JPS6058917A - Carcinostatic agent - Google Patents

Abstract

PURPOSE:A preparation that contains specific glycerides as active ingredients, thus being used as a carcinostatic agent with low toxicity. CONSTITUTION:The objective preparation contains one or more of glycerides of the formula (R1 is residue of fatty acid; R2 and R3 are H, or fatty acid residue) as active ingredients. As the fatty acid residues, are mentioned, e.g., saturated or unsaturated (the number of double bonds is about 1 to 10) radicals of 10- 30 carbon atoms. The active ingredients are substances occurring in air bladders of carps or embryos in rainbow trouts originally or its related compounds, thus being free from anxiety of toxicity, because its embryonic induction from cancer cells to normal cells. The resultant agent can be administered parenterally or orally and the dose is about 10mg/kg in parenteral administration and 0.01-100mg/kg in oral one.

Description

Detailed description of the invention: New Year's Eve ER, -e A-: Cl-1,0-R3 (R1 represents a fatty acid residue, R21R3 represents hydrogen or a fatty acid residue) The present invention relates to an anticancer agent characterized by containing seven or more types of glycerides as active ingredients.

Conventionally, cancer chemotherapy agents include alkylating agents (nightstand diene mustards, ethyleneimines, sulfonic acid esters), antimetabolites (folate antagonists, purine antagonists,
BiIJ midin antagonist), plant fission toxins (colcemid, vinblastine, etc.), antibiotics (delcomycin, carcinophyllin, 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 have searched for substances with low toxicity and anticancer properties, and have newly discovered that the glyceride represented by the above general formula has differentiation-inducing activity against animal tumor cells. The present invention was completed based on the 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 beginning of the present invention is that the above-mentioned substance having anticancer properties is used in animals and plants.
When we investigated a wide range of organisms in the microbial world, we found that carp swim bladders (Carp 5w1m bladder), simonoglyceride, and rainbow trout (Salmagal)
The reason is that sydiglyceride was found in /j~/7-day embryos of .rdnerl). Furthermore, various glycerides were tested for their anticancer activity, and their effects were confirmed, leading to the completion of the present invention #i.

[About monoglycerides in the carp swim bladder] 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, and acetone. Acetone-ether C2: /, v
/v) The differentiation-inducing activity fractions of erythroid leukemia cells and malformed hepatoma cells eluted by high performance liquid chromatography (HPLC) were applied to Nucleos! ;Cog,φ
When diluted with 100% methanol for 2-7 minutes, the retention times were 3 minutes, 9.3 minutes, 9.5 minutes, 10,
! A monoglyceride corresponding to 1 (Rs = -C-CteHst + R = H) was found at the minute position, and IH-NMR, 13
It was identified by instrumental analysis such as C-NMR.

[About diglycerides in rainbow trout embryos] The rainbow trout embryos 2 to 11 days after fertilization were homogenized and treated with acetone, ether, and chloroform-ethanol (2:/
, V/Vl mixture, the three extracts were mixed, concentrated under reduced pressure, and then subjected to silica dull column chromadog 2.
Chlorophon-muhexane (0-700%)
) and methanol-chloroform<o-1oo%).

The active fraction is contained in the chloroform and chloroform-methanol (9:/, V/V) fractions.

This was further subjected to silica gel column chromatography using hexane-ether. The active category is hexane-
Ether; g left: Contained in both parts of 15 (v/v).

This fraction was further prepared and subjected to silica dull thin layer chromatography (solvent: chloroform acetone = 9:4tXV/V
), and of the two obtained fractions, fraction D (Rf value O0) was subjected to reverse phase high performance liquid chromatography (HP LC; Nucleosil) using methanol.

-7 minutes), and the fraction with a retention time of 3'1 minutes was divided into 3.5%
Silica dull thin layer chromatography permeated with boric acid (
Solvent: Chloroform:acetone = 96:Da, V/V) and when purified, the following two types of C substances (DG-/and DG
-2) was obtained.

[Physical properties of DG-/DG-Co] It has been shown.

By Na0CH1 treatment fi, C,,H,,C0OH,
C1, H3, C0OH.

The presence of fatty acids in CHHssCOOH and the presence of siglycerol as a water-soluble component were confirmed by GC-MS by trimethylsilylation (TMS).

Furthermore, by converting this substance into TMS, C18H31COcwt
H3□CO.

The molecular species of diglyceride of C,,H,,Co-C□H3,Co were shown by GC-MS. The present inventors) found that the above substances exhibited differentiation-inducing activity, and when various synthetic mono- or diglycerides were also investigated for their differentiation-inducing activity, they showed remarkable activity.

The active ingredient compound of the present invention will be described below.The active ingredient compound has the above-mentioned general formula, and the fatty acid residue is saturated or unsaturated (the number of double bonds is /
-Able to praise (about 10) things.

These fatty acid residues include saturated ones such as lauryl group (C-H2sCO-), myristyl group (C
tsHzyCO), palmityl group (C11831GO
-) sstearyl group (C1yHasCO). Examples of unsaturated groups include oleyl group or elaidyl group (C1yHssCO-), lylyl group (CuH**CO-), icosamonoenoyl group (C19887GO-), docosahexaenoyl group (C
tyHssCO), and may be the same or different.

Examples of the mono- or diglycerides that are the active ingredients of the present invention are as follows.

Compound RI Rx R3 / C3,H31co H)1 a” CtyHssCOHH 3C,,H,,Co HH 4C13H,,Co C1,H,,COH7C,3H2
, Co HC, 3H2, C0g C, gl-131Co
C1,H31CO)Iq C15H111Go HC
,,H3,C0/θ C17H86cOC1183g
CQ @ // ctt H3,Co HC1,H3,C
0/, 2 C1,)lss co CI, H3, Co
)1 (Oleyl) (Oleyl) 13 C17H3scOHcl, H38c.

(Oleyl) (Oleyl) /4t C,,H,3Co HC,,H33C0 (Elaidyl) (Elayyl) /j C,,) Its co HC1,H2,C0 Compound NIL RI Rz RR The active ingredient of the present invention is , substances that naturally exist in animals such as carp swim bladders or rainbow trout embryos, or compounds similar to these, which are toxic because they exhibit the activity of inducing differentiation from cancer cells to normal cells. There is no need to worry about

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 herbal medicines, such as subcutaneous or intravenous injections such as aqueous suspensions and oil-based preparations, infusions, and solid or suspended viscous liquids to maintain sustained mucosal absorption. .

The formulation of the active ingredient of the present invention includes surfactants, 3FA
1! AI -E customer 11- & customer t -B re
, iγJI r between 1-f IIIL 4M4= 11
In order to form a 111 agent, pharmaceutically acceptable film-forming substances, coating aids, etc. can be used as appropriate, and specific examples thereof are as follows.

In order to clearly display the disintegration and dissolution of the composition of the present invention,
Seven or more types of surfactants, such as alcohols, esters, polyethylene glycol derivatives, sorbitan fatty acid esters, and sulfated fatty alcohols, can be added.

In addition, as excipients, for example, sucrose, lactose, starch, crystalline cellulose, mannitol, light silicic anhydride, magnesium aluminate, magnesium metasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate, Seven types or a combination of two or more types, such as carboxymethyl cellulose calcium, can be added.

As a lubricant, for example, seven or more types of stearin, magnesium stearin, Merck, hydrogenated oil, etc. can be added, and as a flavoring and 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.

Adjuvants such as suspending agents and wetting agents include, for example, 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 (CAP) as carbohydrate derivatives such as sugars,
Examples of I-rivinyl 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 the chemicals from adhering to each other during coating operations. This improved version can make coding operations much easier. Note that a dosage form may be prepared 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.

Particularly preferred range Active ingredients 0.07-90% by weight 0.3-1% by weight Excipients 10-99. g I g left~99. Lubricants or o~20 I Surfactants O~I; Q r (7-Ol film forming substances o, i~60 #0.3-.2θ l Particularly preferred excipients are lactose, Crystalline cellulose, Cal is oxymethyl cellulose calcium.

The dosage should be 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 0.07~/θO■/k191* Tongue/-l, human-
r-b-t /l-/)t, AI')my/
in the range σa=t 1, the upper limit of which is preferably about Sθ
■/kg body weight, more preferably about 10101n/kg body weight, and in the case of parenteral administration, the upper limit is about 10m9
Appropriately is about 9 body weight per STng/kg, preferably 9 body weight per STng/kg, and more preferably 2 m9/kg body weight.

Next, an anticancer test in which the anticancer activity of the compound of the present invention was confirmed will be described.

[l] Friend leukemia cells (mouse erythr
oldleukemia cell, Bg cell)
Test for GIBCO II! ! )-IAM F-/2
2 left
8 g of cells are inoculated, and a predetermined amount of the test compound is added thereto (final volume: 5-).

After culturing for 7 days at 37°C in 7.5% CO3, the cells were stained using Orkin's benzidine staining method.
The number of stained cells, that is, the number of cells that have differentiated into red blood cells and have come to produce syhemoglobin, is measured, and the differentiation induction rate is calculated.

[λ] Mouse teratoc
Teratoma cells (teratoma cells) were used months after transplantation into the peritoneal cavity of mice.

In the peritoneal cavity, gratoma cells form embryoid bodies (e
It exists as a cell mass called mbrold body), and was used without trypsin treatment.

After washing the embryoid bodies obtained by natural settling in the collected ascites three times with Dull 4 Co's modified medium or Hank's solution,
The cells are inoculated into a medium containing 0% tallow serum gold, added with a predetermined amount of the test compound, and cultured at 37°C in air for 1 week under saturated water vapor containing 7.5% to 8% CO2. Centrifugation (ko0θ
θr, p, m, / Q min)
After washing with Naphthol As-MX phosphate and diazo reagent (Fast Violet B
5alt) and left at room temperature for 7 hours. The embryoid bodies are separated by centrifugation (<2000 r, p, m, 70 min), rinsed with ethanol, and left at room temperature for 7 hours.

(Undifferentiated cells are colored red).

This is k, 53! ; Measure the absorption at nm and calculate the alkaline phosphatase activity (degree of differentiation induction).

Hexamethylene bisacetamide (HMB8) 3 mM
The case where HMBAt is added (shows no alkaline phosphatase activity at all) is given a "+10", and the case where HMBAt is not added (shows extremely strong alkaline phosphatase activity).
r--J, and the degree of differentiation induction was shown in the following steps.

++: Shows no alkaline phosphatase activity.

10: Alkaline phosphatase activity fh not shown at all.

- Shows some alkaline phosphatase activity.

m: Strongly exhibits alkaline phosphatase activity.

It shows extremely strong 112-alkaline phosphatase γ-occupancy.

In addition, in the test examples described below, it showed anticancer activity with a differentiation-inducing effect.

The present invention will be specifically explained below using all formulation examples and test examples of the present invention.

Formulation example/(injection/infusion) Compound (1)/Add 3 g of powdered glucose to contain Omft, dispense aseptically into a vial, seal it, fill with inert gas such as nitrogen or helium, and store in a cool, dark place. Save to. Before use, it was dissolved in ethanol and added with 100 gs% physiological saline to make an intravenous injection, and for 7 days.
/θ~100- is administered by intravenous injection or drip depending on the symptoms.

Formulation Example 2 (Injection Drop) Compound 112 m9 was prepared as an intravenous injection for mild symptoms in the same manner as in Formulation Example.
'+Administer by intravenous injection or drip depending on symptoms.

Formulation Example 3 (Enteric Coated Capsule) Compound (/S) Shio y1 Lactose 2.11. Ag and hydroxypropylcellulose 0.0'19f: top p, , respectively
J: (Dry well and sieve to produce granules suitable for bottles, heat seal packaging, etc.) Next, dissolve 5 g of cellulose acetate phthalate θ, left l and hydroxyglobil methylcellulose phthalate θ to form a coating base. Enteric-coated granules are prepared by making the granules floating and coating the base material.This composition is filled into capsules to produce 100 enteric-coated capsule preparations.

Test Example Using the above-mentioned compound, the differentiation induction rate of Friend leukemia cells and the degree of differentiation induction of mouse teratoma cells were investigated using the above-mentioned test methods (1) and [, 2).
The results shown in Table and Table 2 were obtained.

Table I
, Title of the invention Registration agent 3, Relationship with the TM correction case Applicant name (679) RIKEN 4, Agent 5, Date of amendment order January 31, 1980 6. Subject of amendment Details Book 1-),・,1. −τ)

Claims (1)

[Claims] (No General formula: (R1 is a fatty acid residue, and R2 and R3 are hydrogen or a fatty acid residue.) One or more glycerides represented by the following are active ingredients. (J) The anticancer agent according to claim 1 in the form of parenteral administration. (3) The anticancer agent according to claim 1 in the form of oral administration.