JPH05105635A - Antiviral agent - Google Patents

Abstract

PURPOSE:To provide an antiviral agent containing anticathepsin B antibody and expected to be useful as a treating agent for viral disease. CONSTITUTION:Cathepsin B purified from human liver is immunized to an animal (e.g. rabbit, sheep, pig or goat) and the serum of the animal is purified e.g. by chromatography to obtain anticathepsin B antibody. The antibody inhibits the formation of plaque of an envelope virus such as human influenza virus and the proliferation of the virus in chicken egg chorioallantoic membrane liquid. The amount of the anticathepsin antibody as an antiviral agent is 1-500mg/dose and 1-3 doses/day for oral administration, 1-200mg/dose and 1-3 doses/day for intravenous drip infusion, 0.5-10mg/dose and 2-3 doses/day for bronchial aspiration, 0.05-10mg/dose and 2-4 doses/day for nasal cavity administration, 0.01-1mg/dose and 3-4 doses/day for instillation and 0.1-10mg/dose and 1-3 doses/day for ointment.

Description

Detailed Description of the Invention

[0001]

The present invention is an invention useful in the field of medicine. More specifically, the present invention relates to a therapeutic agent for a disease caused by viral infection.

[0002]

2. Description of the Related Art Anti-cathepsin B antibody is a known substance,
It has been used for research to detect and quantify intracellular localization of cathepsin B, which is a lysosomal enzyme [J. Path
logic, vol. 142, p. 87 (1984);
J. Cell Physiol. , Vol. 115,
p. 249 (1983); Urology, vo
l. 144, p. 798 (1990)]. However, there is no report that the anti-cathepsin B antibody exhibits an antiviral effect.

[0003]

Many nucleic acid derivatives are known as antiviral agents, and amantadine and the like are known as antiinfluenza virus agents. Nucleic acid derivative antiviral agents have side effects such as liver dysfunction, mutagenicity and subacute toxicity, and amantadine has adverse effects such as teratogenicity and decreased efficacy due to increased use frequency. Has [Virology, Raven Press (Raven)
Press) pp. 323-348 (1985)
reference]. It is desired to eliminate such problems of known antiviral agents.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that anti-cathepsin B antibody is excellent in antiviral action and safety. The present invention has been completed.

That is, the present invention relates to an antiviral agent containing an anti-cathepsin B antibody as an active ingredient.

The anti-cathepsin B antibody is produced by immunizing an animal (rabbit, sheep, pig, goat, etc.) with cathepsin B purified from human liver by a conventional method and purifying it from the serum by chromatography or the like. Alternatively, it can be obtained from a commercial product.

In the antibody fraction, the main components are IgG and Ig
M, IgA, etc. are included, but these can be used alone or as a mixture.

In order to specifically show the antiviral action of the anti-cathepsin B antibody, test examples will be described.

In the test examples and the examples, The Binding Site Limited (The Bindin)
An anti-cathepsin B antibody derived from lyophilized powdered sheep manufactured by g Site Limited) was used.

1 g of this anti-cathepsin B antibody was added to purified water 10
When dissolved in 0 ml, the protein concentration became 10 mg / ml, and the titer of this antibody solution was 256.

Here, the titer is determined as follows.

The antibody solution is double-diluted with physiological saline, and this is an antibody solution capable of forming a precipitation line (antigen-antibody reaction aggregate) in the agar gel with the antigen cathepsin B solution (protein concentration 0.2 mg / ml). The titer is the maximum dilution factor. Test Example Inhibitory effect on plaque formation of human influenza virus By the method described in Virology, Vol. 29, pp. 84-91 (1966).
Using the normal sheep antibody as a control, the inhibitory effect of the substance A of the present invention and the comparative compound (amantadine) on the plaque formation of human influenza virus A / WSN was measured.

That is, MDCK cells (canine kidney cells) 1 ×
10 ⁶ cells are seeded in a 6 cm plastic dish containing D-MEM medium, incubated at 37 ° C. for 24 hours in a carbon dioxide incubator,
The medium was discarded, and 0.1 ml of a virus diluent containing about 50 human influenza virus A / WSNs was added.
Shake well, leave at room temperature for 30 minutes, add agar overlay solution containing a specified amount of reagent, and solidify the agar at 37 ° C.
The cells were cultured in a carbon dioxide incubator for 3 days. After culturing, the agar layer was removed, the cells were stained with a staining solution, and the number of plaques was counted. Amantadine was used as a comparative compound. The plaque formation blocking rate was calculated by the following formula, and the results are shown in Table 1.

[0014]

[Equation 1] D-MEM medium 1.9% Dulbecco's Modified
Dissolve 9.5 g of EAGLE MEDIUM "Nissui" powder in 1 L of distilled water (hereinafter referred to as 1 * DMEM) and sterilize in an autoclave. 0.584 g of L-glutamine, 0.1 g of kanamycin, 20 ml of 7.5% aqueous sodium hydrogen carbonate solution and immobilized fetal calf serum 11
Add 0 ml. Virus Diluent Phosphate Buffered Saline
1L of calcium chloride 10g, magnesium chloride 10
g and 30% bovine serum albumin 5.8 ml. Agar overlay solution Pure water 16 ml 2 * DMEM + BA 50 ml 1% DEAE dextran 1 ml 7.5% sodium hydrogen carbonate aqueous solution 2 ml 2.0% agar (noble) 30 ml 2 * DMEM + BA 4 * Dulbecco 250 ml 1M HEPES 10 ml 10% bovine serum 20 ml 7. 5% aqueous sodium bicarbonate solution 16 ml 6% glutamine solution 10ml kanamycin aqueous solution (100mg / ml) 1 ml of crystal violet pure water 193ml staining solution 100mg dissolved in ethanol 20 ml, further water is added 80 ml.

[0015]

[Table 1] As is clear from Table 1, the substance A according to the present invention contains 4 μg.
At a concentration of / ml, human influenza virus plaque formation was inhibited by 48%, and at a concentration of 20 μg / ml, human influenza virus plaque formation was inhibited by 92%.
On the other hand, the comparative compound amantadine was 30 μg
It inhibited the formation of the plaque by 60.1% at a concentration of / ml. Therefore, the substance A according to the present invention has a stronger inhibitory effect on the growth of human influenza virus than amantadine.

The substance A according to the present invention is MDCK.
1000 μg / for 3 days cultured cells of (dog kidney cells)
Although no cytotoxicity was shown up to the concentration of ml, amantadine showed a slight but apparent cytotoxicity even at 30 μg / ml. Therefore, the substance A according to the present invention has a wider safety margin than amantadine.

The dose of the anti-cathepsin B antibody used as an antiviral agent varies depending on the administration form and administration route, but in the case of oral administration, it is usually 1 to 500 m once.
g 1 to 3 times a day, once or twice for intravenous drip
00 mg 1 to 3 times a day, and once for inhalation, 0.
05-10 mg is administered 2-3 times a day to the bronchus, 0.05-10 mg is administered 2-4 times in the case of intranasal administration,
As an eye drop, 0.01 to 1 mg may be administered 3 to 4 times a day, and as an ointment, 0.1 to 10 mg may be administered 1 to 3 times, depending on age, sex, weight and symptoms. ,
It can also be used outside these ranges.

The substance according to the present invention can be made into a dosage form for oral or parenteral administration by using the following additives for formulation.

The unit dosage form that can be used for oral administration includes solid preparations such as tablets, troches, powders, pills, granules and capsules. These dosage forms include, for example, gum arabic, gelatin, sorbit, tragacanth, polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropylmethyl cellulose, methyl cellulose, crystalline cellulose or sodium carboxymethyl cellulose, a binder such as lactose, sugar, sucrose, sucrose, Excipients such as mannitol, corn starch, calcium phosphate, sorbitol or crystalline cellulose, lubricants such as magnesium stearate, talc, polyethylene glycol or silica, or potato starch, low-substituted hydroxypropyl cellulose, carboxymethyl cellulose calcium or Additives such as disintegrants such as sodium carboxymethyl starch may be used alone or in combination as appropriate. Further, the soft capsule may contain a commonly used vehicle such as vegetable oil, polyethylene glycol or glycerin, and the following oily suspending agent, liquid agent or wetting agent such as surfactant.

As a liquid preparation for oral administration,
For example, an aqueous or oily suspension, a solution, a syrup, an elixir, or a lyophilizate that can be redissolved in water or another appropriate vehicle at the time of use, etc. can be mentioned. The liquid preparation includes, for example, a suspending agent such as methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, tragacanth, gelatin or sodium alginate, such as lecithin,
Emulsifiers such as sorbitan, fatty acid esters, gum arabic or tragacanth, for example, wetting agents such as polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, hydrogenated castor oil, sesame oil, soybean oil, propylene glycol, polyethylene glycol or ethyl alcohol. For example, preservatives such as methyl p-hydroxybenzoate, propyl p-hydroxybenzoate or sorbic acid, or additives such as sweeteners such as simple syrup, sucrose, sorbit or mannitol, etc. may be used alone or in appropriate combination. May be.

The unit dosage forms that can be used for parenteral administration include injections and aerosols for respiratory administration,
Inhalation liquids, solutions, powders, ointments and the like can be mentioned.

In the case of an aerosol to be administered to the respiratory tract such as the nose and bronchus, for example, a nonionic surfactant such as Alacel or Span 80, an amphoteric surfactant such as lecithin or a dispersant such as oleyl alcohol, and butane, for example. Alternatively, it should be an oil-based aerosol formulation consisting of a propellant such as Freon, or an aqueous aerosol formulation consisting of isotonicity agents such as physiological saline, phosphate buffer or acetate buffer, and purified water or distilled water for injection. be able to.

In the case of liquid preparations, for example, polyethylene glycol, sorbitol, polysorbate, physiological saline or the like can be used as a pharmaceutical carrier. In the case of powders, for example, crystalline cellulose, α-cellulose, crosslinked sodium carboxymethyl cellulose, hydroxypropyl cellulose, carboxymethyl starch or amylose can be used as a pharmaceutical carrier.

In the case of an ointment, for example, polyethylene glycol, hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose or hydroxypropyl cellulose can be used as a pharmaceutical carrier.

These formulations can be prepared by the respective known production methods.

The substance A of the present invention may be contained in these preparations in an amount of about 0.1 to 99%, preferably 0.5 to 90% of the total composition.

[0027]

EXAMPLES The present invention will be specifically described below with reference to examples of the present invention, but the present invention is not limited thereto. Example 1 1 g of freeze-dried powdered anti-cathepsin B antibody, 300 g of polyethylene glycol 400 and polysorbate 80
While stirring 0.5 g, add purified water to a total volume of 1000
The solution was evenly dissolved in ml to prepare a nasal solution. 0.1 ml of this preparation is intranasally administered dropwise. Example 2 2 g of freeze-dried powdered anti-cathepsin B antibody was dissolved in physiological saline to make 1,000 ml to prepare a liquid preparation. This preparation is placed in a nasal drop container and sprayed at a dose of about 0.2 ml. Example 3 2 g of freeze-dried powdered anti-cathepsin B antibody is dissolved in 500 ml of distilled water for injection. To this, 400 ml of a 12.5% sorbitol aqueous solution was added and uniformly mixed, and then 0.1
Adjust to pH 4.0 with normal hydrochloric acid. Then, distilled water for injection was added to make the total volume up to 1,000 ml to prepare a liquid preparation. This solution is put in a nasal drop container and about 0.2 ml is spray-administered once. Example 4 0.1 g of freeze-dried powdered anti-cathepsin B antibody is ground in an agate mortar, and 200 mg of span 85 is added and kneaded uniformly. After cooling the resulting mixture to -40 ° C,
An aerosol formulation is obtained by mixing with 10 g of a 1: 4: 1 mixture of Freon 11, Freon 12, and Freon 114 that has been cooled to -40 ° C in advance, and then low-temperature filling in a pressure resistant container. Example 5 50 mg of freeze-dried powdered anti-cathepsin B antibody and 450 mg of crystalline cellulose are well ground in a mortar to obtain a uniform powdered composition. A capsule preparation for nasal administration was obtained by filling a predetermined capsule with this powdery composition. Example 6 Lyophilized powdered anti-cathepsin B antibody (100 mg) was dissolved in 5% glucose injection solution (500 ml) and sterilized by filtration to obtain an intravenous injection solution. 1 to 1 day of this injection solution for about 2 hours
It can be infused twice intravenously. Example 7 1 g of freeze-dried powdered anti-cathepsin B antibody, 100 g of sucrose (fine powder) and 7 g of gum arabic (fine powder) were mixed, and an appropriate amount of purified water was added and kneaded. After drying the kneaded product, 1 g of magnesium stearate was added and compression-molded by a conventional method to obtain 100 troches.

[0028]

EFFECTS OF THE INVENTION The substance of the present invention inhibits the plaque formation and the growth of the virus in the allantoic fluid of chicken egg chorioallantoic virus against envelope viruses such as human influenza virus. It contributes to the treatment of.

[0029]

Claims (1)

[Claims] 1. An antiviral agent containing an antibody against cathepsin B as an active ingredient.