JPH08173176A - Inhibitor of arterialization - Google Patents

Abstract

PURPOSE: To obtain an inhibitor of arterialization containing borrelidin or its salt as an active ingredient and useful for treatment, etc., of various solid cancers. CONSTITUTION: This inhibitor of arterialization contains borrelidin, having a structural formula represented by the formula and isolated as an antibiotic substance from a culture solution of a microorganism of the genus Streptomyces to determine the structure thereof or its salt as an active ingredient. The borrelidin is preferably obtained by culturing Streptomyces rochei Mer-N 7167 (FERM P-14670) in a nutrient culture medium and collecting the borrelidin from the culture solution from the viewpoint of productivity.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an angiogenesis inhibitor effective for the prevention and treatment of various diseases accompanied by abnormal growth of angiogenesis.

[0002]

2. Description of the Related Art Angiogenesis is a biological phenomenon indispensable for fetal vascular tree formation and morphological and functional development of various organs, but it occurs only in the female sexual cycle in mature individuals. However, it is known that the pathological increase in angiogenesis is involved in the onset or progression of various diseases in mature individuals. Specifically, cancer, rheumatoid arthritis, atherosclerosis, diabetic retinopathy, hemangiomas, psoriasis, etc. are mentioned as diseases accompanied by abnormal angiogenesis (Marsha.
A. et al., Biotechnology, 9 , 630, 1991). In particular, it has been reported that the growth of solid tumors depends on angiogenesis (Folkman J., J. Natl. Cancer Inst., 82 , 4,
1990) Angiogenesis inhibitors are expected to become new therapeutic agents for intractable solid tumors.

Although there have been reports on some angiogenesis-inhibiting substances, no effective substance that can withstand practical use has been found yet (Japanese Patent Laid-Open Publication No. 3-10932).
4, JP-A-3-236324, JP-A-3-2184).

[0004]

DISCLOSURE OF THE INVENTION The present invention is to provide a prophylactic and therapeutic agent for various diseases accompanied by abnormal growth of angiogenesis, which is difficult to treat a novel substance having an angiogenesis inhibitory activity.

[0005]

In view of the above situation, the present inventors have started a search screen for a new angiogenesis inhibitor having a stronger inhibitory activity and less side effects, using a microorganism culture solution as a raw material. As a result, they have found that an angiogenesis inhibitor is produced in the culture solution of a microorganism belonging to the genus Streptomyces. As a result of isolation of this active substance and structural analysis, it was found for the first time that Borrelidin has an angiogenesis inhibitory activity, and the present invention was completed.

[0006] That is, the present invention relates to an angiogenesis inhibitor and an anti-solid cancer agent containing borrelidin or a salt thereof as an active ingredient. The present invention also relates to Streptomyces rochei Mer-N.
The present invention relates to a method for producing borrelidin, which comprises culturing 7167 (FERM P-14670) in a nutrient medium and collecting borrelidin from the culture solution.

Boreridin is a compound whose structure was determined as an antibiotic in 1949 from the culture medium of Streptomyces microorganisms (Berger J. et al., Arch. Biochem. Bio
phys., 22 , 476, 1949). The chemical formula is 2 [7-cyano-
8,16-dihydroxy-9,11,13,15-tetramethyl-
18-oxooxacyclooctadeca-4,6-dien-2-yl] cy
It is clopenta-carboxylic acid, and its structural formula is represented by the following formula (I).

[0008]

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[0009] Borrelidin is known to have an antiviral effect and an anti-ascites tumor (Krebs ascites tumor) effect in addition to the antibacterial effect (Dickinson L., et al., Natur.
e, 206 , 265, 1965). However, the present inventors have for the first time found that it has a completely unexpected action of inhibiting angiogenesis, and borrelidin is expected as a preventive and therapeutic agent for various solid cancers.

[0010] Borrelidin causes ascites cancer (Krebs ascites tumo)
Although it is reported that it has an action of suppressing the proliferation of r), ascites cancer and solid cancer of the lymphatic system are morphologically completely foreign substances. The solid cancer means a solid cancer accompanied by abnormal angiogenesis such as gastric cancer, pancreatic cancer, colon cancer, and lung cancer, and does not include lymphoid cancer such as ascites cancer.

The present invention will be described in detail below. As the microbial species of the microbial culture solution that is the raw material for the extraction of borrelidin,
Streptomyces albovinaceus and Streptomyces rochei were selected as previously reported.
And any microorganism capable of producing borrelidin such as Streptomyces sp. C 2989 can be used (Singh SK et al., Antimicrob. Agents. Ch.
emother. 27 , 239, 1985; Berger J., et al., Arch.B
iochem. Biophys., 22 , 476, 1949; Lumb M. et al.,
Nature, 206 , 263, 1965).

[0012] Examples thereof include Streptomyces rochei ATCC10739 and the like. As a preferable strain with particularly high productivity of borrelidin, an actinomycete isolated from the soil of Ishigaki Island by the present inventors was used.
Mer-N 7167 strain is mentioned. This bacterium has the following mycological properties. Morphology: It is composed of basal hyphae that branch and extend well and aerial hyphae that also well extend, and the tip of aerial hyphae is sporulated. The spore chains are 10 to 50 in series and have a gentle spiral shape, but some are curved. The surface of spores is smooth and the size is about 1 μm × 1 to 2 μm in diameter. Cell component: Diaminopimelic acid includes LL-type, and sugars include galactose, glucose, mannose and ribose.

Growth on various media; ISP-1 growth is moderate and produces pale white aerial mycelia. The reverse side of the culture becomes slightly yellow. ISP-2 grows well and produces a large amount of gray to light white (Light grayishreddish brown) spores on white aerial hyphae. The reverse side of the culture is slightly yellowish brown. ISP-3 Growth is good and the spore color is gray purple (Grayish pu
rple), others are similar to ISP-2 ISP-4 spores are gray (Medium gray) Others are similar to ISP-2 ISP-5 Growth is weak and white aerial hyphae are slightly produced. Modified tyrosine medium Does not produce melanin pigment. Almost no soluble pigment was found in any of the culture media.

Glucose utilization; (+) glucose, inositol, fructose, rhamnose, mannose, arabinose (±) xylose (−) raffinose, sucrose (+); well assimilated (−); not assimilated (± );
Interim identification: Streptomyces roch that carried out the properties of this strain at the same time
Compared with the properties of ei type strain IFO 12908, the differences shown in Table 1 are observed.

[0015]

[Table 1]

[0016] Although there are such differences, they are not so different as to be different in species, and Streptomyces rochei (Strep
Since the range of intraspecific variation of tomyces rochei), the present inventors have determined that this strain is Streptomies rochei (Streptom).
yces rochei) Mer-N 7167 was named. This strain has been deposited as FERM P-14670 at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science on November 28, 1994.

As a screening method for angiogenesis inhibitory activity, the degree of inhibition of lumen formation observed when rat aortic strips were cultured in collagen gel was used as an index (Nicos
ia RF, Lab. Invest., 63 , 115, 1990). In addition, the human colon cancer WiDr cell line was also used as a screening method together with the angiogenesis-inhibiting activity under subcutaneous mouse.

After culturing a microorganism of the genus Streptomyces under ordinary appropriate culturing conditions, the culture solution is clarified and filtered, and then an organic solvent such as butanol or methyl isobutyl ketone is added for extraction, and the organic solvent layer is concentrated under reduced pressure. Then, it is extracted with methanol and treated with petroleum ether (light petroleum) or the like to obtain a crude extract. Then, adsorption chromatography using silica gel or the like, LH 20 gel chromatography, partition chromatography, thin layer chromatography, paper chromatography, etc. are appropriately used for fractionation, and the activity fraction is confirmed by activity screening.
The active substance can be isolated by appropriately combining the above techniques. Solvents used for adsorption chromatography include chloroform, methanol, acetone,
It is possible to use a commonly used organic solvent such as hexane or toluene, and appropriately select and combine the concentrations. As a solvent for crystallization, chloroform and hexane, or chloroform and carbon tetrachloride can be used. One method is M. Lumb's method. (Natu
re, 206 , 263, 1965). Structural analysis of the isolated compound
It can be performed by a conventional method such as elemental analysis, GC-MS, NMR, and melting point.

The isolated compound was found to be a known substance, borrelidin, but it was surprisingly found that borrelidin has a potent angiogenesis-inhibiting activity.
As described above, since abnormal angiogenesis is observed in various diseases, as a preventive agent for those diseases, for example, rheumatoid arthritis, solid cancer, atherosclerosis, diabetic retinopathy, hemangiomas, psoriasis, etc. It is also expected as a therapeutic drug, and is particularly effective as an anti-solid cancer drug.

When the compound is administered as a therapeutic / prophylactic agent for various diseases, it may be orally administered as tablets, powders, granules, capsules, syrups, etc., or sprays, suppositories, injections. Alternatively, it may be parenterally administered as an external preparation or a drip. The dose varies significantly depending on the severity of symptoms, age, type of liver disease, etc., but is usually about 1 per adult per day.
Administer mg to 100 mg once or several times a day.

When formulating, a usual pharmaceutical carrier is used, and it is manufactured by a conventional method. That is, in the case of preparing a solid preparation for oral use, an excipient, and if necessary, a binder, a disintegrating agent, a lubricant, a coloring agent, a flavoring agent, etc. are added to the main drug, and then a tablet is prepared by a conventional method. Coated tablets, granules, powders, capsules, etc. For these tablets and granules, sugar coating, gelatin coating,
Of course, other coating may be appropriately performed if necessary. When preparing an injection, a pH adjusting agent, a buffering agent, a stabilizer, a solubilizing agent, etc. are added to the main drug as necessary, and a subcutaneous, intramuscular or intravenous injection is prepared by a conventional method.

[0022]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. In the examples,% is based on weight unless otherwise specified.

Example 1. Purification and structure confirmation of borrelidin As seed medium, glycerol 2.0%, glucose 2.0
%, Soy flour 2.0%, yeast extract 0.5%, sodium chloride
A medium having a composition of 0.25%, calcium carbonate 0.32% and a trace metal solution (preparation of a solution of copper 0.25%, manganese chloride 0.25% and zinc sulfate 0.25%) 0.2% was used. The production medium was potato starch 2.0% instead of seed medium glycerol 2.0%, and the other composition was the same. At the time of jar culture, an antifoaming agent 0.05% was added.
The pH before sterilization was adjusted to 7.4 before use. Seed medium 100
A 500 ml Erlenmeyer flask in which 1 ml was dispensed was sterilized at 120 ° C for 15 minutes, and 1 platinum loop of a slope agar culture of the Mer-N 7167 strain was inoculated into this, and shake culture was performed at 28 ° C for 3 days to form a seed culture. . 15 L of production medium was dispensed into two 30 L jar fermenters, sterilized at 120 ° C for 30 minutes, 100 ml of each seed culture was inoculated, and aeration (0.5 vvm) at 28 ° C for 5 days with stirring (300 rpm). ) Cultured. After completion of the culture, the supernatant was collected by centrifugation, adjusted to pH 7, adsorbed on an HP-20 column (3 L), washed with water, and washed with water.
After washing with% methanol, elution was performed with 80% acetone. About 3 L of the eluate was concentrated under reduced pressure to remove acetone, water was added to about 1 L, and the mixture was extracted twice with 1 L of ethyl acetate.
The extract was concentrated to dryness under reduced pressure to obtain a blackish brown oily substance. This oily substance was dissolved in a small amount of methanol, placed on the top of Sephadex LH-20 (400 ml), developed with methanol, and the active fraction was concentrated to dryness. Next, dissolve in a small amount of chloroform: methanol = 50: 1 and add silica gel (Kiesel
gel 60) Place it on the top of the column (150 ml) and add chloroform:
Elution was performed with 500 ml of methanol = 50: 1, 20: 1 and 10: 1, respectively. Chloroform: methanol = 50: 1 to 20: 1
The active fractions eluted in (1) were collected and concentrated to dryness. Next, dissolve in a small amount of acetone, mix with silica gel (Kieselgel 60), and concentrate to dryness under reduced pressure.
0) It was placed on the top of a column (150 ml) and eluted with hexane: acetone = 3: 1, 2: 1 and 1: 2, 500 ml each.
The fraction that could not be eluted was extruded with methanol. Active fractions eluted with hexane: acetone = 3: 1 to 2: 1 were collected. The unpurified fraction was dissolved in a small amount of toluene: acetone = 5: 1, placed on the top of a silica gel (Kieselgel 60) column (50 ml), and toluene: acetone = 5:
Elution was carried out with 200 ml each of 1, 4: 1 and 2: 1. Active fractions eluted with toluene: acetone = 5: 1 were collected and combined with the previous active fraction to obtain 611.2 mg of a purified product. The purified product was dissolved in about 30 ml of chloroform and crystallized with hexane to give 420.0 mg of crystals.

The physicochemical properties of the active substance thus purified are as follows. 1) EI Mass spectrum As a result of high-resolution EI Mass spectrum analysis of m / z489 of the active substance, M ⁺ was observed at m / z489, and the molecular formula was C _28.
It was determined to be H ₄₃ NO ₆ . Table 2 shows the results.

[0025]

[Table 2]

2) Ultraviolet absorption spectrum: The measurement results are shown in FIG. 3) Infrared absorption spectrum: The measurement results are shown in FIG. 4) ¹ H nuclear magnetic resonance spectrum: The measurement results are shown in Table 3.

[0027]

[Table 3]

5) ¹³ C nuclear magnetic resonance spectrum: The measurement results are shown in Table 4.

[0029]

[Table 4]

From the above results, this substance was confirmed to be borrelidin.

Embodiment 2 FIG. Angiogenesis Inhibitory Activity Rat aortic strips were cultured in collagen gel, and the observed degree of inhibition of lumen formation was defined as angiogenesis inhibitory activity. That is, the adipose tissue around the aorta extracted from a Sprague-Dawley female rat (8 to 12 weeks old) was carefully removed while washing the aorta with Hanks' solution. After incising the aorta to prepare a 2 mm square section, the section is placed in a 24-well plate with the endothelial cell surface facing upward. Next, neutralized type I collagen gel (Ce
(llmatrixtype IA: Nitta gelatin) 500 μl was poured into each well and left at room temperature for about 20 minutes in a clean bench to solidify the gel. After confirming that the gel has set 5
00 μl of MCDB 131 (manufactured by Chlorella Industrial Co., Ltd.) medium was added to each well and incubated at 37 ° C. in a CO ₂ incubator (5% CO ₂ ). The next day, the culture medium was exchanged with MCDB 131 medium containing borrelidin, and on the fourth day of culturing, MCDB containing borrelidin was added again.
The medium was replaced with 131 medium and the culture was continued. Then, 7 days after the addition of borrelidin, the number of capillaries formed around the aorta was measured using a microscope. The results are shown in Table 5. As is clear from Table 5, borrelidin shows an angiogenesis-inhibitory effect in a concentration-dependent manner, and its IC ₅₀ value is about 0.5 n.
It was g / ml. The inhibitory activity is shown in comparison with the number of capillaries in the borrelidin-free group.

[0032]

[Table 5]

Example 3. In vivo angiogenesis inhibitory activity The above-mentioned effects in mice were examined by the dorsal air sac method (Sa
Kamoto et al., Cancer J., 1 , 55-57, 1986) using a partially improved method. A Millipore ring (manufactured by Japan Millipore) is sealed with a 0.22 μm membrane filter (HAWPO: manufactured by Nihon Millipore) to create a chamber. this
1 × 10 ⁷ human colon cancer W suspended in PBS in chamber W
iDr cells were encapsulated. Next, 6-8 weeks old Balb / c (nu /
nu) Make an air bladder subcutaneously on the dorsal side of female mice and
ported ber. Six hours after the transplantation was completed, borrelidin was intraperitoneally administered, and thereafter, it was continuously administered once a day for 3 days.
4 days after the chamber implantation, ⁵¹ Cr-labeled mouse erythrocytes were injected through the tail vein, and 5 minutes later, the mice were sacrificed. Then ch
After removing the skin from the part in contact with the amber and freezing it, chamb
Only the part in contact with er was accurately separated, and the blood volume was measured by γ-counter. The value obtained by subtracting the blood volume when a chamber enclosing PBS was transplanted from the blood volume was defined as the angiogenic volume (blood volume). The control (CT) was performed with 10 animals per group and the borrelidin-administered group with 5 animals per group. The results are shown in FIG. 3, and the angiogenesis inhibitory action was exhibited in the 1.8 and 6 mg / kg administration groups. However, in the 6 mg / kg administration group, toxicity also appeared and two mice died.

Embodiment 4 FIG. Antitumor activity against human colon cancer WiDr cells 3 × 10 ⁶ human colon cancer WiDr cells were Balb / c (nu / nu)
The mice were subcutaneously transplanted, and from the next day, borrelidin was intraperitoneally administered once a day by a schedule of 5 times a week for 5 weeks. Boreridin was used by dissolving each concentration in 1% NaHCO ₃ water. In addition, the tumor size was measured over time, and the relative tumor volume was calculated. As a result, as shown in Table 6,
It was found that borrelidin is a compound that exhibits an antitumor effect and can be continuously long-term.

[0035]

[Table 6]

Example 5 Antitumor Activity Against Human Prostate Cancer Cell Line PC-3 Cells Balb / c (nu / nu) mice (3 control cells, 6 experimental groups) were treated with 3 × 10 ⁶ PC-3 cells. After 10 days, borrelidin was intraperitoneally administered once a day after 10 days. Boreridin is 1
% NaHCO ₃ dissolved in water, 1.8 mg / kg was administered. As a result, a clear antitumor effect was shown as shown in FIG.

Example 6. Spontaneous metastasis suppressive effect 5 × 10 ⁵ mouse B16BL6X ranoma cells were subcutaneously transplanted into the foot pad of mice (control group 10 animals, experimental group 5), and after 6 days, borrelidin was administered intraperitoneally once a day for 10 days. did.
Twenty-one days after the transplantation, the hind limb with the primary lesion was cut off, and from the 26th day after the transplantation, borrelidine was administered again for 7 days. The number of lung metastases was measured 33 days after transplantation. As a result, as shown in Table 7, mice with 1.8 mg / kg administration group showed a remarkable metastasis-suppressing effect.

[0038]

[Table 7]

[Brief description of drawings]

1 is an ultraviolet absorption spectrum of borrelidin obtained in Example 1. FIG.

2 is an infrared absorption spectrum of borrelidin obtained in Example 1. FIG.

FIG. 3 is a graph showing the effect of borrelidin on the angiogenic potential of the human colon cancer WiDr cell line subcutaneously in mice performed in Example 3.

FIG. 4 is a graph showing the antitumor activity effect of borrelidin on human prostate cancer cell line PC-3 cells subcutaneously in mice carried out in Example 5.

[Procedure amendment]

[Submission date] January 8, 1996

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction content]

[0008]

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 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshiaki Naruse 7-55-5 Minami Ushiku, Ibaraki Prefecture (72) Inventor Yumi Yokoyama 1510-38 Ohata, Shinji-mura, Shinji-gun, Ibaraki (72) Inventor Yoshio Watanabe Kanagawa 2-22-3 Fujigaoka City, Fujisawa Prefecture (72) Inventor Kazuhiko Okamura 3-9-26 Shirahata, Fujisawa City, Kanagawa Prefecture

Claims (3)

[Claims] 1. An angiogenesis inhibitor comprising Borrelidin or a salt thereof as an active ingredient. 2. An anti-solid cancer agent containing Borrelidin or a salt thereof as an active ingredient. 3. Streptoms Roche
yces rochei) Mer-N 7167 (FERM P-14670) is cultured in a nutrient medium, and borrelidin is collected from the culture solution.