JPH0316925B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention has the structural formula: The present invention relates to an anticancer drug containing the compound pinowiltin as an active ingredient. Conventionally, cancer chemotherapy agents include alkylating agents (nitrozene mustards, ethyleneimines, sulfonic acid esters), antimetabolites (folate antagonists, purine antagonists, pyrimidine antagonists), and plant fission toxins (colcemid). , vinblastine, etc.),
Antibiotics (sarcomycin, carcinophilin,
mitomycin, etc.), hormones (adrenal steroids, male hormones, female hormones), and porphyrin complex salts (marphyrin, copp), etc. are used. However, most of them are cytotoxic substances and exhibit serious side effects, so there is a strong desire to develop substances with low toxicity and excellent anticancer activity. The present inventor searched widely for substances with low toxicity and anticancer activity among the metabolites of microorganisms, and found that the compound represented by the above formula has extremely low toxicity and excellent anticancer activity. The present invention has now been completed based on new findings that the substance can exert a remarkable effect on cancer treatment. The active ingredients of the anticancer agent of the present invention include humans, livestock, dogs,
It can be an excellent cancer chemotherapy agent for warm-blooded animals such as cats. The compound represented by the above formula that has anticancer activity and is used in the present invention will be explained below. Pinovirtein has been reported as LL-S491γ isolated from Aspergillus Chevalieri, and is said to have antiviral (Herpes simplex) and antiprotozoal (Tetrahymena pyriformis) effects (Journal of the American Chemical
Society/94:17/August 23, 1972). The present inventors also discovered Ceratocystis densiflora knob sp., which was isolated from the inside of the material that caused pine blight.
nov.sp.) [hereinafter referred to as "Ceratocystis sp."]. The Ceratocystis bacterium was entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 26, 1981, and its microbial accession number is FERM P-6365. Hereinafter, the mycological properties of the Ceratocystis genus bacteria and a production example of pinoirtein will be explained. [Mycological properties of Ceratocystis genus bacteria] (1) Growth status on each medium Tuapetsk agar medium

[Table] Tuapetsk Yeast Extract Peptone Medium Growth is insufficient on the medium used with mycological properties (excluding Tusapetsk agar) as described in the industrial examination standards "Microorganisms and Fermented Products."
The following culture medium was used.

【table】 Pine needle broth medium

[Example of manufacturing Pinot Wiltein]

KCl 0.5g MgSO ₄ 0.5g K ₂ HPO ₄ 1.0g NaNO ₃ 2.0g Water 1 Yeast extract 2.5g Peptone 5.0g Sucrose 5.0g Avicel (cellulose powder) 10.0g Sterile medium with the above composition (divided into flat culture flasks) A total of 10 samples were inoculated with the Ceratocystis bacterium (Feikoken Bacteria No. 6365), and statically cultured at 25°C for 28 days. 100 g of activated carbon was added to the culture solution collected by suction from the flask, followed by deaeration, and the product was adsorbed on the activated carbon, washed with distilled water, and desorbed with methanol. From the obtained water-containing methanol fraction, methanol was distilled off under reduced pressure at 40°C.
The remaining aqueous solution was adjusted to pH 3 with 5% dilute sulfuric acid and extracted with ethyl acetate. The obtained extract was purified by silica gel chromatography and eluted with a mixed solvent of chloroform methanol (10:1).
Crystallization with benzene gave needle-shaped crystals. This was further recrystallized from hexane-ethyl acetate (3:1) to form colorless and transparent plate-like crystals of pino iltein.
Obtained 120mg. [Physical properties of pinoirtein] mp: 194-199℃ (decomposed) [α] ²⁵ _D : +69.9 (MeOH) Molecular weight (FD-mass spectrum, milli-mass spectrum): M ⁺ 348.194; Molecular formula C ₂₀ H ₂₈ O ₅ UVλ ^MeOH _nax nm: 206 (ε=7875) IR ^KBr _nax cm ^-1 : 3510, 3485, 3270, 1738, 1638 NMR ^DMSO _100M Hzδ: 6.55 (1H, br.S), 5.84 (1H, dd , J=9.9, 19.1Hz), 5.68 (1H, br, s), 5.19 (1H, br.S), 4.96 (1H, dd, J=2.0, 19.1Hz), 4.93 (1H, dd, J=2.0 , 9.9Hz), 4.18 (1H, br, d) 1.14 (3H, S), 1.03 (3H, S), 0.89 (3H, S) Toxicity: No effects were observed even after continuous administration of 50 mg/Kg to mice. Not toxic. The above physical properties were consistent with literature values. Next, the anticancer agent of the present invention containing pinovirtein as an active ingredient will be described. The anticancer agent of the present invention can be administered either orally or parenterally.
Administered as hard capsules, tablets, granules, fine granules, or powders; when administered parenterally, injections,
It can be administered in a dosage form such as a suppository so as to maintain sustained mucosal absorption in the form of an infusion, solid form or suspended viscous liquid form. Furthermore, when formulating the active ingredient of the present invention, the compound represented by the above formula can be made into a preparation for subcutaneous or intravenous injection, as well as capsules, tablets, fine granules, etc. It can be formulated into a dosage form and administered orally. The formulation of the active ingredient of the present invention includes surfactants, excipients, lubricants, adjuvants, and pharmaceutically acceptable film-forming substances in order to form an enteric formulation in consideration of the properties of the active ingredient. This can be carried out as appropriate using a coating aid or the like, and specific examples thereof are as follows. In order to improve disintegration and elution of the composition of the present invention, one or more surfactants such as alcohol, esters, polyethylene glycol derivatives, sorbitan fatty acid esters, sulfated fatty alcohols, etc. are used. can be added. In addition, excipients such as sucrose, lactose, starch, crystalline cellulose, mannite, light silicic anhydride, magnesium aluminate, magnesium metasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate, carboxylic Methylcellulose calcium and the like can be added alone or in combination of two or more. As a lubricant, for example, one or more types of magnesium stearate, talc, hydrogenated oil, etc. can be added, and as a flavoring agent and a flavoring agent,
Sweeteners such as salt, saccharin, sugar, mannitrite, orange oil, licorice extract, citric acid, glucose, menthol, eucalyptus oil, and malic acid, fragrances, colorants, preservatives, and the like may be included. Adjuvants such as suspending agents and wetting agents may include, for example, coconut oil, olive oil, sesame oil, fall seed oil, calcium lactate, safflower oil, soybean phospholipid, and the like. Film-forming substances include cellulose, cellulose acetate phthalate (CAP) as a carbohydrate derivative such as sugars, methyl acrylate and methacrylate as polyvinyl derivatives such as acrylic acid copolymers, and dibasic acid monoesters. Examples include copolymers and methyl methacrylate/methacrylic acid copolymers. In addition, when coating the above-mentioned film-forming substance, in addition to commonly used coating aids such as plasticizers, various additives to prevent the chemicals from adhering to each other during coating operations can be added to the film-forming agent. properties and make coating operations easier. In addition,
It is also possible to form a dosage form in which the active ingredient is microencapsulated using a film-forming substance and then mixed with excipients and the like. In particular, the blending ratio in typical dosage forms is as follows.

[Table] Particularly preferred excipients are lactose, crystalline cellulose, and carboxymethyl cellulose calcium. In addition, the dosage is sufficient to effectively treat the target tumor, and depends on the symptoms of the tumor, route of administration, dosage form, etc., but in general, in the case of oral administration, the amount per day for adults is The range is about 0.01 to 100 mg/Kg body weight (0.01 to 60 mg/Kg body weight for dwarfs), and the upper limit is preferably about 50 mg/Kg body weight, more preferably about 10 mg/Kg body weight, and it is suitable for parenteral administration. In this case, the upper limit is approximately 10 mg/Kg body weight, preferably 5 mg/Kg body weight, and more preferably 2 mg/Kg body weight. Next, the anticancer activity testing method for confirming the anticancer activity of the above compound will be described. Yoshida sarcoma cells from rat ascites
sarcoma cells) were removed and placed in DM-160 medium containing 20% fetal bovine serum (no protein or lipids).
10% in 1 ml of pure synthetic tissue culture medium containing essential amino acids as well as dispensable amino acids.
Dilute to ~15 x ¹⁰⁴ cells. Transfer 20 ml of this cell suspension to a TD ₄₀ flask at 37°C.
When cultured for 3 to 4 days in
110-130×10 ⁴ pieces. This proliferated cell suspension was again diluted with the DM-160 medium and 15
Aim for a cell count of ~20 x ¹⁰⁴ . Transfer 20 ml of this diluted cell suspension to a TD ₄₀ flask and
Cells are grown by culturing at ℃ for 3-4 days. In this way, the tissue culture of Yoshida sarcoma cells is maintained for passage by repeating the growth, dilution, and growth sequence using the DM-160 medium in a TD ₄₀ flask. Yoshida sarcoma cells transferred to tissue culture and grown in a TD ₄₀ flask are suspended in the DM-160 medium and adjusted to a number of 20 to 22 x ¹⁰⁴ cells per ml. Dispense 2 ml of this cell suspension into each vial, dissolve the test compound in 10 μl of methanol to a predetermined final concentration, add to each vial, and culture at 37° C. for 4 days. After culturing, take a portion of the cell suspension in the vial and count the number of cells under a microscope using a hemocytometer. The inhibition rate of test cell proliferation was determined by the following formula. Inhibition rate (%) = (1 - (number of cells in vial administered with test compound) / (number of cells in vial without administration of test compound)
) x 100 The present invention will be specifically explained below using formulation examples and test examples. Formulation Example 1 (Injection/Drop) Add 5 g of powdered glucose to contain 10 mg of pinovirus and aseptically dispense into vials.
Seal tightly, fill with inert gas such as nitrogen or helium, and store in a cool, dark place. Before use, add 100 ml of 0.85% physiological saline to make an intravenous injection,
Administer 10 to 100 ml per day by intravenous injection or drip depending on symptoms. Formulation Example 2 (Injection/Drop) Using 2 mg of pinoirtein, prepare an intravenous injection for mild symptoms in the same manner as Formulation Example 1, and administer for one day.
Administer 10 to 100 ml by intravenous injection or drip depending on the symptoms. Formulation Example 3 (Enteric-coated capsules) 5 g of pinouirtein, 2.46 g of lactose, and 0.04 g of hydroxypropyl cellulose were each taken and mixed well, then formed into granules according to a conventional method, dried well, and sieved. We manufacture granules suitable for bottles, heat seal packaging, etc. Next, 0.5 g of cellulose acetate phthalate and 0.5 g of hydroxypropyl methyl cellulose phthalate are dissolved to form a coated base material, and the granules are suspended and fluidized to coat this base material to form enteric-coated granules. This composition is filled into capsules to produce 100 enteric-coated capsule preparations. Test Example When the inhibition rate (%) of the proliferation of Yoshida sarcoma cells was calculated using the above compound and the above test method, the results shown in FIG. 1 were obtained. As is clear from FIG. 1, 50% inhibition of catalysis was found to be approximately 35 ppm. As is clear from the results of the above test examples, it was demonstrated that the above test compound exerts an excellent effect on suppressing the proliferation of cancer cells.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the growth-inhibiting effect of pinovirtein, an active ingredient of the present invention, on cultured Yoshida sarcoma cells.

Claims (1)

[Claims] 1 Structural formula: An anticancer agent characterized by containing a compound represented by the following as an active ingredient. 2. The anticancer agent according to claim 1 in a parenteral administration form. 3. The anticancer agent according to claim 1 in an oral administration form.