JPH09328426A - Therapeutic agent for herpes virus infectious disease and preventing agent for relapse of herpes virus infectious disease caused by herpes virus comprising triterpene derivative as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new therapeutic agent for herpes virus infectious diseases and a new preventing agent for relapse of the herpes virus infectious diseases by the herpes virus, comprising a specific triterpene derivative obtained from a crude drug as an active ingredient and administrable for a long period without causing the emergence of a medicine-resistant strain or manifesting adverse effects. SOLUTION: The therapeutic agent for herpes virus infectious diseases and preventing agent for relapse of the herpes virus infectious diseases caused by the herpes virus comprises a 3-oxo-olean-18-ene-28-oic acid of the formula as an active ingredient. The compound of the formula is moronic acid which is a substance extracted from Rhus javanica L. with hot water. The compound of the formula has suppressing effects on the proliferation of viruses and is capable of especially suppressing even the viruses migrating into the nervous system. The compound of the formula can be used together with acyclovir.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for herpesvirus infections and a preventive agent for recurrence of herpesvirus infections.

[0002]

2. Description of the Related Art Gobaishi is a species of Anacardiaceae.
Aceae) of Rhusjavanica
L. ) The young aphid is parasitic on the young shoots and leaves,
The worm-like insects produced by the stimulation are dipped in hot water for a short time and then dried, and 70-80% of the components
Is known to be tannin, and has been used for astringent antidiarrheal, antitussive, haemostasis, antiperspirant, it was first discovered by the present inventors that Gobaishi has an effect as a herpesvirus therapeutic agent, Based on this, JP-A-6-25003
Filed as an issue.

On the other hand, all drugs used as antiviral agents are nucleic acid derivatives except for interferon. In particular, the product name Acyclovir of Welcome, that is, the following chemical formula (II)

Embedded image 9- (2-hydroxy-ethoxymethyl) guanine [9- (2-hydroxy-ethoxyme
[thyl) guanine] is widely used as a therapeutic agent for herpes.

[0004] Since various substances are contained in the hot water extract of Gobaishi, it has been unclear which of these substances acts as a therapeutic agent for herpesvirus infections.

The most common features of viruses of the Herpesviridae are latent infection and recurrence (relapsing episodes). Moreover, this recurrence surprisingly occurs even in the presence of antibodies.

[0006] Herpes simplex virus is known to recur after the initial infection on the body surface by a route of sensory nerve-latent infection of nervous system-sensory nerve-focal formation of innervated area.

In humans, herpes simplex type 1, which causes oral gingivitis due to primary infection, passes through the trigeminal nerve and reaches the trigeminal ganglion, where it becomes latent. For some reason, the latent virus proliferates in the ganglion, passes through the trigeminal nerve again, and proliferates in cells on the body surface, resulting in herpes labialis. In humans, fever, prolonged sunlight (ultraviolet rays), and
Frequent recurrence is a major problem because of stress, fatigue, menstruation, etc.

On the other hand, herpes simplex virus 2 causes vulva vaginitis. This herpes simplex type 2 virus goes up the sensory nerve from the lumbosacral spinal cord, lurks in the dorsal root ganglion of the lumbosacral spinal cord, proliferates there for some reason, then descends to the sensory nerve again, and genital as a recurrent episode. Causes herpes. In humans, fever, ultraviolet rays, stress, fatigue, menstruation, and the like are factors that cause recurrence, like the herpes simplex virus 1.

When such recurrence of genital herpes occurs in conjunction with pregnancy, two problems arise. One is that the virus can infect the fetus in the womb and cause malformations in the fetus or cause premature stillbirth,
The other is that the fetus is infected in the birth canal at the time of delivery and is a source of neonatal herpes infection.

The birth of a malformed baby due to in utero infection has a very small number of cases, so its effect is not clear, but neonatal herpes due to birth canal infection is a disease with a very high fatality rate, and it can be reliably treated after the onset. Therefore, infection prevention is important.

[0011] There are many unclear points about the mechanisms of latent infection and recurrence. What is the mechanism of recurrent onset in the presence of neutralizing antibodies, how is the growth of the virus suppressed and cured after recurrence? There are various problems such as the mechanism of onset induction and the existing state of the latent virus. Many of these problems remain unsolved, and frequent recurrence is a major problem due to the problem of complications with pregnancy.

[0012] In order to prevent such recurrent onset of herpes virus, it is necessary to suppress the growth of latent virus. For that purpose, it is necessary to administer a drug that suppresses the growth of the virus before the virus is activated.

As a report using the 9- (2-hydroxy-ethoxymethyl) guanine as a preventive agent for recurrence of herpes simplex virus type 2, S. E. FIG. Straus et al. (N. Engl. J. Med., Vol. 310, p.
p. 1545-1550, 1984), J. M. Dou
glass et al. (N. Engl. J. Med., Vol. 3).
10, pp. 1551-1556, 1984), L.S.
G. FIG. Kalowitz et al. (JAMA, Vol. 26).
5, pp. 747-751, 1991) and the like. However, if the administration period of the 9- (2-hydroxy-ethoxymethyl) guanine is long, problems such as the emergence of drug resistant strains and side effects will occur, necessitating a drug holiday. Therefore, the development of a new recurrence preventive agent free from such problems has been desired.

[0014]

DISCLOSURE OF THE INVENTION The object of the present invention is to provide a new herbal medicine-derived herpes that can be administered for a long time without the appearance of drug-resistant strains and side effects, which are the drawbacks of conventional anti-herpes virus agents consisting of nucleic acid derivatives. It is intended to provide a therapeutic agent for viral infections and a preventive agent for recurrence of infectious diseases.

The present inventors have found that Gobai (Rhus.
avanica L. Moronic acid (ie, 3-oxo-olean-18-ene-28-oic acid), which is a hot water extract from), has a therapeutic effect on herpes and a herpes virus recurrence preventive effect without the appearance of drug-resistant strains and side effects. The inventors have found out that, and have reached the present invention.

The active concentration (EC ₅₀ ) of the hot water extract substance (reflux extract of Example 1) from Gobiishi that inhibits virus growth by 50% was 56.7 ± 6.0 μg / ml. Furthermore, the EC ₅₀ values of the ethyl acetate extract fractions due to the difference in pH of this hot water extract substance were 7.4 ± 2.0 μg / ml at pH 10 respectively, showing the strongest antiviral activity and strong cytotoxicity. It was Also, E of pH 3.6
The C ₅₀ value is a concentration of 27.5 μg / ml and a pH of 7.
The EC ₅₀ value of No. 0 was 16.0 μg / ml.
All of these antiviral activities were evaluated by the plaque formation test.

That is, the first aspect of the present invention is the following formula (I):

Embedded image 3-oxo-olean-18-ene-28-
Oic acid (3-Oxo-olean-18-en-2
The present invention relates to a therapeutic agent for herpes virus infection, which comprises 8-oic acid) as an active ingredient.

A second aspect of the present invention is the following formula (I)

Embedded image 3-oxo-olean-18-ene-28-
Oic acid (3-Oxo-olean-18-en-2
The present invention relates to a herpesvirus infection relapse preventive agent, which comprises 8-oic acid) as an active ingredient.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION A test example of the present invention will be described below. The use part of the plant as a crude drug is
The gallworm's gall was used. In addition, all commercially available products were used in the experiments. As a reagent for comparing and contrasting various properties of the 3-oxo-olean-18-ene-28-oic acid of the present invention, 9- (2-hydroxy-ethoxymethyl) guanine [specifically, commercially available from Welcome Co., Ltd. Acyclovir (hereinafter referred to as ACV) tablet].

Example 1 500 g of the fungus gall of Pleurotus cornucopiae was reflux-extracted with 5.2 liters of deionized water or distilled water, and 5N NaOH was added to the extract.
Was added to bring the pH to 10. The extract was mixed with ethyl acetate 6
Extraction was performed using 1 liter, and ethyl acetate was removed to obtain 3.1 g of an extraction residue (hereinafter, EtOAc extract). The same operation was repeated 6 times to obtain 19.1 g of an EtOAc extract.
The residue after removal of ethyl acetate in the above-mentioned ethyl acetate-extracted fraction of pH 10 was about 2% of the hot water extract. 4.85 g of this ethyl acetate extraction residue was chromatographed on silica gel with 3 liters of ethyl acetate: benzene (2: 8).
(Fractions 1 to 5), 700 ml of methanol: chloroform (3: 7) (fraction 6), 300 ml
Were sequentially eluted with methanol (fraction 7), and fractionated into 7 fractions. 3.81 g of the first fraction of the 7 fractions was rechromatographed on silica gel.
8 liters of hexane (fraction 1), 500 ml
Chloroform: hexane (2: 8) (fraction 2), 1 liter of chloroform: hexane (4: 6)
(Fractions 3, 4) 1 liter of chloroform:
Hexane (8: 2) (fractions 5, 6), 1 liter of chloroform (fraction 7), 1 liter of methanol: chloroform (2:98) (fraction 8), 1 liter of methanol: chloroform (5: 9).
5) (Fraction 9), 1 liter of methanol: chloroform (20:80) (Fraction 10) were sequentially eluted, and fractionated into 10 fractions. The 4th to 7th fractions out of 10 fractions were combined and purified by normal phase thin layer chromatography using a developing solvent of ethyl acetate: chloroform (1: 9) to give 3-oxo-olean-18-ene-28. -Oic acid (3-Oxo-olean-18
-En-28-oic acid) 700 mg was obtained.

The outline of the above fractionation processing is shown below.

[Table 1]

The infrared spectrum and melting point of this product were all in agreement with those of literature values, and the structure thereof was confirmed. In FIG. 1, 3-oxo-olean-18-ene-
28-Oic acid (3-Oxo-olean-18-e
The infrared spectrum of (n-28-oic acid) was shown.

[0023] To examine the antiviral activity, (sometimes hereinafter abbreviated as HSV-1) Herpes simplex virus type 1 (7401H Ltd.) as a virus, TK-deficient herpes simplex virus type 1 (B2006 strain) (hereinafter TK ^- Abbreviated), Phosphono aceti
c acid (hereinafter PAA) resistant herpes simplex virus type 1 (7401H-derived PAA resistant strain) (hereinafter sometimes abbreviated as AP ^r 1), herpes simplex virus type 2 (I
to 1262 strain) (hereinafter sometimes abbreviated as HSV-2) was used.

Vero cells were infected with the virus, and after a predetermined period of time, the infected cells were freeze-thawed three times and centrifuged (300
The supernatant (0 rpm, 15 minutes) was used as a virus solution, which was used for the following tests. When storing the virus solution-
Stored at 80 ° C. All cells were cultured in a carbon dioxide incubator at 37 ° C.

The antiviral activity was measured by quantifying the virus using the cytopathic effect CPE as an index. The virus was quantified by the plaque formation test shown below. That is,
Ve cultivated in a single layer in a plastic dish with a diameter of 60 mm
The above virus solution was added to ro cells at 100 plaques / 0.2.
A diluted virus solution (0.2 ml) prepared to a concentration of ml was inoculated and adsorbed on the monolayer Vero cells at 37 ° C. for 1 hour. After the adsorption, a monolayer Vero in which the above-mentioned virus was adsorbed in a MEM medium (5 ml) containing 2% fetal calf serum containing 0.8% methylcellulose and a drug solution having various drug concentrations to be measured.
Plaque formation was attempted by overlaying on cells and culturing at 37 ° C. for 2 to 5 days. 2-5 above to calculate the number of plaques
After monolayer Vero cells cultured for a day were fixed with formalin,
It was stained with 0.03% methylene blue solution. The number of plaques was counted under a stereoscopic microscope. In addition, using the same concentration of the drug solution, the antiviral activity was examined multiple times as described above,
It was confirmed that the measurement result was within the error range of the plaque confirmation judgment.

In Table 2, 3-oxo-olean-18-ene-28-oic acid (3-Oxo-olean-18-
The antiviral effect of en-28-oic acid) against each virus is shown by the 50% plaque formation inhibitory concentration (EC ₅₀ ) determined from the plaque formation test.

A value representing the plaque formation inhibitory concentration of 50% of the plaque formation rate produced by the virus in Vero cells.
The EC ₅₀ value of oxo-olean-18-ene-28-oic acid (moronic acid) was 3.9 ± 1.1 μg / ml.
Concentration. This is 3-oxo-olean-18
It shows that -en-28-oic acid (moronic acid) has a strong antiviral activity.

[0028]

[Table 2] ^{* 1} ACV: Commercially available product name acyclovir ^{* 2} PAA: Phosphonoacetic acid ^{* 3} ND: Not detected All of these data were evaluated by plaque formation test. The EC ₅₀ value is obtained from three independent experiments for each drug.

From Table 2, it can be said that 3-oxo-olean-18-ene-28-euic acid (moronic acid).
Is effective against the virus at the same drug concentration for all strains, and in particular AP ^r which is a drug resistant strain.
It was confirmed that it also had EC ₅₀ values at low concentrations for the 1 and TK ⁻ strains. In addition, 3-oxo-olean-18-ene-28-oic acid (3-Oxo-ol
No cytotoxicity was observed at concentrations at which ean-18-en-28-oic acid) showed anti-herpesvirus activity.

Further, referring to Table 2, acyclovir (ACV) is markedly ineffective against the AP ^r 1 strain, and clinical drug resistance is a problem, whereas 3 -Oxo-olean-18-ene-28
-Oic acid (moronic acid) has been shown to show efficacy against such drug resistant strains.

Next, the virus growth inhibitory activity was measured as follows using the plaque forming unit (PFU / ml) as an index. That is, each of the above viruses was adsorbed on Vero cells monolayer-cultured on a plastic dish having a diameter of 60 mm, and then MEM medium (3 ml) containing 2% fetal bovine serum was added.
And each concentration of 3-oxo-olean-18-ene-28-
Oic acid (3-Oxo-olean-18-en-2
8-oic acid) solution (1 ml) was added, and the temperature was 37 ° C.
The cells were cultured for 24 hours. After 24 hours, the infected cells were freeze-thawed three times, centrifuged (3,000 rpm, 15 minutes), and the supernatant was used as a virus solution. The number of infectious viruses was counted by the above plaque formation test, and plaque forming units (PFU / PFU /
ml) was calculated. Table 3 shows the results.

[0032]

Table 3 Inhibitory effect of 3-oxo-olean-18-ene-28-oic acid (3-Oxo-olean-18-en-28-oic acid) on virus growth ━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━ Drug plaque forming unit (PFU / ml) [Growth inhibition rate ¹⁾ (%)] Concentration ━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ (μg target HSV-1 AP ^r 1 TK ^- HSV-2 / ml) virus ━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 0 9.78 × 10 ⁸ 1.35 × 10 ⁸ 6.18 × 10 ⁸ 5.28 × 10 ⁷ (0.0) (0.0 ) (0.0) (0.0) 5 1.83 × 10 ⁸ 3.55 × 10 ⁷ 7.14 × 10 ⁶ 2.58 × 10 ⁶ (81.3) (73.7) (98.9) (95.1) 10 1.16 × 10 ⁸ 1.63 × 10 ⁷ 2.18 × 10 ⁶ 4.38 × 10 ⁵ (88.1) (87.9) (99.6) (99.2) 20 3.93 × 10 ⁷ 8.75 × 10 ⁶ 2.05 × 10 ⁵ 1.18 × 10 ⁴ (96.0) (93.5) (> 99. 9) (> 99.9) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

[Equation 1]

From Table 3, it can be said that 3-oxo-olean-18-ene-28-oic acid (moronic acid).
At each concentration (5, 10, 20 μg / ml) of 3-oxo-olean-1 compared to the control (water).
As the concentration of 8-ene-28-oic acid (moronic acid) increases, the proportion of plaques formed for each virus strain decreases. This phenomenon is similar to the drug resistant strains of virus, AP ^r strain and TK ⁻ strain.

Specifically, in the case of virus infection, the above 3
In order to prove that -oxo-olean-18-en-28-oic acid (moronic acid) has an effect of suppressing the growth of virus, BAV / c mouse skin cell tissues were infected with HSV-1 strain. After the treatment, the affected area was observed and scored. The score is 2 (topical rash, blisters, erosion) and 6 (mild rash).
The average time (days ± standard deviation) and the number of survival days were taken. In order to examine the effect of difference between various concentrations of control and 3-oxo-olean-18-ene-28-oic acid (moronic acid), 1 mg / kg of mouse body weight was 1 mg / kg in Experiment 1, and 2 in Experiment 2. 0.2 mg
/ Kg, 1 mg / kg, and 5 mg / kg, and in Experiment 3, 1 mg / kg and 10 mg / kg of 3-
Oxo-olean-18-ene-28-oic acid (moronic acid) was orally administered to mice three times a day (0.25 ml per time, the same amount of water as a control), and for 14 days after ingestion of the virus. The changes in skin lesions were observed. The results are shown in Table 4.

[0035]

[Table 4] Effect of moronic acid after HSV-1 infection on skin tissue of BALB / c mouse ━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━ Experiment number Processing average time (days ± standard deviation) ──────────────────────────── Score -2 Score-6 Survival (topical rash, blisters, erosion) (Mild shingles) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━ Experimental control (standard) 3.20 ± 0.42 5.10 ± 0.32 6.20 ± 0.42 1 Moronic acid 3.90 ± 0.70b 5.70 ± 0.68b 7.30 ± 1.16b (1mg / kg) a ─────────── ─────────────────────────── Experimental control (standard) 3.30 ± 0.48 5.33 ± 0.50 6.56 ± 0.73 2 Moronic acid 4.00 ± 0.47d 5.78 ± 0.97 6.89 ± 0.33 (0.2mg / kg) c Moronic acid 3.60 0.70 5.40 ± 0.52 7.00 ± 1.49 (1mg / kg) Moronic acid 3.90 ± 0.74b 5.40 ± 0.53 7.10 ± 1.52 (5mg / kg) ────────────────────── ─────────────── Experimental control (standard) 3.20 ± 0.42 5.20 ± 0.42 6.60 ± 0.52 3 Moronic acid 3.80 ± 0.63b 5.60 ± 0.71 7.70 ± 2.16 (1mg / kg) (ANOVA: 0.056 Repeated measurement) Moronic acid 3.70 ± 0.48b 5.30 ± 0.48 6.80 ± 0.42 (10mg / kg) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━ a: Re-measurement of the control (standard) by ANOVA (Analysis of Variance) showed that the risk factor (P) was 0.01 or less, showing a significant difference. b: Control (standard) by Student's t-test, the risk rate (P) is 0.05 or less, which shows that there is a significant difference. c: Re-measurement of the control (standard) by ANOVA (Analysis of Variance) showed a risk factor (P) of 0.05 or less, showing a significant difference. d: Control (standard) by Student's t-test, the risk rate (P) is 0.01 or less, which indicates that there is a significant difference.

The risk factor (P) and t-test will be described. A statistical method tests whether there are two populations and whether there is a significant difference between the two populations, that is, whether the two populations may be regarded as the same population ( The method of determining) is called t-test, and the probability when the result of this t-test is erroneous is displayed as a risk factor (P). Therefore, the risk factor (P) of 0.01 or less means that t
-Indicates that the probability that the test result is erroneous is 0.01 or less, that is, 1% or less.

In Table 4, "a" is the result of comparison between the control and 1 mg / kg of moronic acid, and "b" and "d" are the numbers in the control column above them. “C” is the comparison result when the control and 0.2 mg / kg of moronic acid were used.

It can be seen from Table 4 that in Experiment 1, Experiment 2 and Experiment 3, the average number of days leading up to score 2 and score 6, and the number of survival days were 3 and 3 respectively.
When the difference in the concentration of -oxo-olean-18-ene-28-oic acid (moronic acid) is compared with each other, 3
The administration of -oxo-olean-18-en-28-oic acid (moronic acid) partially shows a significant difference in the delay of viral expression. Explaining the meanings of the data at the portions marked with a, b, c, and d at the right end of the measurement data, a and c are ANOVA (Analysis of
It shows the safety which means re-measurement by Valience), and b and d determine the probabilities of the risk rate (P) in Student's t-test, and therefore, the difference in the superiority of the treatment group with moronic acid. Shows some data.

According to this, in the data at the portions marked with a and d, the risk factor (P) is 1% or less, and b
And c are 5% or less. As a result, it was confirmed that 3-oxo-olean-18-ene-28-oic acid (moronic acid) had a therapeutic effect on herpesvirus infections.

Next, after the skin cell tissue of a mouse was infected with the HSV-1 strain, the amount of virus transferred from the affected skin area and the skin to the nerve cells in the brain was measured 2 days, 3 days, and 4 days after the infection.
Day and 6 days post infection with control (water only) and 3
3-oxo-olean-18-ene-28-oic acid (moronic acid) was measured when it was administered to mice at various concentrations, and 3-oxo-olean-18-
The effect of en-28-oic acid (moronic acid) on the virus in the animal body was examined. In each experiment of Experiment 1 and Experiment 3, three mice were used as a group, and an average value was obtained using the number of viruses obtained from each mouse, and this was used as the virus amount. In the case of skin, 1 cm ² was cut from the affected area in the case of skin, and the whole amount of brain was used in the case of brain. After 2 ml of physiological saline was added to each tissue and homogenized, plaque assay (plaque measurement method) was carried out using the supernatant as a virus test solution and cultured Vero cells, and the number of plaques was expressed as the amount of virus. The skin / brain ratio is the value calculated as the ratio of the percentages obtained in skin and brain, respectively. The results are shown in Tables 5 and 6.

In the item of viral load (100%),
Numbers in parentheses such as (65.4%) are shown as (100%) with the control virus amount as 100%, and the virus residual rate was 65.4% when 10 mg / kg of moronic acid was administered. Therefore, this is indicated as (65.4%), and the numbers in the parentheses () in the corresponding portions are numbers corresponding to this.

[0042]

Table 5 Effect of viral load of 3-oxo-olean-18-en-28-oic acid (moronic acid) on the brain and skin of HSV-1 infected mice ━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━ Treatment viral load Skin / brain (mean ± standard deviation, PFU / organ) (ratio) ──── ─────────────── Days after infection Skin (log ₁₀ PFU) / cm ² Brain (log ₁₀ PFU) / total amount ━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━ Experiment 1 2 days Control 5.3 ± 0.8 ND (not detected) (100%) Moronic acid 4.9 ± 0.0 ND (not detected) ) (10mg / kg) (91.2%) 4 days Control 6.6 ± 0.1 2.9 ± 0.2 1 (100%) (100%) Moronic acid 6.1 ± 0.4 <2.4 ± 0.0> 1.12 (1mg / kg) (93.5%) <( 83.9%) Moronic acid 6.3 ± 0.3 <2.40 ± 0.0 > 1.15 (10mg / kg) (96.3%) <(83.9%) 6 days Control 6.4 ± 0.3 4.1 ± 0.3 1 (100%) (100%) Moronic acid 6.1 ± 0.2 3.5 ± 0.4 1.12 (1mg / kg) (95.3 %) (85.6%) Moronic acid 6.0 ± 0.1 2.3 ± 0.3 1.67 (10mg / kg) (94.2%) (57.0%)

[0043]

Table 6 Effect of viral load of 3-oxo-olean-18-ene-28-oic acid (moronic acid) on the brain and skin of mice infected with HSV-1 ━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━ Treatment viral load Skin / brain (mean ± standard deviation, PFU / organ) (ratio) ──── ─────────────── Days after infection Skin (log ₁₀ PFU) / cm ² Brain (log ₁₀ PFU) / total amount ━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━ Experiment 2 3 days Control 5.6 ± 0.1 ND (not detected) (100%) Moronic acid 5.5 ± 0.4 ND (not detected) ) (1mg / kg) (98.2%) Moronic acid 5.1 ± 0.4 ND (not detected) (5mg / kg) (91.5%) 6 days control 5.1 ± 0.3 3.7 ± 1.2 1 (100%) (100%) Moronic acid 4.8 ± 0.7 3.0 ± 0.6 1.16 ( 1mg / kg) (93.9%) (80.7%) Moronic acid 5.6 ± 0.2 2.7 ± 0.3 31.9 (5mg / kg) (109.0%) (72.2%) ━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━

It can be said from Tables 5 and 6 that two days after infection,
The number of each virus in the brain tissue and the skin tissue was 3-oxo-olean-18-ene-2, as compared with the control, on both the 3rd day, the 4th day, and the 6th day after the infection.
By treatment with 8-oic acid (moronic acid), the concentration of 3-oxo-olean-18-ene-28-oic acid (moronic acid) decreased almost in a dependent manner, and the virus on the skin of the affected area was decreased. It is clear that not only the growth is suppressed, but especially the amount of virus that goes to the brain is significantly suppressed. This also reduces the transfer of herpesvirus to the nervous system after infection and suppresses the proliferation, and suggests an effect on recurrent disease. That is, the effect of preventing the recurrence of herpes virus infection of moronic acid was confirmed.

Next, in order to examine the toxicity of moronic acid, control (water only) and 3-oxo-olean-18- were used for mice not infected with virus.
Toxicity experiments were carried out in the case where 1 mg, 5 mg, and 10 mg of en-28-oic acid (moronic acid) were administered per 1 kg of mouse body weight, respectively. Immediately before the start of the experiment, the mice used in the experiment were 18 to 21 g / 1 mouse, and 5 mice per group were used. None of the mice died by the end of the experiment. 3-oxo-olean-18-ene-28-oic acid (moronic acid) was orally administered 3 times daily.

[0046]

[Table 7] Toxicity of 3-oxo-olean-18-ene-28-oic acid (moronic acid) to non-infected mice ━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━ Treatment (mean weight ± standard deviation) ────────────────────────── 7 days 21 days 36 days Number of deaths ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Experiment 1 Control 19.5 ± 1.1 No experiment 22.2 ± 1.2 0/5 Moronic acid 19.8 ± 1.4 No experiment 22.6 ± 0.8 0/5 (1mg / kg) Moronic acid 19.9 ± 1.4 No experiment 21.0 ± 0.8 0/5 (5mg / kg) ─── ───────────────────────────────── Experiment 2 Control 20.2 ± 0.8 20.8 ± 0.8 No experiment 0/5 Moronic acid 20.1 ± 1.2 20.4 ± 1.0 No experiment 0/5 (10mg / kg) ━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

It can be seen from Table 7 that the body weight of 5 mice was determined by the control and 3-oxo-olean-18-ene-28-oic acid (moronic acid) at a maximum concentration of 10 mg / kg. There was almost no change with the lapse of time, and all five animals had no deaths after the lapse of 25 days. This means that the toxicity of moronic acid is
It proves that there is almost no for animal cells.

Finally, in order to examine the effect of moronic acid on the virus synthesis ability, J. Tradition
al Med. , 12 , 187-194 (1995), slot hybridization (slot-bl).
The amount of virus in the cultured cells was measured by ot hybridization. Herpesvirus HSV
-1 and AP ^r 1 strains were probed with animal cells (Vero Ce
(11) Immediately after adsorbing the virus to 11) and changing the respective concentrations (0, 5, 10, 20 μg / ml) of 3-oxo-olean-18-ene-28-oic acid (moronic acid) A chart displaying is shown in FIG. 2 (photograph). The intensity of the black portion in FIG. 2 indicates the approximate amount of each herpesvirus. In addition,
For comparison, the virus TK as a reference intensity ^- of DN
A copy number is shown at the right end.

From FIG. 2, it can be said that 3-oxo-olean-18-ene-28-euic acid (moronic acid).
The effect of the drug as a drug was that no inhibition of viral DNA synthesis was observed even when added up to 20 μg / ml. Therefore, the antiviral effect of moronic acid is not due to suppression of DNA synthesis, but it is due to a decrease in virus number. It was confirmed that there is.

[0050]

[Effect] As described above, from the experimental results shown in Table 1 to Table 7,
Oxo-olean-18-ene-28-oic acid (moronic acid) has an effect of suppressing the growth of viruses, and in particular, it also suppresses viruses that migrate to the nervous system, and therefore herpes lurking in the nervous system. Since the recurrence of the virus was suppressed, it was confirmed that in addition to the therapeutic effect on herpes virus infection, it has a remarkable preventive effect on recurrence and that there are almost no side effects. Moreover, 3-oxo-olean-18-ene-28-oic acid (3-Oxo-olean-18-en-28-o of the present invention is used.
ic acid) can be used in combination with acyclovir.

[Brief description of drawings]

FIG. 1 is an infrared spectrum of 3-oxo-olean-18-ene-28-oic acid.

FIG. 2. DN of herpesvirus HSV-1 and AP ^r 1 in animal cells by radiolabeled probe.
5 is a chart showing an A synthesis inhibitory action.

Continuation of the front page (72) Inventor Masahiko Kurokawa 1-8-8 Minamitaikoyama, Kosugi-cho, Imizu-gun, Toyama Prefecture (72) Inventor Tsuneo Namba 41-104 (72) invention, 2556, Gofuku-Suehiro-cho, Toyama City, Toyama Prefecture Person Kadota Shigetori 42-402, 2556, Suehiro Gofuku-cho, Toyama City, Toyama Prefecture

Claims (2)

[Claims] 1. A compound represented by the following formula (I): 3-oxo-olean-18-ene-28-
Oic acid (3-Oxo-olean-18-en-2
8-oic acid) as an active ingredient, a therapeutic agent for herpesvirus infections. 2. The following formula (I): 3-oxo-olean-18-ene-28-
Oic acid (3-Oxo-olean-18-en-2
8-oic acid) as an active ingredient, a herpesvirus infection recurrence preventive agent characterized by the above-mentioned.