JPS6310685B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel anticancer agent.
Traditionally, cancer chemotherapy agents include alkylating agents (nitrozene mustards, ethyleneimines, sulfonic acid esters), antimetabolites (folate antagonists,
purine antagonists, pyrimidine antagonists), plant fission toxins (colcemid, vinblastine, etc.), antibiotics (sarcomycin, cartinophilin, mitomycin, etc.), hormones (adrenal steroids, male hormones, female hormones), and porphyrin complex salts (marphyrin). , COPP), etc. However, the nucleic acid inhibitory effects of anticancer substances are generally highly toxic as they act not only on cancer cells but also on normal cells.
At present, it is difficult to achieve sufficient effects by using large doses of drugs such as chemotherapeutic agents for infectious diseases, as they exhibit serious side effects. The present inventor previously developed a new and useful anticancer agent containing benzaldehyde as an active ingredient (Japanese Patent Publication No. 54
(Refer to Publication No. 962), the action of this active ingredient does not directly attack cancer cells, but is thought to produce therapeutic effects through a mechanism of action that is different from that of conventional chemotherapeutic agents. In addition, as a result of intensive research to search for anticancer active substances, we found that 4,6-O-benzylidene-D-glucopyranose, a benzaldehyde derivative, has a remarkable effect on cancer treatment. We have now completed the anticancer agent of the present invention by obtaining new knowledge that it can exhibit the following effects. Most conventional cancer chemotherapeutic agents are so-called cytotoxic substances, which are more sensitive to transplanted cancers such as Ehrlichi tumors than to cancerous cells caused by the SV ₄₀ oncogenic virus. The active ingredient exhibits high sensitivity to cells transformed into cancer by the SV ₄₀ oncogenic virus.
It is thought to be based on a specific anticancer effect that is different from the mechanism of action of conventional cancer chemotherapeutic agents, and has excellent characteristics as an anticancer agent. The structural formula, melting point, and references of 4,6-O-benzylidene-D-glucopyranose, which is the active ingredient of the present invention, are as follows. mp186-187℃ HGFletcher, Jr.; Methods in Carbohydrate Chemistry, Vol. 2, 307 (1963) 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 a drop, a solid or a viscous liquid suspension. The proportion of the active ingredient in the anticancer drug composition of the present invention may vary depending on the dosage form, but usually approximately 0.3 to 15.0% by weight is appropriate when administered orally or by mucosal absorption, and when administered parenterally, Most of the time,
0.01-10% by weight is suitable. In addition, in formulating the active ingredient of the present invention, it can be made into a subcutaneous or intravenous injection preparation in the form of an aqueous suspension, an oily preparation, etc., as well as a capsule, tablet, or finely divided formulation according to a conventional method. It can be formulated into a dosage form such as granules and administered orally. The surfactants, excipients, lubricants, adjuvants, pharmaceutically acceptable film-forming substances, etc. used in formulating the active ingredient of the present invention 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, mannite, 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 lubricants 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 acetate phthalate (CAP) as a carbohydrate derivative such as cellulose and 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 chemicals from adhering to each other during coating operations can be added to the film-forming agent. properties and make coating operations easier. The anticancer agent of the present invention can be administered as parenteral preparations such as injection (intravenous or subcutaneous), infusion preparations, and suppository preparations, or as oral preparations such as tablets and capsules. Although it may vary depending on the route of administration, dosage form, number of administrations, etc., the effective dose when used for adult treatment is usually about the amount of the above compound as the active ingredient per day for parenterally administered drugs.
In the range of 0.1 to 500 mg/Kg of body weight, preferably 0.5 to 500 mg/Kg of body weight
200mg/kg body weight, more preferably 1-100mg/kg body weight
Kg is appropriate, and for orally administered drugs, per day,
Approximately 0.5 to 5000 mg of the above compounds as active ingredients
Body weight in the range of Kg, preferably 0.5-500 mg/body weight
Kg, more preferably 1 to 200 mg/Kg of body weight. Next, the anticancer activity testing method for confirming the anticancer activity of the above compound will be described. [Test Method 1] W2K-11 cells obtained by turning C3H mouse kidney cells into cancer with the SV ₄₀ oncogenic virus were used as test cells, and were cultured according to the following method. (1) Preparation of growth culture solution 9.4 g of Eagle's MEM medium was dissolved in 900 ml of distilled water, autoclaved at 120°C for 15 minutes, cooled, and 100 ml of calf serum and separately autoclaved at 115°C for 15 minutes.
Add 3 to 5 ml of 10% sodium hydrogen carbonate solution to adjust the pH.
Adjust to 7.1-7.2. Immediately before use, add 10 ml of Millipore filtered L-glutamine (2.92 g/100 ml) solution. In addition, for the preservation of test cells, the final concentration is 10%.
of dimethyl sulfoxide. (2) Preparation of transplanted cells Test cells stored in a Jeep freezer (-80℃) were lysed at room temperature, centrifuged at 670 x g for 5 minutes, the supernatant was discarded, and the precipitated cells were added to the growth medium.
After suspending in 50 ml, transfer to a roux flask and
When cultured at .degree. C., the cells begin to proliferate while adhering to the bottom of the flask, and fully proliferate in 3 to 4 days. Decant the culture solution, then add 0.2% trypsin solution [Eagle MEM medium (Nissui Pharmaceutical Co., Ltd.)
), 0.6 g of baking soda, and 1 g of trypsin in distilled water.
Add 10 ml of the solution dissolved in 500 ml and filtered with a Millipore filter, treat with trypsin for 2 to 3 minutes at room temperature, and then decant the trypsin solution. Furthermore, add 50 ml of fresh growth medium and wash off adhering cells using a Komagome pipette to obtain a cell suspension. A portion is subcultured using Roux flasks. (3) Cell culture and administration of test compound Dispense 1.8 ml of the above cell suspension into a disposable chamber (diameter 35 mm) and incubate at 37°C in a carbon dioxide incubator (5% CO ₂ , 95% air).
Incubate for 24 hours. At this point, 0.2 ml of the test compound solution is administered to continue culturing. The state of cell proliferation is observed every day using an inverted microscope, and the number of surviving cells is counted 48 hours after administration. The test compound is dissolved in distilled water or ethanol (final concentration 2%) and then filtered with a Millipore filter. (4) How to count the number of cells After administering the test compound, decant the shell for 48 hours, discard the supernatant (culture medium), and treat the cells attached to the bottom of the shell with 1.0 ml of the above 0.2% trypsin solution, resulting in single cells. Become. Decant this to remove the trypsin solution, make a cell suspension with physiological saline containing 10 mmol of phosphate buffer (PH7.0), place 1 to 2 drops of this on a hemocytometer, and put it on a cover glass. Count the number of cells under a microscope. The inhibition rate of test cell proliferation was determined by the following formula. Inhibition rate (%) = (Number of cells in a shear plate without test compound administration) - (Number of cells in a shear plate administered with a test compound) / (Number of cells in a shear plate administered with a test compound) x 100 [] Test method 2 against Walker-256sarcoma Antitumor effect test animals were 6-week-old female Wistar rats (Claire Japan), 10 in each group. For transplantation of tumor cells, condensate was collected on the 5th day after passage, and 1×10 ⁶ cells were transplanted subcutaneously into the rat's inguinal region. In intracavitary administration of the test compound, 10, 50 and
At a dose of 100 mg/Kg, 1 day from 24 hours after transplantation.
Efficacy was determined by tumor area (size) and weight and morphological changes of the tumor up to 20 days after transplantation after one daily administration. Tumor inhibition rate (%) = (1 - tumor area or weight of test compound-administered rats/tumor area or weight of non-administered rats x 100) Next, regarding the toxicity of the compound of the active ingredient of the present invention, it is It does not cause any side effects because it is quickly excreted outside the body.The LD ₅₀ values of the active ingredient compound of the present invention are as follows: Mouse Intravenous injection 400mg/Kg Double cavity 1440mg/Kg Oral >3000mg/Kg Subcutaneous >4000mg/Kg Rat Intravenous >400mg/Kg Double cavity 2200mg/Kg Oral >3000mg/Kg 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 500 mg of the above compound, dispense aseptically into vials, seal them, fill with inert gas such as nitrogen or helium, and store in a cool, dark place.Before use, add 0.85% physiological Add 500ml of saline to make an intravenous injection, and administer 10 times a day.
Administer ~500ml by intravenous injection or drip depending on symptoms. Formulation Example 2 (Injection/Drop) An intravenous injection for mild symptoms was prepared in the same manner as Formulation Example 1, except that 50 mg of the above compound was used.
Administer ~500ml by intravenous injection or drip depending on symptoms. Formulation Example 3 (Injection, Capsule) 30 mg of the above compound was dissolved in 1 g of purified sesame oil and 100 mg of aluminum stearate gel and sealed.
Fill with inert gas such as nitrogen or helium and store in a cool, dark place to make a preparation for subcutaneous injection. Administer 1 to 10 ml subcutaneously once a day depending on the symptoms. In addition, the above preparation is dispensed into capsules in 0.5 ml portions to prepare oral capsules, and 1 to 10 capsules are orally administered per day depending on the symptoms. Test Example 1 Cancer was caused by SV ₄₀ oncogenic virus using the above compound as a test compound according to the above anticancer test method 1.
Growth inhibition rate (%) of cancer cells W2K・11 in C3H mice
As a result of calculating the
37%, and 23% after 72 hours treatment at 125mcg/ml concentration. As is clear from the test results, the above compound was proven to have excellent anticancer activity over a wide concentration range. Test Example 2 The anticancer effect on a transplanted tumor (Walker-256) was investigated using the above compound as a test compound using the anticancer test method 2 described above. The results are shown below.

【table】

[Table] As is clear from the test results, the above compound was proven to exhibit excellent anticancer activity in animal tests as well.

Claims (1)

[Claims] 1 An anticancer agent containing 4,6-O-benzylidene-D-glucopyranose as an active ingredient.