JPS63307825A - Antitumor agent and production thereof - Google Patents

Abstract

PURPOSE:To obtain an antitumor agent having excellent effects free from side effects, consisting of a combined material of a polysaccharide of antitumor activity, having beta-1,3 bond glucan as a main chain, comprising repeating units of the main chain under a specific condition, and an anti-cancer chemotherapeutic agent, as an active ingredient. CONSTITUTION:An antitumor agent containing a combined material of a polysaccharide having antitumor activity, which has action function to bring about multiplication inhibition and destruction of tumor cell by activating immunological function of host, and a chemotherapeutic agent which directly acts on tumor cell to inhibit multiplying function of cell and has side effects to have bad influence on normal cell, as an active ingredient. This method not only extremely alleviates cytotoxicity of the chemotherapeutic agent by combination of the medicinal effects of both the agents is used, side effects are relived and frequent administration is made possible since the chemotherapeutic agent can be reduced in organism.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention relates to antitumor agents, and
, 3-bonded glucan as the main chain, and 3 glucose residues in the main chain
The present invention relates to a conjugate of a polysaccharide having a structure in which one β-1,6-linked glucose is branched per month and an anticancer chemotherapeutic agent.

(Prior art) Many polysaccharides have been known that are mainly derived from basidiomycetes and have glucose as their only constituent sugar, and it has also been reported that some of these polysaccharides have antitumor activity. There is 1
For example, polysaccharides obtained from Shiitake mushrooms and Suehirotake mushrooms are clinically used as anticancer agents, and are used in combination with anticancer chemotherapeutic agents or other physical therapy.

(Problems to be Solved by the Invention) Anticancer chemotherapeutic agents are recognized as excellent cancer treatment drugs, but their mechanism of action is direct cell inhibition;
Chemotherapeutic agents have the disadvantage of inhibiting not only the target cancer cells but also normal cells, causing important side effects.Furthermore, chemotherapeutic agents produce resistant cancer cells, a phenomenon similar to drug resistance in microorganisms, and can cause cancer. It is difficult to fully recover.

For this reason, various studies have been carried out to alleviate the cytotoxicity of anticancer chemotherapeutic agents, but the situation remains unsatisfactory.Therefore, it is necessary to use drugs that do not structurally exhibit cross-resistance to resistant cells. Attempts have been made to overcome this condition, but they have only helped prolong life, but have not resulted in a complete cure.As mentioned above, attempts are being made to achieve a complete cure by using anti-tumor-active polysaccharides in combination with chemotherapeutic agents to enhance immune function. Although therapy has shown some clinical efficacy, it has not yet led to a satisfactory solution to the problem.

Even with the use of antibodies, the specific transfer of drugs to cancer by morphological changes, the reduction of 11 effects through sustained release effects, and the use of so-called intensive treatment methods that utilize physical therapy such as hyperthermia and radiation therapy, there is still no cure. At present, the mortality rate has not been reduced significantly, and the emergence of better antitumor agents is desired.

(Means for Solving the Problems) The present invention uses glucose as a constituent sugar as a result of intensive research into antitumor agents that have alleviated cytotoxicity without reducing the efficacy of anticancer chemotherapeutic agents.

Anti-II4 tumor-active polysaccharide and anticancer chemistry with a repeating unit consisting of β-1,3-linked glucan as the main chain and one β-1,6-linked glucose branch for every three glucose residues in the main chain. A conjugate that chemically binds a therapeutic agent provides sustained release without reducing the efficacy of the chemotherapeutic agent, thereby alleviating cytotoxicity and synergizing the efficacy of the drug in conjunction with the antitumor activity of the polysaccharide. Based on this knowledge, they completed this invention.

(Function) The main chain of β-1,3-linked glucan used in this invention is
An antitumor-active polysaccharide whose repeating unit is a structure in which one β-1°6-linked glucose is branched per three glucose residues in the main chain can be obtained, for example, by using basidiomycetes; Obtained from hot water extraction or alkaline solution extraction of fruiting bodies and mycelium obtained by culturing a mushroom fungus, or from a liquid culture filtrate of the mushroom fungus by a precipitation method with a sugar-insoluble organic solvent such as alcohol. Furthermore, by contacting the basidiomycete culture a mycelium developed by the inventors of Hoko with carbohydrates,
can be obtained in almost pure form, but it is not particularly limited to these, and any substance that has multi-Milli structure and antitumor activity as explained above can be used. The other active ingredient, an anticancer chemotherapeutic agent, is not special and is widely used, such as alkylation agents such as melphalan and ACNIJ, and antimetabolites such as cytosin arabinoside and methotrexate. Anticancer antibiotics such as mitomycin C, adriamycin, and daunorubicin hydrochloride can be mentioned. It has been known from numerous immunological studies that the antitumor-active polysaccharide has a mechanism of action that activates the immune function of the host, thereby inhibiting the proliferation or destroying tumor cells.

On the other hand, since chemotherapeutic agents directly act on tumor cells and inhibit their proliferation function, they inhibit not only tumor cells but also normal cells, which is known to have important side effects.

In this invention, based on the medicinal effects of both drugs as described above, by chemically bonding them, surprisingly, the bond not only significantly alleviated the cytotoxicity of the chemotherapeutic drug, but also increased the anti-I.
l! I! It was found that the tumor effect was enhanced.

This invention was made based on this knowledge, and will be explained below.

Now, an antitumor-active polysaccharide, which is one component of the antitumor agent of this invention, is obtained using Maitake mycelium, which belongs to the Basidiomycete family, and the other binding component, an anticancer chemotherapeutic agent, is chemically added to this polysaccharide. to combine. Maitake mycelium grown in advance by culture is added to an acidic (pH about 4) aqueous solution containing only carbohydrates (e.g. glucose), and a contact reaction is carried out at 20 to 30°C for 2 to 8 days with aeration and stirring. Means: For example, when an organic solvent such as a sugar-insoluble alcohol is added to the separated solution after centrifugation using a cloth, the polysaccharide produced will precipitate out, so this precipitate can be separated, and if necessary, After redissolution and solvent precipitation, the resulting precipitate was dissolved in a urea solution and subjected to column chromatography to collect the neutral fraction of the effluent, desalted by tube dialysis, and alcohol was added to the inner solution from which low molecular components had been removed. A precipitate is formed, which is separated and dried, for example by freeze-drying, to obtain a white flocculent polysaccharide. This polysaccharide consists of 1 β-1,6-linked glucose per 3 β-1,3-linked glucose residues in the main chain.
ICR is a polysaccharide whose repeating unit is a branched structure.
- Experimental Sarcoma 180 (solid) transplanted into mice
Antitumor active polysaccharides (hereinafter simply referred to as "polysaccharides") that exhibit strong activity in suppressing cell proliferation and can be used in this invention
The binding of the polysaccharide obtained here and the anticancer chemotherapeutic agent takes into account the chemically bondable reactive groups of both, and the presence of binding aids (e.g. condensing agents, cross-linking agents, etc.) required for this reaction. As explained below, since polysaccharide has glucose as its only constituent sugar, the reactive group involved in the bonding is a hydroxyl group, and chemotherapeutic agents that can be directly bonded have a free carboxyl group, such as melphalan (alkylated M), methotrexate ( The bonding of chemotherapeutic agents that have a hydroxyl group or amino group as a reactive group, which can be easily bonded by an ester bond, can be done using a difunctional group such as glutaraldehyde or diethylmalonimidate. The bonding can be carried out using a crosslinking agent such as, or by introducing a carboxyl group or an amino group into the polysaccharide by chemical modification. Various methods can be considered for introducing carboxyl groups into polysaccharides, but for example, by reacting with a monochloro or monobromo aliphatic carboxylic acid having 1 to 5 carbon atoms, carboxyalkyl (-CH,-
(C1lO), -COOt (, n = 0 to 4) can be introduced. Thus, polysaccharides with free carboxyl groups include melphalan, AC, which has free amino groups.
NU (alkylating agent), cytarabine, methotrexate (antimetabolite), acnanomycin. Mitomycin C
It can be combined with pleomycin monohydrochloride, daunovircin hydrochloride, and adriamycin (anticancer antibiotic). In addition, an aliphatic amine having 3 carbon atoms can be introduced by adding ammonia after the reaction (in order to introduce an amino group into a polysaccharide). Thus, polysaccharides with free amino groups can be conjugated with chemotherapeutic agents with free carboxyl groups, such as melphalan (alkylating agent), methotrexate (antimetabolite). Carboxyl groups can also be introduced by partially decomposing polysaccharides; for example, when polysaccharides are treated with periodate, the carbon number 2 of the pyranose ring of β-1,6-linked glucose (branched glucose) can be introduced. The bond between the positions 1 to 4 is cleaved to form an aldehyde at the end of the cleavage, which can be oxidized and converted into a carboxyl group.

As mentioned above, chemically binding a polysaccharide with a chemotherapeutic agent can be carried out by introducing a free reactive group into the polysaccharide in its original form or by chemical modification. It is undesirable for the value to decrease. As a result of considering this, we found that the degree of introduction of reactive groups (
It has been found that antitumor activity is influenced by the substitution rate (expressed as the percentage of reactive groups introduced per glucose residue). In other words, when carboxymethylated polysaccharides with a substitution rate of 25% and 50% were prepared by reacting monochloroacetic acid with polysaccharides, and these were administered to mice transplanted with Sarcoma 180 (solid) cells, the respective inhibition tendencies were the first. As shown in the table, at a substitution rate of 25%, about 71% of the unmodified activity is retained, but at a substitution rate of 50%, it decreases to about 30%. From this, it is necessary to set the substitution rate of the reactive group by chemical modification in the range of 20% to 30%.

Replacement rate Administration Tumor weight Inhibition rate 1%
/Mouse In! M +
SD %25 250x 5 1.
95±2.211 67.5051 250X
5 4.25±2.85 29.17 Unmodified polyW
s100X5 0.26fO, 6295, 67 pairs
Teru” 6.00±4.0ro
-Test procedure IC111-Series 6 withdrawn mouse (male, weight 27-30g
) Using 10 animals per group, 5XIO'ke of solid sarcoma +80 cells was transplanted subcutaneously in the groin area, and each sample was dissolved in physiological saline on In, +2.14.16 and 18th, with the next day as the starting day was administered intraperitoneally and released on day 35.
and measure.

1) (1-T/C) Table 2 shows the antitumor activity of polysaccharides into which carboxyl groups and amino groups were introduced at a substitution rate of 20 to 30%.

(Margin below) Table 2 The test procedure and display are the same as in Table 1.

The results in Table 2 suggest that in the substitution rate range of 20 to 30%, the length of the linear moiety attached to the free radical does not affect the activity up to about 5 carbon atoms.

From the above results, it is essential in this invention that the substitution rate of the reactive group is in the range of 20 to 30%, but in some cases, depending on the dosage form of the conjugate, it is possible to use a conjugate that is easily soluble in water. It may happen that you want to get it, and in such a case,
As long as the antitumor activity of the conjugate itself is not impaired, the polysaccharide can be chemically modified by increasing the substitution rate of carboxyl groups or by treating the polysaccharide with formic acid or ultrasound to reduce the molecular weight, and then chemically modifying the polysaccharide as described above. The relationship between molecular weight and activity when the molecular weight is reduced is as shown in Table 3, and the molecular weight is approximately 20.00.
0 is the limit for lowering the molecular weight.

(Margin below) Table 3 Same as Table 1 except that two routes of administration were used.

The conditions for chemically bonding a polysaccharide and an anticancer chemotherapy agent have been explained above in detail.Next, we will explain the conjugate between a polysaccharide and mitomycin C, which is a type of anticancer antibiotic. Dissolve in an alkaline aqueous solution, add an excess amount of monochloroacetic acid, react at 40 to 70°C for 3 to 6 hours with stirring, and then dialyze the reaction solution to desalt and remove low molecular components. When frozen and dried, a white carboxymethylated polysaccharide is obtained. Analysis of the obtained carboxymethylated polysaccharide revealed that the substitution rate of carboxymethyl groups was within the range of 20 to 30%. Dissolve this carboxymethylated polysaccharide in water, adjust the pH to 4 to 6, add about 1/+011 mitomycin C, add water-soluble carbodiimide as a condensing agent, and keep the pH at 4 to 6 at 10 to 20°C. Reaction under stirring for 6 hours leads to the formation of a precipitate. This precipitate is centrifuged, washed with water, and then freeze-dried to obtain a purple-colored conjugate of the present invention.

The amount of yaitomycin C bound to this conjugate was measured (
As a result of spectroscopic analysis at a wavelength of 340 nm, the conjugate 10
It was in the range of 3 to 5 μg in 0 μg. This bound amount of mitomycin C is smaller than the theoretical value (approximately 33 μg in 100 μg), but one of the reasons for this is that mitomycin C approaches the carboxyl group due to steric hindrance due to the complex conformation of the polysaccharide. It is possible that it cannot be done. However, it seems possible to increase the amount of mitomycin C bound by controlling the substitution rate of the carboxymethyl group within a limit that does not impair the antitumor activity of the conjugate itself.

The conjugate obtained above shows a tendency to gradually hydrolyze in a pH 6.6 buffer to liberate mitomycin C, and its half-life is estimated to be approximately 6 hours. This situation is shown in Figure 1. Considering that this PH value roughly corresponds to the PH environment in the living body, it is predicted that sustained release of mitomycin C may occur in the living body by forming a conjugate. There is no difficulty in this, and it is fully expected that this sustained release will alleviate the cellular dI properties of mitomycin C.

Therefore, we administered a large amount of this conjugate, mitomycin C 800μ, carboxymethylated polysaccharide, and mitomycin C to normal mice (ff1. weight 27-30 g) so that the dose of mitomycin C was the same, and examined their survival tendency. When investigated, the tendency was as shown in Figure 2, and when mitomycin C alone was administered at 800 μg/mouse (A), 6
Approximately 90% died within a day, and mitomycin C800μ
g/7 mice, carboxymethyl Hojo 31201 g/mouse (b), approximately 60% died on the same day;
In the case of administration (c) of 1000 μg (average 800 μg binding), 100% of the mice survived on the same day, and this state tended to persist. This clearly indicates that the toxicity of mitomycin C was alleviated by the conjugate, which is a major feature that is not observed in the case of combination use.

Next, to explain the results of testing the antitumor properties of this conjugate, ICR-strain mice (9 males weighing 27-30g1
Two groups of 10 animals per group were prepared, and 180 ascites-type sarcoma cells 1X10' were intraperitoneally transplanted into each group. Immediately after transplantation, the conjugate and mitomycin C were dissolved in physiological saline and administered intraperitoneally, and transplanted. As a result of dissection and measurement from the mouth on the 7th day, the inhibition rate by the equivalent amount of mitomycin C is as shown in Figure 3. When comparing the two with the equivalent amount of mitomycin C,
It was observed that the tendency of the inhibition rate (a) by the conjugate far exceeds the tendency of the inhibition rate (b) of mitomycin C alone, and this indicates that the enhancement of the drug efficacy brought about by binding mitomycin C to the polysaccharide. This indicates that the antitumor effects of both conjugates are synergistic. Such activity enhancement is also one of the major features brought about by forming a conjugate.

As is clear from the above examples, the conjugate of a polysaccharide and a chemotherapeutic drug not only does not reduce the antitumor activity of the chemotherapeutic drug, but also synergistically enhances the activity of the chemotherapeutic drug in vivo. It is expected to release the drug in a sustained manner and alleviate cytotoxicity, and together with improving the side effects that have become apparent when administering conventional chemotherapy drugs, it is expected to have high drug efficacy. . As described above, drugs containing a conjugate of a polysaccharide and a chemotherapeutic agent as an active ingredient, which have excellent effects as a drug, are useful antitumor agents for the treatment of various tumors, and can be administered in various forms and routes. Since the polysaccharide itself is a natural product and does not exhibit toxicity, it may be administered according to the usual administration regimen for the chemotherapeutic agent to which it is attached. This will be explained in detail below using Examples.

(Example) (a) 3I of polysaccharides! 58I Maitake fungus strain Gri-fola frondosa pal tocatiana (Gri-fola
frondosa var tokachiana)
(Described in Special Publication No. 56-53351, Microtechnical Research Institute No. 49
Two sections of 3 x 3 mm were taken from the surface of the passage slant medium of No. 79), and these were cultured in three stages as the initial seeds. 3L capacity jar fermentor containing 2L plus ζ air flow 0.5VMM, 28℃, 250r, p, m
After reacting for 2 days, the contents were centrifuged to separate and remove the mycelium. Ethanol was added to the supernatant until the ethanol concentration reached 40% by volume to produce a precipitate, which was collected by centrifugation and dried by freeze-drying. 2.1 g of the product was obtained.

Dissolve 2 g of this dry material in an 8 molar urea aqueous solution, and
- Sephadex A -25 (HCO3- type) column to separate the neutral fraction from the effluent, dialyze it with a cellulose tube (Shirai Matsu Kikai), and add 1.5 times the amount of ethanol to the obtained internal solution. The resulting precipitate was collected and lyophilized to obtain 1.2 g of a white flocculent substance. Analysis of this substance revealed that it was mainly composed of β-1,3-linked glucan with only glucose as its constituent sugar. chain, β-1,6 glucose per 3 main chain glucose residues! It is a polysaccharide whose repeating unit is a branched structure, and it showed a high inhibition rate against solid sarcoma 180 cells transplanted into mice.

(b) JLX of carboxymethylated polysaccharide! The polyp70hg obtained in (a) was dissolved in lN-Na01170111,
Add 5g of Ni monochloroate (special grade reagent) and heat at 60℃ for 3
The reaction was allowed to proceed with stirring for a period of time, and the reaction solution was dialyzed using a cellulose tube (same as above), and the internal solution was freeze-dried to obtain 753 mg of a white dry product. As a result of analyzing this, the substitution rate of carboxymethyl groups was 25%.

(c) Preparation of polysaccharide-mitomycin C conjugate (b) above
Dissolve 30 hg of the dried product obtained in 3 hl of distilled water, adjust the pH to 4.0 with hydrochloric acid, and add 3 ml of mitomycin C (commercial product).
In addition to 0B, water-soluble carbodiimide (EDC) was added as a condensing agent.
) was added and reacted for 4 hours at 15°C while keeping I'H at 6.0. The reaction solution was centrifuged to remove the precipitate, washed with cold water, and lyophilized to obtain a purple dried product.
mg was obtained. As a result of analyzing this dried product, the amount of mitomycin C bound was 4 μg per 100 μg.

(d) Antitumor properties of the conjugate in IRC-type mice (ts1 body m 27-30 gJ l
Six groups of nine animals were prepared, and each group had ascites-type sarcoma +
80 cells I x 10' cells were transplanted intraperitoneally, and the carboxymethylated polysaccharide of (b) above was dissolved in saline on days 1, 2.3.4 and 5, with the next day of transplantation as the starting day. The conjugates of (c) and (c) above were each administered intraperitoneally to investigate survival prolongation. The results are shown in Table 4.

(Margins below.) Table 5 1) (T/CXl0ro) -100C Average survival time of control group (days) T Average survival time of test group (days) 2) The amount in parentheses is the amount of mitomycin C* pro, 05 $41 p<0.001
(Efficacy) When using the antitumor agent of this invention, does it change in vivo? Since the therapeutic agent can be released in a sustained manner, cytotoxicity can be alleviated without reducing the efficacy of the chemotherapeutic agent, resulting in a reduction in side effects, allowing for frequent administration. In addition, since the other component of the conjugate, the polysaccharide, activates the immune function, the two together provide an effective anticancer effect, which is extremely beneficial.

[Brief explanation of the drawing]

Figure 1 shows the hydrolysis of the conjugate in PH6,6wi buffer, Figure 2 shows the survival status when the conjugate was administered to mice at a large dose, and Figure 3 shows the transplanted tumor of the conjugate. It shows a tendency to inhibit.

Claims (6)

[Claims] (1) An antitumor-active polysaccharide with a repeating unit consisting of β-1,3-linked glucan as a main chain and one β-1,6-linked glucose branch for every three glucose residues in this main chain. An antitumor agent characterized by containing a conjugate of a cancer chemotherapeutic agent as an active ingredient. (2) The antitumor active polysaccharide is a polysaccharide obtained by utilizing Maitake mushroom belonging to Basidiomycete (
The antitumor agent described in section 1). (3) The antitumor agent according to claim (1), wherein the anticancer chemotherapeutic agent is selected from the group of alkylating agents, antimetabolites, and anticancer antibiotics. (4) Antitumor-active polysaccharide with β-1,3-linked glucan as the main chain and a repeating unit with a structure in which one β-1,6-linked glucose is branched for every three glucose residues in this main chain. Alternatively, a conjugate of the antitumor active polysaccharide and the anticancer chemotherapeutic agent is produced by reacting a derivative into which a reactive group has been introduced by chemical modification with the anticancer chemotherapeutic agent in the presence of a crosslinking agent or a condensing agent. A method for producing an antitumor agent. (5) The antitumor active polysaccharide is a polysaccharide obtained by utilizing Maitake mushroom belonging to Basidiomycete (
4) The method for producing the antitumor agent described in section 4). (6) The method for producing an antitumor agent according to claim (4), wherein the anticancer chemotherapeutic agent is selected from the group of alkylating agents, antimetabolites, and anticancer antibiotics.