JPS61277617A - Inhibitor of blood platelet aggregation - Google Patents

Abstract

PURPOSE:An inhibitor of blood platelet aggregation promoting dissociation of blood platelet aggregation bulk in organism closely related to diseases such as arteriosclerosis, metastasis of cancer, etc., having excellent preventing effects on thrombosis, containing gliotoxin as an active ingredient. CONSTITUTION:The titled inhibitor containing glitoxin shown by the formula as an active ingredient. Oral administration, intravenous injection, etc. are preferable as administration method. Even if gliotoxin to be administered is not pure but a crude substance from a filtrate of culture mixture, it has no bad influence on inhibitory effect on blood platelet aggregation. Glitotoxin as the active ingredient can be produced by a microorganism or by chemical synthesis.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antiplatelet aggregation agent containing as an active ingredient gliotoxin, which is effective in inhibiting platelet aggregation and is represented by the formula: Gliotoxin.

Thrombotic diseases (cerebral infarction, transient ischemic attack, myocardial infarction, angina pectoris, etc.) caused by the aggregation of platelets are caused by an increase in the elderly population and changes in dietary habits and social environment. It is predicted that the number of cases will continue to increase in Japan as well as in Western countries.

In addition to the above-mentioned diseases, platelet aggregation is also attracting attention in the clinical field, as it has been reported that it is closely related to diseases such as arteriosclerosis and cancer metastasis. Therefore, various studies have been carried out by many researchers in Japan and abroad to develop effective platelet aggregation inhibitors, but the current situation is that nothing satisfactory has been obtained yet. It is.

In view of the above-mentioned current situation, the purpose of the present invention is to treat thrombotic diseases, which are considered to be one of the main causes of these diseases, in particular, considering the fact that sufficient therapeutic effects cannot be expected after they occur. The aim is to provide an anti-platelet aggregation agent to prevent this disease.

As a result of intensive research from the viewpoint of developing a drug that specifically acts on platelets using platelet aggregation inhibition as an indicator, the present inventors discovered that the fungus θrgillus fumigatu (Rugilus 7migatus Freseninus CAIII)
After discovering that the culture supernatant of S. Freseninus (S. Successful.

Furthermore, the present inventors conducted a series of pharmacological and toxicological tests on Gly^6S, and found that this compound has an excellent effect on inhibiting platelet aggregation from a clinical standpoint. The present invention has now been completed.

That is, an object of the present invention is to provide an antiplatelet aggregation agent containing gliotoxin as an active ingredient, which has an excellent effect of inhibiting platelet aggregation.

The above gliotoxin has already been isolated by Weidling et al.
27, P1175 (1937))
Furthermore, there are known compounds chemically synthesized by Fukuyama et al. [tetrahedron 57, P2O45-78 (
1981)).

However, it has not been known until now that gliotoxin has an inhibitory effect on platelet aggregation.

This effect was discovered for the first time by the present inventors in research and development of a substance that has an excellent effect of suppressing platelet aggregation. Therefore, the present invention allows for a substance that has a strong effect in a small amount and can be administered at a low dosage. An excellent anti-platelet aggregation agent which can be expected to have a good possibility of clinical application is provided by selection of the following.The pharmacological effects of the above-mentioned gliotoxin will be specifically explained by showing test examples. The compound (gliotoxin) used in the test was obtained by isolation and purification in Example I described below.

Test Example 1 Antiplatelet aggregation effect Gliotoxin solution (a solution in which the target dose of glyodoxin was dissolved in dimethyl sulfoxide (hereinafter referred to as DMSO))
, 6 animals per group].

After 120 minutes, add 58% sodium citrate (
C6H5Na507・2H20; my 294)
Blood was collected from the inferior vena cava of the mouse under ether anesthesia using a syringe containing 0.08 dO, and
8% sodium citrate aqueous solution to 0.9 volume of blood,
0.1 volume added). Said Kuen Lingmian n, B
- was separated by centrifugation (600 rpm, 5 minutes), and the upper layer liquid (hereinafter referred to as liquid A) was collected. The precipitate was then further separated by centrifugation (3100 rpm, 1
0 minutes], and the upper layer liquid (hereinafter referred to as liquid B) was collected.

Solution A and solution B were mixed to obtain platelet-rich plasma (hereinafter referred to as PRP).Furthermore, a portion of the PRP was placed in a separate container, the remainder was centrifuged (3100 rpm, 5 minutes), and the upper layer was collected. was designated as platelet-poor plasma (hereinafter referred to as PPP).

(For details on the production of PRP and PPP, please refer to the method described in the document "Molecular Pharmacology of Platelets - Basic and Clinical," edited by Hiroyoshi Hidaka, Kodansha Scientific, pp. 74-94.) , platelet aggregation rate (NKK) (EMATRA
0% and 100% of the platelet aggregation rate were measured by PRP and 'PPP, respectively. Furthermore, an AJ)P aggregation experiment was conducted by adding adenosine diphosphate (hereinafter referred to as ADP) to PRPα20m at a final concentration of 5 μM.

In addition, as a control group, ICR male mice (weight 40
9.1 group (6 animals) administered with DMBO Q,04- were used.

figure! As shown in Figure 5, if the maximum aggregation rate is M, the time (minutes) from the start of ADP aggregation to M is t, and the remaining rate of platelet aggregation (undissociated platelet rate) after 2 from the start of ADP aggregation is D, the result is is expressed as in Table 1〇Table 1 1Control group 69.B±5.562.2±5.52.10±
0.15 i68 soil 0.72Hα25ri 1064. rJ
±5.350.0±&61゜57±α15 father 20±23
41[[CL5011%'/% 6&2±1.951.
8±2.71.63±Q, 15 a75±α78IV
1.00111P/Kr 65.3± to 949.2±'
From the table in 5.91.68±α249.70±1.3, compared to the control group, administration of gliotoxin clearly lowered the maximum aggregation rate and also significantly lowered the residual rate of platelet aggregation. That is, the platelet aggregation dissociation rate (M
-D)/, it can be seen that the dissociation rate is significantly suppressed.

Therefore, gliotoxin promotes the dissociation of platelet aggregates generated in vivo, and can be expected to have a significant thrombosis preventive effect.

Test Example 2 Toxicity of Gliotoxin According to Johnson et al. [Journal of the American Chemical Society 66.5 (71 (1944)), the lethal dose of gliotoxin is 45% in rabbits (i, v,).
1'v/Kf, 5019/LI in mouse <1-p-)
, Ratu) (i, p.) is 5U to 65q/Kf.

However, this literature value (LDso) is a value obtained by suspending gliotoxin in water and administering it.

In contrast, in the aforementioned experiments, gliotoxin was dissolved in DMSO since it is insoluble in water.

Therefore, regarding toxicity, gliotoxin J) was administered intraperitoneally to ICR male mice (5 mice per group) as an MSO solution.
When 19-gliotoxin/noodles were administered, all five animals were confirmed to be alive (for one week).

As a result, the toxicity LD5Q value when gliotoxin is dissolved in dimethyl sulfoxide solution is 5'Ilf/9.
It turns out that this is all.

The present invention has been completed based on the above-mentioned novel findings regarding gliotoxin, and provides an antiplatelet aggregation agent that suppresses platelet aggregation and contains the gliotoxin as an active ingredient.

There is no need to use any special method for producing gliotoxin, which is the active ingredient of the antiplatelet aggregation agent of the present invention.
It can be produced by known methods, by microbial production or by chemical synthesis [e.g., Journal of American Chemical Society 65, p.
2oos or tetrahedron 37, see P2O45].

Possible administration methods include oral administration and intravenous injection, but the preferred dosage is 15 to 20 η/Kf for oral administration and 0.05 to 2.0 η/KQ for intravenous injection.

Please note that the gliotoxin administered is not pure;
Even if it is a crudely purified product from culture p fluid, it will not affect the anti-platelet aggregation effect in any way depending on the dosage selection.

Therefore, the antiplatelet aggregation agent according to the present invention is not only excellent in its ability to inhibit platelet aggregation, but also extremely effective in that it has the potential to be used in clinical treatment by selecting the dosage. be. The present invention will be specifically described below with reference to Examples.

Example I Composition shown in Table 2 (yeast extract 2?, NaN033 f1
KH2PO41, 51% to 2! (PO41,9f,
FeSO4”7H20(], CI 1 r.

NaCL 0.2? , glucose 30 f 5Mg5
A medium containing 5 tons of O4.7H20CL and 1 t of purified water was equally dispensed into three 600-sized Sakaro flasks, and Rugilus fumigatus 7reseninus was cultured with shaking at 27° C. for 7 days.

Table 2 After culturing, the culture solution was suction-filtered using an aspirator to obtain a 1500-ml solution. Phosphoric acid was added to the 1500-ml culture solution to adjust the pH to 3. The operation of adding 1,500 ml of ethyl acetate to the liquid and extracting using a separating funnel was repeated twice to obtain an approximately 3,000 ml of extract. The extract was dehydrated with sodium sulfate (Na2804), and the core liquid was dried under reduced pressure using an evaporator to obtain a 764W extract. Next, the extract 30019 was adsorbed on a silica gel column (Wakogel C-200), washed with benzene 7"C, and the active fraction was eluted with 150% dichloromethane and concentrated under reduced pressure using an evaporator.
A crude product of 174M9 was obtained. The crude product was purified by recrystallization in acetonitrile, and subjected to high performance liquid chromatography (column: F'1nepaksilc-18).
, solvent; acetonitrile: Kyoma 30Nia 0) to obtain the pure active substance.

It was confirmed that the active substance was gliotoxin by the following method.

The active substance was subjected to mass spectrometry using a mass spectrometer, and the data was searched on an online system (C Engineering S), and as a result, the relevant substance was limited to one substance, gliotoxin. In addition,
From mass spectrometry data, the molecular weight of this material was determined to be 326, consistent with that of gliotoxin.

In the antiplatelet aggregation agent of the present invention, the above-mentioned gliotoxin may be prepared as it is or in various formulations together with conventionally known and used additives (excipients, dispersants, etc.). I can do it.

Examples of formulations of the antiplatelet aggregation agent of the present invention are shown below.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. I is a diagram showing the results of measuring changes in light transmittance over time using a platelet aggregometer to determine the ADP platelet aggregation rate. The vertical axis shows the light transmittance (-), and the horizontal axis shows the elapsed time (minutes). Patent Applicant: Yaruto Co., Ltd. Head Office Agent: Patent Attorney Takao Minami Figure 1

Claims (1)

[Scope of Claims] An antiplatelet aggregation agent containing gliotoxin as an active ingredient represented by the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼.