JPH07188032A - Influenza-treating medicine - Google Patents

Abstract

PURPOSE:To prepare the subject treating medicine containing glycyrrhizin (salt) as an active ingredient, effective for the therapy of influenza infection, and reduced in side effects. CONSTITUTION:This treating medicine contains glycyrrhizin (salt) of the formula as an active ingredient. The glycyrrhizin is obtained by the extraction of glycyrrhiza. The therapeutic medicine is preferably orally or parenterally administered at a daily dose of 150-225mg for an adult.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a therapeutic drug for influenza, more specifically to a therapeutic drug for influenza containing glycyrrhizin and / or a pharmacologically acceptable salt thereof as an active ingredient.

[0002]

2. Description of the Related Art Influenza is an acute inflammation caused by infection with influenza virus (types A, B, C), and often extends to inflammation of the lower respiratory tract such as the bronchi rather than the upper respiratory tract. Influenza A compared to the other two
It is known to cause a global pandemic based on changes in viral surface antigens about every 10 years. Also, A type, B
It is well known that pneumonia and bronchitis occur together with influenza A, but especially in Japan, with the increase in the number of elderly people, the influenza A and B influenza epidemics have a great influence, and those with cardiopulmonary disease also have the same effect. There is a high risk of becoming severe.

Conventionally, attempts have been made to prevent influenza infection by vaccinating with influenza vaccine against such influenza. Although the vaccine is an important method for preventing the onset of infectious diseases, The fact is that it cannot be a sufficiently effective means because it cannot handle the variable antigenicity of the virus.

In addition, there is currently no effective chemotherapy for influenza patients.
Or, it is in a very limited state, and once the virus infection cannot be prevented with the above vaccine and the person is infected with influenza, they are treated exclusively with symptomatic antipyretic drugs, antitussives, etc. corresponding to various symptoms of influenza. At present, there is no choice but to rely on therapy, and development of an effective therapeutic drug for influenza infection has been desired.

On the other hand, glycyrrhizin, which is an active ingredient of licorice, is a compound of glycyrrhizic acid and two molecules of glucuronic acid, and has been known for a long time as a substance having an anti-inflammatory action, and a therapeutic agent for chronic active hepatitis. Is a compound widely used in clinical areas.

In recent years, the antiviral effect of glycyrrhizin on AIDS patients as well as the intracellular growth inhibitory effect of HIV, for example, 400 mg to 1600 mg (7.2 to 30.8 mg / k) of glycyrrhizin daily to AIDS patients.
g / day) for more than 1 month, HIV-1p
It has been reported that 24 antigens are significantly reduced. In addition, antiviral action of glycyrrhizin against herpes virus is also known, and it is further reported that it has a modifying action on host immune function such as an NK cell activity enhancing action and an interferon inducing action.

[0007] Recently, the activity of suppressor cells derived from burned mice is effectively suppressed by contra suppressor cells induced by glycyrrhizin, and furthermore, in burned mice, herpes simplex virus type 1 is induced by suppressor cells. It has been reported that glycyrrhizin and contra-suppressor cells induced by glycyrrhizin administration are highly effective against (HSV-1) opportunistic infections.

[0008] However, up to now, glycyrrhizin has not been studied as a therapeutic drug for influenza infection, and no report has been made on its effect.

[0009]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an agent for treating influenza which effectively treats influenza infection and has few side effects.

[0010]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has found that glycyrrhizin and / or its pharmacologically acceptable salts are effective in treating influenza. Heading out, the present invention has been completed.

That is, the present invention is a therapeutic drug for influenza containing glycyrrhizin represented by the following general formula (I) and / or a pharmacologically acceptable salt thereof as an active ingredient.

[0012]

[Chemical 2]

The present invention will be described in detail below. The therapeutic agent for influenza of the present invention contains glycyrrhizin and / or a pharmacologically acceptable salt thereof as an active ingredient. Glycyrrhizin is obtained by extracting from licorice. Moreover, you may use what is marketed. Also,
Examples of the pharmacologically acceptable salt include glycyrrhizin ammonium salt, glycyrrhizin alkali metal salt, glycyrrhizin choline salt and the like obtained by reacting glycyrrhizin with an inorganic or organic base in a molar ratio.

The dosage form of the therapeutic agent for influenza of the present invention is:
Although not particularly limited, generally one or several kinds of vehicles, carriers, excipients, binders, disintegrating agents, dispersants, lubricants, coating agents, preservatives, stabilizers, which are generally harmless in terms of formulation, , Tablets, powders, granules, by mixing with flavoring agents, coloring agents, etc.
It may be an oral preparation such as a capsule or a drench, and an injectable preparation such as a sterile solution or suspension. These can be manufactured using a conventionally known technique.

For example, the above-mentioned glycyrrhizin and / or a pharmacologically acceptable salt thereof and a binder such as corn starch and gelatin, an excipient such as microcrystalline cellulose, potato starch, a leavening agent such as sodium alginate, lactose and sucrose. A sweetening agent such as sugar can be prepared into powders, tablets, granules and capsules. In the case of preparing an injection, distilled water for injection, polyethylene glycol or the like is used as a solvent, or a dispersant, a buffer, a preservative, an antioxidant or the like may be added to the solvent, if necessary. Good.

The administration method may be oral or parenteral. The dose varies depending on the age, body weight, symptom, etc. of the patient, but generally, in oral administration, the amount of glycyrrhizin or a salt thereof per adult per day is preferably in the range of 25 to 500 mg, and more preferably, A desired effect can be expected by using it in the range of 150 to 225 mg. In addition, the dose for parenteral administration is suitably 40 to 400 mg per adult per day.

Further, the therapeutic drug for influenza of the present invention is
Not only is it effective for treating influenza infections, but it can also be used effectively for preventing influenza virus infections.

[0018]

[Action] The effect of glycyrrhizin for treating influenza infection was tested using a mouse influenza infection model.

The influenza virus used in the experiment was a mouse-adapted influenza virus (A ₂ Kumamoto strain, H ₂ N ₂ ) that was subcultured from mouse to mouse. Final standard EID ₅₀
The titer (50% hen egg infectivity titer) was 2.6 × 10 ⁸ / ml. This EID ₅₀ value corresponds to 10 ⁴ LD ₅₀ value in nasal spray infection. As the glycyrrhizin, an ammonium salt of glycyrrhizin (manufactured by Minophagen Pharmaceutical Co., Ltd.) was used.

(1) Antiviral action of glycyrrhizin (in vivo) Regarding the antiviral action of glycyrrhizin, an experiment in which the amount of infectious influenza virus was changed, an experiment on the dose dependence of glycyrrhizin, an experiment on the administration schedule of glycyrrhizin, lung consolidation (At the time of pneumonia, it is also referred to as cirrhosis due to the solidification of the tissue found in the lung.) Observation and the amount of virus propagated in the lung were measured.

In the experiments, 8-week-old BALB / c male mice (The Jackson Laboratory, Bar Harbor, ME) were used. All of these mice were infected with influenza virus by nasal spray infection.

Experiments in which the amount of infectious influenza virus was varied: 1 group of 2 mice each containing 15 mice each contained 10 mg / kg of glycyrrhizin dissolved in physiological saline, and the other 1 group (control group) contained physiological saline. 0.2 ml of water alone was intraperitoneally administered to each mouse, and one day after that, 100 LD ₅₀ (50) of influenza A ₂ (H ₂ N ₂ ) virus was given to all mice.
% Lethal dose, nasal spray infection. 1 day after infection and 4
After the day, 10 mg / kg of glycyrrhizin dissolved in physiological saline was administered to the group administered with glycyrrhizin before infection, and 0.2 ml of physiological saline was administered to the group administered with saline alone (control group). , Each was administered intraperitoneally.
Then, the life and death of the mice was observed daily until 21 days after the virus infection.

Further, the same experiment as above was carried out except that the amount of infectious virus was changed from 100 LD ₅₀ amount to 10 LD ₅₀ amount. The results are shown in FIGS. 1 and 2, respectively.
FIG. 1 is a graph showing the time-dependent changes in the survival rates of the glycyrrhizin-administered group and the control group when the infectious virus amount was 100 LD ₅₀ , and FIG. 2 shows the glycyrrhizin-administered group when the infectious virus amount was 10 LD _50. It is a graph which shows the time-dependent change of the survival rate of a control group.

From these results, when a virus as infected as a large amount of 100 LD ₅₀ was infected, the antiviral effect of glycyrrhizin was not observed (FIG. 1), but 10 LD ₅₀
A clear protective effect was confirmed at high viral infections.
That is, in the control group, 50% of the mice died on the 9th day after infection, and on the 14th day, all the mice died, whereas in the glycyrrhizin-administered group, all the mice survived and survived for 21 days or more (FIG. 2). .

It should be noted that this experiment is called a model experiment.
There is 100LD₅₀Very large amount of virus
We are also conducting experiments on infected mice, but 10
LD ₅₀Compared to normal infectious amount of virus infection
Given that the amount is sufficiently large,
Chin is fully effective against influenza virus infections
It can be said to work.

Experiment of Glycyrrhizin Dose Dependence One mouse group consisting of 15 mice each had 0.2 ml of physiological saline alone as a control group, and each other group had glycyrrhizin dissolved in physiological saline. 2.5 mg / kg to 20 m
The dose was changed between g / kg and administered intraperitoneally, and one day later, influenza A was administered to all mice.
₂ (H ₂ N ₂ ) virus was infected by nasal injection in a dose of 10 LD ₅₀ . One day and four days after infection, 0.2 ml of physiological saline alone was given to the control group, and the same amount of glycyrrhizin as the dose before infection was intraperitoneally administered to the group to which glycyrrhizin had been administered before infection. did. Then, the life and death of the mice was observed daily until 21 days after the virus infection.

The results are shown in FIG. FIG. 3 is a graph showing changes in the survival rate of mice with respect to the dose of glycyrrhizin when all mice in the control group died. From this result, when all mice in the control group died, 2.5 mg /
It can be seen that all mice died in the glycyrrhizin administration group of kg amount, and 100% of the mice survived in the glycyrrhizin administration group of 10 mg / kg amount or more.

As described above, the 10
The viral infectious dose, LD ₅₀ , is sufficiently large compared to the usual infectious dose, so in actual use, the dose of glycyrrhizin may be 2.5 mg / kg or less. Is also thought to act effectively against influenza virus infections.

Experiment on Glycyrrhizin Administration Schedule Each group of mice infected with 10 LD ₅₀ of virus was
Glycyrrhizin 10 mg / kg dissolved in physiological saline was administered at various schedules as shown in Table 1, and a control group (1 day before, 1 day, 4 days after infection, 0.2 mg of physiological saline was administered).
(ml administration) and its survival rate were compared. The results are shown in Table 1. The survival rate in Table 1 is the survival rate of the mice in each group when all the mice in the control group died.

[0030]

[Table 1]

As a result, when all the mice in the control group died, 1 day before infection and 2 days after infection in the glycyrrhizin administration group.
The survival rates of the group administered on the day before and the group administered on the first day and the fourth day after the infection were very high at 80% and 70%, respectively, and the remarkable anti-influenza infection protective effect of glycyrrhizin was observed even during the prevention. It was found to be expressed during treatment. In addition, the group to which glycyrrhizin was administered 1 day before, 1 day after, and 4 days after infection had 100% survival, and preventive and therapeutic administration was most effective. On the other hand, all mice died in the group to which glycyrrhizin was continuously administered from day 1 to day 5 after virus infection, indicating that the timing of administration is important for the anti-influenza infection protective effect of glycyrrhizin. .

Observation of Consolidation of Lungs One group of 2 groups of 15 mice each contained 10 mg / kg of glycyrrhizin dissolved in physiological saline, and the other group (control group) contained 0 physiological saline alone. .2ml increments, and each administered intraperitoneally, after one day, influenza a ₂ all mice (H ₂ N ₂₎ virus 10 LD ₅₀ amount, were intranasally injected infection. Glycyrrhizin was administered 1 day and 4 days after infection to glycyrrhizin before infection, and 10 mg / kg of glycyrrhizin dissolved in physiological saline was added to each group. Only 0.2m
Each of them was intraperitoneally administered.

Seven days after virus infection, the consolidation of the control group and the glycyrrhizin administration group was observed under a microscope. Mouse lung consolidation is based on Horsfal
The method (1) (Microbiol. Immunol, 26, 129, 1982) was used for the judgment according to the following criteria.

4 = Consolidation was observed in all lungs 3 = Consolidation site was 75 to 99% of lung 2 = Consolidation site was lung in 50 to 74
% 1 = Consolidation site is 25 to 49% of lung 0 = Consolidation site is less than 25% of lung

The results are shown in FIG. FIG. 4 shows growth of lung consolidation of mice infected with influenza virus in control group and glycyrrhizin-administered group (7 days after infection).
It is the graph which compared. The data of each group are the average value of 15 animals.

From these results, it was found that in the mice of the control group, consolidation was observed in a wide range of lungs, but in the group of glycyrrhizin administration, there was almost no consolidation in the lungs.

Measurement of Virus Amount Proliferated in Lung The virus amount in lung tissue of each of the above-mentioned glycyrrhizin-administered group and control group of influenza-infected mice was measured by 50% hen egg infectivity (EID) every day until 8 days after infection.
₅₀ ).

The results are shown in FIG. FIG. 5 is a graph showing changes over time in the amount of influenza virus in the lung tissue of mice of the control group infected with influenza virus and the glycyrrhizin administration group.

From these results, it was found that the virus amount was 3 × 10 ⁸ EID ₅₀ in the control group 3 days after infection, whereas the virus amount was as low as 1 × 10 ⁷ EID _{50 in the} glycyrrhizin-administered group. It was In the control group, a decrease in the viral load in the lung was observed from the 7th day to the 8th day of the infection, but in the glycyrrhizin-administered group, the lung virus completely disappeared 7 days after the infection.

From the results of the above experiments, it was confirmed that glycyrrhizin had an influenza infection protective effect. It was also confirmed that this influenza infection protective action clearly appears in both prophylactic administration and therapeutic administration.

(2) Virucidal Action and Viral Growth Inhibitory Action of Glycyrrhizin (In Vitro) Next, in order to know the action mechanism, the following two experiments were conducted in vitro.

Experiment on Direct Inactivating Effect of Glycyrrhizin against Influenza Virus 1 ml of phosphate buffer containing 2.6 × 10 ⁶ EID ₅₀ of influenza A ₂ (H ₂ N ₂ ) virus in 5 culture test tubes (PH 7.4) 1ml each, 10% formalin aqueous solution 1ml in one, 2% fetal bovine serum, 1% glutamine, 100U / ml penicillin, 100μg / ml streptomycin in another E contained
MEM (Eagl's Minimum Essential Medium) 1 ml
Glycyrrhizin 10 in the above medium for the remaining 3
Add 1 ml each of the mixture at a ratio of 100 and 1000 μg / ml, mix, and put in a culture tank at 37 ° C.
Culturing was carried out for 1 hour. After that, MDCK (Madin Darby canine
kidney) cells were used to measure TCID ₅₀ (50% tissue culture cell infectivity) / ml.

The analysis results are shown in FIG. In addition, in FIG.
The black bar graph shows the results of culturing by adding 1 ml of 10% formalin aqueous solution, and the white bar graph shows the results of 2% fetal bovine serum, 1% glutamine, 100 U / ml penicillin, and 1%.
EMEM containing 00 μg / ml streptomycin
The results obtained by culturing with 1 ml added are shown, and the line graph shows the relationship between the amount of glycyrrhizin added and the remaining amount of influenza virus after culturing.

As is clear from these results, glycyrrhizin did not have the effect of directly inactivating the influenza virus.

Experiment on Inhibition Effect of Glycyrrhizin on Influenza Virus Growth Using MDCK Cells MDCK cells were treated with 2.6 × 10 ⁴ T on three plates.
Infected with influenza A _{2 of} CID ₅₀ , one hour later,
Glycyrrhizin dissolved in physiological saline was applied to 2 of these plates at 10 μg and 100 μg per 1 ml of cells.
Was added in each proportion. Two days later, the amount of influenza virus in MDCK cells on three plates was determined by TCID.
_It was measured in units of ₅₀ / ml. The results are shown in Table 2.

[0046]

[Table 2]

From these results, the growth of influenza virus was not significantly suppressed by glycyrrhizin, and the fact that glycyrrhizin led the survival of influenza-infected mice was not the result of the direct suppression of growth of infectious virus by glycyrrhizin. found.

(3) Transfer experiment of immunocompetent cells into influenza-infected mice The results of the above-mentioned experiments suggest that the host immunity plays an important role in expressing the protective effect of glycyrrhizin. Various immunocompetent cells obtained from the administered normal mouse were actively transferred to the infected mouse, and the survival rate of the mouse was observed.

Two and four days before obtaining splenic mononuclear cells from mice, 10 mg / kg of glycyrrhizin dissolved in physiological saline was intraperitoneally administered to the mice. Similarly, instead of glycyrrhizin, mice were also prepared by intraperitoneally administering physiological saline alone 0.2 ml each 2 and 4 days before obtaining splenic mononuclear cells.

Next, splenic mononuclear cells were taken out from each of the above-mentioned glycyrrhizin-administered mouse and a mouse to which only physiological saline was administered. The cells (lymphocyte fraction) were separated, and each of the obtained fractions was added to 10 mice at 5 × 10 5 cells / mouse.
Actively populated at a rate of ⁶ . Two hours after that, all mice were infected with 10 LD ₅₀ of influenza virus, and life and death were observed every day until 21 days after infection.

As a control group for this experiment, 10 mice were infected with 10 LD ₅₀ amount of influenza virus and intraperitoneally administered with 0.2 ml physiological saline (one day before infection,
1 day later, 4 days later) was used.

The results are shown in FIG. FIG. 7 is a graph showing the time course of the survival rate of influenza-infected mice into which the various splenic mononuclear cell fractions were transferred. In the figure, □ is
The control group, ○ is a splenic mononuclear cell lymphocyte fraction-transferred mouse group derived from a glycyrrhizin-administered mouse, △ is a splenic mononuclear cell macrophage fraction-transfected mouse group derived from a glycyrrhizin-administered mouse, and ● is a physiological saline solution. The lymphocyte fraction-transferred mouse group derived from the administration mouse and the macrophage fraction-transferred mouse group derived from the physiological saline-administered mouse are shown respectively.

From these results, the macrophage fraction and lymphocyte fraction of the splenic mononuclear cells derived from the saline-administered mouse and the macrophage fraction of the splenic mononuclear cells derived from the glycyrrhizin-administered mouse were completely prolonging the survival in influenza-infected mice. Although no effect was observed, the lymphocyte fraction of splenic mononuclear cells derived from glycyrrhizin-administered mice showed a significant protective effect, and all influenza-transfected mice survived and died 21 days after infection. I understand that I was spared.

Further, the lymphocyte fractions of splenic mononuclear cells derived from the above-mentioned glycyrrhizin-administered mouse and from the saline-administered mouse were respectively classified into a nylon wool adhesive fraction (T cell fraction) and a nylon wool. Non-adhesive fraction (B
Cell fractions), and each fraction is
Active transfer was carried out at a rate of 5 × 10 ⁶ animals / animal. Two hours after that, all mice were infected with 10 LD ₅₀ of influenza virus, and life and death were observed every day until 21 days after infection. As a control group for this experiment, 10 mice were infected with 10 LD ₅₀ of influenza virus,
An intraperitoneal administration of 0.2 ml physiological saline (one day before, one day after, and four days after infection) was used.

The results are shown in FIG. FIG. 8 is a graph showing the time course of the survival rate of influenza-infected mice into which the various splenic mononuclear cell lymphocyte fractions have been transferred. In the figure, □ is a control group, ○ is a splenic mononuclear cell lymphocyte fraction T cell-transfected mouse group derived from glycyrrhizin-administered mice,
△ represents a group of B cell-transfected splenic mononuclear cell lymphocyte fraction B cells derived from a glycyrrhizin-administered mouse, ● represents a group of lymphocyte-fractionated T cell-transfected mouse splenic mononuclear cells derived from a saline-administered mouse, ▲ physiological Each represents a lymphocyte fraction B cell-transfected mouse group of splenic mononuclear cells derived from saline-administered mice.

As can be seen from these results, the control group administered with physiological saline all died on the 14th day after the infection, and the other three groups (B of the splenic mononuclear cell lymphocyte fraction derived from the saline-administered mouse). Cells and T cells, influenza-infected mice transfected with B cells of splenic mononuclear cell lymphocyte fraction derived from glycyrrhizin-administered mice obtained almost the same results as the control group, whereas those derived from glycyrrhizin-administered mice were obtained. 100% of the influenza-infected mice that had been transfected with T cells of the splenic mononuclear cell lymphocyte fraction were spared.

From the above experimental results, it was revealed that glycyrrhizin exerts a remarkable protective effect against influenza infection in mice through activation of splenic mononuclear cell lymphocyte fraction T cells.

Glycyrrhizin was found to exert a remarkable protective effect against infection in a mouse influenza infection model. It was also found that this effect is expressed through the T lymphocyte function activated by the administration of glycyrrhizin.

<Acute toxicity test of glycyrrhizin> LD ₅₀ of glycyrrhizin is as follows and shows low toxicity. (1) Oral administration (rat) 3 g / kg <(2) Subcutaneous injection 5% aqueous solution (mouse) 1873.3 mg / kg (3) Intravenous injection 2% aqueous solution (mouse) 682.5 mg / kg (4) Peritoneal injection 0 .2% aqueous solution (mouse) 225-244 mg / kg

[0060]

EXAMPLES Examples of the present invention will be described below.

[0061]

[Formulation Example 1] Tablets A mixture having the composition shown in Table 3 is made into tablets by a conventional method.

[0062]

[Table 3]

[0063]

[Formulation Example 2] Sugar-coated tablet A mixture having the composition shown in Table 4 is made into a sugar-coated tablet by a conventional method.

[0064]

[Table 4]

[0065]

[Formulation Example 3] Granules A mixture having the composition shown in Table 5 is made into granules by a conventional method.

[0066]

[Table 5]

[0067]

[Formulation Example 4] Injectable solution 200 mg of glycyrrhizin was dissolved in physiological saline to prepare 10
Make up to 0 ml and prepare an injection by a conventional method.

[0068]

[Formulation Example 5] Injections Glycyrrhizin 200 mg, Japanese Pharmacopoeia Aminoacetic acid 20
Dissolve 00 mg and cysteine 100 mg in physiological saline to make 100 ml and prepare an injection by a conventional method.

[0069]

EFFECTS OF THE INVENTION According to the therapeutic agent for influenza of the present invention, influenza infection can be effectively treated and side effects are small.

[Brief description of drawings]

FIG. 1 is a graph showing the time-dependent changes in the survival rate of the glycyrrhizin-administered group and the control group when the amount of infectious virus was 100 LD ₅₀ .

FIG. 2 is a graph showing changes over time in the survival rate of the glycyrrhizin-administered group and the control group when the amount of infectious virus was 10 LD ₅₀ .

FIG. 3 is a graph showing changes in the survival rate of mice against the dose of glycyrrhizin when all mice in the control group died.

FIG. 4 is a graph comparing the growth of lung consolidation (7 days after infection) in mice of the control group infected with influenza virus and the glycyrrhizin administration group.

FIG. 5 is a graph showing changes over time in the amount of influenza virus in the lung tissue of mice in the control group infected with influenza virus and the glycyrrhizin-administered group.

FIG. 6 is a bar graph showing the amount of influenza virus remaining after adding each solution and culturing, and a line graph shows the relationship between the amount of glycyrrhizin added and the amount of influenza virus remaining after culturing.

FIG. 7 is a graph showing changes over time in the survival rate of influenza-infected mice transfected with various splenic mononuclear cell fractions.

FIG. 8 is a graph showing the time course of survival rate of influenza-infected mice into which various splenic mononuclear cell lymphocyte fractions have been transferred.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Makiko Kobayashi 77550 Galveston City, Texas, USA Ferry Road 500, # 414 (72) Inventor Tokuichiro Utsunomiya 77551 Galveston City, United States Central City Boulevard 6315, # 117 ( 72) Inventor Kyozo Utsunomiya 4260 Shimotsuruma, Yamato City, Kanagawa Prefecture

Claims (1)

[Claims] 1. A therapeutic agent for influenza containing glycyrrhizin represented by the following general formula (I) and / or a pharmacologically acceptable salt thereof as an active ingredient. [Chemical 1]