JPH03271222A - Antitumor agent by anti-promoter action - Google Patents

Abstract

PURPOSE:To obtain an antitumor agent, containing a 3-nitro-2,4,6- trihydroxybenzoic acid derivative as an active ingredient, having anti-promoter action and further effects on prevention of tumorigenesis and safely usable for a long period. CONSTITUTION:An antitumor agent containing a compound expressed by formu la I (R is 1-18C straight-chain alkyl, cyclohexyl, substituted phenyl or phenyl-1-4C alkyl) or formula II (R is 1-10C straight-chain alkyl) as an active ingredient. Antitumor effects can be obtained by promoting differentiation of tissues in a precancerous state into normal tissues and preventing conversion of precancer ous cells into malignant cells with the aforementioned compound. The dose of the above-mentioned compound is 1-100mg/g body weight for an adult per day and the administration route thereof is preferably the oral route, subcutane ous injection, intravenous injection or local injection.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] This invention relates to an anti-tumor drug having an active ingredient having anti-tumor activity based on anti-promoter action.

[Prior art]

The mechanisms of cancer development are known to include proliferation of undifferentiated cells, expression of oncogenes by foreign factors, and transformation of cells (introduction of oncogenes) by viruses and the like.

Among these, the following are known as examples of cancers caused by the influence of foreign factors. That is, a small amount of a carcinogenic agent (initiator) is applied to the back skin of a mouse twice. Furthermore, if croton oil or its active ingredient 12-0-tetradecanoylphorbol-13-acetate (TPA) is continuously applied to this area, a tumor will develop.

In this case, application of the initiator alone still does not lead to carcinogenesis, but it does cause a genotoxic effect on the cells. This cell is called a potential tumor cell. Substances such as croton oil and TPA are substances that promote the conversion of potential tumor cells into tumor cells and promote tumor formation, and are called carcinogenic promoters.

On the other hand, antitumor chemotherapy drugs and antitumor immunochemotherapy drugs have been widely used as antitumor drugs. Antitumor chemotherapy drugs include alkylating agents, antimetabolites, and antitumor antibiotics, and antitumor immunochemotherapy drugs include immunoinactive therapeutic drugs.

[Problem to be solved by the invention]

However, the above-mentioned antitumor chemotherapeutic agents that are widely used as antitumor drugs are generally highly toxic and have the disadvantage of causing mutations and other serious side effects.

In view of the above circumstances, the present invention provides an anti-inflammatory drug that is less toxic and exhibits no mutagenicity.

[Means to solve the problem]

As a result of intensive consideration to solve the above issues, we have already found that
3 having the following general formula, which is known as a photosynthesis inhibitor
- Nitro-2,4,8-trihydroxybenzoic acid derivative (I) (Honda et al., 1988 Japanese Society of Agricultural Chemistry Abstracts and Japanese Patent Application Laid-open No. 1-228949) has the above-described anti-promoter action, It has been found that it has the effect of preventing tumor development.

02 (1) (wherein, when X-O, R is straight-chain alkyl and cyclohexyl having from 1 to 18 carbon atoms, substituted phenyl, and phenylalkyl having 1 to 4 carbon atoms, For X-8, R is engineering to 10
3-nitro-2, as described in the Examples below.
The 4,6-dolyhydroxybenzoic acid derivative (1) has the effect of suppressing the action of 12-O tetradecanoylphorbol-13-acetate (TPA), which is known as a strong carcinogenic promoter. That is, Epstein Barr Virus;
In the EBV) early antigen (EA) production inhibition test, this compound (I) inhibited EBV-E produced by TPA.
It was confirmed that it suppresses A expression and has antitumor activity. In addition, in a two-step carcinogenesis test in mouse skin, a
! It was revealed that it suppresses the occurrence of M.

Therefore, this compound (I) can exert an antitumor effect similar to that seen in the action of vitamin A acid and its related compounds. That is, this compound (I) can produce antitumor effects by promoting the differentiation of pre-cancerous tissues into normal tissues or by inhibiting the transformation of pre-cancerous cells into malignant cells.

Note that TPA is one of the promoters with the strongest carcinogenic promotion effect. Therefore, since the action of TPA can be suppressed, it is thought that similar effects can be obtained on other promoters as well. Such other promoters include, for example, HHPA (12-o-hexadecanoyl-16-hydroxy-phorbol-13-acetite), meseraine, teleosidin, etc. (Yakuhin no Ayumi, Vol. 134, No. 13, P11B2.1980
).

The daily dose for an adult adult of the anti-inflammatory drug of this invention is determined as appropriate depending on the symptoms of the patient, but is usually 1 to 100 cm per body weight.

The route of administration is preferably oral, subcutaneous injection, intravenous injection, local injection, etc., but is not particularly limited.

In addition, the dosage form for administration can be prepared into powders, granules, tablets, capsules, injections, external preparations, etc. using a conventional method together with pharmaceutically acceptable excipients.

〔Example〕

Hereinafter, the results of investigating the effects of the anti-inflammatory drug of this invention will be explained.

In order to confirm that the compound of the present invention is useful as an anti-inflammatory drug, the above compound was tested in the following initial antigen production inhibition test <A> and mouse skin carcinogenesis two-stage test <
B> was done.

<A> Epstein-Barr virus (Epste--
(in Barr Virus; EBV) This test was performed using the method of Ito et al. (Cancer Letters).

13 (1981), 29-37).

Preparation of Medium Powdered RPMI-1640 medium was mixed with the composition shown in Table 1, and after desalination was dissolved in twice-distilled water. At this time, dry ice at the tip of the little finger was added to lower the pH to about 6.0 to facilitate dissolution. After the powder is completely dissolved, add sodium bicarbonate (0,56 g / 10100
O) as an antibiotic, penicillin G potassium (20
10,000 units) and streptomycin sulfate (250 units) were added. This solution was sterile filtered and used as RP old-1640 medium. Fetal bovine serum (FBS) was added to this medium at 8% for cell culture and 4% for EBV-EA production suppression test, and used as a basal medium in the experiment.

Culture of Raji cells Raji cells, which are Epstein-Barr virus (EBV) early antigen (EA) producing indicator cells, are cultured. Made of polystyrene 2
Using a 70 ml (75 cJ) cell culture flask,
J including %FBS? Using PMI-J 640 medium,
The cells were cultured in a CO2 incubator at 37°C in the presence of 5% CO2. Cell count is 1-2X106 per ml
When the cells reached 50%, they were diluted approximately 5 times and subcultured. Note that this subplanting operation was performed once every 3 to 4 days. For the EBV-EA production inhibition test, the number of cells is 1 m.
Cells at a stage of 1-2XIO' per liter were used.

Preservation and Thawing of Large Cells Large cells were appropriately cryopreserved by the method shown below. That is, when the number of cells reaches 1 to 2XIO6 per ml, the culture solution containing the cells is diluted at 150X for 10
Centrifuged for minutes. After removing the supernatant, cells were placed in 8% PBS/RPMf containing 10% dimethyl sulfoxide (DMSO).
-1640 medium was added, and the number of cells per ml was 1.5.
The suspension was suspended in a total of 107 cells and stored frozen at -80°C. Thawing of frozen cells was performed on a 30°C water bath. After thawing the cells, add 8% FBS/I? After washing three times with pHl-1840 medium, 5 times the volume of 8% FBS when stored.
/RPMI-1640 medium.

Preparation of test compound solution The test compound solution was prepared immediately before the experiment.

DMSO was used as the solvent, and the final concentration of MSO in the culture solution was
The concentration 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 ml and stored as a stock solution at -20°C.
RPMI-1840 to a concentration of 0 ng/ml.
It was diluted with a medium and used. n-butyric acid, which is used to increase the EA expression rate by TPA and increase the detection sensitivity, has a concentration of 0.
Stored at 4°C as a 5M sterile solution.

EBV-EA expression suppression test N-butyric acid (4mM) and TPA (2ml) were added to RPMI-1640 medium (1ml per tube) containing 4% FBS.
0 ng/ml) was added thereto, and a predetermined amount of the test compound solution was added to the plastic test tube to prepare an assay medium. Large cells containing indicator cells for search, which had been cultured in 8% FBS/RPMI-1640 medium in advance, were collected by centrifugation until the number of cells per ml was 1.
The cells were suspended in an assay medium so that there were 6 xlo cells. After culturing this suspension at 37°C in the presence of 5% CO□ for 48 hours, centrifugation was performed to remove the supernatant, and phosphate buffered saline (
PBS(-)(KC) 200mg/I, KH2PO4
200■/I, NaCl3 g/l, Na2KHPO4
1.15 g/I) in 0.1 ml. This cell suspension was used for an EBV-EA expression inhibition test and an indicator cell viability test.

Measurement of the percentage of cells expressing EA The percentage of cells expressing EBA-EA was measured by indirect fluorescent antibody method as shown below. Cells reacted with 1.0 ml of assay medium were centrifuged at 15DXg for 10 minutes, and after removing the supernatant, 0.1 ml of PBS (-) was added to suspend the cells. This suspension was smeared onto a non-fluorescent slide glass.
After air drying, the slide glass was immersed in acetone for 10 minutes to completely fix the cells on the slide glass surface, and this was used as a specimen for microscopy.

- As the next antibody, use nasopharyngeal cancer with high EBV HA antibody titer (
NPC; nasopharyngeal carcin
oma) patient serum [EA(+), cup cytoantigen (v
iral capsld antigen; VCA)
(+)] was previously diluted with PBS (-) to give 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 vapor towel soaked with water, and an anti-antigen reaction was performed at 37°C for 45 minutes. After the reaction, the slide glasses were immersed in approximately 100 ml of PBS(-), and 3
Washing was performed by shaking for 0 seconds. After performing this washing operation twice, the slide glass was air-dried, and then, as a secondary antibody, FITC (fluorescein isothiocyanate)-labeled human IgG antibody (goat) diluted 20 times with PBS (-) was placed on the same spot. - The reaction was carried out at 37°C for 45 minutes in the same manner as the next antibody reaction.

After the reaction was completed, the cells were washed twice with PBS(-), mounted with PBS(-) containing 20% non-fluorescent glycerin, and observed under a fluorescence microscope. EA-producing cells can be easily identified because they emit PITC fluorescence. Using EA-expressing cells (positive cells) to which only TPA was added as a control, positive cells to which each test compound was added were observed, and the ratio was expressed as a percentage and recorded as an inhibitory effect. For each treatment, a minimum of 250 cells were observed, performed in duplicate, and results are expressed as the average value.

Measurement of cell viability Cell viability was measured by trypan blue staining. That is, 0.05 ml of PBS(-) solution containing 0.25% trypan blue was added to 0.05 ml of cell suspension.
After stirring gently, a portion of the suspension was placed on a hemocytometer, and the number of live cells and the number of dead cells stained with trypan blue were counted. The results are shown as the average value of two series.

The above results are summarized in Tables 2 and 3.

Although the test compounds differed in their activity, it was revealed that all of them suppressed EBV-E^ expression induced by TPA, which is a carcinogenic promoter, and had antitumor properties.

In addition, in the table, the test compound is 1000 times the TPA concentration (mol), 500 times the concentration, and io.

Tests were conducted for double concentration and 10 times concentration.

Table 2 Continuation of the table; Continuation of the phenylalkyl table <B> Mouse skin carcinogenesis two-stage study 7 withdrawal Slc: ICR female mouse mouse part was pricked,
The next day, 0.1 ml of an acetone solution containing 390 nmol of dimethylbenzoanthracene (DMBA) was applied once to the pricked area. DMBA application - 0.1 ml acetone containing 1.7 nmol TPA on the same site a week later
The solution was applied twice a week for 20 weeks. To examine the tumor development suppressing effect of the test compound, an acetone solution containing 85 nm of the above TPA coating was applied, and the same treatment was carried out for 20 weeks to observe the state of tumor development. The number of test animals used was 15 in each group. The inhibitory effect on tumor development was determined by comparing the average number of tumors generated per mouse with that of the untreated group.

Test compounds AI (Vl) and A8 (V2) in Table 2
We conducted tests on the following.

The results are shown in Table 4. All of the test compounds were found to have a remarkable inhibitory effect on the promotion of tumor development by TPA.

Table [Effects of the Invention] The antitumor drug of the present invention has a high antitumor effect, has low toxicity to normal cells, and shows no mutagenicity. 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 presence of cancer is confirmed, and preventing cancer recurrence after cancerous tissue removal.

Claims (2)

[Claims] (1) An anti-tumor drug with anti-promoter action having an active ingredient represented by the following general formula. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R is a straight chain alkyl consisting of 1 to 18 carbon atoms, cyclohexyl, substituted phenyl, and 1
or phenylalkyl having 4 carbon atoms) (2) An antitumor drug with anti-promoter action that has an active ingredient represented by the general formula below ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R is a straight chain alkyl having 1 to 10 carbon atoms. be)