JP2004352712A - Agent for preventing or treating influenza - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an influenza virus resisting agent highly safe for a human body. <P>SOLUTION: This agent for preventing or treating influenza contains a cromoglycic acid or its salt as an active ingredient. A method for preventing or treating the influenza comprises administering the agent to a human or an animal. A clinical symptom of the influenza which is targeted by the agent comprises a systemic symptom, such as pyrexia, malaise, cephalalgia, coxalgia, myalgia, and arthralgia, and a digestive system symptom, to say nothing of a respiratory symptom. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an anti-influenza virus agent. More specifically, the present invention provides a prophylactic or therapeutic agent for influenza characterized by containing cromoglycic acid or a non-toxic salt thereof as an active ingredient, and a prophylactic or therapeutic agent for influenza comprising administering these to humans or animals. Regarding treatment methods.

  Influenza, which is caused by influenza virus, is an infectious disease that repeats a large epidemic due to its strong transmission power even today, and has caused enormous damage to society. While various infectious diseases have been overcome, few drugs are effective and highly safe against influenza viruses, and the emergence of resistant viruses against them has begun to be regarded as a problem, and development of new anti-influenza drugs is expected.

  The influenza virus causes acute respiratory infections, and its clinical symptoms include systemic symptoms such as sudden fever, headache, arthralgia, and general malaise, as well as various respiratory symptoms such as nasal discharge and cough. It is characterized by high heat of over ℃. Normally healed in healthy people in about 1 to 2 weeks, but in infants, elderly people, patients with chronic diseases of the respiratory, circulatory and renal organs, patients with metabolic diseases such as diabetes and immune function decline, In many cases, secondary infections caused by bacteria and pneumonia are accompanied by death. In addition, not only local infection of the respiratory organs but also extremely serious cases such as severe nervous system complications represented by influenza encephalitis and encephalopathy have been reported. In addition, gastrointestinal symptoms such as abdominal pain, nausea / vomiting, and diarrhea may be observed, and special attention is required in children. A major feature to note in diagnosis is that fever and other systemic symptoms are more pronounced than respiratory symptoms.

  Currently, anti-influenza drugs clinically used for treating influenza include amantadine, zanamivir, oseltamivir and the like. Amantadine is effective against influenza A virus, but is ineffective against influenza B virus, easily causes nervous system side effects, and has a relatively early appearance of drug-resistant virus. Have been watched. Therefore, newly developed sialidase inhibitors zanamivir and oseltamivir are newly developed. Both type A and type B viruses are effective and have a low frequency of resistance, and their efficacy has been clarified in recent clinical trial results. However, these drugs also have problems in that they have yet to be used in clinical practice and that their safety for children and the elderly has not been established. In addition, current influenza vaccines have been shown to be effective in preventing illness and complications, but have not been able to completely protect against virus infection or influenza onset. As described above, at present, treatment methods for influenza are not yet well established, and it is the largest plague remaining in humanity. Requested.

  On the other hand, sodium cromoglycate is used clinically as an anti-allergic agent for bronchial asthma and allergic rhinitis, and its side effects are only slightly observed, such as cough sometimes seen when inhaling powder preparations. It is recognized as a highly safe drug. Sodium cromoglycate has not been reported to cause any serious side effects despite its clinical administration for many years, and it should be noted that its efficacy and safety in children have been established.

So far, there is no finding that sodium cromoglycate is effective against influenza, and Loveday et al. Showed no effect of sodium cromoglycate on influenza virus in in vitro and mouse infection experiments. Reported. In this document, in addition to influenza viruses (Influenza A / PR / 8 and Influenza A / Hongkong), five types of viruses (Vaccinia, Herpes simplex, Poliovirus 2, Parainfluenza 1, Respiratory syncytial) have been tested. No effect of sodium cromoglycate was found on the virus (see Non-Patent Document 1).
A document previously published as a reference to the above report is a report on the antiviral activity of sodium cromoglycate by Penttinen et al., Which reports that viruses different from influenza virus (Adenovirus 5, Echovirus 7, Herpes simplex and An experiment using Semliki Forest virus), which states that sodium cromoglycate does not affect virus growth but inhibits cell damage caused by the virus (see Non-Patent Document 2).

  In addition, there is also a patent document that describes that a solution containing cromoglycic acid and salbutamol is also effective for airway diseases such as bronchitis, nasal cavity and bronchial disorders caused by a common cold, in addition to asthma (Patent Documents) 1).

  In addition, the prior art literature describes that treatment with sodium cromoglycate was effective in alleviating the symptoms of upper respiratory tract infections. However, when examining the contents of the experiments shown in these documents in detail, out of 135 subjects exhibiting symptoms of upper respiratory tract infection, only about 10 patients showed clinical signs of influenza, and most Looking at the results of the common cold symptoms, especially the results of each clinical symptom, there is no effect of sodium cromoglycate on symptoms due to fever (chills and pain) which are characteristic of influenza. These documents only state that sodium cromoglycate is effective as a symptomatic treatment limited to clinical symptoms only in the upper respiratory tract in upper respiratory tract infections. Nothing is described as to whether it acts or not (see Patent Document 2 and Non-Patent Document 3).

D. Loveday et al., Brit. Med. J., II, 6086, p557-558 (1977). K. Penttinen et al., Brit. Med. J., I, 6053, p82 (1977). N. Aberg et al., Clin. Exp. Allergy, 26, p1045-1050 (1996). EP 0370632 WO95 / 05816

  The present invention has been made in view of such current state of the art, and an object of the present invention is to provide a highly safe anti-influenza virus agent for the human body. To provide existing drugs as prophylactic or therapeutic agents for influenza.

  As described above, the non-patent document 1 in which the result that sodium cromoglycate does not have an anti-influenza virus effect was described, and the fever symptom that is a characteristic symptom of influenza even when sodium cromoglycate is administered to a subject, Judging from the known circumstances of Patent Document 2 described above, in which the results of no effect were described, the idea of using sodium cromoglycate as an anti-influenza virus agent for the prevention or treatment of influenza was found by those skilled in the art. It was something I could not imagine. However, the present inventors have found that allergic patients who took sodium cromoglycate in clinical settings tended to be less susceptible to influenza than those who did not, and showed the effect in vitro and in mice. The present invention was completed by verifying in an infection experiment.

Therefore, according to the present invention, a prophylactic or therapeutic agent for influenza characterized by containing cromoglycic acid or a salt thereof as an active ingredient shown in the following [1] to [6], and a prophylactic or therapeutic agent for human or animal There is provided a method of preventing or treating influenza comprising administering.
[1] An anti-influenza virus agent comprising cromoglycic acid or a salt thereof as an active ingredient.
[2] An agent for preventing or treating influenza, comprising cromoglycic acid or a salt thereof as an active ingredient.
[3] A method for preventing or treating influenza, which comprises administering the prophylactic or therapeutic agent according to [2] to a human or animal.
[4] A method for preventing the onset or alleviating clinical symptoms of influenza, comprising administering the prophylactic or therapeutic agent according to [2] to a human or animal.
[5] The method according to [4], wherein the clinical symptoms of influenza are fever, malaise, headache, back pain, muscle pain, arthralgia, abdominal pain, vomiting, diarrhea and / or anorexia.
[6] The agent or method according to [1] to [5], wherein the cromoglycic acid or a salt thereof is cromoglycate sodium salt.

According to the present invention, an anti-influenza virus agent highly safe for the human body, more specifically, a prophylactic or therapeutic agent for influenza characterized by containing cromoglycic acid or a salt thereof as an active ingredient, and There is provided a method for preventing or treating influenza comprising administering to a human or animal.
The clinical symptoms of influenza to be targeted in the present invention include, in addition to respiratory symptoms, general symptoms such as fever, malaise, headache, back pain, muscle pain, joint pain, and gastrointestinal symptoms.

  The influenza virus according to the present invention is an RNA virus belonging to the family Orthomyxoviridae, and has a type A, a type B and a type C due to a difference in antigenicity between a nucleoprotein (NP) and a matrix protein (M1 and M2) present inside the virus. are categorized. Furthermore, influenza virus has an envelope composed of a host cell-derived lipid bilayer, on the surface of which hemagglutinin (HA, hemagglutinin) and neuraminidase (NA, sialidase) protrude as spike-like glycoproteins. The two types of spiked glycoproteins are classified into many subtypes depending on the difference in antigenicity. In the influenza A virus, 15 types of hemagglutinin (H1 to H15) and 9 types of neuraminidase (N1 to N9) with different antigenicity are known.

Influenza is an epidemic acute respiratory infection due to influenza virus infection. Influenza recurs every winter and concentrates in a short period of time, causing millions of patients to occur.
As a clinical picture, after a latent period of 1 to 3 days, many patients suddenly have a fever, and are characterized by high fever and systemic symptoms such as general malaise, headache, back pain, muscle pain, and joint pain. Almost simultaneously or slightly later, respiratory symptoms such as nasal discharge, nasal obstruction, sore throat, hoarseness, cough, sneezing, and sputum appear, accompanied by severe feelings that force the patient to sleep for several days . The fever rapidly rises from the onset of the disease, shows the highest body temperature on the first to third days, and often shows 38 to 39 ° C. In addition, gastrointestinal symptoms such as abdominal pain, vomiting, diarrhea, and anorexia may be present. A healthy person usually cures in about 1 to 2 weeks.

  The clinical symptoms of influenza that is the subject of the present invention include respiratory symptoms (upper respiratory inflammatory symptoms) such as nasal discharge, nasal obstruction, sore throat, hoarseness, cough, sneezing, and sputum. It also includes systemic symptoms such as malaise, headache, backache, muscle pain, joint pain, and gastrointestinal symptoms such as abdominal pain, vomiting, diarrhea, and anorexia.

The cromoglycic acid used in the present invention is a known compound 5,5 ′-[(2-hydroxytrimethylene) dioxy] bis (4-oxo-4H-1-benzopyran-2-carboxylic acid).
Salts of cromoglycic acid include, for example, non-toxic salts such as salts with alkali metals such as sodium or potassium, salts with alkaline earth metals such as calcium or magnesium, ammonium salts and quaternary ammonium salts. However, among these, sodium cromoglycate is particularly preferred.

  The prophylactic or therapeutic agent for influenza of the present invention is obtained by blending the active ingredient cromoglycic acid or a non-toxic salt itself or a pharmaceutically acceptable carrier, excipient or diluent with a tablet, capsule or granule. , Powders, syrups, ointments, solutions, suspensions, aerosols, troches and the like.

Examples of the excipient include lactose, sucrose, glucose, sorbitol, mannitol, starch, dextrin, crystalline cellulose, carboxymethylcellulose, gum arabic, gelatin, aluminum silicate, calcium phosphate, calcium carbonate, calcium sulfate, and the like.
The prophylactic or therapeutic agent for influenza of the present invention may optionally contain a binder, a disintegrant, a lubricant, a stabilizer, a flavoring agent (sweetener, acidulant, flavor, etc.), an antioxidant, a preservative (for example, It may contain usual additives such as benzalkonium chloride), dyes, suspending agents, diluents, and formulation solvents (eg, water, ethanol, glycerin, fatty oils, etc.).

  The prophylactic or therapeutic agent for influenza of the present invention can be administered orally or parenterally. However, cromoglycic acid or a salt thereof is the main route of transmission of influenza virus. It is preferable to administer by an administration method that can be delivered to the respiratory tract or lung tissue, for example, intranasal administration, nasal administration, pulmonary administration, oral administration, and the like. Also, intranasal administration and administration by inhalation through the mouth can be combined.

  Cromoglycic acid or a salt thereof may be administered in the form of a solution or suspension or as a dry powder. Solutions and suspensions are aqueous, for example, water alone (eg, sterile or pyrogen-free water), or water and a physiologically acceptable co-solvent (eg, ethanol, propylene glycol, PEG 400, etc.). Polyethylene glycol). Such solutions or suspensions may contain other excipients, for example preservatives (eg, benzalkonium chloride, benzethonium chloride, chlorobutanol, etc.), solubilizers / surfactants such as polysorbate (eg, Tween 80). , Span 80, lecithin, etc.), buffers (eg, boric acid, borax, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc.), isotonicity regulators (eg, sodium chloride, etc.), stabilizers (eg, For example, sodium edetate, glycerin, propylene glycol, polyvinylpyrrolidone, macrogol 600, macrogol 4000, etc., cooling agents (eg, l-menthol, dl-camphor, d-borneol, fennel oil, peppermint oil, peppermint water) And the like) may further contain an absorption promoter and a thickener. The suspension may further contain a suspending agent (eg, microcrystalline cellulose, sodium carboxymethyl cellulose).

Solutions or suspensions of cromoglycic acid or its salts are applied directly to the nasal or oral cavity by conventional means, for example with a dropper, pipette or spray. The formulation may be provided in a single dose or in multiple doses. In the case of an atomizer, this can be done, for example, by means of a metering atomizing spray pump.
For administration to the respiratory tract or lungs, solutions or suspensions may be treated with chlorofluorocarbons (CFC, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, heptafluoropropane), carbon dioxide or other suitable gas. This can be done with an aerosol formulation in pressurized packs with a suitable propellant. The dose of the drug can be controlled by providing a metering valve.

When cromoglycic acid or a salt thereof is in the form of a dry powder, it may be used alone or in the form of a powder mixture mixed with a suitable powder base such as lactose, mannitol, starch, hydroxypropylmethylcellulose or polyvinylpyrrolidone. May be provided.
The powder composition may be in unit dosage form, for example, in a capsule or cartridge of gelatin, or in a container of plastic or blister to which the powder can be administered using an inhaler, or for example, a container for powders. It may be provided in the form of multiple doses to be contained in As a method of administration, it can be inhaled from the mouth by a dry powder inhaler or administered intranasally. The inhaler may be for a single dose or for a multiple dose.

  Cromoglycic acid or a salt thereof may or may not be micronized. In formulations for administration to the respiratory tract or lungs, including intranasal formulations, the compound generally has a small particle size, for example, 5 microns or less. Such a particle size is obtained by methods known in the art, such as by micronization. When desired, certain formulations with sustained release of the active ingredient may be employed.

  Cromoglycic acid or a salt thereof can also be used in combination with other therapeutic agents, for example, anti-influenza drugs such as amantadine, rimantadine, ribavirin, zanamivir, oseltamivir. The individual components of such combinations may be administered sequentially or simultaneously, in separate or combined pharmaceutical formulations.

  The animals to which the method of the present invention is applied are ordinary domestic animals and pets, and include mammals such as cattle, pigs, horses, sheep, goats, dogs, cats, and birds such as poultry.

  The amount of cromoglycic acid or a salt thereof effective for the prevention or treatment of influenza varies depending on the type of the target human or animal, the condition such as the weight and age of the recipient, and the administration method. It can be used at a rate of 1 to 100 mg / kg / day, preferably 0.1 to 10 mg / kg / day. For example, when applied directly to the nasal cavity or oral cavity, 0.1 to 100 mg, preferably 1 to 100 mg / time. It is desirable to administer 20 mg 1 to 10 times, preferably 2 to 5 times per day.

  When the present invention is administered as an influenza prophylactic agent, it can be started during the influenza epidemic season and increased or decreased as needed. That is, the drug is administered when an influenza epidemic is imminent, before or after an action that may cause infection, and before the onset of influenza.

  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Preparation of Inhalation Capsules Hard capsules containing 20 mg of ultrafine sodium cromoglycate powder are prepared. It is commercially available under the trade name Intal® (Fujisawa Pharmaceutical Co., Ltd.).

Preparation of inhalation liquid Sodium cromoglycate 20mg
2 ml of purified water
The above ingredients are mixed and injected into a plastic ampule to form a formulation. It is commercially available under the trade name Intal® Inhalation Liquid (Fujisawa Pharmaceutical Co., Ltd.).

Preparation of aerosol Sodium cromoglycate 200mg
Purified water 10ml
The above components are mixed and filled in an aluminum container to form an aerosol (including heptafluoropropane (HFA-227), polyvinylpyrrolidone K30, and macrogol 600 as additives). It is commercially available under the trade name Intal® Aerosol A (manufactured by Fujisawa Pharmaceutical Co., Ltd.).

Preparation of nasal drops sodium cromoglycate 190mg
9.5 ml of purified water
The above-mentioned components are mixed to give a nasal solution (nasal metered dose inhaler) in a plastic container by a conventional method (containing sodium edetate and benzalkonium chloride as additives). It is commercially available under the trade name Intal® Nasal Drops (Fujisawa Pharmaceutical Co., Ltd.).

Effect of sodium cromoglycate on influenza virus infection (in vitro test)
Quantitative evaluation of in vitro virus infection of cells is performed by measuring the activity of the intracellular enzyme lactate dehydrogenase (LDH) in the culture supernatant to quantify the number of cells with damaged cell membranes. The measurement was carried out by a neutralization activity method, which is a method for quantitative measurement.

I. Test Materials Influenza viruses were used by diluting the five strains shown in Table 1 so that the virus concentrations (HAU) shown in Table 1 were obtained.

HAU was the highest dilution of virus required for guinea pig erythrocytes to agglutinate and was measured as follows (J. Biol. Chem. 260, 1362-1365 (1985)).
Purified influenza virus solution, diluted to 2 ¹⁰ times with 0.01% gelatin-containing PBS, and further repeated byebye diluted wells at (96 U-Bottom, Falcon) above (50μl + 50μl). 50 μl of a PBS solution containing 0.5% (V / V) guinea pig erythrocytes was added, and the mixture was allowed to stand at 4 ° C. for 2 hours. Then, aggregation of erythrocytes was observed. The same experiment was conducted using PBS containing 0.01% gelatin containing no virus as a control.

  For a final concentration of 10, 3 and 1 mM, the sodium cromoglycate solution was prepared by dissolving sodium cromoglycate in purified water so as to have a concentration of 100 mM, and sterilizing by filtration through a 0.2 μm filter. For final concentrations of 0.3 and 0.1 mM, the 100 mM solution was diluted to 30 mM and 10 mM solutions, respectively.

II. Test Method Using a 96-well culture microplate (manufactured by Nunc), put 2 × 10 ⁴ MDCK cells in 100 μl of MEM medium containing 10% fetal bovine serum per well at 37 ° C., 5% After culturing for 16 to 24 hours in the presence of CO ₂ , it was confirmed under a microscope that a monolayer was formed over the entire well.
Next, each virus was diluted with a serum-free medium to the virus concentration shown in Table 1, and the final concentration of sodium cromoglycate was 10, 3, and 100 μl each in a medium containing these viruses and a medium containing no viruses. Sodium cromoglycate solution (10 μl, 3 μl or 1 μl) was added to 1, 0.3 and 0.1 mM. Note that 10 μl of purified water was added to a control containing no drug.
After leaving them at room temperature for 30 minutes, 100 μl was added to the cells washed three times with 100 μl of serum-free medium, and the cells were allowed to stand at 34.5 ° C. for 5 hours in the presence of 5% CO _2. Infected. After completion of the infection, the cells were washed twice with 100 μl of serum-free medium, 100 μl of MEM medium containing 5% fetal bovine serum was added, and the cells were cultured at 34.5 ° C. in the presence of 5% CO ₂ for 20 hours.
After completion of the culture, 10 μl of 5 × PBS was added to another new microplate (96 Flat-Bottom Maxisorp, Nunc), and 40 μl of the supernatant after the culture was added and mixed to make a total volume of 50 μl. After the plate was allowed to stand at 37 ° C for 5 minutes, 50 µl of an LDH measuring reagent (LDH Linear-Neo, manufactured by Sinotest, one bottle of a coloring reagent dissolved in 3.0 ml of substrate solution) was added, mixed, and allowed to stand at 37 ° C for 10 minutes. Was placed. The reaction was stopped by adding 100 μl of 0.5N hydrochloric acid, and the LDH activity was measured by measuring the absorbance of each well at a measurement wavelength of 550 nm and a control wavelength of 630 nm using a microplate reader (MTP-32, Corona).

III. Test results The LDH activity when treated with 0 mM sodium cromoglycate was defined as 100%, and the LDH activities by the sodium cromoglycate treatment at each concentration were relatively expressed and compared. The results are shown in FIG. When the virus was directly treated with 10 mM sodium cromoglycate, the effect of suppressing virus infection to MDCK cells was observed for all virus strains. In particular, H3N2 type A / Memphis / 1/71 and A / Aichi / 2/68 exhibited a 55-65% suppression effect on non-treatment.

Influenza infection protection test by sodium cromoglycate in mice (in vivo efficacy evaluation test)
I. Test Method Influenza virus A / PR / 8 strain was inoculated into embryonated chicken eggs, cultured at 37 ° C., and the collected allantoic fluid was used as a stock solution in the following experiments. The measurement of the virus titer was performed by a plaque assay according to a standard method (Virus Experiment Protocol, Medical View, Inc., p157, 1995). After anesthesia of 4-week-old female BALB / c mice (8 mice per group), 20 μl of a virus suspension (5 × 10 ² PFU / mouse) was intranasally inoculated. Administration of sodium cromoglycate (32 mg / kg) was performed nasally, and the dosing schedule was once 4 hours after infection, twice daily on days 1 and 2, and once on day 3 of infection. 6 times in total. The change in body weight and the course of life and death were observed until 14 days after infection, and the drug efficacy was evaluated based on the life extension effect.

II. Test Results The results of the survival rate are shown in FIG. Looking at the survival rate on day 14 of infection, the group administered sodium cromoglycate (32 mg / kg) showed a survival rate of 62.5% compared to 25% of the non-drug control group, indicating that the protective effect against influenza virus infection was reduced. Admitted.

The effect of suppressing influenza virus infection to cells is shown by the relative value of LDH activity. The protective effect is shown by the survival rate of the mice infected with influenza virus.

Claims (7)

  An anti-influenza virus agent comprising cromoglycic acid or a salt thereof as an active ingredient.   An agent for preventing or treating influenza, comprising cromoglycic acid or a salt thereof as an active ingredient.   An agent for preventing the onset or clinical relief of clinical symptoms of influenza, comprising cromoglycic acid or a salt thereof as an active ingredient.   A method for preventing or treating influenza, comprising administering an effective amount of cromoglycic acid or a salt thereof to a human or animal.   A method for preventing the onset or alleviating clinical symptoms of influenza, comprising administering an effective amount of cromoglycic acid or a salt thereof to a human or animal.   The agent or method according to claim 3 or 5, wherein the clinical symptoms of influenza are fever, malaise, headache, back pain, myalgia, arthralgia, abdominal pain, vomiting, diarrhea and / or anorexia. The agent or method according to any one of claims 1 to 6, wherein cromoglycic acid or a salt thereof is cromoglycic acid sodium salt.

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