JPH01203330A - Carcinostatic agent - Google Patents

Abstract

PURPOSE:To provide the present anticancer agent having low toxicity and carcinostatic properties, by containing docosahexaenoyl-lysophosphatidyl choline as an effective component. CONSTITUTION:The objective carcinostatic agent contains docosahexaenoyl- lysophosphatidyl choline (referred to as D-LPC) as an effective component. The D-LPC is provided, for example, by dissolving docosahexaenoic acid in dehydrated dichloromethane, reacting the acid with N,N'-dicyclohexyl carbodiimide, reacting the reaction product with glycerophosphoryl choline and 4-dimethylaminopyridine in dehydrated dichloromethane, removing a cata lyst from the obtained crude product and subsequently carrying out the removal of the solvent, etc. The D-LPC can be administered by both oral and parenteral administration methods.

Description

Detailed Description of the Invention (Technical Field) The present invention provides docosahexaenoyl-lysophos 77tidylcholine (docosahexaenoyl-lysophos).
The present invention relates to an anticancer agent containing phospha-tidyl choline as an active ingredient.

(Background of the Invention) Conventionally, cancer chemotherapy agents include alkylating agents (night compound mustards, ethyleneimines, sulfonic acid esters), antimetabolites (folate antagonists, purine antagonists,
pyrimidine antagonists), plant nuclear fission, etc. (colcemid, vinblastine, etc.), antibiotics (sarcomycin, cartinophilin, mitomycin, etc.), hormones (adrenal steroids, male hormones, female hormones), and porphyrin complex salts (marphyrin, C0PP) 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.

With this in mind, the present inventors searched for a substance with low toxicity and anticancer properties, and as a result, they discovered that it has a strong ability to induce differentiation of teratoma cells and erythroblastic leukemia cells in living crayfish. We isolated active phosphatidylcholine, analyzed its structure, and discovered that this substance could be used as an excellent anticancer agent.
.

After that, further research was carried out, and various derivatives of the substance were chemically synthesized or semi-synthesized and their anticancer activity (differentiation inducing activity) was investigated, and docosahexaenoyl lysophosphatidylcholine was found to have excellent activity. They discovered this and completed the present invention.

(Objective of the Invention) An object of the present invention is to provide an anticancer agent containing docosahexaenoyl lysophosphatidylcholine as an active ingredient.

(Structure of the invention) can be synthesized by

Synthesis of D-LPG■ Docosahexaenoic acid (DMA) 11.2g (34,
2mM) dehydrated oo methane 28n+j! Dissolved in N
, N'-dicyclohexylcarbodiimide (DCC)
3.4 g (16.3 mM) was added and reacted at room temperature for 24 hours to obtain 7.7 g of DMA anhydride.

■ 7.7 g of DMA anhydride (12.1 mM), 25 ml of dehydrated dichloromethane, and glycerophosphorylcholine (G
PC) 1.25 g (4,8mM) and 4-dimethylaminopyridine (DMAP) 0.58 g (
4,80 IM) was added and reacted at room temperature for 24 hours. After the reaction, the crude phosphatidylcholine (PC) is obtained by filtering and removing the solvent.
I got it.

■ Dissolve crude PC in chloroform-methanol mixture,
The catalyst was removed by passing through a column packed with ion exchange resin Amberlite 200C.

■ After removing the solvent, use 100mj of dehydrated cold acetone! was added and left in the freezer for 3 hours, followed by centrifugation at 3000 RPMXIO minutes. After repeating this operation twice to remove the solvent, a pale yellow oil was obtained.

(2) The oil was applied to a medium pressure column (2.2 cm x 30 cm) of silica gel (CG3, manufactured by Fuji Gel). Developed with methanol-chloroform (0-100%). 1. to the chloroform-methanol (4: IV/V) fraction.
6.0 g of 2-cytocosahexaenoyl phosphatidylcholine (DD-PC) was obtained.

■ Dehydrated ether-dehydrated methanol (98:2v Ha) and Trimeresurus flavovi phospholipase A2 were added to 2.5 g of the obtained DD-PC.
ridis Wako Pure Chemical Industries, Ltd.) 25mg and 100mM calcium chloride solution 50ml and 200mM) Lis-HCl buffer 200+t+j! was added and incubated at 37°C for 24 hours.

(2) Extraction was carried out by the Bligh-Dyer method, and after removing the solvent, 100 ml of cold dehydrated acetone was added and the mixture was left in a freezer for 3 hours. After standing, it was centrifuged at 3000 RPMX for 10 minutes. This operation was repeated twice to obtain a white oil after removing the solvent.

(2) The oil was applied to a medium pressure column (2.2 cm x 30 cm) of silica gel (CG3, manufactured by Fuji Gel). Developed with methanol-chloroform (0-100%). D- in the chloroform-methanol (1:4 v/v) fraction.
9 g of LPGl was obtained.

The analytical values are as follows.

l) Shape of substance: white oil 2) Fatty acid composition analysis by GC (capillary gas chromatography) C22:6 99.6% 3) D-LPC composition analysis by HPLC ODS column, flow rate: l+nf/min SUV205nm: elution Liquid: methanol, single peak.

4) Mass spectrometry: Molecular weight M565 (
CM+H)” 566).

The anticancer agent of the present invention can be administered either orally or parenterally; when administered orally, it is administered as a soft/hard capsule, tablet, granule, fine granule, or powder; when administered parenterally, can be administered in the form of subcutaneous or intravenous injections such as aqueous suspensions, oil-based preparations, infusions, and solid or suspended viscous liquid forms such as lobules to maintain sustained mucosal absorption. .

The active ingredients of the present invention are formulated using surfactants, excipients, lubricants, adjuvants, and, if necessary, pharmaceutically acceptable film-forming substances and coating aids to form enteric-coated formulations. This can be done as appropriate using, for example,
It is as follows.

In order to improve the disintegration and elution of the composition of the present invention,
One or more surfactants such as alcohols, esters, polyethylene glycol derivatives, fatty acid esters of sorbitan, sulfated fatty alcohols, etc. can be added.

In addition, as excipients, for example, sucrose, lactose, starch, crystalline cellulose, mannitol, soft silicic anhydride, magnesium aluminate, magnesium metasilicate aluminate, 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, salt, saccharin, sugar, mannitol, orange oil licorice extract, etc. can be added. ,citric acid,
Sweeteners such as glucose, menthol, eucalyptus oil, and malic acid, fragrances, coloring agents, preservatives, and the like may be included.

Examples of adjuvants such as suspending agents and lubricants include coconut oil, olive oil, sesame oil, peanut oil, calcium lactate,
Safflower oil, soybean phospholipids, etc. can be contained.

Film-forming substances include cellulose, cellulose acetate phthalate (CPA) as carbohydrate derivatives such as sugars,
Examples of polyvinyl derivatives such as acrylic acid copolymers and dibasic acid monoesters include methyl acrylate/methacrylic acid copolymers and methyl methacrylate/methacrylic acid copolymers.

In addition, when coating the above-mentioned film-forming substances, in addition to commonly used coating aids such as plasticizers, the properties of the film-forming agent can be improved by adding various additives to prevent mutual adhesion of chemicals during coating operations. This can improve the coating process and make coating operations easier. In addition, a dosage form may be prepared in which the active ingredient is microencapsulated using a film-forming substance and then excipients and the like are mixed therein.

Next, the compounding ratio in typical dosage forms is as follows.

Active ingredient 0.1-90 Weight% 0.3-1
5 wt% excipient 10-99.8
〃85～99.4 〃Lubricant
O~50 〃 O~20 〃
Surfactant O~50〃0~20〃 Film forming substance 0.1~50〃0.3~20〃
Particularly preferred excipients are lactose, crystalline cellulose, and 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, approximately 0.0 .01-100mg/kg body weight (0.0 for dwarfs)
1 to 60 mg/kg body weight), 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.
kg body weight, more preferably 20 g/kg body weight is suitable.

Next, the anticancer activity test method for confirming the anticancer activity of the compound of the present invention will be described.

Friend leukemia cells (mouse erythroblastic leukemia cells, B8
A test was conducted on cells). HAM's F-12 medium (
GIBCO) with 15% fetal bovine serum and 60 mg/l
of kanamycin plus 2.5 x 10'
B8 cells are inoculated at cell/m j2, and a predetermined amount of the test compound is added thereto (final volume: 5 ml).

After culturing at 37°C for 7 days in 8.0% carbon dioxide gas, the cells were stained using Orkin's benzidine staining method.
The number of stained cells, that is, the number of cells that began to produce hemoglobin due to differentiation into red blood cells, was measured, and the differentiation induction rate was determined from the ratio to the total cells.

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 the compound D-LPClOmg, dispense into vials aseptically, seal them, fill with inert gas such as nitrogen or helium, and store in a cool, dark place. saved. Before use, dissolve in ethanol and add 0.85% physiological saline toom1 to make an intravenous injection.
Sun, 10-100m! ! Administer by intravenous injection or drip depending on the symptoms.

Formulation Example 2 (Injection/Drop) Using 2 mg of the compound D-L P'C, an intravenous injection for mild symptoms was prepared in the same manner as Formulation Example 1, and administered for 10 to 1 day.
100mj! Administer by intravenous injection or drip depending on the symptoms.

Formulation Example 3 (Enteric-coated capsules) After thoroughly mixing 5 g of Compound D-LPG, 2.46 g of lactose, and 0.04 g of hydroxypropyl cellulose, it was formed into granules according to the conventional method 1, and this was thoroughly dried. The mixture was then sieved to produce granules suitable for pin, heat-seal packaging, etc. Next, cellulose acetate phthalate 0.5
g and hydroxypropyl cellulose phthalate) 0
．． 5 g was dissolved to form a coated base material, and the granules were coated on this base material while floating and flowing to give enteric-coated granules.

Enteric-coated capsule preparation 1 by filling this composition into capsules
Manufacture 00 pieces.

Test Example (Anticancer Activity Test) Compound D-LPG was used to investigate the differentiation-inducing activity of Friend leukemia (B8) cells according to the test method described above. The results are shown below.

Claims (1)

[Claims] Anticancer drug containing docosahexaenoyl-lysophosphatidylcholine as an active ingredient