JPH05279252A - Antitumor agent - Google Patents

Abstract

PURPOSE:To obtain an antitumor agent not decomposed in a human body even in the case of oral administration, retaining the antitumor property and simultaneously exhibiting no side effect and having remarkable antitumor effects on various kinds of cancers. CONSTITUTION:The objective antitumor agent contains a ketol type unsaturated fatty acid as an active ingredient. The ketol type unsaturated fatty acid is obtained by making a lipoxygenase and hydroperoxide isomerase to act on an unsaturated fatty acid, e.g. linolic acid, alpha-linolenic acid, gamma-linolenic acid, arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid. A ketol type unsaturated fatty acid obtained by making lipoxygenase and hydroperoxide isomerase to act on the linolic acid is especially preferably used.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antitumor agent containing a ketol-type unsaturated fatty acid as an active ingredient.

[0002]

2. Description of the Related Art Conventionally, the mainstream of research on antitumor active agents has sought substances with higher activity, such as adriamycin, but in reality, the side effects have become stronger accordingly. .. In response to this trend, the antitumor activity of unsaturated fatty acids, which show almost no side effects, has recently attracted attention. For example, it has been reported that fatty acids containing an unsaturated bond such as linoleic acid and oleic acid have an antitumor effect (for example, JP-A-62-1).
No. 2716), none of them has been put into practical use as a medicine.

[0003]

[Means for Solving the Problems] As a result of research and development of an antitumor agent having an excellent antitumor action and having few side effects and high safety, the inventors have found that ketol unsaturated fatty acids are
The present invention was completed by discovering that it has a strong antitumor activity and has almost no side effects.

That is, the present invention is an antitumor agent containing a ketol-type unsaturated fatty acid as an active ingredient.

Another invention is an antitumor agent containing a ketol-type unsaturated fatty acid as an active ingredient, which is obtained by reacting unsaturated fatty acid with lipoxygenase and hydroperoxide isomerase.

Still another invention is a ketol type unsaturated fatty acid obtained by reacting linoleic acid, α-linolenic acid, γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, or docosahexaenoic acid with lipoxygenase and hydroperoxide isomerase. Is an antitumor agent containing as an active ingredient, and the active ingredient is a ketol-type unsaturated fatty acid obtained by allowing lipoxygenase and hydroperoxide isomerase to act on linoleic acid.

The ketol-type unsaturated fatty acid described in the present invention has at least one ketone group and at least one hydroxy group and at least one unsaturated bond, and has 6 to 3 carbon atoms.
6 means a straight chain fatty acid. Usually a ketone group,
And one hydroxy group and one unsaturated bond,
Alternatively, a straight chain fatty acid having 2 or 3 carbon atoms and having 12 to 24 carbon atoms is suitable.

The ketol-type unsaturated fatty acid of the present invention can be obtained by adding a ketone group and a hydroxy group to the carbon chain of the unsaturated fatty acid using lipoxygenase and hydroperoxide isomerase.

The unsaturated fatty acid used as a raw material is
Examples thereof include linoleic acid, α-linolenic acid, γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid.

As the lipoxygenase, those contained in plant seeds are known, and corn-derived lipoxygenase, wheat-derived lipoxygenase, soybean-derived lipoxygenase and the like can be exemplified. These may be isolated and purified, or the seeds may be crushed and the lipoxygenase contained therein may be used for the reaction. Further, commercially available lipoxygenase can also be used.

Hydroperoxide isomerase is
Those contained in plant seeds along with lipoxygenase are known, and corn-derived hydroperoxide isomerase and barley-derived hydroperoxide isomerase can be exemplified, but they can be isolated and purified. It is also possible to utilize the hydroperoxide isomerase contained in crushed seeds for the reaction.

FIG. 1 shows the reaction pathway of ketol-type unsaturated fatty acid synthesis by taking linoleic acid as an example. The numbers in the figure represent the number of carbons counted from the carboxyl carbons of linoleic acid. Plant lipoxygenases such as corn, wheat, and soybeans add one molecule of oxygen to the carbon at the 9th position or the carbon at the 13th position to generate hydroperoxylinoleic acid. That is, when lipoxygenase derived from corn or wheat was used, 9-hydroperoxylinoleic acid was obtained as the main product, and when lipoxygenase derived from soybean was used, 13-hydroperoxylinoleic acid was the main product. Obtained as. Oxygen is transferred from these hydroperoxylinoleic acids by hydroperoxide isomerase contained in plants together with lipoxygenase to produce ketol-type unsaturated fatty acids. That is, 9
Compounds 1 and 2 are produced from hydroperoxylinoleic acid, and compounds 3 and 4 are produced from 13-hydroperoxylinoleic acid. That is, in this reaction system, four types of ketol-type unsaturated fatty acids can be obtained from one unsaturated fatty acid (see J. Lipid Res .. 11, 311 (1970)).

The above reaction formula has been shown by taking linoleic acid as an example, but any unsaturated fatty acid having a substrate specificity for lipoxygenase and hydroperoxide isomerase may be used, for example, α-linolenic acid, γ-linolenic acid. , Arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, etc. can be used for any unsaturated fatty acid, and therefore, the following ketol-type unsaturated fatty acids can be obtained from each. That is, four kinds of compounds obtained by using α-linolenic acid are 13-hydroxy-10-oxo-trans-11-
Cis-15-octadecadienoic acid 9-hydroxy-10-oxo-cis-12,15-
Octadecadienoic acid 9-hydroxy-12-oxo-trans-10-cis-15-octadecadienoic acid 13-hydroxy-12-oxo-cis-9,15-
Octadecadienoic acid, and four kinds of compounds obtained by using γ-linolenic acid are 13-hydroxy-10-oxo-cis-6-trans-11-octadecadienoic acid 9- Hydroxy-10-oxo-cis-6,12-octadecadienoic acid 9-hydroxy-12-oxo-cis-6-trans-10-octadecadienoic acid 13-hydroxy-12-oxo-cis- 4,9-octadecadienoic acid, which is four kinds of compounds obtained when arachidonic acid is used, is 15-hydroxy-12-oxo-cis-5,8-trans-13-eicosatrienoic. Acid 11-hydroxy-12-oxo-cis-5,8,1
4-Eicosatrienoic Acid 11-Hydroxy-14-oxo-cis-5,8-trans-12-Eicosatrienoic Acid 15-Hydroxy-14-oxo-cis-5,8,1
1-eicosatrienoic acid, 4 kinds of compounds obtained by using eicosapentaenoic acid are 15-hydroxy-12-oxo-cis-5,8,1
7-trans-13-eicosatetraenoic acid 11-hydroxy-12-oxo-cis-5,8,1
4,17-Eicosatetraenoic acid 11-hydroxy-14-oxo-cis-5,8,1
7-trans-12-eicosatetraenoic acid 15-hydroxy-14-oxo-cis-5,8,1
1,17-eicosatetraenoic acid, which is obtained when docosahexaenoic acid is used.
A class of compounds is 17-hydroxy-14-oxo-cis-4,7,1
0,19-trans-15-docosapentaenoic acid 13-hydroxy-14-oxo-cis-4,7,1
0,16,19-docosapentaenoic acid 13-hydroxy-16-oxo-cis-4,7,1
0,19-trans-14-docosapentaenoic acid 17-hydroxy-16-oxo-cis-4,7,1
0,13,19-docosapentaenoic acid, which can be expected to have the same antitumor effect.

Since this reaction system is an enzymatic reaction and is substrate-specific, it can be easily separated and produced after the reaction by simply removing unreacted substances, and can be mass-produced in high yield, thus being industrially applicable. Is advantageous to. Furthermore, since this reaction applies not the chemical synthesis but the enzyme reaction system widely existing in the seeds of plant species, the influence on the human body is low.

The ketol-type unsaturated fatty acid of the present invention exhibits an antitumor effect in vitro and in animal experiments and is expected as a pharmaceutical product.

The antitumor effect of the present invention cannot be clearly explained theoretically. However, unlike substances that inhibit DNA biosynthesis, inhibit nucleic acid metabolism to inhibit tumor cell division and growth, and further stimulate the immune function of the body to increase resistance to cancer. By microscopic observation, it can be said that the antitumor effect has a new function of acting on the cell membrane because it destroys a part of the tumor cell membrane and expresses a cell-killing effect.

The ketol-type unsaturated fatty acids used in the present invention include salmonella, typhimurium TA98 and TA10.
No mutagenic effect was observed in the AMES test with 0, and furthermore, it did not induce chromosomal aberrations in human lymphocytes, and thus it can be said that it is a highly safe antitumor agent.

The substance of the present invention is orally or parenterally administered to humans and animals. Oral administration includes sublingual administration. Parenteral administration includes injection administration (eg subcutaneous, intramuscular, intravenous injection, infusion), rectal administration and the like. You may apply.

In some cases, the substance of the present invention may exhibit a good effect when orally administered by mixing with a pharmaceutically acceptable absorption enhancer. As the absorption enhancer, for example, sodium cholate is used.

The dose varies depending on the type of cancer to be treated, symptoms, administration method and the like, and is 1 to 4000 mg / m ² / day, preferably 10 to 3000 mg per body surface area (m ² ) in the case of oral administration. / M ² / day, and when administered by injection, it is in the range of 0.1 to 400 mg / m ² / day, preferably 1 to 200 mg / m ² / day. It is also possible to administer to. It may be administered in 2 to 4 divided doses per day. In addition, depending on the type of cancer and the degree of symptoms, the dose is not limited to the above range, and can be administered in different doses.

The ketol-type unsaturated fatty acids may be used alone or in admixture of two or more. The dosage form may be an emulsion, a tablet, a powder, a capsule, a granule, a syrup, a suspension or a liquid for oral administration, or an injection solution for parenteral administration. The form of suppositories and ointments is also possible.

This substance may be used alone or in combination with a pharmaceutically acceptable diluent, and as a diluent, a solubilizing agent, a solvent, an oil agent, a dispersant, a preservative, a surface active agent, a wetting agent, a bulking agent may be used. , Solid, liquid, semi-solid excipients and the like are used.

Next, the production examples and antitumor effect of the above-mentioned ketol-type compound will be described in more detail, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded.

[0024]

Example 1 13-Hydroxy-10-oxo-trans-11-octadecenoic acid (1) and 9-
Hydroxy-10-oxo-cis-12-octadecenoic acid (2).

7.41 kg of pulverized corn was mixed with 20 liters of water, filtered through a filter cloth, and 2.5 g of linoleic acid was added to the filtrate.
Was added and stirred at 20 ° C to 30 ° C for 1 hour. The reaction solution was filtered through diatomaceous earth, and then passed through a synthetic adsorbent Amberlite XAD-8 column (manufactured by Organo) with a volume of 150 ml, and the adsorbed material was eluted with 95% ethanol. After removing ethanol under reduced pressure, the eluate is suspended in 300 ml of an appropriate amount of water.
It was extracted with ethyl acetate. The obtained ethyl acetate layer was concentrated under reduced pressure and then applied to a normal phase silica gel column (Wako Gel C-300, manufactured by Wako Pure Chemical Industries). The normal phase silica gel column was eluted with a mixed solvent having a hexane: ethyl acetate ratio of 10: 3 to 5: 3. An appropriate amount of the eluate was collected, thin-layer chromatography was performed for each fraction, and fractions having a ketol-type substance spot were collected. When this was cooled to low temperature, the ketol type substance precipitated as white crystals, which were collected and dried under reduced pressure. Thus, 0.3 g of compound 1 and 0.5 g of compound 2 were obtained.

Compound 1: mp: 64 ° C., NMR δpp
m (CD ₃ OD): 0.91 (3H, t, J = 7Hz,
Me), 1.2-1.4 (14H, m , CH 2 × 7),
1.3-1.7 (6H, m, CH ₂ × 3), 2.26
(2H, t, J = 7Hz, 2-H), 2.60 (2H,
t, J = 7 Hz, 9-H), 4.24 (1H, ddt,
J = 5.6Hz, 4.8Hz, 1.6Hz, 13-
H), 6.27 (1H, dd, J = 16Hz, 1.6H
z, 11-H), 6.86 (1H, dd, J = 16H)
z, 12-H).

Compound 2: NMR δ ppm (CD ₃ O
D): 0.90 (3H, t, J = 7Hz, Mo), 1.
2-1.5 (14H, m, CH ₂ × 7), 1.4-1.
8 (4H, m, CH ₂ × 2), 2.0-2.1 (2H,
m, 14-H), 2.28 (2H, J = 8Hz, 2-
H), 4.0-4.2 (1H, m, 9-H), 5.4-
5.7 (2H, m, 12-H, 13-H).

[0028]

Example 2 9-Hydroxy-12-oxo-trans-10-octadecenoic acid (3) and 13-
Hydroxy-12-oxo-cis-9-octadecenoic acid (4).

2.5 g of linoleic acid was dissolved in 200 ml of ethanol, 0.2 M borate buffer pH 9.0 was added, and 2
It was set to 0 liter. To this linoleic acid solution, 1.5 million units of soybean lipoxygenase (manufactured by Sigma) were added and stirred at 20 ° C to 30 ° C while blowing oxygen. Next, the pH was adjusted to 6.0 with acetic acid, and ground barley or malt 3.7 was used.
kg was added and the mixture was further stirred for 1 hour. The reaction solution was filtered through diatomaceous earth, added with 20 liters of ethyl acetate, and poured out.
The obtained ethyl acetate layer was concentrated under reduced pressure. Then, the same purification as in Example 1 was carried out to obtain 0.2 g of Compound 3 and 0.4 g of Compound 4.
g was obtained.

[0030]

INDUSTRIAL APPLICABILITY The present compound has lower toxicity than conventional antitumor agents, has an excellent antitumor effect on many kinds of cancers, and has a stronger activity than ordinary fatty acids.

[0031]

[Use Example 1] In vitro antitumor effect on various tumor cells The in vitro antitumor effect of each compound was compared with that of linoleic acid to determine a 50% cell growth inhibition concentration (IC ₅₀ ).

Various tumor cells were seeded in a 96-well microplate at 2.5 × 10 ³ cells / well, and 24
Specimens were added after time. Then, under 5% CO ₂ , 37
Incubate at ℃ for 3 days, 4 hours before the end of drug inoculation, MTT (3- (4.5
-Dimethylthiazol-2-yl) -2.5-diphenyl-2H-
Tetrazolium bromide), followed by addition of 200 μl / ml of isopropanol containing 0.04 N hydrochloric acid,
The absorbance was measured at 0 nm to determine the IC ₅₀ value. The results are shown in Table 1.
Shown in.

[0033]

[Table 1]

From the above results, the antitumor activity was improved as compared with the substrate linoleic acid, and particularly for human breast cancer cells, the high activity was observed for the ketol unsaturated fatty acid.

[0035]

[Example 2] high antitumor effect cell killing effect in animal experiments Compound 1, i.e. 13-hydroxy-10-oxo - taking trans-11-octadecenoate dichroic acid as an example, the antitumor effects in animal experiments It was measured.

(B) Method for preparing emulsion of test substance for oral administration Administration of one mouse in 0.5 ml of sodium cholate salt (manufactured by Sigma) dissolved in physiological saline at 4% An amount of the test substance was added, and the mixture was heated to 60 ° C. and stirred to obtain an emulsion.

(B) Method for preparing emulsion of test substance for intraperitoneal administration To 0.2 ml of phosphate buffered saline, a test substance of a dose for one mouse was added, and the mixture was heated to 60 ° C. and stirred, An emulsion was obtained.

(C) Method for measuring activity against solid tumors S180 (sarcoma) was obtained by implanting 2 × 10 ⁶ cells / mouse cells subcutaneously in the thigh of 5-week-old ICR female mice. After 14 days, mice having a tumor diameter of 6 to 8 mm were selected, and each group was divided into 7 groups, and the test substance emulsion prepared by the method (a) was used 14 to 18 days after the tumor implantation. Oral administration was continued every day and once every 22nd day. MM46 (breast cancer) was 2 × 10 ⁶ subcutaneously in the thigh of 5-week-old C3H / He female mice.
Cells / mouse cells were transplanted. After 8 days, mice with a tumor diameter of 7-9 mm were selected, and each group was divided into 7 mice, and the emulsion of the test substance prepared by the method (b) was used 8-12 days after the tumor implantation. Was administered intraperitoneally every day.

The result is (major axis × minor axis × height) / 2 of the tumor.
Was used as the estimated volume of the tumor, and the ratio to the volume on the day of administration was calculated daily and shown in the graph. In addition, a significant difference from the control group (test substance 0 mg / mouse / day administration group) was determined by t-test, and the risk rate (p value) was shown in the graph.

FIG. 2 shows the therapeutic results of oral administration of the present compound to an ICR female mouse transplanted with S180 (sarcoma), and it was confirmed that the multiple administrations have an effect of substantially suppressing the growth of tumor.

FIG. 3 shows MM46 in C3H / He female mice.
These are the results of treatment by intraperitoneal administration of the present compound to a transplanted (breast cancer), and tumor atrophy was observed by multiple administrations.

Further, no daily death of the present compound was observed by oral or intraperitoneal administration, and no side effects such as weight loss were observed in oral administration.

[Brief description of drawings]

FIG. 1 is a diagram showing a reaction system of linoleic acid in the present invention.

FIG. 2 is a graph of the tumor volume ratio of the used tumor cells S180 and the number of days after tumor transplantation.

FIG. 3 is a graph of tumor volume ratio of similarly used tumor cells MM46 and days after tumor implantation

FIG. 4 is a graph of the tumor volume ratio and the number of days after tumor implantation.

[FIG. 5] Similarly, a graph of the tumor volume ratio and the number of days after tumor implantation

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[Procedure amendment]

[Submission date] June 18, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction content]

Compound 1: mp: 64 ° C., NMR δp
pm (CD ₃ OD): 0.91 (3H, t, J = 7H
z, Me), 1.2-1.4 (14H , m, CH 2 ×
7), 1.3-1.7 (6H, m, CH ₂ × 3), 2.
26 (2H, t, J = 7Hz, 2-H), 2.60 (2
H, t, J = 7 Hz, 9-H), 4.24 (1H, dd
t, J = 5.6 Hz, 4.8 Hz, 1.6 Hz, 13−
H), 6.27 (1H, dd, J = 16Hz, 1.6H
z, 11-H), 6.86 (1H, dd, J = 16H)
z, 4.8 Hz, 12-H).

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0027

[Correction method] Change

[Correction content]

Compound 2: NMR δ ppm (CD ₃ O
D): 0.90 (3H, t , J = 7Hz, M e), 1.
2-1.5 (14H, m, CH ₂ × 7), 1.4-1.
8 (4H, m, CH ₂ × 2), 2.0-2.1 (2H,
m, 14-H), 2.28 (2H, J = 8Hz, 2-
H), 4.0-4.2 (1H, m, 9-H), 5.4-
5.7 (2H, m, 12-H, 13-H).

[Procedure amendment 3]

[Document name to be amended] Statement

[Name of item to be corrected] Brief explanation of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 is a diagram showing a reaction system of linoleic acid in the present invention.

FIG. 2 is a graph of the tumor volume ratio of similarly used tumor cells S180 and the number of days after tumor implantation

FIG. 3 is a graph of the tumor volume ratio of similarly used tumor cells MM46 and the number of days after tumor implantation

[Procedure amendment 4]

[Document name to be corrected] Drawing

[Name of item to be corrected] Fig. 4

[Correction method] Delete

[Procedure Amendment 5]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 5

[Correction method] Delete

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yuji Oki 1-1-32, Kuronomachi Nishi, Sakai City, Osaka Prefecture Tamano Izu Co., Ltd. 2nd Research Laboratory (72) Inventor Akio Ejima 1-16 Kitakasai, Edogawa-ku, Tokyo No. 13 Daiichi Pharmaceutical Co., Ltd., Tokyo R & D Center (72) Inventor, Fumio Saito 1-132, Nishi, Kuranomachi, Sakai City, Osaka Prefecture Tamano Izu Co., Ltd., 2nd Research Laboratory (72) Inventor, Hiroshi Kuga Edogawa, Tokyo 1-16-13 Kitakasai-ku, Tokyo Daiichi Pharmaceutical Co., Ltd., Tokyo Research and Development Center (72) Inventor Nobuhiro Ishihara 1-132, Kuronomachi Nishi, Sakai City, Osaka Pref. 2nd Research Laboratory, Tamano Izu Co., Ltd.

Claims (4)

[Claims] 1. An antitumor agent containing a ketol-type unsaturated fatty acid as an active ingredient. 2. An antitumor agent containing a ketol-type unsaturated fatty acid as an active ingredient, which is obtained by causing lipoxygenase and hydroperoxide isomerase to act on unsaturated fatty acid. 3. A ketol-type unsaturated fatty acid obtained by allowing lipoxygenase and hydroperoxide isomerase to act on linoleic acid, α-linolenic acid, γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, or docosahexaenoic acid as an active ingredient. Antitumor agent 4. The antitumor agent according to claim 3, wherein the active ingredient is a ketol-type unsaturated fatty acid obtained by allowing lipoxygenase and hydroperoxide isomerase to act on linoleic acid.