JPH0532545A - Anti-neoplastic agent based on anti-promoter effect - Google Patents

Abstract

PURPOSE:To provide an anti-neoplastic agent having a remarkable anti- neoplastic effect, low in toxicity to normal cells, free from mutagenicity and showing its effect owing to an anti-promoter effect. CONSTITUTION:An anti-neoplastic agent containing a 3-nitro-2,4,6- trihydroxybenzoyl ester represented by the formula [X is H or CH3; Y is phenyl having one or more substituents (halogen, trihalomethyl, 1-4C alkyl or 1-4C alkoxy); (n) is 2, 3, or 4] as the active component and showing an anti-neoplastic effect owing to an anti-promoter effect. Since the anti-neoplastic agent has the above-mentioned effect, it can be safely used over a long period for the purpose of preventing a cancer in the case there is a fear of growth of a cancer, treating cancer by its anti-neoplastic effect in the case a cancer is recognized and preventing of relapse of a cancer after removal of the cancer tissue. The dosage of this anti-neoplastic agent for an adult is generally 1-100mg per 1kg body weight a day. Administration is carried out preferably by oral route, subcutaneous injection, intravenous injection, local injection, etc., and the administration method in not limited to special methods.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antitumor drug having an active ingredient having an antitumor activity based on an antipromoter action.

[0002]

2. Description of the Related Art As a cancer development mechanism, proliferation of undifferentiated cells, expression of an oncogene by an exogenous factor, cell transformation by a virus or the like (introduction of oncogene) is known.

Of these, the following is known as an example of the case of carcinogenesis under the influence of an exogenous factor. That is, a small amount of a carcinogen (initiator) is applied once to the back skin of a mouse. Furthermore, if croton oil or its effective component, 12-O-tetradecanoylphorbol-13-acetate (TPA), is continuously applied to this portion, a tumor develops. In this case, application of the initiator alone still does not lead to carcinogenesis, but this causes the cells to undergo a genotoxic effect. This cell is called a latent tumor cell. Substances such as croton oil, TPA, etc. are substances that promote the conversion of these latent tumor cells into tumor cells and promote tumor formation, and are called oncogenic promoters.

On the other hand, as an antitumor drug, an antitumor chemotherapeutic drug and an antitumor immunochemotherapy drug have been widely used. Antitumor chemotherapeutic agents include alkylating agents, antimetabolites, antitumor antibiotics, and antitumor immunochemotherapeutic agents include immunoinactive therapeutic agents.

[0005]

However, the above-mentioned antitumor chemotherapeutic agents, which are widely used as antitumor agents, are generally highly toxic and have the drawback of exhibiting mutations and other serious side effects.

In view of the above circumstances, the present invention provides an antitumor drug which is less toxic and does not show mutagenicity.

[0007]

[Means for Solving the Problems] As a result of extensive studies to solve the above problems, 3-type compounds represented by the general formula (I) shown in Chemical formula 2 already proposed as a photosynthesis inhibitor by the applicant of the present invention are described. The nitro-2,4,6-trihydroxybenzoyl esters (I) (Japanese Patent Application No. 2-157632) have the above-mentioned anti-promoter action and an effect of preventing tumor development. Found out.

[0008]

[Chemical 2] (In the formula, X represents hydrogen or a methyl group, Y is selected from the group consisting of a halogen group, a trihalomethyl group, an alkyl group having 1 to 4 carbon atoms, and an alkoxy group having 1 to 4 carbon atoms. Represents a phenyl group having at least one substituent, and n represents an integer of 2 to 4.) As described in the Examples below, the above 3-nitro-2,4,6-trihydroxybenzoyl esters are used. (I) has an effect of suppressing the action of 12-O-tetradecanoylphorbol-13-acetate (TPA) known as a strong oncogenic promoter. That is, Epstein Bar Virus
Barr Virus; EBV) In the initial antigen (EA) production inhibition test, it was confirmed that this compound (I) inhibits the expression of EBV-EA produced by TPA and has antitumor activity. this
EBV-EA has excellent in vivo correlation with extremely high correlation with in vivo anti-tumor compound assay methods such as the two-step test for mouse skin carcinogenesis.
It is an antitumor compound assay system.

Therefore, this compound (I) can exert an antitumor effect similar to that observed in the action of vitamin A acid and its related compounds. That is, this compound (I)
Thus, an antitumor effect can be obtained by promoting the differentiation of a precancerous tissue into a normal tissue or by preventing the conversion of precancerous cells into malignant cells.

Incidentally, TPA is one of the promoters having the strongest carcinogenic promotion action. Therefore, since the action of TPA can be suppressed, it seems that the same effect can be obtained for other promoters. As such other promoters, for example, HHPA (12-o-hexadecanoyl-16-hydroxy-phorbol-13-acetate), mezeline, teleocidin, etc. are known (medical history, 134, 13). , P1162, 1980).

The daily dose of the antitumor drug of the present invention for an adult is appropriately determined depending on the symptoms of the patient, but is usually 1 mg to 100 mg per 1 kg of body weight.

[0012] The route of administration is oral, subcutaneous injection, intravenous injection,
Local injection and the like are preferable, but not limited thereto.

Further, the dosage form to be administered can be prepared into powder, granules, tablets, capsules, injections, external preparations and the like by a conventional method together with a pharmaceutically acceptable excipient. ..

[0014]

The results of examining the effects of the antitumor drug of the present invention will be described below.

In order to confirm that the compound (I) of the present invention is useful as an antitumor drug,
The initial antigen production suppression test <A> shown below was performed.

<A> Epstein Barr Virus (Ep
ste--in Barr Virus; EBV) This test is based on the method of Ito et al. (Cancer Letters, 13 (1981),
29-37).

[Preparation of Medium] Powder RPMI-1640 medium is shown in Table 1.
The mixture was mixed with the composition shown in (1), desalted, and then dissolved in distilled water that was distilled twice. At this time, add the dry ice just before the little finger to adjust the pH to 6.
It was lowered to about 0 to facilitate dissolution. After the powder was completely dissolved, sodium bicarbonate (0.56 g / 1000
ml) was added with penicillin G potassium (200,000 units) and streptomycin sulfate (250 mg) as antibiotics.
This solution was sterilized and filtered to obtain RPMI-1640 medium. Fetal bovine serum (FBS) was added to this medium at 8% for cell culture and 4% for EBV-EA production inhibition test, and used as a basal medium in the experiment.

[0018]

[Table 1] [Cultivation of Raji cells] Large cells, which are Epstein-Barr virus (EBV) early antigen (EA) production indicator cells, are cultured. Polystyrene 270 ml volume (75 cm
₂ ) Using a cell culture flask, RPMI-1 containing 8% FBS
37 in a CO ₂ incubator using 640 medium
Culturing was carried out at 5 ° C in the presence of 5% CO ₂ . Number of cells per ml
When the number of cells reached 1 to 2 × 10 ^{6, the} cells were diluted about 5 times and subcultured. This transplanting operation was performed once every 3 to 4 days. For the EBV-EA production suppression test, cells at the stage where the number of cells became 1 to 2 × 10 ⁶ per ml were used.

[Storage and Thawing of Large Cells] Large cells were appropriately frozen and stored by the following method. That is, when the number of cells reached 1 to 2 × 10 ⁶ cells / ml, the culture medium containing cells was centrifuged at 150 × g for 10 minutes. After removing the supernatant, the cells were treated with dimethyl sulfoxide (DMSO).
% Of 8% FBS / RPMI-1640 medium was added, and the cells were suspended so that the number of cells was 1.5 × 10 ⁷ cells per ml, and the cells were frozen and stored at −80 ° C. Frozen cells were thawed on a water bath at 30 ° C. After thawing, the cells were washed three times with 8% FBS / RPMI-1640 medium, and then cultured in 5% volume of 8% FBS / RPMI-1640 medium when stored.

[Preparation of test compound solution] The test compound solution was prepared immediately before the experiment. DMSO was used as a solvent, and the final concentration of DMSO in the culture was adjusted to 1.0% or less, which does not affect early antigen production.

[Preparation of TPA solution and n-butyric acid solution] TPA, which is a carcinogenic promoter, was dissolved in DMSO at a concentration of 1 mg / ml and stored as a stock solution at -20 ° C.
It was diluted with RPMI-1640 medium to a concentration of 20 ng / ml before use. N-butyric acid, which is used to increase the EA expression rate by TPA and detection sensitivity, is used as a 0.5 M sterile solution at 4 ° C.
Saved in.

[EBV-EA expression suppression test] RP containing 4% FBS
N-Butyric acid (4 mM) and TPA (20 ng / ml) were added to MI-1640 medium (1 ml per tube), and a predetermined amount of a test compound solution was added to a plastic test tube to prepare an assay medium. Large cells with indicator cells for search, which had been previously cultured in 8% FBS / RPMI-1640 medium, were collected by centrifugation, and this was added to the assay medium so that the number of cells per ml was 1 × 10 ^6. Suspended in. Add this suspension to 37 ° C, 5%
After culturing in the presence of CO ₂ for 48 hours, centrifugation was performed to remove the supernatant, and phosphate buffered saline (PBS (-) (KCl 200 mg / l, KH ₂ PO
₄ 200 mg / l, NaCl 8 g / l, Na ₂ KHPO ₄ 1.15 g / l) 0.1 ml. This cell suspension was used for EBV-EA expression suppression test and indicator cell viability test.

[Measurement of EA-expressing cell rate] The EBA-EA-expressing cell rate was measured by the indirect fluorescent antibody method as shown below.
Cells reacted in 1.0 ml assay medium at 150 xg 1
After centrifuging for 0 minutes and removing the supernatant, 0.1 ml of PBS (-) was added to suspend the cells. This suspension was smeared on a fluorescent-free slide glass, air-dried, and then the slide glass was placed in acetone for 10 minutes.
Immerse for a minute to completely fix the cells on the surface of the glass slide,
This was used as a sample for microscopy.

As a primary antibody, the serum of nasopharyngeal carcinoma (NPC) patients with a high EA antibody titer of EBV [EA
(+), Viral capsid antigen (VCA) (+)]
Was preliminarily diluted with PBS (-) to obtain the optimal antibody titer for the reaction. After placing this on each spot on a slide glass, it was placed in a petri dish containing a paper towel containing water, and an antigen-antibody reaction was carried out at 37 ° C for 45 minutes. After the reaction is complete, slide the slide glass into approx. 100 ml of PBS (-).
Each container was washed by shaking for 30 seconds. After performing this washing operation twice, the slide glass was air-dried, and then FITC (20% diluted with PBS (-) was used as the secondary antibody.
Fluorescein isothiocyanate) labeled human IgG antibody (goat) was placed on the same spot, and the same as for the primary antibody reaction.
The reaction was performed at 45 ° C for 45 minutes. After completion of the reaction, the cells were washed twice with PBS (-), encapsulated with PBS (-) containing 20% non-fluorescent glycerin, and observed with a fluorescence microscope. EA-producing cells emit FITC fluorescence and can be easily identified. Positive cells to which each test compound was added were observed using EA-expressing cells (positive cells) to which only TPA was added as a control, and the ratio was expressed as a percentage and recorded as an inhibitory effect. Minimum for each process
250 cells were observed and performed in duplicate, and the results are shown as the average value.

[Measurement of cell viability] The cell viability was measured by the trypan blue staining method. That is, PBS (-) containing 0.25% trypan blue in 0.05 ml of cell suspension.
After adding 0.05 ml of the solution and gently stirring, a part of the suspension was placed on a hemocytometer to count the number of viable cells and the number of dead cells stained with trypan blue. The results are shown as an average value in duplicate.

Test compounds B-1 to B-5 shown in Table 2 were tested according to the method described above. The results are summarized in Table 3. Although the test compounds had different activities, it was revealed that each of them was a compound having antitumor activity by suppressing the expression of EBV-EA by TPA which is a tumor promoter. In addition, in Table 2, the side chain represents the side chain at the 1-position of the benzoester in the general formula (I), and in Table 3, the test compound is 1000 times concentration, 500 times concentration relative to the TPA concentration (mol). The test was conducted for the case of concentration, 100 times concentration and 10 times concentration.

[0027]

[Table 2]

[0028]

[Table 3]

[0029]

INDUSTRIAL APPLICABILITY The antitumor drug of the present invention has a high antitumor effect, its toxicity to normal cells is weak, and it is not mutagenic. Therefore, it can be safely used for a long period of time for the purpose of preventing cancer when the occurrence of cancer is suspected, antitumor effect when the existence of cancer is confirmed, and preventing cancer recurrence after excision of cancer tissue.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Harukuni Tokuda 3 Shimogamokitaen-cho, Sakyo-ku, Kyoto City, Kyoto Prefecture (72) Inventor Saisuke Tsubasa 1-2-5-2 Makinohonmachi, Hirakata-shi, Osaka Prefecture (72) Inventor Kozuka Mutsuo 458 Kamirei Nakamachi, Kamigyo-ku, Kyoto-shi, Kyoto (72) Inventor Shigeo Yoshida 3-28-15 Kanui, Nerima-ku, Tokyo (72) Inventor Koichi Yoneyama 4-10-5-515 Yonan, Utsunomiya-shi, Tochigi Prefecture

Claims (1)

Claims: 1. An antitumor drug with an antipromoter action, which comprises an active ingredient represented by the general formula (I) shown in Chemical formula 1. [Chemical 1] (In the formula, X represents hydrogen or a methyl group, Y is selected from the group consisting of a halogen group, a trihalomethyl group, an alkyl group having 1 to 4 carbon atoms, and an alkoxy group having 1 to 4 carbon atoms. Represents a phenyl group having at least one substituent, and n represents an integer of 2 to 4.)