution,
Vegetable oil for injection, propylene glycol, polyethylene glycol and the like can be used. Further, if necessary, a tonicity agent, a solubilizing agent, a stabilizer, an antiseptic agent, a soothing agent, etc. may be added. In addition, in the case of this type of dosage form, it is desirable to dissolve in a sterilized injection medium. It goes without saying that the above-mentioned various bases, additives, adjuvants and the like used in the preparation of the preparation should not inhibit the antiviral action of the above oligodeoxynucleotide or polydeoxynucleotide.

The antiviral agent of the present invention is directly applied to a diseased part of a living body infected with a virus, or is administered into a blood vessel so that the above oligodeoxynucleotide or polydeoxynucleotide results in the affected part. Adapt to the living body so that it can reach. The following is an example of an effect confirmation experiment using the mouse for the antiviral agent of the present invention.

[0028]

[Example]

Example 1- (1) 3 weeks old BALB / C mice with various concentrations of 2
To 30 μl of 0 mer MEM solution, HSV-1 (F strain) (1
X 10 ⁴ PFU) was injected, and 200 μl of a 20-mer MEM solution having various concentrations (containing 1% penicillin and streptomycin and 2 mM glutamine) was intraperitoneally injected into HSV-1 (F strain F). From the next day, administration was performed every day for 3 days, and the mortality rate on the 7th day after injection of HSV-1 (F strain) was examined. As shown in FIG. 1, in the 20mer administration group,
The mortality rate due to HSV-1 was reduced in a concentration-dependent manner. The mortality rate was expressed with the mortality rate of the 20mer non-administered group as 100%.

Example 1- (2) 3 weeks old BALB / C mice with various concentrations of 2
To 30 μl of 0 mer MEM solution, HSV-1 (F strain) (1
X10 ⁴ PFU) is included in HSV-1 (F strain) MEM
30 μl of solution (1 × 10 ⁴ PFU) was injected, and 200 μl of 20 mer MEM solution of various concentrations was intraperitoneally injected into the mouse.
(1% penicillin and streptomycin and 2
Contains mM glutamine. ) Was administered every day for 6 days from the day after the HSV-1 (F strain) injection, and the mortality rate was examined at 64-68 hours after the HSV-1 (F strain) injection. The results are shown in Table 1.

[0030]

[Table 1] As shown in Table 1, the mortality rate in the 20mer non-administered group was 5/1.
9 was shown, whereas no death was observed in the 20mer-administered group.

Example 1- (3) 3 weeks old BALB / C mice with various concentrations of 2
HSV-1 (F strain) (5 ×) was added to 30 μl of a 0-mer MEM solution.
Containing 10 ⁴ PFU) and injecting HSV-1 (F strain)
Mortality was examined at 64 to 68 hours and 71 to 78 hours after the injection. Table 2 shows the results.

[Table 2] As shown in Table 2, the mortality rate in the 20-mer non-administered group was 3/1.
While 0 (64 to 68 hours) and 3/10 (71 to 78 hours) were shown, it was confirmed that the 20mer-administered group had a clearly reduced mortality rate.

Example 1- (4) 3 weeks old BALB / C mice were inoculated with various concentrations of 2
HSV-1 (F strain) (5 ×) was added to 30 μl of a 0-mer MEM solution.
10 ⁴ PFU) was injected but the ones include, containing MEM solution 30 [mu] l (1% penicillin and streptomycin and 2mM glutamine 20mer various concentrations intracerebroventricular the mice.) The HSV-1 (F strain ) Administered daily for 2 days from the day after the injection, and after the injection of HSV-1 (F strain) 64
Mortality was examined at ~ 68 hours, 71-78 hours, and 88-92 hours. The results are shown in Table 3.

[Table 3] As shown in Table 3, the mortality rate in the 20-mer non-administration group was clearly decreased by 88 to 92 hours in the 20-mer administration group.

Example 1- (5) Three weeks old BALB / C mice were intraperitoneally injected with 1.12 μg / m 2.
HSV-1 (F
Strain) (1 × 10 ⁶ PFU) was infused, and 200 MEM solution of various concentrations of 200 was intraperitoneally injected into this mouse.
μl (containing 1% penicillin and streptomycin and 2 mM glutamine) was added to HSV-1 (F.
Strain) was administered 3 hours before and 2 hours before the injection and every day for 3 days from the next day, and the mortality at 211 hours and 259 hours after the HSV-1 (F strain) injection was examined. Table 4 shows the results.

[Table 4] As shown in Table 4, in the 20mer non-administered group, the mortality rate was 1 /
While it was 10 (211 hours) and 2/10 (259 hours), no death was observed in the 20mer-administered group.

Example 2 3 weeks old BALB / C under anesthesia, injured 5 times with a sharp needle
In the mouse cornea, 30 μl of MEM solution (containing 1% penicillin and streptomycin and 2 mM glutamine and 5% calf serum) HSV-1 (F).
Strain) (1 × 10 ⁶ PFU / ml) was added dropwise. The above mice were divided into the following 4 groups, and the appearance was observed.

A. 22.4 μg / ml on the cornea of this mouse
10 μl of MEM solution containing 20 mer of HSV-1
(F strain) 2 hours before the dropping and every 3 days from the next day every other day, on the cornea, and 1 day before the dropping of HSV-1 (F strain), 5 hours before and the next day, every 3 days from the cortisone acetate solution (2 mg / kg) ) A group in which 30 μl was intramuscularly injected. B. A group in which 10 μl of a MEM solution containing 22.4 μg / ml of 20 mer was added to the cornea of this mouse 2 hours before the dropping of HSV-1 (F strain F) and every other day from the next day, three times on the cornea.

C. 10 μm MEM solution was applied to the cornea of this mouse.
1 hour before the dropwise addition of HSV-1 (F strain) to the cornea 3 times every other day from the next day, and 1 day before the HSV-1 (F strain) dropwise, 5 hours before and 3 days after the next day, cortisone acetate 3 times A group in which 30 μl of the solution (2 mg / kg) was intramuscularly injected. D. HSV-added 10 μl of MEM solution to the cornea of this mouse.
1 (F strain) 2 hours before dropping and 3 days after administration every other day
The group dropped on the keratoconus.

Attention was paid to the condition of the eyes of the mice in each group and the rash found around the eyes, and the appearance of the mice was examined on the 4th and 7th days,
Observation on the 12th day revealed that the 20-mer-administered group A and the group B had less rash and had better eye condition than the non-administered groups C and D. At this time, the effect of cortisone administration was not observed on the effect of 20mer administration. From Example 1 and Example 2 above, the antiviral agent of the present invention obviously suppresses the growth of the virus in the affected animal, thereby reducing the mortality rate and the symptom. It turns out to be expensive. Hereinafter, formulation examples of the antiviral agent of the present invention are listed.

[0038]

[Formulation example]

Formulation Example 1 The same 20 mer as obtained in Example 3 was synthesized by the liquid layer method, and 2 g of the 20 mer was added to an ampoule by adding an isotonic MEM solution to 1 liter according to a conventional method for producing an injection. Injectable This injection is 20 in 1 ml
Contains 2 mg of mer. This injection is injected in an amount of 1 to 5 ml once according to the symptoms, as close to the affected area as possible.

Formulation Example 2 The same as 20B obtained in Example 4 was synthesized by the liquid layer method, and 10 g of 20B was added with an isotonic MEM solution to make 1 liter to give a 1% eye drop.

This eye drop may be administered once or twice in accordance with the symptoms.
Drop, instill several times a day. Formulation Example 3 The same 20 mer as obtained in Example 3 was synthesized by the liquid layer method, 1 g of the 20 mer was ground into a powder, and 999 g of purified cocoa butter was added thereto and kneaded in a water bath at 60 ° C. And shape it into 2g suppositories. This suppository contains 2 mg of 20 mer in each suppository and is used according to the symptoms. Next, the safety of the antiviral agent of the present invention will be described with reference to test examples.

[0041]

[Test example]

Test Example 1 BALB / C mice were treated with 100 μg / kg of 20mer i.
p. When administered, no deaths were observed. Test Example 2 In the above-described effect confirmation experiment, in which HSV-1 was not used in Example 2 and the other experiments were performed in the same manner, no difference was observed between the 20mer non-administration group and the 20mer administration group. As shown here, it is clear that the antiviral agent of the present invention is safe at the effective dose of the aforementioned oligodeoxynucleotide or polyoxynucleotide. Below, examples of experiments conducted in connection with the present invention are listed.

[0042]

[Experimental example]

Experimental Example 1 Inhibitory action of polyphenylalanine synthesis reaction using polyuridylic acid as messenger RNA by polyadenylic acid and deoxypolyadenylic acid Polyuridylic acid (140 μg), ¹⁴ C-phenylalanine (10 ⁵ cpm, 300 μCi / μmole), Nierenberg and M
Athaei's method (Proc. Nat. Acad. Sci. 47, 1588 (196
1)) E. coli extract, 13 mM magnesium acetate, 1 mM ATP, 1 mM creatine phosphate, creatine phosphokinase (1 μg) were added to 10 mM Tris-HCl (pH 7.
5) was dissolved in 5) to make a reaction system. No. 5 in this reaction system
Add 40 μl of the inhibitors listed in the table to make a reaction in a 1.5 ml Eppendorf tube at 37 ° C. for 30 minutes, and adjust the amount of polyphenylalanine in the solution obtained by the reaction to 10
The amount of radioprotein insoluble in 95% of trichloroacetic acid was defined as the amount of radioprotein, which was measured using a liquid scintillation counter. The degree of inhibition was calculated by comparing the radioactivity of polyphenylalanine when no inhibitor was added with the radioactivity when added.
Table 5 shows the results.

[0043]

[Table 5] As shown in Table 5, polydeoxyadenylic acid (DNA) corresponding to the base sequence of polyuridylic acid (RNA) showed a remarkable polyphenylalanine synthesis inhibitory activity. In this experiment, the DNA chain showed a strong inhibitory activity up to 9 nucleotides or more of oligodeoxyadenylic acid (oligodeoxynucleotide).

Experimental Example 2 Specific Inhibitory Effect of Globin Synthesis in Rabbit Reticulocyte System by Rabbit Single-Stranded DNA This experimental example uses oligodeoxynucleotides or polydeoxynucleotides that hybridize to messenger RNA of eukaryotic cells. It is an experimental example that is confirmed to inhibit protein synthesis under physiological conditions. 4.2 mM calcium phosphate, 2 mM dimethylthiothreitol (DTT), 0.08 mM amino acid (excluding methionine), 6 mM potassium acetate, 8 mM magnesium acetate,
Spermidine 4.5 μg, ³⁵ S-methionine (0.1 μC
i), and the method of Jackson and Pelham et al. (Eur. Biochem.
67. 247-256 (1976)), cell-free reticulocyte protein synthesis system 10 μl, α and β globin messenger RNA 75 μg each were dissolved in 21 mM HEPES
After adding the inhibitors listed in the table to a total volume of 30 μl,
The reaction was carried out at 30 ° C. for 30 minutes in a 1.5 ml Eppendorf tube. The reaction product in the solution obtained by the above reaction was separated into α-globin, β-globin and other proteins by Triton-Acied-Urea gel, and the radioactivity of each fraction was measured by radioautography. The degree of inhibition was calculated by comparison with the radioactivity when no inhibitor was added. Table 6 shows the results.

[0045]

[Table 6]

As shown in Table 6, α-globin genomic DNA specifically inhibits only α-globin synthesis, and β-globin genomic DNA specifically inhibits only β-globin synthesis. Calf thymus DNA showed little inhibitory activity. From this, the presence of DNA corresponding to the nucleotide sequence of messenger RNA of eukaryotic cells causes the messenger R
It can be seen that NA-based protein synthesis is specifically inhibited. Furthermore, 1 corresponding to the N-terminal amino acid of α-globin
Since 5 oligodeoxynucleotides inhibit only the synthesis of α-globin and 15 oligodeoxynucleotides of M13 phage hardly influence the synthesis of α-globin and β-globin, a DNA chain corresponding to the base sequence of messenger RNA is obtained. It was found that the inhibitory activity and specificity are not lost even when is reduced to 15 nucleotides.

Experimental Example 3 Permeability of 20-mer hamster embryonic kidney cells (BHKcell) This experimental example is an experiment confirming the intracellular permeability of a single-stranded oligonucleotide. Hamster embryonic kidney cells (BH
HSV-1 (7.6 × 1) in Kcell, 4.4 × 10 ⁵ cells)
0 ⁴ PFU / ml) for 3 hours. These cells were exposed to 17.66 pmole of ³² P-labeled 20mer for various times. Excluding solvent,
The cells were washed twice with 0.3 ml PBS and further at 36 ° C.
For 30 minutes at 0.1 M NaCl, 33 μM ZnCl ₂ ,
After leaving it in a solution of 33.3 mM sodium acetate buffer (pH 4.5) containing 3360 unit nuclease S1, 2 with the above sodium acetate buffer without nuclease S1
After washing twice, 0.3 ml of 10 mM EDTA, 0.6% sodium dodecyl sulfate and 10 mM Tris buffer (pH 7.
In 5), the cells were lysed. Where TCA in this solution
Insoluble substance and nuclease S1-sensitive 20mer
The amount was measured to be 4.68 per cell in 8 hours.
It was found that × 10 ³ 20 mers were incorporated.

Experimental Example 4 Effect of blending oligodeoxynucleotide (a) Microplate (manufactured by Limbro, hole inner diameter 1.5c
1.5 × 10 ⁵ hamster embryonic kidney cells (BHK cells) per well were placed in m), and the cells were cultured in a 5% CO ₂ incubator at 36 ° C. for 47 to 48 hours. Virus (HSV-1) (7.
160 μl of 6 × 10 ⁴ PFU / ml) was added, and the cells were infected at 36 ° C. for 3 hours. Various amounts of 20mer or a mixture of 20mer and 20B (224: 1) were dissolved in the solvent, and the infected cells were cultured in 0.3 ml (CaCl ₂ concentration of 4.55 mM) for 8 hours.

(B) Separately, in the operation of (a) above, 2
The culture operation was performed in the same manner except that 0 mer or a mixture of 20 mer and 20 B was not used (virus control). Fixing the cultured cells of (a) above, staining,
The number of syncytium was counted. This result was used as the second control, assuming that the number of syncytium in virus control (b) was 100%.
Shown in the figure. As shown in FIG. 2, when the mixture of 20mer and 20B was used, it was recognized that the virus growth inhibitory effect was stronger than that of 20mer alone. From this, it was confirmed that the inhibitory action may enhance the action when different nucleotide sequence portions of the nucleotide sequences constituting the minus-strand DNA of herpes simplex virus are used together.

[Brief description of drawings]

FIG. 1 is a graph showing the results of one experimental example [Previous Example 1- (1)] for confirming the effect of the antiviral agent of the present invention. 2 on the ordinate of the graph for the mortality rate of the 20mer non-administered group
The percentage of mortality in the 0-mer administration group is shown, and the dose of 20-mer (μg / mouse) is shown on the horizontal axis.

FIG. 2 is a graph for comparing the virus growth inhibitory effects of the case of using the 20mer alone and the case of using the mixture with 20B in Experimental Example 4 of the present specification, in which the vertical axis shows that of the virus control group. 2 for syncytium number
The percentage of the number of syncytium in the group using 0 mer or a mixture of 20 mer and 20 B is taken, and the horizontal axis shows the amount of 20 mer used or 20 mer
The amount of the mixture of 20 and 20B used is shown.

Claims (1)

[Claims] 1. An antiviral agent comprising an oligodeoxynucleotide or polydeoxynucleotide equivalent to a partial structure of a DNA sequence capable of hybridizing to a messenger RNA generated during propagation of a pathogenic virus.