HU209625B - Process for producing antiviral agents - Google Patents

Abstract

The present invention relates to a process for the preparation of an antiviral composition comprising the single-stranded DNA of a single-stranded DNA phage prepared in a manner known per se, in admixture with pharmaceutically acceptable additives, into a pharmaceutical composition. EN 209 625 B Description: 8 pages

Description

Scope of the description: 8 pages

EN 209 625 B

EN 209,625 Β

The present invention relates to the preparation of an antiviral agent for the treatment of viral diseases such as viral hepatitis.

Viral diseases include a number of diseases such as viral hepatitis, influenza and herpesvirus-induced cerebral inflammation, as well as other fairly serious diseases such as adult T cell leukemia and acquired immunodeficiency syndrome.

Though certain antidote agents, such as nucleic acid analogues and BRMs (biological reaponse modifier), have been developed against them, none of these conventional drugs can exert a perfect therapeutic effect. In addition, these drugs have serious side effects. Thus, patients with such a disease are often referred to as a so-called "expective" treatment.

BRM is an agent that enhances or regulates the functioning of the immune system and is used in patients whose immune responses are, for example, immunodeficiency syndrome, cancer, radiation therapy, chemotherapy, etc. or in patients with immune disorders such as autoimmune disease. Examples of BRM include cells and components of bacteria, Basidiomycetes, Ascomycetes, a natural product such as a plant component, a low molecular weight compound, a factor produced by an immunocompetent cell, such as interlekuin-2, gamma-interferon, thymine, and the like.

Virology 143: 290-299 (1985) describes the antiviral activity of a double stranded DNA. However, the double stranded DNA examined was not of natural origin.

JNCI 72 (4): 955-962 (1981) describes the antitumor activity of a modified single stranded BCG DNA. However, the modified single-stranded DNA was not derived from phages but from bacterial cells such as BCG.

It is an object of the present invention to provide an antiviral agent which is useful in the treatment of viral diseases and has a minor side effect.

To achieve this goal, several components of a phage (bacteriophage) that are non-infectious and non-pathogenic to eukaryotic cells have been isolated and purified, and antiviral activity of all components has been investigated. As a result, it has been found that DNA of a single-stranded DNA phage has a strong antiviral effect.

The present invention therefore relates to a method for producing an antiviral agent, which is a single-stranded DNA of a single-stranded DNA phage.

Examples of single-stranded DNA of the above single-stranded DNA phages are described in the examples of the invention.

Single-stranded DNA of the single-stranded DNA phage of the present invention exhibits excellent activity as an antiviral agent. Thus, the present invention provides an excellent antiviral agent for the treatment of viral diseases for which no effective and undesirable drug has been developed.

For example, the single-stranded DNA of the single-stranded DNA phage used in the antiviral agent of the present invention is prepared by the following method known per se.

A bacterium is transformed by introducing a commercially available phage-replicative double-stranded DNA or single-stranded phage DNA, for example, treated with calcium ion. The transformed cell thus obtained is incubated and the cells are separated, for example, by centrifugation. The single-stranded DNA phage suspension thus prepared was concentrated and the phage-derived proteins removed from the suspension. For example, ethanol is precipitated by precipitation to obtain the single-stranded DNA of the single-stranded DNA phage.

The single-stranded DNA phage used herein may be derived from any source, but preferably is a phage that is highly contagious to the host bacterium; which causes the single-stranded DNA phage to have a high rate of replication by itself; which does not damage the host cell; which is released from the host cell; and that does not significantly depress host cell proliferation.

The procedure for producing single-stranded DNA of a single-stranded DNA phage is described in detail below.

Examples of commercially available phage-replicative double-stranded DNAs include: M13mp7RFDNA, M13mp8RFlDNA,

M13mp9RFDNA, M13mpl0RFDNA and

M13mpllRFDNA (all from Pharmacia Co. Ltd.); M13mpl8RFDNA, M13pl9DNA and 0X174RFIDNA (all from Takara Co., Ltd.); and M13mp2ORFDNA and M13mp21DNA (all from IBI). Single-stranded phage DNA is M13mp7SSDNA and M13mpl8SSDNA (both from Pharmacia Co., Ltd.); M13mpl9SSDNA and 0X17SSDNA (both from Bethesda Research Laboratories); and M13mp9SSDNA (from Boehringer, Mannheim).

If a double-stranded DNA of a phage-replicative form is specifically used, it can be modified beforehand by conventional recombinant DNA techniques. That is, the double-stranded DNA of phage replication is cleaved with a suitable restriction enzyme and a pre-prepared heterologous gene fragment, using ligase, is inserted, thereby extending, shortening or rearranging the double-stranded DNA chain.

The transformation by the use of alpha-calcium coprecipitation can be carried out in the usual manner, for example using calcium chloride.

Preferably, the host bacterial cell is Escherichia coli or Bacillus subtilis, which are very harmless. The first is more advantageous. Especially preferred are E. coli bacteria having phage-infectious F-piles, since the desired single-stranded phage DNA can thus be readily isolated.

The bacterial cells thus transformed are then incubated in culture medium. The medium must be selected depending on the bacteria used, but any medium may be used if it contains the carbon source, nitrogen source and inorganic materials required for bacterial growth. Examples include YT medium (Difco), Amley Bacto tryptone, Bacto yeast extract, sodium chloride and distilled water.

EN 209 625 áz glaze. Incubation was carried out at 35-39 ° C for 1236 hours.

For example, after incubation, the incubation medium is centrifuged to obtain a single-stranded DNA phage suspension.

The single-stranded DNA phage suspension thus prepared is added to a suitable host cell, which is preferably pre-cultured to the logarithmic growth phase, the latter being preferably infected in a 1-10 fold ratio. The infected cell is then incubated under the conditions described above. Thus, a fresh single-stranded DNA phage suspension is obtained. A single-stranded DNA phage suspension can be produced efficiently by repeating the above procedure.

Alternatively, a commercial single-stranded DNA phage, such as 0X174 or fi (both from the Institute's Fermentation, from Osaka); or M13, fd, S13, OR or ZJ / 2 (ATCC deposit numbers: 15669-B1, 15669-B2, 13706-B5, 13706-B3 or 25298-B2) are added to a suitable host cell, preferably pre-cultured to the logarithmic growth phase , thus, the latter is preferably infested in a ratio of 3-8 times with the former. The infected cell is then incubated under the conditions described above. In this way, a single-stranded DNA phage suspension of the target can be prepared without the use of double-stranded DNA or single-stranded phage DNA in commercial phage replication.

The thus obtained single-stranded DNA phage suspension is treated with polyethylene glycol and salt (s), such as sodium chloride, to precipitate and then concentrate the single-stranded DNA phage.

For removal of proteins from the single-stranded DNA phage, the proteins may be precipitated using, for example, phenol, chloroform or isoamyl alcohol, and then centrifuged.

Finally, single-stranded phage DNA is precipitated by conventional ethanol precipitation. The product thus precipitated is preferably further purified, for example, by dialysis against sterile water or 0.1% saline or by gel filtration column chromatography using a suitable gel such as Sephadex.

The single-stranded phage DNA produced is lyophilized and stored.

Single-stranded DNA phage single-stranded DNAs, which are prepared by the following Examples, are used to prepare the active ingredient of the antiviral agent of the present invention.

Example 1

1.1 ml of E. coli (K-12 JM101 strain, Pharmacia Co., Ltd) suspension was incubated in 200 ml of YT medium prepared by 8 g of Bacto tryptone (Difco), 5 g of Bacto yeast extract (Difco) and 5 g of sodium. chloride was dissolved in distilled water to 1 liter and the pH of the resulting solution was adjusted to 7.2. The incubation is carried out for about 1 hour until the UV absorbance of the culture suspension at 550 nm reaches about 0.2. The culture suspension was then cooled in an ice bath to 4 ° C and centrifuged for 10 minutes at 4 ° C (9000 G). The supernatant was removed to give a precipitate containing the cultured E.coli in the form of a sediment. The pellet was dissolved in 90 ml of a solvent mixture containing 1: 5: 80 aqueous sodium chloride solution, 1 M manganese chloride solution, 12.5 mM aqueous sodium acetate (pH 5.6) and distilled water. : 14 ratio. The resulting solution was incubated in an ice bath at 4 ° C for 20 minutes and then centrifuged for 10 minutes at 4 ° C (9000 G). Thus, the impurities, including the incubation solution, and the supernatant are removed. The residual precipitate was suspended in 9 ml of a solvent mixture containing an aqueous solution of calcium chloride, 1M-manganese chloride solution, 12.5 mM aqueous sodium acetate (pH 5.6) and distilled water. : 32: 1. To the resulting suspension was added 15 µΐ of a solution prepared by M13mp8RF1DNA (Pharmacia, Co., Ltd.) in TE buffer containing 10 mM Tris-hydrochloride (pH 8.0) and 1 mM EDTA. ng / μΐ dissolved to final concentration. The resulting mixture was incubated in an ice bath at 4 ° C for 20 min and then in a water bath at 37 ° C for 2 min, so that the E. coli strain was transfected.

1.2

100 μΐ produced according to Example 1.1 was transformed

The E. coli reaction mixture was added to 3 ml of soft agar medium and 50 µl of 2% dimethylformamide X-gal (Bethesda Research Laboratories) and 10 μΐ of 100 mM aqueous IPTG solution (Bethesda Research Laboratories) were added. The resulting mixture was poured onto YT agar medium and incubated overnight at 37 ° C. Thus, the phage-infected E. coli forms blue plaques. One of these plaques is removed and diluted 100-fold and 10,000-fold. 10 to 10 μΐ of diluted solutions are added to 3 ml of soft agar medium and 50 μΐ of 2% dimethylformamide X-gal (Bethesda Research Laboratories), followed by 10 µl of 100 mM aqueous IPTG (Bethesda Research Laboratories) solution. mix. This mixture was poured onto YT agar medium and incubated overnight at 37 ° C. The surface of the medium, completely covered with blue plaques, is scraped with plaques and centrifuged at room temperature for 10 minutes (10,000 G). Thus, as a supernatant, a phage suspension is obtained. 7.500 µΐ, 10 ⁹ cells / ml E. coli K-12 JM101 (Pharmacia Co., Ltd.) was incubated in 15,000 ml YT medium at 37 ° C for 2-3 hours until the culture suspension was UV absorbed at 550 nm reaches 0.4. The resulting suspension of culture (2x10 ⁸ cells / ml) was then added to the above phage suspension to infect the first 5 times with the latter and then incubate for 7 hours.

1.3

The culture suspension obtained in Example 1.2 was centrifuged at room temperature for 10 minutes (10.000 G). PEG 6000 (Wako Pure Chemicals Co., Ltd.) and sodium chloride were added to the supernatant so obtained to give a concentration of 4% and 0.55 M, respectively. The resulting mixture was allowed to stand at 4 ° C overnight, then centrifuged at 12 ° C for 20 minutes (12.000 G) and the supernatant discarded. For rainfall TE

HU 209 625 B buffer (pH = 8), which is suspended in this way. An equal volume of phenol was added to the resulting suspension. The resulting mixture was thoroughly stirred and centrifuged to divide it into two phases. The phenol phase was removed and a 1: 1 mixture of chloroform and phenol / isoamyl alcohol (1/24) was added to the aqueous phase. The resulting mixture was thoroughly stirred and centrifuged to divide it into two phases. The solvent mixture phase was removed and chloroform containing an equal volume of isoamyl alcohol in 1/24 volume was added to the aqueous phase. The resulting mixture was thoroughly stirred and centrifuged to divide it into two phases. The chloroform layer was removed and diethyl ether was added to the remaining aqueous phase. The resulting mixture was thoroughly mixed and centrifuged to two phases. The diethyl ether phase was then removed. This procedure was repeated five times and the resulting aqueous solution was incubated at 50 ° C for 30 minutes to remove residual diethyl ether. The aqueous solution was then subjected to ethanol precipitation, twice with a 1/10 v / v mixture of cold ethanol and 3M sodium acetate. To the resulting precipitate was added 40 ml of sterile water and the mixture was dialyzed for 46 hours in a dialysis tube, replacing the outer solution (8 liters) seven times. The product was then lyophilized to give 80 mg of M13mp8 single-stranded phage DNA.

Example 2

100 µl, 10 ⁹ cells / ml E. coli (K-12 JM101 strain Pharmacia Co., Ltd.) suspension was incubated in 200 ml YT medium at 37 ° C for 2-3 hours until the culture suspension had a UV absorption of 550 reaches 0.4 at nm. To the resulting culture suspension (2x10 ⁸ cells / ml), the phage solution prepared according to Example 1.2 is added so that the infection is fivefold. The resulting mixture was incubated for 7 hours, then the incubation medium was isolated and the product purified as described in Example 1.3. 100 mg of M13mp8 single-stranded phage DNA were thus obtained.

Example 5

3.1

M13mp8RF1DNA (Pharmacia Co., Ltd.) was digested with EcoRI restriction enzyme (Pharmacia Co., Ltd.) and subjected to ethanol precipitation at -20 ° C. The product is then dissolved in 10 μΐ TE buffer. Separate the EcoRI restriction enzyme Charon 27 phage DNA into which a duck hepatitis B virus (DHBV) gene fragment is inserted as a heterologous gene at the site recognized by EcoRI. The gene fragment was prepared by gel electrophoresis of 0.5% agarose and dissolved in TE buffer. The resulting 200 ng M13mp8RFlDNA EcoRI fragment and 500 ng DHBV gene fragment were mixed with 400 units of T4DNA ligase (New England Bio Labo.) And the resulting mixture was reacted in coupling buffer at 16 ° C for 4 hours. Thus, a reaction mixture containing double-stranded DNA of the phage replicative form into which the DHBV gene fragment is incorporated is obtained.

3.2

E. coli was transformed with the reaction mixture of Example 3.1 in the same manner as described in Example 1.1. The transformed cell-containing reaction mixture thus obtained was added to a soft agar medium and 50 µl of a 2% solution of dimethylformamide X-gal (Bethesda Research Laboratories) and 10 μΐ of 100 mM aqueous IPTG (Bethesda Research Laboratories) were added. The resulting mixture was placed on YT agar medium and incubated overnight at 37 ° C. Thus, white plaques are formed consisting of E. coli transfected with the recombinant phage. Some plaques thus formed were removed and each added to 10 ml of a suspension of E. coli (K-12 JM101, Pharmacia Co., Ltd.) at a concentration of 10 ⁹ cells / ml, diluted 200 times and pre-incubated in YT medium overnight. After overnight incubation, the culture suspension was centrifuged for 10 minutes at room temperature (12.000 G), separating it into supernatant and precipitate. The thus amplified double-stranded DNA of the phage-replicative form is purified in a conventional manner from the E. coli transformed cell in the precipitate and cleaved with EcoRI. The fragment thus prepared was examined by agarose gel electrophoresis and found to be identical to the DHBV gene fragment described in Example 3.1.

3.3

The supernatant obtained in Example 3.2 was diluted 100-fold and 10,000-fold and added to 3-3 ml of soft agar medium. For each mixture, 50 µl of 2% dimethylformamide X-gal (Bethesda Research Laboratories) and 10 µl of 100 mM aqueous IPTG (Bethesda Research Laboratories) are added. The resulting mixture was placed on YT agar medium and incubated overnight at 37 ° C. The soft agar medium, the surface of which is completely covered with white plaques, is then scraped with plaques and centrifuged for 10 minutes at room temperature (12,000 G). Thus, as a supernatant, a phage suspension is obtained.

A suspension of E. coli (K-12 JM101 strain, Pharmacia Co., Ltd.) at 10 ⁹ cells / ml in 7.500 μΐ was incubated in 15,000 ml YT medium at 37 ° C for 2-3 hours until the culture suspension had a UV absorption of 550 µg. reaches 0.4 at nm. To the culture suspension at 2x10 ⁸ cells / ml, the above phage suspension was added in a five-fold ratio. The mixture was incubated for 7 hours, then the phage suspension was separated and purified as described in Example 1.3. Thus, 100 mg of M13mp8 single-stranded phage DNA was obtained, into which the DHBV gene fraction was incorporated as a heterologous gene into the site recognized by the restriction enzyme EcoRI.

Example 4

E. coli (K-12 JM101 strain, Pharmacia Co., Ltd.) was cultured in 15 liters of YT medium until the culture suspension had a UV absorbance of 0.4 at 550 nm. To the culture suspension of 2x10 ⁸ cells / ml, the phage suspension prepared according to Example 3 was added in a five-fold ratio. The mixture was incubated for 7 hours and then the phage suspension in Example 1.3

It is isolated and purified as described in FIG. Thus, 120 mg of M13mp8 single-stranded phage DNA was obtained into which the DHBV gene fraction was incorporated as a heterologous gene into the site recognized by the restriction enzyme EcoRI.

Example 5

E. coli (K-12 JM101 strain, Pharmacia Co., Ltd.) was cultured in 6 liters of YT medium until the UV absorbance of the culture suspension reached 0.2. A suspension of M13 phage wild strain (ATCC 15669-B1) was added to the culture suspension at 2x10 ⁸ cells / ml in a ten-fold ratio. The resulting mixture was incubated for 8 hours, and the phage suspension was isolated and purified as described in Example 1.3. Thus, the wild-type strain of Ml3 is 80 mg single-stranded phage DNA.

In order to illustrate the effect of the antiviral agent of the invention in the treatment of viral diseases, the following examples are provided.

6. Experimental Example

The single-stranded phage produced according to the invention

DNA inhibitory effect on DHBV multiplication

Dip serum infected with DHBV virus is diluted five times with physiological saline. The resulting solution is injected intravenously into 50-day-old ducks for 0 days. Ducks are divided into five groups of 5 to 5 animals. The single-stranded phage DNA prepared according to Examples 2, 4 and 5 was administered to different groups. Next, a single-stranded DNA produced by denaturation of commercial vermin DNA was administered to another group. Each single-stranded DNA is dissolved in physiological saline for intravenous administration, and ducks are administered once daily for 9 days, starting from the administration of DHBV infected duck serum. From day 4, each animal's blood is taken from its heart every other day. The remaining group is used as a control group in which animals are given only physiological saline intravenously.

Table 1 shows the inhibitory effect of individual single-stranded DNAs on the proliferation of viral DNA in the blood following the administration of DHBV infected duck serum.

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Table 1

Inhibitor effect on virus DNA growth in blood (%)

Sample Inhibitor effect on virus DNA growth in blood (%) Single-stranded phage DNA prepared according to Example 2 100 Single stranded phage DNA prepared according to Example 4 100 Single-stranded phage DNA prepared according to Example 5 100 Single-stranded DNA produced by denaturation of Bogutimus DNA 0 control 0

Note: each sample was administered at 2.5 mg / kg.

These results show that all antiviral agents of the invention are effective in the treatment of viral diseases.

In addition, the administration of the products of Examples 2, 4 and 5, which are the active ingredients of the antiviral agent of the present invention, does not produce toxic or side effects to the ducks at the effective doses indicated above.

If the product of Example 2 is administered intravenously to male Balb / c mice at a dose of 37 mg / kg, no animal will die. Thus, it has been shown that the antiviral agent is still harmless at this dose.

From these results, it was found that the effective dose of the antiviral agent of the invention in adults is 0.01 to 50 mg / kg body weight, preferably 0, ΙΙΟ mg / kg body weight.

Thus, the effect of the invention is illustrated in the above examples. However, it is expected that any single stranded DNA will have similar pharmacological properties if it meets the above requirements.

Single-stranded DNA, which is the active ingredient of the antiviral agent of the present invention, can be used to prepare various formulations, including injections, solutions, ointments, and suppositories, using conventional excipients such as solvents, binders, and other excipients as usual.

Other pharmaceutical active ingredients may also be incorporated in the preparation of these compositions.

Injections or solutions can be prepared using conventional diluents such as distilled water for injection, physiological saline, aqueous dextrose solution, vegetable oil for injection, propylene glycol or polyethylene glycol. If necessary, other additives such as toning agents, stabilizers, preservatives or receipts may be used. In this case, the active ingredient is preferably dissolved in a sterile injectable solvent.

Ointments may be prepared, for example, using fat, oil, lanolin, petroleum, paraffin, wax, resin, plastic, glycol, higher molecular weight alcohol, glycerol, emulsifying or suspending agent. If necessary, other additives such as stabilizers or preservatives may be used. In this case, a sterile medium is preferably used to form the ointment.

For example, suppositories are made using cocoa butter or macrogol (polyethylene glycol, Difco). If necessary, other additives such as stabilizers or preservatives may be used. A sterile medium is preferably used to make the suppository.

To further illustrate the present invention, the following examples of use are provided without limiting the invention to them.

Application Example 1

100 mg of M13mp8 single-stranded phage DNA prepared according to Example 2 was filled into a sterile vial and the vial was sealed. When used, the vial contents are dissolved in 5% glucose solution or physiological saline and the patient is administered intravenously over a period of 0.5-2 hours in 500 ml transfusion.

EN 209 625 B

Application Example 2

100 mg of M13mp8 single-stranded phage DNA into which the DHBV DNA fragment of Example 4 is incorporated is filled into a sterile vial and the vial is sealed. When used, the vial contents are dissolved in 5% glucose solution or physiological saline solution and administered to the patient in 500 ml transfusions over 0.5-2 hours, intravenously, dropwise.

Application Example 3

100 mg single-stranded DNA of the wild-type strain M13 of Example 5 was filled into a sterile vial and the vial was sealed. When used, the vial contents are dissolved in 5% glucose solution or physiological saline solution and administered to the patient by intravenous infusion over 0.5-2 hours in 500 ml transfusion.

7. Experimental Example

Single-stranded 0X174 phage DNA antiviral effect

DHBV (duck hepatitis B virus)

DHBV-infected duck serum was diluted ten times with physiological saline. The resulting solution is inoculated into 100 μΐ (1.4x10 ⁸ DHBV) intravenously into freshly duck (0 days) ducks. Ducks are divided into three groups as shown in Table 3. All DNAs were administered intravenously to the animals once a day for 10 days, the first dose on the day of administration of the DHBV-infected serum. Serum samples are taken from the hearts of the ducks, the first on day 4 and then every other day and centrifuged. Only physiological saline is added to the control group. The serum total DHBV DNA content was measured by the method of hybridization and expressed as a percentage of the mean DHBV DNA of the control group (100%). The results are shown in Table 2.

Table 2: Antiviral effect of single-stranded DNA against DHBV

Sample Number of ducks DHBV DNA in serum (%) control 7 100.0 0X174 phage DNA 5 10.6 (1.9-59.4), p < 0.05 M13fágDNS 5 0.1 (0.0-1.1), p <0.01

The dose administered is in each case 2.45 mg / kg. Values in parentheses are values within 95% confidence limits. P represents the significance values of each value relative to the control.

As shown in Table 2, single-stranded 0X 174 phage DNA exhibits antiviral activity against DHBV, similar to single-stranded M13 phage DNA.

8.1. Experimental example

Antiviral effect of single-stranded M13 phage DNA against W (vaccinia virus) in ddY mouse tail vein VV (Lister strain,

2x10 ⁴ PFU / plaque forming unit /). The mice are divided into four equal groups. Single-stranded M13 phage DNA is injected intravenously into the tail of each mouse at the three different doses given in Table 3. Only 10 ml / kg / day of physiological saline was added to the control group for 7 days. Intravenous administration of single-stranded M13 phage DNA was started 3 days prior to VV infection and was performed for seven days. Seven days after infection, antiviral activity was determined by counting the wounds on the tail of each mouse. The results are shown in Table 3.

Table 3: Antiviral effect of phage phage M13 phage DNA against VV

sample number of tail wounds (mean ± S.E) control (saline) 28.8 ± 1.2 M13fágDNS 5.7 mg / kg / day 5.8 ± 1.9 p <0.001 16.7 mg / kg / day 2.3 ± 0.7 p <0.001 50.0 mg / kg / day 2.2 ± 1.6 p <0.001

p represents the significance values of each value relative to the control.

As can be seen from Table 3, single-stranded M13 phage DNA effectively inhibited the proliferation of VV-induced wounds.

8.2. Experimental example

Antiviral activity of single-stranded M13 phage DNA against W in ddY mouse tail vein VV (Lister strain, 2X10 ⁴ PFU / plaque forming unit) is injected. The mice are divided into four equal groups. Single-stranded M13 phage DNA is injected intravenously into the tail of each mouse at the single dose given in Table 4. The control group was treated with 10 ml / kg of saline on the day before the infection. Seven days after infection, antiviral activity was determined by counting the wounds on the tail of each mouse. The results are shown in Table 4.

Experiment 1

The effect of single-stranded M13 phage DNA against VV is evaluated by comparing AGTC (a mixture of AMP, TMP, GMP and CMP corresponding to the single stranded M13 phage DNA composition ratio).

Experiment 2

The effect of M13 phage DNA W is compared to other DNAs. Human placenta DNA and boic thymic DNA were maintained at 100 ° C for 5 minutes. The DNAs are then rapidly cooled and the resulting samples are added.

In Experiment 1, single-stranded M13 phage DNA inhibited the spread of wounds. However, the ATCG mixture had no effect.

In Experiment 2, although single-stranded M13 phage DNA significantly inhibited the spread of wounds, human placen 6

And DNA and calf thymus DNA did not affect the same dose.

A4. Table 1 shows that single-stranded M13 phage DNA has antiviral activity against W and DHBV.

Table 4: Antiviral effect of phage III on VV against VV

sample dose (mg / kg) tail number of injuries (mean ± S. E.) Experiment 1 M13fágDNS 50 4.6 ± 2.2 p <0.001 ATCG mixture 50 33.0 ± l, 5 ATCG mixture 16.7 32.0 ± 2.2 control (salt) 28.0 + 3.8

Experiment 2 M13fágDNS 50 5.8 ± 2.5 p <0.001 human placenta 50 24.2 ± 2.9 calf thymus DNA 50 20.3 ± 2.3 control (salt) 27.9 ± 2.3

p represents the significance values of each value relative to the control.

8.3. Experimental example

Antiviral activity of single-stranded M13 phage DNA against W in ddY mouse tail vein VV (Lister strain,

2x10 ⁴ PFU / plaque forming unit /). The mice were divided into 13 equal groups. Single-stranded M13 phage DNA was injected intravenously at a single dose of 50 mg / kg into the tail of each mouse at the single dose given in Table 5 below. In the table, the sign - and + means how many days before the day before or after the infection. Seven days after infection, antiviral activity was determined by counting the wounds on the tail of each mouse. The results are shown in Table 5.

Experiment 1

The control group was treated with physiological saline at a dose of 10 ml / kg / day for 7 days. Antiviral activity of single-stranded M13 phage DNA is evaluated against multiple VV after multiple dosing.

Experiment 2

The control group was given physiological saline at a dose of 10 ml / kg / day the day before the infection. Antiviral activity of single-stranded M13 phage DNA is evaluated after single administration to VV at different times.

In Experiment 1, single-stranded M13 phage DNA inhibited the spread of wounds, both on single-day, pre-infective, and multiple dosing. Thus, it can be seen that the effect of single stranded Ml3 phage DNA does not depend on the frequency of dosing.

In Experiment 2, single-stranded M13 phage DNA inhibited the spread of wounds at most when dosing was performed on the day before infection. Single-stranded M13 phage DNA was also effective when administered two days before the day of infection or on the day of infection.

Table 5: Antiviral effect of single-stranded M13 phage DNA against VV

Date of dosing DNA of M13 phage Tail number of injuries (mean ± S. E.) Experiment 1 -3 -2 -1 0 +1 +2 +3 2.3 ± 0.8 p <0.001 -3 -1 0 +1 +3 6.7 ± 2.7 p <0.001 -2 0 +2 2.6 ± 0.6 p <0.001 -1 3.7 ± 2.3 p <0.001 control (saline) 29.9 ± 3.2 Experiment 2 -3 25.0 ± 4.5 -2 12.0 ± 3.0 p <0.001 -1 9.0 + 4.1 p <0.001 0 17.0 + 4.3 p <0.01 +1 37.2 ± 3.0 +2 34.5 ± 4.4 +3 34.7 ± 4.0 control 37.4 + 3.8

P represents the significance values of each value relative to the control.

Claims (1)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT DETAILED DESCRIPTION OF THE INVENTION \ t A method of producing an antiviral composition is converted into a pharmaceutical composition. characterized in that a single-stranded DNA phage is self-limiting