JPH0459787A - Complex of N-acetylchitooligosaccharide and mitomycins and antitumor agent - Google Patents

Abstract

NEW MATERIAL:A complex of an N-acetylchitooligosaccharide and mitomycins produced by bonding an N-acetylchitooligosaccharide and mitomycins interposing a spacer. EXAMPLE:A complex of N-acetylchitohexaose-mitomycin C expressed by formula. USE:A drug. It has excellent directional property to a target cell, especially hepatocyte. I has prolonged action, improved antitumor activity and prolonged drug action by the sustained release property and is useful for the remedy of hepatic cancer or leukemia of man and animal. PREPARATION:The objective new substance can be produced by dissolving an N-acetylchitooligosaccharide in water, adding a cyanogen halide, adjusting the pH of the solution to about 10.7 with NaOH, reacting at room temperature for 3-6hr, adding omega-aminoalkanoic acid, reacting at room temperature and about pH 9 for 12-24hr under stirring, adding mitomycins and a condensation agent and reacting at room temperature and pH 5-6 for 12-24hr.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention provides a novel N-acetylchitooligosaccharide in which the imino group of the aziridine ring of mitomycins is bonded to the hydroxyl group of N-acetylchitooligosaccharide via a spacer. and mitomycins, and an antitumor agent containing the complex as an active ingredient.

[Conventional technology]

Mitomycins are antitumor agents that selectively inhibit DNA synthesis and have strong antitumor activity, but they are also highly toxic (for example, typical mitomycin C causes cumulative bone marrow suppression and gastrointestinal disorders).1. Controlling the side effects has become a problem.

Mitomycins, particularly mitomycin C, have a unique structure containing three active groups known to exhibit antitumor activity: an aminoquinone, a carbamoyl group, and an aziridine group (S, K).

Carter, S. T., Crooke (e.
ds, ), “Mitomycin C: Curr.
ent 5tatus and Developme
nt'' (1979) Acadea+ic Press
; and Kin Nakano, Fermentation and Industry, 37°1199 (1
979) ). Among these, the aziridine group has high reactivity and is an active site and also a site that is subject to inactivation. It is known that acylation of the nitrogen at the la position of the aziridine ring masks the activity, and it has been reported that antitumor activity is expressed after the acyl group is eliminated (
Kin Nakano, supra).

A complex is known in which the imino group of the aziridine group of mitomycins and the hydroxyl group of a polysaccharide (for example, dextran, cellulose, or alginic acid) are bonded via a spacer (Japanese Patent Application Laid-Open No. 60-67502). consists of a combination of cyanogen halide and ω-amino lower alkanoic acid (particularly 6-aminohexanoic acid). The effect of this complex is to extend the retention time in the body, that is, to make it sustainable. A similar technique in which the spacer is ω-halo lower carbanoic acid (particularly 6-balohexanoic acid) is also known (Japanese Patent Laid-Open No. 60-67503).

Furthermore, a three-component complex is known, in which an antibody is further bound to the above-mentioned mitomycin-polysaccharide complex via a spacer (Japanese Patent Application Laid-Open No. 60-67433).

This technique aims to impart tropism of antibodies to tumor cells to mitomycin-polysaccharide complexes. Ternary complexes of other antitumor substances (e.g., adriamycin, daunomycin), dextran with the aldohexose ring cleaved, and carriers (antibodies, fibronectin, etc.) are also known (JP-A-60-226819.60-8).
9433.62-283101).

Furthermore, a complex in which baker's yeast mannan and mitomycin C are linked via a spacer using a method similar to that of JP-A-60-67502 has been recently reported [Tatsuji Matsumoto, Kazuhiro Aizawa, Shigeo Suzuki, Masuko Suzuki Biotherap V
, 3 (1) 446-450 (1989): K
, Aizawa.

T., Matsumoto, K., Tsukada, A.
, 1to, H, 5ato.

S, 5uzuki and M, 5uzuki,
I++munophharw+ac, +11+191-
195 (1989)), the polysaccharide mannan used in this technology, unlike the polysaccharides such as dextran used in JP-A-60-67502, has its own antitumor activity, and also has a specific affinity and accumulation property for hepatocytes. We are focusing on the fact that That is, in addition to amplifying antitumor activity, this complex is intended to maintain the above-mentioned sustainability (sustained release) and tropism toward target cells.

[Problem to be solved by the invention]

The above-mentioned mannan-mitomycin C complex is a simple system (binary system) that provides all the properties that have been conventionally sought.
However, there are problems due to the fact that mannan is a class Ii compound6 For example, when administered intravenously to mice, it exhibits an anaphylactic-like lethal effect (
T. Nagase.

T, Mikami, S, 5uzuki, C05
church, and M, 5uzuki+M
icorobio1. I+u+uno1. , 28
．． 997-1007 (1984)), there is generally concern about such effects on humans or animals.

On the other hand, N-acetyl chito-oligo II (synonymous with chitin oligosaccharide) obtained by partial hydrolysis of chitin is known to have immune function light path effect, anti-infectious disease effect, and anti-tumor effect [Separate issue Food Chemical I "Science of Chitin/Chitosan" Published August 20, 1988 Publisher: Food Chemical Newspaper Co., Ltd. pp. 47-53, JP-A-61-130230
, 62-123123).

Surprisingly, the present inventors discovered that N-acetylchito-oligosaccharide has a binding strength to hepatocytes due to the presence of receptors in the cells.
We found that it has affinity and has an organ accumulation property in the liver, and when we used this in place of the polysaccharide-mitomycin C complex described above in place of the polysaccharide-mitomycin C complex, the resulting complex was The present invention was completed based on the discovery that it has excellent target cell targeting and persistence. Since the complex of the present invention uses an oligosaccharide as one component instead of a polysaccharide, it also has the advantage that it is easy to synthesize and purify, and it is easy to obtain a pure product (single product).

[Means to solve the problem]

The present invention relates to a complex of N-acetylchitooligosaccharide and mitomycins, in which N-acetylchitooligosaccharide and mitomycins are bonded via a spacer, and an antitumor agent containing the same as an active ingredient.

Chitin oligosaccharide has N-acetylglucosamine as β-1,
Although it is an oligosaccharide linked with 4 bonds, in the present invention, chitin oligosaccharides with 3 to 6 N-acetylglucosamines linked, namely, N-acetylchitotriose, N-acetylchitotetraose, and N-acetylchitopentaose are used. , N
- Acetylchitohexaose is used. Of these, N-acetylchitopentaose and N
-acetylchitohexa7ose. For example, N-acetylchitohexaose has the following structure: Mitomycins in which the imino group of the aziridine ring is a free imino group are used. Examples include mitomycin C and mitomycin A.

As the spacer, a combination of cyanogen halide and ω-amino lower alkanoic acid is used. Examples of cyanogen halides include cyanogen bromide, cyanogen chloride, cyanogen iodide, etc.
The ω-aminoalkanoic acid is usually ω- having 4 to 8 carbon atoms.
Amino-n-alkanoic acid or a compound in which any hydrogen of its alkylene group is substituted with a lower alkyl group (particularly an alkyl group having 1 to 3 carbon atoms, such as a methyl group or an ethyl group), specifically, for example, 5-amino Pentanoic acid, 6-aminohexanoic acid, 8-aminooctanoic acid, etc. are used. ω-haloalkanoic acids can also be used as spacers. In this ω-haloalkanoic acid, hydrogen of either the ω-haloalkanoic acid having 4 to 8 carbon atoms or its alkylene group is usually substituted with a lower alkyl group (particularly an alkyl group having 1 to 3 carbon atoms, such as a methyl group or an ethyl group). The octoday group includes a chloro group, a bromo group, etc. Specific examples of such ω-haloalkanoic acids include, for example, 5-bromopentanoic acid, 6-bromohexanoic acid, 8-bromooctanoic acid, 6-chlorohexanoic acid, and the like.

When a conjugate of cyanogen halide and ω-aminoalkanoic acid is used as a spacer, the composite can be produced in accordance with the method of JP-A-60-67502 mentioned above. - For example, first, N-acetylchito-oligosaccharide is dissolved in water, cyanogen halide is added thereto, the pH is adjusted to around 10.7 with sodium hydroxide, and the reaction is stirred. Cyanogen halide is usually used in an amount of 50 mg to 100+++ per 10 g of N-acetyl beef tooligosaccharide.

The reaction is completed in 3 to 6 hours at room temperature.

After the reaction is completed, 2 to 5
An equivalent amount of ω-aminoalkanoic acid is added, and the mixture is stirred and reacted at around pH 9 at room temperature for 12 to 24 hours. After the reaction is completed, the spacer-bound N-acetylchito-oligosaccharide is purified and concentrated by ultrafiltration.

Next, 1 to 5 equivalents of mitomycins are added to the bound ω-aminoalkanoic acid, and 10 to 20 equivalents of a suitable condensing agent, such as 1-ethyl-3-(3- Add dimethylaminopropyl) carbodiimide, etc. to pH 5-6, 12-24 at room temperature.
Stir and react for some time. After the reaction is completed, the product is purified by ultrafiltration and freeze-dried to obtain a complex in which the hydroxyl group of N-acetylchitooligosaccharide and the NH group of mitomycin are bonded via a spacer. The structure of such a complex is such that when a cyanogen halide and 6-aminohexanoic acid are used as a spacer, and when an ω-haloalkanoic acid is used as a spacer, the complex can be manufactured by the method of JP-A-60-67503 mentioned above. This can be done in accordance with.

In this complex, usually N-acetylchitooligo 111
Regarding the molecule, one molecule of mitomycins is introduced,
Even more mitomycin molecules may be introduced.

The complex of N-acetyl chito-oligosaccharide and mitomycins obtained in the present invention has excellent targeting to target cells, especially hepatocytes, and is long-lasting. In addition, its antitumor activity is generally superior to that of mitomycins and/or N-acetylchitooligosaccharide alone or in combination. Such conjugates of the invention are believed to be particularly useful in the treatment of liver cancer or leukemia in humans or animals.

The complex of N-acetylchito-oligosaccharide and mitomycins obtained in the present invention can be used as is, or usually at least one
It is used in pharmaceutical compositions mixed with two pharmaceutical auxiliaries.

The complex can be administered parenterally (ie, intravenous injection, rectal administration, etc.) or orally, and can be formulated into a form suitable for each administration method.

The formulation as an injection usually contains a sterile aqueous solution. The above-mentioned formulations also contain buffering agents/pHJ moderating agents (sodium hydrogen phosphate, citric acid, etc.), tonicity agents (sodium chloride, glucose, etc.), preservatives (methyl P-hydroxybenzoate, P-hydroxybenzoic acid, etc.), and preservatives (methyl P-hydroxybenzoate, P-hydroxybenzoic acid, Other pharmaceutical adjuvants other than water may be included, such as propyl (propyl, etc.). The formulation can be sterilized by filtration through a bacteria-retaining filter, by incorporating a disinfectant 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 with the usual pharmaceutical auxiliaries, such as binders (syrup, gum arabic,
gelatin, sorbitol, tragacanth, bonivinylpyrrolidone, hydroxypropyl cellulose, etc.), excipients (
Lactose, sucrose, corn starch, potato starch, turpinto, crystalline cellulose, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, silica, etc.), disintegrants (potato starch, carboxymethyl cellulose, etc.), wetting agents (sodium lauryl sulfate) etc.) can be included.

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 dosage of this antitumor agent is determined by taking into account the dosage of mitomycins and N-acetylchito-oligosaccharide (mitomycin C is in clinical use, and the dosage of the latter is described in JP-A-62-123123). It can be determined as appropriate. For example, when administering the present antitumor agent to humans, the dose of the present complex, which is the active ingredient, per adult is generally 0.
1 to 50 IIIg is suitable.

〔Example〕

In the following examples, N-acetylchitooligo I! (
N-acetylchito-oligosaccha
ride) is abbreviated as NACOS, and for example, N-acetylchitohexaose is referred to as NACOS-6.

Implementation [L (manufacturing example of this complex)
After dissolving 20.20 and 15-g of cyanogen bromide in 200 μl of distilled water at pH 10.7, respectively, was added, and then the pn was diluted with IN sodium hydroxide to 100 μl.
The reaction was carried out for about 6 hours while maintaining the temperature at 7.

Then, after adjusting the pH to 9.0, ε-aminocaproic acid 10
Add 0s+g and adjust the pH to 9.0s+g with IN sodium hydroxide.
The reaction was carried out for 24 hours while maintaining the temperature at 0.

Ultra-free CL after the reaction (manufactured by Nippon Millipore Industries)
It was purified and concentrated by ultrafiltration.

Next, 110 mg of mitomycin C was dissolved in distilled water 10II11 adjusted to pH 5.5, and the pH was adjusted to 5.5.
After adjusting the pH, 200 mg of water-soluble carbodiimide was added, and the mixture was reacted for 24 hours while maintaining the pH at 5 to 6.

After the reaction was completed, the mixture was purified and concentrated using the ultrafiltration described above, and then freeze-dried to obtain a dried complex. The average yield was 60%. Furthermore, the amount of mitomycin C bound was about 4.5% on average.

The bound amount of mitomycin C was measured using a Hitachi Model 200-20 double beam spectrophotometer at 180-400no+, and the absorbance curve of mitomycin C in the complex was compared with the absorbance curve of a certain amount of mitomycin C using the peak area method. It was measured by

Hu2 (In vivo antitumor effect of this complex) C3H/H
e Subcutaneously inject 5X MH134Y hepatoma cells into 1 group of 10 mice.
10' cells were transplanted, and on days 7, 12, and 17 after transplantation, the complex of NACOS-6 and mitomycin C produced in Example 1 (hereinafter referred to as NACO5-6-MMC complex) was added to physiological saline. The dissolved solution was administered intravenously, and 30 days after tumor implantation, the tumor was removed, the tumor weight was measured, and the tumor growth inhibition rate was determined compared to the control test. As a control, an injection solution consisting only of physiological saline was administered in the same manner. For comparison, a physiological saline solution of NACOS-6 or MMC was also administered in the same manner. The results are shown in Table 1.

Table 1. Breincubation. After that, (-)P
Remove the sample by centrifuging twice with BS solution and add 700μ of fresh RPM1 1640 medium! was added and further cultured for 24 hours at 37°C in 15χCO2 (post-incubation). Cell survival was measured by trypan blue staining, and the percentage decrease in viable cells was calculated compared to an untreated control group.

The results are shown in Table 2. The number of tests was four for each test.

Table 2 Urasei ■Yu (In vitro cytotoxic effect of this complex) MH
5 x 10' 134Y hepatoma cells and each concentration of the sample (for example, 10.100 μg/w for NACOS-6-MMC)
elt) RPMI 1640 medium (FBS serum lOx
Mix 700 μl of the solution (including
Insert) (Millipore-HA, Mil
(manufactured by Lipore) into 246 microwells and incubated at 37°C in 5χCO□ for 1 hour *mean value ± S,
E.

(Toxicity test of this complex) The NACOS-6-MMC complex produced in Example 1 was heated with C3H/He.
? 500 mg/kg was administered intravenously to mice (as a physiological saline solution) and observed for 7 days, but no deaths were observed.

〔Effect of the invention〕

A complex of N-acetylchito-oligosaccharides and mitomycins has enhanced anti-tumor activity based on its targeting to target cells, compared to each individual or a mixture, and the effect is sustained due to sustained release. .

Claims (1)

[Scope of Claims] 1. A complex of N-acetylchitooligosaccharide and mitomycins, in which the N-acetylchitooligosaccharide and mitomycins are linked via a spacer. 2. An antitumor agent containing as an active ingredient a complex of N-acetylchitooligosaccharide and mitomycins, which are bonded via a spacer.