JPH0892272A - Therapeutic substance fr177391 for cancer cachexy - Google Patents

Abstract

PURPOSE: To obtain a new substance FR177391, capable of manifesting lipoprotein lipase activities and useful as a therapeutic agent for cancer cachexy, an antitumor agent, etc. CONSTITUTION: This substance NF177391 has the following physical properties: properties: light-yellow powder; specific rotatory power [α]D: +32 deg. (c=0.85; methanol); molecular weight: 470.95, fast atom bombardment-mass spectrometry (FAB-MS)m/z 547 (M+2K-H); infrared absorption spectrum: β KBr max 3400, 2970, 2950, 2930, 1735, 1650, 1570, 1440, 1300, 1230, 1180, 1150, 1060, 1020 and 970cm<-1> ; ultraviolet absorption spectrum: 206 and 203 (sh) nm; solubility: readily soluble in methanol, ethanol, chloroform and acetone, sparingly soluble in water and ethyl acetate and insoluble in hexane; color reaction: positive to the iodine vapor reaction, cerium sulfate-sulfuric acid and the Ehrlich reactions and negative to the Molisch, ninhydrin and ferric chloride reactions; division of acidic and basic: an acidic substance. Furthermore, the substance is obtained by culturing a microorganism, belonging to the genus Serratia and capable of producing the substance FR177391 (FERM P-14475) under aerobic conditions.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a FR177391 substance, a process for producing the same and uses thereof. FR177
The substance 391 is a novel substance that has not been heretofore known and separated and collected from a culture of microorganisms, particularly bacteria, and exhibits an excellent lipoprotein lipase (hereinafter also referred to as LPL) activity recovery action, and is a therapeutic agent for cancer cachexia. It is also useful as various medicines based on the action such as anticancer agents.

[0002]

2. Description of the Related Art Cachexia, also called cachexy, is currently used as a synonym for aggravation that causes a weakened general condition in the cases of tuberculosis, hemophilia, carcinoma, syphilis and the like. And, in particular, cancer cachexia has received clinical attention from the viewpoint of QOL. With cachexia, cancer-bearing patients often suffer from weight loss, anemia, dry skin, edema or anorexia, side effects of chemotherapy and radiation therapy, and secondary immunity due to malnutrition. Infected more easily.

As a drug for preventing and treating such cachexia, an effective screening method has not been established. Therefore, no specific medicine has been developed, which is an excellent drug in the art. The development of liquid therapy is strongly desired.

[0004]

DISCLOSURE OF THE INVENTION The present invention has been made in order to meet such demands in the art, and was made for the purpose of developing a novel and excellent cachexia therapeutic drug. is there.

[0005]

[Means for Solving the Problems] In order to achieve the above object, as a result of examinations from various aspects, first, in patients with cachexia, lipoprotein lipase (L
It was noted that the PL) activity decreased in good correlation with the degree of weight loss.

On the other hand, looking at the LPL activity at the cellular level, 3T3L ₁ cells showed dexamethasone (1 μm).
M), iso-butyl-methyl-xanthine (0.5 m
M) and insulin (10 μg / ml) are added to the cells for culturing to differentiate into adipocytes and induce LPL activity.
In the process, addition of BCG and then LPS-administered rabbit serum (final concentration 2%) suppresses LPL activity expression.

Focusing on these points, the present inventors have developed a screening method for a cachexia therapeutic agent utilizing this system. That is, BCG was added to LPL activity-inducing cells.
(Membrane fraction of Mycobacterium tuberculosis) Then LPS
(E. coli-derived toxoid) -administered rabbit serum was added to suppress LPL activity expression, and then a test sample was added to restore LPL activity, which was recovered to the level to which normal rabbit serum was added. We have succeeded in developing a method for screening as a proper one.

Furthermore, the LPL activity can also be measured by observing the degree of differentiation into adipocytes by a microscope and, if necessary, quantifying the LPL activity by a colorimetric method.
Also confirm that it can be done easily and accurately,
The screening system was completed completely.

On the other hand, the inventors of the present invention continued to search for fermentation products of microorganisms, and newly isolated No. 1 from the soil obtained from New Zealand. When the substance separated and extracted from the culture of the 1821 strain was examined by the above screening method, a new finding was obtained that it has an excellent effect of restoring (or antagonizing) the suppression of LPL activity. After studying the properties in detail, it was confirmed that the substance was a novel substance that was previously unknown, and this substance was newly named FR177391 substance,
As a result of further research, the industrial manufacturing method was established and the present invention was completed.

The FR177391 substance according to the present invention is a novel substance having the physicochemical properties shown in Tables 2, 3 and 4 below.

[0011]

[Table 2]

[0012]

[Table 3]

[0013]

[Table 4]

The FR177391 substance according to the present invention is a No. 1 newly isolated from a soil sample collected in New Zealand, for example. Produced by 1821 strains. No. Although the mycological properties of the 1821 strain are shown, taxonomic studies were mainly conducted according to the methods described in the following references 1) and 2). 1) Krieg, NR and Holt, JG ed .: Bergey's Manua
l of Systematic Bacteriology, Vol.1 Williams and Wi
lkins company, Baltimore, 1984 2) Cowan, ST ed .: Manual for the Identification
of Medical Bacteria, Cambridge University Press, 1
974

(1) Morphological characteristics The strain of this strain was placed on a nutrient agar medium at 30 ° C.
After growing for 24 hours, the morphology was observed with an optical microscope and an electron microscope. The results are shown in Table 5 below.
As is clear from the results, this strain was Gram-negative,
It is a motile bacterium. Colonies of this strain on nutrient agar are smooth, pale yellow, circular, e.
It was ntire-edged. No spore formation was observed. The cells have a size of 0.5 to 0.8 × 1.0 to 2.0 μm.

[0016]

[Table 5]

(2) Physiological characteristics Physiological characteristics of this strain are shown in Tables 6 to 8 below.

[0018]

[Table 6]

[0019]

[Table 7]

[0020]

[Table 8]

As is clear from the above results, the growth temperature range of this strain was 3 ° C to 34 ° C. This strain is positive for all tests of catalase, assimilation of citric acid, reduction of nitrate, hydrolysis of escarin, ONPG test, DNase, VP test, gelatin liquefaction, Tween80 hydrolysis, casein hydrolysis and lysine decarboxylase. Met. The OF test of this strain was fermentative. Oxidase activity, indole formation, and starch degradation were all negative. D-glucose, D-xylose, D
-Fructose, D-mannitol, D-sorbitol, D-mannose, D-trehalose, glycerin,
Acid production was observed from inositol, sucrose and salicin. In addition, D-glucose, D-xylose, D-fructose, D-mannitol, D-sorbitol, D-mannose, D-trehalose, glycerin, inositol, sucrose, and maltose were assimilated.

(3) The same features as the above are described in Bergey's Manual of Systematic Bacterio
As a result of searching from logy (Vol.1), No. The 1821 strain is considered to belong to Serratia, and this strain was designated as Serratia liquefaciens (Serratia).
ratio liquefaciens) No. 182
It was identified as No. 1 and was deposited as FERM P-14475 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry (deposit date: August 18, 1994).

It should be understood that the production of FR177391 material is not limited to the use of the particular microorganisms described herein merely for purposes of illustration. This invention includes artificial mutants and natural mutants that can be obtained from the microorganisms described by X-ray irradiation, ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine and the like. FR177391
It also includes the use of all mutants capable of producing the substance.

The FR177391 substance of the present invention is inoculated into a nutrient medium containing carbon and nitrogen sources capable of assimilating the substance-producing bacterium belonging to the bacterium (for example, No. 1821 strain),
It can be produced by culturing under aerobic conditions (eg, shaking culture, aeration and stirring culture).

As the carbon source, glucose, sucrose, starch, modified starch, fructose, glycerin and other carbohydrates are preferably used.

As the nitrogen source, oatmeal, yeast extract, peptone, gluten meal, cottonseed flour, cottonseed oil meal, soybean flour, corn steep liquor, dried yeast, wheat germ,
Peanut flour, chicken bone meat meal and the like are preferably used, but inorganic and organic nitrogen compounds such as ammonium salts (for example, ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.) urea and amino acids can also be advantageously used.

It is advantageous to use these carbon sources and nitrogen sources in combination, but it is not always necessary to use pure ones. This is because an impure substance may include a growth factor or a trace element, which may be advantageous.

If necessary, the following inorganic salts may be added to the medium; sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate, sodium chloride, potassium chloride, sodium iodide, iodine. Potassium chloride, magnesium salt, copper salt, cobalt salt and the like.

In particular, if the medium strongly foams, liquid paraffin, animal oil, vegetable oil, mineral oil, silicone or the like may be added, if necessary.

For large-scale industrial production of the target substance, it is preferable to carry out aeration-agitation culture as in the case of other fermentation products. For small-volume production, shake culture using a flask is preferable. Also, when culturing in a large tank,
In order to prevent the growth delay of the bacteria in the production process of the FR177391 substance, it is preferable to first inoculate and culture the produced bacteria in a relatively small amount of medium, then transfer the culture to a large tank and perform the production culture there. In this case, the composition of the medium used for the pre-culture and the composition of the medium used for the production culture may be the same or may be different if necessary.

The culture is preferably carried out under aeration and stirring conditions,
For example, known methods such as stirring with a propeller or other machine, rotation or shaking of a fermenter, pumping, blowing of air are appropriately used. Use sterilized air for ventilation.

The culturing temperature may be appropriately changed within the range in which the FR177391 substance-producing bacterium produces this substance, but it is usually 1 to 40 ° C., preferably 23 to 30 ° C. The culturing time varies depending on the culturing conditions and the culturing amount, but is usually about 1 day to 1 week.

After completion of fermentation, the desired FR1 is obtained from the culture.
77391 material is recovered. That is, the cells may be directly extracted with water and / or an organic solvent, or may be disrupted mechanically or using known means such as ultrasonic waves, and then extracted with water and / or an organic solvent, followed by a conventional method. Collect and purify according to In the case of a culture solution, it may be directly recovered and purified by a conventional method.

As a method of recovery and purification, for example, water extraction, organic solvent, solvent extraction with a mixed solvent thereof; chromatography; recrystallization from a single solvent or a mixed solvent, or a conventional method is appropriately used alone or in combination. it can.

The FR177391 substance is recovered and purified by appropriately utilizing the known method as described above. For example, the following method may be used. The culture is extracted with acetone, then with ethyl acetate and then purified by chromatography. The FR177391 substance is an acidic substance and can react with a base to form a salt. The FR177391 substance can be recovered and purified in the free state (FR177391 substance itself), or can be recovered and purified as a salt thereof. Further, these can be exchanged with each other by a conventional method. Examples of salts of the FR177391 substance with a base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt.

The FR177391 substance of the present invention has been confirmed to have an excellent antagonism (recovery) action against LPL activity inhibition, as well as to be clear from the following description, and has low toxicity and high safety. And LPL
It is useful as an antagonist (restoring) agent for suppressing activity and is extremely effective as a cachexia therapeutic agent and an antitumor agent.

Therefore, the substance according to the present invention can be used as an active ingredient of medicines, and such medicines are also included in the present invention. The pharmaceutical composition according to the present invention comprises a FR177391 substance and / or a salt thereof as an active ingredient in addition to an inorganic or organic carrier which is commonly used,
In the solid, semi-solid or liquid form, in addition to oral administration agents, it is formulated into parenteral administration agents such as external preparations.

Preparations for oral administration include tablets, pills, granules, soft and hard capsules, powders, fine granules, powders,
Examples thereof include emulsions, suspensions, syrups, pellets and elixirs. Formulations for parenteral administration include
Injection, drip, infusion, ointment, lotion, tonic,
Sprays, suspensions, oils, emulsions, suppositories and the like can be mentioned.
To formulate the active ingredient of the present invention, a conventional method may be followed, including a surfactant, an excipient, a coloring agent, a flavoring agent, a preservative,
Stabilizers, buffers, suspensions, isotonic agents and other commonly used adjuvants are used as appropriate.

The dose of the pharmaceutical composition according to the present invention varies depending on its type, type of disease to be treated or prevented, administration method, age of patient, symptom of patient, treatment time, etc.
In the case of intravenous administration, the active ingredient (FR
177391 or / and its salt) 0.01-10
00 mg / kg, preferably 0.1-100 mg / kg
In the case of intramuscular administration, 0.01 to 1000 m
g / kg, preferably 0.1 to 100 mg / kg, and in the case of oral administration, the dose is 0.5 to 2000 mg / kg, preferably 1 to 1000 mg / kg.

Hereinafter, the present invention will be described in more detail with reference to Examples.

[0041]

[Example 1: Fermentative production of FR177391]

(1) Culture 120 ml of a seed medium having a composition of yeast extract (0.5%), polypeptone (1%) and sodium chloride (0.5%) was placed in each of 500 ml Erlenmeyer flasks.
Add each and sterilize at 120 ° C. for 30 minutes. Se to them
ratioliquefaciens No. 182
Slope cultures of 1 strain (FERM P-14475) were inoculated with 1 platinum loop each, and shake-cultured at 250 rpm for 30 minutes at 30 ° C. on a rotary shaker. Glycerin (2%), glucose (1%), corn steep liquor (1%), defatted soybean flour (1%), meat extract (0.2%), ammonium sulfate (0.2%), magnesium sulfate 7 100 L of medium (pH 7.0) having a composition of hydrate (0.01%) and calcium carbonate (0.2%) was injected into 5 units of 30 L jar fermenter, 20 L each, and sterilized at 120 ° C. for 30 minutes. And then add 3 to the above culture.
Inoculate 60 ml each, and incubate for 3 days at an aeration rate of 1 VVM, a stirring speed of 200 rpm, and a culture temperature of 25 ° C.

(2) Purification After completion of the culture, an equal amount of acetone is added to the culture (100 L) and extraction is carried out for 1 hour at room temperature. Add 4 kg of diatomaceous earth and filter. The obtained filtrate (160 L) is concentrated under reduced pressure to 100 L. After adjusting the pH of this concentrate to 7, an equal amount of ethyl acetate is added and extraction is performed. The extraction residue is re-added with an equal volume of ethyl acetate and the extraction is repeated. The obtained extract is concentrated under reduced pressure, and the obtained oily substance (30 g) is subjected to silica gel chromatography (1 L). This column was mixed with n-hexane (3 L), a mixture of n-hexane and ethyl acetate (1: 1; v / v) (3 L), ethyl acetate (3 L), ethyl acetate and acetone (1: 1; v / v).
v) mixed solution (3 L), washed with acetone (3 L), then mixed solution (3: 1) of acetone and methanol (1: 1; v / v)
L) elutes.

The active eluate is concentrated under reduced pressure and the resulting oily substance (2.6 g) is again subjected to silica gel chromatography (100 ml). The column is filled with dichloromethane (30
0 ml), dichloromethane and methanol (50:
1; v / v) mixture (300 ml), dichloromethane and methanol (20: 1; v / v) mixture (300 m)
l), dichloromethane and methanol (10: 1; v
/ V) mixed solution (300 ml), followed by a mixture of dichloromethane and methanol (5: 1; v / v) (300
ml), followed by dichloromethane and methanol (2:
1; elute with a mixed solution (300 ml) of v / v). The active eluate was concentrated under reduced pressure, and the obtained oily substance (180 mg) was dissolved in 10 ml of methanol. This methanol solution is applied to a column (350 ml) packed with a reverse phase resin “YMC ODS-AM” (trademark, manufactured by YMC) (ODS-AM 120-S-50 type). The column was loaded with 35% aqueous acetonitrile-50 mM phosphate buffer (pH 7.
0), the active fraction is concentrated under reduced pressure, the solvent is removed, and the mixture is extracted with ethyl acetate. The obtained extract is concentrated under reduced pressure to obtain 102 mg of a coarse powder.

The crude powder is subjected to silica gel chromatography (10 ml). This column was loaded with n-hexane
ml), n-hexane and acetone (1: 1; v /
v) mixed solution (30 ml), acetone (30 ml), mixed solution of acetone and methanol (3: 1; v / v) (30
ml) and then acetone and methanol (1:
1; elute with a mixture of v / v) (30 ml) and then methanol (30 ml). The active fraction is concentrated under reduced pressure, and 36 mg of the obtained crude powder is dissolved in 1 ml of methanol. This methanol solution is a reverse phase resin "YMC ODS-AM"
(Trademark, manufactured by YMC) (ODS-AM 120-S-5
HPLC column AM-303 (i.
d. 250 × 4.6 mm). The column was loaded with 35% aqueous acetonitrile-50 mM phosphate buffer (pH 7.
0), the active fraction is concentrated under reduced pressure, the solvent is removed, and the mixture is extracted 3 times with ethyl acetate. The obtained extracts are combined, concentrated under reduced pressure, and dried to obtain FR177391 substance powder (25 mg).

[0046]

Example 2: Biochemical properties of FR17731

(1) Antagonistic (recovery) action against suppression of LPL activity of 3T3L ₁ cells by rabbit serum (2%) administered with BCG and LPS ”FR1773 against suppression of LPL activity as follows
The antagonism or recovery of 91 substances was measured.

I) Method BCG and LPS-administered rabbit serum: White rabbit New Z
40 m of dry BCG (Japan BCG Manufacturing Co., Ltd.) for percutaneous injection from ear vein of ealand White (12 weeks old, female)
g (/ ml / head) was administered, and after breeding for 2 weeks, LPS (E. coli B8, D) was further administered through the ear vein.
Ifco) 100 μg (/ ml / head) is administered. 1 hour and 30 minutes after administration, whole blood was collected from the rabbit ear vein, and the blood was allowed to stand at 37 ° C. for 2 hours and then centrifuged (3
A serum fraction is obtained at 000 rpm, 10 min).
Serum was inactivated by heat treatment at 56 ° C. for 30 minutes, and then sterilized by filtration with a Millipore filter and used for the experiment.

3T3L ₁ cells subcultured in an F75 flask (Corning) using MEM medium (10% fetal bovine serum FBS added) were trypsinized to obtain a cell suspension of 2 × 10 ^4. A 96-well microtiter plate was seeded so as to have cells / well. 37
Cultivation was carried out at 5 ° C in a 5% CO ₂ incubator for 2 days.
After 2 days of culturing, the culture medium (MEM + 10% FBS) was extracted, and 100 μl of a differentiation-inducing medium (MEM medium containing dexamethasone 1 μM, iso-butyl-methyl-xanthine 0.5 mM and insulin 10 μg / ml) was newly added and further cultured. Continued. After 2 days of culturing (4th day of culturing), the culture medium was removed and the rabbit serum (1.
Sample liquid 1 diluted with MEM medium containing 5 to 2%)
After adding 00 μl and culturing for another 5 days, 3T3L ₁
The degree of differentiation of cells into adipocytes was measured by quantifying LPL activity or by observing the degree of cell differentiation.

By this measurement, it became possible to examine the recovery effect of the sample on the suppression of LPL activity expression associated with the differentiation of 3T3L ₁ cells into adipocytes by the rabbit serum administered with BCG and then LPS.

Measurement of LPL activity: The LPL activity was obtained by freeze-thawing LPL 96-well cells and eluting the cells with heparin from the cells to obtain an enzyme solution (10 μl), which was added to bovine serum albumin (2%). Ammonium sulfate (0.05%) and triglyceride emulsion (1%) as a substrate were added, and FFA was quantified by the NEFAC Test Wako kit using a total reaction volume of 50 μl to measure LPL activity.

Ii) Results 3T3 with rabbit serum administered BCG followed by LPS
The antagonistic (recovery) action concentration ED ₅₀ : ng / ml of the FR177391 substance for the suppression of LPL activity expression associated with the differentiation of L ₁ cells into adipocytes was as follows. FR177391 substance: 0.01 to 0.02

(2) Antagonism of suppression of LPL activity of 3T3L ₁ cells by TNF-α i) 3T3L ₁ cells were cultured according to the method of (1) above, and after addition of a differentiation induction medium, human TNF-α (2
5 ng / ml) was added to the sample diluted with MEM medium to suppress differentiation, and the cells were further cultured for 5 days to obtain TNF-
The degree of recovery of α-induced inhibition of 3T3L ₁ cell differentiation into adipocytes was measured.

Ii) Results The antagonistic (restoring) action concentration (ED ₅₀ : ng / ml) of the FR177391 substance for suppressing LPL activity was as follows. FR177391 substance: 0.5 to 1.0

(3) Antitumor activity (in vitro)
(Cytotoxicity) FR177391 human breast adenocarcinoma (MCF-7), human lung adenocarcinoma (A549), human T cell leukemia (Jurka)
t), human histiocytic lymphoma (U937), SV40-transformed human fibroblasts (293SV40), mouse leukemia cells (P388), and a control for cell growth inhibitory activity against mouse bone marrow cells were examined. Although the activity assay was performed by a conventional method, the culture was started at a cell concentration of 5 × 10 ³ / well, and the number of viable cells was quantified by the MTT method on the 4th day. However, for mouse bone marrow cells, 1.5 × 10 ⁴ / wel
A cell concentration of 1 was used. The results are summarized in Table 9.

[0056]

[Table 9]

As is clear from the above results, cancer cells MCF-7, Jurkat, U937, 293SV4
The FR177391 substance shows excellent antitumor activity against 0 and P388.

(4) Toxicity test FR177739 was added to 5 ICR female mice aged 5 weeks.
The substance was intraperitoneally injected once daily at a dose of 5 mg / kg for 3 consecutive days, but there were no deaths and weight gain was exactly the same as in the non-administered mouse group, confirming the high safety of FR177391 substance. It was

[0059]

[Example 3: Production of injection] (1) FR177391 substance produced in Example 1 5g (2) Salt 9g (3) Sodium hydrogencarbonate 1g

All the components (1) to (3) were added to 100 m of distilled water.
After dissolving in 1 l, 1 ml each was dispensed into ampoules to manufacture 100 injections.

[0061]

INDUSTRIAL APPLICABILITY The present invention provides a FR177391 substance, which is a novel substance which has hitherto been unknown, and which exhibits an excellent antagonism or recovery action against LPL activity inhibition and is useful for treating cachexia of cancer. It can be used for various pharmaceuticals as an LPL activity recovery pharmaceutical preparation such as an agent and an antitumor agent.

[Brief description of drawings]

FIG. 1 is a chart showing ¹ H nuclear magnetic resonance spectrum of FR177391 substance.

FIG. 2 is a chart showing a ¹³ C nuclear magnetic resonance spectrum of FR177391 substance.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI technical display location C12R 1: 425) (72) Inventor Hiro Terano 2600-3 Manabe, Tsuchiura City, Ibaraki Prefecture

Claims (6)

[Claims] 1. A FR177391 substance having the physical properties shown in Table 1 below. [Table 1] 2. A method for producing a FR177391 substance, which comprises culturing a FR177391 substance-producing bacterium to produce the FR177391 substance, and collecting the FR177391 substance. 3. The production method according to claim 2, wherein the FR177391 substance-producing bacterium is a microorganism belonging to the genus Serratia. 4. A pharmaceutical preparation for recovering lipoprotein lipase (LPL) activity, which comprises FR177391 substance as an active ingredient. 5. The pharmaceutical preparation according to claim 4, wherein the pharmaceutical preparation for recovering LPL activity is a therapeutic agent for cachexia of cancer. 6. The pharmaceutical preparation according to claim 4, wherein the LPL activity-restoring pharmaceutical preparation is an antitumor agent.