JP2001031575A - Inhibition of side effect of 5-fluorouracil by chitin chitosan and formulation therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To effectively attain prevention of the side-effect of 5-flurouracil(5-FU), for example hypoleukocytosis by administering chitin chitosan in a specific form in a specific carcinostatic method using 5-FU and chitin chitosan. SOLUTION: In a carcinostatic method in which (A) 5-FU and (B) chitin chitosan are orally used in combination, at least the component B is given in the enteric form to suppress the side-effect of the 5-FU. The amount of the component B is 3-40-fold in weight, particularly 12-60-fold based on the component A in weight. In a preferred embodiment, the enteric form of the component B is prepared as capsules, tables or resinates. In the capsule preparation, the component A is used as a core capsule component, and the core component A is surrounded with the outer capsule of the component B to form a double capsule structure. Or, in another embodiment, a capsule is divided into two sections and these sections are filled with the components A and B separately.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for the combined use of 5-fluorouracil (hereinafter abbreviated as 5-FU) and chitin / chitosan for cancer therapy. The present invention relates to a method for preventing 5-FU side effects by combined use.

[0002]

2. Description of the Related Art In general, the history of anticancer drugs is based on the poison gas (Iperit) used by the German army in World War I.
Is known to have started to show that there is a movement to suppress the hematopoietic effect of bone marrow.

[0003] Thereafter, studies on the effects of poisonous gas on living bodies became active, and it was discovered in 1942 during World War II that nitrogen mustard was effective against cancer. This toxic gas binds to the guanian of the gene-DNA,
Cut the strands. In order for cancer cells to divide, it is necessary to increase the amount of DNA. However, it has been found that if the DNA strand is cut, synthesis of the DNA cannot be performed, and growth is suppressed or cancer cells die. .

Since then, many anticancer drugs have been developed, one of which is 5-FU. 1957, He
40 since Idberger et al. discovered 5-FU
Years have passed. However, even at present, 5-FU is indispensable as a single agent or a combination agent for treating various solid cancers including advanced / recurrent gastrointestinal cancer. 5-FU is a white crystal and is slightly soluble in water.
Inhibits biosynthesis of DNA in competition with uracil and exhibits antitumor effect. Gastrointestinal cancer such as stomach cancer, rectal cancer, colon cancer, breast cancer,
It is used as an injection, dry syrup, hydrophilic ointment for uterine cancer, lung cancer, skin cancer, various other carcinomas, reticulum sarcoma, Hodgkin's disease and the like. poison. Extreme amount: 0.8 g / day (bore, intravenous). The structural formula is shown below.

[0005]

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[0006] On the other hand, chitin and chitosan also have N
K (the action of lymphocytes to kill cancer cells) and LAK (YAC-
It is known that there is an enhancing effect on the activity (killing one cancer cell) (Takumichi Okuda: Chitin and Chitosan Basics and Pharmacology, pp. 85-86, (1998), published by Pharmacy Shimbun).

Thus, chitin-chitosan is a mixture of chitin and chitosan. Chitin is N-acetyl-β
It is a polysaccharide having a molecular weight of 1,000,000 or more in which 1 to 4 D-glucosamine residues are bonded in a number of 5000 or more. This chitin can be found in crustaceans such as crabs and shrimp, krill skins, beetles,
It is a polysaccharide that exists in living organisms such as the shells of insects such as grasshoppers, clams and shells such as oysters, squid bones, and cell walls of fungi. It is estimated that 100 billion tons are produced annually in the living world.

At present, chitin and chitosan have been isolated from crab and shrimp shells. When about 5% hydrochloric acid solution is added to the crust or shrimp shell, CaCO ₃ contained in the shell is added.
Changes to CaCl ₂ and CO ₂ , and carbon dioxide gas is generated. Soaking the demineralized crust in about 5% sodium hydroxide causes the protein to dissolve. The remaining insolubles are chitin. When a 40-45% sodium hydroxide solution is added to this chitin and the mixture is treated at 80-120 ° C., as shown by the following chemical structural formula, the acetyl group bonded to the amino group of the chitin is removed and the chitin is converted to chitosan. The yield from crab and shrimp shells is 15-30%.

[0009]

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However, chitosan prepared by such a treatment is not 100% pure but usually contains 10-20% chitin. Chitin is said to be non-toxic. Chitosan is also said to be significantly less toxic than glucose and sugar. The lethal dose of glucose and sugar is said to be 8-12 g / kg in dogs, but Professor Hirano reports that no toxicity was observed when mice were orally administered 18 g of chitosan / kg body weight (Shigehiro Hirano,
"Separate volume food chemical" I, pp. 1-4 (198
7)).

[0011]

Since the discovery that nitrogen mustard is effective in treating cancer, many anticancer drugs have been discovered, one of which is "5-fluorouracil" (5-FU). is there. This was discovered by Heidelberger et al. In 1958, focusing on the fact that uracil tends to collect in cancer tissues (uracil is a base but not contained in DNA;
Base present in NA).

DNA has four types of nucleotides (dAT)
P, dGTP, dCTP, dTTP), the smallest of which is dTTP. Therefore, without dTTP, DNA will not be synthesized no matter how many other nucleotides are present. 5-FU
Inhibits the synthesis of dTTP,
It reduces the synthesis of A and exerts an anticancer effect.

As shown in the following reaction scheme 1, 5-FU is phosphorylated to form F-dUMP, which binds to dTMP synthase together with folic acid, thereby rendering this enzyme inoperable. dTMP synthase is a key and important enzyme in the production of dTTP.

[0014]

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As described above, uracil is one of the bases constituting RNA.
FU is also incorporated into RNA. Intracellular RNA is primarily involved in protein synthesis. Proteins are a series of amino acids, and D is used to indicate the sequence.
Carrying (transmitting) NA commands from the cell nucleus to the cytoplasm
Is messenger RNA. Messenger RN
Ribosomes provide a place to make proteins from amino acids based on the instructions of A, and ribosomes contain large amounts of RNA.

Therefore, when 5-FU is incorporated into RNA, the function as a messenger RNA or ribosome cannot be performed, protein synthesis is inhibited, and cancer cells cannot survive. That is, 5-FU is
It exerts an anticancer effect by inhibiting the synthesis of DNA and protein. Cancer cells repeatedly proliferate and proliferate. Before a cell divides into two cells,
DNA must be doubled and proteins and lipids need to be increased. At that time, 5-FU becomes DN
If the increase in A or protein is prevented, cancer cells cannot divide.

It is well known that administration of 5-FU to cancer patients causes side effects such as hair loss, leukopenia and diarrhea. Hair root cells, bone marrow cells, intestinal mucosal cells, and the like are actively dividing cells in the body like cancer cells, and therefore, 5-F attacks the actively dividing cells.
If U disrupts cell division of hair root cells, hair loss
Leukopenia is caused by bone marrow cells and diarrhea is caused by impaired division of intestinal mucosal cells. In this case, a decrease in leukocytes reduces the resistance to cancer,
FU impairs the resurrection of surviving cells and reduces the resistance to infection, thereby posing the risk of susceptibility to pneumonia and the like.

The present invention has been made in view of such conventional disadvantages, and its purpose is to provide an appropriate modulator.
Is intended to provide a method for preventing side effects caused by oral administration of 5-FU.

[0019]

Means for Solving the Problems The present inventors have proposed a 5-FU.
Has been studied diligently to prevent side effects caused by oral administration of chitin and chitin / chitosan, effectively preventing the side effects of 5-FU administration such as leukopenia and reduction of small intestinal mucosal enzyme activity. To complete the present invention.

That is, according to the present invention, the following method for preventing the side effect of 5-FU by chitosan and a compounding agent therefor are provided, and the above object of the present invention has been achieved.

(1) An anticancer method for oral administration of 5-FU and chitin / chitosan in combination, wherein at least chitin / chitosan is administered in the form of an intestine.
-How to prevent side effects of FU. (2) The method according to the above (1), wherein the enteric form is a capsule. (3) The method according to (1) or (2), wherein the capsule is either a hard capsule or a soft capsule. (4) The capsule according to any one of (1) to (3), wherein the capsule is a double capsule, and the inner capsule is filled with 5-FU, and the outer capsule surrounding the inner capsule is filled with chitin / chitosan. The method described in Crab. (5) The method according to any one of (1) to (3), wherein the capsule is a two-chamber fraction capsule, and each compartment is separately filled with 5-FU or chitin / chitosan. (6) The method according to (1), wherein the enteric form is a tablet. (7) The method according to (1), wherein the intestinal form is a resinate. (8) The method according to (1), wherein 5-FU and chitin / chitosan are simultaneously administered. (9) The method according to (1) above, wherein 5-FU is orally administered after oral administration of chitin / chitosan. (10) The method according to the above (1), wherein chitin / chitosan is used in combination with 5-FU in a weight ratio of 3 to 240 times. (11) The method according to the above (1), wherein chitin / chitosan is used in combination with 5-FU in a weight ratio of 12 to 60 times.

(12) 5 prepared in an intestinal form for use in the method for preventing side effects of 5-FU described in (1) above.
-A combination of FU and chitin / chitosan. (13) The combination preparation according to (12), wherein the combination preparation of 5-FU in the enteric form and chitin / chitosan is a capsule. (14) The formulation according to (12) or (13), wherein the capsule is either a hard capsule or a soft capsule. (15) The capsule is an internal capsule, and the capsule is 5-FU.
The combination according to any one of the above (12) to (14), wherein in the filled capsule, the outer capsule surrounding the inner capsule is a chitin / chitosan filled capsule. (16) The capsule agent has 5-FU in one compartment,
The above-mentioned (12) to (1), which are two-room fraction capsules in which the other compartment is separately filled with chitin and chitosan, respectively.
The combination according to any one of 4). (17) The combination preparation according to the above (12), wherein the combination preparation of 5-FU in the enteric form and chitin / chitosan is a tablet. (18) The combination according to the above (12), wherein the combination of the intestinal form of 5-FU and chitin / chitosan is a resinate. (19) The combination according to the above (12), wherein chitin / chitosan is added in a weight ratio of 3- to 240-fold relative to 5-FU tin in the intestinal combination. (20) The combination according to the above (12), wherein chitin / chitosan is added in a weight ratio of 12 to 60 times with respect to 5-FU in the intestinal combination. (21) The combination according to any of (12) to (20), wherein both the 5-FU and chitin / chitosan are powder.

[0023]

Embodiments of the present invention will be described below in detail. Anti-cancer agent 5-FU used in the present invention
Has the chemical structure and side effects described above, and the mechanism of its antitumor activity and side effects is considered as follows.

5-FU is a strongly time-dependent antimetabolite, and its mechanism of antitumor activity and side effects has been fundamentally elucidated, and its clinical significance seems to be clear. As shown in Reaction Scheme 2, the main action of 5-FU is that FdUMP assimilated and metabolized in vivo and reduced folate (5,10-CH ₂ -THF) are dTMP synthase (a rate-limiting enzyme for DNA synthesis). TS) and inhibits TS by forming a three-way covalent conjugate, thereby inhibiting DNA synthesis and consequently exhibiting cell killing activity. Pyrimidine nucleotidi and 5-F
The metabolism of U is shown in the following reaction scheme 2.

[0025]

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The basis of the development of 5-FU was based on two findings that the uracil utilization of tumor tissue was higher than that of normal tissue and that the biological activity was enhanced by the introduction of fluorine. It is said that 5-FU has a wide spectrum of anti-cancer spectrums, ranging from solid cancers such as stomach cancer, colon cancer, liver cancer and breast cancer to malignant lymphomas. ) Also shows clinical effects. However, since this drug is an active substance, it has strong side effects, and may exhibit myelosuppression and gastrointestinal disorders, as well as hair loss, pigmentation, and neurosis in the case of long-term administration. In particular, dry syrup has a drawback of easily causing stomatitis, diarrhea, anorexia, esophagitis, gastritis and the like. Therefore, tablets are currently being formulated and used to reduce such side effects.

In order to maintain the anticancer action while suppressing such side effects, it is easily considered that 5-FU needs to be present in the blood at a low concentration for a longer time. By the way, it was discovered that chitin / chitosan binds to chlorine in salt and is excreted in feces, so that serum chlorine does not rise, thereby preventing blood pressure from rising (Takumichi Okuda,
Chitin and Chitosan Fundamentals and Pharmacology, pp. 18-35, 199
Published by Pharmacy Shimbun on June 20, 2004).

Therefore, the present inventors have proposed that Na ⁺ Cl ⁻
5-F having a halogen F in the structure thereof as in
The possibility of chitin / chitosan as a U modulator was examined. The interaction between the two is considered as shown in the following chemical formula.

[0029]

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Although the above reaction scheme shows the chemical structure of 5-FU, it appears that most of the negative charge has been lost since fluorine is attached to the fifth carbon of uracil. However, the slight negative charge left can loosely bind the positively charged chitosan. Since this is a loose bond, it does not bind firmly like fluorine alone as in the case of salt,
It is not excreted in feces, and at best only keeps 5-FU in the intestinal tract. And this is the 5-F
It is thought that this leads to the presence of U in the blood at a low concentration for a longer time, and to maintain the anticancer action while suppressing side effects.

The chitin / chitosan used in the present invention needs to be in the form of an intestine in order to prevent wasteful consumption by chlorine in the stomach during oral administration. Chitin and chitosan in this intestinal form include capsules,
Tablets and resinates are included. Further, the capsules include both hard capsules according to prescription and soft capsules manufactured in advance at a pharmaceutical factory.

Also, the shape of the capsule is cap and body.
Capsule consisting of 5-F in small inner capsule
If U is filled and formed into a large outer capsule filled with chitin / chitosan so as to surround the small inner capsule as shown in FIG. 8, the chitin / chitosan first dissolves in the intestine, Since 5-FU dissolves, 5-FU
It is convenient to keep in the intestinal tract. However, as shown in FIG. 9, the capsule body may be appropriately divided into two by a partition wall, and each section may be formed so as to be separately filled with 5-FU and chitin / chitosan. In addition, it is possible to mix and fill 5-FU and chitin / chitosan at an appropriate weight ratio in a simple capsule, or to put only the latter in a capsule and leave the former as it is, or wrap it in an oblate, etc. The administration according to the patient's preference, for example, oral administration of 5-FU afterwards, can be performed without any limitation.

The tablet may be either a compressed tablet or a wet tablet, and any tablet can be used without any limitation as long as it can be made into an intestinal form by applying a coating. In addition, a drug and an ion-exchange resin are combined to form a sustained-release preparation. The ion is gradually exchanged with alkali in the intestinal fluid to release the drug, and to sustain its release using its release property. It can be used without any limitation as long as it is applied to a sexual preparation.

In the present invention, chitin / chitosan is
-FU is preferably used in a combination of 3 times to 240 times by weight, more preferably 12 times to 60 times in combination with FU. If the weight ratio of chitin / chitosan to 5-FU is less than three-fold, the retention of 5-FU in the intestinal tract, and thus the ability of 5-FU to remain in the blood at lower concentrations and longer, will decrease, and the -It is not preferable from the viewpoint of preventing the side effects of FU. On the other hand, if the ratio is 240 times or more, the amount of waste chitin / chitosan is undesirably increased, if limited to the prevention of side effects. However, this does not apply if health effects such as hypertension, hyperlipidemia, appetite enhancement, obesity countermeasures, and stiff shoulder / low back pain are also desired.

The shapes of 5-FU and chitin / chitosan used in the present invention are not particularly limited, but in order to use them as sustained-release sustained-release preparations such as capsules and tablets, both may be powders. Convenient and convenient.

[0036]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. All percentages in Examples and Comparative Examples are by weight unless otherwise specified.

Experimental materials: Chitin / chitosan used was a product of Fuji Bio Co., Ltd., but the chitosan was converted to a hydrochloride and had an intrinsic viscosity of about 100 cP. The average molecular weight by the viscosity method was about 100-200 kilodaltons and the degree of acetylation was 14%. Hereinafter, it is simply abbreviated as chitosan for the sake of simplicity, but more precisely, it is composed of 86% chitosan and 14% chitin. Chitosan was suspended in distilled water and used as it was. For 5-FU, a product of Wako Pure Chemical Industries was used. [6- ³ H] 5-FU (specific activity: 462.5
GBq / mmol) is NEN Life Science
eProducts (Boston, Mass., USA) chemicals were used. The mouse lymphocyte separation medium (Lympholytes-Mouse) used the product of Dainippon Pharmaceutical. Fluorescein isothiocyanate (FITC) -labeled anti-mouse CD8 and phycoerythrin (PE) -labeled anti-mouse NK1.1 were Serotec
Ltd. (Oxford, UK). Other chemicals were reagent grade. Sarcoma 180 cancer cells used were stored in Ehime University, Faculty of Medicine, and Medical Chemistry Department II.

Experimental animal: Male ICR strain mouse (6 years old)
Weeks) and male C57BL / 6 strain mice (5 weeks after birth) were purchased from Clare Japan and Charles River Japan. Both mice were incubated at 25 ± 1 ° C., 6
They were bred for 1 week in a room maintained at 0% RH and had free access to water and food. The room was lit at 7 am and lit for 12 hours a day.

Measurement of antineoplastic activity and side effects: On day 0, 1.5 × 10 ⁶ solid sarcoma 180 cancer cells were implanted subcutaneously on the back of the mouse. 12 hours after transplantation of the cancer cells, 5-FU (1.25 mg) was used as a comparative example.
/ Kg body weight) or 5-FU (1.25
mg / kg) + chitosan (150 mg / kg, 375 m
g / kg or 750 mg / kg body weight) was dissolved and suspended in distilled water, respectively, and twice a morning and evening (7 am and 7 pm).
) For 8 consecutive days. Control mice received only distilled water orally according to the same schedule.

On day 9, heparin was collected from the vein under diethyl ether anesthesia, and then the cancer, small intestine, liver, adipose tissue and spleen were excised and weighed for evaluation of antitumor activity and side effects. The blood sample was refrigerated in a test tube containing heparin, and the number of leukocytes was measured using a Coulter counter (manufactured by Nippon Kagaku Kiki Co., Ltd.).

Measurement of Sucrase Activity of Small Intestinal Mucosa of Sarcoma 180 Tumor-bearing Mouse The small intestine was washed with cold 0.9% NaCl to remove the contents. Then, the mucous membrane was scraped off with a slide glass,
Using a tron homogenizer (Kinematica, Switzerland), the mixture was homogenized to a final volume of 2 ml with 80 mmol (mM) of phosphate buffer (pH 7.0). Sucrose activity was determined by the method of Kimura et al. (Chem. Ph.
arm. Bull. , 30 , 4444-447 (19
82) and Planta Med. , 50 , 465-4
68 (1984)). In short, the assay is 5 mM sucrose in a total volume of 0.5 ml,
A reaction mixture containing homogenate (50 microliters) in 80 mM sodium phosphate buffer (pH 7.0) was performed at 37 ° C. for 30 minutes. Released glucose was quantified using a glucose oxidase reagent.

Determination of 5-FU Phosphate Esters Oxygen solutions to be assayed were prepared from Sarcoma 180 cancer cells. Cancer cell pellet was added with 2-mercaptoethanol in 5
Homogenized with 4 volumes of 5 mM Tris hydrochloride (pH 8.0) containing mM. The homogenate was centrifuged at 105,000 g at 4 ° C. for 60 minutes, and the supernatant fraction was subjected to a method such as in a pond (Jpn. J. Cancer Res., 70 ,
353-359 (1979)).

Statistical analysis and pharmacokinetics All values were expressed as mean ± standard deviation (SE).
Statistical analysis was performed with Dunnet's test to determine significance using Super ANOVA software. Pharmacokinetic parameters were determined using PK for Mac ver 2.0 software (Meiji Pharmaceutical Pharmaceutical Research Center).

Examples and Comparative Examples The following measurements were carried out using 5-FU alone as a comparative example and using 5-FU and chitosan as examples.

In the sarcoma 180 tumor-bearing mouse,
Measurement of FU-derived diarrhea appearance day On day 0, 1 × 10 ⁶ solid sarcoma 180 cancer cells were implanted subcutaneously on the back of the mouse. 12 hours after transplantation of the cancer cells, 5-FU (25 mg / kg body weight) or 5-FU (12.5 mg / kg body weight) + chitosan (15
0 mg / kg or 750 mg / kg body weight) was dissolved and suspended in distilled water, respectively, and administered twice daily in the morning and evening (7 am and 7 pm) until diarrhea appeared. Control mice received only distilled water orally according to the same schedule.

[0046] C57BL / 6 T cell population in mice ^{(CD 8+} and NK1.1 ⁺ T cells) and white blood cell count measured 5-FU (12.5mg / kg body weight) or 5-FU (1
2.5 mg / kg body weight) + chitosan (150 mg / kg
Or 750 mg / kg body weight) were dissolved and suspended in distilled water, respectively, twice a day and evening (7 am and 7 pm), 7
Oral administration for one day. Control mice received only distilled water orally according to the same schedule. On day 8, the neck of the mouse was dislocated and killed, and the spleen was quickly removed. The organs were gently scratched using cold phosphate buffered saline (PBS, pH 7.4) to dislodge the cells with dissecting forceps. 5 ml of cell suspension is added to 5 ml of L
placed on ympolytes-Mouse, 1500
Centrifuged at g for 30 minutes. The leukocyte band was collected at the interface and the cells were rinsed three times with PBS (pH 7.4). Leukocyte counts were determined using a Coulter counter. After adjusting the cell concentration to 2 × 10 ⁶ cells / 100 microliter, 10 microliter of FITC-labeled anti-mouse CD
8 or PE labeled anti-mouse NK1.1 was added to 100 microliters of cell suspension. After incubation at 4 ° C. for 30 minutes, leukocytes were rinsed three times with 1 ml of PBS,
Centrifuge at 0 g for 5 minutes, then CD ⁸⁺ and NK1.
¹⁺ T cell populations were collected using FACS Calibur (Bect
on & Dickinson, Mountain View, CA).

Measurement of 5-FU in mouse blood 5-FU (12.5 mg / kg) or 5-FU (12.
Mice were orally administered 5 mg / kg) + chitosan (750 mg / kg). Blood collected from the vein under anesthesia at 5, 15, 30, 60, 90 and 120 minutes after administration of 5-FU and 5-FU + chitosan was centrifuged at 1500 g for 10 minutes at 4 ° C. to separate serum. . Serum samples (1 ml) were shaken with 5 ml of chloroform for 10 minutes. Mix the mixture at 4 ° C for 10 minutes 1
The organic phase was removed by centrifugation at 500 g. 5-FU in the residual aqueous phase was extracted twice with 4 ml of ethyl acetate,
The ethyl acetate extract was concentrated at 40 ° C. under a stream of nitrogen gas.
The residue was then dissolved in distilled water and the 5-FU content was determined by reverse-phase high performance liquid chromatography (HPLC) under the following chromatographic conditions. Monitoring wavelength 280n
m, flow rate 1 ml / min, mobile phase 5 mM tetrabutylammonium solution containing 2% methanol adjusted to pH 5 with 5 mM dilute formic acid.

Cancer in sarcoma 180 tumor-bearing mice,
5 to PCA soluble and RNA fraction of small intestine and spleen tissue
-FU measuring uptake [6- ³ H] 5-FU (12.5mg / kg, 18.
5 MBq / kg) or [6- ³ H] 5-FU (12.
5 mg / kg, 18.5 MBq / kg) + chitosan (1
50 mg / kg or 750 mg / kg) was administered to Sarcoma 180 tumor-bearing mice 9 days after transplantation into cancer cells. 1
Alternatively, 4 hours later, the mice were sacrificed, and the cancer, small intestine and spleen tissues were excised, quickly frozen, and stored at -80 ° C until use. Cancer tissue (500 mg), small intestine (1.0 g) and spleen (100 mg) were homogenized with 5 volumes of 10% perchloric acid (PCA), and centrifuged at 1500 g for 10 minutes at 4 ° C. The radioactivity of 5-FU, a mixture of 5-FU and its nucleosides and nucleotides, incorporated into PCA-soluble fractions of cancer, small intestine and spleen was measured using a liquid scintillation counter. The radioactivity incorporated into the RNA fraction present in the PCA-precipitable material was slightly modified for the quantification of the amount of 5-FU incorporated into the RNA by a method such as Schneider et al. (J. Biol Chem. ,
164 , 747-751 (1946)).

[0049] 5-
Antitumor Activity of Combination of FU and Chitosan FIG. 1 shows changes in body weight of each group during the experimental period. Sarcoma 180 tumor-bearing mice (control group), 5-FU treated group and 5
There was no significant difference between the -FU + chitosan treatment groups (see FIG. 1). As shown in FIG. 2, 5-FU is Zarcoma 1
The tumor weight of 80 tumor-bearing mice was reduced. 5-FU + chitosan (150 mg, 375 mg and 750 mg / body weight ×
2 times / day) also suppressed cancer growth (see FIG. 2). However, the 5-FU treatment group and 5-FU + chitosan (1
50 mg, 375 mg and 750 mg / kg x 2 times /
D) There was no significant difference between the groups. These results indicate that chitosan maintains the anti-tumor activity of 5-FU.

In the sarcoma 180 tumor-bearing mice,
Effects of chitosan on FU-derived bone marrow disorders and gastrointestinal disorders Next, the effects of chitosan on some 5-FU-derived side effects: bone marrow disorders, gastrointestinal disorders and reduced immune function were tested. Treatment with 5-FU reduced white blood cell count and small intestine weight (see FIGS. 3 and 4). These facts
5-FU has been shown to cause bone marrow and gastrointestinal disorders. As shown in FIG. 3, the leukocyte reduction caused by 5-FU administration was reduced by chitosan (150 mg or 750 mg).
(mg / kg x 2 times / day). The small intestinal weight loss caused by 5-FU was also suppressed by chitosan (750 mg / kg x 2 times / day) (see Figure 4). Sucrase activity of small intestinal mucosa was reduced by oral administration of 5-FU in Sarcoma 180 tumor-bearing mice (see FIG. 5), indicating that 5-FU damages small intestinal mucosa. 5-FU + chitosan (150
mg, 375 mg or 750 mg / kg x 2 times / day)
Significantly suppressed the decrease in sucrase activity in the small intestinal mucosa. In addition, as shown in Table 1, a two-fold higher dose (25 mg
/ Kg x 2 times / day) of 5-FU resulted in the development of diarrhea after 5 days. The occurrence of diarrhea caused by 5-FU (25 mg / kg x 2 times / day) was delayed by oral administration of chitosan (150 mg or 750 mg / kg x 2 times / day) (see Table 1).

[0051]

[Table 1]

Inhibition of reduced immunity of 5-FU-derived spleen
Effect of chitosan 5-FU is immunosuppressive and immune with reduced spleen and thoracic weight
Many researchers report causing epidemics.
You. As shown in FIG. 6, 5-FU (12.5 mg / kg
× 2 times / day), the weight loss of the spleen
Chitosan (750m) in Lucoma 180 tumor-bearing mice
g / kg x 2 times / day). I
Thus, the immune dysfunction caused by 5-FU
To clarify prevention by chitosan, C57BL
5-FU (12.5 mg /
kg × 2 times / day) CD of spleen after administration⁸⁺And NK. 1.
1⁺ Study the effect of chitosan on T cell and lymphocyte numbers
Studied. As shown in Table 2, chitosan (150 mg and
750 mg / kg x 2 times / day) is C57BL / 6 mouse
Lymphocytes and CD⁸⁺And NK. 1.1⁺T
There was no effect on cell number or spleen weight. On the other hand, spleen
CD of⁸⁺The number of T cells and lymphocytes is
It decreased more significantly. Spleen weight and N. K. 1.1
⁺The number of T cells is also not significantly reduced, but 5-F
It tends to decrease with the administration of U. Chitosan is 5-
Lymphocytes CD produced by FU⁸⁺Cells and
N. K. 1.1⁺The decrease in the number of T cells was suppressed (Table 2).
reference). In addition, spleen weight and lymphocytes, CD⁸⁺as well as
N. K. 1.1⁺Number of T cells, increase not significant
Increased by chitosan (750 mg / kg x 2 times / day)
(See Table 2).

[0053]

[Table 2]

5-FU concentration in mouse plasma after oral administration of 5-FU in combination with chitosan As shown in FIG.
5 minutes and 15 minutes after oral administration of FU (12.5 mg / kg)
In minutes, the drug 160 ng / ml and 200
ng / milliliter, then dropped rapidly. Table 2
Is T _1/2 , Tmax, Cm for 5-FU administration
ax and AUC (0 to 120 minutes) are each 30.9
Min, 10.1 min, 195.4 ng / ml and 1
68.5 ng · h / ml.
On the other hand, the concentration of 5-FU in the blood of mice is 5-FU (1
2.5, 5, 30 and 60 minutes after the combined administration of 2.5 mg / kg) + chitosan (750 mg / kg)
2 ng / ml, 129 ng / ml, 1
They were 10 ng / ml and 69 ng / ml (see FIG. 7). T for 5-FU + chitosan
_1/2 , Tmax, Cmax and AUC (0 to 120
Pharmacokinetic parameters such as
6 minutes, 7.5 minutes, 136.4 ng / ml and 1
It was 59.2 ng · h / milliliter (see Table 3). Therefore, Cmax of the 5-FU concentration of blood is
Although decreased by the combined oral administration of 5-FU and chitosan,
The AUC of blood 5-FU concentration (0-120 min) was not affected.

[0055]

[Table 3]

PCA soluble and RNA fraction of cancer in Sarcoma 180 tumor-bearing mice, 5-F in small intestine and spleen
Effect of Chitosan on U Uptake As shown in Table 4, chitosan (750 mg / kg)
^{3 H-5-FU (12.5mg} / kg, 18.5MB
q / kg) 1 hour and 4 hours after oral administration of PC
A soluble and RNA fractions and ³ H-5-F in the small intestine
U uptake was suppressed. On the other hand, PCA solubility and R
The incorporation of ³ H-5-FU into the NA fraction was not suppressed by administration of chitosan (150 mg and 750 mg / kg) (see Table 4). These results indicate that chitosan does not suppress 5-FU uptake in cancer cells but suppresses 5-FU uptake in normal cells.
It suggests preventing 5-FU-induced gastrointestinal and immunocompetent disorders without reducing antitumor activity.

[0057]

[Table 4]

It should be noted that chitosan should be administered in the form of an intestine such as a capsule in order to prevent unnecessary waste due to chlorine in the gastrointestinal tract. These mice were small mice weighing -40 g and were orally administered in the form of a suspension because they were not feasible.

The results shown in the above examples show that the combination of chitin and chitosan with 5-FU can maintain the leukocyte reduction and the small intestinal mucosal enzyme by 5-FU while maintaining or enhancing the antitumor effect of 5-FU alone. It was shown to prevent a decrease in activity. In addition, the appearance of diarrhea due to 5-FU administration was also reduced by chitin /
It was shown to be delayed by chitosan administration. In addition, it was shown that administration of chitin / chitosan prevented 5-FU from decreasing the immune function. First, the present inventors assumed that chitin
It is reported that chitosan has an NK activity enhancing effect. In addition, they have found that chitin / chitosan has an effect of enhancing NK-like activity of lymphocytes between intestinal epithelial cells. Therefore, the above results suggest that the effect of chitosan is due to the selective suppression of 5-FU incorporation into small intestine and spleen tissue.

[0060]

According to the present invention, chitin / chitosan and 5
Since the combination of -FU is useful for preventing leukopenia, reduction of small intestinal mucosal enzyme activity and reduction of immune function by 5-FU, chitin / chitosan may be used as a side effect preventive agent for cancer chemotherapeutic agents comprising 5-FU With 5 in the blood
-Lowering the FU concentration allows the cells to be taken up by cancer cells but not easily taken up by normal cells, and the presence of 5-FU for a longer period of time may increase the effect on cancer cells. Also occurs.

[Brief description of the drawings]

FIG. 1 is a graph showing changes in body weight of Sarcoma 180 tumor-bearing mice due to the combined use of 5-FU and chitin / chitosan. The results are shown as the mean ± standard deviation of 9 mice (the same applies to FIGS. 2 to 6).は: Sarcoma 180 tumor-bearing mouse (control), は: 5-FU (12.5 mg / kg × 2 times / day), □: 5-FU + chitosan (150 mg / kg ×
2 times / day) is 5-FU + chitosan (375 mg / kg)
× 2 times / day), Δ represents 5-FU + chitosan (750 mg / day)
kg x 2 times / day).

FIG. 2 is a bar graph showing the effect on antitumor activity of a combination of 5-FU and chitosan. 1 is Zarcoma 18
0 tumor-bearing mice (control), 2 was 5-FU (12.5
mg / kg × 2 times / day) administration, 3 was 5-FU + chitosan (150 mg / kg × 2 times / day) administration, 4 was 5-FU +
Chitosan (375 mg / kg x 2 times / day) administration, 5 for 5
In the case of -FU + chitosan (750 mg / kg x 2 times / day) administration.

FIG. 3 is a bar graph showing the inhibitory effect of chitosan on bone marrow injury by 5-FU. 1 is a Sarcoma 180 tumor-bearing mouse (control), and 2 is 5-FU (12.5 mg).
/ Kg × 2 times / day), 3 was 5-FU + chitosan (1
Administration of 50 mg / kg × 2 times / day, 4 administration of 5-FU + chitosan (375 mg / kg × 2 times / day), administration of 5
This is the case of administration of U + chitosan (750 mg / kg × 2 times / day).

FIG. 4 is a graph showing the inhibitory effect of chitosan on gastrointestinal disorders (reduction of small intestinal weight) by 5-FU. 1 is a Sarcoma 180 tumor-bearing mouse (control), 2 is 5-F
U (12.5 mg / kg x 2 times / day), 3 is 5-FU +
Chitosan (150 mg / kg x 2 times / day), 4 is 5-F
U + chitosan (375 mg / kg x 2 times / day), 5 for 5
In the case of -FU + chitosan (750 mg / kg x 2 times / day) administration.

FIG. 5 is a bar graph showing the inhibitory effect of chitosan on gastrointestinal disorders (reduction of sucrase activity in small intestinal mucosa) by 5-FU. 1 is a Sarcoma 180 tumor-bearing mouse (control), and 2 is 5-FU (12.5 mg / kg ×
3 times 5-FU + chitosan (150 mg / k)
g × 2 times / day), 4 is 5-FU + chitosan (375 mg
/ Kg × 2 times / day), 5 is 5-FU + chitosan (750
mg / kg x 2 times / day).

FIG. 6 is a bar graph showing the inhibitory effect of chitosan on immune-induced organ damage (reduction of spleen weight) by 5-FU. 1 is a Sarcoma 180 tumor-bearing mouse (control),
2 is 5-FU (12.5 mg / kg × 2 times / day), 3 is 5-FU + chitosan (150 mg / kg × 2 times / day),
4 is 5-FU + chitosan (375 mg / kg × 2 times /
Days), 5 is 5-FU + chitosan (750 mg / kg × 2
Once / day) administration.

FIG. 7 is a graph showing changes in the concentration of 5-FU in plasma of mice after oral administration of 5-FU and chitosan in combination. The results are shown as the mean ± standard deviation of 5 mice. * 5
-Significantly different from FU alone administration, P <0.05;
-FU (12.5 mg / kg), and ● represents 5-FU +
Chitosan (750 mg / kg).

FIG. 8 is a schematic sectional view of a double capsule.

FIG. 9 is a schematic cross-sectional view of a two-chamber fractionation type capsule, in which (a) shows a two-end cap type capsule and (b) shows a one-end cap type capsule.

[Explanation of symbols]

 1 Movable intermediate partition plate

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4C076 AA29 AA36 AA53 AA54 AA56 AA63 BB01 CC27 EE37 FF25 FF31 4C086 AA01 AA02 BC43 EA23 MA02 MA05 MA35 MA37 MA43 MA52 NA06 NA13 ZB26

Claims (21)

[Claims] 1. A method for the anticancer treatment of oral administration of 5-fluorouracil and chitin / chitosan in combination, wherein at least chitin / chitosan is administered in intestinal form. 2. The method of claim 1, wherein said enteric form is a capsule. 3. The method according to claim 1, wherein the capsule is a hard capsule or a soft capsule. 4. The capsule according to claim 1, wherein the capsule is a double capsule, and the inner capsule is filled with 5-fluorouracil, and the outer capsule surrounding the inner capsule is filled with chitin / chitosan. The method of claim 1. 5. The method according to claim 1, wherein the capsule is a two-compartment capsule, and each compartment is separately filled with 5-fluorouracil or chitin / chitosan. 6. The method of claim 1, wherein said enteric form is a tablet. 7. The method of claim 1, wherein said enteric form is a resinate. 8. The method according to claim 1, wherein 5-fluorouracil and chitin / chitosan are co-administered. 9. The method according to claim 1, wherein 5-fluorouracil is orally administered after oral administration of chitin / chitosan. 10. The method according to claim 1, wherein chitin / chitosan is used in combination with 5-fluorouracil in a weight ratio of 3 to 240 times. 11. The method according to claim 1, wherein chitin / chitosan is used in combination with 5-fluorouracil in a weight ratio of 12 to 60 times. 12. A combination preparation of 5-fluorouracil and chitin / chitosan prepared in an enteric form for use in the method for preventing side effects of 5-fluorouracil according to claim 1. 13. The combination preparation according to claim 12, wherein the combination preparation of 5-fluorouracil and chitin / chitosan in an enteric form is a capsule. 14. The combination according to claim 12, wherein the capsule is a hard capsule or a soft capsule. 15. The capsule according to claim 5, wherein the internal capsule is 5
The combination according to any one of claims 12 to 14, wherein the outer capsule surrounding the inner capsule in the fluorouracil-filled capsule is a chitin-chitosan filled capsule. 16. The capsule according to any one of claims 12 to 14, wherein the capsule is a two-chamber fraction capsule in which 5-fluorouracil is separately filled in one compartment and chitin / chitosan is separately filled in the other compartment. The combination as described. 17. The combination preparation according to claim 12, wherein the combination preparation of 5-fluorouracil and chitin / chitosan in an enteric form is a tablet. 18. The combination according to claim 12, wherein the combination of 5-fluorouracil and chitin / chitosan in the enteric form is a resinate. 19. The intestinal form of the combination preparation contains chitin.
13. The combination preparation according to claim 12, wherein chitosan is added in a 24- to 3000-fold weight ratio with respect to 5-fluorouracil. 20. The intestinal form-containing composition contains chitin.
Chitosan was added to 5-fluorouracil in a weight ratio of 120.
13. The compounding agent according to claim 12, which is added by a factor of 1 to 600 times. 21. The combination preparation according to claim 12, wherein said 5-fluorouracil and chitin / chitosan are both powders.