JPS63183536A - Antitumor agent - Google Patents

Abstract

PURPOSE:To obtain an antitumor agent useful for treating malignant tumor, having antitumor activity, immune enhancing activity, safety in terms of toxicity to human body and side effects, rapidly showing medicinal effects, containing a polysaccharide comprising galactosamine as an active ingredient. CONSTITUTION:An antitumor agent having a polysaccharide comprising galactosamine (e.g. galactosamine polymer produced by Aspergillus parasticus) as an active ingredient. The antitumor agent shows excellent antitumor activity even with a small amount of does and a few times of administration. The antitumor agent is effective against tumor cell of sarcoma 180, Ehrlich carcinoma, etc. A dose is 0.1mug/kg-100mg/kg, preferably 0.1mug/kg-100mug/kg daily per adult.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a novel antitumor agent containing a polysaccharide consisting of galactosamine as an active ingredient. The antitumor agent of the present invention is particularly useful as a therapeutic agent for malignant tumors.

(Prior Art) Conventionally, as a polysaccharide consisting of galactosamine, Aspergillus balaciticus (Aspergillus spar.
galactosamine polymer produced by P. asticus [Journal of Biological Chemistry (
Journal of Biological Che
mistry) Vol. 235, pp. 2538-2541, 1
969] and Paecilomyces
A bacterial strain belonging to the genus Benicillomyces (Paeci)
PF-101 (Japanese Patent Publication No. 12639/1983, Agricultural and Biological Chemistry)
logical chemistry) Volume U, No. 31
59-3164, 1985], etc. are known.

It is also known that PF-101 has a flocculating activity useful as a flocculant [Japanese Patent Publication No. 12639/1983, Agricultural and Biological Chemistry]
Logical Chemistry) Volume 49, No. 31
51-3164, 1985].

Furthermore, as a polysaccharide with antitumor activity, lentinan (Japanese Patent Publication No. 47-3
No. 7002, No. 49-484), PSK (trade name Krestin), a protein polysaccharide whose main chain is β-(1→4) bonds of glucose extracted from C. versicolor
(Japanese Patent Publications No. 55-23271, No. 57-40159), and N-acetylglucosamine is β-(1→4
) bound polysaccharide chitin (JP-A-59-27826
Publication No.) etc. are known.

(Problems to be Solved by the Invention) However, although antitumor agents containing polysaccharides as active ingredients have excellent safety with few side effects, It is still not fully satisfactory as an anti-tumor agent because of the large amount of ingredients administered and the number of times it is administered, and there has been a desire to develop an anti-tumor agent that is even safer and more effective.

(Means for Solving the Problems) The present inventors have conducted extensive research in order to solve the problems of conventional anti-tumor agents containing polysaccharides as active ingredients and to provide even better anti-tumor agents. As a result of repeated studies, it was discovered that a polysaccharide consisting of galactosamine unexpectedly has antitumor activity and immunoenhancing activity, and that it also shows excellent antitumor agent activity even when the dose and frequency of administration are reduced.The present invention I was able to complete it.

The polysaccharide composed of galactosamine used as an active ingredient of the antitumor agent of the present invention is a substance that can be described as polygalactosamine, and a polymer formed by binding galactosamine molecules to each other through α-(1→4) bonds. The amino group of the galactosamine moiety of these polysaccharides may be partially acetylated.

As an example of the polysaccharide consisting of galactosamine used in the present invention, specifically, the Journal of Biological Chemistry (Journal of Biological Chemistry)
cal Chemistry) Volume 235 No. 2538-
2541, (1969).
galactosamine polymer produced by Agricultural and Biological Chemistry (Agr.
cultural and biologicalC
hemistry) Vol. 49, pp. 3159-3164 (
1985)
Known bacterial strains belonging to the genus Benicillomyces s
p, l-1 (FERN-P No. 39
Polysaccharide PF-1 consisting of galactosamine produced by 28)
01 and PF-102, and the like.

The above PF-101 is a known substance, but PF-10
2 is a new substance disclosed in Japanese Patent Application No. 134799 (Applicant: Higeta Soy Sauce Co., Ltd., filed on June 12, 1986), and is produced from galactosamine by the method described below and used in Rikaku as described below. At least one polysaccharide such as polygalactosamine, PF-101, PF-102, etc. is blended with a known pharmaceutically acceptable liquid or solid carrier in a conventional manner, and if necessary, a solvent, dispersant, emulsifier, etc. It is used by adding buffering agents, stabilizers, excipients, binders, disintegrants, lubricants, etc. to formulate injections, tablets, granules, powders, powders, capsules, etc.

The antitumor agent of the present invention can be formulated as an injection and administered intravenously, and can also be administered subcutaneously, subcutaneously, intramuscularly, or intraperitoneally. In addition, the solution can be formulated as a tablet, granule, powder, or powder and used by oral administration.6 Tumor cells to which the antitumor agent of the present invention is effective include, for example, Sarcoma 180, Ehrlich carcinoma, Leukemia L-1210, Leukemia P-3゛88, Nto46, Math-A, Lewis
Examples include experimental tumors such as s lung cancer and BC47 bladder cancer, X-5536 plasmacytoma, AC-755, and B-16.

Furthermore, the dosage of the active ingredient of the antitumor agent of the present invention is 0.1 μg/kg to 100 μg/kg per day for adults. mg/kg preferably in the range of 0.1 μg/kg to 100 μg/kg.

(Actions and effects of the present invention) Antitumor f! of the present invention! 1M acts via the immune system and has an immune-enhancing effect.

The antitumor agent of the present invention has practically no toxicity or side effects to the human body, is extremely safe, is easy to formulate and administer as an injection, and has a long expression of medicinal efficacy. Furthermore, it has excellent advantages such as exhibiting antitumor activity with an extremely small dose of active ingredient.

Since the above-mentioned PF-102, which can be used as an active ingredient in the antitumor agent of the present invention, is a new substance, the physical properties and manufacturing method of this substance will be explained below.

PF-102 dissolves in aqueous solutions of almost all acids and forms acid salts. Although it is possible to isolate PF-102 as a free basic substance, it is difficult to handle because it is sparingly soluble in water. Therefore, among the physical and chemical properties of PF-102, the hydrochloride of PF-102 was used to measure aggregation activity, aggregation activity pH range, aggregation activity temperature range, ultraviolet absorption spectrum, and color reaction.

PF-162 is a substance containing an amino group and has the following physical and chemical properties.

(1) Agglomerating activity: Even when added in an extremely small amount, suspended fine particles in water are agglomerated.

(2) Aggregation activity pH range: Stably exhibits aggregation activity at p82-9.

(3) Aggregation activity temperature range; aggregation activity is observed at 0 to 100°C.

(4) Coagulation active ionic strength; carbonic acid and Fe2 (SO4
)3 inhibits the aggregation activity of PF-102, but other various ions and ionic strengths have no effect on the aggregation activity, and NaCl and K2SO4 do not affect IM at all.

(5) Elemental analysis: Nitrogen 8.64%, carbon 42.80%,
Hydrogen 6.87% General formula: (CGH11NO4-XI20) (6) Ultraviolet absorption spectrum; as shown in Figure 1 of the attached drawings.

(7) Infrared absorption spectrum; as shown in Figure 2 of the attached drawings.

(8) Color reaction; ninhydrin reaction 2;: dexanthoprotein reaction 21 Ehrlich reaction 2 - Morisch reaction 2 - Phenol-sulfuric acid method: ± Relent test: - (9) Electrophoresis; density gradient isoelectric point It was confirmed as a single substance by electrophoresis, and its isoelectric point (pI) is 8.5.

(10) Color of substance; light brown (11) Distinction between basic, acidic, and neutral; pH when suspended in water at a concentration of 0.5% w/v is 7.
5 (pH 5.8 of deionized water).

(12) Solubility in solvents: Slightly soluble in hot water, slightly soluble in cold water, but resistant to dilute acid aqueous solutions. Slightly soluble in dilute alkaline hydroxide solution, alcohols,
Insoluble in acetone, chloroform, benzene, and n-pentane.

(13) Average molecular weight: 160,000 or more As the acid salt of PF-102, phosphate, hydrochloride, acetate, lactate, citrate, etc. are preferable. The infrared absorption spectrum of the hydrochloride of PF-102 is shown in FIG.

The new substance PF-102 was published in Japanese Patent Publication No. 56-12639 and Agricultural and Biological Chemistry.
Logical Chemistry) Volume 49, No. 31
59-3164 (1985), a known strain belonging to the genus Paecilomyces (Paecilomyces sp.
! It can be produced by culturing RM-P 3928; which has the mycological properties described in Japanese Patent Publication No. 56-12639 and can be distributed by Kaikoken) using a conventional liquid culture method for filamentous fungi.

That is, Benicillomyces sp. I-1 strain (FE
RM-P Nα3928) spores or mycelium are inoculated into a liquid medium and cultured aerobically to form PF-
102 will be produced. The carbon source of the medium is glucose,
Maltose, sucrose, starch, blackstrap molasses, etc. can be used, but glucose is preferably used.

As the nitrogen source, inorganic nitrogen sources such as ammonium sulfate and sodium nitrate, and organic nitrogen sources such as peptone and yeast extract can be used.

Although the culture temperature can be changed as appropriate within the range in which the PF-102-producing bacteria produce this substance, it is usually preferable to culture at 20 to 25°C. Culture time varies depending on culture conditions, but
It usually takes about 4 to 5 days, and it can be finished at an appropriate time by estimating the time when the accumulation of this substance reaches its maximum. If the culture filtrate of the culture solution obtained here is concentrated by a method such as vacuum concentration or ultrafiltration, and the concentrated liquid is precipitated by adding an organic solvent such as ethanol, the known method described in Japanese Patent Publication No. 56-12639 can be obtained. The substance PF-101 is obtained.

PF-102 can be obtained by adding various salts to the above culture filtrate or filtrate concentrate and precipitating by salting out, separating the precipitate, washing with water, and dissolving it in a dilute acid aqueous solution. , by adding salt again or by adding alkali etc.
Highly purified PF-102 can be obtained by precipitation with H of 7 to 9 or more.

The salt added to the solution containing PF-102 may include one or more of the following salts.

Namely, chlorides such as potassium chloride, sodium chloride, calcium chloride, and ammonia chloride; nitrates such as potassium nitrate and sodium nitrate; acetates such as sodium acetate; sulfates such as dipotassium sulfate, ammonium sulfate, calcium sulfate, and copper sulfate; Examples include phosphates such as dipotassium phosphate, monopotassium phosphate, disodium phosphate, and monosodium phosphate.

Any amount of salt can be added as long as it is in a dissolved state, but it is preferable to add 0 to PF-102 containing liquid.
．． It is about 5 to 50%, more preferably about 2 to 40%.

Depending on the type of salt added, the pH of the solution may be 7 or higher, so in this case, PF
-102 is precipitated, so it is sufficient to separate the precipitate.

If the pH is below 7 even after adding salt, it is necessary to use an alkali such as sodium hydroxide to adjust the pH to 7 to 9, preferably around 8 or 5, which is the isoelectric point. be.

By adding salt to the PF-102-containing solution and adjusting the pH to 7 to 9, PF that did not easily precipitate due to interference from impurities can be removed.
-1,02 causes precipitation and separates from impurities. This precipitate can be separated by centrifugation or filtration with a filter cloth.

Since the separated precipitate contains a large amount of salt, it is washed with water or a solvent to desalt it, and then dissolved in an acid.

The acid may be any acid such as an organic acid such as acetic acid or an inorganic acid such as hydrochloric acid, and the concentration is preferably about 0.01 to 3 mol. After dissolving the precipitate in acid in this way, the precipitate will be easily precipitated only by treatment near the isoelectric point of pH 7 to 9, so add an alkali such as sodium hydroxide and adjust the pH to 7 to 9, preferably The pH was adjusted to pH 8.5 to obtain a precipitate.

Furthermore, in order to purify this precipitate, wash it with water etc.
A precipitate can be obtained by dissolving in acid again and adjusting the pH to 7 to 9. This purification treatment can be repeated any number of times, and when the purification is completed, the precipitate becomes almost pure and PF-102 is obtained.

(Example) In the present invention, the above-mentioned PF used as an active ingredient
Production examples of -101 and PF-102 are shown below in Reference Example 1 and Reference Example 2.

Reference Example I Production of PF-101 PF-101 was produced in the following manner according to Example 4 of Japanese Patent Publication No. 12639/1982. 300g of glucose, 45g of casamino acids in a 20fl jar fermenter,
110 g of CaO was added and adjusted to pi (6.0) with hydrochloric acid.

After that, the final liquid volume was adjusted to] 5Q, heat sterilized by heating at 120°C for 15 minutes, and Venicillomyces I cultured in a 500mQ Erlenmeyer flask with shaking in 100n+Q of G and Ye medium for 5 days.
-1 strain (FERM-P392g)' was inoculated and cultured with aeration for 5 days (aeration rate 10Q/min, 200r
, p, m). After culturing, the culture solution is filtered with a filter cloth, the culture filtrate is concentrated using a flash evaporator, and the concentrated solution is heated to 2.3Q.
Obtained. Next, an equal amount of ethanol (95%) was added to the concentrate, and the precipitate was collected by centrifugation, washed with 75% ethanol, and freeze-dried to obtain 5.2 g of a powder specimen.

Add 3g of the obtained powder sample to 0.5Ω bottled water,
The mixture was further heated for 15 minutes to dissolve it. Because there are some insoluble materials,
A slide was obtained after centrifugation. Based on the absorbance of this product at 490 mμ (A490) determined by the phenol-sulfuric acid method, the concentration is approximately 4 mg.
/mQ.

100 mR of this solution was gel-filtered through a Sepharose 6B column. First, impurities were eluted through a 1/40 molar Tris-HCl buffer, and then the target product was eluted with a 1740 molar Tris-maleic acid buffer.

Next, 250 mQ of ethanol was added to this to obtain a precipitate, and the precipitate was further washed three times with 75% ethanol and freeze-dried to obtain a freeze-dried specimen of polysaccharide PF-101 consisting of galactosamine.

Reference Example 2 Production of PF-102 Glucose 600g, Polypeptone 60g, CaCQ2
・Dissolve 125g of 2H20 in 17Q tap water and add concentrated Na
After adjusting the pH to 7.0 with an OH solution, the mixture was transferred to a 30Ω jar fermenter.

This medium solution was pressurized and heat sterilized (121°C, 20 minutes) by injecting steam, and the cooled medium (final liquid volume 20Q) was placed in a 500 mfl Erlenmeyer flask with 150
Medium with the same mQ composition (glucose 3%, polypeptone 0.
3%, CaCQ 20.5%, pH 7.0) at 26°C, 4
Benicillomyces I-1 strain (FER
M-P, No.

3928) was aseptically inoculated at a volume ratio of approximately 10%. After inoculation, the temperature was 27°C, the aeration rate was 5 VVM, and the number of stirring was 20 ORPM (71
The cells were cultured for 5 days under these conditions.

After the culture was completed, the culture was filtered through a filter cloth to obtain a culture filtrate Ill. While heating this culture filtrate to 50°C to 60°C, an ultrafiltration membrane (Mitsubishi Rayon,
The low molecular weight fraction was removed by passing through a UF membrane tubular module F type manufactured by Engineering Co., Ltd., and concentrated until the liquid volume was approximately 3Q. Furthermore, about 14,000
Cell residue and heat-denatured proteins were removed by centrifugation at XG.

After centrifugation, approximately 4.3 kg of table salt (approximately 2
5% concentration) was added and stirred, and after dissolving, the pH was adjusted to 7.0 to 8.5 with concentrated NaOH. - After leaving to stand overnight to extract ten samples of salted-out material, the salted-out material was collected on a cloth made of Saran (a copolymer of vinylidene chloride and vinyl chloride).

Furthermore, a large amount of slightly alkaline water (p) is poured over this salted-out product.
17.0 or higher) to wash away excess common salt, neutral sugars and other impurities mixed in the culture solution at the same time.

Next, approximately three times the volume of 0.1M hydrochloric acid solution was added to the salted out product after washing with water and dissolved. A concentrated NaOH solution was added to this solution to adjust the pH to 8.5, which is the isoelectric point of polygalactosamine. - After leaving it overnight to precipitate the ten-analyte precipitate, the precipitate was collected on Saran cloth in the same manner as above and washed with a large amount of tap water. This washed product was dissolved once again in 0.1M hydrochloric acid, subjected to isoelectric precipitation, and purified by repeated washing with water.

This purified precipitate was sterilized at 121° C. for 15 minutes and then freeze-dried to obtain 7 g of purified powder of polysaccharide PF-102 containing polygalactosamine as a main component (about 99% purity as polygalactosamine). □ Depending on the purpose, a part of the above purified powder may be added to 01.0%. Ultrafiltration membrane dissolved in M hydrochloric acid and having a molecular weight cutoff of 300,000 (molecular sieve membrane type XM manufactured by Amicon)
300) to separate those with an average molecular weight of 160,000 to 300,000 and those with an average molecular weight of 300,000 or more.

Next, formulation examples of the antitumor agent of the present invention will be explained.

Formulation Example 1 Polysaccharide PF- consisting of galactosamine obtained in Reference Example 1
Dissolve 0.25 g of 101 in heated distilled water for injections, and then add physiological saline for injections to make the entire volume 100.
An injection of 0.25% in mΩ was obtained.

Polysaccharide PF- consisting of galactosamine obtained in Reference Example 2
0.25gt for 10'2! -0,05N hydrochloric acid aqueous solution 90
+nQ and left to dissolve for 6 hours, then tris(hydroxymethyl)aminomethane (solubilizing agent) was added dropwise little by little while stirring to adjust the pH to 6.8, and then physiological saline for injection was added to the solution. Water was added to bring the total volume to 100n+Q to obtain a 0.25% injection.

Next, the antitumor activity and immune enhancing activity of the antitumor agent of the present invention will be illustrated by experimental examples.

Experimental Example I Antitumor effect test on MM-46 solid tumor 5 x 10' MM-46 tumor cells were implanted subcutaneously in the inguinal region of C3H/He5lc male mice (10 mice per group), and the tumor was transferred to the mouse. Seven days after the day of complete engraftment, that is, on the 14th day after tumor cell transplantation, administer an injection containing polysaccharide PF-102 made of galactosamine as an active ingredient prepared according to Formulation Example 2 at a dose of 1 μg/kg of the active ingredient. was administered into the tail vein of mice. On the 27th day after tumor cell transplantation, the tumor was removed, the tumor weight was measured, and the tumor inhibition rate was determined. Note that a control test was conducted in the same manner using an injection solution containing only physiological saline. The results obtained are shown in Table-1.

Experimental Example 2 Antibody Production Enhancement Test C3H/)leslc strain male mice (4-6 weeks old, 1
Immunization was performed by administering IX 108 sheep red blood cells per mouse as an antigen into the tail vein of a group of 4 mice, and at the same time, polysaccharide PF-102 consisting of galactosamine prepared according to Formulation Example 2 was administered as an active ingredient. active ingredient 1
After 4 days, the lungs were removed and the number of antibody-producing cells per lung was determined by Cunning.
Calculated by ham's plaque forming cell assay method. In addition, 45% of mice were also immunized with sheep red blood cells and simultaneously administered with physiological saline (control test).
After 6 days, the lungs were removed and the number of antibody-producing cells was calculated using the same plaque-forming cell measurement method as described above.The effect of enhancing antibody production was examined using the following formula.

However, T: number of antibody-producing cells in the lungs of mice administered with PF-102; C: number of antibody-producing cells in the lungs of mice administered with physiological saline.

Similarly, using different groups of mice, PF-10
On the 5th day, 100th day or 155th day after administration of PF-102, I x 10' sheep red blood cells were administered per mouse, and on the 4th day, that is, on the 9th day, 144th day and 199th day after administration of PF-102. Lungs from each eye were removed to examine the sustainability of the effect of enhancing antibody production.

Note that a control test was conducted in the same manner using an injection solution containing only physiological saline. The results obtained are shown in Table-2.

Table 2 Antibody Production Enhancement Effect Experimental Example 3 Cellular Immunity Enhancement Test C3H/He5lc male mice (4-6 weeks old, 4 mice per group) were subcutaneously injected with sheep red blood cells as an antigen at 1 x 10' per mouse. At the same time, polysaccharide PF-- consisting of galactosamine prepared according to Formulation Example 2 - was administered for immunization.
Injection containing 102 as active ingredient, 1μg/kg of active ingredient
Four days later, IX 10" sheep red blood cells were administered subcutaneously to the other mouse's paw as an antigen. One day later, swelling of the mouse's footprint was observed. The edema increase rate was calculated by measuring with a caliper, and the cell-mediated immunity enhancing effect was investigated.

Note that a control test was conducted in the same manner using an injection solution containing only physiological saline. The results obtained are shown in Table 3.

q Antitumor effect test of preperitoneal administration of PF-101 against MM-46 solid tumor Polysaccharide PF-101 consisting of galactosamine prepared according to Formulation Example 1 was administered into the peritoneal cavity of C3H/He5lc male mice (7 mice per group). An injection containing 101 as an active ingredient was administered three times a day, 5 days before, 3 days before, and 1 day before tumor cell transplantation, at a dose of 50 mg/jcg of the active ingredient per mouse. Next, on the day after the 3@ administration of PF-101, 1×10 5 Ato46 tumor cells were transplanted into the abdominal cavity of the mice, and the number of mice alive and dead was observed for 60 days. As a result, all seven of the seven mice administered with PF-101 were still alive 60 days after tumor cell transplantation.

In a control test conducted in the same manner using an injection containing only physiological saline, all seven out of seven mice died 28 days after tumor implantation.

Experimental Example 5 PF-101 against MM-46 solid tumor
Antitumor effect test of intraperitoneal administration of C3H/He5lc male mice (7 mice per group). 1 x 105 Hto46 tumor cells were transplanted and inoculated into the peritoneal cavity, and the next day, preparations were made according to Formulation Example 1. An injection containing the polysaccharide PF-101 consisting of galactosamine as an active ingredient was intraperitoneally administered to mice at a dose of 50 mg/kg of the active ingredient per mouse, once every other day for a total of 3 times. After tumor cell transplantation 6
The number of live and dead mice was observed for 0 days. As a result, all seven of the seven mice administered with PF-101 were still alive 60 days after tumor cell transplantation.

In a control test conducted in the same manner using an injection containing only physiological saline, all seven out of seven mice died 28 days after tumor implantation.

Experimental Example 6 Acute Oral Toxicity Polysaccharide PF-10 consisting of galactosamine was administered to ddY-N male mice (approximately 5 weeks old, 8 mice per group) weighing 26 to 28 g.
1 or PF-102 was orally administered once by gavage to mice at a dose of 300 mg/kg using a gastric probe. After administration 1
Although the animals were observed for weeks, there were no deaths and no notable symptoms of poisoning were observed.

Shou Nezuki I Acute Oral Toxicity Polysaccharide PF-10 consisting of galactosamine was administered to ddY-N male mice (approximately 5 weeks old, 8 mice per group) weighing 26 to 27 g.
1 or PF-102 was administered intraperitoneally to mice at a dose of 300 mg/kg. Observation was made for one week after administration.
There were no fatalities in either case, and no notable poisoning symptoms were observed.

[Brief explanation of the drawing]

Figure 1 is an ultraviolet absorption spectrum diagram of the hydrochloride of PF-102, Figure 2 is an infrared absorption spectrum diagram of PF-102, and Figure 3 is an infrared absorption spectrum diagram of PF-102.
The figure is an infrared absorption spectrum diagram of the hydrochloride of PF-102.

Claims (1)

[Claims] An antitumor agent characterized by containing a polysaccharide consisting of galactosamine as an active ingredient.