WO2003074049A1

WO2003074049A1 - Antitumor agents

Info

Publication number: WO2003074049A1
Application number: PCT/JP2003/002716
Authority: WO
Inventors: Shiro Sasakuri; Rei Asaumi; Tokio Yamaguchi
Original assignee: Japan Science And Technology Agency
Priority date: 2002-03-07
Filing date: 2003-03-07
Publication date: 2003-09-12
Also published as: JP2003261448A

Abstract

It is intended to provide drugs containing a therapeutically effective amount of bilirubin, its precursor or derivatives thereof which are antitumor agents showing the toxicity exclusively on tumor cells with little side effects.

Description

Description Antitumor agent

TECHNICAL FIELD The invention of this application relates to an antitumor agent. More specifically, the present invention relates to an antitumor agent comprising pyrirubin, an analogous compound thereof or a derivative thereof, which is an in vivo metabolite and selectively shows toxicity only to tumor cells, as an active ingredient.

BACKGROUND ART Antitumor agents (antineoplastic agents, or care inostatic agents) are originally selectively toxic to tumor cells only, and it is desirable to inhibit their division and proliferation. However, currently used antineoplastic agents have spread to normal cells, tissues and organs, and patients suffered intolerable pain and fatal side effects during the course of chemotherapy. In the clinical setting, the following five types of antitumor agents are used in combination with multiple drugs or in combination with other treatments: (1) Alkylating agents such as night diene mustards (2) antimetabolites such as antifolates; (3) antitumor antibiotics such as actinomycin D; (4) hormones such as antiestrogens; and (5) other antitumor agents such as plant alkaloids. . On the other hand, bilirubin is the end product of heme from hemoglobin, myoglobin, or respiratory pigments (cytochrome, catalase, etc.). That is, the heme is converted to bilubeldin-IXα by the microsomal heme enzyme-added enzyme, and is further converted to pyrilrubin-1IXα by the bilubeldin reductase. The resulting unconjugated (indirect) virbergine (lipid-soluble) binds to albumin in the serum and is transported to the Dysse cavity from sinusoids in the liver. Away from albumin in hepatocytes It is transported by binding to Y protein and Ζ protein, and undergoes glucuronidation by pyrilrubin UDP-dalcuronyltransferase contained in the smooth endoplasmic reticulum in microsomal fractionation in hepatocytes, and is conjugated (direct type) It becomes pyrilrubin (water-soluble) and is secreted into the bile duct in the form of diglucuronide. This abnormal metabolism of pyrilrubin causes diseases such as hyperpyrilvinemia and pyrilrubin encephalopathy. For this reason, methods for quantifying the amount of pyrilrubin in blood and urine and reagents therefor have been developed for a long time (for example, JP-B-51-5600, JP-B-54-12840, and JP-B-57-59497). No.). In addition, a therapeutic agent for jaundice (for example, Japanese Patent Publication No. Hei 07-59514) or a method for preventing jaundice (for example, Japanese Patent Application Publication No. 3-505864) for the purpose of increasing pyrilirubin excretion and suppressing pyrilrubin production, or bilirubin excretion A therapeutic agent for liver disease for the purpose of promotion (for example, JP-A-10-330265) is known. However, no drug containing pyrirubin itself as a component is known, and the antitumor effect of bilirubin is not known at all. The inventors of the present application have diligently searched for a substance that is selectively toxic to only tumor cells. The inventors have found that they exhibit toxicity and completed the present invention. The purpose of this application is to provide a new antitumor agent based on the above-described novel findings by the inventors.

DISCLOSURE OF THE INVENTION This application provides an antitumor agent containing a therapeutically effective amount of pyrilubiine, a similar compound thereof or a derivative thereof, as an invention to solve the above-mentioned problem. Hereinafter, embodiments of the present invention will be described, and the antitumor agent of the present invention will be described in detail. Various techniques used for practicing the present invention are described in well-known literature (eg, Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing), except for the technique whose source is specifically indicated. Co., Eastern, PA, 1990) can be easily and reliably implemented by those skilled in the art.

BEST MODE FOR CARRYING OUT THE INVENTION The active ingredient of the antitumor agent of the present invention is a known pyrilvin, its analogous compound pyriberdine or a derivative thereof (hereinafter sometimes collectively referred to as “pyrilvins”). ). Pyrirubin (pyrurubin-1IXα) is represented by the following chemical formula (1).

As the pyrilrubin α, a powder product isolated and purified from bile of the gall bladder of the sea or bush is commercially available (for example, manufactured by Nacalai Tesque, Inc.), and this commercially available product can be used. Alternatively, a compound chemically synthesized by a known method may be used. The derivative of pyrirubin α can be represented by the following chemical formula (2)

(2)

In the chemical formula (2), R1, R2, R3 and R4 are the same or different and each represents a hydrocarbon group or a hydrogen group which may have a substituent, and XI or X2 is the same or different. A hydrogen group or a hydrocarbon group which may have a substituent is shown. Examples of the hydrocarbon group include a saturated or unsaturated chain-like or cyclic hydrocarbon, or any of these hydrocarbons having a substituent such as an amino group, an alkoxy group, or a sulfonyl group. You. Specifically, for example, 36 kinds of derivatives shown in Table 1 are exemplified.

table 1

1: R ^: one Cri3,

2: R ^: one CH3,

3:

Four:

Five :

CH ₂ -CH ₂ -NH ₂

6: R, one H, one H

C00H

7: R:-C, = Η,

8: R _r ^: one CH one CH3, one CH3, one H,

CH ₂ -CH ₂ -NH ₂ CH ₂ -CH ₂ -NH ₂

^9: R,: - CH- CH

3, one CH3, R3 ~ -CH3, Cii— CH3, X | ⁼ — H,

CH _r CH-NH ₂ CH ₂ -CH-NH ₂

C00H C00H

For 10 to 9, Xr-CH ₃ , ¾ = -H (9 types)

X, =-H, X ₂ =-CH ₃ (9 types) for 11 to 9

For 10 to 9, X, =-CH ₃ , X ₂ = -CH ₃ (9 types)

These pyriluvine α derivatives can be chemically synthesized by a known method.

Pyrivudine-IXα, which is a similar compound of bilirubin-IXα, can be represented by the following chemical formula (3), and also chemically synthesized by a known method can be used. C0 ₂ H No C0 ₂ H

(3)

Further, the derivative of pyriveridine-ΙΧα can be represented by the following chemical formula (4).

In the chemical formula (4), Rl R23 and R4 are the same or different and each represents a hydrocarbon group or a hydrogen group which may have a substituent, and XI or X2 is the same or different. And represents a hydrogen group or a hydrocarbon group which may have a substituent. Examples of the hydrocarbon group include a saturated or unsaturated chain or cyclic hydrocarbon, or any of these hydrocarbons having a substituent such as an amino group, an alkoxy group, or a carbonyl group. . Specifically, for example, 36 kinds of derivatives having the same hydrocarbon group and hydrogen group as those shown in Table 1 are exemplified. These derivatives can also be chemically synthesized by a known method. The antitumor agent of the present invention can be formulated by uniformly mixing the above pyrilvins and a pharmacologically acceptable carrier. The carrier can be appropriately selected from a wide range depending on the dosage form of the drug, but the antitumor agent of the present invention is desirably in a unit dosage form that can be administered orally or by injection. . In particular, in the case of administration by injection, local injection, intra-abdominal injection, selective intravenous injection, intravenous injection, subcutaneous injection, organ perfusion fluid injection, and the like can be employed. Oral liquid preparations such as suspensions and syrups include water, sugars such as sucrose, sorbitol, and fructoses; glycols such as polyethylene glycol; oils such as sesame oil and soybean oil; Preservatives such as benzoate Trobeli. It can be manufactured using flavors such as flavor and peppermint. Powders, pills, capsules and tablets include lactose, glucose, sucrose, mannitol and other excipients, starch, sodium alginate and other disintegrants, magnesium stearate, talc and other lubricants, polyvinyl chloride It can be formulated using a binder such as alcohol, hydroxypropylcellulose and gelatin, a surfactant such as fatty acid ester and the like, a plasticizer such as glycerin and the like. Tablets and capsules are preferred unit dosage forms in the formulations of the present invention in that they are easy to administer. When manufacturing tablets and capsules, a solid pharmaceutical carrier is used. Further, the solution for injection can be formulated using a carrier comprising a salt solution, a glucose solution, a mixture of a saline solution and a glucose solution, various buffers and the like. Alternatively, the composition may be formulated in a powder state, and mixed with the liquid carrier before use to prepare an injection solution. The dose of the antitumor agent of the present invention varies depending on the patient's age, body weight, symptoms, administration route and the like. It is only necessary to administer an amount that is about 5 thigh ol. The antitumor agent of the present invention having the above constitution is effective for, for example, the following benign or malignant tumors. a) Epithelial tumor

Benign epithelial tumor: papilloma, adenoma

Malignant epithelial tumors: squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, undifferentiated cancer, hepatocellular carcinoma, renal cell carcinoma, choriocarcinoma, seminoma, undifferentiated germ cell type, fetal carcinoma

b) Non-epithelial tumor Benign non-epithelial tumors: fibromas, fibromatosis, fibrous histiocytomas, lipomas, chondromas, osteomas, leiomyomas, rhabdomyomas, hemangiomas, lymphangiomas, mesotheliomas, neurofibromas, Schwannoma, ganglionoma,

Pheochromocytoma, glioma, meningioma, pigmented nevus

Malignant non-epithelial neoplasia: fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, chondrosarcoma, osteosarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, synovial sarcoma, malignant mesothelioma, malignant peripheral nerve sheath Tumor, neuroblastoma, medulloblastoma, retinoblastoma, glioblastoma, malignant melanoma, malignant lymphangioma, leukemia, osteogenic myeloma

c) Epithelial non-epithelial mixed tumor: pleomorphic adenoma, carcinosarcoma, mesenchymal tumor, teratoma Among these various tumors, the antitumor agent of the present invention is particularly effective against cancer peritoneal dissemination and solid cancer. is there. In other words, in vivo pyrilvin immediately binds to albumin in the blood and neutralizes its toxicity, but when administered to almost serum-free cancer peritoneal dissemination and solid tumors, these tumor cells Exhibits strong toxicity to Pyrirubin is a substance that exists in a certain amount in the living body, and is a low-molecular-weight substance (molecular weight of about 600) with negligible antigenicity, so its safety is guaranteed.

EXAMPLES Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples relating to the antitumor effect of pyrilrubin, but the invention of this application is not limited by the following examples. Example 1

In vivo testing Materials and methods (1) Cells

Human gastric cancer disseminated cells TMK-1 (transferred from the Department of Pathology, Hiroshima University) were used. TMK-1 cells were cultured in RPMI medium containing 1M FBS. (2) Pyrirubin solution

Pyrirubin (manufactured by Nacalai Task) was dissolved in PBS to a concentration of 200 ig / ml, and the pH was adjusted to 7.9.

(3) Tumor formation

(3-1) Intraperitoneal

0.05¾ Trypsin-Adjust the TMK-1 cells collected by treatment with 0.5 mM EDTA to 5 x 10 ⁶ cells with PBS, and inject 0.5 ml intraperitoneally to each of 9 9-week-old nude mice. Entered. Immediately after TMK-1 cell injection, 5 mice were intraperitoneally administered 1 ml of the pyrilrubin solution, and the same pyrilvin administration was performed three times every two hours. Controls (4 animals) received PBS (pH 7.9) in the same manner and on the same schedule as pyrilrubin administration. Two weeks later, the mice were sacrificed under general anesthesia, and the intraperitoneal tumor weight (wet weight) was measured.

(3-2) subcutaneous

The TMK-1 cells collected in the same manner as described above were adjusted to 5 × 10 ⁶ cells / ^ ΟΟι 1 with PBS, and 0.5 ml was injected subcutaneously into the proximal axilla of each of 9 9-week-old nude mice. After one week, subcutaneous tumor formation was confirmed in 8 mice. Four of these mice received 1 ml of the pyrilrubin solution twice a day at the tumor site, for a total of 20 doses. The remaining four (control) mouse tumors were administered PBS (pH 7.9) on the same schedule.

Seven days after the final administration, the mice were sacrificed under general anesthesia, and the subcutaneous tumor weight (wet weight) was measured. Result

(1) Effect of pyrilvin on intraperitoneal tumorigenesis

The effects of the pyrilrubin solution and PBS on nude mice transplanted with TMK-1 cells intraperitoneally are shown in Table 2. Table 2

As is evident from Table 2, all the control mice to which PBS was administered showed intraperitoneal tumors, whereas no tumor formation was observed in 3 out of 5 mice in the pyrilrubin administration group. In addition, the tumor weight (average 0.105 g) of the two pyrilrubin-treated groups in which tumors were observed was smaller than the tumor weight (average 0.145 g) of the control group.

(2) Effect of pyrilvin on subcutaneous tumor formation

The effects of the pyrilrubin solution and PBS on nude mice transplanted with TMK-1 cells subcutaneously are shown in Table 3. Table 3

As is clear from Table 3, the subcutaneous tumor remained in all of the control mice to which PBS was administered, whereas the tumor formed before administration of one mouse in the group treated with pyrilrubin disappeared. In addition, the tumor weight of the three animals receiving pyrilrubin with residual tumor (mean 0.0173 g) was smaller than the tumor weight of the four animals in the control group (mean 0.0203 g). In comparison, pyrilrubin treatment reduced tumor weight by 50% to 90% when compared individually. From the above results, the antitumor effect of pyrilrubin was confirmed at the animal individual level.

Example 2

In vitro test Materials and methods

(1) cells

Cells isolated from the peritoneum of a 20-week-old rat by the explant method were used as normal cells. Cells were cultured in DMEM medium containing 10% FBS. Cells that reached subconfluence were treated with 0.05% trypsin-0.5 mM EDTA, passaged, and up to the third generation were used for the test.

As the tumor cell, a 4B2 cell line isolated from the ovarian tumor by the inventor of this application was used.

The 4B2 cell line was cultured in DMEM medium containing 10% FBS, recovered by 0.05% trypsin-0.5 mM EDTA treatment and subcultured. The TMK-1 cells used in Test Example 1 were also used.

(2) Pyrirubin treatment

Inoculate 1 × 10 ⁴ Zwell cells in a 96-well plate, and incubate at 37 ° C overnight in a C02 incubator at 0 / N.Pirilubi so that the final concentration is 1, 3, 10 or 30 ig / ml. Of the solution (pH 7.9) was added to the culture solution.

(3) MTT Atsushi

Thirty hours after the addition of the pyrilrubin solution, Diphenyltetrazolium Bromide was dissolved in PBS at a concentration of 5 mg / ml and passed through a filter to obtain an MMT solution. A 1/10 volume of the MMT solution is added to the culture, and pre-incubation is performed for 2 hours, and the formed formazan is reduced to 10% SDS-50¾ equivalent to the culture. It was dissolved in Dime thylf ormami de and the absorbance at 570 nm was measured. result

The cytotoxicity 30 hours after the addition of pyrilrubin was examined by MMT assay. As a result, normal cells (primary cultured cells derived from rat peritoneum) maintained about 85% of the cell activity even after addition of a 10 μg / ml pyrilrubin solution, but the 4B2 cell line, which is a tumor cell, And TMK-1 cells had reduced cell activity to less than 50%. When a 30 g / ml pyrilrubin solution was added, normal cells maintained about 80% of cell activity, whereas 4B2 cell line and TMK-1 cells had cell activity of 20% or less. Decreased. From the above results, it was confirmed that bilirubin, which is an active ingredient of the antitumor agent of the present invention, shows little toxicity to normal cells and selectively shows toxicity to tumor cells.

INDUSTRIAL APPLICABILITY According to the invention of this application, an antitumor agent having an extremely low side effect, comprising an in vivo substance bilirubin which exhibits selective toxicity only to tumor cells, is provided.

Claims

The scope of the claims

1. An anti-tumor agent comprising a therapeutically effective amount of pyrilvin, a similar compound or a derivative thereof.