JP2002255821A - Medicine for treating cancer resistant to anticancer medicine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a camptothecin derivative which has a strong anti-tumor activity and exhibits a cytocidal action also against CPT-11 resistant strain due to the excessive expression of BCRP and MRP2. SOLUTION: This medicine against cancer resistant to anticancer medicines contains a camptothecin derivative represented by formula (1) (X is H, a halogen atom, amino, a lower alkyl, a lower alkoxyl or OH; Y is H, a halogen atom, or OH; the compound wherein X is OH and Y is H is excluded) as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a camptothecin derivative having a strong antitumor activity against cancer cells exhibiting resistance to various anticancer drugs, and relates to a therapeutic agent using the same.

[0002]

2. Description of the Related Art Camptothecin (hereinafter referred to as C)
PT (abbreviated as PT) is a Chinese native tree (Camptotheca ac)
uminata) 7-ethyl-1 which is an alkaloid contained in leaves, bark, etc., and a semi-synthetic derivative of CPT.
0-piperidinopiperidinocarbonyloxycamptothecin (hereinafter abbreviated as CPT-11;
No. 7) is a particularly important substance as a compound that maintains the high antitumor activity of CPT and has reduced toxicity. This CPT-11 is metabolized in the living body into 7-ethyl-10-hydroxycamptothecin, which is also a semi-synthetic derivative of CPT (hereinafter, abbreviated as SN-38; see JP-B-62-47193). It is said that activity appears.

[0003] That is, CPT-11 can be said to be a pro-drug of SN-38. This CPT-
The mechanism by which 11 is metabolized to SN-38 and exhibits antitumor activity has been reported in detail in several documents (Kaneda et al., Cancer Res., 50, 1715 (1990), Nagata et al.,
J. Aichi Med. Univ. Assoc., 15, 683 (1987), Tricoli
Res., 158, 1 (1985); Nagata et al., Cance.
r Treatment Reports, 71, 341 (1987)). CPT,
All of the CPT derivatives represented by SN-38, CPT-11 and the like are known to have strong antitumor activity. Among them, CPT-11 is currently widely used in clinical practice as an antitumor agent. ing.

On the other hand, as a problem in cancer chemotherapy,
Cancer cells have acquired resistance to chemotherapeutic agents.
Tumor cells with acquired resistance to PT-11 were also found. This limits the chemotherapy of cancer with CPT-11. As the main mechanism of cancer cell resistance acquisition,
Topoisomerase I, a target molecule of CPT-11 (to
po I) point mutation ¹⁾ , decreased expression of topo I, breast cancer resistant protein (BCR
P) ²⁾ , or Multi resistance protein 2 (MRP2) ³⁾
Increased expression of molecules from cells due to overexpression of. Therefore, obtaining an effective CPT derivative even for cancer cells that have acquired resistance without being a substrate for BCRP or MRP2 is extremely important from the viewpoint of overcoming drug resistance.

[0005]

Accordingly, an object of the present invention is to provide a CPT derivative having a strong antitumor activity and exhibiting a cell killing effect even on a CPT-11 resistant strain due to overexpression of BCRP and MRP2. It is in.

[0006]

Means for Solving the Problems Accordingly, the present inventors have:
While conducting intensive research to solve the above problems, various CP
As a result of studying the T derivative, it was found that C represented by the formula (I)
The present inventors have found that a PT derivative has an excellent antitumor effect against an anticancer drug-resistant cancer, and have completed the present invention. That is, the present invention relates to formula (1)

Embedded image (Wherein X is hydrogen, a halogen atom, an amino group, a lower alkyl group, a lower alkoxyl group or a hydroxyl group, Y is hydrogen, a halogen atom or a hydroxyl group, X is a hydroxyl group and Y
A camptothecin derivative represented by the formula (1) except for hydrogen) as an active ingredient. In the present invention, the lower alkyl group in the formula (I) means an alkyl group having 1 to 5 carbon atoms, particularly an alkyl group having 1 to 3 carbon atoms, and the lower alkoxyl group is a carbon atom An alkoxyl group having 1 to 5 carbon atoms, especially an alkoxyl group having 1 to 3 carbon atoms, is meant. The present invention also relates to the therapeutic agent, wherein the anticancer drug-resistant cancer is a cancer resistant to irinotecan hydrochloride (CPT-11).

[0007] The CPT derivative as a therapeutic agent for anticancer drug-resistant cancer according to the present invention has been confirmed to have excellent antitumor effects in in vitro and in vivo test systems, as described below. Compared to CPT-11 and SN-38, it has been confirmed that it has a characteristic of being less likely to be a substrate for BCRP or MRP2, and is effective against resistant cancers that have acquired resistance to anticancer drugs.

[0008]

Embodiments of the present invention will be described below. The therapeutic agent for anticancer drug-resistant cancer according to the present invention comprises a CPT derivative having an ethyl group at the 7-position of B ring and various substituents at the 10- and / or 11-position of the A ring of CPT as an active ingredient. . These derivatives can be produced by a known total synthesis method (see JP-A-1-186892) or a semi-synthesis method from a natural CPT derivative having a substituent on the A ring. For example, a known compound (S) -4-ethyl-7,8-dihydro-4-hydroxy-1H-pyrano [3,4-f] indolizine-3,6,10 (4
Reaction of (H) -trione with 2-amino-4-hydroxypropiophenone substituted at position 4 by Y and position 5 by X (P &
U route (Henegar, KE; Ashford, SW; Baughman,
TA; Sih, JC; Gu, RLJ Org. Chem. 1997,
62, 6588-6597.).

The method of administration of the therapeutic agent according to the present invention is appropriately selected. For example, it can be administered subcutaneously, intravenously or intramuscularly. In addition, it can be processed into various preparations by a conventional method and used, for example, can be formulated into an injection or the like together with pharmaceutically acceptable auxiliaries such as a stabilizer, an osmotic pressure adjusting agent and a pH adjusting agent. In addition, a form may be used in which each drug is mixed and used at the time of use. The dose of the CPT derivative used in the therapeutic agent according to the present invention may be appropriately selected depending on the type and condition of the tumor.
0.1-100 mg / kg of body weight is preferred, especially 0.5-20 mg
/ Kg of body weight is preferred. Also, combination with other chemotherapeutic agents is effective.

[0010]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

1. Test of antitumor effect in vivo A 7-week-old CDF1 mouse (female) was used. 6 or 1 group
0 mice and L1210 cells (10 ⁵ cells) intraperitoneally
Was transplanted. Specimens were intraperitoneally administered on days 1, 5, and 9 after tumor implantation, and the number of days after which the mice survived was examined. The survival days were expressed as the survival rate (T / C) according to the following formula. T / C = average surviving days of the administration group / average surviving days of the control group × 100 The survival of the mice was observed for 40 days. The specimen is saline,
Alternatively, it was dissolved or suspended in distilled water for injection and administered.
The control group received only physiological saline or distilled water for injection. The structural formula of the CPT derivative subjected to the test is shown in Table 1.
1 and Table 2 and Table 3 show the results of the antitumor effect test.

[0012]

[Table 1]

[0013]

[Table 2]

[0014]

[Table 3]

With the exception of SN-444, all CPT derivatives showed a T / C of more than 100% at a given dose. Therefore, except for SN-444, the CPT derivatives used in the present invention show antitumor activity at an in vivo level, and they can be used as anticancer agents. Since SN-444 showed cytocidal activity against tumor cells containing L1210 in the following in vitro test, the derivative can be accurately delivered to tumor tissues by modifying the administration route. Thus, this can also be used as an anticancer agent.

2. Test of cell killing effect in vitro (1) Cells used and cell culture method KB (Dainippon Pharmaceutical Co., Ltd.) derived from human nasopharyngeal carcinoma was E-MEM (Nissui) supplemented with 10% bovine serum (Gibco). And L1210 human leukemia cells (Dainippon Pharmaceutical Co., Ltd.) were cultured in RPMI1640 medium (Nissui Pharmaceutical) supplemented with 10% fetal calf serum under conditions of 37 ° C. and 5% carbon dioxide. Human leukemia cells; L1210, human small cell lung cancer cells; PC-6,
An SN-38 resistant strain; PC-6 / SN2-5H (obtained from Nagasaki University School of Medicine), and a human liver cancer cell; HepG2 were used. PC-
6 is kanamycin sulfate (Meiji Seika, 50 mg / l), and 10
The cells were cultured in RPMI1640 (SIGMA) medium containing 5% fetal bovine serum (Gibco) at 37 ° C. under 5% carbon dioxide. PC-6 / SN2-5H
Was further cultured in the above medium supplemented with SN-38 (final concentration 25 nM) under the conditions of 37 ° C. and 5% carbon dioxide. HepG2 contains kanamycin sulfate (50mg / l) and 10% fetal calf serum
The cells were cultured in a D-MEM (SIGMA) medium at 37 ° C. under 5% carbon dioxide.

(2) Cell growth inhibitory activity measurement method In the system using KB, cells in the logarithmic growth phase were 2 × 10 ^{4 on} Day -1.
Cells / ml were diluted with the culture solution, and 3 ml was seeded in a 60 mm plastic dish (6 × 10 ⁴ cells / dish). After culturing this overnight, it was replaced with a culture solution containing an appropriate concentration of each drug on Day 0, and culturing was continued for 3 days. After washing the cells with PBS (Nissui Pharmaceutical) on Day 3, the cells were detached from the dish using 0.5 ml of 0.25% trypsin solution (Gibco), and the whole amount was diluted to 10 ml with physiological saline. In the system using L1210, Da
Cells in the logarithmic growth phase were diluted with medium to 2 x 10 ⁴ cells / ml in y 0, were implanted 0.5 ml per well in 24-well plastic plates (10 ⁴ cells / well). After culturing this for 3 hours, a culture solution containing a double concentration of each drug was added at 0.5 ml / well, and culturing was continued for 3 days. On Day 3, all wells were diluted to 10 ml with saline. Cell suspension diluted in physiological saline was prepared using Coulter Counter (Type ZM, Coulter Electron
ics), the number of cells per 0.5 ml was measured twice each. At this time, the growth inhibition rate was calculated by subtracting the number of cells in Day 0 from the number of cells in Day 3 and the number of cells in each concentration-treated dish (well) with respect to the number of cells in the drug-untreated dish (well). The ratio was subtracted from 1 and calculated as the average value of two dishes (wells) for each concentration. The 50% growth inhibition concentration (GI ₅₀ ) was calculated from the concentration-growth inhibition curve of each drug by interpolation. Specific activity to CPT (relativ
e activity (hereinafter RA) was also tested as a reference control
The CPT of GI ₅₀ values expressed in divided by the GI ₅₀ values of each agent.

In the system using PC-6, PC-6 / SN2-5H and HepG2, the MTT method was used. Cells are 0.05% Trypsin-EDTA
(GIBCO BRL), and a cell suspension prepared at 5.5 × 10 ³ cells / ml was added to a 96-well microplate (180 μl / well). After culturing overnight, the drug (20 μl / well) diluted in each step was added. After culturing for 72 hours, 5 m
g / ml of 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyltetrazolium bromide (MTT, Nacalai Tesque) at 20 μl /
Add well and incubate for 4 hours, then centrifuge (400 xg, 10 minutes)
Then, 200 μl / well of dimethyl sulfoxide was added to the cells. After that, wavelength 570 nm, reference wavelength 655 nm
The absorbance at was measured. At this time, the 50% growth inhibitory concentration (IC
₅₀ ) was calculated from the concentration-growth rate curve of each drug by interpolation.

(3) Results Cell killing effect of various derivatives on 1 KB cells All CPT derivatives showed RA values of 1 or more (Table 4). Therefore, all CPT derivatives show stronger cytocidal action against KB than existing CPT, and they can be used as anticancer agents.

(4) Result 2 Cell killing effect of various derivatives on L1210 All CPT derivatives showed RA values of 1 or more (Table 4). Therefore, all CPT derivatives are
It shows stronger cell killing effect than existing CPT, and these can be used as anticancer agents.

[0021]

[Table 4]

(5) Result 3 Cell killing action of various derivatives on PC-6 IC of CPT derivative used in the present invention on PC6
₅₀ was less than that of SN-38 (Table 5). Therefore, the CPT derivative used in the present invention is an active substance of CPT-11, which is an existing potent anticancer agent, with respect to PC-6.
It shows stronger cell killing effect than SN-38, and these can be used as anticancer agents.

3. Effects of Various Derivatives on BCRP and MRP2 (1) Cell Killing Action of Various Derivatives on SN-38 Resistant Strain PC-6 / SN2-5H The CPT derivative used in the present invention against PC6 / SN2-5H which is a BCRP overexpressing cell The IC ₅₀ was smaller than that of SN-38, and the specific resistance value (RR) to resistant cancer cells was small (Table 5). Therefore, the CPT derivative used in the present invention is less likely to be a BCRP substrate than SN-38,
It can be used as an anticancer agent having an effect on cancer cells having resistance to SN-38.

[0024]

[Table 5]

[0025] (2) IC ₅₀ on HepG2 is MRP2-overexpressing cells of CPT derivative used in the cell killing invention various derivatives on HepG2 was the equal to or less than the SN-38
(Table 6). Therefore, the CPT derivative used in the present invention is
It can be used as an anticancer drug that is unlikely to be a substrate for MRP2.

[0026]

[Table 6]

As described above, the CPT derivative used in the present invention has a strong cell killing effect, and exhibits BCRP and MRP2
It became clear that it was difficult to become a substrate for the. Therefore, the CPT derivative used in the present invention can be expected to have an effect on cancer cells that have acquired resistance to an anticancer agent, and can be used as a therapeutic agent for resistant cancer.

[0028]

According to the present invention, a therapeutic agent having a strong antitumor activity against cancer cells having resistance to an anticancer agent can be obtained.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yaegashi ▼ Takashi 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha (72) Inventor Tomio Furuta 1-1-1, Higashi-Shimbashi, Minato-ku, Tokyo No. 19 Yakult Honsha (72) Inventor Hiroshi Nagata 1-119 Higashi-Shimbashi, Minato-ku, Tokyo (72) Inventor Shinzo Tanabe 2- Kitahoncho, Funabashi-shi, Chiba 25-6 (72) Inventor Tomohisa Ishikawa 4-17-30 Kirigaoka, Midori-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Emi Yoshikawa 3,133 Kanaicho, Machida-shi, Tokyo 3-8-406 Fujinodai Estate 3-8-406 (72) Inventor Shinya Hayasaka 1-35-35, Nishikan, Furukawa-shi, Miyagi (72) Inventor Yoji Ikegami 4-10-213, Kurihara, Niiza-shi, Saitama F-term (reference) 4C050 AA01 BB04 CC07 DD02 EE02 FF02 GG02 GG03 GG04 4C086 AA01 AA02 AA03 CB22 MA01 MA04 NA14 ZB26

Claims (2)

[Claims] (1) Formula (1) (Wherein X is hydrogen, a halogen atom, an amino group, a lower alkyl group, a lower alkoxyl group or a hydroxyl group, Y is hydrogen, a halogen atom or a hydroxyl group, X is a hydroxyl group and Y
A therapeutic agent for an anticancer drug-resistant cancer, comprising a camptothecin derivative represented by the following formula: 2. The method according to claim 1, wherein the anticancer drug-resistant cancer is irinotecan hydrochloride (CP
The therapeutic agent according to claim 1, which is a cancer resistant to T-11).