JPH0367051B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel compound CSd-1 and an anticancer agent containing this substance as an active ingredient. Conventionally, alkylating agents (nitrodiene mustards, ethyleneimines,
sulfonic acid esters), antimetabolites (folate antagonists, purine antagonists, pyrimidine antagonists), 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 the development of anticancer agents with low toxicity and excellent anticancer activity is strongly desired. In view of the above-mentioned purpose, the present inventors searched for substances with low toxicity and anticancer properties from a wide range of organisms including animals, plants, and microorganisms.
They isolated an active substance from the swim bladder and discovered that it is a completely new substance with unique physical and chemical properties that have not been described in literature.
The present invention was completed by newly discovering that it has differentiation-inducing activity against animal tumor cells, and by obtaining new findings that it has extremely low toxicity and excellent anticancer activity. The active ingredient of the anticancer agent of the present invention can serve as an excellent cancer chemotherapeutic agent for warm-blooded animals such as humans, livestock, dogs, and cats. The novel compound CSd-1 of the present invention will be explained below. [Separation and purification of compound CSd-1] Homogenize the carp swim bladder and extract with acetone. Concentrate the acetone extract and extract with ethyl acetate. The yellow oily substance obtained by concentrating the ethyl acetate extract was subjected to silica gel column chromatography, and sequentially eluted with n-hexane, ether, acetone, and methanol. Fractions eluted with n-hexane-ether (1:1, v/v) were subjected to preparative silica gel thin layer chromatography (TLC).
and purified by high performance liquid chromatography (HPLC) to obtain the compound CSd-1 of the present invention. The physicochemical properties of the compound CSd-1 thus obtained are as follows. [Physical and chemical properties of compound CSd-1] (1) Form of substance: white plate-like crystals (2) Melting point: 140-145℃ (3) Molecular weight: 386 (by FD-MS) (4) Molecular formula: C ₂₇ H ₄₆ O (by high-resolution MS) (5) IR spectrum: ν ^KBr _nax cm ^-1 (see Figure 1) 2920
，
2850, 1742, 1460, 1380 (6) ¹ H-NMR spectrum: CDCl ₃ , 400MHz
(See Figure 2) (7) Reaction to reagent: Anthrone reagent Positive H ₂ SO ₄ -HOAc (1:1) Positive (8) TLC (CHCl ₃ ): Rf=0.2 (9) HPLC (100% methanol 2ml/min): R _T =27
Next, the anticancer agent of the present invention will be explained. The anticancer agent of the present invention is characterized by containing the compound CSd-1 as an active ingredient. Compound CSd-1 is a substance isolated from substances naturally present in animals, such as the swim bladder of carp, and exhibits an effect of inducing differentiation from cancer cells to normal cells, so there is no concern about toxicity. The anticancer agent of the present invention can be administered either orally or parenterally.
Administered as hard capsules or tablets, granules, fine granules, or powders, and for parenteral administration, subcutaneous or intravenous injections such as aqueous suspensions and oil-based preparations, infusions, and solid and suspended viscosity. It can be administered in the form of a suppository so that sustained mucosal absorption can be maintained in the form of a thick liquid. The formulation of the active ingredient of the present invention includes surfactants, excipients, lubricants, adjuvants, and, if necessary, pharmaceutically acceptable film-forming substances and coating aids to form enteric-coated formulations. 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 alcohols, esters, polyethylene glycol derivatives, sorbitan fatty acid esters, sulfated fatty alcohols, etc. are added. Can be added. Examples of excipients include sucrose, lactose, starch, crystalline cellulose, mannitol, light silicic anhydride, magnesium aluminate, magnesium aluminate metasilicate, synthetic aluminum silicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate, Carboxymethyl cellulose 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, peanut 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, acrylic acid copolymers, and polyvinyl derivatives such as dibasic acid monoesters such as methyl acrylate and methacrylic acid copolymers. Examples include polymers 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 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 carboxymethylcellulose calcium. 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, when administered orally, adults receive approximately The range is 0.01 to 100 mg/Kg body weight (0.01 to 60 mg/Kg body weight for children), and the upper limit is preferably about 50 mg/Kg body weight, more preferably about 10 mg/Kg body weight, and in the case of parenteral administration. The upper limit is about 10 mg/Kg body weight, preferably 5 mg/Kg body weight, more preferably 2 mg/Kg body weight. An acute toxicity test of CSd-1 by intraperitoneal administration in mice showed that the LD ₅₀ was greater than 100 mg/Kg. Next, the anticancer activity tests that confirmed the anticancer activity of compound CSd-1 will be described. mouse myeloid leukemia cells
test for leukemia cell, M1) GIBCO Eagle MEM medium containing 10% horse serum and 60mg/
of kanamycin and 5.0×10 ⁴ cells/
ml of M1 cells, and a predetermined amount of the test compound is added thereto (final volume: 5 ml). After culturing for 7 days at 37°C in 7.5% CO ₂ , lysozyme activity induced by differentiation into phagocytic cells or granulocytes is examined. In addition, one unit of lysozyme activity is defined as the amount of lysozyme activated by using a suspension of Micrococcus lysodeikticus cells as a substrate to increase the pH.
6.24, the amount of lysozyme that reduces the absorbance at a wavelength of 450 mμ by 0.001 per minute when measured at a temperature of 25°C. The present invention will be specifically explained below using manufacturing agents, formulation examples, and test examples. Production Example [Separation and Purification of Compound CSd-1] 500 g of carp swim bladder was homogenized, and the homogenate was extracted with acetone. The obtained acetone extract was concentrated under reduced pressure, and extracted with ethyl acetate. The yellow oily substance obtained by concentrating this ethyl acetate extract was subjected to silica gel column chromography (Kieselgel 60, manufactured by Merck & Co.) and developed sequentially with n-hexane, ether, acetone and methanol. The active fraction of compound CSd-1 is contained in the n-hexane-ether (1:1, v/v) elution fraction. Collect this fraction and prepare silica gel.
When subjected to TLC (Kieselgel 60F-254, manufactured by Merck & Co.) (developing solvent: chloroform), a single spot is obtained. This Rf=0.2 fraction was collected and HPLC
(Nucleosil5C ₁₈ , manufactured by Hitachi) (developing solvent: methanol, 2 ml/min) to purify the compound CSd-1.
10 mg of crystals were obtained. Formulation example 1 (injection/infusion) Add 5 g of powdered glucose to contain compound CSd-1 (10 mg), dispense aseptically into vials, seal, and fill with inert gas such as nitrogen or helium. Store in a cool, dark place. Before use, it is dissolved in ethanol and added with 100 ml of 0.85% physiological saline to form an intravenous injection, and 10 to 100 ml is administered per day by intravenous injection or drip depending on the symptoms. Formulation example 2 (injection/infusion) Compound CSd-1 (2 mg) was prepared as an intravenous injection for mild symptoms in the same manner as in formulation example 1.
Administer 10 to 100 ml per day by intravenous injection or drip depending on symptoms. Formulation Example 3 (Enteric-coated capsules) Compound CSd-1 (5 g), 2.46 g of lactose, and 0.04 g of hydroxypropyl cellulose were mixed well, then formed into granules according to a conventional method, and dried thoroughly. and sieves to produce granules suitable for bottles, heat-seal packaging, etc. Next, 0.5 g of cellulose acetate phthalate and 0.5 g of hydroxypropyl cellulose phthalate are dissolved to form a coated base material, and the base material is coated while the granules are suspended and fluidized to form enteric-coated granules. This composition is filled into capsules to produce 100 enteric-coated capsule preparations. Test Example When compound CSd-1 was used to investigate the lysozyme activity induced by differentiation of mouse myeloid leukemia cells using the test method described above, it was found that compound CSd-1 had a strong effect on M1 cells by inducing differentiation at 0.36 μg/ml. It was found that lysozyme activity (6196U/ml) was exhibited. As described above, the compound CSd-1 of the present invention exhibits an effect of inducing differentiation of cancer cells into normal cells, and thus has been demonstrated to exhibit excellent anticancer activity with little toxicity.

[Brief explanation of drawings]

Figure 1 is a graph showing the infrared absorption spectrum of compound CSd-1, and Figure 2 is a graph showing the infrared absorption spectrum of compound CSd-1.
It is a graph showing the ¹ H-NMR spectrum of.

Claims (1)

[Claims] 1. A compound CSd- having the following physical and chemical properties.
1. (1) Shape of substance: white plate-like crystals (2) Melting point: 140-145℃ (3) Molecular weight: 386 (by FD-MS) (4) Molecular formula: C ₂₇ H ₄₆ O (by high-resolution MS) ( 5) 1R spectrum: ν ^KBr _nax cm ^-1 2920, 2850, 1742,
1460, 1380 (6) Reaction to reagent: Anthrone reagent Positive H ₂ SO ₄ -HOAc (1:1) Positive 2 Compound CSd-1 with the following physical and chemical properties
An anticancer agent characterized by containing as an active ingredient. (1) Shape of substance: white plate-like crystals (2) Melting point: 140-145℃ (3) Molecular weight: 386 (by FD-MS) (4) Molecular formula: C ₂₇ H ₄₆ O (by high-resolution MS) ( 5) 1R spectrum: ν ^KBr _nax cm ^-1 2920, 2850, 1742,
1460, 1380 (6) Reaction to reagent: Anthrone reagent Positive H ₂ SO ₄ -HOAc (1:1) Positive 3 The anticancer agent according to claim 2 in a parenteral administration form. 4. The anticancer agent according to claim 2 in an oral administration form.