JP2014040401A - Therapeutic agent for hepatocellular carcinoma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel anticancer agent and therapeutic method having an excellent anticancer activity for hepatocellular carcinoma.SOLUTION: A therapeutic agent for hepatocellular carcinoma containing miriplatin and another therapeutic agent for hepatocellular carcinoma has a more excellent anticancer activity by applying transcatherter arterial chemo-embolization (TACE) with respect to hepatocellular carcinoma. As another therapeutic agent for hepatocellular carcinoma, doxorubicin hydrochloride, epirubicin hydrochloride, mitomycin C, or 5-fluorouracil is preferable, with mitomycin C being the most preferable. Further, formation of platinum-DNA crosslinking with miriplatin can be promoted by a combined use of another therapeutic agent for hepatocellular carcinoma.

Description

  The present invention belongs to the field of therapeutic methods for hepatocellular carcinoma, and relates to the combined use of miriplatin and other therapeutic agents for hepatocellular carcinoma.

  Hepatocellular carcinoma is one of the tumors that develop in the liver and is a malignant tumor derived from hepatocytes. Pharmacotherapy for hepatocellular carcinoma is broadly divided into intrahepatic arterial infusion chemotherapy and systemic chemotherapy. Intrahepatic arterial infusion chemotherapy, also called hepatic arterial infusion chemotherapy, is a treatment method in which a catheter is inserted into the hepatic artery and an anticancer agent is administered directly. Examples of existing hepatocellular carcinoma therapeutic agents used for hepatic arterial chemotherapy include doxorubicin hydrochloride, epirubicin hydrochloride, mitomycin C, 5-fluorouracil (5-FU), cisplatin ( Cisplatin) and the like.

  Transarterial chemoembolization (TACE), which is one of the treatment methods for advanced hepatocellular carcinoma, is the insertion of a catheter into the hepatic artery that feeds the tumor in advanced hepatocellular carcinoma, This is a method of injecting a therapeutic agent for cell cancer and an embolic substance, embolizing the hepatic artery, and causing tumor cells to become necrotic. For TACE, the aforementioned doxorubicin hydrochloride, epirubicin hydrochloride, mitomycin C, 5-fluorouracil, and cisplatin were used, and dinostatin stimamarer (Zinostatin timamarmer) was also used. However, TACE for unresectable hepatocellular carcinoma in particular does not yet have a standard treatment.

  In pharmacotherapy for cancer, multi-drug combination therapy in which a plurality of anticancer agents are administered is generally performed. In multi-drug combination therapy, the mechanism of action is aimed at further enhancing the antitumor effect when anticancer drugs are administered alone, reducing side effects, and preventing the acquisition of drug resistance by tumor cells. Two or more types of anticancer agents with different types are used in clinical practice. However, the selection of the anticancer agent to be used in combination does not always achieve the above-mentioned object easily by simply performing vaguely. In order to find an enhancing effect or synergistic effect on the antitumor action, trial and error needs to be examined for the type, dosage, administration time, etc. of the anticancer agent to be used in combination. Clinical studies using two or more anticancer agents in combination include cisplatin and 5-fluorouracil (see Non-patent Document 1), cisplatin and epirubicin hydrochloride (see Non-patent Document 2), epirubicin hydrochloride and mitomycin C. Combination use has been reported (see Non-Patent Document 3). However, there is still no established standard combination therapy for the treatment of hepatocellular carcinoma.

  As an example, there are multiple reports on studies on the combination of cisplatin and mitomycin C. For example, when a Chinese hamster cell line V79-171B is used, there is a report that a synergistic effect was confirmed in this combination (see Non-Patent Document 4). On the other hand, there is a report that the effect was antagonistic when the mouse leukemia cell line P388 was used (see Non-Patent Document 5). Furthermore, there is a report that this combination was synergistic when a human gastric cancer cell line was used for the combination (see Non-Patent Documents 6-7). That is, the usefulness of the combined use of cisplatin and mitomycin C has been suggested for leukemia and gastric cancer, but is unclear for hepatocellular carcinoma.

  Cisplatin belongs to the platinum-containing antineoplastic drug because of its chemical structure. Other platinum-containing antineoplastic agents include carboplatin, nedaplatin, oxaliplatin and the like. However, the combined effect of carboplatin and mitomycin C, the combined effect of nedaplatin and mitomycin C, or the combined effect of oxaliplatin and mitomycin C has not yet been reported.

  Milliplatin is a new therapeutic agent for hepatocellular carcinoma that was approved in Japan in 2009 for the efficacy and effect of lipiodization in hepatocellular carcinoma with hydrate as an active ingredient (see Patent Document 1). Lipidation means intrahepatic arterial administration of a therapeutic agent for hepatocellular carcinoma suspended in iodized poppy oil fatty acid ethyl ester. The chemical name for miriplatin is (SP-4-2)-[(1R, 2R) -cyclohexane-1,2-diamine-N, N ′] bis (tetradecanoato-O) platinum. Due to this chemical structure, miriplatin also belongs to platinum-containing anticancer drugs. With regard to miriplatin, which is a novel therapeutic agent for hepatocellular carcinoma, it is desired to discover and develop a concomitant drug that can further enhance the effect. However, there are still no reports on the combined use of existing therapeutic agents for hepatocellular carcinoma used for hepatic arterial chemotherapy and miriplatin, and the effects obtained by this combination.

Japanese Patent Laid-Open No. 11-315088

Journal of Surgical Oncology, 2002; 80; 143-8 Hepatology Research, 2011; 41; 303-9 Japan Journal of Radiology, 2012; 30; 263-70 International Journal of Cancer, 1989; 44; 911-7 Japan Journal of Cancer Chemotherapy, 1989; 16; 2275-82. Journal of Surgical Oncology, 1993; 54; 98-102. Journal of Surgical Oncology, 1994; 56; 242-5

  It is an object of the present invention to provide a novel anticancer agent and treatment method for hepatocellular carcinoma, and a treatment having an additive effect or a synergistic effect of an antitumor effect by using a combination of a conventional therapeutic agent for hepatocellular carcinoma and miriplatin. Is to provide an agent.

  As a result of intensive studies to solve the above problems, the present inventors have found that the combination effect of the combination of miriplatin and a conventional therapeutic agent for hepatocellular carcinoma is obtained by a combination index method in a model using a rat or human hepatocellular carcinoma cell line. As a result of the test, it was found that there is an additive or synergistic effect of antitumor action by combining miriplatin and a conventional therapeutic agent for hepatocellular carcinoma, and the present invention was completed.

  That is, the present invention relates to the following.

Item 1. Selected from the group consisting of miriplatin and doxorubicin or a pharmaceutically acceptable salt thereof, epirubicin or a pharmaceutically acceptable salt thereof, mitomycin C or a pharmaceutically acceptable salt thereof and 5-fluorouracil or a pharmaceutically acceptable salt thereof A therapeutic agent for hepatocellular carcinoma, comprising one or more other therapeutic agents for hepatocellular carcinoma.

Item 2. The therapeutic agent according to Item 1, wherein the other therapeutic agent for hepatocellular carcinoma is doxorubicin hydrochloride, epirubicin hydrochloride, mitomycin C or 5-fluorouracil.

Item 3. Item 3. The therapeutic agent according to Item 1 or 2, wherein the other therapeutic agent for hepatocellular carcinoma is mitomycin C.

Item 4. A method of promoting the formation of a platinum-DNA crosslink by miriplatin by using another therapeutic agent for hepatocellular carcinoma in combination.

Item 5. Item 5. The method according to Item 4, wherein the other therapeutic agent for hepatocellular carcinoma is mitomycin C.

  In one embodiment of the invention, the invention includes administering a therapeutically effective amount of miriplatin to a human or mammal in need of treatment, including concomitant use with other therapeutic agents for hepatocellular carcinoma. The present invention relates to a method for treating cell cancer.

  In another aspect of the invention, the invention comprises administering to a human or mammal in need of treatment a therapeutically effective amount of miriplatin and a therapeutically effective amount of other hepatocellular carcinoma therapeutics. The present invention relates to a method for treating hepatocellular carcinoma.

  In another aspect of the invention, the invention relates to a pharmaceutical product for the treatment of hepatocellular carcinoma comprising miriplatin and other hepatocellular carcinoma therapeutics.

  In another aspect of the invention, the invention relates to a kit for the treatment of hepatocellular carcinoma comprising miriplatin and other hepatocellular carcinoma therapeutics.

  In another aspect of the invention, the invention relates to the use of miriplatin and other hepatocellular carcinoma therapeutics for the manufacture of a pharmaceutical composition for the treatment of hepatocellular carcinoma.

  In another aspect of the invention, the invention relates to a pharmaceutical composition comprising miriplatin and other hepatocellular carcinoma therapeutics for use in the treatment of hepatocellular carcinoma.

  In another aspect of the present invention, the present invention relates to the use of miriplatin for the manufacture of a pharmaceutical composition for the treatment of hepatocellular carcinoma, characterized by being used in combination with other therapeutic agents for hepatocellular carcinoma.

  In another aspect of the present invention, the present invention relates to the use of another therapeutic agent for hepatocellular carcinoma for producing a pharmaceutical composition for the treatment of hepatocellular carcinoma, characterized by being used in combination with miriplatin.

  In another aspect of the invention, the invention relates to the use of miriplatin to improve the effects of other hepatocellular carcinoma therapeutics in the treatment of hepatocellular carcinoma.

  In another aspect of the invention, the invention relates to the use of other therapeutic agents for hepatocellular carcinoma to improve the effects of miriplatin in the treatment of hepatocellular carcinoma.

  In another aspect of the invention, the invention relates to the use of miriplatin for the manufacture of a medicament that enhances and / or improves the effects of other hepatocellular carcinoma therapeutics in the treatment of hepatocellular carcinoma.

  In another aspect of the invention, the invention relates to the use of other therapeutic agents for hepatocellular carcinoma to produce a medicament that enhances and / or improves the effect of miriplatin in the treatment of hepatocellular carcinoma.

  In each of the aforementioned aspects of the present invention, other therapeutic agents for hepatocellular carcinoma include doxorubicin or a pharmaceutically acceptable salt thereof, epirubicin or a pharmaceutically acceptable salt thereof, mitomycin C or a pharmaceutically acceptable salt thereof, and Examples include one or more drugs selected from the group consisting of 5-fluorouracil or a pharmaceutically acceptable salt thereof, preferably doxorubicin hydrochloride, epirubicin hydrochloride, mitomycin C or 5-fluorouracil, most preferably mitomycin C. .

  According to the present invention, it was possible to further enhance the effect of treating hepatocellular carcinoma by administering miriplatin together with other therapeutic agents for hepatocellular carcinoma rather than administering it alone. In addition, this combination is expected to reduce the doses of other hepatocellular carcinoma therapeutic agents and miriplatin and, as a result, reduce the side effects of conventional hepatocellular carcinoma therapeutic agents. Furthermore, it is expected that acquisition of drug resistance by tumor cells against conventional therapeutic agents for hepatocellular carcinoma can be prevented by this combination.

FIG. 1 shows the results of testing the combined effect of miriplatin and doxorubicin hydrochloride in an in vitro model using the rat liver cancer cell line N1-S1 in Example 1. The mixing ratio of the drugs used in this combination is doxorubicin (as titer) concentration in the culture medium: milliplatin concentration in the suspension solution = 1: 100. The abscissa fa is a value calculated from the formula: fa = 1−f = 1 − {(average fluorescence intensity at each drug concentration) / (average fluorescence intensity when no drug is added)}. The combination index (CI) on the vertical axis is calculated from the IC ₅₀ value (drug concentration that inhibits cell growth by 50%) obtained by applying the dose of concomitant drug and cell growth rate to the logistic curve and the slope of the curve. Value. FIG. 2 is a diagram showing the results of testing the combined effect of miriplatin and mitomycin C in an in vitro model using the rat liver cancer cell line N1-S1 in Example 1, as in FIG. The mixing ratio of the drugs used in this combination is mitomycin C (as titer) concentration in the culture medium: milliplatin concentration in the suspension solution = 1: 10. FIG. 3 is a diagram showing the results of testing the combined effect of miriplatin and epirubicin hydrochloride in an in vitro model using the rat liver cancer cell line N1-S1 in Example 1, as in FIG. The mixing ratio of the drugs used in this combination is epirubicin (as titer) concentration in the culture medium: milliplatin concentration in the suspension solution = 1: 100. FIG. 4 is a diagram showing the results of testing the combined effect of miriplatin and 5-fluorouracil in Example 1 using the rat hepatoma cell line N1-S1 in the same manner as FIG. 1. The mixing ratio of the drugs used in this combination is 5-fluorouracil concentration in the medium: milliplatin concentration in the suspension solution = 1: 1. FIG. 5 is a diagram showing the results of testing the combined effect of miriplatin and mitomycin C in an in vitro model using the rat liver cancer cell line AH109A in Example 2. The mixing ratio of the drugs used in this combination is mitomycin C (as titer) concentration in the culture medium: milliplatin concentration in the suspension solution = 1: 50. The fa on the horizontal axis and the CI on the vertical axis are the same as those described above with reference to FIG. FIG. 6 is a diagram showing the results of testing the combined effect of miriplatin and mitomycin C in an in vitro model using the human liver cancer cell line Li-7 in Example 2, as in FIG. The mixing ratio of the drugs used in this combination is mitomycin C (as a titer) concentration in the medium: milliplatin concentration in the suspension solution = 1: 150. FIG. 7 is a graph showing the results of testing the combined effect of miriplatin and mitomycin C in Example 3 on a platinum-DNA adduct formation model using the rat liver cancer cell line N1-S1. The mitomycin concentration on the horizontal axis represents the concentration of mitomycin (μg / mL) combined with 100 μg / mL of miriplatin. Pt-DNA addts on the vertical axis represents the amount of platinum-DNA adduct formed (pg / μg DNA), and is expressed as the average value ± standard deviation of three experiments. Compared with miriplatin alone by Dunnet's multiple comparison test, a combination group with significantly higher platinum-DNA adduct formation was marked with *. FIG. 8 is a graph showing the results of testing the combined effect of miriplatin and mitomycin C in Example 3 on a platinum-DNA adduct formation model using the rat hepatoma cell line AH109A, as in FIG.

  Hereinafter, embodiments of the present invention will be described in detail.

  “Milliplatin” in the present invention is (SP-4-2)-[(1R, 2R) -cyclohexane-1,2-diamine-N, N ′] bis (tetradecanoato-O) platinum, Although hydrates and solvates are also included, hydrates or anhydrides are preferred.

  From the structure represented by the above chemical name, miriplatin belongs to a platinum-containing antineoplastic agent, and is lipophilic in which 1,2-diaminocyclohexane (DACH) is coordinated as a carrier ligand and myristic acid is coordinated as a leaving group. It is a platinum complex. Miliplatin has a high affinity for iodinated poppy oil fatty acid ethyl ester, and is suspended in iodized poppy oil fatty acid ethyl ester and administered into the hepatic artery (lipiodization), so that it stays locally in the tumor and is a platinum component. It has the feature of releasing slowly. As the action site and action mechanism of miriplatin, miriplatin is converted into dichloro 1,2-diaminocyclohexaneplatinum (DPC) in which a leaving group is mainly substituted with a chloride ion in a living body, and then DNA in cancer cells. It is believed that platinum-DNA crosslinks (adducts) covalently bonded to the chains are formed and apoptosis is induced, thereby acting as an anticancer agent. The “formation of platinum-DNA cross-link” in the present invention means the above action mechanism.

The “other therapeutic agent for hepatocellular carcinoma” in the present invention is a therapeutic agent for hepatocellular carcinoma excluding miriplatin, that is, a drug used for the treatment of hepatocellular carcinoma excluding miriplatin. Specifically as "other hepatocellular carcinoma therapeutic agents", doxorubicin or a pharmaceutically acceptable salt thereof, epirubicin or a pharmaceutically acceptable salt thereof, mitomycin C or a pharmaceutically acceptable salt thereof and 5-fluorouracil or Examples thereof include pharmaceutically acceptable salts, and two or more of these can be combined. As the “other therapeutic agent for hepatocellular carcinoma”, doxorubicin hydrochloride, epirubicin hydrochloride, mitomycin C or 5-fluorouracil is preferable, and mitomycin C is most preferable.
The “hepatocellular carcinoma” in the present invention is one of tumors occurring in the liver, and is a malignant tumor derived from hepatocytes.

  In the present invention, “doxorubicin” means (2S, 4S) -4- (3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyloxy) -2,5,12-tri Hydroxy-2-hydroxyacetyl-7-methoxy-1,2,3,4-tetrahydrotetracene-6,11-dione. The pharmaceutically acceptable salt thereof can also be used in the present invention, and doxorubicin hydrochloride represented by CAS number 25316-40-9 is preferable. Doxorubicin belongs to an anthracycline antitumor agent, and the antitumor effect of doxorubicin is based on inhibition of topoisomerase II activity. Doxorubicin hydrochloride can be purchased from Kyowa Hakko Kirin Co., Ltd.

  In the present invention, “epirubicin” means (2S, 4S) -4- (3-amino-2,3,6-trideoxy-α-L-arabino-hexopyranosyloxy) -2,5,12-tri Hydroxy-2-hydroxyacetyl-7-methoxy-1,2,3,4-tetrahydrotetracene-6,11-dione. A pharmaceutically acceptable salt thereof can also be used in the present invention, and a free form represented by CAS number 56420-45-2 or epirubicin hydrochloride represented by CAS number 56390-09-1 is preferred, and epirubicin Hydrochloride is more preferred. Epirubicin belongs to an anthracycline antitumor drug, and the antitumor effect of epirubicin is based on inhibition of topoisomerase II activity. Epirubicin hydrochloride can be purchased from Kyowa Hakko Kirin Co., Ltd.

  In the present invention, “mitomycin C” means (1aS, 8S, 8aR, 8bS) -6-amino-4,7-dioxo-8a-methoxy-5-methyl-1-1a, 2,8,8a, 8b- Hexahydroazirino [2,3: 3,4] pyrrolo [1,2-α] indol-8-ylmethyl carbamate. Although the pharmaceutically acceptable salt can also be used in the present invention, a free form represented by a CAS number of 50-07-7 is preferred. Mitomycin C belongs to an alkylating agent, and the antitumor effect of mitomycin C is based on binding to cancer cell DNA, inhibiting DNA replication, and suppressing cancer cell division. Mitomycin C can be purchased from Kyowa Hakko Kirin Co., Ltd.

  In the present invention, “5-fluorouracil” (5-FU) is 5-fluoro-2,4 (1H, 3H) -pyrimidinedione, and the CAS number is represented by 51-21-8. The pharmaceutically acceptable salt can also be used in the present invention, but the free form is preferred. 5-FU belongs to the fluoropyrimidines among the antimetabolite pyrimidine antimetabolites, and the anti-tumor effect of 5-FU is based on inhibition of DNA precursor synthesis. 5-FU can be purchased from Kyowa Hakko Kirin Co., Ltd.

  The “therapeutically effective amount” in the present invention means an amount of a drug or pharmaceutical agent that elicits a biological or pharmaceutical response required by a researcher or doctor in a tissue, system, animal or human.

  “Treatment” in the present invention means any treatment of a disease (eg, improvement of symptoms, reduction of symptoms, suppression of progression of symptoms, etc.) and any prevention of diseases (eg, suppression of onset and / or progression of disease, recurrence) Suppression). In the present invention, “enhancement” and “improvement” refer to an increase in the effect of treatment as a therapeutic agent for hepatocellular carcinoma, reduction of side effects as a therapeutic agent for hepatocellular carcinoma, and resistance to drug by tumor cells against the therapeutic agent for hepatocellular carcinoma. It means prevention or suppression of acquisition.

  The term “pharmaceutical product” in the present invention is intended to encompass a product containing a specific component in a specific amount and any product that results directly or indirectly from a combination of specific components in a specific amount. .

  In the combination or combination of the miriplatin of the present invention and another therapeutic agent for hepatocellular carcinoma, both agents may be administered separately or may be administered together as a single pharmaceutical composition. Also, one component of the combination of the present invention may be administered to the other component first, simultaneously or later. These ingredients may be prepared into pharmaceutical products in a single dosage form or in separate dosage forms. As for miriplatin, for example, with reference to the pamphlet of International Publication No. WO2003 / 026642, the lyophilized preparation can be used as an injection preparation suspended in iodized poppy oil fatty acid ethyl ester. In humans, it is desirable to administer TACE. The components of the combination of the present invention are preferably administered in TACE for humans.

In the combination or combination of the miriplatin of the present invention and other therapeutic agents for hepatocellular carcinoma, the dose of miriplatin varies depending on the patient's symptoms, age, weight, etc. A range of about 1 mg to about 1000 mg per day, preferably a range of about 5 mg to about 300 mg, more preferably a range of about 10 mg to about 200 mg, and even more preferably a range of about 50 mg to about 120 mg. The dose of other hepatocellular carcinoma therapeutic agents also varies depending on the type of drug, patient symptoms, age, weight, etc., but ranges from about 2 mg to about 30 mg per day per human adult or about 9 mg / m ² to about 2600 mg. A range of / m ² is mentioned. For example, is preferably in the range of about 9mg / ^{m 2} ~80mg / ^m 2 if other hepatocellular carcinoma therapeutic agent is doxorubicin hydrochloride, in the range of from about 15mg / ^{m 2} ~100mg / ^m 2 when it is epirubicin hydrochloride Preferably, a range of about 2 mg to about 30 mg is preferable for mitomycin C, and a range of about 5 mg / kg to 20 mg / kg is preferable for 5-FU. More preferably, the other hepatocellular carcinoma therapeutic agent is in the range of about 10 mg (0.2 mg / kg) to 30 mg (0.6 mg / kg) when it is doxorubicin hydrochloride, about 60 mg when it is epirubicin hydrochloride, mitomycin C In the case of 5-FU, the range is about 5 mg / kg to 10 mg / kg.

In the combination and combined use of the miriplatin of the present invention and other therapeutic agents for hepatocellular carcinoma, the number of administrations of miriplatin varies depending on the patient's symptoms, age, body weight, etc., but it is 4 in the case of repeated administration once a day. It is desirable to have an observation period of more than a week. The frequency of administration of other therapeutic agents for hepatocellular carcinoma also varies depending on the type of drug, patient symptoms, age, weight, etc., but if it is administered once a day, it will have an observation period of about 1 week to 4 weeks or more. Can be mentioned. For example, when the other therapeutic agent for hepatocellular carcinoma is doxorubicin hydrochloride, in the case of repeated administration once a day, single to 6 days, 7 days to 4 weeks are preferred, and epirubicin hydrochloride In some cases, once a day for repeated administration, a single to 7-day continuous treatment for 4 days to 4 weeks is preferred. For mitomycin C, once a day for repeated administration, In the case of 5-FU, once a day, in the case of repeated administration, a single to 20 day continuous daily, 1-3 week rest is preferable. More preferably, when the other therapeutic agent for hepatocellular carcinoma is doxorubicin hydrochloride, once a day for 2 to 6 days, 7 days to 18 days of rest, epirubicin hydrochloride once a day Once, 3 weeks to 4 weeks of withdrawal, mitomycin C, once a day to once a week to consecutive days, 1 week to 3 weeks of withdrawal, 5-FU, once a day 1 to 2 times a week.

  Hereinafter, the present invention will be described in more detail with reference to Examples, Reference Examples, and Test Examples, but the present invention is not limited to these.

Example 1
In vitro combination effect of miriplatin

Using a rat liver cancer cell line, the in vitro combination effect with 4 existing drugs for treating liver cancer (doxorubicin hydrochloride, mitomycin C, epirubicin hydrochloride, 5-fluorouracil) was examined. The combined effect was determined by calculating a combination index (CI) by the method of Chou and Talalay (Adv Enzyme Regul 1984; 22; 27-55).
Rat hepatoma cell line N1-S1 was obtained from DS Pharma Biomedical and subcultured in DMEM (Dulbecco's Modified Eagle Medium) medium containing fetal bovine serum 10% and antibiotics penicillin and streptomycin. The culture was performed in an incubator at 37 ° C. and 5% CO ₂ . This medium was also used in the subsequent experiments.

The rat liver cancer cell line N1-S1 being passaged was suspended in a medium so as to be 1 × 10 ³ cells / mL, and 0.25 mL was seeded in a 24-well plate (Day 0). After sowing, the cells were cultured overnight in an incubator at 37 ° C. and 5% CO ₂ .

On the next day (Day 1), the test substance was prepared as follows. Doxorubicin hydrochloride was dissolved in a physiological saline solution in a 10 mg vial obtained from Kyowa Hakko Kirin Co., Ltd. to give a stock solution. Mitomycin C was dissolved in physiological saline so as to be 0.4 mg / mL in a 2 mg vial obtained from Kyowa Hakko Kirin Co., Ltd. to prepare a stock solution. For epirubicin hydrochloride and 5-fluorouracil, 2 mg / mL and 50 mg / mL solutions obtained from Kyowa Hakko Kirin Co., Ltd. were used, respectively. These were serially diluted with medium and added at 0.25 mL per well. In addition, 0.25 mL of the medium alone was added to the miriplatin single agent group. The final concentration of each test substance is based on the IC ₅₀ value of each test substance alone for the N1-S1 cell line, and a titer of 0.1-33 ng / mL for doxorubicin hydrochloride and epirubicin hydrochloride, and a potency for mitomycin C The value was 0.33 to 100 ng / mL, and 5-fluorouracil was 0.01 to 3.3 μg / mL. Furthermore, miriplatin was suspended in iodinated poppy oil fatty acid ethyl ester (hereinafter also abbreviated as a suspension liquid) so as to be 20 mg / mL in a 70 mg vial. This was serially diluted with a suspension solution, and 0.5 mL was added to each cell culture insert (manufactured by Becton Dickinson) placed on the well. The final concentration of miriplatin was 0.0033-100 μg / mL. Each test substance single agent group was added with 0.5 mL of the suspension only in the cell culture insert. The cells were cultured in an incubator up to Day 8 at 37 ° C. and 5% CO ₂ .

On Day 8, the cell culture insert was removed, and 40 μL of alamarBlue reagent (Wako Pure Chemical Industries) was added to each well. Thereafter, the cells were cultured in an incubator at 37 ° C. and 5% CO ₂ for about 3 hours, and the fluorescence intensity was measured with a fluorescence microplate reader Flx800TBI (Bio-TEK). The measurement conditions were an excitation wavelength of 540 nm and a fluorescence wavelength of 590 nm.

The drug dose and cell growth rate were fitted to a logistic curve, and the combination index was used for fa in the range of 0.1 to 0.9 using the obtained IC ₅₀ (drug concentration that inhibits cell growth by 50%) and the slope of the curve. (CI) was calculated. Here, fa = 1−f, f = (average fluorescence intensity at each drug concentration) / (average fluorescence intensity when no drug was added). The larger the value of fa, the stronger the cell growth inhibitory action, and the smaller the fa value, the weaker the cell growth inhibitory action. That is, a high fa range means a high chemical treatment concentration range, and a low fa range corresponds to a low chemical treatment concentration range. Average value of three experiments when fa is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9 For CI calculated as a ± 95% confidence interval, according to the usual method, when CI is less than 1, the combined effect is synergistic, and when CI is 1, the combined effect is additive. When is greater than 1, the combined effect was determined to be antagonistic.

  The results are shown in FIGS. FIG. 1 shows a combination of miriplatin and doxorubicin hydrochloride (drug mixing ratio is doxorubicin (as titer) concentration in medium: concentration of miriplatin in suspension solution = 1: 100), FIG. 2 shows miriplatin and mitomycin C (Drug mixing ratio is mitomycin C (as titer) concentration in the medium: milliplatin concentration in the suspension solution = 1: 10), FIG. 3 is a combination of miriplatin and epirubicin hydrochloride (drug mixing ratio is , Epirubicin (as titer) concentration in the medium: milliplatin concentration in the suspension solution = 1: 100), FIG. 4 is a combination of miriplatin and 5-FU (drug mixing ratio is the concentration of 5-fluorouracil in the medium) : Represents the test result of each effect of miriplatin concentration in the suspension solution = 1: 1). In each figure, CI whose mean value ± 95% confidence interval is less than 1 is indicated by ●. In rat liver cancer cell line N1-S1, the combination of doxorubicin hydrochloride, epirubicin hydrochloride or 5-fluorouracil and miriplatin was additive, but the combination with mitomycin C had a synergistic effect in the high fa range. Admitted.

Example 2
In vitro effect of miriplatin and mitomycin C

  In Example 1, when the rat liver cancer cell line N1-S1 was used, synergistic effects were observed in the combination of mitomycin C and miriplatin among the existing four drugs for liver cancer. Also examined this combination.

Rat hepatoma cell line AH109A and human hepatoma cell line Li-7 were obtained from Kumamoto University and Tohoku University Institute of Aging Medicine, Medical Cell Resource Center, respectively, and contained fetal bovine serum 10% and antibiotics penicillin and streptomycin Subculture in RPMI 1640 (Rosewell Park Memorial Institute 1640) medium. The culture was performed in an incubator at 37 ° C. and 5% CO ₂ . These media were used in subsequent experiments.

The rat hepatoma cell line AH109A or the human hepatoma cell line Li-7 being passaged was suspended in a medium so as to be 4 × 10 ³ cells / mL or 2 × 10 ⁴ cells / mL, respectively. 25 mL each was seeded (Day 0). After sowing, the cells were cultured overnight in an incubator at 37 ° C. and 5% CO ₂ .

The next day (Day 1), 0.4 mg / mL mitomycin C stock solution was serially diluted with medium and added at 0.25 mL per well. The final concentration of mitomycin C was 0.2 to 66 ng / mL as a titer. Furthermore, 20 mg / mL of miriplatin was serially diluted with the suspension solution, and 0.5 mL was added to each cell culture insert placed on the well. The final concentration of miriplatin was based on the IC ₅₀ value of miriplatin alone for each cell line, and was 0.01-3.3 μg / mL for the AH109A strain and 0.033-10 μg / mL for the Li-7 strain. mL.

On Day 8, the cell culture insert was removed, and 50 μL of alamarBlue reagent was added to each well. Thereafter, the cells were cultured in an incubator at 37 ° C. and 5% CO ₂ for 2 to 6 hours, and the fluorescence intensity was measured with a fluorescence microplate reader Flx800TBI. In the same manner as in Example 1, the combination index (CI) was calculated for the range of fa of 0.1 to 0.9 using IC ₅₀ and the slope of the logistic curve.

  The results are shown in FIGS. FIG. 5 shows a combination of miriplatin and mitomycin C against the rat hepatoma cell line AH109A (drug mixing ratio is mitomycin C (as titer) concentration in the medium: milliplatin concentration in the suspension solution = 1: 50), FIG. Each effect of combined use of miriplatin and mitomycin C on human liver cancer cell line Li-7 (drug mixing ratio is mitomycin C (as titer) concentration in medium: concentration of miriplatin in suspension solution = 1: 150) Represents the test results. In both rat liver cancer cell line AH109A and human liver cancer cell line Li-7, the combined use of mitomycin C and miriplatin had a synergistic effect. Therefore, it was suggested that a synergistic effect can be obtained with this combination regardless of the difference in liver cancer cell lines.

Example 3
Enhancement of platinum-DNA adduct formation by mitomycin C

  In order to examine the effect of mitomycin C combined use on the platinum-DNA adduct formation action of miriplatin, DNA was purified from cells that had been treated with miriplatin and mitomycin C for 3 days, and the amount of platinum contained in the DNA was quantified.

Suspended rat hepatoma cell line AH109A or N1-S1 is suspended in a medium to 3 × 10 ⁵ cells / mL or 5 × 10 ⁵ cells / mL, respectively, and seeded at 1.0 mL in a 6-well plate (Day 0). After sowing, the cells were cultured overnight in an incubator at 37 ° C. and 5% CO ₂ .

  On the next day (Day 1), 0.4 mg / mL mitomycin C stock solution was serially diluted with medium and added at 1.0 mL per well. The final concentration of mitomycin C was 0.67 and 2 μg / mL as the titer in the case of the AH109A strain, and 1 and 10 μg / mL as the titer in the case of the N1-S1 strain. Furthermore, 20 mg / mL of miriplatin was serially diluted with the suspension solution, and 2.0 mL was added to each cell culture insert placed on the well. The final concentration of miriplatin was 100 μg / mL for all cell lines.

  On Day 4, the cell culture insert was removed and the cells were collected. After washing the cells with phosphate buffered saline, the DNA was purified using DNAzol Reagent (manufactured by Invitrogen) and dissolved in Tris buffered EDTA solution (pH 8.0). The DNA concentration was quantified using an intercalate reaction between double-stranded DNA and the fluorescent dye Hoechst 33258, and the platinum concentration was measured using an atomic absorption photometer Z-9000 or Z-2710 (manufactured by Hitachi High-Tech). Based on the obtained DNA concentration and platinum concentration, the amount of platinum-DNA adduct formation was calculated as the amount of platinum (pg) contained in 1 μg of purified DNA.

  The results are shown in FIGS. FIG. 7 shows the results of the test using the rat liver cancer cell line N1-S1, and FIG. 8 shows the results of the test using the rat liver cancer cell line AH109A. In any rat liver cancer cell line, the amount of platinum-DNA adduct formed by miriplatin increased in a mitomycin C concentration-dependent manner. In addition, the combined dose of mitomycin C corresponding to the drug mixing ratio in which a synergistic effect was observed significantly increased the amount of platinum-DNA adduct formation compared to the case of miriplatin alone. From these results, it was suggested that the mitomycin C combination exhibits a synergistic effect by enhancing the platinum-DNA adduct forming action of miriplatin.

Example 4
In vivo effect of miriplatin and mitomycin C

  The in vivo combined effect of mitomycin C and miriplatin is examined using a liver cancer model animal prepared by transplanting the rat liver cancer cell line N1-S1 or the human liver cancer cell line Li-7 into the rat liver.

  Miriplatin is suspended in the suspension solution to a concentration of 20 mg / mL in a 70 mg vial. Mitomycin C is dissolved in physiological saline or a water-soluble contrast agent. Thereafter, the miriplatin suspension and the mitomycin C aqueous solution are mixed and administered once into the hepatic artery of the cancer-bearing rat.

  The antitumor effect is evaluated based on the tumor size at the time of administration and the tumor size 1 to 2 weeks after administration.

  The combination or combined use of the miriplatin of the present invention and other therapeutic agents for hepatocellular carcinoma makes it possible to further enhance the therapeutic effect of hepatocellular carcinoma than when other therapeutic agents for hepatocellular carcinoma or miriplatin is administered alone. It was. In addition, this combination is expected to reduce the side effects of conventional therapeutic agents for hepatocellular carcinoma and prevent acquisition of drug resistance by tumor cells against conventional therapeutic agents for hepatocellular carcinoma.

Claims (5)

  Selected from the group consisting of miriplatin and doxorubicin or a pharmaceutically acceptable salt thereof, epirubicin or a pharmaceutically acceptable salt thereof, mitomycin C or a pharmaceutically acceptable salt thereof and 5-fluorouracil or a pharmaceutically acceptable salt thereof A therapeutic agent for hepatocellular carcinoma, comprising one or more other therapeutic agents for hepatocellular carcinoma.   The therapeutic agent according to claim 1, wherein the other therapeutic agent for hepatocellular carcinoma is doxorubicin hydrochloride, epirubicin hydrochloride, mitomycin C or 5-fluorouracil.   The therapeutic agent according to claim 1 or 2, wherein the other therapeutic agent for hepatocellular carcinoma is mitomycin C.   A method of promoting the formation of a platinum-DNA crosslink by miriplatin by using another therapeutic agent for hepatocellular carcinoma in combination.   The method of claim 4, wherein the other therapeutic agent for hepatocellular carcinoma is mitomycin C.

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