WO2003066052A1

WO2003066052A1 - Anticancer agent comprising nitroimidazole and topoisomerase inhibitor as active ingredients

Info

Publication number: WO2003066052A1
Application number: PCT/KR2003/000276
Authority: WO
Inventors: Jong-Won Lee; Sun-Ha Lim; Hong-Tae Kim; Hun-Suk Suh; Sung-Hwan Park; Yang-Il Kim
Original assignee: Hypoxi Co., Ltd.
Priority date: 2002-02-07
Filing date: 2003-02-07
Publication date: 2003-08-14
Also published as: AU2003208037A1; KR20030067275A

Abstract

The present invention relates to an anticancer agent comprising active ingredients of a nitroimidazole compound such as pimonidazole, misonidazole, etanidazole or ornidazole and a topoisomerase inhibitor such as doxorubicin, mitomycin C, camptothecin, novobiocin, epirubicin, dactinomycin or etoposide, and their pharmaceutically acceptable carrier. The anticancer agent induces apoptosis by the activity of topoisomerase inhibitor under the state of normoxia as well as by the activity of nitroimidazole compound under the state of hypoxia. Accordingly, the anticancer agent can be practically applied for the treatment of almost all kind of cancer, inter alia, solid cancer promoting the hypoxic condition regardless of the presence of oxygen.

Description

ANTICANCER AGENT COMPRISING NITROIMIDAZOLE AND TOPOISOMERASE INHIBITOR AS ACTIVE INGREDIENTS

BACKGROUND OF INVENTION

Field of the invention

The present invention relates to an anticancer agent comprising active ingredients of nitroimidazole compound and topoisomerase inhibitor, more specifically, to an anticancer agent comprising active ingredients of nitroimidazole compound such as pimonidazole, misonidazole, etanidazole and ornidazole, and a topoisomerase inhibitor such as doxorubicin, mitomycin C, camptothecin, novobiocin, epirubicin, dactinomycin and etoposide, and their pharmaceutically acceptable carriers.

Description of the Prior Art

Anticancer agent is the general term for those drugs that act upon various metabolic pathways of a cancer cell, thereby having cytotoxic or cytostatic effects on the cancer cell. Anticancer agents developed so far can be classified into metabolic antagonist, vegetable alkaloid, topoisomerase inhibitor, al ylating agent, anticancer antibiotic, hormonal agent and the others, depending on their mode of action and chemical structure. Anticancer agents have a variety of intracellular targets: for example, some agents block DNA replication, transcription and translation, and others inhibit the action of protein' s essential for cell survival. These activities on intracellular targets, lead the cells to death by way of necrosis or apoptosis.

Topoisomerase is an enzyme essential for changing the topology of DNA during DNA replication, which is classified into type I-A, type I-B and type II. Topoisomerase type I- A includes bacterial ω protein, topoisomerase III and reverse DNA gyrase, topoisomerase type I-B includes eukaryotic topoisomerase, and topoisomerase type II includes prokaryotic DNA gyrase, topoisomerase IV and mammalian topoisomerase Ilα and II β . Topoisomerase inhibitors for anticancer agents act specifically upon the above topoisomerase species. For example, topoisomerase II inhibitor includes anthracyclines such as doxorubicin, quinolones such as ofloxacin, and flavones, topoisomerase I-B inhibitor includes camptothecin, and actinomycin D act upon both types of topoisomerase.

The topoisomerase inhibitors lead the cell to death by way of inhibiting the activity of topoisomerase which is expressed much in cancer cell relative to normal cell. Therefore, it has been known that they cause less harmful effects and possess much more potent anticancer activities than any other anticancer agents.

Some types of cancers can be completely cured over 90% if they are diagnosed early and treated properly, while solid cancers, such as lung cancer, liver cancer and rectum cancer, although various anticancer agents are clinically used, are hard to be cured. Major anticancer therapy includes surgical extirpation, radiotherapy and chemotherapy using anticancer agents. Among them, though the chemotherapy with oral administration or injection of anticancer agent can be easily performed, while not imposing a heavy burden to the patient and not giving fear and discomfort. The chemotherapy is, however, proven to be less satisfactory in the senses that it provides almost no or about 1% therapeutic effect for solid cancers, or even propagates the cancer cell finally to reduce the life span of the patient. Thus, it has been known that surgical extirpation and radiotherapy are more effective in the treatment of solid cancers. Under the circumstances, there are strong reasons for exploring and developing an anticancer agent effective for solid cancers to be used in chemotherapy that may alleviates bodily and mental discomfort of the patient.

SUMMARY OF THE INVENTION

The present inventors have made an effort to develop an anticancer agent effective for solid cancer, and found that: an anticancer agent comprising a topoisomerase inhibitor, though it maintains the survival of caner cells under the state of hypoxia, can induce cell death regardless of oxygen condition if co-administered with nitroimidazole compounds.

A primary object of the present invention is, therefore, to provide an anticancer agent comprising active ingredients of nitroimidazole compound and topoisomerase inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and the other objects and features of the present invention will become apparent from the following descriptions given in conjunction with the accompanying drawings, in which:

Figures la and lb are graphs showing cell survival rates depending on the changes of doxorubicin concentration, under normoxic and hypoxic condition, respectively.

Figures 2a and 2b are graphs showing the concentrations of glucose in cell culture depending on the changes of doxorubicin concentration, under normoxic and hypoxic condition, respectively.

Figures 3a and 3b are graphs showing the concentrations of lactose in cell culture depending on the changes of doxorubicin concentration, under normoxic and hypoxic condition, respectively.

Figures 4a, 4b and 4c, and 5a, 5b and 5c are photographs of electrophoresis showing DNA fragmentation patterns depending on the changes of doxorubicin concentration, under normoxic and hypoxic condition, respectively.

Figures 6a and 6b are graphs showing cell survival rates depending on the changes of pimonidazole concentration, under normoxic and hypoxic condition, respectively.

Figures 7a and 7b are graphs showing the concentrations of glucose in cell culture depending on the changes of pimonidazole concentration, under normoxic and hypoxic condition, respectively.

Figures 8a and 8b are graphs showing the concentrations of lactose in cell culture depending on the changes of pimonidazole concentration, under normoxic and hypoxic condition, respectively. Figures 9a and 9b are graphs showing cell survival rates depending on the changes of pimonidazole and doxorubicin concentrations, under normoxic and hypoxic condition, respectively.

Figures 10a and 10b are graphs showing the concentrations of glucose in cell culture depending on the changes of pimonidazole and doxorubicin concentrations, under normoxic and hypoxic condition, respectively.

Figures 11a and lib are graphs showing the concentrations of lactose in cell culture depending on the changes of pimonidazole and doxorubicin concentrations, under normoxic and hypoxic condition, respectively.

Figures 12a, 12b and 12c, and 13a, 13b and 13c are photographs of electrophoresis showing DNA fragmentation patterns depending on the changes of pimonidazole and doxorubicin concentrations, under normoxic and hypoxic condition, respectively. DETAILED DESCRIPTION OF THE INVENTION

The anticancer agent of the present invention comprises active ingredients of nitroimidazole compound and topoisomerase inhibitor, and their pharmaceutically acceptable carriers. For this anticancer agent, 100 to 1000 ug/mL of nitroimidazole and 0.1 to 100 ug/mL of topoisomerase inhibitor are effective for 2.5 x 10⁵ cell/mL of cancer cell, where the nitroimidazole compound can be pimonidazole, misonidazole, etanidazole or ornidazole and the topoisomerase inhibitor can be doxorubicin, mitomycin C, camptothecin, novobiocin, epirubicin, dactinomycin or etoposide, respectively.

The present invention is further illustrated in more detail as follows.

The basic reason why solid cancer cannot be cured by chemotherapy using anticancer agent is that its therapeutic effect cannot reach the inside of cancer lump which is one of the features of solid cancer. In addition, insufficient supply of oxygen and nutrients may create the state of hypoxia because blood vessels are not properly generated inside of solid cancer. In this regard, for the purpose of developing an anticancer agent employing topoisomerase inhibitor which has been known to be most harmless so far, the present inventors administered topoisomerase inhibitor such as doxorubicin into the cell. From these experiments, the present inventors have found that topoisomerase inhibitor which induces cell death under the state of normoxia, plays a role of maintaining the survival of cancer cells under the state of hypoxia. This finding indicates that the administration of topoisomerase inhibitor into solid cancer even cause to the propagation of the cancer. Under the circumstance, they made every efforts to improve its anticancer activity under the state of hypoxia, and found that it can efficaciously induce cell death under the states of both normoxia and hypoxia if co- administered with nitroimidazole which had been considered that it does not cause cell damage under normoxic condition and induce cell death by generating free radical under hypoxic condition.

The anticancer agent of the invention comprising nitroimidazole and topoisomerase inhibitor induces cell death by the activity of topoisomerase inhibitor under the state of normoxia as well as by the activity of nitroimidazole under the state of hypoxia. Accordingly, the anticancer agent can be practically applied for the treatment of almost all kinds of cancer, inter alia , solid cancer creating the state of hypoxia.

The present invention is further illustrated in the following examples, which should not be taken to limit the scope of the invention.

Example 1 : Effects of topoisomerase inhibitor on survival and metabolism of cells

Under the state of normoxia and hypoxia, it was investigated whether doxorubicin, an anticancer agent acting as a topoisomerase inhibitor, has an effect on the survival and metabolism of cells. Hereinafter, the terms of "normoxia" and "hypoxia" are employed to mean the environmental conditions of 21% oxygen and 1% oxygen, respectively, if they are not specifically defined in the specification.

Example 1-1; Maintenance of cells

HepG2 (human hepatocelluar carcinoma cell line, ATCC HB 8065) was cultured in MEM (minimum essential medium,

Gibco BRL, USA) containing 10% FBS ( fetal bovine serum,

Gibco BRL, USA) , lOOUnits/L penicillin G sodium and lOOμg/L streptomycin sulfate (Gibco BRL, USA) at the temperature of 37 "C, under a normoxic condition of 5% C0₂ and 95% air with replacements of medium every two or three days. When the density of cells occupied 70-80% of plate area during culture, cells were maintained healthy by treating trypsin and subculturing on a new plate.

Example 1-2; Effects of doxorubicin on survival and metabolism of cells

Example 1-2-1: Effects on survival of cells

To investigate the effects of doxorubicin on the survival of cells, trypan blue tests were carried out on the cells which were treated with doxorubicin and cultured under the state of normoxia and hypoxia, respectively: 2.5^χl0⁵cells/ml of HepG2 cells maintained healthy were attached to 60mm plate containing 4mL culture medium, cultured at the temperature of 37 ^°C under a normoxic condition of 5% C0₂ and 95% air for 48 hours, replaced into a new culture medium, and added 0(1 ), 0.01(1 ), 0.1 (A ), 1 (▼ ) , 10(φ) and 100(#)μg/mL of doxorubicin (Ildong Pharmaceutical Co., Ltd., Korea), respectively. Then, the cells were cultured under a hypoxic condition of 5% C0₂ and 1% 0₂. After culturing cells, culture medium was removed from culture solution every 24 hours. Cells were rinsed with PBS, detached from the plate by treating trypsin and harvested by centrifugal separation. The cells thus obtained were suspended on a culture medium, mixed with 0.4% trypan blue (Gibco BRL, USA) at a ratio of 1:1 (v/v) and cultivated for 5 minutes. The number of living cells was counted by hemocytometer . Figures la and lb are the graphs showing the survival rates of cells depending on the changes of doxorubicin concentration under the state of normoxia and hypoxia. As shown in Figures la and lb, it was found that: under the state of normoxia, the cell survival rates began to decrease at the concentration of doxorubicin of O.lμg/mL, and they were decreased, as the concentration of doxorubicin increased; and, under the state of hypoxia, the cell survival rates at the doxorubicin concentrations of 0.1, 1 and lOμg/mL were higher than that with no treatment. Therefore, it could be concluded that doxorubicin, an anticancer agent, plays a role of maintaining the survival of cells under the state of hypoxia.

Example 1-2-2: Effects on metabolism of cells

To investigate the effects of doxorubicin on the metabolism of cells, the glucose consumption and the production and consumption of lactic acid were measured by the concentrations of glucose and lactic acid in a culture solution, respectively: Cells attached to 60mm plate as in Example 1-2-1 were treated with a certain concentration of doxorubicin under the state of normoxia or hypoxia, and cultures were collected from the culture solution every 24 hours and kept freezing at the temperature of -70°C. The concentration of glucose was measured by using a reagent for AsanTech GLU II autoanalyzer (Hitachi 747, Hitachi, Japan) . The concentration of lactic acid was measured by using LAC slide (VITROS, Ortho-Clinical Diagnostics, Inc., USA) and Ektachem 750 (Rochester, USA). Figures 2a and 2b are graphs showing the concentrations of glucose in cell culture depending on the changes of doxorubicin concentration under the state of normoxia and hypoxia, respectively. As shown in Figures 2a and 2b, it was found that: under the state of normoxia, as the concentration of doxorubicin increased, the survival rate of cells was decreased and the concentration of glucose was lowered; under the state of hypoxia, the concentration of glucose was lowered rapidly after 24 hours, regardless of the concentration of doxorubicin. Figures 3a and 3b are the graphs showing the concentrations of lactic acid in cell culture depending on the changes of doxorubicin concentration under the state of normoxia and hypoxia, respectively. As can be seen in Figures 3a and 3b, it was found that: under the state of normoxia, the concentrations of lactic acid, at the concentration range of doxorubicin of 0.01 and O.lμg/mL, increased to the elapsed time of 24hours and decreased after 24hours, and the concentration of lactic acid, at the other concentration ranges, increased a little consistently; and, under the state of hypoxia, the produced lactic acid, in all the cases, was hardly consumed until 24hours. From the results of Figures la, lb, 2a, 2b, 3a and 3b, it could be concluded that:

(1) Under the state of normoxia, as the concentration of doxorubicin increased, the survival rate of cells and the consumption of glucose were decreased. In a case that the concentration of doxorubicin was low, the consumption of glucose increased consistently. However, if glucose was depleted, lactic acid produced was used as energy source by oxidative phosphorylation. If lactic acid was depleted, the survival rate of cells was decreased. (2) Under the state of hypoxia, with the treatment of doxorubicin of 0.1, 1 and lOμg/mL, the survival rates of cells were higher than that with no treatment. In all the cases, glucose was completely depleted in 24hours, while lactic acid produced was not used at all, since the oxidative phosphorylation was not operated owing to the lack of oxygen.

Example 1-3: Cell death by doxorubicin

To investigate the effects of doxorubicin, an anticancer agent acting as a topoisomerase inhibitor, on cell death under the state of normoxia and hypoxia, DNA fragmentation, a major phenomenon of apoptosis was examined, respectively: Cells attached to 60mm plate were treated by 0 (see: Figures 4a and 5a), 0.1 (see: Figures 4b and 5b) and lμg/mL (see: Figures 4c and 5c) of doxorubicin and cultivated under the state of normoxia and hypoxia. After cultivating cells for 0, 12, 24, 30, 36, 48, and 72hours, cells were collected from culture medium to extract DNA by treating lysis buffer, protease K and P LT , etc., and electrophoresis was carried out on 1.5% agarose gel. Figures 4a, 4b and 4c, and 5a, 5b and 5c are photographs of electrophoresis showing the patterns of DNA fragmentation depending on the changes of doxorubicin concentration under the state of normoxia and hypoxia, respectively. In Figures 4a, 4b, 4c, 5a, 5b and 5c, M indicates lOObp DNA marker and 1, 2, 3, 4, 5, 6 and 7 represent DNAs extracted from cells at the elapsed time of Ohour, 12hours, 24hours, 30hours, 36hours, 48hours and 72hours after treatment of doxorubicin, respectively. As can be seen in Figures 4a, 4b and 4c, it was found that: under the state of normoxia, DNA fragmentation was observed after 72hours with no treatment of doxorubicin, and it was observed earlier, as the concentration of doxorubicin increased. Also, Figures 5a, 5b and 5c have shown that: under the state of hypoxia, DNA fragmentation began to be observed from the elapsed time of 24hours in all cases; and, it was further observed to the elapsed time of 30hours with no treatment of doxorubicin, to the elapsed time of 72hours with O.lμg/mL treatment, to the elapsed time of 48hours with lμg/mL treatment, respectively, and then, it was not observed any more. Therefore, it could be concluded that doxorubicin can induce apoptosis under the state of normoxia, while it suppresses apoptosis under the state of hypoxia.

Example 2 : Effects of other anticancer agents on survival and metabolism of cells under the state of hypoxia

It was investigated whether other anticancer agents besides doxorubicin, acting as topoisomerase inhibitors or expressing anticancer activities with other mechanisms, repress apoptosis under the state of hypoxia, and have the effects of maintaining cell survival in a similar manner as in Examples 1-2-1 to 1-2-3. As anticancer agents such as doxorubicin (Ildong Pharmaceutical Co., Ltd., Korea), epirubicin (Ildong Pharmaceutical Co., Ltd., Korea), mitomycin C (Korea United Pharm. Co., Ltd., Korea), etoposide (Dong-a Pharmaceutical Co., Ltd., Korea) and dactinomycin (Merck & Co., Inc., USA) were used in the experiments. Table 1 shows the effects of anticancer agents acting as topoisomerase inhibitors on the repression of apoptosis. As shown in Table 1 below, under the state of hypoxia, all of the anticancer agents acting as topoisomerase inhibitors, though their effective amounts are different from one another, possessed a biological activity of repressing apoptosis and mitomycin C, an alkylating agent, possessed the same activity, while the other anticancer agents possessed little activities. Therefore, it was clearly demonstrated that anticancer agents acting as topoisomerase inhibitors repress apoptosis under the state of hypoxia.

Table Effects of anticancer agents acting as topoisomerase inhibitors on repression of apoptosis under the state of hypoxia

Example 3 : Effects of pimonidazole on survival and metabolism of cells

Example 3-1: Effects on survival of cells

To investigate the effects of pimonidazole on the survival of cells, cells were treated with pimonidazole (National Cancer Institute, Korea) at the concentrations of 0(1 ), 1(1 ), 10 (A ) , 100 (▼ ) and 1000 (♦) μg/mL, and cultured under the state of normoxia and hypoxia in a similar manner as in Example 1-2-1, and then the survival rates of cells were measured, respectively. Figures 6a and 6b are the graphs showing the survival rates of cells depending on the changes of pimonidazole concentration under the state of normoxia and hypoxia, respectively. As can be seen in Figures 6a and 6b, lOOμg/mL and less pimonidazole had no effect on the survival of cells under the state of normoxia, while all the cells under the state of hypoxia were dead after 1 day of cultivation, regardless of the concentration of pimonidazole.

Example 3-2: Effects on metabolism of cells

To investigate the effects of pimonidazole on the metabolism of cells, cell culture was treated with pimonidazole at the concentrations of 0 (• ) , 1(1 ), 10 (A ) , 100 (▼ ) and 1000 (♦) μg/mL in a similar manner as in Example 1-2-2, and the concentrations of glucose and lactic acid were measured, respectively. Figures 7a and 7b are the graphs showing the glucose concentrations of cell culture depending on the changes of pimonidazole concentration under the state of normoxia and hypoxia, respectively. As shown in Figures 7a and 7b, it was observed that: under the state of normoxia, lOOμg/mL of pimonidazole had a little effect on the glucose concentration of cell culture, while less than lOOμg/mL of pimonidazole had little effect on it, similarly as in the above results of the survival rate of cells; under the state of hypoxia, lOOOμg/mL of pimonidazole had a similar effect to the case under the state of normoxia, while in case of lOOμg/mL of pimonidazole, the extent of decrease in the concentration of glucose was lower than that of the state of normoxia. Figures 8a and 8b are the graphs showing the lactic acid concentrations of cell culture depending on the changes of pimonidazole concentration under the state of normoxia and hypoxia, respectively. As can be seen in Figures 8a and 8b, it was observed that: under the state of normoxia, lOOμg/mL and less pimonidazole had little effect on the variation of lactic acid concentration, similarly as the above results of the survival rate of cells and the concentration of glucose; under the state of hypoxia, lOOμg/mL and more pimonidazole only had an effect on the variation of lactic acid concentration, similarly as the above results of glucose concentration. From the results of Figures 6a, 6b, 7a, 7b, 8a and 8b, it was clearly demonstrated that lOOμg/mL pimonidazole does not have a large effect on the survival and metabolism of cells under the state of normoxia, while it inhibits the metabolism of cells under the state of hypoxia.

Example 4 : Effects of pimonidazole and doxorubicin on survival and metabolism of cells

Based on the results of Examples 3-1 and 3-2, it was investigated the effects of lOOμg/mL pimonidazole and doxorubicin of various concentrations on the survival and metabolism of cells under the state of normoxia and hypoxia, respectively.

Example 4-1: Effects on survival of cells

To investigate the effects of pimonidazole and doxorubicin on the survival of cells, cells were treated with Oμg/mL of doxorubicin and pimonidazole (• ), O.lμg/mL of doxorubicin (I ), lμg/mL of doxorubicin (A ), lOOμg/mL of pimonidazole (▼ ), O.lμg/mL of doxorubicin and lOOμg/mL of pimonidazole (♦) and lμg/mL of doxorubicin and lOOμg/mL of pimonidazole (•) , and cultured under the state of normoxia and hypoxia in a similar manner as in Example 1-2-1, and then the survival rates of cells were measured, respectively. Figures 9a and 9b are the graphs showing the survival rates of cells depending on the changes of pimonidazole concentration under the state of normoxia and hypoxia, respectively. As shown in Figures 9a and 9b, the results of treating both pimonidazole and doxorubicin were very similar to those of treating only doxorubicin under the state of normoxia, while the former showed more rapid decrease of cell survival rate than the latter. Accordingly, these results indicated that: treatment of both lOOμg/mL of pimonidazole and doxorubicin whose concentration is within the range for maintaining cell survival under the state of hypoxia has no significant effect on cell survival under the state of normoxia, while it induces cell death under the state of hypoxia.

Example 4-2: Effects on metabolism of cells

To investigate the effects of pimonidazole and doxorubicin on the metabolism of cells, cell culture was treated with pimonidazole and doxorubicin at the same concentrations as in Example 4-1, and the concentrations of glucose and lactic acid were measured in a similar manner as in Example 1-2-2. Figures 10a and 10b are the graphs showing the glucose concentrations of cell culture depending on the changes of pimonidazole and doxorubicin concentrations under the state of normoxia and hypoxia, respectively, and Figures 11a and lib, graphs showing the lactose concentration of cell culture depending on the changes of pimonidazole and doxorubicin concentrations under the state of normoxia and hypoxia, respectively. As shown in Figures 10a, 10b, 11a and lib, it was clearly demonstrated that: under the state of normoxia, treatment of both pimonidazole and doxorubicin, gave very similar results to those of treating only doxorubicin; under the state of hypoxia, pimonidazole has more critical effects on metabolism than doxorubicin similarly as the effects on cell survival of Example 4-1. Example 4-3: Cell death by pimonidazole and doxorubicin

To investigate the effects of pimonidazole and doxorubicin on cell death, each DNA was extracted from cells treated with lOOμg/mL of pimonidazole ( see : Figures 12a and 13a), O.lμg/mL of doxorubicin and lOOμg/mL of pimonidazole (see: Figures 12b and 13b), and Iμg/mL of doxorubicin and lOOμg/mL of pimonidazole ( see : Figures 12c and 13c) , respectively, and then DNA fragmentation was examined in a similar manner as in Example 1-3. Figures 12a, 12b and 12c, and 13a, 13b and 13c are photographs showing DNA fragmentation patterns depending on the changes of pimonidazole and doxorubicin concentrations under the state of normoxia and hypoxia, respectively. As shown in Figures 12a, 12b, 12c, 13a, 13b, and 13c, only doxorubicin had an effect under the state of normoxia, while only pimonidazole had an effect under the state of hypoxia.

Example 5 : Effects of doxorubicin and pimonidazole depending on oxygen concentration

To investigate the effects of oxygen concentration on the biological activities of doxorubicin and pimonidazole, cells were treated with Oμg/mL of doxorubicin and pimonidazole (control) , O.lμg/mL of doxorubicin (Doxo 0.1), lμg/mL of doxorubicin (Doxo 1), lOOμg/mL of pimonidazole (Pimo 100), O.lμg/mL of doxorubicin and lOOμg/mL of pimonidazole (Doxo 0.1 + Pimo 100) and lμg/mL of doxorubicin and lOOμg/mL of pimonidazole (Doxo 1 + Pimo 100), under an environment of 1, 3, 5, 10 and 21% of oxygen, respectively. And then, the survival rates of cells were measured, respectively. Table 2 shows the survival rates of cells with symbols of ®, O, Δθ, Δ, Δx and x in a descending order, at various concentrations of oxygen, doxorubicin and pimonidazole. As shown in Table 2, in the case of doxorubicin treatment only, O.lμg/mL of doxorubicin had stronger activity of maintaining cell survival than that of control only under the condition of less than 3% oxygen, and lμg/mL of doxorubicin did only under the condition of 1% oxygen. Further, in the case of pimonidazole treatment only, pimonidazole had stronger activity of inducing cell death under the condition of less than 10% oxygen, than that of control. Also, in the case of co-treatment of doxorubicin and pimonidazole, treatment of O.lμg/mL of doxorubicin and lOOμg/mL of pimonidazole had similar activity to those of pimoniazole treatment only under the condition of less than 10% oxygen, and doxorubicin treatment only under the condition of 21% oxygen, treatment of lμg/mL of doxorubicin and lOOμg/mL of pimonidazole had similar activity to those of pimoniazole treatment only under the condition of 1% oxygen, and doxorubicin treatment only under the condition of more than 21% oxygen, and it had strong synergic effects under the oxygen concentration of 3%. Accordingly, it was clearly demonstrated that: the effects of doxorubicin on cell death are oxygen-dependent and, its effects on cell survival can be completely inhibited with co-treatment of pimonidazole.

Table 2: Cell survival rates at various concentrations of oxygen, doxorubicin and pimonidazole

Example 6: Effects of co-treatment of nitroimidazole and other anticancer agents

Besides doxorubicin and pimonidazole, to investigate whether the interaction of other anticancer agents and other kinds of nitroimidazole compounds can induce cell death under the state of hypoxia, the survival rates of cells which were treated with various kinds of anticancer agents and nitroimidazole compounds were measured, respectively. Nitroimidazole compounds used herein include metronidazole (Keunhwa Chem. Co., Korea), ornidazole (Keunhwa Chem. Co., Korea), etanidazole (Sigma Chem. Co., USA), tinidazole (Sigma Chem. Co., USA), dimetridazole (Sigma Chem. Co., USA), misonidazole (National Cancer Research Center, Korea) and pimonidazole (National Cancer Research Center, Korea) . Table 3 shows the effects of nitroimidazole with co-treatment of other anticancer agents, where each numeral indicates the concentration (μg/mL) of a nitroimidazole compound sufficient for inhibiting the activity of an anticancer agent for maintaining cell survival, each value in ^λ ( ) ' indicates the time of showing the activity at the above concentrations of nitroimidazole, each value after V indicates the concentration of an anticancer agent, at which it has the strongest activity of maintaining cell survival, and x' indicates almost no effect. As shown in Table 3, lOOμg/mL of pimonidazole, if co-treated with other anticancer agents besides doxorubicin, which maintain cell survival under the state of hypoxia, can lead all cells to death within 24 hours. In addition to pimonidazole, misonidazole and etanidazole possessed similar activity at the high concentration of lOOOμg/mL, and lOOOμg/mL of ornidazole did the same if co-treated only with doxorubicin, camptothesin or dactinomycin. Interestingly, all of nitroimidazole compounds except the aboves had no activity unless they were co-treated with dactinomycin.

Table 3 : Effects of nitroimidazole with co-treatment of other anticancer agents(unit : ug/mL)

As clearly illustrated and demonstrated as above, the present invention provides an anticancer agent comprising active ingredients of nitroimidazole compound such as pimonidazole, misonidazole, etanidazole and ornidazole, and a topoisomerase inhibitor such as doxorubicin, mitomycin C, camptothecin, novobiocin, epirubicin, dactinomycin and etoposide, and their pharmaceutically acceptable carriers. The anticancer agent induces cell death by the activity of topoisomerase inhibitor under the state of normoxia as well as by the activity of nitroimidazole compound under the state of hypoxia. Accordingly, the anticancer agent can be practically applied for the treatment of almost all kinds of cancer, inter alia , solid cancer creating the state of hypoxia regardless of oxygen condition.

While the present invention has been shown and described with reference to the particular embodiments, it will be apparent to those skilled in the art that many changes and modifications may be made without departing from the spirit and scope of the invention as defined in the claims.

Claims

WHAT IS CLAIMED IS:

1. An anticancer agent comprising active ingredients of nitroimidazole compound and topoisomerase inhibitor, and their pharmaceutically acceptable carriers.

2. The anticancer agent of Claim 1, wherein the nitroimidazole compound is pimonidazole, misonidazole, etanidazole or ornidazole.

3. The anticancer agent of Claim 1, wherein the topoisomerase inhibitor is doxorubicin, mitomycin C, camptothecin, novobiocin, epirubicin, dactinomycin or etoposide .

4. The anticancer agent of Claim 1, wherein the effective dose on cancer cell of 2.5 x 10⁵ cells/ml is 100 to lOOOug/ml for nitroimidazole compound and 0.1 to lOOug/ml for topoisomerase inhibitor, respectively.

5. The anticancer agent of claim 1, which is effective for cancer creating a hypoxic condition of 0 to 5.0% 0₂.