JPH0447648B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antitumor agent, and more particularly to an antitumor agent containing trichostatin A represented by the following structural formula [] as an active ingredient. Until now, it was not known that trichostatin A exhibits antitumor activity, but the present inventors have discovered that trichostatin A has antitumor activity.
mouse erythroid cell Friend cells transformed with Murine Sarcoma virus, mouse fibroblast M-MSV Balb3T3 transformed with Murine Sarcoma virus Moloney strain, human cervical cancer cell Hella (HeLa) cells, human leukemia cells, HL-
It was discovered for the first time that it has a strong growth inhibiting effect on 60 cells and ML-1 cells, and based on this discovery, the present invention was completed. Trichostatin A represented by the above structural formula []
Especially Friend cells, M-MSV/Balb3T3 cells,
It has a strong growth inhibitory effect on HeLa cells, HL-60 cells, and ML-1 cells, and can be used as an antitumor agent. Trichostatin A represented by the above structural formula can be produced by the method discovered by the present inventor.
That is, the microorganism used in the present invention is a microorganism that has the ability to produce trichostatin A, and specifically, a microorganism isolated from soil is used. This microorganism was
Of Determinative Bacteriology 8
(1974), it was identified as Streptomyces sioiaensis FERM P-7296. In the present invention, not only the above-mentioned strains and their artificial and natural mutant strains, but also all trichostatin A-producing bacteria belonging to the genus Streptomyces can be used. The medium used to produce trichostatin A using this microorganism is a conventional liquid medium containing a carbon source, a nitrogen source, inorganic salts, and, if necessary, organic micronutrients. Examples of carbon sources include glucose, fructose, maltose, sucrose, starch,
Carbohydrates such as dextrin, starch hydrolyzate, and blackstrap molasses, organic acids such as citric acid, succinic acid, fumaric acid, and acetic acid, and alcohols such as glycerin are used. Examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, and ammonium acetate, nitrates such as sodium nitrate and potassium nitrate, urea, aqueous ammonia, ammonia gas, amino acids, and peptone, soybean whey, and soybean flakes. and proteins such as their hydrolysates, rice bran, etc. are used. As other inorganic salts, for example, manganese salts and phosphates are used as appropriate, and as organic micronutrients, amino acids, vitamins, peptones containing these, yeast extracts, etc. are used as appropriate.
Culture conditions vary depending on the medium composition and other factors, but for example, culture with shaking at a pH of 4.0 to 9.0 and a temperature of 15 to 40°C,
Culture is performed under aerobic conditions such as aerated agitation culture. The trichostatin A of the present invention is present in the culture solution obtained by culturing in this way and in the bacterial cells, and methods for separating and collecting trichostatin A from the culture solution include an organic solvent extraction method such as ethyl acetate, a normal phase extraction method, etc. and known separation methods such as adsorption chromatography using adsorbents such as reversed-phase silica gel and cellulose, gel filtration methods, and methods that utilize differences in solubility in various solvents.
Purification methods are appropriately combined. On the other hand, trichostatin A produced within the bacterial cells is extracted with an organic solvent such as chloroform, and then purified and separated according to the above-mentioned method. Next, a manufacturing example will be shown. Dispense 100ml of the medium (PH7.2) shown in Table 1.
Sterilize a 500ml flask at 120℃ for 20 minutes, and add Streptomys sioiaensis FERM・P to it.
-7296 culture solution (1 ml) was inoculated and cultured at 27°C for 4 days. On the other hand, put 18 of the above medium into a sterilized 30-volume stainless steel jar fermenter, inoculate the above seed mother 2, stir (350 rpm), aerate (1/2 vvm), and culture at 27°C for 3 days. continued. Furthermore, the 20 was used as a secondary seed mother to inoculate the sterilized medium 280 with the above composition in a 300-volume stainless steel tank, stirred (310 rpm), and aerated (1/2
vvm) and cultured at 27°C for 3 days. Table 1 Glucose 1.0% Yeast extract 0.2% KH ₂ PO ₄ 0.1% MgSO ₄・7aq 0.1% Bactosoitone 0.7% Bactopeptone 0.5% Starch 2.0% Adecanol 0.05% (PH7.2) The culture solution of 280 was centrifuged and the hyphae Obtained 13 kg and 260 sterilization liquid. To obtain a segment containing trichostatin A from hyphae, the following method may be followed. To 13 kg of this mycelium, 60 g of a mixture of chloroform and methanol (1:1) was added and stirred at room temperature for 4 hours, and then the mycelium was removed by filtration. The chloroform-methanol mixture containing trichostatin A was concentrated to obtain an oily substance. To obtain the segment containing trichostatin A from the disinfectant solution 260, the following method may be followed. The sterilizing solution 260 was poured into an adsorption chromatography column ("Amberlite XAD-7" manufactured by Organo) (15 cm φ x 67 cm) to remove substances not adsorbed to the adsorption chromatography column. Thereafter, 100% methanol 75 was poured into an adsorption chromatography column, and a solution containing trichostatin A eluted with 100% methanol was collected. Concentrate the fraction at room temperature using an evaporator, 3.1
A concentrated solution was obtained. The oily substance obtained by chloroform-methanol extraction of mycelium and the substance adsorbed to the adsorption chromatography column in the sterilizing solution.
For the segment eluted with 100% methanol,
The common trichostatin A isolation and purification process described below can be used. Hereinafter, a process for isolating and purifying trichostatin A from a concentrated solution containing trichostatin A obtained from a sterilizing solution will be described. Add ethyl acetate 6 to the above concentrated solution 3.1, shake vigorously for 20 minutes at room temperature, and let stand.
was fractionated. Then add 6 ethyl acetate to the residual liquid.
The mixture was again shaken vigorously at room temperature for 20 minutes and allowed to stand, and ethyl acetate fraction 5 was collected. After combining these ethyl acetate fractions, the ethyl acetate fractions were concentrated and dried at room temperature using an evaporator. This concentration
The dried fraction containing trichostatin A was dissolved in 200 ml of 100% methanol. Next, gel permeation chromatography (“LH-20” manufactured by Pharmacia)
After pouring the 100% methanol solution containing trichostatin A into the column (30 cmφ x 800 cm), add a new
Pour 30% of 100% methanol and perform gel filtration.
The liquid was separated into 100 ml portions. Fraction 1, which has a growth inhibiting effect on Friend leukemia cells, was collected from these fluids. After concentrating this fraction,
Mixed solvent of ethyl acetate and methanol (7.5:1)
Dissolve in 10 ml and perform silica gel column chromatography. Ethyl acetate/methanol (7.5:
After development in step 1), 100 ml of the fraction having a growth inhibiting effect on Friend leukemia cells was collected. This fraction was concentrated to obtain approximately 500 mg of trichostatin A crude material. Furthermore, this crude substance was separated and purified by silica gel thin layer chromatography to obtain approximately trichostatin A.
Got 200mg. The physicochemical properties of trichostatin A are as follows. Due to this property, this substance has been identified by Tsuji et al., Journal of Antibiotics 19 , 1.
(1979) (J. Antibiotics, N. Tsuji et al 19 ,
1 (1976)). Melting point m.p150~151℃ Molecular weight 302 (by FD-MASS method) Elemental analysis C, 67.28%, H, 7.40%, N,
9.43%, UV absorption spectrum λ ^EtOH _nax 253nm, 267nm, 341nm Solubility in solvents Soluble in chloroform, ethyl acetate, acetone, benzene Insoluble in water Color reaction Dragendorff reaction Positive ¹ H-NMR spectrum See Figure 1 ¹³ C- NMR spectrum See Figure 2 Next, we will show the antitumor activity of trichostatin A (1) Growth inhibitory effect on Friend cells Ham'S F-12 powder medium (Gibco cell culture medium component) 10.4 g and _1.4g of NaHCO3
Dissolved in 1.0 μl distilled water and sterilized through a Millipore filter with a pore size of 0.22μ, and added aseptically prepared fetal bovine serum (manufactured by Flow labo) to this solution.
A medium for cell culture was prepared by adding 100 ml. Pre-cultured Friend leukemia cells (see Yoji Igawa, Metabolism, 15 , 145 (1978)) were added to this medium (cell concentration: 1 x 40 ⁴ /ml), and the cell suspension was placed in a microtest plate (Nunc). (manufactured by company, 96 holes)
Dispense aseptically to 0.1ml and place in a carbon dioxide incubator (carbon dioxide concentration 5%, temperature 37℃).
Cultured for 24 hours. To this culture solution, trichostatin A isolated and purified from the fermentation solution of Streptomyces sioiaensis FERM P-7296 was added.
Add 0.1ml of the above medium containing a certain amount of
The culture was continued for an additional 3 days, and the growth inhibitory effect of trichostatin A on Friend's leukemia cells was investigated (by counting the number of viable cells by a cell staining method using trypan blue). The results are shown in Table 2. In the table, (-) indicates no growth inhibition, and (+++) indicates that all cells die. [Table] As is clear from Table 2, trichostatin A has a remarkable cell growth inhibiting effect on Friend's leukemia cells and is cytotoxic to Friend's leukemia cells. (2) Effect on M-MSV Balb 3T3 cells Next, Balb 3T3 cells transformed with M-MSV virus (Aaronson and Rowe,
Virology. 42 . 9 (1970))) are shown in Table 3. In this experiment, as a base
MEM Dulbecco's base (manufactured by Gibco) was used. The results are shown in Table 3. In addition, (-) in the table
indicates no growth inhibition, and (+++) indicates that all cells die. [Table] As is clear from Table 3, trichostatin A has a remarkable cell growth inhibitory effect on M-MSV-Balb3T3 cells.
It has been revealed that it exhibits cytotoxicity to cells. (3) Effect on human cervical cancer cells HeLa MEM Dulbecco powder medium (manufactured by Gibco) 1
After dissolving g in 100ml of double-distilled water, 0.14g
of NaHCO ₃ was added and dissolved, passed through a Millipore filter (0.22μ), and added in advance to a medium to which 11 ml of fetal bovine serum (manufactured by Flow Lab.) was added.
HeLa cells cultured for 3 days at 37°C in the presence of 5% _CO2 (Gey, Kubicelc and Coffman Cancer Res,
12, 264 (1952)) to 5 × 10 ⁴ cells/ml, dispensed 0.2 ml of the medium into a microplate (96 wells manufactured by Nunc), and incubated at 5% for 3 hours.
Cultured at 37°C in the presence of _CO2 . 0~500μ for this
Trichostatin A was added at a concentration of g/ml and cultured for 5 days. Thereafter, the amount of growth was determined by counting viable cells using trypan blue staining.
As is clear from Table 4, trichostatin A
It has been revealed that it has a significant cell growth inhibiting effect on HeLa cells and is cytotoxic to HeLa cells. Therefore, trichostatin A
It can be seen that it is cytotoxic to HeLa cells. [Table] (4) Effect on human leukemia cells HL-60 and ML-1 1 g of RPMI-1640 powder medium (manufactured by Gibco)
After dissolving in 100ml of double-distilled water, 0.14g of
Add NaHCO ₃ to dissolve, pass through a Millipore filter (0.22μ), and add fetal bovine serum (Flow
Lab. Co., Ltd.) in advance at 37°C.
HL-60 cells cultured for 3 days in the presence of 5% _CO2 (Collins, et al, Nature, 270 , 347-349
(1977) and ML-1 cells (J. Minowada et al.
al，International Symposium on New
Trends in Human Immunology and Cancer
Immunotherapy pp188-199 (1980)) was dispersed at 5 x 10 ⁴ cells/ml, and 0.2 ml of each medium was added to a microplate (Nunc 96).
wells) and cultured at 37°C in the presence of 5% CO ₂ for 3 hours. Trichostatin A was added to this at a concentration of 0 to 500 μg/ml and cultured for 5 days. Thereafter, the production amount was determined by counting viable cells using trypan blue staining. Table 5 shows the degree of cell growth inhibition based on the criteria shown in Table 4. [Table] As is clear from Table 5, trichostatin A has a remarkable cell growth inhibitory effect on HL-60 cells and ML-1 cells, and is cytotoxic to HL-60 cells and ML-1 cells. It has become clear that this shows that The above results revealed that trichostatin A inhibits the growth of cancer cells and can be an effective antitumor agent. The antitumor agent of the present invention containing the substance represented by the structural formula [ ] as an active ingredient is useful as an antitumor agent for treating mammals with tumors, including humans, and can be administered orally in tablets, capsules or elixirs. It can be used to suppress tumors in living organisms by formulating it as a preparation such as a drug or as a sterile solution or suspension for parenteral administration. The active ingredients used in the present invention are generally administered in divided doses to patients (animals and humans) in need of such treatment in a dosage range of 0.2 to 500 mg per patient, thus a total daily dose of 1 to 2000 mg per day. can be administered. Dosage will vary depending on the severity of the disease, the weight of the patient and other factors recognized by those skilled in the art. The substances used in the present invention include physiologically acceptable vehicles, carriers, excipients, binders, preservatives,
It is mixed with stabilizers, flavoring agents, etc. in unit dosage form as required by generally accepted pharmaceutical practice.
The amount of active substance in these compositions or preparations is such that a suitable dosage within the indicated range will be obtained. Specific agents that can be incorporated into tablets, capsules, etc. are: binders such as tragacanth, gum arabic, cornstarch or gelatin; excipients such as microcrystalline cellulose; cornstarch, pre-gelatinized starch,
leavening agents such as alginic acid; lubricants such as magnesium stearate; sweetening agents such as sucrose, lactose or saccharin; flavoring agents such as peppermint, redwood oil or thiery, when the dosage unit form is a capsule. Materials of the type described above may further contain a liquid carrier such as an oil or fat. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets may be coated with syrup, sugar or both. A syrup or elixir is an active compound,
It may contain sucrose as a sweetening agent, methyl and propyl parapenes as preservatives, dyes and flavoring agents such as cherry or orange flavor. Sterile compositions for injection include the active substance dissolved or suspended in a vehicle such as water for injection, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil and the like or a synthetic fatty vehicle such as ethyl oleate and the like. It can be disposed of in accordance with normal pharmaceutical practice. Buffers, preservatives, antioxidants, etc. can be combined as necessary. Trichostatin A of the present invention is highly safe;
_LD50 in ICR mice is 100 when administered i.p.
mg/Kg or more.

[Brief explanation of the drawing]

FIG. 1 is a ¹ H-NMR spectrum of trichostatin A. Figure 2 shows trichostatin A
¹³ CH-NMR spectrum.

Claims (1)

[Claims] 1. An antitumor agent containing a substance represented by the following structural formula: