WO1993023056A1

WO1993023056A1 - Preventive or therapeutic agent for infectious diseases containing mizoribine as active ingredient

Info

Publication number: WO1993023056A1
Application number: PCT/JP1993/000615
Authority: WO
Inventors: Yoshinori Kosugi
Original assignee: Asahi Kasei Kogyo Kabushiki Kaisha
Priority date: 1992-05-15
Filing date: 1993-05-11
Publication date: 1993-11-25
Also published as: JPH05310578A

Abstract

A preventive or therapeutic agent for infectious diseases caused by viruses belonging to the human-infecting family Orthomyxoviridae or Paramyxoviridae, which agent contains mizoribine (chemical name: 4-carbamoyl-1-β-D-ribofuranosylimidazolium 5-oleate) as the active ingredient. As mizoribine has a potent antiviral activity against specified RNA viruses belonging to the abovementioned families even when administered in a minute amount, it can provide a useful preventive or therapeutic agent for infectious diseases caused by these viruses.

Description

Description Preventive or therapeutic agent for infectious diseases containing mizoribine as an active ingredient

The present invention relates to a virus belonging to the family Orthomyxoviridae or Paramyxoviridae, which comprises Mizoribine (Miz 0 ribine: chemical name; 4 1-Rubamoyl-1 1-D-ribofuranosyl-Imidazolium-5-year-old) as an active ingredient. And a prophylactic or therapeutic agent for infectious diseases caused by Conventional technology

Mizoribine is a nucleic acid-related substance that was found in the culture solution of Euvenicillium breff Eldianum M-2166 (FERM P-114) belonging to the genus Euvenicillium. It is a weakly acidic substance that decomposes brown in the vicinity of C. Various methods for its production are known, such as the fermentation method and the chemical synthesis method using the above strains (J. Antibiotics, 27 , (10), 775 (1974), Chem. Pharm. B υ11.1, 23, 245 (1975). JP-A-48-56894, JP-A-50-12 No. 1275, Japanese Patent Application Laid-Open No. 50-121276, and Japanese Patent Publication No. 51-1693).

Mizoribine has an immunosuppressive effect, for example, it is useful for suppressing rejection in renal transplantation.Typically, the initial dose per kg of body weight is 2 to 3 mg, and the maintenance dose is 1 to 2 mg. Anhydrous crystals are used as tablets of Bredinin (registered trademark: manufactured by Asahi Kasei Kogyo Co., Ltd.), which are orally administered in an equivalent amount per day. Mizoribine is known to exhibit strong anti-viral activity against vaccinia virus, a DNA virus (Japanese Patent Application Laid-Open No. 48-56894). It has been reported that it has no effect on the infectious type 1 parainfluenza HVJ virus ゃ poliovirus (J. Antibiotics, 27, (10), 775 (1974)). Issues to be solved

In recent years, the development of chemotherapeutic agents for herpes virus and lys virus has made it possible to provide causal therapy for viral diseases. Orthomyxoviridae (for example, influenza A, B, and C influenza viruses) or Paramyxoviridae (for example, measles virus, mumbus virus, parainfluenza virils or respiratory virus). Various types of RNA viruses, such as RS virus, are the most remarkable viruses as causative viruses for multiple infectious diseases including viral respiratory infections. Clinical differentiation of the pathogenic viruses of these two viral families is limited to measles with characteristic rashes, mumps with typical parotid swelling and influenza in extreme epidemics Difficult. Therefore, antiviral agents having a broad spectrum for these viruses are eagerly awaited (chemotherapy, Vol. 7, No. 10 1991).

To date, the above-mentioned orthomyxoviridae and paramyxoviridae have been treated with amanthodin hydrochloride, rimantadine hydrochloride and rivapirin (1-yS-D-ribofuranosysy I—1, 2, 4-triazo 1 e—3 -carbox am ide) is reported to be effective (J. Med., 281: 578-584 (1969), 381: 1443-1447 (1983), J. pedtar., 1 17, 313 1 320 (1 990)). However, the former two are effective only for influenza A in the Orthomyxoviridae family, and are ineffective for viruses in the B and C strains of Influenza and Paramyxoviridae. Ribavirin, on the other hand, is Has broad antiviral activity and is approved as the only antiviral agent against RS virus in the United States, but has various restrictions such as strong cytotoxicity and necessitating long-term treatment by aerosol inhalation . Therefore, a safer preparation with stronger antiviral activity has been desired. Means for solving the problem

As mentioned earlier, mizoribine is effective against some DNA viruses, but not against RNA viruses! : J. Antibiotics, 27, (10), 775 (1974)] surprisingly, against the virus belonging to the myzoribine-powered, human-infectious Balamixoviridae or orthomyxoviridae family. As a result, they have found that they show stronger antiviral activity than the conventionally used rivapirin and have low cytotoxicity. As a result, it has become possible to provide a better prophylactic or therapeutic agent than ribavirin.

The present invention has been completed based on the above findings, and is an agent for preventing or treating an infectious disease caused by a virus belonging to the human infectious orthomyxoviridae or paramyxoviridae family containing mizoribine as an effective component.

Mizoribine of the present invention, already filed by the immunosuppressive agent commercially available, acute toxicity (LD _5.) Is orally to male and female mice> 4883 mg / kg, sc> 4883mg / kg, i.v.> 3042 mg / kg,> 2800 mgZkg intramuscularly,> 310 OmgZkg orally to male rats,> 41 61 mg / kg subcutaneously,> 2572 mgZkg intravenously,> 2800 mgZkg intramuscularly, orally to female rats >2847mgZkg,> 3795mgZkg subcutaneously,> 2608mgZkg intravenously,> 280OmgZkg intramuscularly, and its toxicity to various tissue culture cells is as follows. / 23056 P

Four

Cytotoxicity

experimental method

Derbecco Minimum Essential Medium (hereinafter abbreviated as D-MEM medium) and 10% fetal bovine serum are added to the medium. Cells and MDCK cells (above, manufactured by Omoto Pharmaceutical Co., Ltd.) were cultured. G—HeLa cells (Anti-mi-crobial Agentsent C hemother apy Vol. 36, No 2., 4 35-4 39.1 992) contain more glucose 1 in this medium. The cells were cultured using a medium supplemented with 6 gZl. 9 Dispense each cell 2 x 10 ce 1 l / we 11 into a 6-well plastic plate (Falcon 3402), add a medium containing mizoribine diluted to a predetermined concentration, and then in 37, 5 The cells were cultured for 3 days in a carbon dioxide culture device. Toxicity to cells was examined by the MTT method (metabolism, Vo 1.28,. No. 12, page 33, 1991).

G—He La cells (cells derived from human cervix)> 500 fig / m 1 Ver0 cells (cells derived from african green monkey kidney)> 500 gZm1 MDCK cells (cells derived from canine kidney cell)> 50 Ojug / ml This mizoribine is easy to use as an oral preparation (registered trade name: Bredinin tablet), and is appropriately administered in oral preparations such as capsules and granules, suppositories, and aerosol inhalation It can be formulated as a preparation, transdermal absorption preparation or injection by a conventional formulation technique.

Formulations for oral administration include, for example, anhydrous lactose, crystalline cellulose, dextran, starch, etc. as excipients, carboxymethylcellulose sodium, methylcellulose, ethylcellulose, etc. before conjugation, and disintegrants such as ruboxymethylcellulose, calcium carbonate, etc. Lubricants such as methylcellulose and stearic acid, magnesium stearate, talc, etc. Oral preparations such as tablets and capsules can be prepared by a selective combination or a conventional method.

In addition, for example, a solution for aerosol and a preparation for injection administration can be produced by dissolving mizoribine in an aqueous solvent, and the concentration of mizoribine in the solution is 0.1 to 10 WZV based on the aqueous solvent. %, Preferably about 1 to 10%. Particularly for preparations for injection administration, the preparations may be prepared by sterilizing or removing bacteria after adjustment. Examples of the aqueous solvent include distilled water for injection and sterilized purified water.

Further, additives which are usually appropriately selected and used in liquid preparations, such as buffer for pH adjustment (for example, phosphate buffer, borate buffer, citrate buffer, tartrate buffer, acetate buffer, etc.), etc. Tonicity agents (eg, sorbitol, glycerin, polyethylene glycol, propylene glycol, glucose, sodium chloride, etc.), stabilizers (eg, sulfites, bisulfites, metabisulfites, etc.), preservatives / disinfectants ( For example, benzalkonium chloride, paraoxybenzoic acid esters, benzyl alcohol, parachloromethaxinol, chlorcresol, phenethyl alcohol, sorbic acid or its salts, thimerosal, chlorobutanol, etc., chelating agents ( For example, sodium edetate, sodium citrate, condensed sodium phosphate Etc.), viscosity formulations (such as polyvinylpyrrolidone, methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, polyvinyl alcohol, sodium port Riakuriru acid) and the like can and child blended additives in the usual amount to be employed.

Examples of the human infectious virus belonging to the orthomyxoviridae family include viruses belonging to the genus Influenza virus, such as influenza A, B, and C viruses belonging to the genus influenza virus. Viruses belonging to the human infectious paramyxoviridae family include those belonging to the genus Paramyxovirus, the genus Morbidillus, and the genus Newvirus. For example, type 2 and 3 parainfluenza virus belonging to the genus Paramyxovirus, mumps virus, measles virus belonging to the genus Morbivirus, and RS virus belonging to the genus Pneumovirus. Infectious diseases caused by these viruses are defined as diseases caused by these viruses such as common cold symptoms, bronchial inflammation, lung inflammation, measles-like rash, parotid swelling, meningeal inflammation, encephalitis, etc. The names of the onset diseases include influenza, measles, respiratory tract infections, respiratory infections, subacute sclerosing panencephalitis, aseptic meningitis, parotitis, and common cold.

In addition, the amount of mizoribine used in the present invention is, for example, 1 to 20 mg / kg (body) as an adult 1 B dose in 1 to 3 divided doses a day, or 5 to 50 mg mg as appropriate. It may be administered by inhalation as a contained aerosol preparation. For example, preferably, oral administration of mizoribine to adult patients weighing 50 to 6 Okg: 30 mg of mizoribine at a time using 25 mg or 5 Omg tablets, divided into 3 times a day, or 5 mg 3 to 18 hours Z day aerosol administration. Example

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Example 1

Antiviral activity against measles virus (Mease1svirus) belonging to the Paramyxoviridae family

The antiviral activity was measured according to a plaque reduction method (Plaquere duction method: Antimi crobial Agegents and Chemopheral Vol. 36, No. 2, 435-439.1992). Specifically, D-M was prepared by adding Vero cells to 24-well plastic plates (Falcon 3702) and adding 10% fetal bovine serum. After culturing for 18 hours at 37 ° C in 5% CO2 incubator using EM medium to form monolayer cultured cells, remove the culture solution and remove 20 ~ 10 O PFU (p 1 a qu efo r ng un its) measles virus Sugiyama strain (Meas 1 es Virus S υ giyamastrain; Ant im icrobia 1 Agents and d Ch emo thera py V o 1.36, No 2., 435-439.1992) Inoculated. A D-MEM medium containing a prescribed concentration of anhydrous crystalline mizoribine, 2% fetal bovine serum, and 0.75% methacel (Methocel A-4M premium: manufactured by Dow Chemical Company, hereinafter abbreviated to metcell). After 0 ml was added and the cells were cultured in a 35 ° 5% carbon dioxide gas culture apparatus for 2 days, the cells were fixed with formalin, stained with crystal violet, and the number of plaques was measured under a microscope. The antiviral effect (EC ₅ ) was calculated from the measured number of plaques according to the following equation.

Anti-virus effect _{(EC 5.) = 1 0} A

(Where A is the following equation

50% -B

A = 1 o g 2 x + 1 o g D

C-B

(Where B is the maximum inhibition below 50%, C is the minimum inhibition above 50%, and D is the maximum drug concentration below 50%). Value)

The above results are shown in Table 1 below. Table 1

Example 2

Antiviral activity against paramyxoviridae type 3 parainfluenza virus (ParainI1 uenzaa ΐype3C243stranin)

Antiviral activity was measured as described in the previous section. Specifically, Vero cells were cultured for 18 hours at 37 ° C and 5% CO 2 in a D-MEM medium containing 10% fetal bovine serum on a 24-well plastic plate. (4) After the formation of the culture cells, remove the culture medium and remove 20 to 10 PFU of the PFU type 3 paraline bruenza virus C243 strain (P arainf 1 uenzatye 3C243H-1 strain; Antimicrobia 1 Agentsand 36, No 2., 435-439.19.92) was inoculated. 1.0 ml of D-MEM medium containing specified concentration of mizoribine, 2% fetal calf serum and 0 75% medocel 35 with the addition of 5% carbon after formalin fixed after 2 days of culture in a gas culture apparatus Crystal Bio Re' preparative stained conducted since _c measured number of plaques were measured bra one click number under a microscope Example 1 Similarly, anti-viral effect and (EC _5.) the Chico 0

The above results are shown in Table 2 below.

Table 2

Example 3

Antiviral activity against RS virus belonging to Paramyxoviridae The antiviral activity was measured in the same manner as in the previous section. Specifically, G-HeLa cells were placed in a 24-well plastic plate at 37 ° C and 5% CO 2 in a D-MEM medium containing 10% fetal bovine serum and 16 g / 1 glucose. After culturing for 18 hours to form 80% monolayer cultured cells, remove the culture solution and remove 20 to 100 PFU of RS virus long strain (Anti micr obial Agen tsa nd Ch emo theray Vo l) . 36, No 2., 435-439. 1 992). D-MEM medium containing the specified concentration of mizoribine, 2% fetal bovine serum, 0.75% methocel and 16 g / 1 glucose 1. Add Oml, and incubate at 35 ° C and 5% CO2 After culturing for 4 days, the cells were fixed with formalin, stained with crystal violet, and the number of plaques was measured under a microscope. The antiviral effect (EC ₅ ) was calculated from the measured number of plaques in the same manner as in the previous section.

The above results are shown in Table 3 below.

Table 3

Example 4

Antiviral activity against influenza B belonging to the Orthomyxoviridae family

The antiviral activity was measured according to the MTT-microplate method (MTT-Microplate method, metabolism, Vol. 28,. No. 12, see page 333, 1991). Specifically, MDCK cells were cultured in a 96-well plastic plate (Falcon 3402) in D-MEM medium supplemented with 8% fetal bovine serum in a 5% CO2 incubator at 37 ° C for 36 hours, and 80% After the culture of the monolayer column, the culture solution is removed and 2 Q 0 xTC ID 50 (Tis sue Cu l tur e e n f ect on dose 50) of influenza B virus strains of Singapore (i η ί 1 enza enzavirus Β / Singap orestrai η; Ant im icror obial Agents and Chemo t he rapy Vo l. 36, No 2. 435—439. 1992). Add D-MEM medium 1001 containing the specified concentrations of mizolipin, 2% bovine albumin, and 0. OS SmgZm1 tribcine, and incubate for 5 days in a 5% CO2 culture device at 35 ° C and 5 mg / ml. Add 1 ml of MTT solution to 10 〃 1 and react at 37 ° C for 4 hours.Add 5% Triton X-100 and isopropanol 1001 containing 0.04 N hydrochloric acid and add at room temperature for 18-24 hours After standing, the absorbance of each well was measured with a microplate reader (Bio-Rad clay, Model-3550), and the antiviral effect (EC ₅ ) was calculated from the measured absorbance.

The above results are shown in Table 4 below.

Table 4

Example 5

Mizoribine (anhydrous crystals) 50 mg lactic anhydride 126 mg

ί¾ Cellulose 20 mg Carboxymethylcellulose calcium 1 Omg

Magnesium stearate 2mg

, Dry granulation, sieving, adding 2 mg of magnesium stearate to the granulated powder and tableting to obtain 21 Omg per tablet (containing 50 mg of mizoribine). Used as a prophylactic or therapeutic agent for viral infectious diseases. Example 6

Mizoribine 3: 0 g

Sodium sulfite 1.0

0.5 g of disodium hydrogen phosphate

Bersalkonium chloride 0.0 1 g

Suitable amount of sodium hydroxide (pH 8.0) Sterilized purified water Total volume 100 mI

To about 80 ml of sterilized purified water, 1.0 g of sodium sulfite, 0.5 g of sodium sodium phosphate 'I dihydrate, and 3.0 g of mizoribine were sequentially added and dissolved. To this solution was added and dissolved benzalkonium chloride. Then, sodium hydroxide was added to adjust the pH to about 8, and sterile purified water was added to make a total volume of 100 ml. This solution is sterilized by filtration and then aseptically filled into an aerosol inhalation container to obtain an aerosol inhalation preparation, which is used as a preventive or therapeutic agent for the infectious disease caused by the virus. The invention's effect

Mizoribine has about 10 times stronger antiviral activity against measles virus belonging to Paramyxoinfluenzae family and type 3 parainfluenzaviras than Ribavirin as shown in Tables 1 and 2 above. As shown in Tables 3 and 4, among the various RNA viruses, It also has strong antiviral activity against viruses belonging to the family members of the viridae and orthomyxoviridae. Therefore, based on these results, mizoribine is a drug exhibiting strong antiviral activity in a very small amount against a specific RNA virus belonging to the family Orthomyxoviridae or Paramyxoviridae, and has low cytotoxicity. It is useful as a prophylactic or therapeutic agent for infectious diseases such as influenza, measles, respiratory infection, subacute sclerosing panencephalitis, aseptic meningitis, parotitis, and common cold caused by the virus.

Claims

The scope of the claims

1. An agent for preventing or treating infectious diseases caused by viruses belonging to small infectious orthomyxoviridae or paramyxoviridae, which comprises mizoribine as an active ingredient.

2. The preparation according to claim 1, wherein the prophylactic or therapeutic agent is a preparation for oral administration, a preparation for injection administration or a preparation for aerosol inhalation.