JPH03218317A - Anti-retrovirus agent - Google Patents

Abstract

PURPOSE:To obtain an anti-retrovirus agent useful for preventing and treating retrovirus infection because of low side effects, having strongly inhibitory action on virus multiplication, containing a sulfuric ester of curdlan low polymer. CONSTITUTION:A sulfuric ester of curdlan low polymer having about 10-400, especially about 10-200 average polymerization degree comprises as an active ingredient to give an anti-retrovirus agent useful for preventing and treating infection of retrovirus, especially HTLV (human T cell leukemia)-I, HTLV-II, HIV (human immunodeficiency virus)-1 or HIV-2 because the compound has high water solubility, low toxicity and relatively high inhibitory activity against human retrovirus multiplication. A dose of the compound is generally 0.1-20mg/kg per adult daily and administered once-six times daily by oral medica tion or injection.

Description

[Detailed description of the invention] Industrial applications The present invention relates to antiretroviral agents.

Conventional technology Retroviruses belong to the Retroviridae family, have RNA as their genes, and use their own reverse transcriptase (RNA-dependent DNA polymerase) to self-synthesize DNA using RNA as a template. A general term for viruses that perform the first stage of replication. The Retroviridae family consists of three subfamilies: Oncoviruses, Lentiviruses, and Submaviruses. Retroviruses are known to infect and multiply in various animals, including birds and mammalian mammals, and cause sarcoma, leukemia, cancer, and the like. Adult T-cell leukemia virus (adult T-cell leukemia virus) is a retrovirus that uses humans as a host.
leukemia virus, ATLV), human T-cell leukemia virus type I (human T-ce
lleukemia virus type I
, HTL V-I) and human immunodeficiency virus (human immunodeficiency virus).
virus, HIV), etc. have been reported.

ATLV was proven to be the same virus as HTLV-1. In addition to this, HTLV has T cell lineage h
There is a type 2 that induces airy cell leukemia. HIV is classified into HIV-1 and HIV2, and HIV-1 causes acquired immunodeficiency syndrome, a serious immunodeficiency disease.
ficiency syndrome, AI
It is known that the virus causes DS.

Furthermore, HIV-2 is also considered to be the causative virus of AIDS, but it is not as clearly understood as HIV=1.

Adult T-cell leukemia caused by ATLV is
It is a type of leukemia that is commonly seen in the southwestern region of Japan, particularly in the Kyushu region, and was first reported in 1977. In adult T-cell leukemia patients, T4-positive T cells become cancerous and cause skin lesions. This may lead to lymph node enlargement, hepatoma, etc. There is no effective treatment for adult induced cell leukemia, and most cases of 1
Died within a year.

AIDS caused by HIV-1 is a disease that has recently attracted attention as an immunodeficiency disease with a poor prognosis, and is characterized by clinical manifestations such as Pneumocystis carinii pneumonia and Kabosi sarcoma. HIV-1 infects T4-positive cells, invades and proliferates inside the cells, and destroys them, thereby causing immunodeficiency. Acitothymidine (AZT) is currently used as a therapeutic agent for AID-3S, and has been shown to improve symptoms and prolong life.

Problems to be Solved by the Invention Although AZT is used as an antiretroviral agent as described above, this substance has drawbacks such as bone marrow suppression as a side effect, and there is a need for a more effective treatment with fewer side effects. It is hoped that a drug will emerge.

Means for Solving the Problems In the process of studying the physiological properties of various derivatives of curdlan low polymers, the present inventors discovered that these sulfonated products (sulfuric esters) were isolated from T cells of patients with adult artificial cell leukemia. The present inventors have discovered that the established cell line MT-4 can be strongly inhibited from being infected with HIV and the proliferation of the virus without causing cell damage, and through further intensive research, they have completed the present invention.

The present invention provides a pharmaceutical composition containing a sulfonated product (sulfuric ester) of a curdlan low polymer as an active ingredient, which is useful for the prevention and treatment of retrovirus infection in chewed milk.

That is, the present invention provides (1) an antiretroviral agent containing a sulfate ester of curdlan low polymer; (2) a retrovirus containing HTLV-I, HTLV-II, HIV-
1 or HIV-2'7 virus, (3) the average degree of polymerization of the curdlan low polymer is about 10 to 40.
The formulation according to item (1) or (2) using a sulfuric ester having a sulfate of 0 and (4) the average degree of polymerization of the curdlan low polymer is about 10 to 20.
The invention relates to the formulation according to claim (1), (2) or (3), which uses a sulfuric ester having a concentration of 0.

Retroviruses as used in the present invention refer to all viruses that have RNA as a gene, including those mentioned above.

Diseases targeted by the formulation of the present invention include all diseases caused by retroviruses, including those mentioned above.

Curdlan (also known as heat-coagulable polysaccharide PSJ) is a water-insoluble straight chain β-1.3 having only one bond, produced by a microbial strain belonging to the genus Alcaligenes or Agrobacterium. It is known as a heat-coagulable glucan [Special Publication No. 7,000 of 1970,
No. 8-32,673 and Japanese Patent Publication No. 48-32.674].

Curdlan producing bacterium Alcaligenes faecalis variety myxogenes
var. myxogenes) NTK-u strain, Agrobacterium radiobacter (Agrobacterium
erium radiobacter) strains and Agrobacterium radiobacter (Agrobac
terium radiobacter ) U-1
The nine stocks are American Type Culture as ATCC=21680, -6466 and 21679, respectively.
Collection Catalog e of Strains (AT
CC-Catalogue of Strains
) I, 15th edition. It is a listed strain described in 1982.

Next, the properties of curdlan low polymer and its production method are as follows.
JP-A-53-66442 and JP-A-55-837 have already been published.
It is described in detail in Japanese Patent No. 98.

Curdlan low polymers with a degree of polymerization of 400 or less can be produced by hydrolyzing curdlan obtained by culturing according to a conventional method, and also have linear β-1,3
-Has a glucan structure.

The curdlan low polymer in the present invention is a compound represented by the formula [wherein n represents an integer from 4 to about 400], and the sulfuric acid ester of the curdlan low polymer used in the present invention is The three hydroxyl groups of the middle monosaccharide and the hydroxyl groups of the monosaccharides at both ends of the compound are sulfonated,
The average degree of substitution (DS: Degree of
A substance having a DS of 0.5 to 3 per each monosaccharide is usually used, and preferably a DS of 1 to 2 is advantageously used.

In addition, the above-mentioned curdlan low polymer has an average degree of polymerization (
DP: Degree of Polymeri
zation) of about 400 or less, but especially those whose DP, which is a partial hydrolyzate, is about 1
0 to 400, especially about 10 to 200 are advantageous.

Note that n and DP in equation (1) have a relationship of DP-2=n.

The sulfuric ester of curdlan low polymer in the present invention is
It can also be used as its salt, such as ammonium salt, alkali metal salt (sodium salt, potassium salt, etc.)
Examples include salts with basic inorganic substances such as.

A method for producing the sulfuric ester of compound (T) will be described below.

Examples of methods for hydrolyzing curdlan include conventionally well-known acid hydrolysis methods, alkaline hydrolysis methods, and enzymatic decomposition methods using β-1,3-glucanase.

In order to separate the curdlan low polymer from the reaction mixture, various methods used for the purification and fractionation of polysaccharides and oligosaccharides, such as precipitation in acidic conditions, precipitation by adding ethanol, gel filtration, etc., can be used. . By such means, various low polymers having a desired average degree of polymerization can be separated.

The average degree of polymerization of curdlan and its low polymer is
Manners et al.'s method [Carbohydrate Research (
Carbohydrate Res, ), ↓ Yu, 10
9, (1971)].

The average degree of polymerization of curdlan (natural type) is often 30-800, and low polymers with an average degree of polymerization of 2 to about 400 can be produced by changing the reaction conditions.

Curdlan low polymers can be prepared by reacting a sulfating agent such as chlorosulfonic acid or sulfuric anhydride in the presence of pyridine, formamide or dimethylformamide, or by reacting sulfuric anhydride with an organic base such as pyridine, dimethylformamide, etc.
Sulfation can be achieved by reacting a complex with trimethylamine dimethylaniline or the like. [Journal of Bio-Oral Chemistry (J.B.
iolChem. ), 239.2986 (1964)]. The reaction product is purified and obtained by adding an organic solvent such as alcohol or acetone to the reaction solution and separating the resulting precipitate, or by a method such as gel filtration using Sephadex G-25 or dialysis. can do.

The method for producing the sulfuric acid ester of the curdlan low polymer mentioned above has already been described in detail in Japanese Patent Application No. 1-172539.

Curdlan low polymer sulfate ester has high solubility in water, low toxicity as evidenced by the test examples described below, and high relative human retrovirus proliferation inhibitory activity, so it is advantageously used as a pharmaceutical product. be able to.

The antiretroviral agent of the present invention is useful for preventing and treating retrovirus infections in mammals, and can be administered orally or parenterally. The sulfate ester in the present invention can be made into tablets, capsules, powders, granules, troches, suppositories, etc., as desired, and may also be used in injections, suspension injections, and the like.

The dosage of the curdlan low polymer sulfate ester of the present invention is:
The amount may vary as long as it is sufficient to produce the desired prophylactic and therapeutic effects in the body, and may vary depending on the disease to be prevented or treated, the patient's weight, administration route, dosage form, etc. For example, it is generally administered orally or by injection in an amount of 0.1 to 20 mg/kg per day for adults. The frequency of administration is appropriately selected within the range of 1 to 6 times a day.

Effects and Examples The present invention will be explained in more detail by referring to Reference Examples, Test Examples, and Examples, but the present invention is not limited thereto.

Reference Example 1 After suspending 2.5 g of curdlan with an average degree of polymerization of 540 in 100 g of dimethylformamide, 12.5 g of triethylamine sulfonic acid synthesized from 13.5 g of chlorosulfonic acid and 117 g of triethylamine was added, and the mixture was poured into ice water. The reaction was allowed to take place while stirring for 24 hours. Reaction liquid to 0
．． After thorough dialysis against 5M ammonium bicarbonate, the product was freeze-dried to obtain 44 g of the desired product. Average fff conversion of the obtained curdlan sulfate ammonium salt (DS>c)
．． 04 (sulfur content 127%).

Reference Example 2 2.5 g of curdlan low polymer (average degree of polymerization 131) obtained by partial hydrolysis with 85% bovine acid (80°C, 30 minutes) of curdlan having an average degree of polymerization of 540 was added to 100 m
After suspending in 12 ml of dimethylformamide, 12.
5 g of tosoethylamine-sulfonic acid was added and reacted in ice water with stirring for 24 hours. 0.5 of the reaction solution
After thorough dialysis against ammonium bicarbonate of M, lyophilization was performed to obtain 3.9 g of curdlan sulfate ammonium salt.

1I The DS of the obtained target product was 1.46 (sulfur content 1
5.4%).

Reference Example 3 2.5 g of curdlan low polymer (average degree of polymerization 68) obtained by partial hydrolysis with 4N sulfuric acid (60°C, 4 hours) of curdlan having an average degree of polymerization of 540 was suspended in 100 d of dimethylformamide. After this, 12.5 g of triethylamine-sulfonic acid was added, and the mixture was reacted with stirring in ice water for 24 hours. 2.5ρ Sephadex equilibrated with 0.02M ammonium bicarbonate.
It was loaded onto a G-25 (fine) column and subjected to gel filtration. The sugar content in the eluate was analyzed using the phenol-sulfuric acid method.
The sugar fractions were collected and freeze-dried to obtain 4.0 g of curdlan sulfate ammonium salt. DS of the obtained target
was 1.61. (Sulfur content 16.2%).

Reference Example 4 2.5 g of curdlan low polymer (average degree of polymerization 45) obtained by partial hydrolysis with 85% formic acid (85° C., 30 minutes) of curdlan having an average degree of polymerization of 540 (average degree of polymerization 45) was mixed with 100 d of dimethyl L suspended in formamide
2.5 g of triethylamine-sulfonic acid was added and reacted in ice water for 24 hours with stirring. The reaction solution was reduced to 0.
2. equilibrated with 0.02M ammonium bicarbonate. It was loaded onto a 5ff Sephadex G-2 5 (fine) column and gel filtration was performed. The sugar content in the eluate was analyzed by the phenol-sulfuric acid method. Collect and lyophilize the sugar fraction; 2.
7 g of curdlan sulfate ammonium salt was obtained.

The DS of the obtained target product was 1.41 (sulfur content 1
51%).

Reference Example 5 After 1.0 g of curdlan with an average degree of polymerization of 540 was suspended in 50 g of dimethylformamide, 50 g of triethylamine-sulfonic acid was added and reacted with stirring at 24° C. for 24 hours. The reaction solution was dialyzed against a 10% sodium bicarbonate solution overnight, and further dialyzed against pure water for 2 days.

After centrifugation, the dialyzed fluid was concentrated to about 50 g, a small amount of sodium acetate solution was added, and 4 times the volume of ethanol was added.

The precipitate was washed with ethanol and dried under reduced pressure to obtain 1.9 g of curdlan sulfate sodium salt. The DS of the obtained target product was 1.63 (sulfur content 15.9%).
).

Reference Example 6 1.0 g of a curdlan low polymer (average degree of polymerization 131) obtained by partial hydrolysis of curdlan with an average degree of polymerization of 540 with 85% formic acid (80°C, 30 minutes) was added to 50 d of dimethylformamide. After suspending, 5.0 g of triethylamine-sulfonic acid was added and the mixture was incubated at 24°C for 24 hours.
The reaction was allowed to take place while stirring for a period of time. The reaction solution was treated in the same manner as in Reference Example 5 to obtain 2.0 g of curdlan low polymer sulfate ester sodium salt. The DS of the obtained target is 1.7
2 (sulfur content 163%).

Reference Example 7 1.0 g of curdlan low polymer (average degree of polymerization 82) obtained by partial hydrolysis of curdlan with an average degree of polymerization of 540 with 85% formic acid (83°C, 30 minutes) was added to 50 d of dimethylformamide. After suspending, 5.
Add 0 g of triethylamine-sulfonic acid and heat at 24°C.
The mixture was reacted with stirring for 24 hours. Reference example 5
2.4 g of curdlan low polymer sulfate ester sodium salt was obtained. The DS of the obtained target is 1
．． 99 (sulfur content 17.4%).

Reference Example 8 Curdlan low polymer obtained by partial hydrolysis of curdlan with an average polymerization degree of 540 with 85% formic acid (85°C, 30 minutes) (average polymerization degree of 45N.Og was suspended in dimethylformamide with an average polymerization degree of 50%). After making it cloudy, 5.0 g of triethylamine-sulfuric acid was added and reacted with stirring at 24°C for 24 hours.The reaction solution was treated in the same manner as in Reference Example 5, and 2.5 g of curdlan A polymeric sulfate ester sodium salt was obtained.The DS of the obtained target product was 2.07 (sulfur content 17.8%).

Reference Example 9 3.0 g of curdlan low polymer (average degree of polymerization 37) obtained by partial hydrolysis with 85% formic acid (88°C, 30 minutes) of curdlan having an average degree of polymerization of 540 was added to 15M dimethylformamide. After suspending, 15 g of triethylamine-sulfonic acid was added and reacted at 24°C for 24 hours with stirring.

The reaction solution was treated in the same manner as in Reference Example 5 to obtain 7 g of curdlan low polymer sulfate ester sodium salt.

The DS of the obtained target product was 2.09 (sulfur content 1
7.8%).

Reference Example 10 3.0 g of curdlan low polymer (average degree of polymerization 16) obtained by partial hydrolysis of curdlan having an average degree of polymerization of 540 with 85% formic acid (90'C, 30 minutes) was mixed with 150-dimethylformamide. 15.0 g of triethylamine-sulfonic acid was added thereto, and the mixture was reacted with stirring at 24° C. for 24 hours. The reaction solution was treated in the same manner as in Reference Example 5 to obtain 5.8 g of curdlan low polymer sulfate ester sodium salt. The DS of the obtained target is 1.89
(sulfur content 17.0%).

Test Example 1 The human retrovirus growth inhibition performance of the curdlan sulfate esters and sulfate esters of curdlan low polymers obtained in Reference Examples 1 to 4 was measured as follows.

(1) Materials (a) Cells: HTLV-1 persistently infected cell line MT4 was used. Cells were treated with RPMI-1 from which phenol red had been removed.
The cells were cultured in 640 medium (Kyokuto Co., Ltd.) supplemented with 10% fetal bovine serum and 10 mM HEPES.

(b) Virus/HIV is Molt-4/H
Obtained from the culture supernatant of TLV■.

(2) Method MT-4 cells were cultured in a 96-well flat bottom plate at a density of 8 x 10' cells/50 μI2/well, and HIV was cultured at MO.
I. (multi plicity offinfe
ction) 0.002 and incubated at 37°C for 1 hour to allow virus 18 to adsorb to the cells. After the adsorption operation, the cells were washed, and the samples synthesized in Reference Examples I to 4 were added at various concentrations and cultured for 6 days at 37°C and 5% CO. On the other hand, MTT was dissolved in a small amount of DMSO and diluted with a medium to prepare a 0.5 mg/d solution. P in Kono solution lm
20 μC of MS solution (0.153 mg/d) was mixed. Add 50 μρ of each of these mixtures to the wells of the plate cultured for 6 days, and leave at 37°C for 4 hours.
Absorbance at 50 nm was measured (blank was prepared by adding 100 μg of medium to the wells of the plate). The H r V inhibitory activity of a sample is determined by the concentration of curdlan sulfate and curdlan low polymer sulfate required to suppress cell death by 50%, ED. It is expressed as (μ<!/d).

Test Example 2 Since antiviral agents often exhibit toxicity to host cells, the toxicity of the samples was investigated as follows.

MT-4 cells were cultured under the same conditions as in the above test example, and various concentrations of curdlan sulfate ester and sulfate ester of curdlan low polymer obtained in Reference Examples 1 to 4 were added to ensure that the cells were not infected with viruses. After further culturing for 6 days, Test Example-1
Colorimetric determination was carried out in the same manner as above. Cytotoxicity is ID5o (μg/d
).

HIV of curdlan sulfate ester and sulfate ester of curdlan low polymer obtained in Test Examples 1 and 2
The test results for inhibitory activity and cytotoxicity are shown in Table 1.

Table 1 Card Run ED511 1D
50540 (natural type): 1.04 10.9
>100131 :1.46 2.
34 >10068 +1.61
2.39 From the results of >100 or more, it is clear that curdlan low polymer sulfate ester has high human retrovirus proliferation inhibitory activity. Curdlan low polymer sulfate ester has high solubility in water, low toxicity as shown in Table 1, and high relative human retrovirus growth inhibitory activity, so it can be advantageously used in pharmaceuticals, etc. I can do it.

Test Example 3 The ability to suppress human retrovirus proliferation was measured according to the method of Test Example 1 for the curdlan sulfate esters and curdlan low polymer sulfate esters obtained in Reference Examples 5 to 10.

Test Example 4 Cytotoxicity TDso (μg/m) was measured according to the method of Test Example 2 for the curdlan sulfate esters and curdlan low polymer sulfate esters obtained in Reference Examples 5 to 10.

The test results of HIV suppressive activity and cytotoxicity obtained in Test Examples 3 and 4 are shown in Table 2.

Table 2 Curdlan and Curdlan ED5o ID5o 540 (natural type): 131°82 ・ 45 ・ 37 l. 63 1,72 1.99 2 07 2.09 10.9 4.09 2,76 2,08 3.30 >100 >100 >100 >100 >100 From the above results, curdlan low polymer sulfate ester It is clear that it has high human retrovirus growth inhibitory activity. In particular, curdlan low polymer sulfate esters with a DP of between 16 and 131 have particularly high activity.

Curdlan low polymer sulfate ester has high solubility in water, low toxicity as shown in Table 2, and high relative human retrovirus proliferation inhibitory activity.
It can be advantageously used in medicines, etc.

Example 1 00 mg of the curdlan low polymer sulfate ammonium salt obtained in Reference Example 2, 9 g of sodium chloride, and 9 g of henzyl alcohol were dissolved in distilled water for injection to make a total volume of +000 d. This solution is sterile-filtered using a Membrane filter with a pore size of 0.2 μm, filled into a vial with an internal capacity of 50 tl2 by aseptic operation, sealed with a rubber stopper, and tightly wrapped with an aluminum cap to prepare an injection.

Example 2 Curdlan low polymer sulfate ester ammonium salt obtained in Reference Example 3 400 mg, milk m 195 mg
and 5 mg of magnesium stearate are thoroughly mixed by a conventional method and filled into hard gelatin capsules to prepare capsules.

Effects of the Invention The curdlan low polymer sulfate ester of the present invention has a strong retrovirus growth inhibiting effect and can be used for the prevention and treatment of retrovirus infection.

Claims (4)

[Claims] (1) An antiretroviral agent containing a sulfate ester of curdlan low polymer. (2) Retroviruses are HTLV-I and HTLV-II
, HIV-1 or HIV-2 virus (
1) The formulation described. (3) The average degree of polymerization of curdlan low polymer is about 10 to 40
Claim (1) or (2) using a sulfuric ester having a
). (4) The average degree of polymerization of the curdlan low polymer is about 10 to 20
The preparation according to claim (1), (2) or (3), which uses a sulfuric ester having a concentration of 0.