JPS62252730A - Antitumor agent - Google Patents

Abstract

PURPOSE:An antitumor agent having high antitumor action, alleviating or preventing side effects, obtained by blending an interleukin 2 activator with a specific glucan as active ingredients. CONSTITUTION:An antitumor agent containing a blend obtained by combining interleukin 2(IL-2 for short) with a straight-chain beta-1,3-glucan (e.g. sizofilan) which may contain glucose residue by beta-1,6-bond as a side chain, a partial hydrolyzate or a carboxymethylated substance thereof. Both IL-2 and the gluan have been used as an antitumor substance, a method of increasing dose is applied to raise antitumor effects, but manifestation of side effects such as pyrexia, headache, rash, etc., is sometimes found. Use of both the substances raises antitumor effects and alleviates side effects. In the antitumor substance, both the substances are blended and applied as one drug or may be separately or simultaneously administered or in a time lag.

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to novel antitumor agents. BACKGROUND OF THE INVENTION In recent years, attempts have been made to use so-called lymphokines such as interleukin-2 (hereinafter sometimes abbreviated as rl'L-2J) to enhance immunity and treat tumors and various viral infections. [Journal of Immunology (J, Immunol, )]. +25, 1904 (1980)]. By making full use of the above-mentioned polymer technology, high-purity products can now be produced in large quantities at relatively low cost [Japanese Patent Application Laid-Open No. 60-115
Publication No. 528]. On the other hand, linear β-1,3-glucan is a water-insoluble, heat-coagulable glucan with a linear β-1,3-bond as a bonding mode produced by alkaline earth metals or microorganisms belonging to the genus Agrobacterium. (abbreviated as TAK-N) and a partially hydrolyzed low polymer (TAK-N).
AK-D) and their carboxymethylated derivatives (CMTAK) are known to have antitumor effects [JP-A-53
-66442 publication].・Also, linear β-1,3-glucans having glucose residues as side chains due to β-1,6-bonds, such as the basidiomycete Sch 1zophyllum
sizorilan, which is obtained from the culture filtrate of A. commune, is used as an antitumor agent. 01111 /I< vAitL l, l'l
J-17. lQl! , Ili In order to enhance the antitumor effect, methods such as increasing the dosage of the above drugs are known, but on the other hand, treatment with large doses is not recommended due to the occurrence of various side effects such as fever, headache, and rash. Have difficulty. Means for Solving the Problem The present inventors, in the course of developing the application of IL-2 as an antitumor agent, discovered that IL-2 had a glucose residue in its side chain due to a β-1°6 bond. The antitumor effect is enhanced by using it in combination with linear β-1,3-glucan, its partially hydrolyzed product, or its carboxymethylated product, which may have a strong antitumor effect that cannot be obtained by using it alone. They also found a way to reduce or prevent the above-mentioned side effects, and conducted further intensive research to complete the present invention. The present invention provides an interleukin-2 active substance and β-1,
A linear β-1,3-glucan which may have a 6-linked glucose residue as a side chain. It is an antitumor agent formed by combining its partial hydrolyzate or its carboxymethylated product. The interleukin-2 active substance has IL-2 activity,
In other words, any substance may be used as long as it maintains 'r cells for generations while maintaining their function and has a vasogenic effect. For example, natural IL produced in the animal body or in animal cells.
Examples include -2, II produced by genetic recombination technology, -2, and related substances. When the IL-2 and its related substances are proteins, they may or may not have sugar chains. Specifically, for example, it may be a polypeptide (human IL-2) having the amino acid sequence shown in FIG. 1, or a fragment consisting of a partial amino acid sequence necessary for its biological or immunological activity. , a fragment lacking several amino acids at the carboxyl terminal portion [JP-A-60
-126088 Publication], and furthermore, polypeptides having the amino acid sequence shown in Figure 1 in which some of the constituent amino acids are deleted or substituted with other amino acids, such as the cysteine residue at position 125. JP-A-59-93093] may be used if the group is substituted with a serine residue. Moreover, the above-mentioned IL-2 is one chemically modified with a polyethylene glycol derivative or the like [for example, JP-A-60-22682
Publication No. 1] may be used. In particular, in the present invention, it is preferable to use human L-2 having the amino acid sequence shown in FIG.
In this case, an additional methionine residue (M
mixtures with and without et) [Unexamined Japanese Patent Publication No. 6
No. 0-115528], but especially Japanese Patent Application No. 1987-205873 (filed on September 17, 1985), which does not have Met at the amino terminus and begins with alanine (Ala). , corresponds to Japanese Patent Application Laid-Open No. 61-78799. ] is preferred. Among the polysaccharides used in the present invention, as a linear β-1°3-glucan, Japanese Patent Publication No. 1977-7QOQ is preferred. 4B-32673, 4B-32674 and British Patent No. 1352938.
-N/7< can be mentioned. Linear β-1,3-glue 66
TAK-D described in Japanese Patent No. 442 is mentioned. As carboxymethylated products of linear β-1,3-glucan and carboxymethylated products of partial hydrolyzate of linear β-1,3-glucan, GMT and AK described in JP-A-53-66442 are used. Can be mentioned. 'I''AK-N has a straight chain β-1,3-bond as a bonding mode, is insoluble in water, and most of them gel and solidify when heated with water. It has properties not found in glucans, and its degree of polymerization may vary depending on the manufacturing method, but Manners et al.'s method [Carbohydrate Research (Carbohydrate Research)
Polymers having an average degree of polymerization of from about 70 to 1000°, especially from about 400 to 700, as measured by Re5earch, Dan, 109 (1971), are advantageously used. 'r8 -D l:l, the above 'r A K -N,
For example, acid hydrolysis, alkaline hydrolysis or β-1
, 3-glycanase and the like, but those having an average degree of polymerization of about 5 to 300, especially about 50 to 2 QO, can be advantageously used. CMTAK is TAK-N or HTAK-
D is prepared by conventional means for carboxymethylating sugars, such as by reacting with monochloroacetic acid, and has the formula [wherein at least one tJ of R represents -CH,C00II, and the remainder indicates if , n represents 0 or a positive integer. It can also be expressed as Further, C M'rΔ may be a salt with three kinds of bases, such as a physiologically acceptable salt such as sodium, potassium, calcium, aluminum, or magnesium salt. The average degree of polymerization of CMTAK is -
N is the same as -D in 'l゛Δ, and its preferred embodiment is also the same. The carboxymethyl group content in C M i' A K is preferably 3 or less per glucose residue in the molecule. In the present invention, linear β-1,3-glucan. Among the partial hydrolysates or carboxymethylated products thereof, carboxymethylated products of linear β-1,3-glucan are particularly advantageously used, and those having an average degree of polymerization of about 400 to 700 are particularly preferred. As the linear β-1°3-glucan having a glucose residue as a side chain due to a β-1,6=bond used in the present invention, for example, schizophylrane (s 1zof i
tan). Schizophyllan is a type of basidiomycete.
zophyllum commune Fr1es
) is a polysaccharide obtained from the culture filtrate of mycelium, and β-
Glucose is branched from a linear glucose residue forming a 1,3-bond via a β-1°6-bond [Special Publication No. 4]
2-12000, JP 52-57335, 9Tai Sugar Research Institute Report 23.77 (1965), GANN,
60. 137~! 44 (1969), U.S. Patent No. 3,
See Publication No. 987,166. ]. Its structure is
No. 2-57335, U.S. Patent No. 3,987,166
Examples include those described in the publication No. Partial hydrolyzate of linear β-1゜3-glucan having glucose residue with β-1,6-linkage as a side chain used in the present invention; glucose residue with β-1,6-linkage The carboxymethyl compound of a linear β-1,3-glucan or a partial hydrolyzate thereof having as a side chain is a carboxymethyl compound of the above-mentioned linear β-1,3-glucan or a partial hydrolyzate thereof. It can be manufactured in the same way as when In the present invention, an IL-2 active substance, a linear β-1,3-glucan which may have a glucose residue as a side chain due to a β-1゜6-linkage, and a partial product thereof (hereinafter referred to as these) "
Antitumor agents made in combination with ``glucans'' (sometimes collectively referred to as ``glucans'') have low toxicity and can therefore be used safely. For example, the minimum lethal doses (MLDs) of IL-2 obtained by the method described in JP-A-60-115528 are lo
mg/mouse or more (Img=3.5X10'unit
s/mgX intraperitoneal administration). In addition, glucans have low toxicity, such as T AK-N, T AK-D
The toxicity of CM1''AK and CM1''AK is extremely low; for example, the LD when administered orally and intraperitoneally to mice or rats in an acute toxicity test is 5 g/kg or more and 2 g/kg or more, respectively. LD of schizophyllan in mice was 200 p.p.
On+g/kg or more, 300 mg/k by intravenous administration
g or more. In the present invention

[The amount of L-2 active substance and glucan used varies depending on the method and purpose of use, etc.!
Protein mtmcg (IL-2 activity: 35 units (U), 41
+! /I'14+11', Ya+-Ti1l 1 Full+-nomc n-115528. 1
A unit (U) corresponds to 2 g, Gmcg of pure recombinant IL-2 II,-2 activity. ),
It is preferable to use glucans in a proportion of about 0.5 mcg to looomcg, particularly about 50 mcg to 40 mcg.
0 mcg and administered orally or parenterally. Although the dosage of the antitumor agent of the present invention varies depending on the type of 1L-2 glucan used, it is generally administered to warm-blooded mammals (e.g., mice, cats, dogs, cows, sheep, goats, rabbits, etc.). When administered as an injection to humans (based on the amount of IL-2 protein), it is approximately 0.1-500 mcg for mice and approximately 0.001-4IIICg for mammals other than mice. , about 0.01 to 20 mcg/kg when administered as a suppository, and about o, oot to 2 mcg/kg when administered as an infusion.
g is approximately 0°2 to 4 when administered as a transdermal absorption agent.
mcg/kg, respectively, is preferred. To prepare the formulation of the present invention as an injection, examples of the carrier include distilled water, physiological saline, and physiological saline containing human serum albumin. For preparation as a raw material, carriers include, for example, saturated triglycerides, hydrogenated triglycerides, gelatin, glycerin, polyethylene glycol monostearate, and the like. For preparation as a drop, examples of the carrier include distilled water, physiological saline, and aqueous dextran sulfate solution. For preparation as a transdermal absorption agent, carriers include, for example, oily bases such as glycerin and sodium lauryl sulfate, polyethylene glycol oily bases, and white wax. In manufacturing the formulation of the present invention, conventional methods commonly used are employed. The antitumor agent of the present invention which is a combination of an IL-2 active substance and glucans can be prepared by combining the above two substances with a known pharmaceutical production method, and optionally with a pharmaceutically acceptable carrier (diluent). It can be administered as a single drug by mixing them together. Moreover, each substance can be formulated separately and administered as a single drug using a diluent or the like at the time of use. Furthermore, as described above, each of the separately formulated preparations can be administered to the same subject at the same time or at different times. The antitumor agent of the present invention is useful for treating or preventing tumors in mammals, and is particularly effective in prolonging the life of mammals harboring tumors, for example. Targeted diseases include various types of leukemia, malignant lymphoma, osteosarcoma, malignant melanoma, malignant villous epithelium, sarcoma, ovarian cancer, uterine cancer, prostate cancer, pancreatic cancer, gastrointestinal cancer such as stomach and intestine cancer, and lung cancer. Examples include esophageal cancer, neck and head tumors, and brain tumors. EXAMPLES The present invention will be specifically explained by the following experimental examples and examples, but the present invention is not limited thereto. In addition, IL-2 used in Experimental Examples 1 to 3 and Example 1.2 below was derived from Escherichia coli (Escherichia coli).
cbLa coli) Dll I/p'rl? 4(
I FO14299, FFRM r3 ['-628)
(Japanese Patent Application Laid-Open No. Sho GO-1155
IL-2(A) used in Examples 3 and 4 below.
la molecular species) is Escherichia coli N4830/p
T['(285(IFO14437°FERM DI
)-852), IL-2 was produced and separated into molecular species having Uo and Ala at the amino terminus (Example 5 of the specification of JP-A-61-78799). Carboxymethylated linear β-1,3-glucan (CMTA) used in Experimental Example 1.2 and Example 1.3 below.
K) is obtained as a sodium salt by reacting linear β-1,3-glucan with an average degree of polymerization of 540 with monochloroacetic acid [JP-A-53-66442]. The schizophyllum used in the following Experimental Example 3 and Example 2.4 was produced by Schizophyllum c.
It is obtained from the culture filtrate of the mycelia of P. omllune Fr1es), and β-1,G-
It has a bonded glucose residue as a side chain, and its weight average molecular weight is approximately 450,000. +xlO”pI subcutaneously on the flank
Fibrosarcoma cells (Mcth-A111g cells) were transplanted into Math- using a syringe, and on the 7th day after tumor transplantation, those whose tumors reached a certain size were selected, divided into groups, and drug administration started. did. The drug was administered subcutaneously to the flank opposite to the tumor implantation site once a day for 10 consecutive days. Each drug was dissolved in physiological saline (dissolution solution) to which 5% normal mouse serum was added, and the drug was prepared as a single drug so that the amount of solution administered was O, lyf/20 g of mouse body weight. To evaluate the antitumor effect, the amount of tumor ffi was measured on the 211th day after tumor transplantation, and the average tumor weight of each experimental group was determined. ML with 5-10 fish)
The tumor weight ratio (170%) was determined. In addition, drug 1
The dose per 0 is the drug type FA (I
I+cg). ! Table 1 shows the results of administering Shi-12 alone and administering the antitumor agent consisting of IL-2 and carboxymethylated linear β-1,3-glucan (CMTAK) of the present invention. Table 1 Experimental Example 2 (Antitumor effect by intravenous administration) Body weight 20
txto'' 1AeLh-A tumor cells were implanted subcutaneously in the flank of a female 13ALB/c mouse using a syringe, and 1lii tumor reached a certain size 7 days after tumor implantation. The mice were divided into groups and drug administration was started. Drug administration was carried out once a day through the tail vein of the mice for 10 consecutive days. All drugs were administered in physiological saline (solution solution) supplemented with 5% normal mouse serum. Dissolved in 0.2 as the dose volume
It was prepared as a single drug to give a mouse weight of d/20g. The tumor weight was measured on the 177th day after tumor transplantation, the average tumor weight of each experimental group was determined, and the tumor weight ratio (
170%) and evaluated the antitumor effect. 1 shi 1 2
Table 2 shows the results of single administration and administration of the antitumor agent consisting of IL-2 and carboxymethylated linear β-1,3-glucan (CMTAK) of the present invention. In addition,
The daily dose of drug is the weight of drug per mouse.
(mcg). [Table 2] Experimental Example 3 Tumor effect by intramuscular administration IXIO' MeLh-A tumor cells were transplanted subendothelially in the flank of a female BALB/c mouse weighing 20 g, and tumor transplantation was performed. Seven days later, those whose tumors had reached a certain size were selected and divided into groups, and drug administration was started. Drug administration was performed intramuscularly in the thigh once a day for 10 consecutive days. All drugs were dissolved in physiological saline (dissolution solution) to which 5% normal mouse serum was added, and the amount of solution administered was 0.11n1/
It was prepared as a single drug to give a mouse weight of 20 g. The antitumor effect was evaluated by measuring the tumor weight 21 days after tumor transplantation.
The average tumor weight of each experimental group was determined, and the tumor weight ratio (170%) between the drug-administered group (T, 5 animals per group) and the drug-untreated control group (C, 10 animals per group) was determined. The daily dose of the drug is 11 (mcg) per mouse.
). 1L-2 alone administration and the present invention!
Table 3 shows the results of administering an antitumor IQ agent consisting of L-2 and schizophyllane. Table 3: Out of 10 animals, the tumor died on the day before autopsy. Example 1 (Injection preparation) Carboxymethylated β-1,3-glucan (average degree of polymerization 540) (CMTAK) 160 mg sorbitol 200 mg carboxymethyl cellulose・Sodium 0mg, -2-Yo30, total 400ag After mixing the above ratio, dissolve in distilled water for injection, aseptically dispense 1 ha into vials, freeze-dry, and use for injection. An antitumor agent is prepared. This injection preparation is dissolved in 1 hour of distilled water for injection before use. Example 2 (Injection preparation) Schizophyllan 160 mg Sorbit 200 mg Sodium carboxymethylcellulose 0 mg [L-230 mg Total 400 mg Above After mixing the solution with the following ratio, dissolve it in distilled water for injection, aseptically dispense 1 liter into vials, and freeze-dry.
An antitumor 1a agent for injection is prepared. This injection preparation is dissolved in 1 rall of distilled water for injection. Example 3 (Injection formulation) Carboxymethylated β-! , 3-glucan (average degree of polymerization 540) (CMTAK) 160+++g Sorbit 200IIIg Sodium carboxymethyl cellulose 1 (lng [L-2 (Ala) child i) 30mg total 4
00 mg After mixing the four components in the above proportions, the mixture was dissolved in distilled water for injection, and aseptically dispensed into vials of Id each and freeze-dried.
Prepare an injectable antitumor agent. This injection preparation is dissolved in 1 cup of distilled water for injection before use. Example 4 (Preparation for injection) Schizophylrane 160+g Sorbit 200B Sodium carboxymethylcellulose 10+ng IL-2 (Ala molecular species) 30mg
A total of 400 mg of injectable anti-tumor agent was prepared by mixing the surrounding ingredients in the above ratio, dissolving in distilled water for injection, aseptically dispensing each Lndl into vials, and freeze-drying to prepare an injectable anti-tumor agent. The preparation is dissolved in 1 ml of distilled water for injection before use. Effects of the Invention The IL-2 active substance of the present invention is combined with a linear compound that may have a glucose residue as a side chain due to a β-1, f3-bond. Antitumor agents made by combining chain β-1,3-glucans, their partial hydrolysates, or their carboxymethylated products exhibit strong antitumor effects that cannot be obtained by using each alone, and have few side effects. , can be effectively used as an antitumor agent.

[Brief explanation of drawings]

FIG. 1 shows an example of the amino acid sequence of IL-2 used in the present invention.

Claims (1)

[Claims] An interleukin-2 active substance, a linear β-1,3-glucan which may have a glucose residue as a side chain through a β-1,6-linkage, a partial hydrolyzate thereof, or a carboxymethylated product thereof An antitumor agent consisting of a combination of.