WO2002013826A1

WO2002013826A1 - Anti-cancer composition composed of anti-cancer and anti-malarial drugs

Info

Publication number: WO2002013826A1
Application number: PCT/KR2001/001314
Authority: WO
Inventors: Ki-Chang Keum; Nae-Choon Yoo; Won-Min Yoo
Original assignee: Keum Ki Chang; Yoo Nae Choon; Yoo Won Min
Priority date: 2000-08-02
Filing date: 2001-08-02
Publication date: 2002-02-21
Also published as: KR20020011528A; EP1313475A1; AU2001275821A1; KR100390332B1; WO2002013826A9; EP1313475A4

Abstract

The present invention relates to complex compositions of anticancer drugs, which are administrated in combination with antimalarial drugs for reducing a minimum IC50 of anticancer drugs and inhibiting development of drug resistance of cancer cells caused by anticancer drugs, thereby enhancing the effect of the anticancer drugs. More particularly, the present invention relates to complex compositions of anticancer drugs, which are administrated in combination with antimalarial drugs for a minimun IC50 of anticancer drugs and inhibiting development of drug resistance of cancer cells caused by anticancer drugs, thereby enhancing the effect of the anticancer drugs; in which the anticancer drugs are selected from the group consisting of doxorubicin, cisplatin, and the like and the antimalarial drugs are selected from the group consisting of hydroxchloroquine, chloroquine, primaquine and the like.

Description

ANTICANCER COMPOSITION COMPOSED OF ANTICANCER AND ANTIMALARIAL DRUGS

Technical Field The present invention relates to a complex composition of an anticancer drug in combination with an antimalarial drug for reducing a minimum IC₅₀ of the anticancer drug and inhibiting development of drug resistance in cancer cells caused by exposure to the anticancer drug, thereby enhancing the effect of the anticancer drug. More particularly, the present invention relates to a complex composition of an anticancer drug in combination with an antimalarial drug for reducing a minimum IC₅₀ of the anticancer drug and inhibiting development of drug resistance in cancer cells caused by exposure to the anticancer drug, thereby enhancing the effect of the anticancer drugs; in which the anticancer drug is selected from the group consisting of doxorubicin, cisplatin, and the like and the antimalarial drug is selected from the group consisting of hydroxychloroquine, chloroquine, primaquine and the like. According to the. present invention, the efficacy of the anticancer drugs can be increased by approximately 3 times for breast cancer, approximately 10 times for gastric cancer, approximately 10 times for colon cancer and approximately 10 times for sarcoma, as compared with the treatment with of the anticancer drugs only.

Background Art Recently, chemotherapy exhibits significant progress in treating acute leukemia, malignant lymphoma, testicular tumor, and the like. However, there are still problems to be solved in that some cancers show resistance to anticancer drugs from the early stage of chemotherapy, or tend to relapse after stopping the therapy, leading to death. The drug resistance of cancer cells is one of the major obstacles in the treatment of the cancer. It is suggested that the mechanism through which cancer cells come to develop resistance to anticancer drugs involves pharmacokinetic factors, types and biological features of pathologic cells, immunity of subjects, administration route of drugs, and drug resistance at a cellular level. Among these, drug resistance at a cellular level is considered to be the most important. Multidrug resistance (MDR) refers to a phenomenon, whereby cancer cells which have acquired a resistance to a first drug, also show resistance to other drugs having chemical structures completely different from the first drug.

Late in 1960s, Kessel et al . observed that P388 leukemia cell line, made to be resistant to vinblastin by continuous exposure in a low dosage, exhibits cross- resistance against dactinomycin, vincristine and daunorubicin. Such phenomenon was termed "Multidrug Resistance (MDR)" by Biedler et al . Ling and Thomson discovered that surface glycoproteins of 170 kD were increased in membranes of Chinese hamster ovarian tumor cells which had acquired multidrug resistance. They called the proteins "p-glycoproteins" and reported that these p-glycoproteins function in multidrug resistance by acting to reduce concentrations of drugs in the cells. The p-glycoproteins function as a pump to actively force anticancer drugs out of the cells using calcium and ATP, inhibiting the drugs from accumulating in the tumor cells. Consequently, the concentration of the drugs in the cells is reduced and the effect of the drugs is thus decreased. Anticancer drugs which have been found to be associated with the multidrug resistance include anthracycline family drugs such as adriamycin and daunorubicin; vinka alkaloid family drugs such as vincristine and vinblastin; epipodophllotoxin family drugs such as etoposide; and others such as actinomycin-D and taxol . Also, it was found that these drugs induce cross-resistances with each other. Such anticancer drugs are commonly characterized in that they are hydrophobic materials having a molecular weight of 300 to 900; that they comprise complicated ring structures and have a nitrogen group of a positive charge; and that they are passively diffused into the cells. In 1986, Gros et al . examined the cDNA base sequence of a gene for p-glycoprotein, and made the first identification of MDR1 (Multidrug Resistance 1) gene, which is associated with multidrug resistance.

The p-glycoprotein includes 1280 amino acids and has a structure similar to those of transport proteins such as hemolysin B, leukotoxin, histidine, and the like. It was also found that transporting proteins having structures similar to that of the p-glycoprotein exist in bacteria and yeast. Thus, it is noted that the p-glycoprotein can be found in normal cells of the small and large intestines, adrenal glands, kidneys, liver, etc. as well as tumor cells and is a variety of transporting proteins to play a general role for transporting intracellular cytotoxin out of cells. Particularly, the p-glycoprotein mainly distribute in the biliary tract in the liver, luminar surface in proximal tubules of kidneys, columnar cells in luminar mucous membrane of small and large intestines, and adrenal cortex and medulla. Also, it was shown that levels of mdrl mR A are high at diagnosis in some cancers including colon cancer, kidney cancer, liver cancer, chronic leukemia, adrenal carcinoma and non- small cell cancer, which indicates endogenous multidrug resistance (see, Fojo AT, Ueda K, Slamon DJ, Poplack DG and Gottesman MM, Pastan I: Expression of a multidrug resistance gene in human tumors and tissues, Proc. Natl. Acad. Sci. USA, 84:265 (1987); and Glodstein LJ, Glaski H, Fojo A, Willingham M, Lai SL, Gazdar A, and Pirker R, Expression of a multidrug resistance gene in human cancers, J. Natl. Cancer Inst., 81:116 (1989)).

In many cancers, the overexpression of mdrl mRNA and p-glycoprotein are considered to be a major cause of failure of the treatment. These overexpressions are sometimes referred to as indicators or factors forecasting the efficacy of the treatment and prognosis. Such being the case, there have been attempts to develop a treatment aimed at inhibiting the expression of the multidrug resistant gene and controlling the function of p-glycoprotein in order to overcome the multidrug resistance. Tsuro et al . disclosed a use of a calcium channel blocker, for example, verapamil, based on the fact that the cancer cells showing multidrug resistance contains a large amount of calcium in plasmalemma and cytoplasm, compared to sensitive cancer cells. They reported that the calcium channel blocker suppresses the elimination of anticancer drugs out of the tumor cells and increases the accumulation of the anticancer drugs, thereby overcoming the multidrug resistance (Tsuruo T. Fidler IJ, Differences in drug sensitivity among tumor cells from parental tumors, selected variants, and spontaneous metastases, Cancer Research, 41 (8) : 3058-64, Aug. 1981 (1981)). Other than verapamil, cyclosporin-A, calmodulin, phenothiazine, quinine and tamoxifen have been examined for their utility as calcium channel blockers. However, these drugs have limitations in their clinical application in that they can hardly reach the concentration sufficient to overcome the multidrug resistance in the body. Even when their concentrations in the body are sufficient to overcome the multidrug resistance, they show certain side effects such as cardiotoxicity, nephrotoxicity, and so on. These drugs have been reported to be so effective so as to be able to expect a perfect cure in hematologic malignancies such as leukemia, malignant lymphoma and multiple myeloma. However, their efficacy in most of solid tumors is insignificant.

Hydroxychloroquine is one of well known antimalarial drugs and is sometimes used as an anti- inflammatory agent for various rheumatic diseases. It is known that antimalarial drugs including hydroxychloroquine exhibit pharmacological effect by increasing the pH of intracellular organelles including the lysosome, endosome and the trans -Golgi network (see, Fox RI, Mechanism of Action of Hydroxychloroquine as an Antirheumatic Drug, Seminars in Arthritis & Rheumatism, 23 (2 Suppl. 1):82-91, 1993; Fox R, Antimalarial drugs: Possible Mechanisms of Action in Autoimmune Disease and Prospects for Drug Development, Lupus, 5 Suppl. 1.S4-10, 1996) . Also, it was reported that alkalinization of intracellular organelles affects the secretion of proteins synthesized intracellularly and also inhibits the synthesis of DNA and RNA.

Disclosure of Invention In order to overcome multidrug resistance to the existing anticancer drugs in the course of cancer chemotherapy, the present inventors studied the effectiveness of combined administration of an anticancer drug with an antimalarial drug to inhibit the development of drug resistance in cancer cells. We treated stomach cancer and colon cancer cell lines by administration of an anticancer drug in combination with an antimalarial drug in prescribed proportions and examined IC₅₀ of respective combinations of the anticancer drug and the antimalarial drug. The used anticancer drug includes doxorubicin and cisplatin. The used antimalarial drug includes hydroxychloroquine, chloroquine and primaquine. As a result, it was discovered that the combined administration of an anticancer drug with an antimalarial drug can inhibit multidrug resistance in the cancer cells caused by exposure to the anticancer drug, thereby enhancing the effect of the anticancer drug.

Brief Description of Drawings

The above object and other objects, features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings, in which:

Fig. 1 is a graph depicting the changes in cytotoxicity of doxorubicin (ADR) and cisplatin (DDP) when combined with hydroxychloroquine at concentrations of 15 and 30 μg/ml in colon and gastric cancer cell lines, respectively (The X axis represents administered concentrations of the anticancer drug. The Y axis represents cell viability) ; and

Fig. 2 is a graph depicting the changes in cytotoxicity of ADR and DDP when combined with either chloroquine at concentrations of 20 μM (10.318 μg/ml) and 40 μM (20.636 μg/ml), or primaquine at concentrations of 1.5 μM (0.683 μg/ml) and 3 μM (1.366 μg/ml) in breast cancer, gastric cancer and fibrosarcoma cell lines, respectively (The X axis represents administered concentrations of anticancer drug. The Y axis represents cell viability) .

Best Modes for Carrying out the Invention

Culture of cell lines and tumor cells Colon cancer cell lines of HT-29 (ATCC HTB38, human colonic adenocarcinoma, moderately well differentiated grade II) and HCT-15 (ATCC CCL225, human colonic adenocarcinoma) , gastric cancer cell lines of KHH (YCC-2, human gastric adenocarcinoma), PHB (YCC-3, human gastric adenocarcinoma), KMB (YCC-7, human gastric adenocarcinoma) and AGS (ATCC CRL 1739, human gastric adenocarcinoma) , a fibrosarcoma cell line of HT 1080 (ATCC CCL 121, human fibrosarcoma) , and breast cancer cell lines of SK-BR-3 (ATCC HTB 30, human breast adenocarcinoma, malignant pleural effusion) are maintained in RPMI 1640 medium (Gibco, U.S. ) supplemented with 10% fetal calf serum (Commonwealth Serum Lab., Australia; hereinafter referred to as FCS) , which has been thermally inactivated (56 °C, 30 min.), containing 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, U.S.A) in a 37 °C incubator under 5% C0₂. Agents used in experiments

The anticancer drugs used in the experiments are adriamycin (ADR, generic name doxorubicin) , supplied by Farmitalia Carlo Erba Ltd. (Italy) , and diaminodichloro platinu (DDP, generic name cisplatin), supplied by Pharmachemie B.V. (Holland) . The antimalarial drugs used in the experiments were hydroxychloroquine, chloroquine, and primaquine. The anticancer drugs are administered in combination with the antimalarial drugs varying proportions and are examined for cytotoxicity according to MTT assay using 3- (4 , 5-dimethylthiazol-2-yl) -2 , 5- diphenyl-1-butene (Sigma, U.S.A.). Particularly, ADR is used at concentrations of 10, 1, 0.1 and 0.01 μg/ml by cascade dilutions in each column on a microplate while DDP is assayed at 50, 5, 0.5, 0.05 μg/ml. For the antimalarial drugs, hydroxychloroquine is examined at concentrations of 15 and 30 μg/ml, chloroquine at 10.3 and 20.6 μg/ml, and premaquine at 0.68 and 1.36 μg/ml. Cytotoxicity (anticancer effect) test by MTT assay

The principle of the MTT assay is based on the phenomenon that succinate dehydrogenase, an mitochondrial enzyme located at cytochrome b and c of a living cell, cleaves tetrazolium ring of 3- [4,5- dimethylthiazol-2-yl] -2, 5-diphenyltetrazolium bromide (MTT) , whereby the yellow color of the MTT salt changes to the purple color of formazan, a reduced product. That is, the MTT assay cannot be observed in media of dead cells or tissues, but can be observed selectively only in the viable cells. The color change is measured by counting living cells by means of a spectrophotometer . The MTT assay is now one of the in vitro test methods for sensitivity of human tumor cells to anticancer drugs which are now recommended by the National Cancer Institute in U.S.A., due to its excellent reproducibility on repeated experiments.

According to the present invention, the MTT assay is performed as described by Carmichael et al . In brief, first, the standard growth curve of each cultured cancer cell line is generated to determine the exponential growth period of each cell line. Then, each exponentially dividing cell line is treated with 0.25% trypsin-EDTA, suspended in a single cell population and washed three times with RPMI 1640 medium containing 10% FCS . Cells in each culture are counted by staining with Trypan blue (Gibco, U.S.A.) . 180 μl of cultures at the exponential phase are aliquoted on a 96 well plate (Costar, U.S.A), followed by an incubation at 37°C under 5% C0₂. After 24 hours incubation, anticancer drugs at various concentrations are added either alone or in combinations with antimalarial drugs, each dissolved in saline at a volume of 20 μl, followed by a further 4 days incubation. 50 μl of MTT (2 mg/ l stock) is added to the wells and further incubated for 4 hours. Control cultures include equivalent amounts of saline instead of antimalarial drug. After final incubation, the plate is centrifuged at 450 g for 10 minutes. Supernatants are carefully removed from the well plate with the aid of digital multichannel pipette (Flow Titertck, Finland) to leave a 30 μl of culture in each well. To each well was added 150 μl dimethyl sulfoxide (Sigma, U.S.A), followed by shaking the plate at 36 °C for 10 minutes until formazan was completely solubilized. The plate was subject to multi-well ELISA automatic spectrophotometer reader (Behring ELISA Processor II, Germany) to measure the absorbance at 540 nm. Cell viability was calculated by the following equation:

% cell viability average absorbance of test group-standard absorbance average absorbance of control group-standard absorbance

Example 1

According to the described method above, each IC₅₀ of doxorubicin (ADR) and cisplatin (DDP) was measured when combined with hydroxychloroquine (HCQ) at concentrations of 15 μg/ml and 30 μg/ml in cancer cell lines. The results are shown in Table 1 below. Table 1

As seen from the table 1, the combinations of ADR with HCQ showed about 10 fold increased anticancer effects in colon cancer cell lines, HT-29 and HCT-15, about 3 to 10 fold in gastric cancer cell lines, YCC-2, YCC-3 and YCC-7. Also, the combinations of DDP with HCQ showed anticancer effects increased 2 to 5 fold in colon cancer cell lines, about 4 to 15 fold in gastric cancer cell lines, compared with the treatment with anticancer drug only.

Example 2

According to the same method as in Example 1, each IC₅₀ of doxorubicin (ADR) and cisplatin (DDP) was measured when combined with chloroquine (CQ) at concentrations of 20 μM (10.318 μg/ml) and 40 μM (20.636 μg/ml) in cancer cell lines. The results are shown in Table 2 below.

Table 2

As seen from the table 2, the combinations of ADR with CQ showed anticancer effects improved by 1.4 to 3 fold in a breast cancer cell line, SK-Br-3, about 6 to 10 fold in gastric cancer cell lines, AGS and YCC-7, and about 4 to 10 fold in fibrosarcoma cell line, HT1080. Also, the combinations of DDP with CQ showed anticancer effects about 2 to 2.3 fold in the breast cancer cell line, about 1 to 5 fold in the gastric cancer cell lines, and about 2.3 to 12 fold in the fibrosarcoma cell line, compared with the treatment with anticancer drug only.

Example 3

According to the same method as in Example 1, each IC₅₀ of doxorubicin (ADR) and cisplatin (DDP) was measured when combined with primaquine (PQ) at concentrations of 1.5 μM (0.683 μg/ml) and 3 μM (1.366 μg/ml) in cancer cell lines. The results are shown in Table 3 below.

Table 3

As seen from the table 3, the combinations of ADR with PQ showed anticancer effects increased by about 3 fold in a breast cancer cell line, SK-Br-3, about 10 fold in gastric cancer cell lines, AGS and YCC-7, and about 3 to 4 fold in fibrosarcoma cell line, HT1080. Also, the combinations of DDP with PQ showed anticancer effects improved by about 1.2 fold in the breast cancer cell line, about 1 to 6 fold in the gastric cancer cell lines, and about 1.3 fold in the fibrosarcoma cell line, compared with the treatment with anticancer drug only. These results demonstrate that the combinations of an anticancer drug with an antimalarial drug showed markedly improved anticancer effects on many cancer types.

Administration of the antimalarial drug may be performed by either oral or parenteral routes in accordance with the anticancer drugs. Preferably, hydroxychloroquine, chloroquine and primaquine are administered to humans at a dose of 0.1 to 500 mg/kg, 0.1 to 700 mg/kg, and 0.1 to 800 mg/kg, respectively, together with an anticancer drug in the amount which has been conventionally used for chemotherapy by practitioners skilled in the art. More preferably, 10 to 100 mg/kg for hydroxychloroquine, 10 to 300mg/kg for chloroquine, and 50 to 500 mg/kg for primaquine are administered.

Industrial Applicability

As clearly described and demonstrated above, the present invention provides a complex composition comprising an anticancer drug including doxorubicin or cisplatin in combination with an antimalarial drug including hydroxychloroquine, chloroquine or primaquine. The composition of the invention lowers the IC₅₀ of anticancer drug and inhibits the development of drug resistance in cancer cells. The composition is therefore capable of enhancing the effectiveness of chemotherapy about 3 fold in breast cancer, about 10 fold in gastric cancer, colon cancer, and sarcoma, respectively, compared with the treatment with anticancer drug only.

Claims

1. A complex composition comprising an anticancer drug in combination with an antimalarial drug for enhancing the effect of the anticancer drugs.

2. The complex composition of claim 1, wherein the antimalarial drug inhibits development of drug resistance in cancer cells, thereby enhancing the effectiveness of chemotherapy for cancer.

3. The complex composition of claim 1, wherein the antimalarial drug is selected from the group consisting of hydroxychloroquine, chloroquine and primaquine and the anticancer drug is selected from the group consisting of doxorubicin and cisplatin.

4. The complex composition of claim 3, wherein the antimalarial drug is hydroxychloroquine, and the anticancer drug is doxorubicin.