JPH02304025A - Anti-retrovirus agent - Google Patents

Abstract

PURPOSE:To obtain a safe anti-retrovirus agent effective as a remedy for AIDS by using a sulfate or phosphate of cyclodextrin as an active component. CONSTITUTION:Cyclodextrin sulfate, cyclodextrin phosphate or its salt is used as an active component. The compound can be administered orally or parenterally at a daily dose of preferably 0.1 to 300-mg/kg, especially 0.5 to 100mg/kg.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to antiretroviral drugs.

(Prior art) AIDS (acquired immunodeficiency syndrome) is a fatal or extremely malignant disease caused by infection with the human immunodeficiency virus (IV), a type of retrovirus, and prevention and eradication are currently a worldwide challenge. It has become the most important problem for humanity to overcome at this level.

Conventionally, attempts have been made to treat AIDS using antiretroviral agents that block the reverse transcriptase possessed by HrV, and typical examples of such antiretroviral agents include azidothymidine and the like. Known [Medical History, Volume 142, No. 9, 61
9-622 (1987)].

However, it has not yet been fully verified and confirmed whether the conventionally known anti-herovirus-related drugs are effective and safe for the treatment of AIDS.

In addition, cyclodextrin sulfate has anti-inflammatory effects,
Although it is known to have an anti-arteriosclerotic effect and a lipid serum clarifying effect (Japanese Patent Application Laid-Open No. 50-36422), its antiretroviral effect is completely unknown. Furthermore, it is known that cyclodextrin phosphate esters are useful as catalysts [e.g., Chemistry Letters (
Chemistry Letters), 1063
-1066, 1980], but its pharmacological effects are unknown.

(Structure and Effects of the Invention) The present invention relates to an antiretroviral drug comprising a sulfuric acid or phosphoric acid ester of Schiflobuchis I. phosphorus, or a salt thereof as an active ingredient.

The sulfuric acid or phosphoric acid ester of Sifurobukis 1-phosphorus, which is the active ingredient of the present invention, has an excellent anti-herovirus effect, especially an excellent anti-proliferation effect against HIV, and has low toxicity and is highly safe as a medicine. There is also a characteristic. In addition, some conventionally known anti-HIV agents have excellent anti-HIV effects, but on the other hand, they have anticoagulant effects (administration of drugs with this effect is difficult to prevent HIV in hemophilia patients).
However, cyclodextrin sulfate or phosphate esters, especially cyclodextrin phosphate esters, are also characterized by extremely weak anticoagulant effects. Therefore, the anti-rovirus drug of the present invention can be used for the prevention and/or treatment of retrovirus infections such as AIDS (AIDS).

Specific examples of the sulfuric acid or phosphoric acid ester of cyclodextrin, which is an active ingredient of the antiretroviral drug of the present invention, include the sulfuric acid or phosphoric acid ester of α-cyclodextrin, β-cyclodextrin, or T-cyclodextrin. Although these esters may be in free form, it is usually preferable to use them in the form of pharmacologically acceptable salts, such as salts such as
・Alkali metal salts such as lithium salts, potassium salts, and lithium salts; alkaline earth metal salts such as calcium salts, magnesium salts, and barium salts; trimethylamine salts, triethylamine salts, pyridine salts, ethanolamine salts, and basic amino acid salts. Examples include organic amine salts.

The active ingredient of the present invention, cyclodextrin sulfuric acid or phosphoric acid ester or a salt thereof, can be administered orally or parenterally (
For example, it can be administered intravenously, intramuscularly, or subcutaneously, and can be used in the form of appropriate pharmaceutical preparations such as tablets, granules, capsules, powders, and injections by conventional methods.

The dosage of the active ingredient compound of the present invention varies depending on the age, weight, condition, and type of disease of the patient, but is usually 1.
Approximately 0 per day. 1 to 300 mg/kg is appropriate, especially about 0. It is preferably about 5 to 100 mg/kg.

In addition, the sulfuric acid or phosphoric acid ester of Sifurobukis 1-phosphorus, which is the active ingredient of the present invention, can be produced, for example, by reacting cyclodextrin with a sulfonating agent or a phosphorylating agent in an appropriate solvent; The phosphate ester of was prepared by the method of Breslon et al. [
Journal of the AmericanCh
emical 5ocieLy, + vol. 99, 230
9, (1977)].

As the sulfonating agent, for example, sulfur trioxide-pyridine complex, sulfur trioxide-trialkylamine complex, sulfuric anhydride, concentrated sulfuric acid, chlorosulfuric acid, etc. can be used.

As the phosphorylating agent, for example, phosphorus oxychloride, phosphoric anhydride, etc. can be suitably used. As the solvent, for example, tertiary amines (eg, pyridine, picoline, lutidine, N,N-dimethylaniline), etc. can be suitably used. The amount of the sulfonating agent or phosphorylating agent used is preferably in excess of the cyclodextrin. For example, the sulfonating agent is a sulfur dioxide-pyridine complex or the sulfur trioxide-trialkylamine complex is used as the phosphorylating agent. When using phosphorus oxychloride, it is preferable to use about 5 to 30 moles per mole of cyclodextrin. This reaction can be carried out under cooling to heating, preferably under heating when reacting with a sulfonating agent, and at room temperature when reacting with a phosphorylating agent.

After the reaction is completed, the target product can be isolated and purified by conventional methods. For example, the above reaction may be carried out using pyridine as the solvent and a sulfur trioxide-pyridine complex or a sulfur trioxide-trialkylamine complex as the sulfonating agent.
When using a complex, the target product is usually obtained as a candy-like substance containing impurities such as pyridine used as a solvent and unreacted sulfur trioxide/pyridine complex, but these impurities can be removed as follows. Can be removed. That is, the above candy-like substance is dissolved in water, treated with a strongly acidic ion exchange resin to remove pyridine as a basic substance, and then barium hydroxide and/or barium carbonate are added to precipitate sulfate ions as barium sulfate. Then, the barium ions are removed by treatment with a strongly acidic ion exchange resin, and an alkali metal hydroxide (e.g. potassium hydroxide) is added thereto to remove insoluble matter, followed by freeze-drying to achieve the desired goal. can be obtained as alkali metal salts.

Furthermore, when the reaction between cyclodextrin and a phosphorylating agent is carried out using, for example, pyridine as a solvent and phosphorus oxychloride as a phosphorylating agent, insoluble matter is filtered out after the reaction is completed, and the filtrate is evaporated with ether. The desired product can be obtained as a pyridine salt by pouring it into a solvent such as the following, filtering the precipitate that precipitates out, and treating this precipitate with a water-ether mixture or the like.

Actual Situation - Examples - (Principle) Human T-cell leukemia virus
Leukemia virus, HTLV-1 (A
TLV)) II to MT-4 cells, a persistently infected cell line.
It is known that when infected with IV, HIV rapidly proliferates, and MT-4 cells die in 5 to 6 days due to cytotoxicity. Therefore, the IIIV proliferation inhibitory effect of the sample can be investigated using the number of viable MT-4 cells infected with old V as an indicator.

(Method) MT-4 cells (IV (TALL, -1/LAV) culture supernatant) were incubated at 0.001 TCIDso (median t
issue culture 1nfectious
After infecting at 37°C for 1 hour at a dose of 50% tissue culture contagious infection/cell, the cells were washed and transferred to RPMI-1640 medium (Fe2 (fe
tal calf serum: 10% fetal bovine serum)
The cells were suspended at a concentration of 1X'1O5 cells/ml. Transfer this cell suspension to a flat bottom culture plate for 200 min.
tt I! , put /ue11fJ, 37°C15
% carbon dioxide for 5 days. After culturing, the number of living cells in the cell suspension was counted by trypan blue staining. The HIV proliferation inhibitory effect of the specimen was determined as the concentration of the specimen that inhibited 100% of the infectivity and cytopathic effects of HIV on MT-4 cells.

(result) The results are shown in Table 1 below.

Table 1 ■20D represents cyclodextrin. (The same applies hereafter) Experimental Example 2 (Principle) Mol t-4 cells and Mol persistently infected with old V
When t-4/111V cells are mixed, giant cells are formed in 1 to 2 days. This phenomenon is due to CD on the surface of Molt-4 cells.
This occurs when gp120, an old V envelope protein expressed on the surface of Molt-4/HIV cells, binds to the Molt-4 receptor. Therefore, the inhibitory effect of the specimen on IIIV and CD4 molecule binding (adsorption of HIV to lymphocytes) can be determined by the presence or absence of giant cell formation.

(Method) Mol t-4 cells and Mo persistently infected with illv
Cell concentration:
5 x 105 cells/ml) was placed in a culture plate at 1 ml/nell and cultured for 24 hours. As a control, uninfected AOI t-4 cells (cell concentration: 5
X105 cells/mR) were similarly cultured in a medium containing various concentrations of the specimen. After culturing, the presence or absence of giant cell formation was observed using a microscope, and at the same time, the number of viable cells was counted. The inhibitory effect on giant cell formation is calculated from the following formula. Using the giant cell formation inhibition rate (%) as an index, it was determined as the sample concentration when the weight was 50%.

Giant cell formation inhibition rate (%) (result) The results are shown in Table 3 below.

Table 3 Experimental Example 3 (Method) The experiment was carried out according to the method of LEE (J, Clnc, Microbio, Vol. 25, p. 1717, 1987).

That is, Mol t-4/, which is a persistently infected cell of old V,
1. 100 ml of the AV cell culture supernatant was subjected to ultracentrifugation, and 10 ml of virus solubilization buffer was added to the precipitate to obtain a solution with reverse transcriptase activity.

This solution is 50/7p, and the serially diluted sample solution is 50μ! was added and reacted for 30 minutes, then reverse transcriptase (reverse
transcriptase; RT) reaction buffer at 90 μl. added. Of this, 95 μl was added to 10 μC+ of 311 thymidine triphosphate and the temperate primer [po
ly(rA) ・p(dT) +2-18) 2-5μ
In addition to the duplex containing g15μ℃ (total 1: 100μ
The mixture was then covered with a lid and allowed to react at 37°C for 22 hours.

After terminating the reaction by adding a 10% aqueous solution of I.trichloroacetic acid, the mixture was filtered through a glass filter, and unreacted 3I+thymidine triphosphate was removed by washing with a 5% aqueous solution of trichloroacetic acid. The glass filter was dried, and radioactivity was measured using a liquid scintillation counter to examine reverse transcriptase activity. The inhibitory effect of the sample on reverse transcriptase activity was investigated as the reverse transcriptase activity inhibition rate (%) calculated from the following formula.

1″I Pl: Enzyme activity when no sample is added P2: Enzyme activity when sample is added (results) As a result, the reverse transcriptase activity inhibition rate (%) of β-cyclodextrin phosphate ester pyridine salt (Production Example 8) is 1
000μg/i! , the concentration was 80%.

Production Example 1 2.0g of β-cyclodextrin and 200mf of pyridine
20 g of sulfur trioxide-pyridine complex and 200 g of pyridine were heated and stirred at 70°C.
ml and stirred at 70°C for 6 hours. The supernatant liquid was left to cool, the residue was dissolved in 30 m2 of water, and 200 m of strongly acidic ion exchange resin (Dowcx 50x8 (H type)) was added.
j! and stir for 30 minutes at room temperature. The resin is filtered and washed with water, and the filtrate (including the washing liquid) is adjusted to pH 6 with a 3% aqueous barium hydroxide solution, and then adjusted to pH 7.5 with powdered barium carbonate. Centrifugation (7,000 rpm.

45 minutes), the supernatant obtained was treated with activated carbon, and then treated with a strongly acidic ion exchange resin [trade name: Diaion 5K-IB (
Type H) manufactured by Mitsubishi Kasei Co., Ltd. (40 ts) was added, and the mixture was stirred at room temperature for 20 minutes. The resin is filtered off, the filtrate is concentrated, and then filtered through a membrane filter.

The filtrate was adjusted to pH 7.3 with a 1% aqueous potassium hydroxide solution and freeze-dried to obtain β having the following physicochemical properties.
-Cyclodextrin sulfate potassium salt 3.1
Obtain 6g.

(1) Infrared absorption spectrum (Nujol) 3470,
1640.1240.1158.1038.1000.
940.880.810°740+690+580c
m-' (2) Actual elemental analysis value (%"): C, 14,96; H, 2,65;
:S, 14. ．． 66 K, 19.16 (3) Specific optical rotation [α] + 62.6° (C・0.2. Water) (4) Rt (retention time) of main peak in high performance liquid chromatography (minutes) 7 10. 22
Nearby [conditions] (same below) Stationary phase: Polyvinyl alcohol 500 mm x 7.6 mm 1.0° Mobile phase: , 0.
01M phosphate buffer (pH 7,0) Column temperature: 40°C Detector: Refractometer Flow rate: 1.0 m 11 minutes (5) Differential thermal analysis (heating rate: 5°C/min) Initial drop temperature: 219° C Production Example 2 Dissolve 2.0 g of β-cyclodextrin in pyridine 200, heat and stir at 50°C, add 20g of sulfur trioxide-pyridine complex and 200m of pyridine, and stir at 100°C for 6 hours. . After cooling, Production Example 1
By treating in the same manner as above, 3.44 g of β-cyclodextrin sulfate potassium salt having the following physicochemical properties is obtained.

(1) Infrared absorption spectrum (Nujol) 3500,
1640.1240.1160.1040.100B,
943.810°740.582 cm-' (2) Actual elemental analysis value (%); C, 13,62; H, '2.27
;S, 15.33, 16.44 Production Example 3 β-cyclodextrin 2.0g, pyridine 400mB
and 20 g of sulfur trioxide-pyridine complex at 50°C for 6 hours, and then treated in the same manner as in Production Example 1 to obtain β-cyclodextrin sulfate potassium salt having the following physicochemical properties. Obtain 81 g.

(1) Infrared absorption spectrum (Nujol) 3500,
1640.1240.1160.100B, 940,
880.810.690°630.582 cm (2) Actual elemental analysis value (%), C, 16,08; H+2.83;
,S,14. ．． 30, 17.14 Production Example 4 β-Cyclodextrin2. Og, pyridine 400m and sulfur trioxide-trimethylamine complex 1
After reacting 7.5 g at 70°C for 6 hours, the same treatment as in Production Example 1 was carried out to obtain β-cyclodextrin sulfate potassium salt 3 having the following physicochemical properties.
．． Obtain 40g.

The main island contains small amounts of trimethylamine.

(1) Infrared absorption spectrum (KBr) 3450, 16
40.1240.1160.1038.1000.94
0.878.810°620+580 cm-' (2) Elemental analysis value: Actual value (%); C, 18,38; H, 3,43;
N+1-29; S, 14.73 K, 15.96 Production Example 5 2.2 g of β-cyclodextrin was dissolved in 100 m of pyridine, and sulfur trioxide-trimethylamine.
After adding 7.7 g of the complex and stirring at 00°C for 6 hours, the mixture was treated in the same manner as in Production Example 1 to obtain 3.61 g of β-cyclodextrin sulfate potassium salt having the following physicochemical properties.

(1) Infrared absorption spectrum (KBr) 3400.16
35,1230.1000,800.580 as-'
(2) Actual elemental analysis value (%>; C, 14,88; 11.2.42
; S, 15.74, 20.75 Production Example 6 2.0 g of α-cyclodextrin was added to 200 yr of pyridine.
r1. 70° after adding 20 g of sulfur trioxide-pyridine complex and 200 ml of pyridine.
Stir at C for 6 hours. After cooling for 1 hour, the same treatment as in Production Example 1 produced α-
Cyclodex 1 to phosphorus sulfate/potassium salt 2.6
Obtain 8g.

(1) Infrared absorption Nujol 3460
, 1635.1240.1160.1042.1000
．． 955.878°803.735,690,580
cm-' (2) Actual elemental analysis value (%): c, 14.60. ll+2.88
; s, 15.30 to 17.46 (3) Specific optical rotation [α): '+52.0° (C = 0.2.water) (4) Rt (retention time) of the high performance liquid chromatography main peak (minutes): 10.3
Around 2 (5) Differential thermal analysis (heating rate: 5°C/min) Initial drop temperature: 239°C Production Example 7 By treating 1 to 2.0 g of phosphorus in the same manner as in Production Example 6, the following was obtained. γ-Cyclodegistrin sulfate potassium salt with physicochemical properties 1.76g
get.

(1) Infrared absorption spectrum (KBr) 3450, 16
30.1232.1160.1038.1003.94
0.880°803.740,692,582 cm-
'(2) Actual elemental analysis value (%); C, 13,73; H, 2,73;
S, 15.45, 16.05 (3) Specific rotation [α] n0 + 78.2° (C = 0.4. water) (4) Rt (retention time) of the main peak in high performance liquid chromatography (min) : 10.0
Near 0 (5) Differential thermal analysis (heating rate: 5°C/min) Initial drop temperature: 230°C Production example 8 Add 9.3rd phosphorus oxychloride dropwise to 100 mA of pyridine at -5 to -10''C , add 2.27 g of β-cyclodextrin at -20°C, and stir at room temperature for 20 hours. Insoluble materials are filtered off, the filtrate is added to ether, and the precipitate is collected by filtration. Dissolve in 100 mfi of water, add 150 m1 of ether and separate the liquid.

By filtering the precipitate from the aqueous layer, the precipitate (
0.33 g) is obtained.

This precipitate (0.33 g) was dissolved in 30 ml of water, and a strongly acidic ion exchange resin [trade name: Diaion 5K-IB (H
Type) manufactured by Mitsubishi Chemical Corporation] 10mff1 was added, stirred for 30 minutes, the aqueous layer was adjusted to pH 7.3 with 0.18N potassium hydroxide, filtered and freeze-dried to obtain β-cyclo, which has the following physicochemical properties. Dextrin phosphate ester
Obtain 0.32 g of potassium salt.

On the other hand, the filtrate was concentrated under reduced pressure to a volume of about 30 ml, dropped into methanol, and the precipitate was collected by filtration.
0.81 g of pyridine salt is obtained.

I Nikku l Dan 1 also Su JIJ-7MMμjisagimisuni table ↓晟-
Mu seat (1) Infrared absorption spectrum (KBr) 3400,
1630.1520.1150.1040.910.8
40.740.510cm -' (2) Elemental analysis value 2C
4゜++620358P (hlk 1511□0 calculated value (%); C, 21,47; IL4.29; K
, 13.31P, 10.58 Actual value (%); C, 21,31; H, 3,78:
K, 13.06P, 10.13 (3) Specific rotation [α] +82.4° (C = 0.2.water) (4) Rt (retention time) of high performance liquid chromatography main peak (min) : Approximately 10 (5) Differential thermal analysis (heating rate: 5°C/min) Initial drop temperature = 228
°C
1) Infrared absorption spectrum (KB r) 3400, 16
35.1490.1150.1040.750.680
．． 490cl' (2) Actual elemental analysis value (%); C, 33,66; H, 4,42;
N, 2.47P, 11.94 (3) m, p, : 210°C (decomposition) (4) Specific optical rotation [α), +91.4° (C・0.2.water) (5) High speed Liquid chromatography Main peak Rt (retention time) (min): Approximately 10 (6) Differential thermal analysis (heating rate = 5°C/min) Initial drop temperature: 199
°C

Claims (6)

[Claims] (1) Antiretroviral drugs containing cyclodextrin sulfate or phosphate esters or salts thereof as an active ingredient. (2) The antiretroviral drug according to claim (1), wherein the active ingredient is cyclodextrin sulfate or a salt thereof. (3) The antiretroviral drug according to claim (1), wherein the active ingredient is a cyclodextrin phosphate ester or a salt thereof. (4) cyclodextrin is α-cyclodextrin,
The antiretroviral drug according to claim (2) or (3), which is β-cyclodextrin or γ-cyclodextrin. (5) An antiretroviral drug containing β-cyclodextrin sulfate or its salt as an active ingredient. (6) An antiretroviral drug containing β-cyclodextrin phosphate or a salt thereof as an active ingredient.