JPH08208482A - Virus protease inhibitor - Google Patents

Abstract

PURPOSE: To obtain the subject inhibitor containing, as active ingredient (s), Methyl Orange, Toluidine Blue, etc., having marked inhibitory action on virus protease, esp. retrovirus protease, thus useful for treating (retro)virus infectious diseases. CONSTITUTION: This inhibitor contains, as active ingredient(s), Methyl Orange, Toluidine Blue, Nile Blue sulfate, Nile Blue chloride, Quinaldine Red, N,N- dimethyl-indoaniline, 2,3',6-trichloroindophenol sodium salt and/or 6-amino-2- naphthyl-p-guanidinobenzoate dimethanesulfonate. The formulation of this inhibitor is e.g. tablets, pills, powder, liquid, suspension, emulsion, granules, or capsules. The daily dose of this inhibitor, in terms of the active ingredient, is 0.1-20mg/kg.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for viral infections, and more particularly to a viral protease inhibitor useful for treating viral infections.

[0002]

2. Description of the Related Art Regarding the characteristics of viral protease, especially retroviral protease, see, for example, Japanese Clinical Volume 47, No. 1 (January 1989), Supplement, 197-2.
06, Cell Engineering Vol. 7, Suppl. 1,198
8, S67-S77, Biochemistry No. 60, No. 9, September 1988, pp. 1053-1059.
According to this, human T-cell leukemia virus (HTLV)
Reproducible, ie, non-defective, retroviruses such as and immunodeficiency virus (HIV) have a gene for a virus-specific protease.

This protease is required to cleave the precursor forming the immature particle and convert it into a mature protein during the production of virus particles to produce mature particles. Therefore, it is possible that a retrovirus may be defective in its maturation process, lose its infectivity, and prevent its transmission by deleting its viral protease gene or inhibiting its viral protease activity. [Japanese Clinical Vol. 47, No. 1 (1989)
(January, 1st issue), separate pages 197 to 206, Cell Engineering Vol.
7, Suppl. 1, 1988, S67 to S77, Biochemistry No. 60, No. 9, September 1988, No. 1053 to 1059.
page].

The protease isolated and purified from virus particles is an enzyme similar to aspartic protease due to the detailed study of its biochemical characteristics. However, it is substrate-specific with conventionally known fungal or cell-derived aspartic protease. It has been found that they are novel with different properties, molecular construction, optimum pH, etc. [Japanese Clinical Vol. 47, No. 1 (January 1989), Supplement, pp. 197-206, Cell Engineering Vol. 7, Suppl. 1,1988, S67
~ S77, Biochemistry Vol. 60, No. 9, September 1988, 10th
53-1059].

Studies have been carried out on such protease inhibitors, and some compounds such as pepstatin A have been confirmed to have a retroviral protease inhibitory action. However, the details of the relationship between the structure and activity of retroviral protease have not been completely elucidated, and generally, there are few effective inhibitors for aspartic prostheses (pepsin, renin, etc.). Due to the fact that it has a different property from that of known aspartic proteases, it is not easy to search for compounds that have a retrovirus protease inhibitory action, and it is the current situation that we have to resort to trial and error. Retrovirus protease is a newly discovered protease, and the development of compounds with its inhibitory action is just beginning,
In general there are no reports yet.

[0006]

SUMMARY OF THE INVENTION The main object of the present invention is to provide a protease inhibitor of viral protease, particularly retrovirus.

[0007]

[Means for Solving the Problems] The present inventors have conducted extensive studies to achieve the above object, found that certain compounds are effective as protease inhibitors of retroviruses, and completed the present invention. It was

That is, the present invention provides methyl orange, toluidine blue, nile blue sulfate, nile blue chloride, quinaldine red, N, N-dimethyl-indoaniline, 2,3 ', 6-trichloroindophenol sodium salt and 6 -Amino-2-naphthyl p-guanidino benzoate dimethane sulfonate is provided as an active ingredient, and a viral protease inhibitor is provided.

The above-mentioned compounds found to have a viral protease inhibitory action in the present invention are all known compounds and are readily available.

Among these compounds, 6-amino-2
-Naphthyl p-guanidinobenzoate dimethanesulfonate is a water-soluble compound and is easily noticed because it is easily taken up by cells.

Examples of viruses for which the viral protease inhibitors of the present invention are effective include various retroviruses. For example, human T-cell leukemia virus (HT).
LV), human immunodeficiency virus (HIV), bovine leukemia virus (BLV), mouse leukemia virus (ML)
V), avian myeloblastosis virus (AMV) and other viruses belonging to the retroviridae family.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The viral protease inhibitor of the present invention is usually used in a form according to its intended purpose,
Typical examples thereof include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules and the like. In the case of molding in the form of tablets, those conventionally known in this field can be widely used as carriers, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, etc. Excipient,
Water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, binders such as polyvinylpyrrolidone, dry starch,
Sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, disintegrating agent such as lactose, sucrose, stearin, cocoa butter, hydrogenated oil Disintegration inhibitors such as quaternary ammonium base, absorption promoters such as sodium lauryl sulfate, humectants such as glycerin and starch, starch, lactose, kaolin, bentonite,
Examples thereof include adsorbents such as colloidal silicic acid, purified talc, stearates, boric acid powders, and lubricants such as polyethylene glycol. Further, the tablets are tablets coated with a usual coating as necessary, for example, sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets,
It can be a film-coated tablet, a double tablet, or a multilayer tablet. In the case of molding in the form of pills, those conventionally known in this field can be widely used as carriers, for example, glucose, lactose, starch, cocoa oil, hydrogenated vegetable oil,
Examples include excipients such as kaolin and talc, gum arabic powder, tragacanth powder, binders such as gelatin, and disintegrating agents such as laminaranthantene.

Of the above administration forms, liquid formulations are particularly preferable.

The concentration of the compound as an active ingredient in each preparation in the above dosage forms is not particularly limited, but is usually 0.1.
It may be about 70% by weight.

In formulating the inhibitor of the present invention,
Various conventionally used methods can be widely adopted.

The method of administration of the inhibitor of the present invention is not particularly limited, and it may be administered according to various dosage forms, age of the patient, sex and other conditions, degree of disease, etc. Since it targets cells, it is administered intravenously by injection.

The dose of the inhibitor of the present invention can be appropriately determined according to the usage, the age of the patient, the sex and other conditions, the degree of the disease, etc. Usually, the amount of the compound as an active ingredient is 1
It may be about 100 μg to 20 mg per 1 kg of body weight per day. The formulation may be administered in 2 to 4 divided doses per day.

[0018]

The present invention will be described in more detail below with reference to formulation examples and test examples of the inhibitor of the present invention.

Reference formulation example 1 A tablet having the above composition was produced in one tablet by a conventional method.

Formulation Example 1 The parabens, sodium metabisulfite and sodium chloride are dissolved in about half of the above distilled water at 80 ° C. with stirring. The resulting solution was cooled to 40 ° C. and the inhibitor of the invention, then polyethylene glycol and polyoxyethylene sorbitan monooleate were dissolved in the solution. Next, distilled water for injection is added to the solution to adjust the final volume, and the solution is sterilized by sterilizing filtration using an appropriate filter paper and dispensed in 1 ml aliquots to prepare an injection.

Test Example 1 The action of the inhibitor of the present invention on retroviral protease was measured by the following method.

Purification of Protease The method for purifying bovine leukemia virus (BLV) and mouse leukemia virus (MLV) is detailed in the literature [Yoshinakawai. (Yoshinaka Y.) et al., Proc. Natl. Acad. Sc
i. USA: 82 : 1618-1622, J. Viral., 5
5 : 870-873 (1985) and Yoshinaka Wai.
(Yoshinaka Y.) et al., J. Viral., 57 : 826-832.
(1986)]. According to the method described in these documents, the virus was separated from the culture supernatant of the virus-infected cell and dissolved in a detergent (NP40 or Triton X-100) or a buffer solution (10 mM Tris-HCl, 1M in acetone powder).
After extracting the protein with NaCl, 1 mM EDTA, pH 7.6), the gel filtration method and high performance liquid chromatography method are used in combination and purified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) until a single band is obtained. did.

The gel filtration method was performed under the following conditions. That is, using Sephadex G75 (90 x 1.5 cm),
STE (0.13M NaCl, 1 mM EDTA, 1
Elute with 0 mM Tris-HCl, pH 7.6) and fractionate into 3 ml fractions.
It was analyzed or measured by DS-PAGE.

The high performance liquid chromatography method was carried out under the following conditions. That is, the fractions containing the protease fractionated by the gel filtration method were pooled, completely dissolved with 6M-guanidine hydrochloride, 10 ml was loaded on a Waters Microbonder column C ₁₈ , and acetonitrile (0-
Elution with a gradient of 60%). Fractions of 5 ml each were separated, lyophilized, and then dissolved in 1 ml of water, and a part thereof was examined for protein composition and protease activity.

The SDS-polyacrylamide gel electrophoresis was carried out under the following conditions. That is, the Laemmli system (Laemmli, UK 1970, Nature (London) 22
7 : 680-685), electrophoresis was performed at 150 V for 5 hours using a gradient gel having a polyacrylamide concentration of 8 to 18%.

Commercially available avian myeloblastosis virus (AMV) (Life Science Inc. St. Petersburg, Flori
da, USA) protease was also purified by the same method. The HIV viral protease expressed in E. coli used a gel-filtered fraction, but the inhibition test gave comparable results.

Substrate of Protease As a substrate, protease deficient mouse sarcoma virus (M
Hamster cell HTG-2 (Gazdar) producing SV
Et al. (1971), Nature. New Biol., 324, 69-.
72, obtained from Dr. Gazdar (NIH, USA)) with 0.5% nonidet NP-40 (hereinafter referred to as “NP-40”) which is a neutral surfactant. A solubilized product was used.

This virus is mainly composed of a protein (Pr65) having a molecular weight of 65000 which is a precursor of a protein (group specific antigen) constituting the internal structure of particles. The Pr65 is AMV, MLV, BLV, HI
It can serve as a substrate for homologous and heterologous retroviral proteases such as V and ATLV.

Protease activity measurement Optimal pH of the activity of each retrovirus-derived protease
Is slightly different depending on the virus species, but generally pH 6.0-
It is within the range of 7.0. P by viral protease
The cleavage site in the r65 molecule is slightly different depending on the virus species, but there are about 3 to 4 sites including common sites. The experiments were mainly carried out using AMV protease because of the ready availability of materials.

0.02 with varying concentrations of protease
M MES (that is, 2- (N-morpholino) ethanesulfonic acid, Sigma), 0.5% NP-40, concentration 0.
In 100 μl of 3 μg / ml leupeptin solution, 3 μl of the substrate (5 μg protein, 0.5% NP-40, 0.1%) was added.
3M NaCl, 1 mM EDTA, 0.01M Tr
is-HCl, pH 7.4) was added, and the mixture was kept at 22 ° C for 16 hours. After the reaction, the protein composition in the reaction solution was analyzed by SDS-PAGE according to a conventional method, and the activity intensity was measured from the composition change. That is, the decrease of Pr65 band and the increase of specific product band were measured with a densitometer.

Measurement of Inhibitory Effect and Concentration of Inhibitor The protease concentration at which the Pr65 band disappears by 95% in the above reaction was measured, and various concentrations of the protease inhibitor of the present invention (10 μm) were added to the reaction solution having this concentration.
l) was added. The inhibitor was prepared by dissolving a compound found to have a protease inhibitory activity according to the present invention in water or dimethylsulfoxide (DMSO). As a control, only each solvent was used. DM
SO has no effect on protease activity at concentrations below 20%. The activity was measured by the method described in the above "Measurement of protease activity".

As an example, FIG. 1 shows the cleavage pattern (SD) of Pr65 by a typical retroviral protease.
S-PAGE). When AMV protease is serially diluted 2-fold with the reaction solution, a certain amount of substrate is added and the reaction is carried out, Pr65 decreases depending on the amount of protease, while an increase in the reaction product is observed. 95% Pr65
The concentration of the protease that decreases the amount was determined and used to measure the inhibitory effect of the following inhibitors.

FIG. 2 shows the pattern of AMV protease activity inhibition by neomycin sulfate (neomycin-S) and G418. Concentrations of neomycin sulphate or G418 that inhibit Pr65 reduction by 50% (50
% Inhibition concentration, IC ₅₀ ) was calculated to be 0.00, respectively.
It was 3 mM and 0.01 mM. The 50% inhibitory concentration was determined by analyzing the sample by SDS-PAGE, staining the gel with Coomassie Brilliant Blue R-250, and measuring the intensity of the Pr65 band with a densitometer (KEMIC) manufactured by Atago Co., Ltd. In comparison, the cleavage of Pr65 is 5
The concentration at which 0% inhibition was obtained was determined.

Similarly, the IC ₅₀ of the compound found to have a protease inhibitory activity in the present invention was determined.

The results are shown in Table 1.

Table 1 Test Compounds IC ₅₀ (mM) Methyl Orange 1 Toluidine Blue 0.01 Nile Blue Sulfate 0.1 Nile Blue Chloride 0.1 Quinaldine Red 0.003 N, N-Dimethyl-Indoaniline 1 2,3 ', 6-trichloro India phenol sodium salt 1 6-amino-2-naphthyl p- guanidino benzoate dimethacrylate Nsuruhoneto 0.5 as is apparent from table 1, an inhibitor of the present invention AMV
It is found that it effectively inhibits the protease.

Other retroviruses such as human T-cell leukemia virus (HTLV), human immunodeficiency virus (H
IV), bovine leukemia virus (BLV), mouse leukemia virus (MLV) and other proteases
Since it exhibits the same activity as V protease, it was confirmed that these retrovirus proteases are similarly inhibited by the inhibitor of the present invention.

[0038]

INDUSTRIAL APPLICABILITY As compared with conventionally known viral protease inhibitors, the inhibitor of the present invention exerts an equal or greater remarkable inhibitory action on retroviral protease.

Therefore, the inhibitor of the present invention may cause the infectivity of viruses, particularly retroviruses, to be lost and prevent their transmission.

[Brief description of drawings]

FIG. 1 is a photograph as a substitute for a drawing, which shows the result of examining the cleavage pattern of Pr65 by a typical retrovirus AMV protease by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

FIG. 2 Neomycin sulfate (neomycin-S) and G
The activity inhibition pattern of 418 AMV protease was
DS-polyacrylamide gel electrophoresis (SDS-P
It is a photograph instead of a drawing showing a result of examination by AGE). In FIG. 2, the arrows (→) and the numerical values of 0 to 4 have the following meanings. →… Position of product band that appears when protease cleaves Pr65 Numerical value from 0 to 4… Value of n

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number in the agency FI technical display location A61K 31/535 // C12N 9/99 (72) Inventor Shoichi Adachi At 3493 Ishihara-cho, Takasaki-shi 9

Claims (1)

[Claims] 1. Methyl orange, toluidine blue, Nile blue sulfate, Nile blue chloride, quinaldine red, N, N-dimethyl-indoaniline,
1 selected from the group consisting of 2,3 ′, 6-trichloroindophenol sodium salt and 6-amino-2-naphthyl p-guanidinobenzoate dimethanesulfonate
A viral protease inhibitor, which comprises a seed as an active ingredient.