JPH02167092A - Anti-tumor agent containing lisolipin x and production of lisolipin x - Google Patents

Abstract

PURPOSE:To obtain an anti-tumor agent which is useful as an intraperitoneal, subcutaneous and intravenous injections and local injection by using lisolipin X as an active ingredient. CONSTITUTION:The subject anti-tumor agent contains, as an active ingredient, lisolipin X of the formula. The lisolipin X is obtained by isolating it from the culture mixture of a microorganism, capable of producing lisolipin X, in Streptomyces platencis, BTO98 strain (FERM BP-2076) in a suitable culture medium under aerobic conditions.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] Technical Field The present invention relates to antitumor agents, and more particularly, to solipin
The present invention relates to an antitumor agent containing the following.

Many prior art antitumor substances have already been put into practical use as medicines. Generally, the physiological activity of a chemical substance largely depends on its chemical structure, so it can be said that there is a constant desire for compounds with antitumor properties.

[Summary of the invention]

SUMMARY The present invention meets the above needs. That is, the present inventor has Jf! As a result of a wide range of natural searches for substances that suppress the growth of f+ tumor cells, we discovered that solipin X, produced by actinomycetes, strongly suppresses the growth of tumor cells and is also effective as an antitumor agent. , we have completed the present invention.

Therefore, the anti-ulcer agent according to the present invention is characterized in that it contains the compound risoripin X represented by the following formula as an active ingredient.

The invention also relates to a method for producing this compound risoripin X. That is, the method for producing the compound lysoripin The present invention is characterized by obtaining a compound risoripin X.

[Detailed description of the invention]

Resoripin X The compound Resoripin X used as the active ingredient of the antitumor agent according to the present invention is a compound known per se.

That is, lysoripin
No. 478).

Overview of the production of risoripin Since the molecular structure of lysoripin In addition to those used in the method, we can exemplify strain 87098, which is an actinomycete (belonging to the genus Streptomyces) that the present inventors isolated from the soil of Hokkaido.

It may also be possible to use genetic engineering techniques.

That is, it would also be possible to introduce the lysoripin X-producing gene into a suitable microorganism, culture such a transformed microorganism, and obtain it from this culture.

Strain 87098 The present inventors have discovered strain 87098 as a Streptomyces strain capable of producing lysoripin X.
The contents are as follows.

1) Origin and Deposit Number Strain 87098 was isolated from soil collected in Hokkaido as mentioned above, and was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology on September 30, 1988, and was designated as the It has a number of Article No. 2076 J (FERMBP-2076).

2) Mycological properties The mycological properties of strain 87098 are as follows.

(1) Type 87098 strain grows extremely poorly on glucose-asparagine agar and relatively poorly on nutrient agar, but grows well on other media. The attachment of aerial mycelia was poor on glucose-asparagine agar and nutrient agar, moderate to slightly poor on oatmeal agar, but generally good on other media. Aerial hyphae generated from basal hyphae form simple branches and elongate, but spore chains are generally difficult to form. On oatmeal agar medium and starch inorganic salt agar medium, the number of spore chains is about 10 to 50, and they often form a compact spiral (3 to 4 turns) at the tip.
Incomplete spirals or loops are also seen. In addition, the shape of the spore is cylindrical or oval, and the size is 0.4 to 0.6
μm×0, 7 to 1.0 μm, and its surface is smooth.

Specialized forms such as sporangia, flagellated spores, and sclerotia are not observed. However, bulges resembling spore aggregates may be observed on the aerial mycelia on oatmeal agar media.

(2) Growth status on various media The results of culturing 87098 strains on various media at 27°C for 3 weeks are shown in Table 1.

(3) Physiological properties The physiological properties of strain 87098 are shown in Table 2.

(4) Carbon source utilization The carbon source utilization of strain 87098 (on Bridham-Gotlieb agar medium) is as shown in Table 3.

(5) Analysis of diaminopimelic acid As a result of analyzing diaminopimelic acid, which is one of the amino acids constituting the cell wall of strain 87098, LL-diaminopimelic acid was detected.

From the above mycological properties, strain 87098 was determined to be a strain of the genus Streptomyces, and has the following characteristics.

(1) Spore chains are relatively difficult to form, their tips are mainly spiral or loop-shaped, and the surface of the spores is smooth.

(11) Aerial mycelium is yellowish white to yellowish gray, and the color of the underside is pale yellow to dull yellow to yellowish orange to dark yellow to dark orange.

(iii) No melanin-like pigment is produced on tyrosine agar, peptone yeast/iron agar, or tryptone yeast liquid medium, and there is a slight yellow color in glycerol/asparagine agar, tyrosine agar, or oatmeal agar. ~Produces a yellowish-brown pigment.

(1v) Swellings resembling spore aggregates may be observed on aerial mycelium on oatmeal agar medium.

Based on the above properties, ISP (International Streptomyces Project) describes (E, B).

5h1r+tng and D, Gottlicb:
+nt,'', 5yst, nact, 1g09-189
．． 279-392 (1968): 19391-51
2 (1969):22265-394 (1972)
) and Bercy's Manual of Determinative Bacteriology (f3ergcy's Manual of Determinative Bacteriology)
null ofDeterminative Bact
When searching for related known bacterial species using the 8th edition (174) of Streptomyces platensis as a reference, we found that Streptomyces platensls
) (Ink, J, 5ysL.

Back: 1g, 380 (19(i8)) seemed to be the most similar.

Therefore, when comparing strain 87098 and Streptomyces platensis, we found that they have morphological characteristics that form spiral spore chains and spores with smooth surfaces, that they do not produce melanin-like pigments on peptone, yeast, iron agar media, etc. The two species are in good agreement in that they produce a yellowish pigment in oatmeal agar media, the color tone of aerial mycelia, the ability to assimilate sugar, and the fact that swellings like spore aggregates are observed in oatmeal media. The difference is that the color tone of the pigment produced by strain 87098 does not change with 0.05N hydrochloric acid.

As mentioned above, although there are some differences, the 87098 strain is quite similar to Streptomyces platensis in its basic characteristics, and therefore
It is appropriate to identify it as P. platensis. therefore,
Streptomyces platensis (SL) strain 87098
reptoIlycesplaLensis ) B
It shall be named T n 98.

Table 1 Table 2 Table 3 +: Utilization - Dual non-utilization Culture/Production of lysoripin X Compound lysoripin can be produced by collecting the target product from the culture.

The medium can contain any nutrient source that can be utilized by the lysoripin X-producing bacteria. Specifically, for example, glucose, sucrose, maltose, starch, fats and oils can be used as a carbon source, and organic substances such as soybean flour, cottonseed meal, dried yeast, yeast extract, and cornstarch liquor can be used as a nitrogen source. Inorganic ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride can be utilized. Moreover, inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, phosphates, and heavy metal salts can be added as necessary. In order to suppress foaming during fermentation, suitable antifoaming agents such as silicone oil can also be added according to conventional methods.

The most suitable culture method is the aerobic liquid culture method, which is the same as the commonly used antibiotic production method. The culture temperature is suitably 8 to 37°C, preferably 20 to 30°C.

In this way, a culture in which lysoripin X is accumulated is obtained. In this culture, lysoripin
Exists in liquid.

Any suitable method can be used to collect soripin X from such a culture.

One method is based on the principle of extraction,
Specifically, soribin X in the culture filtrate may be extracted with a suitable water-immiscible solvent for soribin X, such as chloroform or ethyl acetate. The culture itself can also be subjected to the above extraction operation without separating the bacterial cells. Countercurrent distribution methods using suitable solvents can also be included in the scope of extraction.

Another method for collecting soripin A suitable adsorbent such as silica gel,
Resoripin X can be obtained by adsorbing the desired paste solipin X using activated carbon, Diaion HP-20J (manufactured by Mitsubishi Kasei Corporation), and then eluting it with an appropriate solvent. By concentrating the soripin X solution to dryness under reduced pressure, a crude specimen of soripin

In order to further purify the crude preparation of lysoripin For example, high-performance liquid chromatography and countercurrent distribution using adsorbents such as silica gel, column chromatography rYMC back using gel filter agents such as Sephadex LH-20J (manufactured by Pharmacia) (manufactured by Yamamura Kagaku Co., Ltd.), etc. Laws may be implemented in appropriate combinations. Specifically, for example, a crude sample of Lisoripin After concentration to dryness, a pure product of Solipin X is obtained.

Biological Activity of Resoripin X The compound of the present invention, Resoripin X, is useful in that it has anti-Ill tumor activity.

Antitumor activity Lysoripin
In vivo tumor activity was observed in experimental animals against 8+11 tumors (mountain blood disease) or human tumors such as human breast cancer (MX-1).

Specifically, a suspension of P388 tumor cells was intraperitoneally administered to CDF mice, and on the first day after transplantation, a solution containing lysoripin X was intraperitoneally administered to the mice (j [
1 number, as shown in Table 1, P 388
A life-prolonging effect of risoripin X on fi-implanted mice was observed. be). The details of this experiment are described in Experimental Example 2 below.

Table 1 Survival-prolonging effect of risoripin
．． 52 Risolipi>X 5mg/kg (B) Liquid 0.2m
l/7us 9.2+0.82 103 Lisoripin X 2
mg/kg (C) solution 0.2ml/mouse 11.3±
0.52 121 Note I T/C-100X (Average survival days of treatment group/Average survival days of control group) In addition, human breast cancer (MX-
The tumor pieces from 1) were transplanted under the synthetium of BDF mice, and a solution containing risoripin As shown in Table 2, the tumor growth inhibitory effect was observed (Il! I! The tumor growth inhibitory effect is the tumor growth inhibition rate (T).

G.1. R).

The details of this experiment are only described in Experimental Example 3 below.

Table 2 Experimental group Dose T, G, 1. R.

Resoripin X 2mg/kK (C) solution 0.2ml
/mouse/mouth 73% Nosolipin X Img/kg
(D) ifk 0.2ml/mouse/day 52%
Antitumor agent Thus, the compound risoripin X in the present invention is an antitumor agent or I+41! It can be used as a tumor treatment.

The compound risoripin
can be administered. The drug is usually in a diluted form with a pharmaceutically acceptable carrier or diluent.

When the compound risoripin X is actually administered as an antitumor agent, one typical method is to dissolve it in distilled water for injection or physiological saline and inject it. Specifically, in animals, methods such as intraperitoneal injection, subcutaneous injection, vascular injection into a vein or artery, and local administration by injection, and in humans, vascular injection or injection into a vein or artery. There are methods such as local administration.

The dosage of the compound risoripin X in the present invention is determined in consideration of the results of animal tests and various situations so that the total dose does not exceed a certain amount when administered continuously or intermittently. The specific dosage will vary depending on the method of administration, the circumstances of the patient or treated animal, such as age, weight, sex, sensitivity, diet, time of administration, concomitant drugs, and the degree of the patient or his or her disease. Needless to say, the appropriate dose and frequency of administration under certain conditions must be determined by a specialist's appropriate dose determination test based on the above guidelines. Specifically, it is about 0.1-1 g per day for an adult.

In addition, in acute toxicity tests on mice, no deaths were observed when compound risoripin X was administered intraperitoneally at 10 mg/kg.

EXPERIMENTAL EXAMPLES The following experimental examples are intended to explain the present invention in more detail. The present invention is not limited by these experimental examples.

Experimental Example 1: Preparation of Risoripin X 2.5% glucose, 1.5% soybean flour, 0.
A liquid medium having a composition of 2% calcium carbonate and 0.4% calcium carbonate was adjusted to pH 7.4, and 100 ml of this was dispensed into a 500 ml Erlenmeyer flask with warts and sterilized. In addition, Streptomyces platensis BTO98 strain (FERM BP
-2076) and cultured with shaking at 27°C for 4 days.

One liter of the culture solution was extracted twice with 500 ml of chloroform, and the solvent was distilled off under reduced pressure to obtain a crude extract. This was subjected to silica gel (Klesel Gel 60 manufactured by Merck) column chromatography 3.0 (φ) x 30 cm and eluted with chloroform/methanol (40:1) a solution. The lysoripin
) x 50cm and chloroform/methanol (1
: 471 mg of pure lysoripin X was obtained by elution with step 1). Ultraviolet absorption spectrum, mass spectrum, 'H-
NMR spectra, and! This was confirmed to be Lisoripin X by 3C-NMR spectrum.

Experimental Example 2: Antitumor effect of risoripin X on mouse tumors 1) Preparation method of risoripin
．． 9 ml of physiological saline was added to obtain a long/ml glue soribin X solution (A). In addition, (A) was diluted using physiological saline containing 1% DMSO as a solvent, and 0.5 g/g of copper was added.
ml (B), O82mg/ml (C), 0.
A 1 mg/ml (D) solution was obtained.

2) Antitumor effect on P2S5 & stereotropic P 388
1. eukemia (murine leukemia) cells 1 x 106
Mice that received Cells intraperitoneally (CDF, ♀,
Drug (B) on the 1st day and 5th month after P388 transplantation (7 weeks old)
and (C) were each administered intraperitoneally at 0.2 ml/mouse.

3) Results As shown in Section 1 above, risoripin
It was found that it has anti-fMi tumor effect against P388.

Experimental Example 3: Anti-IN of risoripin X against human tumors!
fI tumor effect 1) Preparation method of risoripin A solution (A) was obtained. In addition, (A) was diluted using physiological saline containing 1% DMSO as a solvent, and 0.
2mg/ml (C), O, lag/ml (D),
0. 01 +ng/kg (E) solution was obtained.

2) Method for determining the IP1 antitumor effect of risoripin X on human breast cancer (MX-1) ubrenal capsule assay
; 5RCA) A human breast cancer (MXl) that has been passaged in nude mice is excised to create a flat cylindrical tumor piece with a diameter of 0.8++ os and a height of about 0.3 mm. This is transplanted under the growth membrane of an anesthetized mouse (BDF1, female, 6 weeks old), and the long axis and short axis of the transplanted tumor piece are measured using a stereoscopic microscope. From day 1 to day 5 after transplantation, 0.2 ml of risoripin xl is intravenously administered every day for 5 days. On the 60th day, the mice were sacrificed, the kidneys were removed, and the major and minor axes of the transplanted tumor pieces were measured using a stereoscopic microscope. The antitumor effect is determined by the tumor growth inhibition rate (T, G).

1, R) is determined as follows.

Tumor Growth Inhibition Rate
Part I? ate; T, G, 1. I? )−(
1-T/C) Volume I)! f! Tumor mass - major axis x minor axis 2/2 3) Results As shown in Table 2, lysoripin X was found to have a growth-inhibiting effect on human breast cancer transplanted under the mouse renal capsule.

Claims (1)

[Scope of Claims] 1. An antitumor agent containing the compound risoripin X represented by the following formula as an active ingredient. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ 2. A strain belonging to the genus Streptomyces platensis and capable of producing risoripin X is cultivated aerobically in an appropriate medium, and the compound risoripin X is obtained from the culture. A method for producing lysolipin X, which is characterized by the following formula. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼