JPH04202197A - Reduced hexa-n-acetyl-chitohexaose and antineoplastic agent therefrom - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to reduced hexa-N-acetyl-chitohexaose and an antitumor agent containing the same as an active ingredient.

[Conventional technology]

In recent years, anti-malignant tumor agents containing polysaccharides obtained from various bacterial cells or anti-inflammatory products as active ingredients have been provided.
For example, an antitumor agent containing lentinan, a polysaccharide extracted from the fruiting body of shiitake mushrooms, as an active ingredient (Special Publication No. 47-37002
(Japanese Patent Publication No. 1984, Japanese Patent Publication No. 49-484), an antitumor agent containing protein polysaccharide extracted from Corsicolor versicolor as an active ingredient (Japanese Patent Publication No. 55-23271, Japanese Patent Publication No. 57-40159), antitumor agents from Mycobacterium tuberculosis Antitumor agents containing extracted ribopolysaccharide as an active ingredient (Japanese Unexamined Patent Publication No. 18619/1983) have been provided.

The present inventors have also previously developed an antitumor agent containing chitin, a polysaccharide formed by bonding many N-acetylglucosamine molecules that exist in large quantities in nature, as an active ingredient (Japanese Patent Laid-Open No. 59-27
No. 826), and water-soluble chitin oligo 1 obtained by hydrolyzing chitin! The present invention provides an antitumor agent (Japanese Unexamined Patent Publication No. 123123/1983) containing as an active ingredient a chitosan oligosaccharide (also referred to as N-acetyl chitooligosaccharide) or chitosan oligosaccharide (also referred to as chitooligosaccharide). In addition, the present inventors have disclosed an invention regarding an anti-infective agent containing chitin oligosaccharide or chitosan oligosaccharide as an active ingredient (Unexamined Japanese Patent Publication No. 61-1302).
30) is also carried out.

Regarding the antitumor activity of chitin, chitosan, and their oligosaccharides, see Shigeo Suzuki, Bessatsu Food Chemical Lou ■1, Food Chemical Newspaper, published August 20, 1988, 47-53.
The antitumor activity of chitin, chitosan, and their derivatives is also described in Tokura Kiyoshi-5 Special Edition Food Chemistry Volume 2, Food Chemical Newspaper, published August 20, 1988, 5-11. In addition, as a review of the manufacturing method, physical properties, and uses of chitin oligosaccharides and chitosan oligosaccharides, Kazuo Sakai, Bessatsu Food Chemical-I1, Food Chemical Newspaper, published August 20, 1986,
There are 106-111.

Further, reduced chitosan oligosaccharide is described in JP-A-64-79194, and it is said that it can be widely used like chitosan oligosaccharide.

For the reduction of chitin oligosaccharides, see AkiraOht.
akara and Masaru Mitsutomi
, Methods in Bnzymology, 1
6L 453-457. (198B), and reduced chitin oligosaccharides are described as intermediates for chemical conversion in a paper on a method for measuring the degree of polymerization of chitin oligosaccharides (C, S, Tsai.

Analytical Biochemistry 3
6.114-122 (1970)).

(Problems to be Solved by the Invention) The above-mentioned antitumor agents containing chitin as an active ingredient have excellent antitumor activity, but since chitin is a water-insoluble polymer, it is difficult to formulate liquid preparations such as injections. There have been practical problems such as difficulty in administration and inability to administer intravenously.Furthermore, antitumor agents containing water-soluble chitin oligosaccharides or chitosan oligosaccharides as active ingredients have excellent antitumor activity; ,
There were problems with the stability of the active ingredient, especially in aqueous solutions.

[Means to solve the problem]

The present inventors have conducted intensive research to solve the above-mentioned problems of chitin oligosaccharides and provide more practical antitumor agents. Among the reduced chitin oligosaccharides obtained by reducing chitin oligosaccharides, especially Reduced chitin oligosaccharide consisting of 6 monosaccharide units, i.e., reduced hexa-N-acetyl-chitohexaose, exhibits antitumor activity equivalent to or greater than that of the starting material hexa-N-acetyl-chitohexaose, and is stable. They have found that it has particularly excellent stability in aqueous solutions, and have completed the present invention. This reduced hexa-N
-Acetyl-chitohexaose is represented by the following formula: This compound is formally known as 2-acetamido-2-deoxy-
Although it is called 4-glucidoyl penta-N-acetyl-β-chitopentanoside, it will be referred to hereinafter as reduced hexa-N-acetyl-chitohexaose for convenience as described above.

Thus, the present invention relates to reduced hexa-N-acetyl-chitohexaose and an antitumor agent containing it as an active ingredient.

Although reduced hexa-N-acetyl-chitohexaose is nominally included in the claims of the above-mentioned JP-A-64-79194, it is not specifically described at all, and its antitumor properties are Activity is also not described. That is, the invention of JP-A No. 64-79194 essentially relates to a reduced chitosan oligosaccharide containing reduced chitohexaose,
Furthermore, there is no specific description of the physiological activity of reduced xatone oligosaccharides. Reduced hexa-N-acetyl-chitohexaose can also be prepared using Methods i
nEnzymology+ 161 453-45
7 (1988), but in this paper, a sample containing N-acetylglucosamine obtained by acid hydrolysis of chitin and a chitin oligosaccharide mixture was subjected to a reduction reaction to obtain N-acetylglucosamitol. and a reduced chitin oligosaccharide mixture, and neither the reduced hexa-N-acetyl-chitohexaose nor the reduced chitin oligosaccharide mixture was separated from the reaction mixture. Reduced hexa-N-acetyl-chitohexaose is further synthesized in the above-mentioned Analytical Biochemistry.
36.114-122 (1970), but this paper specifically deals only with the reduction of tri-N-acetyl-chitotriose regarding the reduction of chitin oligosaccharides; -Acetyl-chitotriose is also subjected to the next conversion step and converted without being isolated. The above two papers do not describe anything about the physiological activity of reduced chitin oligosaccharides.

As mentioned above, reduced hexa-N-acetyl-chitohexaose was either included only nominally in the prior literature or was present in solution as a mixture with other congeners and starting materials. Too small, therefore not isolated. Moreover, its antitumor activity is also not recognized. Therefore, it is considered that reduced hexa-N-acetyl-chitohexaose under such circumstances can be regarded as a new substance.

Reduced hexa-N-acetyl-chitohexaose can be obtained by reduction of hexa-N-acetyl-chitohexaose: ---→Reduced hexa-N-acetyl-chitohexaose The above reductions can be carried out in various ways known to those skilled in the art. For example, an aqueous solution of hexa-N-acetyl-chitohexaose is placed in an autoclave, pressurized hydrogen is introduced into the autoclave, and the reaction is carried out at room temperature or under heating in the presence of a Raney-nickel catalyst or a ruthenium catalyst. This can be done by keeping it at room temperature. A specific example of production and physical properties are shown in Example 1 below.

M-hexa-N-acetyl-chitohexaose exhibits excellent antitumor activity equivalent to or better than hexa-N-acetyl-chitohexaose against mouse Sarcoma 180 solid tumor and mouse Sarcoma-A solid tumor, and Stability, especially stability in aqueous solutions, is superior to hexa-N-acetyl-chitohexaose. In addition, as shown in Example 3 below, the homologs reduced tetra-N-acetyl-chitotetraose and reduced penta-N-acetyl-
Since chitopentaose exhibits virtually no antitumor activity, the antitumor activity exhibited by reduced hexa-N-acetyl-chitohexaose is remarkable in that sense as well. Furthermore, reduced hexa-N-acetyl-chitohexaose is hexa-N-
Compared to acetyl-chitohexaose, its solubility in water is dramatically increased. By improving water solubility, it is possible to increase the concentration and therefore reduce the amount of liquid when preparing an injection, and this also enables subcutaneous injection and intramuscular injection; preparative separation by HPLC and GPC During purification, the amount of water used can be reduced due to the high water solubility, which has the advantage of reducing the burden of water concentration in subsequent steps and improving production efficiency. Furthermore, the starting material hexa-N-acetyl-chitohexaose is a known substance and can be obtained by partial hydrolysis of chitin (J, Chem, Soc.
, 1654-1655 (1970): l, it is relatively difficult to separate it from other chitin oligosaccharides (for example, the peaks in HPLC separation tend to overlap), whereas reduced hexa-N-acetyl-chitohexaose Hexa-N mixed with other chitin oligosaccharides as a synthetic raw material
- Even when using acetyl-chitohexaose or a partial hydrolyzate of chitin, it has the advantage that it can be easily separated from other reduced chitin oligosaccharides produced at the same time (for example, the peaks in HPLC separation are clearly distinguished). be.

This reduced hexa-N-acetyl-chitohexaose has excellent antitumor activity against experimental tumors in mice and is expected to be effective in treating tumors in humans or animals (livestock, zoo animals, pets, etc.). Ru.

The reduced hexa-N-acetyl-chitohexaose is used as such or in pharmaceutical compositions, usually with at least one pharmaceutical auxiliary.

The reduced hexa-N-acetyl-chitohexaose can be administered parenterally (i.e., intravenously, subcutaneously, subcutaneously, intramuscularly, rectally, etc.) or orally, and formulated into a form suitable for each administration method. I can do it.

Among these, intravenous, subcutaneous,
Intramuscular administration, particularly intravenous administration, is preferred.

The formulation as an injection usually includes a sterile aqueous solution. Preparations of the above form can also be used with buffers pl (regulators (sodium hydrogen phosphate, citric acid, etc.), tonicity agents (sodium chloride, glucose, etc.), preservatives (methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, etc.). ).The formulation can be sterilized by filtration through a bacteria-retaining filter, by incorporating a bactericide into the composition, by irradiating or heating the composition.

The preparation can also be prepared as a sterile solid composition and used by dissolving it in sterile water or the like before use.

Orally administered preparations are formulated in a form suitable for absorption by the gastrointestinal tract. Tablets, capsules, granules, granules and powders are prepared using conventional pharmaceutical auxiliaries, such as binders (syrups, gum arabic,
gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, hydroxypropylcellulose, etc.), excipients (
lactose, sugar, corn starch, calcium phosphate, sorbitol, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, silica, etc.), disintegrants (potato starch, carboxymethyl cellulose, etc.), wetting agents (sodium lauryl sulfate, etc.)
can be included. Tablets can be coated by conventional methods.

Oral liquid preparations can be made into aqueous solutions or dry products. Such oral solutions may contain conventional additives such as preservatives (methyl or propyl p-hydroxybenzoate, sorbic acid, etc.).

The amount of reduced hexa-N-acetyl-chitohexaose in this antitumor agent can be varied, but is usually 1 to 1.
100χ (tv/w) is appropriate. The dosage of this antitumor agent varies depending on the species to be administered, age, tumor type, symptoms, administration method, etc., but for adults, the active ingredient is 0.
．． 1-500mg/kg/day, especially 1-10m
g/kg/day is appropriate. The acute toxicity of reduced hexa-N-acetyl-chitohexaose is L'D s o
(ddy mouse, iv administration) >3 g/kg.

〔Example〕

Next, examples of the present invention will be shown.

Practical Flame Synthesis Examples and Physical Properties Hexa-N-acetyl-chitohexaose (purity 75.
9%) 2.0g (1.92mmol) in 100mj of water
! 10.0 g of Raney nickel was added, and the mixture was heated under a hydrogen pressure of 32-37 kg/cm'' in an autoclave at 50° C.
Stirred at ~70°C for 8 hours.

After Raney nickel filtration, the contents were lyophilized to obtain a crude product of reduced hexa-N-acetyl-chitohexaose.
6 g (yield 80.0%, purity 72.4%) was obtained.

This was subjected to HPLC preparative separation (ODS column, 20 mm (φ)
x 1 m, column temperature: room temperature, eluent: water] to reduce hexa-N-acetyl-chitohexaonic acid O: 6
4g (yield 31.9%, purity 100%) was obtained. This was further added to the Pellicon cassette system (UF membrane = 10.0O
ON MW L) The pyrodiene was removed using an ffl external filtration system manufactured by Nippon Millibore Ltd., and the filtrate was freeze-dried to obtain a white powder of reduced hexa-N-acetyl-chitohexaose. Quantification of pyrodiene was performed using “EndotoxineTest-D” manufactured by Seikagaku Kogyo Co., Ltd.
(LPS quantitative kit)'' was used.

In addition, in the above series of operations, tracking of the target product and analysis of purity were performed by HPLC under the following conditions = (column) Y
MC-Pack PA-03 (manufactured by YMC Co., Ltd.) 4
．． 6 X 250mm (eluent) CH3CN:H2O (65:35)
0.8ml/mln (detector) UV (220nm).

The physical properties of the finally obtained reduced hexa-N-acetyl-chitohexaose were as follows.

Melting point 〉300°C Properties White powder Specific rotation [α)'' (1χ, water) -18,2° Solubility > 100mg10.2d (water, 22°C) (Hexa-N-acetyl-chitohexaose in water The solubility is 1.4g/100ml at 20°C ± 1°C) IR Figure 1 (KBr method) “C-NMR (in D20) δ (ppm) 177.600
．． 177.44L 177.25L 104.376°104.194.104.156.103.974.
82.177°82.116. 81.957. 78
．． 838. 77.433°77.266, 76.3
94. 75.172. 75.073°75.012
．． 74.246. 72.690. 70.990°64.827. 64.023. 63.522. 6
2.907°58.52B, 58.126. 57
．． 98L 57.852°55.735. 25.0
73 (Figure 2) Disaster I Synthesis example Hexa-N-acetyl-chitohexaose (purity 93.
0%) 2.38g (1.92mmol) in 220d of water
40,146 g of NaBH was added, and the mixture was heated at room temperature for 5.
Stir for hours.

After adding acetone 3 to the reaction solution and stirring for 30 minutes,
A small amount of the solvent was evaporated using a rotary evaporator, and then several drops of hydrochloric acid were added to make the mixture weakly acidic. Methanol 101d was then added and the mixture was concentrated to about half its volume. This solution was treated with ion exchange resins HCR-W2@ (The Dow Chemical Company) and IRA-4000 (Rohm & Haas Company), and then lyophilized to give 2.07 g of white powder (yield: 87. 0%, purity 95.
3%). This was purified by HPLC in the same manner as in Example 1 to obtain reduced hexa-N-acetyl-chitohexaose with a purity of 98.6% (recovery rate 78.1%).
.

Implementation 1 Antitumor activity against Sarcoma 180 solid tumor 1 x 106 Sarcoma 180 tumor cells were subcutaneously transplanted into the inguinal region of 5pp-day I mice (8 mice per group), and 14 days after the transplant, physiological saline for injection was administered. 0.1% reduced hexa-N-acetyl-chitohexaose dissolved in
d was injected. 30 days after transplantation, the tumor was taken out and weighed, and the inhibition rate was determined by comparing it with the control (physiological saline administration).

For comparison, the antitumor activity of related substances was also determined. When the related substances were chitin and chitosan, they could not be injected intravenously, so a suspension in physiological saline for injection was administered intraperitoneally.

The results are shown in Table 1.

2. Antitumor activity against solid tumors of Meth-A (Meth-A). 3 x 10 5 female A tumor cells were transplanted subcutaneously into the scratches of G mice (8 mice per group), and after transplantation. Reduced hexa-dissolved in physiological saline for injection on day 14
N-acetyl-chitohexaose into the tail vein (J, 1 m
I injected 1. On the 27th day after transplantation, the tumor was taken out, its weight was measured, and the inhibition rate was determined in comparison with the control (physiological saline administration).

For comparison, the antitumor activity of related substances was also determined.

The results are shown in Table 2.

Incidentally, C and D were produced in the same manner as in Example 1 from tetra-N-acetyl-chitotetraose and penta-N-acetyl-chitopentaose, respectively.

Apical administration Intravenous injection reduced hexa-N-acetyl-chitohexaose 20~
The solution is dissolved in 100 times (volume/weight) sterile physiological saline and aseptically filtered through a filter (pore size: 0.45 μm), and the filtrate is used as an injection.

Implementation ■ Tablet Hydroxypropyl cellulose 1 part Lactose 10.9 parts Potato starch
1 part magnesium stearate
20 parts of a 60% ethanol aqueous solution containing 0.1 part of hydroxypropylcellulose and 1 part of hydroxypropylcellulose was prepared, 7 parts of reduced hexa-N-acetyl-chitohexaose and 10.9 parts of lactose were added, thoroughly kneaded, and then mixed under reduced pressure. 1 part of potato starch and 0.1 part of magnesium stearate are added to the obtained dried product, mixed, and tableted using a tablet machine.

〔Effect of the invention〕

The reduced hexa-N-acetyl-chitohexaose of the present invention exhibits an antitumor activity equal to or greater than that of hexa-N-acetyl-chitohexaose, and is stable, especially in an aqueous solution. Better than hexaose.

[Brief explanation of the drawing]

FIG. 1 shows an infrared absorption spectrum (KBr method) of reduced hexa-N-acetyl-chitohexaose of the present invention. FIG. 2 shows the "C-NMR spectrum (in D20) of reduced hexa-N-acetyl-chitohexaose of the present invention.

Claims (1)

[Claims] 1,2-acetamido-2-deoxy-4-glucitoylpenta-N-acetyl-β-chitopentanoside. An antitumor agent containing 2,2-acetamido-2-deoxy-4-glucitoylpenta-N-acetyl-β-chitopentanoside as an active ingredient.