KR100390332B1 - anti-cancer composition composed of anti-cancer and anti-malarial drugs - Google Patents
anti-cancer composition composed of anti-cancer and anti-malarial drugs Download PDFInfo
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Abstract
본 발명은 항말라리아제와 항암제의 병용투여에 의해 항암제의 최소 유효농도를 낮추고 항암제에 의한 암세포의 약제 내성을 억제하여 항암제의 항암효과를 상승시킨 항암제 복합 조성물에 관한 것이다. 구체적으로는, 항말라리아제로 흔히 사용되는 하이드록시클로로퀸, 클로로퀸, 프리마퀸 등과 대표적인 항암제인 독소루비신, 시스플라틴 등을 병용투여하면 항암제의 최소 유효농도(IC50)을 낮추고 항암제에 의한 암세포의 약제 내성을 억제하여 유방암의 경우 약 3배, 위암의 경우 약 10배, 대장암의 경우 약 10배, 육종암의 경우 약 10배까지 항암 효과를 상승시킬 수 있는 항암제 복합 조성물에 관한 것이다.The present invention relates to an anticancer complex composition in which the antimalarial agent and the anticancer agent are administered in combination, thereby lowering the minimum effective concentration of the anticancer agent and inhibiting drug resistance of the cancer cells by the anticancer agent, thereby increasing the anticancer effect of the anticancer agent. Specifically, in combination with hydroxychloroquine, chloroquine, primaquine, etc., which are commonly used as antimalarial agents, doxorubicin and cisplatin, which are representative anticancer agents, lower the minimum effective concentration (IC 50 ) of anticancer agents and inhibit drug resistance of cancer cells by anticancer agents. By about 3 times in the case of breast cancer, about 10 times in the case of stomach cancer, about 10 times in the case of colorectal cancer, and about 10 times in the case of sarcoma, the present invention relates to an anticancer complex composition.
Description
본 발명은 항말라리아제와 항암제의 병용투여에 의해 항암제의 최소 유효농도를 낮추고 항암제에 의한 암세포의 약제 내성을 억제하여 항암제의 항암효과를 상승시킨 항암제 복합 조성물에 관한 것이다. 구체적으로는, 항말라리아제로 흔히 사용되는 하이드록시클로로퀸, 클로로퀸, 프리마퀸 등과 대표적인 항암제인 독소루비신, 시스플라틴 등을 병용투여하면 항암제의 최소 유효농도(IC50)을 낮추고 항암제에 의한 암세포의 약제 내성을 억제하여 유방암의 경우 약 3배, 위암의 경우 약 10배, 대장암의 경우 약 10배, 육종암의 경우 약 10배까지 항암 효과를 상승시킬 수 있는 항암제 복합 조성물에 관한 것이다.The present invention relates to an anticancer complex composition in which the antimalarial agent and the anticancer agent are administered in combination, thereby lowering the minimum effective concentration of the anticancer agent and inhibiting drug resistance of the cancer cells by the anticancer agent, thereby increasing the anticancer effect of the anticancer agent. Specifically, in combination with hydroxychloroquine, chloroquine, primaquine, etc., which are commonly used as antimalarial agents, doxorubicin and cisplatin, which are representative anticancer agents, lower the minimum effective concentration (IC 50 ) of anticancer agents and inhibit drug resistance of cancer cells by anticancer agents. By about 3 times in the case of breast cancer, about 10 times in the case of stomach cancer, about 10 times in the case of colorectal cancer, and about 10 times in the case of sarcoma, the present invention relates to an anticancer complex composition.
최근 급성 백혈병, 악성 임파종 및 고환암 등의 일부 고형암에서 항암화학요법에 의한 치료성적이 괄목할만하게 향상되었다. 그러나, 아직도 많은 종류의 암에서는 초기 치료에서부터 항암제에 내성을 보이거나, 치료후 재발하여 사망에 이르게 된다. 따라서, 항암제에 대한 암세포의 내성은 암치료에 있어 중요한 장애요인중의 하나이다. 암세포가 항암제에 대해 내성을 가지게 되는 기전에는 약동학적인 요인, 병리세포형과 생물학적 특성, 개체의 면역반응, 약제투여경로 및 세포단위에서의 약제내성이 제시되고 있다. 이중에서 세포단위에서의 약제내성이 가장 중요한 기전으로 생각되며, 한가지 약제에 내성이 생긴 암세포가 화학적 구조가 전혀 다른 항암제에 대해서도 동시에 내성을 가지는 경우를 다중약제내성(multidrug resistance, MDR)이라고 한다.Recently, some solid cancers, such as acute leukemia, malignant lymphoma and testicular cancer, have improved remarkably. However, many types of cancer are still resistant to anticancer drugs from early treatment, or relapse after death, leading to death. Therefore, the resistance of cancer cells to anticancer drugs is one of the important obstacles in the treatment of cancer. The mechanism by which cancer cells become resistant to anticancer agents has suggested pharmacokinetic factors, pathologic types and biological characteristics, individual immune responses, route of drug administration, and drug resistance at the cellular level. Among these, drug resistance at the cellular level is considered to be the most important mechanism, and when a drug-resistant cancer cell is simultaneously resistant to anticancer drugs with completely different chemical structures, it is called multidrug resistance (MDR).
1960년대말 Kessel등은 vinblastine을 저용량으로 지속적으로 노출시켜 vinblastin에 내성을 갖게 만든 P388 백혈병 세포주가 dactinomycin, vincristine 및 daunorubicin에 교차내성을 나타내는 것을 관찰하였고, Biedler등은 이러한 현상을 다중약제내성이라고 명명하였다. Ling 및 Thomson은 다중약제내성을 가진 Chinese hamster 난소암 세포의 막에서 170kD 크기의 표면 당단백질이 증가되어 있음을 발견하여 P-당단백질이라고 명명하였으며, 세포내 약물의 농도를 감소시키는 작용을 하여 항암제의 다중약제내성과 관련이 있음을 보고하였다. P-당단백질의 기능은 칼슘과 ATP를 이용하여 세포내로 들어온 항암제를 세포밖으로 능동적으로 내보내는 펌프 역할을 하여 세포내 항암제의 유입을 억제하여 농도를 낮추고, 이로인해 항암제의 항암효과를 감소시키게 된다. 다중약제내성에 관련되는 항암제로는 adriamycin과 daunorubicin등의 anthracycline계 약물, vincristine과 vinblastin등의 vinka alkaloid계 약물, etoposide등의 epipodophllotoxin계 약물 및 그외 actinomycin-D, taxol등의 항암제가 있으며, 이들 항암제간에는 교차내성을 일으킨다. 이들 항암제들의 특징으로는 300-900D의 분자량의 약물들로 소수성이며, 복잡한 환구조로 이루어져 있고, 양전하의 질소 그룹을 가지고 있으며, 수동적으로 세포내로 확산되는 공통점이 있다. 1986년 Gros 등은 P-당단백질 유전자에 대한 cDNA의 염기배열순서를 규명하여 다중약제내성과 관련된 mdrl(multidrug resistance) 유전자의 존재가 밝혀지게 되었다. P-당백질은 1280개의 아미노산으로 구성되어 있으며, hemolycin B, leukotoxin, histidine등의 수송을 담당하는 수송단백질과 구조가 유사하며, 박테리아나 효모에서도 유사한 수송단백질이 존재함이 알려져 있다. 즉, P-당단백질은 암세포에만 특이하게 발견되는 것이 아니라, 대장 및 소장, 부신, 신장, 간 등의 정상세포에서도 발견되며, 세포내로 들어온 세포독성물질을 세포밖으로 내보내는 일반적인 역할을 하는 수송단백질의 일종이라는 것이 알려졌다. 특히 P-당단백질은 간세포의 담도쪽, 신장 근위세관의 내강표면, 대장 및 소장 내강점막의 원주세포, 부신피질과 수질에 주로 분포하며, 대장암, 신장암, 간암, 만성 백혈병, 부신암 및 비소세포성암 등의 암에서는 진단 당시부터 mdrl 유전자의 mRNA 양이 증가되어 있어 내인성 다중약제내성 나타낸다(Fojo AT, Ueda K, Slamon DJ, Poplack DG, Gottesman MM, Pastan I : Expression of a multidrug resistance gene in human tumors and tissues. Proc Natl Acad Sci USA 84:265, 1987; Goldstein LJ, Galski H, Fojo A, Willingham M, Lai SL, Gazdar A, Pirker R : Expression of a multidrug resistance gene in human cancers. J Natl Cancer Inst 81:116, 1989).In the late 1960s, Kessel et al. Observed that P388 leukemia cell lines that sustained low-dose vinblastine resistance to vinblastin exhibited cross resistance to dactinomycin, vincristine and daunorubicin. Biedler et al. . Ling and Thomson discovered that the surface glycoprotein of 170kD size was increased in the membrane of multi-drug resistant Chinese hamster ovarian cancer cells and named it P-glycoprotein, and it acted by reducing the concentration of intracellular drug. It has been reported to be related to multi drug resistance. The function of P-glycoprotein acts as a pump to actively release the anticancer agent into the cell using calcium and ATP, thereby inhibiting the influx of the intracellular anticancer agent to lower the concentration, thereby reducing the anticancer effect of the anticancer agent. Anticancer agents related to multi-drug resistance include anthracycline drugs such as adriamycin and daunorubicin, vinka alkaloid drugs such as vincristine and vinblastin, epipodophllotoxin drugs such as etoposide, and other anticancer drugs such as actinomycin-D and taxol. Cause cross resistance. These anticancer drugs are characterized by a molecular weight of 300-900D, which are hydrophobic, have complex ring structures, have a positively charged nitrogen group, and have a common passive diffusion. In 1986, Gros et al. Identified cDNA sequencing sequences for the P-glycoprotein gene and revealed the presence of the mdrl (multidrug resistance) gene associated with multidrug resistance. P-glycoprotein is composed of 1280 amino acids and is similar in structure to the transport proteins responsible for transporting hemolycin B, leukotoxin, histidine, and similar transport proteins in bacteria and yeast. That is, P-glycoprotein is not only found in cancer cells but also in normal cells such as colon and small intestine, adrenal gland, kidney and liver, and is a transport protein that plays a general role in transporting cytotoxic substances into cells. It is known that. In particular, P-glycoprotein is mainly distributed in the biliary tract of hepatocytes, lumen surface of proximal renal tubules, columnar cells of colon and small intestinal luminal mucosa, adrenal cortex and medulla, colon cancer, kidney cancer, liver cancer, chronic leukemia, adrenal cancer and In cancers such as non-small cell cancer, the mRNA level of the mdrl gene has been increased since diagnosis (Fojo AT, Ueda K, Slamon DJ, Poplack DG, Gottesman MM, Pastan I: Expression of a multidrug resistance gene in human tumors and tissues.Proc Natl Acad Sci USA 84: 265, 1987; Goldstein LJ, Galski H, Fojo A, Willingham M, Lai SL, Gazdar A, Pirker R: Expression of a multidrug resistance gene in human cancers.J Natl Cancer Inst 81: 116, 1989).
각종 암에서 mdrl mRNA과 P-당단백질의 과발현(over expression)은 치료실패의 중요한 요인으로 생각하게 되었고, 일부 암에서는 이들의 과발현이 치료성적 및 예후를 예측하는 인자로 제시되었다. 이러한 사실에 근거하여 다중약제내성을 극복하기 위하여 다중약제내성유전자의 발현을 억제하거나 P-당단백질의 기능을 조절하는 것을 목표로 한 치료법이 시도되었다. Tsuro등은 다중약제내성을 보이는 암세포들이 감수성 암세포에 비해 세포막과 세포질에 더 많은 양의 칼슘이 함유되어 있다는 사실에 근거하여 verapamil등의 칼슘통로차단제를 사용하였고, 이 약제들이 세포내 약물배출과정을 억제하여 항암제의 세포내 축적을 증가시켜 다중약제내성을 극복할 수 있다고 보고하였다(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, 1981 Aug. 1981). Verapamil 이외에도 cyclosporin-A, calmodulin, phenothiazine, quinine, tamoxifen등이 시도되고 있으나, 제 1상 및 제 2상 임상시험에서 대부분의 약제들이 체내에서 다중약제내성을 극복할만한 농도에 이르기 어렵거나, 혹은 그러한 농도에 이른다 하더라도 심독성, 신독성 등의 심각한 부작용이 발생하여 임상에 사용되는데는 아직 제한점이 있다. 또한 백혈병, 악성 임파종 및 다발성 골수종 등의 혈액암에서는 높은 항암효과가 관찰되어 일부 완치의 가능성을 보이기도 했으나, 대부분의 고형암에서는 그 효과가 아직은 미미한 실정이다.Overexpression of mdrl mRNA and P-glycoprotein in various cancers is considered to be an important factor of treatment failure, and in some cancers, their overexpression has been suggested as a predictor of therapeutic outcome and prognosis. On the basis of this fact, in order to overcome the multi-drug resistance, a therapy aimed at suppressing the expression of the multi-drug resistance gene or modulating the function of P-glycoprotein has been attempted. Tsuro et al. Used a calcium channel blocker such as verapamil based on the fact that multi-drug resistant cancer cells contained more calcium in the cell membrane and cytoplasm than susceptible cancer cells. (Tsuruo T. Fidler IJ.Differences in drug sensitivity among tumor cells from parental tumors, selected variants, and spontaneous metastases. Cancer Research. 41 (Tsuruo T. Fidler IJ.Differences in drug sensitivity among tumor cells, selected variants, and spontaneous metastases. 8): 3058-64, 1981 Aug. 1981). In addition to verapamil, cyclosporin-A, calmodulin, phenothiazine, quinine, and tamoxifen have been tried. However, in phase 1 and phase 2 clinical trials, most drugs are difficult to reach or exceed the levels in the body that can overcome multi-drug resistance. Even if it reaches to serious side effects such as cardiotoxicity, nephrotoxicity is still limited to use in clinical. In addition, hepatic cancers such as leukemia, malignant lymphoma, and multiple myeloma have been shown to have high anti-cancer effects, and some cures have been shown, but the effects are still insignificant in most solid cancers.
Hydroxychloroquine은 대표적인 항말라리아제로 최근에는 각종 류마치스 질환에서 항염증약물로도 사용된다. 이들 약물은 세포내에서 산성을 유지하는 기관인 lysosome, endosome 및 trans-Golgi network 등 세포내 기관들의 pH를 상승시켜 약리작용을 나타낸다고 알려져 있다(Fox RI.Mechanism of action of hydroxychloroquine as an antirheumatic drug.Seminars in Arthritis Rheumatism. 23(2 Suppl 1):82-91, 1993 Oct.; Fox R.Anti-malarial drugs: possible mechanisms of action in autoimmune disease and prospects for drug development. Lupus. 5 Suppl 1:S4-10, 1996 Jun.). 세포내 기관들의 알칼리화는 합성된 단백질의 분비에 영향을 미치게 되고, 또한 DNA와 RNA 합성을 저해한다는 보고도 있다.Hydroxychloroquine is a representative antimalarial agent and has recently been used as an anti-inflammatory drug in various rheumatic diseases. These drugs are known to exhibit pharmacological action by raising the pH of intracellular organs such as lysosome, endosome and trans-Golgi network, which maintain acidity in cells (Fox RI.Mechanism of action of hydroxychloroquine as an antirheumatic drug.Seminars in Arthritis Rheumatism. 23 (2 Suppl 1): 82-91, 1993 Oct .; Fox R. Anti-malarial drugs: possible mechanisms of action in autoimmune disease and prospects for drug development.Lupus. 5 Suppl 1: S4-10, 1996 Jun.). Alkalization of intracellular organs has been shown to affect the secretion of synthesized proteins and also inhibit DNA and RNA synthesis.
본 발명자는 다중약제내성을 나타내는 위암 및 대장암 세포주에 항말라리아 제제인 하이드록시클로로퀸, 클로로퀸, 프리마퀸 등과 항암제인 독소루비신, 시스플라틴 등을 일정약물 농도로 병용투약한 후 각각의 조합에 의한 항암효과를 나타내는 최소 유효농도 조사하여 하이드록시클로로퀸등 항말라리아제제가 항암제에 의한 암세포의 약제 내성을 억제하여 항암제의 항암효과를 상승시키는지를 연구하였다.The present inventors co-administered antimalarial agents hydroxychloroquine, chloroquine, primaquine and anticancer agents doxorubicin, cisplatin and the like at a certain drug concentration in gastric and colorectal cancer cell lines exhibiting multi-drug resistance, and then have anti-cancer effects by each combination. We investigated whether the antimalarial agents such as hydroxychloroquine increase the anticancer effect of anticancer drugs by inhibiting drug resistance of cancer cells by anticancer drugs.
도 1a 내지 도 1c는 대장암과 위암 세포주에서 하이드록시클로로퀸 15㎍/㎖과 30㎍/㎖ 사용에 따른 Doxorubicin(ADR)과 DDP의 세포독성도의 변화를 나타낸 것이다(X축은 항암제의 투여농도, Y축은 세포 생존율).Figures 1a to 1c shows the change in cytotoxicity of Doxorubicin (ADR) and DDP according to the use of hydroxychloroquine 15 ㎍ / ㎖ and 30 ㎍ / ㎖ in colorectal cancer and gastric cancer cell lines (X axis is the concentration of the anticancer drug, Y Axis is cell viability).
도 2a 내지 도 2b는 유방암, 위암, 섬유육종암 세포주에서 클로로퀸 20μM(10.318㎍/㎖)과 40μM(20.636㎍/㎖) 또는 프리마퀸 1.5μM(0.683㎍/㎖)과 3μM(1.366㎍/㎖) 사용에 따른 Doxorubicin(ADR)과 DDP의 세포독성도의 변화를 나타낸 것이다(X축은 항암제의 투여 농도, Y축은 세포 생존율).2A-2B show 20 μM (10.318 μg / ml) and 40 μM (20.636 μg / ml) or Primaquin 1.5 μM (0.683 μg / ml) and 3 μM (1.366 μg / ml) of chloroquine in breast, gastric and fibrosarcoma cell lines. The change in cytotoxicity of Doxorubicin (ADR) and DDP according to the use was shown (X axis shows anticancer drug concentration, Y axis shows cell viability).
도 1a 내지 도 1c는 대장암과 위암 세포주에서 하이드록시클로로퀸 15㎍/㎖과 30㎍/㎖ 사용에 따른 Doxorubicin(ADR)과 DDP의 세포독성도의 변화를 나타낸 것이다. 도 2a 내지 도 2b는 유방암, 위암, 섬유육종암 세포주에서 클로로퀸 20μM(10.318㎍/㎖)과 40μM(20.636 ㎍/㎖) 또는 프리마퀸 1.5μM(0.683㎍/㎖)과 3μM(1.366㎍/㎖) 사용에 따른 Doxorubi cin(ADR)과 DDP의 세포독성도의 변화를 나타낸 것이다.Figures 1a to 1c shows the change in cytotoxicity of Doxorubicin (ADR) and DDP according to the use of hydroxychloroquine 15 ㎍ / ㎖ and 30 ㎍ / ㎖ in colorectal cancer and gastric cancer cell lines. 2A-2B show 20 μM (10.318 μg / ml) and 40 μM (20.636 μg / ml) or primaquin 1.5 μM (0.683 μg / ml) and 3 μM (1.366 μg / ml) chloroquine in breast, gastric and fibrosarcoma cell lines. The changes in cytotoxicity of Doxorubicin (ADR) and DDP were shown.
세포주 및 암세포의 배양Culture of Cell Lines and Cancer Cells
HT-29 (ATCC HTB38, Adenocarcinoma, colon, moderately well differentiated grade II, human), HCT-15 (ATCC CCL225, colon adenocarcinoma, human)의 대장암 세포주와, KHH (YCC-2, Human gastric adenocarcinoma),PHB (YCC-3, Human gastric adenocarcinoma), KMB (YCC-7, Human gastric adenocarcinoma),AGS (ATCC CRL 1739, Human gastric adenocarcinoma)의 위암 세포주,HT 1080 (ATCC CCL 121, Fibrosarcoma, Human)섬유육종 세포주, SK-BR-3 (ATCC HTB 30, Adenocarcinoma, breast, malignant pleural effusion human) 유방암세포주를 가열 비활성화(56℃ 30분) 되어진 10% 우태아혈청 (Commonwealth Serum Laboratories, Australia; 이하 FCS로 약함)이 함유된 RPMI 1640 배지(GIBCO, U.S.A.)에 penicillin(100U/ml; GIBCO, U.S.A)과 streptomycin(100㎍/㎖; GIBCO, U.S.A)을 첨가하여 5% CO2의 존재하에 37℃ 항온배양기에서 배양하였다.Colon cancer cell lines of HT-29 (ATCC HTB38, Adenocarcinoma, colon, moderately well differentiated grade II, human), HCT-15 (ATCC CCL225, colon adenocarcinoma, human), KHH (YCC-2, Human gastric adenocarcinoma), PHB Gastric cancer cell line (YCC-3, Human gastric adenocarcinoma), KMB (YCC-7, Human gastric adenocarcinoma), AGS (ATCC CRL 1739, Human gastric adenocarcinoma), HT 1080 (ATCC CCL 121, Fibrosarcoma, Human) SK-BR-3 (ATCC HTB 30, Adenocarcinoma, breast, malignant pleural effusion human) contains 10% fetal bovine serum (Commonwealth Serum Laboratories, Australia; hereinafter weakly FCS) inactivated (56 ° C 30 min) breast cancer cell line Penicillin (100 U / ml; GIBCO, USA) and streptomycin (100 µg / ml; GIBCO, USA) were added to RPMI 1640 medium (GIBCO, USA) and cultured in a 37 ° C. incubator in the presence of 5% CO 2 .
실험에 사용된 약제Drug used in experiment
실험에 사용된 항암제는 adriamycin(일반명: 독소루비신(Doxorubicin), Farmitalia Carlo Erba Ltd, Italy; 이하 ADR로 약함), cisplatin(diaminodichloro platinum: Pharmachemie B.V., Holland; 이하 DDP로 약함). 항암제에 의한 세포 살상능의 검사는 MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-1-butene; Sigma, U.S.A}를 사용하였다. MTT 검사에 이용한 항암제의 농도는 ADR의 경우 10, 1, 0.1, 0.01㎍/㎖로, DDP는 50, 5, 0.5, 0.05㎍/㎖의 농도로 하여 microplate의 각각의 column에서 단계적으로 희석하여 사용하였다. Hydroxychloroquine의 농도는 15, 30㎍/㎖, Chloroquine의 농도는 10.3, 20.6 ㎍/㎖, Primaquine의 농도는 0.68, 1.36㎍/㎖를 사용하여 실험하였다.The anticancer agents used in the experiment were adriamycin (common name: Doxorubicin, Farmitalia Carlo Erba Ltd, Italy; hereinafter abbreviated as ADR), cisplatin (diaminodichloro platinum: Pharmachemie B.V., Holland; hereinafter abbreviated as DDP). Examination of cell killing ability by the anticancer agent was performed by MTT {3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-1-butene; Sigma, U.S.A}. The concentration of anticancer agent used for MTT test was 10, 1, 0.1, 0.01 μg / ml for ADR and 50, 5, 0.5, 0.05 μg / ml for DDP. It was. Hydroxychloroquine concentration was 15, 30µg / ml, Chloroquine concentration was 10.3, 20.6µg / ml, Primaquine concentration was 0.68, 1.36µg / ml.
MTT 방법에 의한 세포독성도(항암효과)의 검사Examination of cytotoxicity (anticancer effect) by MTT method
MTT검사는 살아있는 세포의 경우 cytochrome b 및 c 부위에 있는 미토콘드리아의 효소인 succinate dehydrogenase가 3-[4,5-dimethylthiazol-2-yl]- 2,5- diphenyltetrazolium bromide (MTT)의 tetrazolium환을 분절시켜 황색조의 MTT salt를 자주색의 formazan으로 환원시키는데, 그 원리를 두고 있으며, 죽은 세포나 조직 배양액에서는 이러한 변화가 일어나지 않고, 산 세포에서만 선택적으로 일어나는 이 색조 반응을 분광흡도계로 측정하여 살아있는 세포의 수를 알 수 있는 검사법으로, 여러번의 반복 실험에서 재현성이 높아, 현재 미국 국립암연구소에서 인체 종양세포주 에 대한 체외 항암제감수성 검사방법으로 추천되고 있다.MTT test showed that in living cells, succinate dehydrogenase, a mitochondrial enzyme at the cytochrome b and c sites, splits the tetrazolium ring of 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT). Its principle is to reduce the yellow-toned MTT salt to purple formazan. This change does not occur in dead cells or tissue cultures. As a test that can be seen, it is highly reproducible in several repeated experiments and is currently recommended as an in vitro anticancer susceptibility test for human tumor cell lines by the National Cancer Institute.
본 연구에서는 Carmichael등의 방법에 따라 MTT 검사를 시행 하였으며, 약술하면 다음과 같다. 각각의 배양된 암세포주에서 성장곡선을 그려, 지수성장을 보이는 세포수를 결정 한 후 세포성장곡선상 지수성장기에 해당하는 세포수를 0.25% trypsin-EDTA를 처리하여 단일 부유 암세포로 만든후, 10% FCS가 함유된 RPMI 1640 배지로 3회 세척하고, trypan blue (GIBCO, U.S.A) 염색으로 세포수를 세어 180μl의 배양액에 지속성장기에 해당하는 세포수를 부유하여 96 well plate (Costar, U.S.A)에 분주하였다. 각각의 세포주는 96 well plate에 분주하여 370C, 5% CO2배양기에서 24시간 배양한 후, 여러 농도의 항암제를 단독 또는 조합하여 20μl의 생리식염수에 용해시켜 96 well plate에 투여하였다. 이후 microplate를 4일 추가 배양한 후 MTT 50μl (2mg/ml)를 각well에 첨가하고 이를 4시간 더 추가 배양하였다. 이때 대조군의 well에는 항암제 대신 동량의 생리식염수를 사용하였다. 배양이 끝난 microplate는 450g에서 10분간 원심분리한 후, 바닥에 있는 formazan 결정이 제거되지 않도록 주의하면서, 30μl 정도의 배양액이 남도록 digital multichannel pipette (Flow Titertck, Finland)을 사용하여 상층액을 제거하였다. 여기에 다시 150 μl의 dimethyl sulfoxide (Sigma, U.S.A)를 각 well에 첨가하고 진탕기(plate shaker)에서 formazan결정이 용해될 때까지 360C에서 10분간 진탕한 후 multi-well ELISA automatic spectrometer recorder (Behring ELISA Processor II, Germany)를 이용하여 540nm의 파장에서 판독한 실험군의색소흡수율(absorbance, optical density)을 대조군의 색소흡수율과 비교하여 아래의 공식으로 생존율을 구하였다.In this study, MTT test was performed according to Carmichael's method. Draw a growth curve in each cultured cancer cell line, determine the number of cells showing exponential growth, and make the single floating cancer cells by treating 0.25% trypsin-EDTA with the number of cells corresponding to the exponential growth phase on the cell growth curve. Washed three times with RPMI 1640 medium containing% FCS, counted the cells by trypan blue (GIBCO, USA) staining, and suspended the cell number corresponding to the sustained growth period in 180 μl of culture medium in 96 well plate (Costar, USA). Busy. Each cell line was divided into 96 well plates, incubated for 24 hours in a 37 0 C, 5% CO 2 incubator, and then dissolved in 20 μl of saline alone or in combination with various concentrations of anticancer agents. Thereafter, the microplate was further incubated for 4 days, and then 50 μl of MTT (2 mg / ml) was added to each well, followed by an additional 4 hours of incubation. At this time, the same amount of physiological saline was used for the well of the control group instead of the anticancer agent. The cultured microplate was centrifuged at 450g for 10 minutes, and the supernatant was removed using digital multichannel pipette (Flow Titertck, Finland) to leave about 30μl of the culture solution while being careful not to remove the formazan crystals on the bottom. Again, 150 μl of dimethyl sulfoxide (Sigma, USA) was added to each well, shaken at 36 0 C for 10 minutes until the formazan crystals were dissolved in a plate shaker, and then multi-well ELISA automatic spectrometer recorder ( Using the Behring ELISA Processor II, Germany), the absorption rate (absorbance, optical density) of the experimental group read at the wavelength of 540nm was compared with the pigment absorption rate of the control group to calculate the survival rate by the following formula.
본 발명의 구체적인 구성과 작용을 실시예를 들어 보다 구체적으로 설명하면 다음과 같다.When explaining the specific configuration and operation of the present invention in more detail as follows.
실시예 1.Example 1.
위에 기술한 방법에 따라 아래 암세포주에 하이드록시클로로퀸을 15㎍/㎕, 30㎍/㎕을 각각 독소루비신, 시스플라틴(DDP: cisplatin)과 병용투여하여 각각의 최소유효농도 (IC50)을 측정하여 표 1에 기재하였다.According to the method described above, 15 μg / μl of hydroxychloroquine and 30 μg / μl of chlorochloroquine were combined with doxorubicin and cisplatin (DDP) in the following cancer cell lines to determine the respective minimum effective concentrations (IC 50 ). It described in 1.
표 1에서 보는 바와 같이, Hydroxychloroquine(HCQ)의 경우 50%세포사망률을 측정한 결과 대장암의 경우 HCQ실험농도에서 항암제 Doxorubicin는 약 10배의 약효증대를 보여주고있으며, DDP의 경우 평균 2-5배의 약효증대 효과를 보였다. 위암세포주의 경우에는 HCQ실험농도에서 항암제 Doxorubicin은 약3-10배의 약효증대를 보여주고있으며, DDP의 경우 평균 4-15배의 약효증대 효과를 보여주었다.As shown in Table 1, 50% cell death rate was measured for hydroxychloroquine (HCQ), and the anticancer drug Doxorubicin showed about 10-fold increase in HCQ test concentration in colon cancer, and average 2-5 in DDP. It showed an effect of increasing pear efficacy. In gastric cancer cell lines, the anticancer drug Doxorubicin showed about 3-10-fold increase in the HCQ concentration, and DDP showed an average of 4-15-fold increase in efficacy.
실시예 2.Example 2.
상기 실시예 1과 같은 방법으로 아래 암세포주에 클로로퀸 20μM, 40μM를 각각 독소루비신, 시스플라틴(DDP: cisplatin)과 병용투여한 후 각각의 최소유효농도를 측정하여 하기 표 2에 기재하였다.In the same manner as in Example 1, 20 μM and 40 μM of chloroquine were dosed in combination with doxorubicin and cisplatin (DDP: cisplatin) in the following cancer cell lines, respectively, and the respective minimum effective concentrations were measured and described in Table 2 below.
표 2에서 보는 바와 같이, Chloroquine(CQ)의 경우 50% 세포사망률을 측정한 결과 유방암의 경우 CQ 실험농도에서 항암제 Doxorubicin는 약 1.4-3배, 위암의 경우는 약 6-10배, 육종암의 경우는 약 4-10배의 약효증대를 보여주고 있으며, DDP의 경우 유방암의 경우에서는 약 2-2.3배, 위암의 경우는 약 1-5배, 육종암의 경우는 약 2.3-12배의 약효증대를 보여주고 있다.As shown in Table 2, 50% cell death rate was measured for chloroquine (CQ), and the anticancer drug Doxorubicin was about 1.4-3 times higher at CQ concentrations for breast cancer, about 6-10 times higher than for sarcoma. In case of DDP, it shows about 4-10 times increase in efficacy.In case of DDP, it is about 2-2.3 times in breast cancer, about 1-5 times in gastric cancer, and about 2.3-12 times in sarcoma cancer It is showing an increase.
실시예 3.Example 3.
실시예 1과 같은 방법으로 아래 암세포주에 프리마퀸 1.5μM, 3μM을 각각 독소루비신, 시스플라틴(DDP: cisplatin)과 병용투여한 후 각각의 최소 유효농도를측정하여 하기 표 3에 기재하였다.In the same manner as in Example 1, 1.5 μM and 3 μM of Primaquin in the following cancer cell lines were combined with doxorubicin and cisplatin (DDP: cisplatin), respectively, and the respective minimum effective concentrations were measured and described in Table 3 below.
표 3에서 보는 바와 같이, Primaquine(PQ)의 경우 50%세포사망률을 측정한 결과 유방암의 경우 PQ실험농도에서 항암제 Doxorubicin는 약 3배, 위암의 경우는 약 10배, 육종암의 경우는 약 3-4배의 약효증대를 보여주고 있으며, DDP의 경우 유방암에서는 약 1.2배, 위암의 경우는 약 1-6배, 육종암의 경우는 약 1.3배의 약효증대를 보여주고 있다.As shown in Table 3, 50% cell mortality was measured for Primaquine (PQ) and about 3 times as anticancer drug Doxorubicin, 10 times for gastric cancer, and 3 for sarcoma cancer at PQ concentrations for breast cancer. It shows a 4-fold increase in efficacy, DDP shows about 1.2 times in breast cancer, 1-6 times in stomach cancer, and 1.3 times in sarcoma cancer.
이상 살펴본 바와 같이 본 발명에서 항말라리아제와 항암제의 병용투여시 암종류에 관계없이 항암제의 항암효과가 증대하는 것을 알 수 있으며, 이 경우 기존 항암제의 투여방법에 따라 항말라리아제는 경구, 비경구 어느 경우라도 좋으며, 하이드로클로로퀸 0.1~500mg/kg, 클로로퀸 0.1~700mg/kg, 프리마퀸 0.1~800mg/kg을 기존 항암제의 투여용량과 병용투여하는 것이 바람직하며, 하이드로클로로퀸 10~100mg/kg, 클로로퀸 10~300mg/kg, 프리마퀸 50~500mg/kg을 기존 항암제 투여용량과 병용투여하는 것이 보다 바람직하다.As described above, it can be seen that the anti-cancer effect of the anti-cancer agent is increased regardless of the type of cancer when the anti-malarial agent and the anti-cancer agent are used in combination in the present invention. It is preferable to use a hydrochloroquine 0.1 ~ 500mg / kg, chloroquine 0.1 ~ 700mg / kg, Primaquine 0.1 ~ 800mg / kg in combination with the dosage of the existing anticancer drugs, hydrochloroquine 10 ~ 100mg / kg, chloroquine 10 ~ 300mg / kg, Primaquin 50 ~ 500mg / kg is more preferably administered in combination with the existing anticancer drug dose.
이상 설명하고 실시예를 통하여 알 수 있는 바와 같이, 본 발명은 항말라리아제로 흔히 사용되는 하이드록시클로로퀸, 클로로퀸, 프리마퀸 등과 대표적인 항암제인 독소루비신, 시스플라틴 등을 병용투여하면 항암제의 최소 유효농도(IC50)을 낮추고 항암제에 의한 암세포의 약제 내성을 억제하여 유방암의 경우 약 3배, 위암의 경우 약 10배, 대장암의 경우 약 10배, 육종암의 경우 약 10배까지 항암 효과를 상승시킬 수 있는 효과가 있다.As can be seen above description and through the examples, the present invention provides anti-malarial often hydroxy chloroquine used agent, chloroquine, Prima Queen as a representative anticancer drug doxorubicin, when administered in combination with Cisplatin, such as a minimum effective concentration of the anticancer drug (IC 50 ) And increase the anticancer effect by inhibiting the drug resistance of cancer cells by anticancer drugs, up to about 3 times for breast cancer, about 10 times for stomach cancer, about 10 times for colon cancer, and about 10 times for sarcoma cancer It works.
Claims (4)
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| KR10-2000-0044851A KR100390332B1 (en) | 2000-08-02 | 2000-08-02 | anti-cancer composition composed of anti-cancer and anti-malarial drugs |
| PCT/KR2001/001314 WO2002013826A1 (en) | 2000-08-02 | 2001-08-02 | Anti-cancer composition composed of anti-cancer and anti-malarial drugs |
| AU2001275821A AU2001275821A1 (en) | 2000-08-02 | 2001-08-02 | Anti-cancer composition composed of anti-cancer and anti-malarial drugs |
| EP01953364A EP1313475A4 (en) | 2000-08-02 | 2001-08-02 | Anti-cancer composition composed of anti-cancer and anti-malarial drugs |
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Cited By (3)
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| WO2010151005A2 (en) | 2009-06-24 | 2010-12-29 | Yeo Oh-Young | Injectable composition containing hydroxychloroquine for local administration for treating cancer |
| KR20160122310A (en) | 2015-04-13 | 2016-10-24 | 영남대학교 산학협력단 | Anti-cancer pharmaceutical composition comprising doxorubicin and irinotecan and manufacturing method thereof |
| KR101698003B1 (en) | 2016-06-20 | 2017-01-19 | 여오영 | Injectable composition for topical administration for cancer treatment that comprising a quinine salt suspension |
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| US6916627B2 (en) | 2002-11-27 | 2005-07-12 | St. Jude Children's Research Hospital | ATM kinase compositions and methods |
| US7108992B2 (en) | 2002-11-27 | 2006-09-19 | St. Jude Children's Research Hospital | ATM kinase compositions and methods |
| AU2012201742B2 (en) * | 2005-01-19 | 2015-05-14 | The Trustees Of The University Of Pennsylvania | Regulation of autophagy and cell survival |
| WO2008038291A1 (en) * | 2006-09-27 | 2008-04-03 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Combination of liposomal anti-cancer drugs and lysosome/endosome ph increasing agents for therapy |
| KR101067443B1 (en) * | 2009-06-23 | 2011-09-27 | 여오영 | Local injection composition comprising hydroxychloroquine for treatment of hemorrhoids |
| KR101068202B1 (en) * | 2009-10-27 | 2011-09-28 | 조재용 | Anti-cancer agent composition for stomach cancer comprising vorinostat |
| TWI482621B (en) | 2009-12-23 | 2015-05-01 | Sigma Tau Ind Farmaceuti | Anticancer combinations of artemisinin-based drugs and other chemotherapeutic agents |
| WO2014052550A1 (en) * | 2012-09-27 | 2014-04-03 | Thomas Jefferson University | Use of parp inhibitors to treat breast cancer |
| FR3076711B1 (en) * | 2018-01-15 | 2020-10-30 | Inoviem Scient | COMPOSITION INCLUDING HYDROXYCHLOROQUINE AND THERAPEUTIC USE |
| US20210361643A1 (en) * | 2018-06-19 | 2021-11-25 | Armaceutica, Inc. | Bifunctional compositions for the treatment of cancer |
| WO2021105761A1 (en) * | 2019-11-29 | 2021-06-03 | Institut National De La Santé Et De La Recherche Médicale (Inserm) | Compositions and their uses for treating cancers |
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- 2001-08-02 EP EP01953364A patent/EP1313475A4/en not_active Ceased
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| Biochemical Pharmaco;ogy, 59(10), 1245-1252, 2000 May 15 * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010151005A2 (en) | 2009-06-24 | 2010-12-29 | Yeo Oh-Young | Injectable composition containing hydroxychloroquine for local administration for treating cancer |
| WO2010151005A3 (en) * | 2009-06-24 | 2011-05-19 | Yeo Oh-Young | Injectable composition containing hydroxychloroquine for local administration for treating cancer |
| US8506973B2 (en) | 2009-06-24 | 2013-08-13 | Oh-Young Yeo | Injectable anticancer composition for local administration containing hydroxychloroquine |
| KR20160122310A (en) | 2015-04-13 | 2016-10-24 | 영남대학교 산학협력단 | Anti-cancer pharmaceutical composition comprising doxorubicin and irinotecan and manufacturing method thereof |
| KR101698003B1 (en) | 2016-06-20 | 2017-01-19 | 여오영 | Injectable composition for topical administration for cancer treatment that comprising a quinine salt suspension |
| EP3260138A1 (en) | 2016-06-20 | 2017-12-27 | Oh Young Yeo | Injectable anticancer composition for local administration containing suspension of quinine salt |
| US10441582B2 (en) | 2016-06-20 | 2019-10-15 | Oh Young Yeo | Injectable anticancer composition for local administration containing suspension of quinine salt |
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| KR20020011528A (en) | 2002-02-09 |
| WO2002013826A9 (en) | 2003-11-13 |
| EP1313475A1 (en) | 2003-05-28 |
| EP1313475A4 (en) | 2004-08-18 |
| AU2001275821A1 (en) | 2002-02-25 |
| WO2002013826A1 (en) | 2002-02-21 |
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