JP2001114692A - Multiple drug resistance-overcoming agent containing aureolic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject medicament capable of resolving the problem of developing drug resistance in cancer chemotherapies. SOLUTION: This medicament is such as to contain at least one kind of aureolic acid selected from the group consisting of mithramycin, chromomycin and chromocyclomycin. The other objective composition for cancer therapy is obtained by combining the above aureolic acid with other anticancer agent(s).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a multidrug resistance overcoming agent containing aureoleic acid and a composition for treating cancer characterized by combining aureoleic acid with another anticancer agent.

[0002]

2. Description of the Related Art In recent years in Japan, cancer has become one of the most socially ill diseases, accounting for about one-quarter of the Japanese deaths. Currently, treatments for cancer include surgery, radiation therapy, and chemotherapy.
These methods alone have not yielded a sufficient therapeutic effect. In chemotherapy for cancer, it has been a serious problem that cancer cells show resistance to drugs used, that is, the development of drug resistance.

One of the major causes of drug resistance is p-
Glycoproteins. p-glycoprotein is a protein present in the cell membrane that acts as a pump to pump drugs out of the cell,
It has the function of lowering the intracellular drug concentration. It is known that some cancer cells highly express the MDR1 gene encoding this p-glycoprotein and increase the amount of p-glycoprotein on the cell membrane. In such cancer cells, the intracellular drug concentration decreases and becomes lower than the effective concentration, resulting in resistance to the drug. In addition, p-glycoprotein has low substrate specificity, and in cancer cells in which MDR1 is highly expressed, not only the previously used drug but also other drugs are simultaneously acquired, and chemotherapy is acquired. It is often impossible. Such a phenomenon is called multidrug resistance. Drugs in which cancer cells develop resistance due to multidrug resistance include adriamycin, vinblastine, vincristine, actinomycin D, colchicine, colcemide, puromycin, emetine, anthracycline, daunomycin, maytansine, etc. Different drugs are mentioned. Since these are typical drugs frequently used in chemotherapy, the seriousness of the multidrug resistance problem can be understood.

[0004]

In order to solve these problems, attempts have been made to search for and develop substances that inhibit the function of p-glycoprotein. For example, it has been reported that calcium antagonists such as verapamil and immunosuppressants such as cyclosporin A and FK506 inhibit the function of p-glycoprotein. However, when they are administered for the purpose of overcoming drug resistance, their inherent effects of calcium antagonism and immunosuppression appear as strong side effects, and are not suitable for actual administration. Also, the cyclosporin inducer SDZPSC833 and the quinoline derivative MS-2
09 is under development, but has not yet been applied to clinical applications due to its effects and toxicity.

[0005] In recent years, as the analysis of cell functions at the gene level has progressed, the development of drugs that inhibit the expression of harmful proteins themselves, rather than inhibiting the functions of harmful proteins already expressed on cells, has been increasing. It is thriving. For example, p-
Regarding glycoproteins as well, Japanese Patent No. 214500 discloses that quercetin, which is one of the plant components, suppresses an increase in the expression of p-glycoprotein due to arsenic acid stimulation. Kim et al. Also report that quercetin increases the sensitivity of multidrug-resistant cancer cells to vinblastine and vincrestin (Kim et al., Exp. Mol. Me
d. , 30, 87-92 (1998)). However, quercetin is a flavonoid having no sugar in its structure, and is hardly dissolved in water, so that it is a substance that is difficult to handle in preparations. Furthermore, it is unknown whether the concentration of these in vitro experimental results will provide sufficient effects on humans, such as the clinical summary.

[0006] Humans have a variety of genes, and the properties of cancer cells caused by genetic abnormalities also vary widely. In fact, effective drugs differ greatly from one cancer cell to another, and there are many cancer types for which effective drugs have not yet been found, such as certain solid cancers. Clinically, overcoming cancer requires a wider variety of drugs. That is, when a substance that effectively suppresses the expression of the MDR1 gene and reduces the amount of p-glycoprotein is newly found, its industrial utility is immense.

[0007]

Means for Solving the Problems As a result of intensive studies on the above problems, the present inventors have found that aureolic acids such as mitramycin, chromomycin, and chromocyclomycin suppress the expression of the MDR1 gene. The present invention has been completed. Accordingly, a first aspect of the present invention is a multidrug resistance overcoming agent characterized by containing aureolic acid. A second aspect of the present invention is a composition for treating cancer, which comprises combining aureolic acid with another anticancer agent.

[0008]

DETAILED DESCRIPTION OF THE INVENTION Aureolic acids are Streptom
It can be isolated from actinomycetes such as yces tanashiensis using conventional means such as solvent extraction and chromatography. It can also be obtained by ordinary chemical synthesis. Also, commercially available products can be used. Mitramycin, chromomycin and chromocyclomycin are administered to cancer tissues that have acquired drug resistance,
It can suppress the expression of the MDR1 gene and p-glycoprotein to overcome drug resistance, enhance the therapeutic effect of the anticancer drug, or maintain the effect. When the compound of the present invention is administered in combination with another anticancer agent, the compound can be administered in an appropriate dosage form such as a tablet, capsule, soft capsule, powder, injection, or patch. Excipients, solubilizers, binders, stabilizers, flavoring agents, and the like can be used in various formulations using these dosage forms.

Specific examples of adjuvants that can be incorporated into tablets, capsules and the like are as follows. Binders such as tragacanth gum, gum arabic, corn starch or gelatin, excipients such as microcrystalline cellulose, disintegrants such as constarch, fully gelled starch, alginic acid, lubricants such as magnesium stearate, sucrose A sweetener such as lactose or saccharin, a flavoring agent such as peppermint, reddish oil or cherry, and, when the unit use form is a capsule, containing a liquid carrier such as a fatty oil in addition to the above type of material. Can be. Various other materials may be present as coatings or to otherwise alter the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.

[0010] Sterile compositions for injection include water for injection, sesame oil,
By dissolving or suspending the active substance in excipients, such as naturally occurring vegetable oils, such as coconut oil, peanut oil, cottonseed oil, and the like, can be formulated according to conventional pharmaceutical practice. Buffers, preservatives, antioxidants and the like can be mixed as necessary. The drug of the present invention can be performed by various injections such as intravenous injection, subcutaneous injection, intramuscular injection or oral administration, and various methods such as transdermal administration.
It is desirable to make it liquid and administer it as injection or infusion. A particularly preferred form of administration is intravenous administration, and the dose is generally preferably 1 to 1000 μg / kg per day, and may be administered several times a day. By administering another anticancer agent 1 to 4 days after administration of mitramycin, or simultaneously administering mitramycin and another anticancer agent, the therapeutic effect of the anticancer agent can be enhanced or the effect can be maintained. For example, in the case of adriamycin, the dose is 1 to 300 mg / m ² per day.
Is preferred intravenously. The dose generally varies depending on the type and symptoms of the disease caused by drug resistance, the method of administration, and the like, and the dose can be administered outside the above range.

[0011]

It is already known that aureolic acids have antitumor activity, especially that mitramycin is applied to testicular tumors and is a useful drug against hypercalcemia. However, the effect of suppressing the expression of the MDR1 gene and p-glycoprotein and the effect of overcoming multidrug resistance are activities newly discovered by the present inventors. In particular, mitramycin is a drug that is actually used in the clinic and can be used immediately in the clinic, so that it can be expected to give a great contribution to clinical cancer treatment by administering it in combination with other anticancer drugs. .

[0012]

The present invention is not limited to the following examples. In the following examples, mitramycin was purchased from SIGMA-ALDRICH Mit
hramycin A and chromomycin A ₃ purchased from SIGMA-ALDRICH were used for chromomycin.

Example 1 (Preparation of Chromocyclomycin) The soil collected in Moriya-cho, Kita-Soma-gun, Ibaraki Prefecture was dried, ground in a mortar, suspended in physiological saline, and then placed on an AV agar plate (Eiken Chemical Co., Ltd.). Company), actinomycetes strain KS12571
Got the strain. After culturing at 30 ° C. for 1 week, the grown actinomycete colonies were selected, and cotton seed meal liquid medium (2% corn starch (Wako Pure Chemical Industries, Ltd.), 0.5% cotton seed meal (SOUTHERN COTTON OIL COMPANY), 0.05% yeast Extract (DIFCO LABORATORIES), 0.1% NaC
l, 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ ) 500 ml and inoculate at 30 ° C.
After weekly shaking culture, 4.0 g of a culture supernatant was obtained. This 1.5 g was subjected to reverse phase HPLC under the following HPLC conditions, and the peak shown in FIG. 1 was collected and freeze-dried to obtain 22 mg of a yellow powder. As a result of structural analysis by instrumental analysis, this compound was identified as one of aureolic acids, chromocyclomycin (Reference: Berlin)
Yu A. et al., Chem. Commun ., 762-763 (1968))
Was confirmed. HPLC conditions: System, Shimadzu LC
-8A; column, Tosoh ODS-80 (60 mm ID × 30 cm); flow rate,
80 ml / min .; mobile phase, solution A, 10% CH ₃ CN; solution B, 60% CH ₃ CN, and a straight line from (A: B) = (70:30) to (10:90) in 30 minutes Gradient; detection, UV 220 nm.

Example 2 (tablets, capsules) Mitramycin 0.1 Lactose 0.75 Magnesium stearate 0.15 Total 1.0 The above parts by weight (g) are uniformly mixed, and the tablets are mixed according to a conventional method. And capsules. Chromomycin and chromocyclomycin were similarly made into tablets and capsules. Mitramycin and chromomycin were commercially available.

Example 3 (Powder, granule) Mitramycin 0.2 Starch 0.3 Lactose 0.5 Total 1.0 The above parts by weight (g) were uniformly mixed, and the powder and granules were mixed according to a conventional method. Agent. Chromomycin and chromocyclomycin were also used as powders and granules.

Example 4 (Injection) Mitramycin 0.1 Surfactant 0.9 Physiological saline 9.0 Total 10.0 The above parts by weight (ml) are mixed by heating, sterilized and mixed with an injection. did. Chromomycin and chromocyclomycin were also used as injections.

Example 5 (Effect of Inhibiting MDR1 Expression) Human small cell lung cancer cell line SBC-3 / ADM, which is an adriamycin-resistant cell line overexpressing MDRI (Katsuyuki Kimura et al.)
l., Anti-Cancer Drug Design , 7, 463-470 (1992))
Containing 0.01 to 0.1 mg / ml of mitramycin or chromomycin or chromocyclomycin, respectively.
The cells were cultured in an I1640 (Gibco BRL) medium at 37 ° C. in an atmosphere containing 5% carbon dioxide for 48 hours. After RT-PCR culture, cells were collected and RNA was extracted according to a standard method. RT-PCR
After amplifying MDR1 and β ₂ -m (control) RNA by the
After electrophoresis, the expression levels of MDR1 were compared. The result is shown in FIG. Mitramycin, chromomycin and chromocyclomycin suppressed the expression of MDR1, but did not suppress the expression of the control β ₂ -m. That is, MDR
1 was selectively and nontoxicly suppressed.

Example 6 (Suppressing Effect of p-Glycoprotein Expression) A human small cell lung cancer cell line SBC-3 / ADM, which is an adriamycin-resistant cell line overexpressing MDRI, was treated with 5 μg of mitramycin.
The cells were cultured for 96 hours in an RPMI1640 medium containing 1 / ml at 37 ° C. in an atmosphere containing 5% carbon dioxide. After the culture, the cells were collected, and the expression levels of p-glycoprotein were compared by FACS analysis.
As shown in FIG. 3, mitramycin clearly suppressed the expression of p-glycoprotein. That is, it was revealed that the present compound has an activity of releasing multidrug resistance caused by p-glycoprotein.

Example 7 (Effect of suppressing cancer cell drug excretion) 5 μg of mithramycin was obtained from human small cell lung cancer cell line SBC-3 / ADM, which is an adriamycin-resistant cell line overexpressing MDRI.
The cells were cultured for 96 hours in an RPMI1640 medium containing 1 / ml at 37 ° C. in an atmosphere containing 5% carbon dioxide. Calcein-AM, a kind of fluorescent dye, was added at 5 μg / ml and exposed for 30 minutes. The cells were collected, and the amount of calcein-AM incorporated into the cells was measured by FACS analysis. As shown in FIG. 4, mitramycin clearly suppressed the drug efflux mechanism of cancer cells.

Example 8 (Effect of inhibiting growth of cancer cells) Human small cell lung cancer cell line SBC-3 / ADM, which is an adriamycin-resistant cell line overexpressing MDRI, was cultured at 1.0 × 1 per flask.
0 I've got ^five . The cells were treated with 2 μg of adriamycin /
in RPMI 1640 medium containing 5 ml / ml or mitramycin 5 μg / ml or both at 37 ° C., 5% carbon dioxide
The cells were cultured in the containing atmosphere for 96 hours. As a control, an experiment without drug was also performed. As shown in Table 1, the cell number did not decrease with mitramycin alone or with adriamycin alone (growth rate> 100%). However, by administering the combination of mitramycin and adriamycin, the cell number was lower than at the time of seeding. Decreased (proliferation rate <100
%).

Table 1 Number of mitramycin ADM cells Proliferation rate (5 μM) (2 μM) (cells / ml) (%) 1 − − 8.0 × 10 ⁵ 800 2 + − 2.5 × 10 ⁵ 250 3 − + 1.0 × 10 ⁵ 100 4 + 0.5 × 10 ⁵ 50

Example 9 (Effect of Inhibition of Growth of Cancer Cells) Human small cell lung cancer cell line SBC-3 / ADM, which is an adriamycin-resistant cell line overexpressing MDRI, or SBC-, an adriamycin-sensitive cell line that does not express MDRI 3 against 37% in RPMI 1640 medium containing 5 μg / ml of mitramycin and / or 1 to 20 μl / ml of adriamycin.
The cells were cultured in an atmosphere containing 5% of carbon dioxide at 96 ° C for 96 hours. After culturing, CellTiter 96 Non-Radioactive Cell Proliferatio
n A cell growth inhibition test was performed according to the protocol of Assay (Promega). As shown in FIG. 5, by administering a combination of mitramycin and adriamycin,
The growth of MDR1-high expressing cells was clearly suppressed.

[Brief description of the drawings]

FIG. 1 is a reversed-phase HPLC chromatogram of the culture supernatant of the KS12571 strain.

FIG. 2: 1, 5 is control, 2 is mitramycin 0.01 mg / ml, 3 is mitramycin 0.1 mg / ml
ml, 4 are electrophoresis diagrams showing the case where chromomycin 0.01 mg / ml and 6 are chromocyclomycin 0.1 mg / ml.

FIG. 3 is a FACS analysis diagram showing that the horizontal axis represents the fluorescence intensity and the vertical axis represents the number of cells, and the rightward of the frequency distribution curve, the more the p-glycoprotein is expressed. In the figure, 1 indicates a cell group without drug addition, 2 indicates a cell group with mitramycin addition, and 3 indicates a cell group without mitramycin addition.

FIG. 4 is a FACS analysis diagram showing that the horizontal axis is the fluorescence intensity and the vertical axis is the number of cells, and that the more the frequency distribution curve is to the right, the more fluorescent dye is taken up into the cells. In the figure, 1 is a cell without addition of mitramycin, 2 is a cell with addition of mitramycin,
3 shows cells that do not highly express p-glycoprotein.

FIG. 5 is a graph showing the relationship between the survival rate of cancer cells and ADM (μM). ○ and ● represent cells with high MDR1 expression (SBC-3 / ADM), showing the case without mitramycin and the case with 5 μg / ml of mithramycin, respectively. Δ indicates adriamycin-sensitive cells SBC-3.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Mika Nakao 1-1-21 Midori, Moriya-cho, Kitasoma-gun, Ibaraki Asahi Breweries Co., Ltd. Food and Drug Research Laboratories (72) Yasuyuki Otake Midori, Moriya-cho, Kitasoma-gun, Ibaraki 1-1-21 Asahi Breweries, Ltd. Food and Drug Research Laboratories (72) Inventor Naoko Nozato 1-1-21 Asahi Breweries, Ltd. Liquor Research Institute in Asahi Breweries, Ltd. (72) Inventor Kazunori Ochiai Setagaya, Tokyo 1-25-6, Shinmachi, Ward (72) Inventor Seiji Isonishi 3-5-7, Narita Nishi, Suginami-ku, Tokyo (72) Inventor Aiko Okamoto 4-5-2 Igusa, Suginami-ku, Tokyo F-term (reference) 4C084 AA19 AA20 NA14 ZB011 ZB012 ZB261 ZB262 4C086 AA01 AA02 BC30 CB14 EA10 GA07 MA01 MA02 MA04 NA14 ZB01 ZB26

Claims (8)

[Claims] 1. An agent for overcoming multidrug resistance, which comprises an aureolic acid. 2. The agent for overcoming multidrug resistance according to claim 1, comprising one or more aureolic acids selected from the group consisting of mitramycin, chromomycin, and chromocyclomycin. 3. A composition for treating cancer, comprising combining aureoleic acid with another anticancer agent. 4. The composition for treating cancer according to claim 3, wherein one or more aureolic acids selected from the group consisting of mitramycin, chromomycin, and chromocyclomycin are combined with another anticancer agent. 5. The method according to claim 1, wherein the other anticancer agent is one or a combination of adriamycin, vinblastine, vincristine, actinomycin D, colchicine, colcemide, puromycin, emetine, anthracycline, daunomycin, and maytansine. The composition for treating cancer according to claim 3 or 4, wherein the composition is used for treating cancer. 6. A composition for treating cancer, which comprises administering a combination of mitramycin and adriamycin. 7. A composition for treating cancer, which comprises administering a combination of chromomycin and adriamycin. 8. A composition for treating cancer, which comprises administering a combination of chromocyclomycin and adriamycin.