WO2009107322A1

WO2009107322A1 - Pharmaceutical composition for treatment of cancer

Info

Publication number: WO2009107322A1
Application number: PCT/JP2009/000310
Authority: WO
Inventors: 坂井田功
Original assignee: 国立大学法人山口大学
Priority date: 2008-02-25
Filing date: 2009-01-27
Publication date: 2009-09-03
Also published as: JP2009196959A

Abstract

Disclosed is a pharmaceutical composition which is effective as a therapeutic agent for cancer which requires an iron ion for its expansion, particularly liver cancer. Specifically disclosed is a pharmaceutical composition which comprises, as an active ingredient, deferoxamine which is one of the iron chelating agents that have been used as therapeutic agents for hemochromatosis. The pharmaceutical composition is effective as a therapeutic agent for liver cancer.

Description

Pharmaceutical composition for cancer treatment

The present invention relates to a pharmaceutical composition that can be used as a therapeutic agent for cancer that requires iron ions for growth, and more specifically, has been used as a therapeutic agent for hyperchromatosis. A pharmaceutical composition for the treatment of cancers that require iron ions for growth, particularly a composition for the treatment of liver cancers such as hepatocellular carcinoma, comprising deferoxamine, an iron chelator, as an active ingredient It relates to things.

Liver cancer is a general term for cancer that occurs in the liver, and is classified into primary liver cancer that occurs when liver cells become cancerous and metastatic liver cancer that results from metastasis of cancer that occurs elsewhere. . “Hepatocellular carcinoma (HCC)”, which occurs when hepatocytes become cancerous, accounts for 90% of primary liver cancer, and most hepatocellular cancers are known to occur from chronic hepatitis through cirrhosis. Yes. Onset is common among men in their 50s and 60s, and about 95% of hepatocellular carcinomas are characterized by infection with hepatitis B virus (HBV; 25%) or hepatitis C virus (HCV; 70%). It is.

Liver cancer is the fourth most common cancer in Japan and the third highest number of deaths (2004: National Cancer Center statistics). Efforts have been made. There are two types of treatment: surgical treatment to remove the affected area and medical treatment such as drug administration. The internal treatment is percutaneous ethanol injection (direct injection of pure ethanol into the liver and cancer tissue). Necrosis), percutaneous radiocautery therapy (necrosis of cancer tissue by direct irradiation of radio waves), hepatic artery embolization therapy (embedding the hepatic artery and cutting off blood supply to the cancer) Necrosis), chemotherapy by administration of various anticancer agents, and the like, and in fact, treatments combining these as necessary are performed. However, in surgical treatment, there is a problem that cancer newly arises from the liver remaining after resection (particularly in the case of virus-infected liver), and although medical treatment methods are also effective, they are still not sufficient. In particular, various effective substances have been reported for chemotherapy including anticancer agents, but there are few that have been studied with a control group as in the case of other malignant tumors. Chemotherapy has not yet been established as a treatment for liver cancer. In fact, liver cancer is recognized as an indication for liver cancer in 2007 (insurance coverage) anti-metabolites such as alkylating agents cyclophosphamide and 5-fluorouracil, doxorubicin, mitomycin C, etc. In order to establish an effective treatment for liver cancer, there are only a limited number of antibiotics and platinum preparations such as cisplatin. Development was anxious.

Deferoxamine and some of its derivatives are drugs that have an iron chelating action and are known to be effective against hemochromatosis, a disease in which iron (iron ions) are present in excess in the body ( Patent Documents 1 and 2). Furthermore, a metal chelating agent containing deferoxamine is used for cancer treatment as an apoptosis-inducing compound (Patent Documents 3 and 4), a ribonucleotide reductase inhibitor (Patent Document 5), or an inhibitor of fibrous adhesions (Patent Document 6). The possibility of use is suggested. In Patent Document 7, as a result of examining the effects of a cytostatic and deferoxamine on a 6-week-old congenital acute leukemia patient in whom the effect of chemotherapy was not observed and a mouse transplanted with Lewis lung cancer cells, deferoxamine or cell proliferation was examined. When the inhibitor was administered alone, no effect was observed, but it was clarified that a synergistic antiproliferative effect was observed by administering deferoxamine in combination with a cell growth inhibitor. In Patent Document 8, several types of established tumor cells (breast cancer, colorectal cancer, erythroleukemia, sarcoma cancer, scaly cancer, testicular cancer, ovarian cancer, and bladder cancer-derived cell lines) are cultured in vitro. As a result of examining changes in the number of cells when an antigen fork (preferably a bispecific antibody) having a disorder is combined with deferoxamine or cisplatin and added to the culture solution, the cytotoxic effect of the antigen fork on cultured cells However, the effect of deferoxamine alone is to obtain an effect of stopping or delaying the growth of cultured cancer cells or an effect of reducing by about 10-20% (317G5-454A12 strain). Yes, the effect of deferoxamine is only to increase the effect of the antigen fork by using it together with the antigen fork. It was. As described above, several studies have been conducted on the effectiveness of deferoxamine and other chelating agents for cancer treatment, but most of them are in vitro experiments using cultured cells. However, little is known about the action of deferoxamine at the biological level. In addition, the effect of deferoxamine on liver cancer has not been clarified at the level of cultured cells.

The present inventors have so far conducted research on liver cells and liver cancer by means of internal medicine, and among them, induced by TBHP (Tert-butyl hydroperoxide), which is a drug that causes oxidative stress damage to cells. It was clarified that iron ions are necessary for cell death of cultured hepatocytes, and that cell death is inhibited by deferoxamine (Non-patent Document 1). In addition, the present inventors clearly show that deferoxamine inhibits and reduces liver damage caused by acetaminophen and pre-tumor lesions in a rat diet model (Cholin-deficient L-amino acid defined diet) even in the rat body. (Non-Patent Documents 2 and 3).
Special table flat 7-507288 Japanese Patent Fair 8-013795 Special table 2004-532245 Special table 2005-514322 Special table 2004-523497 Special table 2007-504273 JP 62-223130 Special table hei 8-510116 Sakaida I. et al. 1990. Molecular Pharmacology 37 (3): 435-442. Sakaida I. et al. 1995. Scandinavian J. Gastroenterology. 30 (1): 61-67. Sakaida I. et al. 1999. Digestive diseases and Sciences. 44 (3): 560-569.

In view of the above situation, the present invention focuses on deferoxamine as a therapeutic agent for cancer (particularly liver cancer) that requires iron ions for growth, and includes deferoxamine, a derivative thereof, or a salt thereof as an active ingredient. The purpose is to provide a pharmaceutical composition for the treatment of cancer.

In order to solve the above problems, the present inventor may progress from cirrhosis to liver cancer in patients with hemochromatosis, which is a disease in which iron is excessively deposited in the liver, without treatment. Focusing on the findings, he found that hepatocytes around carcinogenic lesions had excessive iron deposition, whereas hepatic cancer sites (inside liver cancer cells) no longer had excessive iron deposition. . In addition, it is hypothesized that iron is necessary for the growth of hepatoma cells, and deferoxamine, which has an iron chelating effect, is effective as an inhibitor of liver cancer. As a result, the present invention has been completed.

That is, the present invention includes (1) deferoxamine (Deferoxamine), a derivative thereof, and a pharmacologically acceptable salt thereof as an active ingredient, and has an iron ion requirement for growth. A pharmaceutical composition for treating cancer, (2) a pharmaceutical composition according to (1) above, wherein the cancer requiring iron ion for growth is liver cancer, and (3) the liver However, the pharmaceutical composition according to (1) or (2) above, which is a hepatocyte cancer, or (4) a pharmacologically acceptable salt is an organic acid salt The pharmaceutical composition according to any one of (1) to (3) above, or (5) the organic acid salt is an organic acid salt having 2 or 1 carbon atoms, 4) The pharmaceutical composition according to any one of (4) and (6) Deferoxamine mesylate (Deferoxamine) mesilate), the pharmaceutical composition according to (5) above, and (7) the anticancer agent further comprising (1) to (6) above (1) to (7), wherein the pharmaceutical composition or (8) the anticancer agent is one or more anticancer agents selected from 5-fluorouracil, adriamycin, cisplatin, and mitomycin The pharmaceutical composition according to any one of the above.

The present invention also provides (9) a method for treating cancer having an iron ion requirement for growth, comprising administering the pharmaceutical composition according to any one of (1) to (8) above, 10) The method of the above-mentioned (9), wherein the cancer requiring iron ion for proliferation is liver cancer, or (11) the liver cancer is characterized by hepatocellular carcinoma. The above (9) or (10) relates to the treatment method.

Furthermore, the present invention relates to (12) use of the pharmaceutical composition according to any one of (1) to (8) above in the manufacture of a medicament for treating cancer having an iron ion requirement for proliferation, The use according to (12) above, wherein the cancer having an iron ion requirement for proliferation is liver cancer, or (14) the above, wherein the liver cancer is hepatocellular carcinoma ( The use according to 12) or (13).

By using the present invention, it is possible to provide an effective drug for liver cancer for which there has been no effective chemotherapy. Deferoxamine, which is an active ingredient of the present invention, has already been established in safety, such as being used as a hemochromatosis therapeutic drug, and is expected to greatly shorten clinical trials, and far more than existing anticancer drugs. Therefore, it is possible to effectively proceed with treatment without lowering the quality of life (QOL) of the patient. Furthermore, combining with other cancer treatments other than chemotherapy is expected to increase the cancer treatment effect.

The effect of deferoxamine (DFO) on Huh-7 cells is shown. The effect of DFO on HepG2 cells is shown. The effect of DFO on HLF cells is shown. The effect of 5-FU on Huh-7 cells is shown. The effect of 5-FU on HepG2 cells is shown. The effect of 5-FU on HLF cells is shown. The effect of ADM on Huh-7 cells is shown. The effect on Huh-7 cells by the combined use of DFO and 5-FU is shown. The effect to HepG2 cell by combined use of DFO and 5-FU is shown. The effect on HLF cells by the combined use of DFO and 5-FU is shown. The effect on Huh-7 cells by the combined use of DFO and ADM is shown. The time-dependent change of the expression level of the tumor marker by DFO processing is shown. The CT image before the treatment in case 1 is shown. The CT image after the treatment in case 1 is shown. The transition of the tumor marker after DFO administration in case 1 is shown. The transition of the tumor marker after DFO administration in case 2 is shown. The transition of the tumor marker after DFO administration in case 3 is shown. The transition of the tumor marker after DFO re-administration in case 3 is shown.

The pharmaceutical composition for the treatment of cancer requiring iron ions for growth of the present invention includes at least one of deferoxamine, its derivatives, and pharmacologically acceptable salts as an active ingredient. The deferoxamine is not particularly limited, and the deferoxamine is a deferoxamine-producing strain belonging to the genus Streptomyces, for example, Streptomyces ４０３ pilosus (stored as JCM4403 in RIKEN and ATCC 19797 in the United States). In addition to being obtained by culturing, it can also be synthesized by the method of Proleg et al. (Helv Chim Acta, 45, 31, 1962). The deferoxamine derivative has any structure as long as it is a compound represented by the following chemical formula (I) and has an inhibitory effect on cancer, particularly liver cancer. There may be.

In the chemical formula (I), the substituent R1 may be hydrogen or other group, that is, sulfonyl group, oxy group, thio group, sulfinyl group, imino group, oxycarbonyl group, aromatic group, etc. Furthermore, an organic acid, a hydrocarbon chain, or the like may be bonded through these bonds. In the formula, X represents an adduct such as an organic acid adduct, and the adduct is preferably an organic acid adduct, more preferably an organic acid adduct having 1 or 2 carbon atoms. .

The salt of deferoxamine or a derivative thereof is not particularly limited as long as it is a conventional non-toxic acid addition salt that is pharmacologically acceptable, and specifically, an inorganic acid addition salt (for example, hydrochloride, odor) Hydrohalates, sulfates, phosphates, etc.), organic carboxylic acid addition salts or organic sulfonic acid addition salts (eg formate, acetate, trifluoroacetate, maleate, tartrate, methanesulfonate, Examples thereof include benzene sulfonate, p-toluene sulfonate, etc., salts with basic amino acids or acidic amino acids (eg arginine, aspartic acid, glutamic acid, etc.). It is preferable that it is an organic acid salt having 1 or 2 carbon atoms. Deferoxamine mesylate which is a particularly preferred example is shown as the salt of deferoxamine of the present invention in the following chemical formula (II). Deferoxamine mesylate is commercially available as a reagent or pharmaceutical.

Cancers that require iron ions for growth are not particularly limited as long as they require iron ions to grow or are affected by excessive deposition of iron or iron ions. Not precancerous or cancerous, for example, liver cancer (hepatocellular carcinoma, cholangiocellular carcinoma), melanoma (melanoma), fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteogen Sarcoma, chordoma, angiosarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, colon cancer, colon Cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell cancer, basal cell cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary cancer, papillary gland Cancer, cystadenocarcinoma, bone marrow cancer, bronchogenic cancer, renal cell cancer, ureteral cancer, bile duct cancer Choriocarcinoma, seminoma, fetal cancer, Wilms tumor, cervical cancer, endometrial cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, glial Tumor, astrocytoma, myeloblast, craniopharyngeal cancer, laryngeal cancer, tongue cancer, ventricular ependymoma, pineal gland tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, marrow Meningiomas, peritoneal dissemination, teratomas, neuroblastoma, medulloblastoma, retinoblastoma, acute myeloid leukemia, acute promyelocytic leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, Preferred examples include Hodgkin disease, non-Hodgkin lymphoma, adult T-cell leukemia lymphoma, multiple myeloma and the like, and particularly effective against liver cancer. Here, liver cancer includes hepatocellular carcinoma and cholangiocellular carcinoma, and the pharmaceutical composition of the present invention is particularly hepatocyte among liver cancers as shown in the following examples. It is effective against cancer.

The pharmaceutical composition for the treatment of cancer requiring iron ions for proliferation of the present invention may further contain an anticancer agent or the like as a secondary component, and the anticancer agent includes an existing anticancer agent. Or any anticancer agent developed in the future, for example, 5-Fluorouracil (5-FU), Adriamycin (ADM), Mitomycin, Cisplatin (Cisplatin), paclitaxel (Paclitaxel), docetaxel (Docetaxel), etoposide (Etoposide), lomustine (Melphalan), mercaptopurine (Mercaptopurine), etc. can be specifically mentioned, among them 5- FU or ADM can be preferably mentioned. As shown in the Examples below, when deferoxamine mesylate is used in combination with 5-FU or ADM, it is 1/2 to 1/10 of the normal dose for 5-FU, and the normal dose for ADM. Since the effect was recognized even in an amount of 1/10 to 1/100, the effect was obtained by combining the anticancer agent as a secondary component with the main component deferoxamine having an effect as a single agent. It is possible to develop drugs for the treatment of cancer that have a synergistic effect with large side effects.

In addition, the anticancer agent contained as an accessory component in the pharmaceutical composition for treating cancer that requires iron ions for growth of the present invention is an amount of 1/2 to 1/100 of the amount used as a single agent. For example, in the case of systemic administration, the dose of 5-fluorouracil is 600 mg / m ² or less at a time, preferably 50-300 mg in total, more preferably 240-260 mg in total, and administration of adriamycin The amount is 500 mg / m ² or less per dose, preferably 50-450 mg / m ² , more preferably 300-400 mg / m ^{2. For} topical administration, the dose of 5-fluorouracil is The total amount per patient is 600 mg or less, preferably 50-450 mg, more preferably 100-300 mg. The dose of adriamycin is The amount 600 mg / ^{m 2} or less, preferably 50-500 mg / ^{m 2,} more preferably 200-300 mg / ^{m 2,} the dose of mitomycin is total 60mg or less at a time, preferably 5-50 mg, more preferably It is 10-30 mg, and these anticancer agents are preferably included in combination, and more preferably all three types of anticancer agents are included.

The method for treating cancer having an iron ion requirement for growth according to the present invention is not particularly limited as long as it is a therapeutic method for administering the pharmaceutical composition of the present invention, and the pharmaceutical composition of the present invention includes: Various pharmaceutical compounding ingredients such as usual pharmaceutically acceptable carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, solubilizers, solubilizers, isotonic agents, etc. Can be added and administered. The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it is a commonly used dosage form. For example, oral, rectal, intravaginal, parenteral, intramuscular, intraperitoneal, intraarterial In addition, it can be appropriately selected from administration routes such as subarachnoid, intrabronchial, subcutaneous, intradermal, intravenous, intranasal, buccal, or sublingual. It is preferred to administer the product parenterally in the form of an injection. When the pharmaceutical composition for cancer treatment of the present invention is administered by injection, it may be systemic administration or local administration (local injection) to the affected area, but it may be local administration. preferable. Here, the topical administration means that the therapeutic agent is administered directly to the affected part, that is, the cancerous lesion part, and is not particularly limited to the route, but is preferably transhepatic arterial administration, more preferably transcutaneous. Catheter administration can be mentioned as an example.

When using an injection used for systemic administration, deferoxamine is continuously used for 12-24 hours, more preferably 24 hours, depending on the administration route selected from any one of arterial injection, intravenous injection, and subcutaneous injection. One administration, 1-5 times a week, 2 weeks of administration and 1 week of non-administration (withdrawal) combined into one course, 10-80 mg / kg per dose, more preferably Is preferably administered in an amount of 40-60 mg / kg, and preferably 1-30 minutes (within 30 minutes) when an injection used for local administration (local injection) to the affected area is used. More preferably, 10 to 15 minutes of local administration to the affected area is performed once, and deferoxamine in an amount of 10 to 80 mg / kg suspended in 1 to 10 ml (10 ml or less) of an oily contrast medium at a time. months Once, or at appropriate dosing schedule depending on the condition, the form preferably administered by transhepatic arterial pathways. The oil-based contrast agent in the above embodiment is used for the purpose of stagnating deferoxamine, which is the main component, in cancer cells by local administration, and is used in cancer treatment, particularly in embolization treatment of liver cancer. The composition is not particularly limited as long as stagnation is observed, but iodinated poppy oil fatty acid ethyl ester is a suitable example as a commonly used oily contrast agent. Furthermore, the pharmaceutical composition of the present invention can be used in the manufacture of a medicament for the treatment of cancer having an iron ion requirement for proliferation, and in particular, a medicament for the treatment of liver cancer such as hepatocellular carcinoma. It can be suitably used in production.

Examples of the present invention are shown below, but the present invention is not limited to the examples.

(Basic safety trials)
First, in order to confirm the safety of deferoxamine to cells, human cultured hepatocytes are divided into two groups, and one group is deferoxamine mesylate (trade name Desferal; hereinafter referred to as DFO). Was added at a concentration of 50 μM and cultured for 4 days, and the cell mortality was compared with the non-added group. The culture method is Williams E medium (Gibco Laboratories) with 10% calf serum, 10 IU / ml penicillin, 10 μg / ml streptomycin, 0.05 mg / ml gentamicin, 0.02 U / ml insulin to form Complete Williams E medium. Then, 2 × 10 ⁵ cultured hepatocytes and 3 ml of the medium were added to the culture dish 1Dish and cultured at 37 ° C., 5% CO ₂ , 95% Room Air. Cell mortality was confirmed by trypan blue staining. As a result, the mortality rate in the DFO addition group was 5 ± 2%, whereas in the non-addition group, it was 6 ± 3% (n = 3), and there was no significant difference between the two. This indicates that 50 μM deferoxamine has no harmful effect on cultured human hepatocytes.

(Effect of deferoxamine on tumor cells)
Three types of cultured hepatoma cells, Huh-7, HepG2, HLF, RPMI 1640 medium (Gibco Laboratories) supplemented with 10% calf serum and 1 mM Na pyruvate, 1 × 10 ⁶ cells per dish in dish Was cultured in a culture system under the same conditions as in Example 1, and DFO was added thereto at various concentrations to examine the antitumor effect. Two independent cell culture experiments were performed, and the cell viability assay was performed to determine the relative value when the survival rate of the control group without addition was defined as 100. For the cell viability assay, MTT [3- (4,5-Dimethylthiazol-2-yl) -2,5-Diphenylthetrazolium bromide] assay widely used in cultured cell survival experiments was used. The principle of this assay is that MTT is specifically taken up into the mitochondria of living cells and measured by fluorescence at a wavelength of 570 nm. Assay results are expressed as the average of two experiments. Figures 1 to 3 show the results of these experiments.

FIG. 1 is a graph showing the effect on Huh-7 cells. The horizontal axis of the graph represents the time (hour) after addition of DFO, and the vertical axis represents the survival rate at each time when the control group is 100. Compared with the control group (-●-), the survival rate decreased in the 5 μM DFO addition group (-▲-), and further decreased in the 10 μM DFO addition group (-■-). The survival rate after 96 hours of the 10 μM DFO addition group was 24% of the control. Even when 100 μM (...,...), 500 μM (...,...), Or 1000 μM (...,...) DFO was added, the same effect as the addition of 10 μM was observed. That is, DFO showed an antitumor effect on cultured hepatoma cells in a time-dependent and concentration-dependent manner.

FIG. 2 is a diagram showing the effect of DFO on HepG2, the horizontal axis of the graph shows the time after the start of culture (hour), and the vertical axis shows the survival rate at each time when the control group is 100. Compared with the control group (-●-), the survival rate decreased in the 5 μM DFO addition group (-▲-), and further decreased in the 10 μM DFO addition group (-■-). Even when 100 μM (...,...), 500 μM (...,...), Or 1000 μM (...,...) DFO was added, the same effect as that with 10 μM was observed. The survival rate after 96 hours in the 100 μM DFO addition group was 28% of the control. Also for HepG2 cells, DFO showed a time-dependent and concentration-dependent antitumor effect.

FIG. 3 is a diagram showing the effect of DFO on HLF, where the horizontal axis of the graph shows the time after the start of culture (hour), and the vertical axis shows the survival of each time when the control group is 100. Compared with the control group (-●-), the survival rate decreased in the 5 μM DFO addition group (-▲-), and further decreased in the 10 μM DFO addition group (-■-). Even when 100 μM (...,...), 500 μM (...,...), Or 1000 μM (...,...) DFO was added, the same effect as the addition of 10 μM was observed. The survival rate after 96 hours in the 100 μM DFO addition group was 36% of the control. Also for HLF cells, DFO exhibited a time-dependent and concentration-dependent antitumor effect.

(Examination of combined use of deferoxamine and other drugs)
For 5-fluorouracil (5-FU) and adriamycin (ADM), which are widely used as existing antitumor agents, the effect of combination with DFO was verified. Prior to verification, 5-FU and ADM were added alone to cultured hepatoma cells, and the effects were examined. The cultured cell line experiment was performed by the method described in Example 2, and the value was calculated as an average value of two independent cultured cell line experiments. 4 to 6 show the 5-FU results, and FIG. 7 shows the ADM results.

FIG. 4 shows the effect of 5-FU on Huh-7 cells. The horizontal axis of the graph is the time after addition of 5-FU, and the vertical axis is relative to the control group (no drug added) as 100. A good survival rate. Compared with the control group (-●-), when 5-FU was added to a concentration of 0.1 µg / ml (-▲-), almost no effect was seen, and 0.5 µg / ml (- (2) The effect was small even at 1.0 μg / ml (... ○ ...). The survival rate at the addition of 1.0 μg / ml was 75% of the control.

FIG. 5 shows the effect of 5-FU on HepG2 cells. The horizontal axis of the graph shows the time after addition of 5-FU, and the vertical axis shows the relative survival rate when the control group is 100. Compared with the control group (-●-), when 5-FU was added to a concentration of 0.1 µg / ml (-▲-), the effect was small, and the survival rate after 96 hours was 81 times that of the control. %Met. A certain antitumor effect was observed at 0.5 μg / ml (-■-) and 1.0 μg / ml (... ○ ...). The survival rate at the addition of 1.0 μg / ml was 43% of the control.

FIG. 6 shows the effect of 5-FU on HLF cells. The horizontal axis of the graph shows the time after addition of 5-FU, and the vertical axis shows the relative survival rate when the control group is 100. Even when compared with Huh-7 and HepG2 cells, no clear effect of 5-FU was observed with the control group (-●-). When added to a concentration of 0.1 μg / ml (-▲-), the survival rate after 96 hours was 95% of the control. Even at 0.5 μg / ml (-■-) and 1.0 μg / ml (... ○ ...), the survival rate after 96 hours is as high as 83% and 79%. From these results, it is widely used as an anticancer agent. 5-FU is not expected to be very effective for hepatocellular carcinoma, and there are cell types with little effect, whereas deferoxamine provided by the present invention can be used as a single agent. It showed excellent antitumor effect exceeding 5-FU, and it was shown that the effect does not vary so much depending on the cell type.

FIG. 7 shows the effect of ADM on Huh-7 cells. The horizontal axis of the graph shows the time after addition of ADM, and the vertical axis shows relative survival when the control group is 100. Compared to the control group (-●-), when added at a concentration of 10 nM (-▲-), almost no effect is seen, but at 100 nM (-■-), a certain effect is seen, and further 1 μM ( ... ○ ...), the effect increased, and the survival rate after 96 hours was 40% of the control. At 10 μM, a more prominent effect was observed, and the survival rate after 96 hours was 9% of the control. However, ADM is known as an anticancer agent having a very strong side effect such as myocardial injury and equivalent to 10 μM. Is a numerical value that should be used as a reference value because it is a clinically impractical number.

From the results of the addition experiment with DFO, 5-FU, and ADM alone, in order to verify the effect of the combined use, the concentration at which the effect was not seen with the single agent, that is, the concentration of 5-FU of 0.1 μg / ml, The combined use with DFO having a concentration of 5 μM was verified under the condition of ADM concentration of 10 nM. Since ADM is a drug having a strong action on the cell itself, the combined effect at lower concentrations of 0.1 nM and 1 nM was also verified. The cultured cell system experiment was performed in the same procedure as described above, and the average value was calculated by performing two independent experiments. Figures 8-11 show these results.

FIG. 8 is a graph showing the combined effect of DFO and 5-FU on Huh-7 cells. The horizontal axis of the graph represents the time after the addition of each drug (hour), and the vertical axis represents the relative value when the control group is 100. A good survival rate. The black bar graph is the control, the dark gray is the one with the addition of 5 μM DFO alone, the light gray is the one with the addition of 0.1 μg / ml 5-FU, and the white bar graph is the addition of DFO and 5-FU at the same time Is the result of things. As shown in the graph, when DFO and 5-FU were used in combination, an effect higher than that of the single addition was observed, and the survival after 96 hours was 61% of the control. When the effect of the combined use is compared in more detail with the case of DFO alone, it is assumed that 0.1 μg / ml DFO is added to 5 μM (3.338395 μg / ml) DFO instead of 5-FU. The amount added was equivalent to 5.15 μM DFO, and the expected value with 5.15 μM addition was 74.25% of the control from the decrease rate from 0 → 5 μM, and 61% obtained with the combination of DFO and 5-FU was this The value was smaller than the value, and it was considered that there was an effect of combined use.

FIG. 9 is a graph showing the combined effect of DFO and 5-FU on HepG2 cells. The horizontal axis of the graph is the time after the addition of each drug (hour), and the vertical axis is the relative survival when the control group is 100. Indicates the rate. The black bar graph is the control, the dark gray is the one added with 5 μM DFO alone, the light gray is the one added with 5-FUｇ0.1 μg / ml alone, the white bar graph is the one added with DFO and 5-FU at the same time It is a result. The effect similar to Huh-7 cell was seen also in HepG2 cell, and the survival rate after 96 hours was 68% of control. When the effect of the combined use is compared in more detail with the case of DFO alone, when it is assumed that 0.1 μg / ml DFO is added to 5 μM DFO instead of 5-FU, the addition amount is equivalent to 5.15 μM DFO. From the decrease rate from 0 to 5 μM, the expected value when 5.15 μM was added was 80.43% of the control, and 68% obtained with the combination of DFO and 5-FU was smaller than this value. It was considered effective.

FIG. 10 is a graph showing the combined effect of DFO and 5-FU on HLF cells. The horizontal axis of the graph is the time after the addition of each drug (hour), and the vertical axis is the relative survival when the control group is 100. Indicates the rate. The black bar graph is the control, the dark gray is the one added with 5 μM DFO alone, the light gray is the one added with 5-FUｇ0.1 μg / ml alone, the white bar graph is the one added with DFO and 5-FU at the same time It is a result. HLF cells were less effective than Huh-7 and HepG2 cells, and survival after 96 hours was 79% of controls. When the effect of the combined use is compared in more detail with the case of DFO alone, when it is assumed that 0.1 μg / ml DFO is added to 5 μM DFO instead of 5-FU, the addition amount is DFO 5.15 μM Correspondingly, from the rate of decrease from 0 to 5 μM, the expected value when 5.15 μM was added was 83.52% of the control, and 79% obtained with the combination of DFO and 5-FU was smaller than this value. It was thought that there was an effect.

FIG. 11 is a graph showing the combined effect of DFO and ADM on Huh-7 cells, where the horizontal axis of the graph is the time after the addition of each drug (hour), and the vertical axis is the relative survival when the control group is 100. Indicates the rate. Black bar graph is a control, dark gray is ADM 10 nM added alone, white is DFO 5 μM added alone, light gray is DFO 5 μM and ADM 0.1 nM added simultaneously, lighter The gray color is the result of adding DFO 5 μM and ADM 1 nM at the same time, and the light gray color is the result of adding DFO 5 μM and ADM 10 nM at the same time. As shown in the results, a remarkable antitumor effect was observed after 72 hours in combination with DFO even at a concentration at which the effect of ADM was not observed or at a concentration lower than that, and the cell viability after 96 hours was When DFO 5 μM and ADM 10 nM were used in combination, it decreased to 55% of the control. This value is much smaller than the theoretical value 68.25% of the simple additive effect of DFO 5 μM single agent (75%) and ADM 10 nM single agent (91%), and the combined effect is a synergistic effect It has been shown.

(Detection of tumor markers)
In order to clarify the antitumor effect of deferoxamine on liver cancer cells, the expression of tumor marker protein in cultured hepatoma cells after addition of DFO was detected by the antigen-antibody method. Extraction of proteins from cells and detection of marker proteins by antigen-antibody reaction followed the method of Jin H. et al. (J. Gastroenterol. 42: 475-484, 2007). Specifically, Huh-7 cells and HepG2 cells cultured by the above method are washed with ice-cooled Phosphate-buffered saline (PBS) and put into a Cell lysis buffer containing 1 mM Phenylmethanesulfonyl fluoride (manufactured by Cell Signaling Technology). Suspended. The cell membrane was crushed with an ultrasonic crusher, and the membrane components were removed by centrifugation to obtain a whole cell protein sample. Mitochondrial protein and cytoplasmic protein were each separated and purified by Mitochondria / cytosol fractionation kit (manufactured by Biovision), and the expression levels of each tumor marker were compared by Western blotting. Primary antibodies for each tumor marker were obtained as follows: Cyclin D1, Cyclin D3 (manufactured by Cell Signaling Technology); Cytochrome c, cdk4, p-Rb (manufactured by Santa Cruz Biotechnology); β-actin (manufactured by Abcam) ). As the secondary antibody, one obtained from Amersham Bioscience was used. Using these antibodies, expression of Cyclin D1, Cyclin D3, cdk4, Cytochrome c, p-Rb and β-actin (control) for Huh-7 cells, and Cyclin D1, cdk4, Cytochrome c, p- for HepG2 cells. Rb expression was compared at the protein level.

FIG. 12 shows the results of comparison of tumor marker protein expression. The left lane in the figure represents the expression level of each marker in Huh-7 cells (the marker name is shown at the left end), and the right lane represents the expression level of each marker in HepG2 cells. In Huh-7 cells, there was no change in the expression level of β-actin as a control, whereas the expression of Cyclin D1, Cyclin D3, cdk4, and p-Rb decreased, whereas the amount of Cytochrome c protein increased. did. Cyclin D1, Cyclin D3, and cdk are proteins related to the cell cycle, and the expression level of these proteins is reduced by the addition of DFO, so DFO suppresses cell growth of cultured hepatoma cells. It was shown that. On the other hand, Cytochrome c is known to increase in cell death, and this result showed that DFO also induced cell death of hepatoma cells. Since p-Rb also decreased with time, it was shown that DFO stopped the cell cycle at the G0 / G1 phase. In HepG2 cells, the expression of CyclinD1, Cyclin D3, cdk4, and p-Rb decreased and Cytochrome c tended to increase in the same manner as Huh-7 cells, supporting the results in Huh-7 cells.

(DFO administration to animals for clinical trials)
In order to ensure the safety of clinical deferoxamine administration, an overdose experiment using rats was conducted. Ten rats were administered intraperitoneally with 400 mg / kg / day, which is 5 times the dose of 80 mg / kg / day, which is the upper limit dose of DFO in humans, and the course of 24 hours was observed. Even after 24 hours, all the rats survived, confirming the safety of administration to the living body.

The following clinical trials are all conducted at the Yamaguchi University Hospital, and are approved by the Ethics Committee of the Yamaguchi University Hospital, complying with relevant laws and regulations, and informed consent is given to the subjects. It was done with thorough consent in writing.

(Clinical trial-Case 1, 63 years old, male)
In September 2005, hepatocellular carcinoma frequently occurring in the liver (hereinafter sometimes referred to as HCC) was recognized, and thereafter transarterial chemoembolization (hereinafter also referred to as TACE) was repeated for HCC. In February 2006, the reservoir was placed and intraarterial chemotherapy (Low dose FP therapy + Isovorin) was not effective, but multiple lung metastases were observed in April. Therefore, treatment with DFO administration was started in May.
Blood data at the start of treatment were as follows: TP 7.8 g / dl, Alb 2.5 g / dl, FBS 144 mg / dl, BUN 9 mg / dl, Cre 0.64 mg / dl, T.P. Bil 1.3 mg / dl, D.I. Bi 0.4 mg / dl, ALP 762 IU / l, γ-GTP 85 IU / l, ChE 126 IU / l, AST 49 IU / l, ALT 27 IU / l, LDH 181 IU / l, WBC 3840 / μl (Netro 48.0%, Eosino 7.0%, Baso 0.3%, Mono 6.3%, Lymph 40.4%), RBC 359 × 104 / μl, Hb 11.7 g / dl, Ht 35.6%, MCV 99.2fl, MCH 32.6pg, MCHC 32.9%, Plt 9.6 × 10 4 / μl, PT 13.8 (11.3) sec / 65.8%, APTT 36.4 (27.2) sec, BT 5. 5min, 24Ccr 85.3mg / min
The tumor marker data at the start of treatment were as follows: AFP (L3) 36.4 ng / ml (L3 is 0.5% or less normal), PIVKA2 603 AU (normal 40 AU or less) Background liver cirrhosis (B Type) and Child-Pug B (7 points).

The administration schedule of the weekly DFO intraarterial administration therapy is as follows: One administration is 24 hours and administration is 1 to 5 administrations per week. 2 weeks is 1 course, and at least 2 courses are repeated, and this course is repeated as appropriate for the cases where the effect can be expected. Effectiveness is determined by tumor markers or diagnostic imaging. Specifically, chemotherapy is performed from a gripper needle punctured in the reservoir port. DFO (deferoxamine mesylate; trade name Desferral; manufactured by Novartis; common in clinical trials) 80 mg / kg or less is adjusted to a total volume of 240 ml with physiological saline and administered with an infusion pump for 24 hours. After completion, heparin Na 5000 units / A is injected to finish.

In this case, DFO 50 mg / kg was administered 3 times / week (24 hours of continuous administration at a time), and 4 weeks were taken as 1 course (2 weeks administration, 1 week off and 2 weeks administration). The results of the CT examination are shown in FIGS. FIG. 13 is a CT image before treatment. In the abdominal dynamic CT image of A, an intrahepatic tumor is observed in the part indicated by the white circle and the black arrow, and in the simple CT images of B and C, the part indicated by the white arrow in the figure. Lung metastases were confirmed. FIG. 14 is a CT image after 3 months from the start of treatment. In the abdominal dynamic CT image of A, a marked reduction of the intrahepatic tumor was confirmed in 3 months as indicated by white circles and black arrows, and B The disappearance of lung metastases was also confirmed in C and CT images. The transition of the tumor marker during the treatment period is shown in FIG. In the graph, the horizontal axis represents the time elapsed from the start of treatment, the vertical axis represents the marker value, and the DFO administration period is indicated by a gray bar on the graph. After the start of administration, AFP and PIVKA2 decreased rapidly, and the L3 fraction also decreased from 4 months later, and both decreased to normal values (FIG. 15). As of the end of February 2008, he has been alive for 1 year and 10 months and no pulmonary metastases have appeared.

(Clinical trial-Case 2, 63 years old, male)
HCC was first launched in October 2003, and TACE and radiofrequency ablation (RFA) were performed. Thereafter, TACE was performed in December 2005, but in March 2006, multiple reservoirs were placed in the reservoir and intraarterial infusion chemotherapy (Low dose FP therapy + Isovorin) was performed. The tumor increased from the end of 2006, and it was decided to perform DFO treatment.
Blood data at the start of treatment were as follows: TP 7.8 g / dl, Alb 2.5 g / dl, FBS 184 mg / dl, BUN 13 mg / dl, Cre 1.03 mg / dl, T.P. Bil 3.3 mg / dl, D.I. Bi 1.7 mg / dl, ALP 449 IU / l, γ-GTP 69 IU / l, ChE 60 IU / l, AST 82 IU / l, ALT 48 IU / l, LDH 468 IU / l, IV7S 12.0 ng / ml, PIIP 1.00 U / Ml, HA 1120 ng / ml, Ferritin 691.0 ng / ml, Fe 168 μg / dl, WBC 6850 / μl (N seg. 54.5%, N. band 4.0%, Eosino 2.0%, Baso 0. 5%, Mono 2.0%, Lymph 27.0%), RBC 269 × 10-4 / μl, Hb 10.6 g / dl, Ht 30.9%, MCV 114.9 fl, MCH 39.4 pg, MCHC 34 .3%, Plt 8.6 × 10 −4 / μl, PT 13.5 (11.7) sec / 67 9%, APTT 34.1 (28.1) sec, BT 4.5min
The tumor marker data at the start of treatment were as follows: AFP (L3) 1.6 ng / ml (L3 is normal at 5% or less), PIVKA2 4942 AU (normal 40 AU or less) Background liver is cirrhosis (C type) And Child-Pugh C (11 points).

DFO (50 mg / kg / day) 4 times (once 24 hours continuous administration) / week was started. At the time of administration for 2 weeks, mild appetite decline and mild renal dysfunction (Cre increased to 1.8 mg / dl, but soon normalized) were observed without major side effects. The tumor marker PIVKA2 rapidly decreased and normalized after 2 weeks in the second half after a 1-week withdrawal. The transition of the tumor marker is shown in FIG. The horizontal axis of the graph shows the time elapsed from the start of treatment, the vertical axis shows the marker value, and the DFO administration schedule is shown on the graph. As is clear from the graph, it was shown that the PIVKA2 marker was rapidly decreased by DFO administration. He later died in February 2007 due to a sudden rupture of esophageal varices when he was not treated with DFO.

(Clinical trial-Case 3, 65 years old, male)
In January 2004, the patient was diagnosed with multiple HCC (Vp3, Vv3) and right intra-atrial tumor plug, and started intraarterial infusion chemotherapy (low dose FP therapy + Isovorin + IFN) after reservoir placement. Although once effective, no effect was observed with the October treatment. In this case, arterial injection chemotherapy had strong side effects of thrombocytopenia, and was treated intermittently thereafter. However, in February 2006, HCC was also increasing and lung metastases appeared. Therefore, it was judged that there was no effect and DFO treatment was performed.
Blood data at the start of treatment were as follows: TP 7.2 g / dl, Alb 3.1 g / dl, BUN 15 mg / dl, Cre 1.05 mg / dl, T.P. Bil 2.0 mg / dl, D.D. Bil 0.7 mg / dl, ALP 299 IU / l, γ-GTP 35 IU / l, ChE 105 IU / l, AST 83 IU / l, ALT 86 IU / l, LDH 236 IU / l, T-chol 120 mg / dl, CRP 0.14 mg / Dl, Ferritin 222.7 ng / ml, Fe 216 μg / dl, Na 136 mmol / l, K 3.7 mmol / l, Cl 104 mmol / l, WBC 3600 / μl (Netro 41.7%, Eosino 2.2%, Baso 1.1%, Mono 12.2%, Lymph 42.8%), RBC 314 × 10 −4 / μl, Hb 11.1 g / dl, Ht 32.8%, MCV 104.5 fl, MCH 35.4 pg, MCHC 33.8%, Plt 10.6 × 10-4 / μl, PT 2.5 (11.6) sec / 82.4%, APTT 32.6 (29.3) sec
Tumor marker data at the start of treatment were as follows: FP (L3) 52.5 ng / ml (<0.5%), PIVKAII 23703 AU (normal 40 AU or less) Background liver cirrhosis (C type), Child -Pugh A (5 points).

In April 2006, DFO was started to be administered at 50 mg / kg, but there was a slight decrease in renal function before treatment, and 25-50 mg / kg / day was intermittently administered depending on the renal function. After the start of treatment, PIVKA2, which was 23000, once decreased to 2500 after 2 months. Although AFP recognized a slight decrease, there was no significant change. FIG. 17 shows the transition of the tumor marker. The horizontal axis of the graph represents the time elapsed from the start of treatment, and the vertical axis represents the marker value. The timing of DFO administration is shown in the upper part of the graph. As shown in the graph, it was clarified that the marker rapidly decreased by administration of DFO.

In this case, the CT image was SD, and no increase was observed in lung metastatic lesions. However, when treatment was stopped, the tumor marker PIVKA2 was re-elevated, and when DFO was treated again, PIVKA2 decreased in response (DFO was administered at 25-50 mg / kg for 24 hours at a time, 1-5 As needed at the rate of times / week). FIG. 18 shows the transition of the tumor marker at the time of DFO re-administration. The horizontal axis of the graph represents the time elapsed from the start of treatment, the vertical axis represents the marker value, and the timing of DFO administration is shown at the top of the graph. The tumor marker rapidly increased when DFO administration was stopped at home temporarily, the number of metastases in the lung increased, the tumor in the liver also increased, and the treatment resumed, but died at the end of April 2007. Survived 1 year and 1 month after DFO administration. Compared to the usual course of no treatment, both hepatic and pulmonary metastases did not increase rapidly, and the growth was slow. Case.

By using the pharmaceutical composition provided by the present invention, it is possible to develop a drug effective against liver cancer, particularly hepatocellular carcinoma, for which there has been no effective chemotherapy. Deferoxamine has already been established as a safe drug for iron overload, which leads to the development of a highly effective liver cancer drug with few side effects.

Claims

A pharmaceutical composition for treating cancer having an iron ion requirement for growth, comprising at least one of deferoxamine, a derivative thereof, and a pharmacologically acceptable salt thereof as an active ingredient .
The pharmaceutical composition according to claim 1, wherein the cancer requiring iron ion proliferation is liver cancer.
3. The pharmaceutical composition according to claim 1 or 2, wherein the liver cancer is hepatocellular carcinoma.
The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmacologically acceptable salt is an organic acid salt.
The pharmaceutical composition according to claim 1, wherein the organic acid salt is an organic acid salt having 2 or 1 carbon atoms.
The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition is deferoxamine mesilate.
The pharmaceutical composition according to any one of claims 1 to 6, further comprising an anticancer agent.
The pharmaceutical composition according to any one of claims 1 to 7, wherein the anticancer agent is one or more anticancer agents selected from 5-fluorouracil, adriamycin, cisplatin, and mitomycin.
A method for treating cancer having an iron ion requirement for growth, comprising administering the pharmaceutical composition according to any one of claims 1 to 8.
The method according to claim 9, wherein the cancer having an iron ion requirement for proliferation is liver cancer.
The method according to claim 9 or 10, wherein the liver cancer is hepatocellular carcinoma.
Use of the pharmaceutical composition according to any one of claims 1 to 8 in the manufacture of a medicament for treating cancer that requires iron ions for growth.
The use according to claim 12, wherein the cancer having an iron ion requirement for proliferation is liver cancer.
The use according to claim 12 or 13, wherein the liver cancer is hepatocellular carcinoma.