JP2997848B2 - Complex of N-acetylchitooligosaccharide and mitomycins and antitumor agent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to 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 a complex of 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 at the same time have strong toxicity (eg, in the case of typical mitomycin C, accumulation of bone marrow suppression and gastrointestinal disorders, etc.). Control is a problem.

Mitomycins, especially mitomycin C, have a unique structure containing an aminoquinone, a carbamoyl group, an aziridine group and three active groups known to exhibit antitumor activity in the molecule [SKCarter. STCrooke (ed.
s.), “Mitomycin C: Current Status and Developmen
t "(1979) Academic Press; and Kin Nakano, Fermentation and Industry, 3
7 , 1199 (1979)]. Among them, the aziridine group has high reactivity, is a site where an activity is expressed, and is a site which is inactivated. It is known that the acyl modification of the la-position nitrogen of the aziridine tube 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 is linked to the hydroxyl group of a polysaccharide (for example, dextran, cellulose or alginic acid) via a spacer (Japanese Patent Application Laid-Open No. 60-67502). Here, the spacer comprises a conjugate of a cyanogen halide and an ω-amino lower alkanoic acid (particularly, 6-aminohexanoic acid). The utility of this complex is to prolong the retention time in the body, that is, to maintain it.
A similar technique using ω-halo lower carbanoic acid (particularly, 6-halohexanoic acid) as a spacer is also known (Japanese Patent Application Laid-Open No. Sho 60/1985).
-67503).

In addition, a three-membered complex in which an antibody is further bound to the above-described complex of mitomycin and polysaccharide via a spacer is known (Japanese Patent Application Laid-Open No. 60-67433). This technique aims to impart the mitomycins-polysaccharide conjugate with the antibody's directivity to tumor cells. A ternary complex of another antitumor substance (for example, adriamycin, daunomycin), dextran in which the aldohexose ring is cleaved, and a carrier (antibody, fibronectin, etc.) are also known (JP-A-60-226819, 60-89433, 62-283101).

More recently, a complex in which baker's yeast mannan and mitomycin C are bound via a spacer in the same manner as in the above-mentioned JP-A-60-67502 has been reported (Tatsuji Matsumoto, Kazuhiro Aizawa, Shigeo Suzuki, Masuko Suzuki) Biotherapy, 3 (1) 446-
450 (1989); K. Aizawa, T. Matsumoto, K. Tsukada, A. Ito,
H.Sato, S.Suzuki and M.Suzuki, Immunopharmac., 11 , 191
-195 (1989)]. This technique focuses on the fact that the polysaccharide mannan used has antitumor activity by itself, unlike polysaccharides such as dextran used in JP-A-60-67502, and has specific affinity and accumulation properties on hepatocytes. ing. That is, the complex is intended to maintain the sustainedness (sustained release) and the directivity to target cells as described above, in addition to the amplification of the antitumor activity.

[Problems to be solved by the invention]

The above-mentioned mannan-mitomycin C complex has the advantage that the properties imparted conventionally required can be summarized and embodied in a simple system (binary system), but there is a problem that mannan is a polysaccharide. is there. For example, when administered to mice intravenously, they exhibit anaphylactic-like lethal effects (T. Nagase, T. Mikami, S. Suzuki, C. Schuerch, and M. Suzuki).
ki, Micorobiol. Immunol., 28 , 997-1007 (1984)], and thus there is concern about such effects on humans or animals in general.

On the other hand, it is known that N-acetylchitooligosaccharide (synonymous with chitin oligosaccharide) obtained by partially hydrolyzing chitin has an immune function-enhancing effect, an anti-infectious disease effect, and an antitumor effect [separate volume Food Chemicals] I, "Science of chitin / chitosan", issued August 20, 1987. Publisher: 47-53, Food Science Newspaper Co., Ltd., JP-A-61-130230, 62-123123].

The present inventors have surprisingly found that N-acetylchitooligosaccharide has binding ability and affinity for hepatocytes due to the presence of the receptor in hepatocytes, and has N-acetyl chitooligosaccharide accumulation in organs in the liver. Were used in place of the polysaccharide of the polysaccharide-mitomycin C complex described above, and the resulting complex had excellent directivity to target cells,
The present invention was found to have sustainability and completed the present invention. Since the conjugate of the present invention uses an oligosaccharide as one component instead of a polysaccharide, it also has the advantage that synthesis and purification are easy and a pure product (single product) is easily obtained.

[Means for solving the problem]

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

Chitin oligosaccharides are N-acetylglucosamine β-1,
In the present invention, chitin oligosaccharides having 3 to 6 N-acetylglucosamines linked thereto, ie, N-acetylchitotriose, N-acetylchitotetraose, and N-acetylchitopentaose are oligosaccharides linked by four bonds. ,
N-acetylchitohexaose is used. Among these, N-acetylchitopentaose and N-acetylchitohexaose give the complex a particularly excellent antitumor activity as an antitumor activity for target cells. For example, N-acetylchitohexaose has the following structure: As mitomycins, those in which the imino group on the aziridine ring is a free imino group are used. For example, mitomycin C and mitomycin A are included.

A conjugate of a cyanogen halide and an ω-amino lower alkanoic acid is used as the spacer. As the cyanogen halide, cyanogen bromide, cyanogen chloride, cyanogen iodide and the like, and as the ω-aminoalkanoic acid, usually ω-amino-n-alkanoic acid having 4 to 8 carbon atoms or any hydrogen of the alkylene group thereof is used. Compounds 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-aminopentanoic acid, 6-aminohexanoic acid, 8-aminooctanoic acid, etc. Used. As the spacer, an ω-haloalkanoic acid can also be used. As the ω-haloalkanoic acid, usually, an ω-haloalkanoic acid having 4 to 8 carbon atoms or any hydrogen of the alkylene group thereof is a lower alkyl group (particularly, 1 to 8 carbon atoms).
Compounds substituted with an alkyl group (e.g., a methyl group or an ethyl group) are used, and the halo group includes a chloro group, a bromo group and the like. 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.

In the case where a conjugate of a halogenated cyanine and ω-aminoalkanoic acid is used as a spacer, the production of the present complex can be carried out according to the above-mentioned method of JP-A-60-67502. As an example, first, N-acetylchitooligosaccharide is dissolved in water, a cyanogen halide is added thereto, the pH is adjusted to about 10.7 with sodium hydroxide, and the mixture is stirred and reacted. Cyanogen halide is N-acetylchitooligosaccharide 100m
Usually, 50 mg to 100 mg is used for g. The reaction is completed at room temperature for 3 to 6 hours.

After completion of the reaction, 2 to 2
Five equivalents of ω-aminoalkanoic acid are added, and the mixture is stirred and reacted at about pH 9 at room temperature for 12 to 24 hours. After completion of the reaction, the spacer-bound N-acetylchitooligosaccharide 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, for example, 1-ethyl-3- (3- (Dimethylaminopropyl) carbodiimide and the like are added and reacted under stirring at pH 5 to 6 at room temperature for 12 to 24 hours. After completion of the reaction, the mixture is purified by ultrafiltration and lyophilized to obtain a complex in which the hydroxyl group of N-acetylchitolide sugar and the NH group of maletomycins are bound via a spacer. The structure of such a complex is obtained by using, for example, a cyanogen halide and 6-aminohexanoic acid as spacers. When the ω-haloalkanoic acid is used as the spacer, the production of the composite can be carried out according to the method described in the above-mentioned JP-A-60-67503.

In this complex, N-acetylchitooligosaccharide 1
One molecule of mitomycins is introduced for the molecule,
More mitomycin molecules may be introduced.

The complex of N-acetylchitooligosaccharide and mitomycins obtained in the present invention has excellent directivity to target cells, particularly to hepatocytes, and has long-lasting properties. In addition, its antitumor activity is generally superior to that of a single or mixed administration of mitomycins and / or N-acetylchitooligosaccharides.
Such conjugates of the invention are believed to be particularly useful for treating human or animal liver cancer or leukemia.

The complex of N-acetylchitooligosaccharide and mitomycins obtained in the present invention is used as it is or in a pharmaceutical composition usually mixed with at least one pharmaceutical auxiliary. 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.

Formulation forms for injection usually contain a sterile aqueous solution. The above-mentioned preparations also include preservatives (methyl P-hydroxybenzoate, P-hydroxybenzoate) such as buffers / pH regulators (sodium hydrogen phosphate, citric acid, etc.), tonicity agents (sodium chloride, glucose, etc.). (Eg, propyl acid). The preparation can be sterilized by filtration through a bacteria-retaining filter, by mixing a bactericide into the composition, or by irradiating or heating the composition. The preparation can also be produced as a sterilized solid composition and used after dissolving in sterilized water or the like at the time of use.

Oral formulations are formulated in a form suitable for absorption by the gastrointestinal tract. Tablets, capsules, granules, fine granules and powders are usually pharmaceutical auxiliaries such as binders (syrup, gum arabic, gelatin, sorbit, tragacanth, polyvinylpyrrolidone, hydroxypropylcellulose, etc.), excipients (lactose) Sucrose, corn starch, potato starch, sorbite, crystalline cellulose, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, silica, etc.), disintegrants (potato starch, carboxymethyl cellulose, etc.), lubricants (sodium lauryl sulfate, etc.) ) Can be included. The oral solution can be made into an aqueous solution or the like or a dry product.
Such oral solvents are commonly used with additives such as preservatives (P-
Methyl or propyl hydroxybenzoate, sorbic acid, etc.).

The dose of the antitumor agent is determined in consideration of the doses of mitomycins and N-acetylchitooligosaccharide (mitomycin C is used clinically, and the latter dose is described in JP-A-62-123123). Can be determined appropriately. For example, when the present antitumor agent is administered to a human, the dose is 1 adult.
Generally 0.1 to 50m as the complex, which is an active ingredient per day
g is appropriate.

〔Example〕

In the following examples, N-acetylchito-oligosaccharide is abbreviated as NACOS, and for example, N-acetylchitohexaose is referred to as NACOS.
-6.

Example 1 (Production example of this complex) After dissolving 100 mg of NACOS-6 in 10 ml of distilled water having a pH of 10.7, 20, 20, and 15 mg of cyanogen bromide were each dissolved in 200 µ of distilled water having a pH of 10.7.
, And then reacted for about 6 hours while maintaining the pH at 10.7 with IN sodium hydroxide.

Then, after adjusting the pH to 9.0, 100 mg of ε-aminocaproic acid was added, and the pH was maintained at 9.0 with IN sodium hydroxide.
The reaction was performed for 4 hours.

After the reaction is completed, Ultrafree CL (manufactured by Nippon Millipore Industries)
The mixture was subjected to ultrafiltration and purified and concentrated.

Next, a solution prepared by dissolving 10 mg of mitomycin C in 10 ml of distilled water adjusted to pH 5.5 was added to adjust the pH to 5.5, and then 200 mg of water-soluble carbodiimide was added. Thereafter, the mixture was reacted for 24 hours while maintaining the pH at 5 to 6. Was. After completion of the reaction, the mixture was purified and concentrated by the above-mentioned ultrafiltration, and then freeze-dried to obtain a dried complex. The yield was on average 60%. The binding amount of mitomycin C was about 4.5% on average.

The binding amount of mitomycin C is measured at 180-400 nm using a Hitachi 200-20 double beam spectrophotometer, and the absorbance curve of mitomycin C in the complex is compared with the absorbance curve of a certain amount of mitomycin C by the peak area method. Was measured.

Example 2 (In vivo antitumor effect of this complex) MH134Y hepatocarcinoma cells 5 × 10 5 subcutaneously into 10 mice per group of C3H / He mice
^Five cells were transplanted, and on days 7, 12, and 17 after transplantation, the complex of NACOS-6 and mitomycin C prepared in Example 1 (hereinafter referred to as NACOS
A solution obtained by dissolving OS-6MMC complex) in physiological saline was intravenously administered, and the tumor was removed 30 days after tumor implantation, the tumor weight was measured, and the tumor growth inhibition rate as compared with the control test was determined. Was. As a control, an injection consisting only of physiological saline was similarly administered. For comparison, a physiological saline solution of NACOS-6 or MMC was similarly administered. Table 1 shows the results
Shown in

Example 3 (Ex vivo cytotoxic effect of this complex) 5 × 10 ⁵ MH134Y hepatoma cells and each concentration of the sample (for example, N
In ACOS-6-MMC, 700 μl of RPMI1640 medium (containing 10% of FBS serum) of 10, 100 μg / well) was mixed, and a culture plate insert (Culture Plate Insert) having a diameter of 12 mm was mixed.
(Millipore-HA, manufactured by Millipore) was dispensed into 24-well microwells, and cultured at 37 ° C. in 5% CO ₂ for 1 hour (preincubation). Thereafter, the sample was removed by centrifugal washing twice with a (-) PBS solution, and RPMI was newly added.
700 μm of 1640 medium was added, and the cells were further cultured (post-incubation) at 37 ° C. in 5% CO ₂ for 24 hours. Cell survival was measured by trypan blue staining, and the percentage of viable cells was calculated as compared to the untreated control group. Table 2 shows the results.
The number of tests was four for each test.

Example 4 (Acute toxicity test of this complex) The NACOS-6-MMC complex produced in Example 1 was intravenously administered to C3H / He mice at 500 mg / kg (as a saline solution).
Observed for 7 days, no deaths were found.

[Effects of the Invention] The complex obtained by binding N-acetylchitooligosaccharide and mitomycins has an enhanced antitumor activity based on its directivity to target cells as compared to each alone or a mixture, and has an effect due to sustained release. Is maintained.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C07H 15/26 C08B 37/08 A61K 31/70 A61K 31/715 CA (STN) REGISTRY (STN) BIOSIS (DIALOG )

Claims (2)

(57) [Claims] 1. A complex of an N-acetylchitooligosaccharide and mitomycins, wherein N-acetylchitooligosaccharides having 3 to 6 N-acetylglucosamine units and mitomycins are linked via a spacer. 2. A complex of mitomycins and N-acetylchitooligosaccharides in which N-acetylchitooligosaccharides having 3 to 6 N-acetylglucosamine units are linked to mitomycins via a spacer is contained as an active ingredient. Antitumor agent.