JPH0367055B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel choline-containing phospholipid B and an anticancer agent characterized by containing the 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 a substance with low toxicity and anticancer properties from a wide range of organisms in the animal, plant, and microbial worlds. separated the choline-containing phospholipid contained in
Furthermore, the present invention was completed by newly discovering that the substance has differentiation-inducing activity against animal tumor cells, and by obtaining new findings that the substance has extremely low toxicity and excellent anticancer activity. It came to this. 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. Below, the novel choline-containing phospholipid B of the present invention
(hereinafter referred to as "phospholipid B") will be explained. [Separation and its construction method] Crayfish (2.7Kg) was extracted with chloroform-methanol (2:1), and the extract was subjected to silica gel column chromatography (solvent: chloroform-methanol).
The active fraction (chloroform-methanol = 2:8 eluate) was further subjected to high-performance liquid chromatography (Nucieosil 5C18, solvent:
95% MeOH, flow rate: 2 ml/min). An active fraction was obtained at a retention time of 17.5 minutes, and this fraction was then evaporated and concentrated to yield 5 mg of the single active substance as a colorless oil. The physicochemical properties of the phospholipid B thus obtained are as follows. [Physical and chemical properties of phospholipid B] (1) Form of substance: Colorless oil (2) Mass spectrometry: Molecular weight determined by FD-MS.FAB-MS
509 (3) UV spectrum λ ^MeOH _nax : 233 nm (ε: 770) (4) IR spectrum λ ^KBr _nax cm ^-1 : 3000, 2930, 1730, 1430,
1260, 1145 (5) Solubility: Soluble in chloroform and methanol (6) Reaction to reagents: Dragendorff reagent positive Deitmer-Lester reagent positive KMnO ₄ positive Anthrone reagent negative Considering the above physical and chemical properties, phospholipid B
It is positive for Dragendorff's reagent and Deitmer-Lester's reagent, so it has choline and phosphoric acid in its molecule. It is negative for Anthrone's reagent, so it does not contain amino acids and sugars. The spectrum shows a maximum value of 233 nm, so it has a conjugated double bond.
The IR spectrum shows an absorption band at 1730 cm ^-1 , which indicates that it contains an ester bond, that the amide bond of sphingomyelin is not found, and that it is phospholipase A ₁ as described below.
Fatty acids are found by hydrolysis of Phospholipase _A No fatty acids are found by hydrolysis of Phospholipase C, Phospholipase D
Since choline phosphoric acid or choline was found in the treatment, it was concluded that the substance of the present invention is a choline-containing phospholipid in which a fatty acid is ester-bonded to the end of glycerin. Next, in order to determine the position of this fatty acid, processing was performed using the following method. Phospholipid B is hydrolyzed by Rhizopus delemar lipase having phospholipase A ₁ activity (selectively hydrolyzing the ester bond at position 1),
As a result of GC-MS analysis after esterification with diazomethane, a fatty acid ester corresponding to C ₂₀ H ₃₁ COOCH ₃ was identified. As a result, it was found that a fatty acid corresponding to C ₂₀ H ₃₁ COOCH was bound to the 1st position. Next, the anticancer agent of the present invention will be explained. The anticancer agent of the present invention is characterized by containing the above-mentioned phospholipid B as an active ingredient.
Phospholipid B is a substance isolated from those naturally present in animals such as crayfish, 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, powders, and for parenteral administration, subcutaneous or intravenous injections such as aqueous suspensions and oil-based preparations, infusions, and solid or suspended viscosity. It can be administered in a dosage form such as a suppository so that sustained mucosal absorption can be maintained as a 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. In addition, excipients such as 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, 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, peanut oil, calcium lactate, safflower oil, soybean phospholipid, and the like. Film-forming substances include cellulose, cellulose acetate phthalate (CAP) as a carbohydrate derivative of saccharides, acrylic acid copolymers, and methyl acrylate/methacrylic acid copolymers as polyvinyl derivatives such as dibasic acid monoesters. Examples include methyl methacrylate/methacrylic acid copolymer. 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] Substance
Particularly preferred excipients are lactose, crystalline cellulose, calcium carboxymethyl cellulose. 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 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. As a result of an acute toxicity test conducted in mice by intraperitoneal administration, the LD ₅₀ was greater than 52 mg/Kg. Next, an anticancer test that confirmed the anticancer activity of phospholipid B will be described. [1] Friend leukemia cells (mouse erythrold
Test GlBCO against leukemia cell B8 cells)
15% bovine serum and 60mg/kanamycin were added to F-12 medium manufactured by HAM.
B8 cells are inoculated at 2.5×10 ⁴ cells/ml, 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% _CO2 , the cells were stained using Orkin's benzidine staining method, and the number of stained cells, that is, the number of cells that began to produce hemoglobin due to differentiation into red blood cells, was determined. Measure and determine the differentiation induction rate. Differentiation induction rate (%) = number of stained cells/total number of cells x 100 [2] Mouse myeloid leukemia cells
leukemia cell, M1) test M1 cells were inoculated at 5.0 x 10 ⁴ cells/ml into GlBCO's Eagle MEM medium to which 10% horse serum and 60 mg/kanamycin were added. Add a fixed amount of test compound (5 ml final volume). 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. Note that 1 unit of lysozyme activity refers to Micrococcus lysodicteichus.
lysodeikticus) using a suspension of bacterial cells as a substrate.
This refers to the amount of lysozyme that causes the absorbance at a wavelength of 450 mμ to decrease by 0.001 per minute when measured at pH 6.24 and temperature 25°C. The present invention will be specifically explained below using formulation examples and test examples. Formulation example 1 (injection/infusion) Add 5 g of powdered glucose to contain 10 mg of phospholipid B, dispense aseptically into vials, seal, and store in a cool, dark place with inert gas such as nitrogen or helium sealed. do. Before use, dissolve in ethanol and add 100 ml of 0.85% physiological saline to make an intravenous injection, and administer 10 to 100 ml per day by intravenous injection or infusion depending on the symptoms. Formulation Example 2 (Injection/Drop) Using 2 mg of phospholipid B, make an intravenous injection for mild symptoms in the same manner as Formulation Example 1, and administer for 10 to 10 days.
Administer 100ml by intravenous injection or drip depending on symptoms. Formulation Example 3 (Enteric-coated capsules) 5g of phospholipid B, 2.46g of lactose, and 0.04g of hydroxypropyl cellulose were each taken and mixed well, then formed into granules according to a conventional method, dried well, sieved, and bottled. , manufactures granules suitable for 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 Using phospholipid B, the differentiation induction rate of Friend leukemia cells,
When lysozyme activity was investigated by inducing differentiation of mouse myeloid leukemia cells, it was found that lysozyme activity was induced at 10 to 100 μg/ml against Friend leukemia cells (ELC) and at 50 μg/ml against mouse myeloid leukemia cells (MLC). I found out that it shows. 1.5% DMSO (dimethyl sulfoxide) was used as a positive control. The results are shown in Table 1.

[Table] As is clear from the results of the above test examples, phospholipid B exhibits an effect of inducing differentiation of cancer cells into normal cells, and therefore has been proven to exhibit excellent anticancer activity with little toxicity. Ta.

Claims (1)

[Scope of Claims] 1. Choline-containing phospholipid B having the following physical and chemical properties. (1) Form of substance: Colorless oil (2) Mass spectrometry: Molecular weight from FD-MS.FAB-MS
509 (3) UV spectrum λ ^MeOH _nax : 233 nm (ε: 770) (4) IR spectrum ν ^KBr _nax cm ^-1 : 3000, 2930, 1730, 1430, 1260,
1145 (5) Solubility: Soluble in chloroform and methanol (6) Reactions to reagents: Dragendorff reagent Positive Deitmer-Lester reagent Positive KMnO ₄ Positive Anthrone reagent Negative 2 Choline-containing phospholipid B with the following physicochemical properties An anticancer agent characterized by containing it as an active ingredient. (1) Form of substance: Colorless oil (2) Mass spectrometry: Molecular weight from FD-MS.FAB-MS
509 (3) UV spectrum λ ^MeOH _nax : 233 nm (ε: 770) (4) IR spectrum ν ^KBr _nax cm ^-1 : 3000, 2930, 1730, 1430, 1260,
1145 (5) Solubility: Soluble in chloroform and methanol (6) Reactions to reagents: Dragendorff reagent, positive Deitmer-Lester reagent, positive KMnO ₄ , positive Anthrone reagent, negative 3 Claim 2 according to parenteral administration form anticancer drug. 4. The anticancer agent according to claim 2 in an oral administration form.