JP3330127B2 - Solid cancer treatment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an anticancer compound and a method for producing the same, and more particularly, to a new regulated cancer compound contained in lupus venom, a Chinese herbal medicine, and a method for producing the same.

[0002]

2. Description of the Prior Art It can be said that Chinese herbal medicine is a collection of wisdom of humankind with an old history. The present inventors have searched for anticancer herbal medicines whose anticancer active substance has not been elucidated, and found that venom (Stellera chamaejasme. L, Euphorbia f.
ischeriana Steud., E. ebiacteola Hayata) was confirmed to have strong anticancer activity, and a method for collecting an antitumor substance was first filed (Patent Application No. 58126).
However, in that application, it has not been possible to efficiently isolate a compound having an anticancer activity and confirm its efficacy against solid cancer.

[0003]

Means for Solving the Problems The present inventors have further studied on the antitumor active ingredient contained in the antitumor substance of lupus venom, and as a result, isolated and obtained the component responsible for the antitumor activity of lupus venom. .
And it discovered that the novel compound represented by a following formula was also contained in it.

That is, a first object of the present invention is to provide the following formula (I) or (II)

Embedded image The present invention is to provide stellarmacrin A and stellarmacrin B represented by

It is a second object of the present invention to provide a method for producing stellarmacrin A and stellarmacrin B from wolf venom. It is a further object of the present invention to provide an anticancer agent containing stellarmacrin A or stellarmacrin B. Furthermore, object of the present invention is to provide a solid cancer therapeutic agent comprising as an active ingredient Gunidimakurin or Pimerea factor P _2.

[0006]

DETAILED DESCRIPTION OF THE INVENTION The formula (I) or (II) of the present invention
Are extracted from the above-ground or underground (rhizome) wolf venom using an organic solvent. It is obtained by purification.

Specifically, for example, first, the above-ground or underground portion of venom is extracted using a lower alcohol such as methanol, ethanol or propanol, and the extract is extracted using a water-insoluble organic solvent such as petroleum ether. To obtain a crude extract. Next, the crude extract is purified using silica gel column chromatography using hexane-ethyl acetate or the like as an eluent, or ODS column chromatography using methanol-water or the like to obtain some active fractions. Furthermore, by subjecting it to high performance liquid chromatography and preparative thin layer chromatography to purify it,
Formula (I) or (II) as a compound having an anticancer activity
Can be isolated.

[0008] In the separation and purification steps by the above-mentioned methods, anticancer compounds other than stellarmacrin, for example, gnidimacrin [gnidimacrin; J. Nat. Prod. 1985,
48 (3), 440-445], Pimelea factor P ₂
P ₂ : Abstracts of Natural Organic Compounds Symposium, 27, 734-74
1), Subtoxin A (Summary of Natural Organic Compounds Symposium, 27, 734-741), Huratoxin (hur
atoxin; Proceedings of the Symposium on Natural Organic Compounds, 27, 734-74
1), simplexin (simplexin; Aust. Vet. J., 51)
(6), 325-326) etc., but these are obtained as fractions different from these, and their structure should be analyzed using ultraviolet absorption spectrum, proton nuclear magnetic resonance spectrum, mass spectrum, etc. Is determined by

[0009] Incidentally, Gunidimakurin is another anticancer compound obtained from Okamidoku, Pimerea factor P _2, etc. are known substances, these known substances are to be effective in improving the conventionally ascites leukemia I was

However, a correlation between the action on leukemia and the anti-cancer activity on solid tumors has not been recognized as easily understood from the examples of carbazylquinone and bleomycin. It was not known whether they had anticancer activity against s.

As a result of the study of the present inventors, it has been newly found that gnidimacrine and pimelea factor P ₂ also have a strong inhibitory action against solid cancers such as gastric cancer, lung cancer and liver cancer.

[0012] The use of the present invention as a carcinostatic agent and the use of gniramacrin or pimeria factor P ₂
When is used as a therapeutic agent for solid cancer, it may be administered to animals and humans as it is or together with a conventional pharmaceutical carrier.

[0013] Sutereramakurin and Gunidimakurin, no particular restrictions on the dosage form of Pimerea factor P _2, can be selected as needed. For example, it may be an oral preparation such as tablets, capsules, granules, fine granules and powders, or a parenteral preparation such as injections and suppositories.

The novel compound produced according to the present invention, stellarmacrin A, has an LD ₅₀ value of 5 mg / kg, and the latter has a LD ₅₀ value of 12 mg / kg,
When these are used as oral preparations, in order to exert the intended effect, the age, weight, and degree of the disease vary depending on the patient. It is appropriate to administer about 20 mg in several divided doses. Also, Gnydi Maclin,
In the case of Pimerea factor P _2, adult per day 0.01mg~1
It is appropriate to administer about 0 mg.

Oral preparations include, for example, starch, lactose, sucrose,
Mannitol, carboxymethylcellulose, corn starch, inorganic salts and the like may be used as excipients in accordance with a conventional method. In order to prepare an oral preparation, in addition to the above-mentioned excipients, a binder, a disintegrant, a surfactant, a lubricant,
A fluidity promoter, a flavoring agent, a coloring agent, a fragrance and the like can be blended, and specific examples of each are as follows.

Examples of the binder include starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol and the like.

Examples of the disintegrant include starch, hydroxypropyl starch, sodium carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, low-substituted hydroxypropylcellulose and the like.

Examples of the surfactant include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80 and the like.

Examples of the lubricant include talc, waxes,
Examples include hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol and the like.

Examples of the fluidity promoter include light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate and the like.

The compounds of the present invention can also be administered as suspensions, emulsions, syrups and elixirs. These various forms may contain flavoring agents, coloring agents and the like. Good.

When the compounds of the present invention are used as a parenteral preparation, the patient should be able to achieve the desired effect by adjusting the patient's age, weight,
Although it depends on the degree of the disease, it is usually advisable for an adult to intravenously, intravenously, intravenously, subcutaneously, or intramuscularly in an amount of about 0.005 mg to 5 mg per day as the weight of the compounds of the present invention.

These parenteral preparations are manufactured according to a conventional method, and generally include, as diluents, distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil,
Soybean oil, corn oil, propylene glycol, polyethylene glycol and the like can be used.

Further, if necessary, a bactericide, a preservative and a stabilizer may be added.

As a preferred method from the viewpoint of stability, this parenteral preparation is preferably frozen after filling in a vial or the like, removing water by a conventional freeze-drying technique to obtain a lyophilized product, and then preparing a liquid preparation immediately before use. It is also possible to readjust.

Further, if necessary, an isotonic agent, a stabilizer, a preservative, a soothing agent and the like may be appropriately added.

Other parenteral preparations include liquid preparations for external use, ointments and other suppositories, and suppositories for rectal administration.
It can be produced according to a conventional method.

Now, the present invention will be described in further detail with reference to Examples. Example 1 1kg of underground part of Mizuoka wolf poison (Stellera chamaejasme.L)
Was ground, 3 l of methanol was added thereto, and the mixture was heated under reflux for 5 hours to extract. The extraction residue was extracted under the same conditions, and both extracts were combined and filtered under reduced pressure to obtain 153.6 g of a methanol extract. Further, this methanol extract was extracted with petroleum ether for 5 hours using a Soxhlet extractor to obtain 15.8 g of petroleum ether extract (MMP).

6.5 g of this MMP was subjected to silica gel column chromatography (diameter 5 cm, length 16.5 cm), and hexane-ethyl acetate was eluted with a solvent [1: 4 (v / v)].
v), 1: 2 (v / v), 1: 1 (v / v)]. Hexane-ethyl acetate = 1:
1 g (v / v) of the eluted fraction 1 g was subjected to high performance liquid chromatography using an ODS reverse phase column (diameter 0.2 cm, length 25 cm) to obtain methanol-water [10: 1 (v / v).
v)]. Retention time 9.5 minutes, 11.4 minutes, 1
It was confirmed that a substance having strong anticancer activity was present in each of the fractions at 4.8 minutes, 17.2 minutes, 19.8 minutes and 21.0 minutes. Each fraction was designated as fraction 1, fraction 2, fraction 3, fraction 4, fraction 5 and fraction 6 in ascending retention time.

Next, each fraction was subjected to preparative thin-layer chromatography [silica gel column, hexane-ethyl acetate = 1: 2 (v / v)] and preparative high performance liquid chromatography [ODS column, methanol-water = 10: 1 (v
/ V)] to separate a single compound as needed. The yield of the isolated single compound was 1.90 mg from Fraction 1, 3.29 mg from Fraction 2, and 2.0 from Fraction 3.
6 mg, fraction 4 was 8.23 mg, and fraction 5 was 1.23 mg. (When the raw material was changed to the above-ground portion of venom, fraction 1 was 1.35 mg, fraction 2 3.61 mg were obtained).

When the chemical structure of the isolated compound was analyzed, the compound isolated from fraction 1 was a novel compound represented by the formula (I). A new compound is called Stellaramclin A (stelleram)
acrin A)]. Further, the compound isolated from fraction 2 was gnidimacrine, the compound isolated from fraction 3 was pimarea factor P ₂ , the compound isolated from fraction 4 was subtoxin A, and the compound isolated from fraction 5 The compound isolated from Huratoxin and Fraction 6 was Simplexin.

The physicochemical properties of a novel compound, stellaramacin, among the isolated compounds are shown below. Physicochemical properties of Sterellamacrine: molecular formula: C ₃₇ H ₅₀ O
₁₁ Properties: white powder Mass spectrometry: M + H = 671 benzoyl group: 1 daphnan system: having a diterpene skeleton. Proton nuclear magnetic resonance spectrum (δ ppm in CDC
l ₃ ) ¹ H-NMR; 2.59 (1H, dd, J = 12.5), 1.
65 (1H, m), 3.81 (1H, d), 3.72 to 3.9
5 (1H), 3.39 (1H, s), 3.03 (1H, d, J
= 2.9), 2.91 (1H, d, J = 12.5), 2.76
(1H, m), 2.02 (1H, d, J = 7.5), 2.32
(1H, d, J = 14.7), 4.40 (1H, d, J = 2.
9), 5.19 (1H, s), 4.95 (1H, s), 1.84
(3H, s), 4.92 (1H, bd, J = 10.0), 4.3
7 (1H, t, J = 10.0), 1.17 (3H, d, J = 7.
6), 3.89 (1H, d, J = 10.0), 3.73 (1H,
d, J = 11), 2.26 (1H, m), 3.78-3.89
(1H, d, J = 7.3), 1.00 (3H, d, J = 7.
6), 8.07 (1H, m), 7.57 (1H, m), 7.47
(2H, m) Carbon nuclear magnetic resonance spectrum (δ ppm in CDC
l ₃ ): ¹³ C-NMR; 14.58, 18.20, 19.05,
22.81, 23.11, 23.61, 24.19,
25.18, 27.25, 28.52, 29.51,
36.53, 37.62, 40.85, 48.01,
48.13, 61.28, 63.18, 65.12,
68.27, 70.81, 72.08, 78.47,
79.42, 81.03, 81.34, 84.42, 1
11.89, 118.50, 128.43, 129.73,
130.30, 133.13, 145.56, 167.09.

Example 2 2 g of MMP obtained by the method described in Example 1 was
(LC-Sorb, SP-A-ODS, manufactured by Chemco) and chromatographed on an open 9 column (ODS column, methanol-water 10: 1, v / v) using methanol-water 9: 1 (v / v). eluted in v).

When this eluate was detected at 254 nm, 6
Two fractions were obtained, among which fractions 1, 2 and 3 showed strong anticancer activity. Among them, the fraction 2 exhibiting the strongest anticancer activity was further subjected to high-performance liquid chromatography for separation (column: Shim-Pack PREP-ODS, 20 mm × 2
5 cm), and methanol-water (10: 1, v / v)
Was used as an elution solvent, and elution was performed while monitoring at UV 228 nm. As a result, at the elution time of 17.5 minutes, Sterellamacrin A, at the elution time of 27.9 minutes, Gnidimacrin,
At an elution time of 44.4 minutes, each of stellarmacrine B was eluted.

1 and 2 show proton NMR of the obtained stellarmacrins A and B, respectively. Further, from the above fractions 1 and 3, stellalacmarin was similarly obtained.

The murine leukemia cell P-388 in Test Example 1 BDF ₁ mice intraperitoneally
Were transplanted at 1 × 10 ⁶ per animal, and the test drug was intraperitoneally administered once a day for 5 days from the next day, and the number of survival days was compared with that of the control group to determine the survival rate. Table 1 shows the results.

[0037]

[Table 1]

Test Example 2 Gastric cancer, lung cancer, liver cancer, and leukemia cells isolated from a human were placed in RPM-1640 medium containing 10% calf serum at a concentration of 10 ⁴ cells / ml, and 5% carbonated. Incubate at 37 ° C. for 4 days in a gas incubator. The concentration of the culture was varied and then added to the mixture, and the sample concentration (IC ₅₀ ) at which human cancer cells were inhibited by 50% was determined. Table 2 shows the results.

[0039]

[Table 2]

[0040] 1 × 10 ⁶ cells of Lewis lung carcinoma cells in Test Example 3 BDF ₁ per mouse, BALB / C per mouse of 5 × 10 ⁵ cells clones 26 (Clon26) colon cancer cells, BDF ₁ per mouse 0.25 ml of 20% homogenate of B-16 melanoma solid cancer was implanted subcutaneously to form solid cancer, and from the next day, gnidimacrine and pimeria factor P ₂
Was administered intraperitoneally once a day for 5 days to compare the survival rate with the control group. Table 3 Results for Gunidimakurin respectively show results for Pimerea factor P ₂ in Table 4.

[0041]

[Table 3]

[0042]

[Table 4] Gnidimacrine showed a survival rate of 47.8% at 0.06 mg / Kg when administered intravenously in Lewis lung cancer.

Test Example 4 Gastric cancer, lung cancer, liver cancer, and leukemia cells isolated from a human were placed in an RPM-1640 medium containing 10% calf serum at a concentration of 10 ⁴ cells / ml and 5%. Incubate at 37 ° C. for 4 days in a carbon dioxide incubator. Was added Gunidimakurin or Pimerea factor P ₂ by changing the concentration of the culture, human cancer cells was determined sample concentration (IC ₅₀₎ that is 50% growth inhibition. Table 5 shows the results.

[0044]

[Table 5]

Test Example 5 The effect of gnidimacrin on solid tumors was examined using experimental animals. That is, first, 10% fetal bovine serum of Meth A adenosarcoma cells (fibrosarcoma) adjusted to 1 × 10 ⁵ cells / ml was added subcutaneously to female BALB / c mice (6 mice per group; 17 to 21 g). RPMI 164
0 ml of 0.01 ml was inoculated. Seven days after inoculation, solid tumors are formed. To this, 0.001 mg of gnidimacrine dissolved in physiological saline containing 0.5% CMC is added.
/ Kg by injection. This dose should be taken once daily
Times for 5 days. Meth A adenosarcoma cell administration 21
On the day, tumors were removed and their weights were compared to a control group that did not receive gnidimacrine. As a result, the average tumor weight of the group treated with gnidimacrine was 0.4 g,
The average tumor weight of the control group was 2.1 g, and gnidimacrine inhibited the growth of solid tumors by 81%.

Formulation Example 1 Tablets: (Preparation) Corn starch 48 g Crystalline cellulose 42 g Carboxymethyl cellulose calcium 8 g Light anhydrous silicic acid 0.5 g Magnesium stearate 0.5 g Stellarmacrin A 1 g Total 100 g

(Preparation method) In accordance with the above-mentioned formula, (1) was uniformly mixed, and the mixture was compression-molded with a tableting machine to give 200 mg per tablet.
Tablets were obtained. One tablet contains 2 mg of the stereramacrine A obtained in Example 1 and is used for one adult.
Take 1 to 10 tablets daily in several divided doses.

Formulation Example 2 Condyle Granule: (Preparation) Corn starch 88 g Magnesium stearate 1.5 g Carboxymethyl cellulose calcium 8 g Light anhydrous silicic acid 1.5 g Stellarmacrine B 1 g Total 100 g

(Preparation method) According to the above-mentioned formula, (1) and (2) were uniformly mixed, compression-molded by a compression molding machine, crushed by a crusher and sieved to obtain granules. 1 g of this granule contains the stereramacrine B 1 obtained in Example 2.
It contains 0 mg, and 0.1 to 2 g of adult daily is divided into several doses.

Formulation Example 3 Encapsulation agent: (Preparation) 98.5 g of corn starch 1.0 g of light anhydrous silicic acid 0.5 g of stellalamacrine A 0.5 g in total 100 g

(Preparation method) According to the above-mentioned formula, (1) was evenly mixed, and 200 mg was filled in a No. 2 capsule.
One capsule of this capsule contains 1 mg of stellarmacrin A obtained in Example 1, and 1 to 20 capsules per day of an adult is divided into several doses.

Formulation Example 4 Injection: (Preparation) 89.5 g of distilled water for injection 5 g of soybean oil 2.5 g of soybean phospholipid 2.5 g glycerin 2 g 1 g of stereramacrine A 100 g in total

(Preparation method) In accordance with the above-mentioned formula, and was dissolved in, and the resulting solution was added and emulsified.
An injection was obtained.

[0054]

As described above, the stellarmacrin of the present invention has a high anticancer activity, so that it can be used as an anticancer agent having a wide range of application. Further, the solid Ganchi療剤containing Gunidimakurin and Pimerea factor P ₂ is stomach cancer,
It is useful for treating solid cancers such as lung cancer, liver cancer and breast cancer.

[Brief description of the drawings]

FIG. 1 shows the proton NM of the present invention.
It is a drawing which shows R.

FIG. 2 Proton N of the present invention
It is a drawing which shows MR.

Continuation of the front page (56) References Japan Association of Pharmacists, edited by the Japanese Society of Pharmacy, April 30, 1985, Journal of Pharmaceutical Sciences, Vol. 72, No. 11, p. 1285-1287 Naturewissenchaften, Vol. 72, No. 7, p. 373-375 (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 31/357 A61K 35/78 C07D 493/18 CA (STN) REGISTRY (STN) MEDLINE (STN) BIOSIS (STN)

Claims (2)

(57) [Claims] (1) A therapeutic agent for solid cancer comprising gnidimacrine as an active ingredient. 2. The therapeutic agent for solid cancer according to claim 1, wherein the solid cancer is gastric cancer, lung cancer or liver cancer.