WO2014021408A1 - Procédé de traitement du cancer par mise en œuvre d'agents anticancéreux combinés - Google Patents

Procédé de traitement du cancer par mise en œuvre d'agents anticancéreux combinés Download PDF

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WO2014021408A1
WO2014021408A1 PCT/JP2013/070825 JP2013070825W WO2014021408A1 WO 2014021408 A1 WO2014021408 A1 WO 2014021408A1 JP 2013070825 W JP2013070825 W JP 2013070825W WO 2014021408 A1 WO2014021408 A1 WO 2014021408A1
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paclitaxel
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cancer
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幸規 荒井
金子　直樹
香菜子 井口
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アステラス製薬株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/433—Thidiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a pharmaceutical, particularly a pharmaceutical composition for treating cancer, and particularly to a pharmaceutical composition for treating cancer for use in combination with another antitumor agent, which contains a NO production inhibitor as an active ingredient.
  • the present invention also relates to a combination therapy of an NO production inhibitor and an antitumor agent.
  • taxane antitumor agents are used as effective drugs in several cancer types.
  • Taxane antitumor agents are known to inhibit the division of cancer cells by inhibiting the depolymerization of microtubules necessary for cell division.
  • Paclitaxel is used to treat ovarian cancer, non-small cell lung cancer, breast cancer, gastric cancer, etc.
  • Docetaxel is used to treat prostate cancer, breast cancer, non-small cell lung cancer, gastric cancer, head and neck cancer, ovarian cancer, esophageal cancer, endometrial cancer, etc. Has been.
  • multi-drug combination therapy combining a taxane anti-tumor agent with another anti-tumor agent having a different mechanism of action is performed.
  • a combination of paclitaxel and carboplatin cancer, 1996, 77: 2458-63.
  • a combination of docetaxel and cisplatin is known (Eur. J. Surg. Oncol., 2006, 32 (3) 297- 302).
  • the maximum tolerated dose is set from the viewpoint of side effects, and the dose is limited.
  • paclitaxel “injects 210 mg / m 2 once a day for 3 hours by intravenous infusion. The administration is repeated for at least 3 weeks, and the administration is repeated as a course.
  • paclitaxel has been specified in addition to the dose, as described in the following. .
  • taxotere injection registered trademark: trade name of docetaxel
  • the maximum single dose is 75 mg / m 2 ” and the dose is indicated.
  • Nitric oxide is a gaseous radical produced from various cells in vivo, and L-arginine is oxidized in a NADPH-dependent manner by a homodimer of nitric oxide synthase (NO synthase, NOS) It is produced in the process of conversion to L-citrulline (Biochem. Pharmacol. 2008; 75: 923-30, Mol. Pharmacol. 2009; 76: 153-62).
  • NOS includes neuronal nitric oxide synthase (neuronal NO synthase, nNOS), inducible nitric oxide synthase (inducible NO synthase, iNOS), and endothelial nitric oxide synthase (endothelial NO synthase, eNOS).
  • nNOS neuronal NO synthase
  • iNOS inducible NO synthase
  • eNOS endothelial nitric oxide synthase
  • nNOS and eNOS are mainly expressed constitutively, and their activity is regulated by Ca 2+ -dependent calmodulin, producing a small amount of NO.
  • nNOS is involved in neurotransmission
  • eNOS is involved in the regulation of vascular tonus or immune mechanism.
  • iNOS is expressed in various types of cells such as epithelial cells, smooth muscle cells, macrophages, etc., and its expression is induced at the transcriptional level by cytokines induced by infection, inflammation, etc., and produces a large amount of NO. .
  • iNOS plays a central role as a mediator in infection and defense, while a large amount of NO produced by the constant expression of iNOS is involved in various inflammatory diseases ( J. Med. Chem., 1994; 37: 3886-8, Biochem. Pharmacol., 2008; 75: 923-30).
  • Non-Patent Document 1 Physiological research on cancer has been conducted from both aspects of production suppression, and a certain opinion has not been obtained as to whether NO production promotion or NO production inhibition is good for application to cancer treatment.
  • Non-patent documents 4 and 5 describe that in human ovarian cancer cells and cervical cancer cells, low concentrations of NO tend to suppress apoptosis by cisplatin and paclitaxel, and high concentrations of NO promote apoptosis. ing. These documents also describe that the combination of iNOS inhibitor 1400W and survivin RNAi promotes apoptosis by not only paclitaxel but also cisplatin, and it can be said that the cytoprotective effect of NO is not a paclitaxel selective action.
  • Non-Patent Document 6 reports that NO alone inhibits microtubule polymerization, but does not describe the physiological significance in cancer cells or the relationship with anticancer agents.
  • Non-Patent Document 7 in human lung cancer cells, NO suppresses the degradation of Bcl-2 by the proteasome by inhibiting S-nitrosylation of Bcl-2, an oncogene that suppresses apoptosis, and induces apoptosis by cisplatin. It has been reported to suppress the action. It has also been reported that NO is inactivated by direct S-nitrosylation of caspase-3, which is a major enzyme constituting the signal transduction system for inducing apoptosis (Non-patent Document 8). HIF-1 ⁇ is normally degraded by the proteasome under normoxic conditions and does not function as a transcription factor.
  • Patent Document 1 NO is subjected to S-nitrosylation of HIF-1 ⁇ , so that it does not function under normoxic conditions. It has been reported that it inhibits the degradation of HIF-1 ⁇ by the proteasome and is involved in neovascularization of chemotherapeutic agents and tumors after radiation injury.
  • Patent Document 2 is effective in combination with chemotherapeutic agents and radiotherapy by regulating immature tumor blood vessels by regulating NO production (increase of eNOS in vascular endothelium and inhibition of iNOS in tumors). Insist, but the combined effect with chemotherapeutic agents has not been studied.
  • Patent Document 3 describes a method for increasing the activity of a compound that is inactivated by NO in combination with a NO inhibitor or a NO scavenger. Specific examples of taxane antitumor agents and NO inhibitors are described in Patent Document 3. Combinations are not listed.
  • Patent Document 4 published after the filing of Japanese Patent Application No. 2012-171017 (filing date: August 1, 2012), which is the basis for claiming priority of the present application, includes one or more selected from taxane antitumor agents A composition for cancer treatment containing FK330 or a salt thereof as an active ingredient for use in combination with an antitumor agent is described.
  • Taxane antitumor agents are useful for the treatment of various tumors, but their dosage and usage are limited in terms of side effects, and treatment methods that increase the efficacy of various tumors without increasing the dose are now available. It is still sought after. In addition, although effective at the beginning of treatment, there is a problem of acquired resistance in which the antitumor effect decreases as treatment is continued.
  • the present inventor unexpectedly achieved a remarkable antitumor effect enhancement by using a NO production inhibitor in combination, and The present invention was completed by finding out the mechanism of the enhancing action. That is, the present invention relates to the following.
  • a pharmaceutical composition for cancer treatment containing a NO production inhibitor (except for FK330 and its salts) as an active ingredient, which is administered in combination with a taxane antitumor agent.
  • a NO production inhibitor except for FK330 and its salts
  • the NO production inhibitor is an iNOS inhibitor.
  • the iNOS inhibitor is KD-7040 or GW-274150.
  • Use of the NO production inhibitor according to [9] wherein the NO production inhibitor is an iNOS inhibitor.
  • the iNOS inhibitor is KD-7040 or GW-274150.
  • [12] Use of the NO production inhibitor according to [9] to [11], wherein the taxane antitumor agent is paclitaxel or docetaxel. [13] The NO according to [9] to [12] for producing a pharmaceutical composition, which is administered simultaneously with the taxane antitumor agent, separately, sequentially or at intervals. Use of production inhibitors. [14] Use of the NO production inhibitor according to [9] to [12] for producing a pharmaceutical composition for treating cancer using a taxane antitumor agent. [15] Use of the NO production inhibitor according to [9] to [12] for producing a pharmaceutical composition for treating breast cancer, ovarian cancer, prostate cancer, gastric cancer, or non-small cell lung cancer.
  • a NO production inhibitor (except for FK330 and its salts) for cancer treatment, which is administered in combination with a taxane antitumor agent.
  • It is further characterized by being administered in combination with one or more antitumor agents selected from platinum antitumor agents, anthracycline antitumor agents, cyclophosphamide, fluorouracil, trastuzumab and capecitabine
  • antitumor agents selected from platinum antitumor agents, anthracycline antitumor agents, cyclophosphamide, fluorouracil, trastuzumab and capecitabine
  • the NO production inhibitor is an iNOS inhibitor.
  • the iNOS inhibitor is KD-7040 or GW-274150.
  • the taxane antitumor agent is paclitaxel or docetaxel.
  • the taxane antitumor agent is paclitaxel or docetaxel.
  • [29] The method according to [26], wherein the taxane antitumor agent is paclitaxel or docetaxel. [30] The method according to [24] to [29], wherein the taxane antitumor agent and the NO production inhibitor are administered simultaneously, separately, sequentially or at intervals. [31] The method of [24] to [29], wherein a cancer using a taxane antitumor agent is treated. [32] The method according to [24] to [29], wherein breast cancer, ovarian cancer, prostate cancer, gastric cancer, or non-small cell lung cancer is treated.
  • the “subject” is a human or other animal that needs the prevention or treatment, and as a certain aspect, it is a human that needs the prevention or treatment.
  • the present invention also relates to the following.
  • An antitumor effect enhancer of a taxane antitumor agent comprising a NO production inhibitor as an active ingredient.
  • a pharmaceutical composition for cancer treatment for treating a subject undergoing treatment with a taxane antitumor agent comprising an NO production inhibitor as an active ingredient.
  • a taxane-based antitumor agent resistance-suppressing agent or resistance-preventing agent comprising a NO production inhibitor as an active ingredient.
  • a taxane antitumor agent sensitivity enhancer comprising a NO production inhibitor as an active ingredient.
  • the pharmaceutical composition for cancer treatment described in the present application can be used in combination with a taxane antitumor agent for the treatment of various cancers for which existing taxane antitumor agents are known to be applied.
  • Applicable cancers include solid cancers and lymphomas in which taxane antitumor agents are used.
  • FIG. 1 is a graph showing the antitumor effect in Example 1 when FK330 and doxorubicin are administered alone or in combination. Dox indicates doxorubicin.
  • FIG. 2 is a graph showing the antitumor effect of FK330 and gemcitabine administered alone or in combination in Example 1. Gem indicates gemcitabine.
  • FIG. 3 is a graph showing the antitumor effect in Example 1 when FK330 and irinotecan are administered alone or in combination. Iri indicates irinotecan.
  • FIG. 4 is a graph showing the antitumor effect in Example 1 when FK330 and carboplatin are administered alone or in combination. CBDCA indicates carboplatin.
  • FIG. 5 is a graph showing the antitumor effect in Example 1 when FK330 and paclitaxel are administered alone or in combination.
  • PTX indicates paclitaxel.
  • FIG. 6 is a graph showing the antitumor effect in Example 1 when FK330 and docetaxel are administered alone or in combination.
  • Doce indicates docetaxel.
  • FIG. 7 is a graph showing the results of Example 2 in which the antitumor effect enhancing effect of FK330 upon multiple cycles of paclitaxel in mice bearing human non-small cell lung cancer (Calu-6) was examined.
  • PTX indicates paclitaxel.
  • FIG. 8 is a graph showing the results of Example 3 in which the dose dependence of the antitumor effect enhancing effect of paclitaxel of FK330 in mice bearing human non-small cell lung cancer (Calu-6) was examined.
  • PTX indicates paclitaxel.
  • FIG. 9 is a graph showing the results of Example 4 in which the administration timing of FK330 in mice bearing human non-small cell lung cancer (Calu-6) was examined.
  • PTX indicates paclitaxel.
  • FIG. 10 is a graph showing the results of Example 5 in which the antitumor effect enhancement effect of paclitaxel by FK330 in human gastric cancer (AZ-521) tumor-bearing mice was examined.
  • PTX indicates paclitaxel.
  • FIG. 11 is a graph showing the results of Example 5 in which the antitumor effect enhancement effect of paclitaxel by FK330 in human gastric cancer (MKN-28) tumor-bearing mice was examined.
  • PTX indicates paclitaxel.
  • FIG. 12 is a graph showing the results of Example 5 in which the antitumor effect enhancement effect of paclitaxel by FK330 in human gastric cancer (NCI-N87) tumor-bearing mice was examined.
  • PTX indicates paclitaxel.
  • FIG. 13 is a graph showing the results of Example 6 in which the antitumor effect potentiating action of paclitaxel by FK330 in human breast cancer cell (MDA-MB-231) -bearing mice was examined.
  • FIG. 14 is a graph showing the results of Example 7 in which the inhibitory action of S-nitroso-N-acetyl-D, L-penicillamine® (SNAP), which is a NO donor, on the taxane-induced tubulin polymerization action was examined.
  • FIGS. 14A and 14B show a representative example of the inhibitory action of SNAP on paclitaxel-induced tubulin polymerization and the aggregated value for 60 minutes after the start of tubulin polymerization, respectively.
  • PTX indicates paclitaxel.
  • FIG. 15 is a graph showing the results of Example 7 in which the inhibitory action of S-nitroso-N-acetyl-D, L-penicillamine® (SNAP), which is a NO donor, on the taxane-induced tubulin polymerization action was examined.
  • FIGS. 15A and 15B show a representative example of the inhibitory action of SNAP on docetaxel-induced tubulin polymerization and the aggregated value for 60 minutes after the start of tubulin polymerization, respectively.
  • DTX indicates docetaxel.
  • FIG. 16 is a graph showing the results of Example 8 in which the effects of paclitaxel alone and FK330 in combination on tumor tissue iNOS protein expression in human lung cancer cell line Calu-6 tumor-bearing mice were examined.
  • FIG. 17 is a graph showing the results of Example 8 in which the effect on macrophage (F4 / 80) infiltration in tumor tissues when paclitaxel alone and FK330 were combined in human lung cancer cell line Calu-6 tumor-bearing mice was shown.
  • PTX indicates paclitaxel.
  • FIG. 18 shows the results of Example 8 in which the effect on expression of nitrotyrosine (the main final product of NO) in tumor tissue when paclitaxel alone and FK330 were used together in human lung cancer cell line Calu-6 tumor-bearing mice is shown. It is a graph.
  • PTX indicates paclitaxel.
  • FIG. 17 is a graph showing the results of Example 8 in which the effect on macrophage (F4 / 80) infiltration in tumor tissues when paclitaxel alone and FK330 were combined in human lung cancer cell line Calu-6 tumor-bearing mice was shown.
  • PTX indicates paclitaxel.
  • FIG. 18 shows the results of Example 8 in which the effect
  • FIG. 19 is a graph showing the results of Example 9 in which the antitumor effect enhancing effect of paclitaxel by FK330 in mice bearing human hormone resistant prostate cancer cells (PC-3) was examined.
  • PTX indicates paclitaxel.
  • FIG. 20 is a graph showing the results of Example 10 in which the antitumor effect-enhancing effect of docetaxel by FK330 in mice bearing human hormone-resistant prostate cancer cells (PC-3) was examined.
  • DTX indicates docetaxel.
  • FIG. 21 is a graph showing the results of Example 11 in which the antitumor effect enhancing effect of docetaxel by FK330 in mice bearing human breast cancer cells (MDA-MB-468) was examined. DTX indicates docetaxel.
  • FIG. 22 is a graph showing the results of Example 12 in which the antitumor effect enhancing effect of paclitaxel by FK330 in mice bearing human ovarian cancer cells (MCAS) was examined.
  • PTX indicates paclitaxel.
  • FIG. 23 shows S-nitroso-N-acetyl-D, L-penicillamine (SNAP), which is a NO donor for the reduction in cell viability of cancer cells induced by various chemotherapeutic agents in human lung cancer cell line Calu-6. It is a graph which shows the result of Example 13 which examined the inhibitory action of No ..
  • FIG. 24 shows S-nitroso-N-acetyl-D, L-penicillamine (SNAP), which is a NO donor for reduction in cell viability of cancer cells induced by various chemotherapeutic agents in human gastric cancer cell line MKN-28. It is a graph which shows the result of Example 13 which examined the inhibitory action of No ..
  • FIG. 25 shows S-nitroso-N-acetyl-D, L, a NO donor for caspase-3 / 7 activation induced by taxane in human lung cancer cell line Calu-6 and human gastric cancer cell line MKN-28. It is a graph which shows the result of Example 14 which examined the inhibitory effect of -penicillamine (SNAP).
  • SNAP L-penicillamine
  • FIG. 26 is a graph showing the results of Example 15 in which the antitumor effect enhancement effect of paclitaxel by the iNOS inhibitor KD-7040 in mice bearing human non-small cell lung cancer (Calu-6) was examined.
  • PTX indicates paclitaxel.
  • Example 27 is a graph showing the results of Example 15 in which the antitumor effect enhancing effect of paclitaxel by the iNOS inhibitor GW-274150 in mice bearing human non-small cell lung cancer (Calu-6) was examined.
  • PTX indicates paclitaxel.
  • cancer in “pharmaceutical composition for cancer treatment” includes all solid cancers and lymphomas.
  • Some embodiments include breast cancer, endometrial cancer, ovarian cancer, prostate cancer, lung cancer, stomach (gastric gland) cancer, non-small cell lung cancer, pancreatic cancer, squamous cell carcinoma of the head and neck, esophageal cancer, bladder cancer, melanoma, colon cancer, Examples include renal cell carcinoma, non-Hodgkin lymphoma, urothelial cancer, and the like.
  • breast cancer, ovarian cancer, prostate cancer, stomach (gastric gland) cancer, non-small cell lung cancer, etc. and in another aspect, breast cancer, stomach (gastric gland) cancer, non-small cell lung cancer, etc.
  • Embodiments include prostate cancer, and yet another embodiment includes non-small cell lung cancer.
  • breast cancer for example, HER2-positive metastatic breast cancer, HER2-negative metastatic breast cancer, HER2-positive breast cancer, or metastatic breast cancer can be mentioned, and as another aspect, HER2-negative metastatic breast cancer can be mentioned, Another embodiment includes HER2-positive breast cancer.
  • non-small cell lung cancer for example, locally advanced non-small cell lung cancer or metastatic non-small cell lung cancer can be mentioned, and as another aspect, non-squamous cell carcinoma in non-small cell lung cancer can be mentioned.
  • Another aspect includes non-squamous cell carcinoma in metastatic non-small cell lung cancer, and yet another aspect includes squamous cell carcinoma in metastatic non-small cell lung cancer.
  • gastric cancer metastatic gastric cancer, metastatic gastroesophageal junction cancer and the like can be mentioned.
  • the prostate cancer include metastatic castration-resistant prostate cancer, hormone-resistant prostate cancer, and the like. Another embodiment includes hormone-resistant prostate cancer, and another embodiment includes metastatic. Castration resistant prostate cancer.
  • the “NO production inhibitor” means a substance that inhibits NO production, and may be in any form such as a low molecular weight compound, peptide, natural product, RNAi, antisense RNA, and the like.
  • One embodiment is a “compound having NO production inhibitory action”.
  • Another embodiment of the “NO production inhibitor” is an NO production inhibitor excluding FK330.
  • Other embodiments of the “NO production inhibitor” or “compound having NO production inhibitory action” include NOS transcription inhibitors, NO scavengers, NOS inhibitors and the like.
  • Examples of the “NO production inhibitor excluding FK330” include NOS transcription inhibitors, NO scavengers, NOS inhibitors other than FK330, and the like.
  • NOS transcription inhibitor examples include, for example, CR-3294, PMI-001, and PMI-005.
  • Another embodiment of the NOS transcription inhibitor includes a NOS transcription factor inhibitor, and specific examples include an NFkB inhibitor, an IL-1 inhibitor, a TNF inhibitor, and an IFN- ⁇ inhibitor.
  • Another embodiment of the NOS transcription inhibitor includes various drugs that suppress inflammation and indirectly suppress NOS protein production, and include, for example, steroids and non-steroidal anti-inflammatory agents.
  • ⁇ NO scavenger '' include non-heme iron-containing peptides, non-heme iron-containing proteins, porphyrins, metalloporphyrins, dithiocarbamates, dimercaptosuccinic acid, phenanthroline, desanthroline, Ferrioxamine (desferrioxamine), pyridoxal isonicotinoyl hydrazone (PIH, NSC-77674), 1,2-dimethyl-3-hydroxypyridin-4-one (L1), [+] 1,2-bis (3,5 -Dioxopiperazin-1-yl) propane (ICRF-187), 2- (4-carboxyphenyl 9-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy- Examples include 3-oxide (carboxy-PTIO).
  • NOS inhibitor means a substance that inhibits the NO production function of NOS, and in one embodiment, is a “compound having NOS inhibitory action”. Another embodiment of the “NOS inhibitor” includes a substance that selectively inhibits NOS. Specifically, the inhibitory action on NOS is, for example, 10 times the inhibitory action on a target other than NOS. As described above, another embodiment includes a substance having 100 times or more activity.
  • NOS inhibitors include NG-monomethyl L-arginine (NMMA), NG-nitro-L-arginine methyl ester (NAME), NG-nitro-L-arginine (NNA), NG-amino-L.
  • NAA -Arginine
  • NG NG
  • NG-dimethylarginine asymmetric dimethylarginine
  • ADMA L-thiocitrulline
  • S-methyl-L-thiocitrulline diphenyleneiononium chloride (diphenyleneiononium chloride)
  • Examples include canavanine, ebselen, S-methyl-L-citrulline, S-methylisourea, 2-mercaptoethylguanidine and the like.
  • the “NOS inhibitor” includes iNOS inhibitors, nNOS inhibitors, eNOS inhibitors, and other embodiments include iNOS inhibitors.
  • the “iNOS inhibitor” means a substance that inhibits the NO production function of iNOS. In one embodiment, it is a “compound having an iNOS inhibitory action”.
  • the “eNOS inhibitor” means a substance that inhibits the NO production function of eNOS. In one embodiment, it is a “compound having an eNOS inhibitory action”.
  • the “nNOS inhibitor” means a substance that inhibits the NO production function of nNOS, and in one embodiment, is a “compound having an nNOS inhibitory action”.
  • NOS inhibitor excluding FK330 iNOS inhibitor excluding FK330, nNOS inhibitor, eNOS inhibitor and the like can be mentioned, and another variety includes iNOS inhibitors excluding FK330.
  • nNOS inhibitor examples include L-NPA, 7-nitroindazole, ARL17477, vinyl-L-NIO, TRIM and the like.
  • FK330 (FR260330): The chemical name is N 2 -[(2E) -3- (4-chlorophenyl) -2-propenoyl] -N- [2-oxo-2- (4- ⁇ [6- (trifluoromethyl ) Pyrimidin-4-yl] oxy ⁇ piperidin-1-yl) ethyl] -3-pyridin-2-yl-L-alaninamide, the method described in WO 02/055541 or for those skilled in the art It is readily available by obvious methods or variations thereof. It has been reported to show excellent iNOS inhibitory activity (European Journal of Pharmacology, 2005, 509: 71).
  • KD-7040 Chemical name is N'-benzo [1,3] dioxy-5-ylmethyl-N- (3-imidazol-1-yl- [1,2,4] thiadiazol-5-yl) -N- Methyl-propane-1,3-diamine, which can be easily obtained by the method described in WO 2006/060424, the method obvious to those skilled in the art, or a modification thereof.
  • GW-274150 The chemical name is (s)-[2- (1- (iminoethylamino) ethyl) -L-homocysteine, and the method described in International Publication No. 98/30537 pamphlet or those skilled in the art It is readily available by obvious methods or variations thereof.
  • SD-6010 (Cindunistat): The chemical name is (s)-[2- (1- (iminoethylamino) ethyl) -2-methyl-L-cysteine, and the method described in International Publication No. 2001/072702 pamphlet Or readily available by methods obvious to those skilled in the art or variations thereof. In addition to the above, the following may also be mentioned.
  • Gingivex L-NIL-TA (see WO 1996/0151202 pamphlet), ONO-1714 (see European patent application 0870763), CR3558, 2-Iminobiotin (international publication) 2011/49349 pamphlet), aminoguanidine disulfide carbonate (Guanidinoethyldisulfide bicarbonate, GED bicarbonate) (see US Pat. No. 5929063), KLYP-961 (see WO 2009/029592 pamphlet), GW273629 (See pamphlet of International Publication No. 1998/030537), 1400W, and aminoguanidine (AG).
  • FK330, KD-7040, GW-274150, SD-6010 and Gingivex can be mentioned, and as another aspect, FK330, KD-7040, GW-274150 and SD-6010 can be mentioned, Yet another embodiment includes FK330, KD-7040, and SD-6010. Another embodiment includes KD-7040, GW-274150 and SD-6010, and another embodiment includes KD-7040 and GW-274150.
  • the “compound having iNOS inhibitory action”, “compound having eNOS inhibitory action”, or “compound having nNOS inhibitory action” is, for example, a method known to those skilled in the art or the British Journal of Pharmacology, 2005, 145, 301. It can be obtained by screening the compound for iNOS inhibitory activity, eNOS inhibitory activity or nNOS inhibitory activity according to the experimental method described in -312 or an evaluation method obvious to those skilled in the art according to them. At this time, HTS (High-throughput screening) well known to those skilled in the art is conventional and efficient as a screening technique.
  • HTS High-throughput screening
  • a compound to be evaluated a newly synthesized compound, a commercially available compound, a compound synthesized by combinatorial chemistry, a compound registered in a compound library composed of these, or a known compound can be used.
  • various natural compounds and their derivatives isolated from microorganisms or animals and plants can be used.
  • further iNOS inhibitors can be obtained by developing peripheral derivatives of compounds having iNOS inhibitory activity obtained by HTS evaluation.
  • the 50% inhibitory concentration (IC50) of “a compound having NOS inhibitory activity”, “a compound having iNOS inhibitory activity”, “a compound having nNOS inhibitory activity”, or “a compound having eNOS inhibitory activity” is about It may be less than 10 ⁇ M, in another embodiment less than about 1 ⁇ M, in another embodiment less than 100 nM, and in yet another embodiment less than 10 nM.
  • the antitumor effect enhancing effect of the taxane antitumor agent which is a NO production inhibitor, can also be confirmed by a cancer-bearing animal model test, as shown in Examples described later. Furthermore, it can be confirmed by other appropriate in vitro or in vivo experiments, or by clinical trials for cancer patients.
  • the “compound having NO production inhibitory action” may exist as a tautomer or a geometric isomer depending on the type of substituent.
  • the “compound having NO production inhibitory activity” may be described in only one form of an isomer, but the present invention includes other isomers, and the isomers are separated. Or mixtures thereof.
  • the “compound having NO production inhibitory action” may have an asymmetric carbon atom or axial asymmetry, and optical isomers based on this may exist.
  • the present invention also includes isolated optical isomers of “compound having NO production inhibitory action” or a mixture thereof.
  • the present invention includes pharmaceutically acceptable prodrugs of “compounds having NO production inhibitory activity”.
  • a pharmaceutically acceptable prodrug is a compound having a group that can be converted to an amino group, a hydroxyl group, a carboxyl group, or the like by solvolysis or under physiological conditions. Examples of groups that form prodrugs include those described in Prog. Med., 5, 2157-2161 (1985) and “Development of pharmaceuticals” (Yodogawa Shoten, 1990), Volume 7, Molecular Design 163-198. Is mentioned.
  • the “compound having NO production inhibitory action” may form an acid addition salt or a salt with a base, and is included in the present invention as long as such a salt is a pharmaceutically acceptable salt. Specifically, it is a pharmaceutically acceptable salt of a “compound having NO production inhibitory action” and may form an acid addition salt or a salt with a base depending on the type of substituent.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid Acid addition with organic acids such as lactic acid, malic acid, mandelic acid, tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, aspartic acid, glutamic acid Salts, salts with inorganic bases such as sodium, potassium, magnesium, calcium and aluminum, salts with organic bases such as methylamine, ethylamine, ethanolamine, lysine and ornithine, salts with various amino acids and amino acid derivatives such as acetyll
  • the present invention also includes “compounds having NO production inhibitory activity” and various hydrates and solvates of the salts thereof, and polymorphic substances.
  • the present invention also includes compounds labeled with various radioactive or non-radioactive isotopes.
  • a pharmaceutical composition containing one or more of “NO production inhibitors” as an active ingredient uses excipients usually used in the art, that is, pharmaceutical excipients, pharmaceutical carriers, etc. And can be prepared by commonly used methods. Administration is orally by tablets, pills, capsules, granules, powders, solutions, etc., or injections such as intra-articular, intravenous, intramuscular, suppositories, eye drops, ophthalmic ointments, transdermal solutions, Any form of parenteral administration such as an ointment, a transdermal patch, a transmucosal liquid, a transmucosal patch, and an inhalant may be used.
  • a solid composition for oral administration tablets, powders, granules and the like are used.
  • one or more active ingredients are mixed with at least one inert excipient.
  • the composition may contain an inert additive such as a lubricant, a disintegrant, a stabilizer and a solubilizing agent according to a conventional method. If necessary, tablets or pills may be coated with a sugar coating or a film of a gastric or enteric substance.
  • Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs and the like, and commonly used inert diluents such as purified water. Or it contains ethanol.
  • the liquid composition may contain solubilizers, wetting agents, auxiliaries such as suspending agents, sweeteners, flavors, fragrances and preservatives in addition to the inert diluent.
  • the injection for parenteral administration contains a sterile aqueous or non-aqueous solution, suspension or emulsion.
  • aqueous solvent include distilled water for injection or physiological saline.
  • Non-aqueous solvents include alcohols such as ethanol.
  • Such compositions may further contain isotonic agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers, or solubilizing agents. These are sterilized by, for example, filtration through a bacteria-retaining filter, blending with a bactericide or irradiation. These can also be used by producing a sterile solid composition and dissolving or suspending it in sterile water or a sterile solvent for injection before use.
  • the daily dose of the NO production inhibitor is about 0.001 to 100 mg / kg, preferably 0.1 to 30 mg / kg, more preferably 0.1 to 10 mg / kg per body weight. This is administered once or divided into 2 to 4 times.
  • the appropriate daily dose is about 0.0001 to 10 mg / kg per body weight, and is administered once to several times a day.
  • a transmucosal agent about 0.001 to 100 mg / kg per body weight is administered once to several times a day. The dose is appropriately determined according to individual cases in consideration of symptoms, age, sex, and the like.
  • the pharmaceutical composition of the present invention is 0.01 to 100% by weight, and in one embodiment, 0.01 to 50% by weight of the active ingredient. Or more "NO production inhibitor" is contained.
  • taxane antitumor agent examples include a compound having a taxane ring or a similar structure thereof, and another embodiment includes a compound having a taxane ring (taxane). .
  • taxane includes a compound having a microtubule depolymerization inhibitory action.
  • Another embodiment of the taxane antitumor agent is selected from the group consisting of paclitaxel, docetaxel, cabazitaxel, abraxane, tesetaxel, and ortataxel. Taxanes or combinations thereof.
  • paclitaxel paclitaxel
  • docetaxel docetaxel
  • abraxane abraxane
  • paclitaxel paclitaxel
  • docetaxel docetaxel (docetaxel) is mentioned.
  • the main existing taxane antitumor agents that can be used in combination with the “pharmaceutical composition for cancer treatment” are shown in the table below together with main indication cancer types or candidate cancer types.
  • the applicable cancer types of these antitumor agents are not limited thereto.
  • Paclitaxel, docetaxel, cabazitaxel, or Abraxane can be purchased and used commercially, or can be easily obtained by methods obvious to those skilled in the art or modifications thereof.
  • Cabazitaxel can also be easily obtained by the production method disclosed in WO 96/30355 or a modification thereof.
  • Tesetaxel can be easily obtained by a method obvious to those skilled in the art, a production method disclosed in WO 01/27115, or a modification thereof.
  • Ortataxel can be easily obtained by methods obvious to those skilled in the art, or by the production method disclosed in US Pat. No. 5,705,508 or a modification thereof.
  • the package insert of Taxol Injection (registered trademark: trade name of paclitaxel) on the website for providing information on pharmaceuticals and drugs in Japan includes “[Effectiveness or effects] Lung cancer, breast cancer, gastric cancer; [Dosage and administration]: 1. In general, for adults, instillate 210 mg / m 2 as paclitaxel once a day for 3 hours, and take a rest for at least 3 weeks. The administration is repeated as a cool. The dosage is appropriately reduced depending on the age and symptoms.
  • the package insert of Taxotere Note (registered trademark: trade name of docetaxel) on the website for providing information on pharmaceuticals and drugs in Japan includes “1. Breast cancer, non-small cell lung cancer, Head and neck cancer; indication or dosage for each effect; 1. In general, once a day for adults, 60 mg / m 2 as docetaxel is infused intravenously at intervals of 3 to 4 weeks over 1 hour. It is increased or decreased as appropriate symptoms, however, the highest dose once is described as "the 75 mg / m 2.
  • the therapeutically effective dose when used in the combination treatment of the taxane antitumor agent described in the present specification is the same or lower than that usually administered alone depending on the administration route usually administered (for example, , 0.10 to 0.99 times the maximum dose when administered alone).
  • the administration cycle can be appropriately adjusted so as to be suitable for combined use with the pharmaceutical composition for cancer treatment described in the present specification.
  • Specific administration frequency, dosage, administration cycle, and the like are appropriately determined according to individual cases in consideration of patient symptoms, age, sex, and the like.
  • the administration form in the case of administering a NO production inhibitor and a taxane antitumor agent in combination is not particularly limited as long as a suitable administration route, administration frequency and dosage are adopted, for example, (1) NO production A composition containing an inhibitor and a taxane antitumor agent, that is, administration as a single preparation, (2) two types obtained by separately formulating an NO production inhibitor and a taxane antitumor agent Simultaneous administration by the same administration route of the preparation, (3) Administration of the two preparations obtained by separately formulating the NO production inhibitor and the taxane antitumor agent with a time difference in the same administration route (for example, Administration in the order of NO production inhibitor and taxane antitumor agent, or administration in the reverse order), (4) two types obtained by separately formulating NO production inhibitor and taxane antitumor agent Simultaneous administration by different administration routes of the preparation, (5) NO production inhibitor and Administration of two types of preparations obtained by separately formulating xanthine antitumor agents at different time intervals
  • the NO production inhibitor is formulated separately from the taxane antitumor agent, the taxane antitumor agent is administered intravenously, intravenously or intravenously, and the NO production inhibitor is administered orally or parenterally. Is done. In another embodiment, the taxane antitumor agent is administered intravenously or intravenously, and the NO production inhibitor is administered orally.
  • the preferred administration form of the “pharmaceutical composition for cancer treatment” is a method in which the pharmaceutical composition for cancer treatment is orally or parenterally administered on at least one day in one cycle of administration of the taxane antitumor agent.
  • cycle means a treatment cycle or treatment course with a certain regimen. For example, in paclitaxel and docetaxel, 3 to 4 weeks from the start of administration until the next administration after the drug withdrawal period. About the unit dosage period.
  • a pharmaceutical composition for cancer treatment containing an NO production inhibitor is orally administered on at least one day on the day of administration of the taxane antitumor agent, orally on the entire day of administration of the taxane antitumor agent.
  • a method of administering, a method of oral administration on at least one day of a taxane antitumor drug holiday, a method of oral administration on all days of a taxane antitumor drug holiday, at least a day of administration of a taxane antitumor agent A method of oral administration on at least one day on the first day and a drug holiday, a method of oral administration on all days in one cycle of taxane antitumor agent administration, or intermittent during one cycle of taxane antitumor agent administration ( Orally once every two days, once a week, etc.).
  • it is the method of orally administering on the whole day in 1 cycle of taxane type
  • the pharmaceutical composition for cancer treatment may be administered from several days before to the day before the administration of the taxane antitumor agent, and this embodiment is included in the administration of the drug holiday of the taxane antitumor agent.
  • the administration time can be divided to avoid the interaction and / or the administration can be performed at a necessary interval.
  • the present invention may be in the form of a “pharmaceutical composition for cancer treatment for treating a subject undergoing treatment with a taxane antitumor agent, containing an NO production inhibitor as an active ingredient”.
  • a taxane antitumor agent containing an NO production inhibitor as an active ingredient.
  • Other antitumor agents include those described below.
  • the “subject” may be administered with a taxane antitumor agent at the same time, separately from the pharmaceutical composition for cancer treatment containing an NO production inhibitor as an active ingredient, continuously, or at intervals.
  • the present invention may be in the form of “a kit including a combination of (a) an NO production inhibitor and (b) a taxane antitumor agent”.
  • This includes a preparation containing the active ingredient (a) (first preparation) and a preparation containing the active ingredient (b) (second preparation) in combination so that they can be used in combination therapy of these active ingredients.
  • the kit may be a packaged product including an additional formulation or a display member that facilitates administration in accordance with each administration time, such as a placebo agent.
  • the two types of preparations can be administered with the same or different administration routes under the administration conditions such as formulation formulation, administration route, administration frequency, etc. suitable for each formulation, taking into consideration the bioavailability, stability, etc. of each formulation.
  • first formulation and the second formulation are administered together by the same administration route
  • first formulation and the second formulation are the same or different administration routes. It means that they are administered separately at the same or different dosing frequency or dosing interval.
  • subsequentially means that the first formulation and the second formulation are administered by the same or different routes of administration, in the order of the first formulation, followed by the second formulation, or in the reverse order.
  • At an interval means that the first preparation and the second preparation are administered by the same or different routes of administration, followed by the first preparation at a certain interval in the order of the second preparation or in the reverse order. Means that.
  • the present invention includes a pharmaceutical composition containing (a) a NO production inhibitor as an active ingredient and (c) a package insert indicated to be used in combination with a taxane antitumor agent. It may be in the form of “medicine”. This includes a composition (a) containing a therapeutically effective dose of an active ingredient for use in combination therapy and a package insert relating to the composition of (a), which is indicated to be used in combination with a taxane antitumor agent. (c) means a medicine for cancer treatment packaged including both.
  • antitumor agents used for chemotherapy can be used in combination as desired.
  • antitumor agents that are conventionally known to be used in combination with taxane antitumor agents, such as platinum antitumor agents, anthra
  • antitumor agents selected from cyclin antitumor agents, cyclophosphamide, fluorouracil, trastuzumab and capecitabine are included, and in one embodiment, cisplatin or carboplatin, and in another embodiment, doxorubicin .
  • the present inventors have clarified the following as a mechanism for enhancing the antitumor action of a taxane antitumor agent by an NO production inhibitor. That is, the taxane antitumor agent enhances the expression of iNOS protein in the tumor tissue, and as a result, enhances NO production.
  • the produced NO directly suppresses the microtubule depolymerization inhibitory action of the taxane, thereby causing attenuation of the antitumor effect via apoptosis of the taxane antitumor agent.
  • the NO production inhibitor was able to suppress the attenuation of the effects of the taxane anti-tumor agent by inhibiting NO production enhanced by the taxane anti-tumor agent, thereby enhancing the anti-tumor action. is there. From the above, it can be said that the NO production inhibitor can enhance the action of all taxane antitumor agents that target the microtubule depolymerization inhibitory action, and the present invention provides an NO production inhibitor containing an iNOS inhibitor. However, the in vivo data revealed for the first time that the effects of taxane antitumor agents can be enhanced.
  • FK330 free body dihydrate which is an iNOS inhibitor used in all the following examples is FK330 free body obtained by the method described in Example 41 of WO 02/055541 pamphlet, By recrystallizing in an alcohol / water solvent system such as ethanol / water, isopropanol / water, etc., and performing a humidity control operation on the free body anhydride obtained by drying the obtained crystals under reduced pressure, Obtainable.
  • the dose of FK330 is indicated by the weight of the dihydrate.
  • Docetaxel hydrate injection (Taxotere TM intravenous drip infusion) was purchased from Sanofi-Aventis Co., Ltd. and used as docetaxel.
  • Paclitaxel injection (paclitaxel injection “Sawai”) was purchased from Sawai Pharmaceutical Co., Ltd. and used as paclitaxel.
  • Irinotecan hydrochloride (Topotecin (trademark) Note) was purchased from Daiichi Sankyo Co., Ltd. and used as irinotecan.
  • Gemcitabine hydrochloride for injection (Gemzar (trademark) for injection) was purchased from Eli Lilly Japan and used as gemcitabine.
  • Doxorubicin hydrochloride for injection (Adriacin (trademark) injection 10) was purchased from Kyowa Hakko Kogyo Co., Ltd. and used as doxorubicin.
  • Carboplatin injection solution (Paraplatin (trademark) injection solution) was purchased from Bristol-Myers Co., Ltd. and used as carboplatin. 2) Preparation and administration of test substance FK330 was suspended in a 0.5% aqueous methylcellulose solution (0.5% MC) (20 mg / mL). Docetaxel was adjusted to 4 mg / L with the attached dissolution solution, and then diluted to 1 mg / mL with physiological saline (Otsuka Pharmaceutical).
  • Paclitaxel, irinotecan, gemcitabine, doxorubicin and carboplatin were prepared at the time of injection in physiological saline for injection to 1 mg / mL, 3 mg / mL, 16 mg / mL, 1 mg / mL and 6 mg / mL, respectively.
  • 3) Cells Human lung cancer-derived Calu 6 (HTB-56) was obtained from the American Type Culture Collection (VA, USA). The cells were cultured under the conditions of 37 ° C. and 5% CO 2 using RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells collected using trypsin were suspended in PBS at 6 ⁇ 10 7 cells / mL and mixed with an equal amount of Matrigel TM Basement Membrane Matrix (Becton Dickinson Co., Bedford, MA, USA).
  • FBS heat-inactivated fetal bovine serum
  • Control group untreated FK330 single administration group: FK330 100mg / kg Twice a day (bid) (200 mg / kg / day, administered daily from the start to the end of the experiment)
  • Docetaxel alone administration group docetaxel 10mg / kg / day (Day 1, 5 and 9)
  • Paclitaxel alone administration group Paclitaxel 10mg / kg / day (from day 1 to 7)
  • Irinotecan single administration group Irinotecan 30mg / kg / day (Day 1 to 7)
  • Gemcitabine alone administration group Gemcitabine 160 mg / kg / day (Days 1, 4 and 7)
  • Doxorubicin alone administration group doxorubicin 10mg / kg / day (Day 1 and 8)
  • Carboplatin alone administration group Carboplatin 60mg / kg / day (Day 1 and 2)
  • Combination group FK330 100 mg / kg bid + docetaxel 10 mg / kg / day (
  • Each chemotherapeutic agent was administered intravenously (10 mL / kg) via the tail vein 2 to 4 hours after administration of FK330. Tumor diameter was measured every 2-4 days using body weight and digital calipers. The tumor volume was calculated from an elliptic volume calculation formula (major axis x [minor axis] 2 x 0.52).
  • FK330 did not affect the antitumor effects of gemcitabine and carboplatin, and some enhancement of the antitumor effects was due to doxorubicin (tumor volume reduction rate of 33.5% compared to doxorubicin alone group on Day29) and irinotecan (irinotecan alone on Day29) The tumor volume reduction rate for the group (30.8%) was observed, but it was not significant. 8) Conclusion FK330 alone enhanced the antitumor effect of paclitaxel and docetaxel, although no antitumor effect was observed. On the other hand, the antitumor effects of irinotecan, gemcitabine, doxorubicin and carboplatin were not affected. These results suggest that FK330 selectively enhances the antitumor effect of taxane antitumor agents.
  • Example 2 Test substance The dose of FK330 was expressed as the weight including hydrate.
  • Paclitaxel injection (paclitaxel injection “Sawai”) was purchased from Sawai Pharmaceutical Co., Ltd. and used as paclitaxel.
  • Preparation and administration of test substance FK330 was suspended in a 0.5% aqueous methylcellulose solution (0.5% MC) (20 mg / mL).
  • Paclitaxel was prepared at the time of use at 1 mg / mL with physiological saline for injection (Otsuka Pharmaceutical).
  • Control group untreated FK330 single administration group: FK330 100mg / kg bid (200mg / kg / day, continuous administration from the start of the experiment to the end of the experiment)
  • Paclitaxel alone administration group Paclitaxel 10mg / kg / day (7 days of continuous administration in 1 cycle, 6 days of continuous administration after 2 cycles)
  • Combination group FK330 100 mg / kg bid (200 mg / kg / day, continuous administration from the start of the experiment to the end of the experiment) + paclitaxel 10 mg / kg / day (7 days of continuous administration in 1 cycle, 6 days of continuous administration after 2 cycles)
  • FK330 was administered by oral gavage (5 mL / kg) twice a day at intervals of 6 to 8 hours.
  • Paclitaxel was administered as an intravenous bolus (10 mL / kg) from the tail vein 2 to 4 hours after administration of FK330. Tumor diameter was measured every 2-4 days using body weight and digital calipers. The tumor volume was calculated from an elliptic volume calculation formula (major axis x [minor axis] 2 x 0.52).
  • Example 3 Preparation of the test substance and cell preparation were performed in the same manner as in Example 2.
  • Control group untreated Paclitaxel alone administration group: Paclitaxel 10mg / kg / day (Consecutive administration from Day 0 for 6 days in 1 cycle, continuous administration from Day 27 to 6 days in 2 cycles)
  • Paclitaxel was administered as an intravenous bolus (10 mL / kg) from the tail vein 2 to 4 hours after administration of FK330. Tumor diameter was measured every 2-4 days using body weight and digital calipers. The tumor volume was calculated from an elliptic volume calculation formula (major axis x [minor axis] 2 x 0.52).
  • FIG. 8 shows the results of examining the dose dependence of the antitumor effect enhancing effect of paclitaxel of FK330.
  • Tumor volume at the end of the study (Day59) paclitaxel alone group in 1640.1 ⁇ 902.8 mm 3, FK330 in 30 mg / kg bid combination group, 955.9 ⁇ 231.0 mm 3, FK330 60mg / kg bid The combination group 706.2 ⁇ 256.1 mm 3, In the FK330 100 mg / kg bid combination group, it was 297.9 ⁇ 101.4 mm 3 , and FK330 enhanced the antitumor effect of paclitaxel in a dose-dependent manner (tumor volume reduction rate compared to paclitaxel alone group: 41.7% at FK330 30 mg / kg bid, FK330 (56.9% at 60mg / kg bid, 81.8% at FK330 100mg /
  • Example 4 Preparation of the test substance and cell preparation were performed in the same manner as in Example 2.
  • Control group untreated Paclitaxel alone administration group: Paclitaxel 10mg / kg / day (Day 0 to Day 5 and Day 28 to Day 32)
  • Continuous administration FK330 100 mg / kg bid (200 mg / kg / day) + paclitaxel 10 mg / kg / day (Day 0 to Day 5 and Day 28 to Day 32)
  • FK330 was administered by oral gavage (5 mL / kg) twice a day at intervals of 6 to 8 hours.
  • FK330 is on the day of paclitaxel administration (Day 0 to Day 5 and Day 28 to Day 32). It was administered on all days until the day.
  • Paclitaxel was administered intravenously (10 mL / kg) from the tail vein 2 to 4 hours after administration of FK330. Tumor diameter was measured every 3 to 4 days using body weight and digital calipers. The tumor volume was calculated from an elliptic volume calculation formula (major axis x [minor axis] 2 x 0.52).
  • Example 5 The test substance was prepared in the same manner as in Example 2.
  • Cells Human gastric cancer-derived NCI-N87 (CRL-5822) is from the American Type Culture Collection (VA, USA)
  • MKN-28 (JCRB0253) is from the Pharmaceutical Research Laboratory (HSRRB)
  • AZ-521 (RCB2087) is from RIKEN The one obtained from BRC was used.
  • NCI-N87 and MKN-28 cells use RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), AZ-521 contains DMEM medium containing 10% heat-inactivated FBS was cultured under the conditions of 37 ° C. and 5% CO 2 .
  • FBS heat-inactivated fetal bovine serum
  • AZ-521 contains DMEM medium containing 10% heat-inactivated FBS was cultured under the conditions of 37 ° C. and 5% CO 2 .
  • Tumor volume (major axis x [minor axis] 2 x 0.52) from 386.6 to 766.6 mm 3 in AZ-521 tumor-bearing mice, tumor volume from 186.0 to 319.2 mm 3 in MKN-28 tumor-bearing mice, NCI-N87 tumor bearing
  • nude mice whose tumor volume was 192.1 to 364.2 were divided into groups using SAS so that the variation in tumor volume between groups and within groups was reduced.
  • Drug administration and measurement With Day 0 as the first day of administration, observation was performed until Day 46 for AZ-521-bearing mice, until Day 84 for MKN-28-bearing mice, and until Day 56 for NCI-N87-bearing mice.
  • FK330 was administered by oral gavage at 5 mL / kg, and paclitaxel was administered by tail vein at 10 mL / kg.
  • Paclitaxel was administered in multiple cycles under the following conditions.
  • Control group untreated FK330 single administration group: FK330 100mg / kg bid (200mg / kg / day, continuous administration from the start of the experiment to the end of the experiment)
  • Paclitaxel alone administration group Paclitaxel 10mg / kg / day AZ-521 tumor-bearing mice: Paclitaxel was administered continuously for 6 days from Day 0 to Day 5, followed by a 22-day drug holiday (1 cycle), and paclitaxel was administered continuously for 5 days from Day 28 to Day 32 (2 cycles).
  • MKN-28 tumor-bearing mice Paclitaxel was administered continuously for 6 days from Day 0 to Day 5, and after a 22-day withdrawal period, it was administered continuously for 5 days from Day 28 to Day 32, followed by a 23-day withdrawal period (2 cycles). Furthermore, continuous administration for 5 days from Day 56 to Day 60 was performed (3 cycles).
  • NCI-N87 tumor-bearing mice Paclitaxel was administered continuously for 6 days from Day 0 to Day 5, and then administered continuously for 5 days from Day 32 to Day 36 after a 26-day drug holiday (2 cycles).
  • Combination group FK330 100 mg / kg bid (200 mg / kg / day, continuous administration from the start of the experiment to the end of the experiment) + paclitaxel 10 mg / kg / day FK330 was administered by oral gavage (5 mL / kg) twice a day at intervals of 6 to 8 hours. Paclitaxel was administered intravenously (10 mL / kg) from the tail vein 2 to 4 hours after administration of FK330. Tumor diameter was measured every 2 to 4 days using body weight and digital calipers. The tumor volume was calculated from an elliptic volume calculation formula (major axis x [minor axis] 2 x 0.52).
  • FIGS. 10 to 12 show the results of examining the combined use effect of FK330 with paclitaxel in mice bearing human gastric cancer (AZ-521, MKN-28, NCI-N87).
  • 10 shows the combined effect of FK330 and paclitaxel in AZ-521 tumor-bearing mice
  • FIG. 11 shows the combined effect in MKN-28-bearing mice
  • FIG. 12 shows the combined effect in NCI-N87-bearing mice.
  • FK330 enhanced the antitumor effect of paclitaxel.
  • the tumor volume of the paclitaxel alone group on the last day of the experiment was 3285.4 ⁇ 359.4 mm 3
  • the paclitaxel and FK330 combination group was 795.0 ⁇ 223.3 mm 3
  • the tumor volume was 75.8 compared to the paclitaxel alone group % (P ⁇ 0.01) decrease.
  • Example 6 Test substance The dose of FK330 was converted to the weight of a single substance (non-hydrate).
  • Paclitaxel was purchased from LC Laboratories (Woburn, MA, USA) and used as paclitaxel.
  • Preparation and administration of test substance FK330 was suspended in a 0.5% methylcellulose aqueous solution (0.5% MC) (20 mg / mL) and orally administered.
  • a 10 mg / mL stock solution in ethanol / cremophor solution (mixed 1: 1), then dilute to 1 mg / mL with saline for injection (Otsuka Pharmaceutical) just before administration. It was administered via the tail vein.
  • HTB-26 Human breast cancer-derived MDA-MB-231 was obtained from the American Type Culture Collection (VA, USA). The cells were cultured under conditions of 37 ° C. and 5% CO 2 using DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells collected using trypsin were suspended in PBS at 6 ⁇ 10 7 cells / mL and mixed with an equal amount of Matrigel TM Basement Membrane Matrix (Becton Dickinson, Bedford, MA, USA).
  • FBS heat-inactivated fetal bovine serum
  • Control group untreated FK330 single administration group: FK330 100 mg / kg bid (200 mg / kg / day, continuous administration from the start of the experiment to the end of the experiment)
  • Paclitaxel alone administration group Paclitaxel 10 mg / kg / day (continuous administration for 4 days in each cycle)
  • Combination group FK330 100 mg / kg bid (200 mg / kg / day, continuous administration from the start of the experiment to the end of the experiment) + paclitaxel 10 mg / kg / day (continuous administration for each cycle for 4 days)
  • FK330 was administered by oral gavage (5 mL / kg) twice a day at intervals of 6 to 8 hours.
  • Paclitaxel was administered as an intravenous bolus (10 mL / kg) from the tail vein 1 to 4 hours after administration of FK330. Tumor diameter was measured twice a week using body weight and digital calipers. The tumor volume was calculated from the ellipse volume calculation formula (major axis x [minor axis] 2 x 0.5).
  • FIG. 13 shows the results of examining the combined effect of FK330 with paclitaxel in human breast cancer-bearing mice.
  • human breast cancer (MDA-MB-231) tumor-bearing mice FK330 markedly enhanced the antitumor effect of paclitaxel, and the tumor volume reduction rate relative to the paclitaxel alone group on Day 28 was 59.6% (P ⁇ 0.01), The tumor volume reduction rate for the paclitaxel alone group was 82.8% (P ⁇ 0.01).
  • Conclusions in human breast cancer (MDA-MB-231) tumor-bearing mice combined use with FK330 enhanced the antitumor effect of paclitaxel after multiple cycles and markedly suppressed tumor regrowth.
  • Example 7 Test substance S-nitroso-N-acetyl-D, L-penicillamine (SNAP), which is a NO donor, was purchased from Cayman Chemical (Ann Arbor, MI, USA). The tubulin polymerization measurement kit was purchased from Cytoskeleton (Denver, CO, USA) and used. As paclitaxel, paclitaxel enclosed in the measurement kit was used as paclitaxel. Docetaxel was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as docetaxel. 2) Test method The tubulin polymerization reaction measurement by the fluorescence method was performed according to the instruction manual of the kit.
  • SNAP S-nitroso-N-acetyl-D, L-penicillamine
  • porcine brain-derived tubulin final concentration 2 mg / mL
  • SNAP final concentration 200 or 500 ⁇ mol / L
  • paclitaxel final concentration 3 ⁇ mol / L
  • docetaxel final concentration 1 ⁇ mol / L
  • GPT final concentration 1 mmol / L
  • the fluorescence intensity was measured every minute with a microplate reader (SpectraMax, Molecular Devices, Sunnyvale, CA, USA). The value of the fluorescence intensity was 60 minutes after the start of the reaction corresponding to the equilibrium stationary phase, expressed as a percentage of the SNAP and taxane-untreated group, and the average value obtained three times was used as the value.
  • FIGS. 15A and 15B Results A representative example of the inhibitory action of SNAP on paclitaxel-induced tubulin polymerization and the aggregated value for 60 minutes after the start of tubulin polymerization are shown in A) and B) of FIG. A representative example of this and the total value 60 minutes after the start of tubulin polymerization are shown in FIGS. 15A and 15B, respectively.
  • Paclitaxel or docetaxel significantly promoted tubulin polymerization by 152.7% (P ⁇ 0.05) and 184.1% (P ⁇ 0.001), respectively, compared to each taxane untreated.
  • NO donor SNAP significantly inhibited tubulin polymerization reactions promoted by paclitaxel and docetaxel.
  • SNAP also significantly suppressed the spontaneously induced tubulin polymerization reaction.
  • Conclusion The NO donor directly inhibited the tubulin polymerization reaction promoted by paclitaxel and docetaxel.
  • Example 8 Test substance The dose of FK330 was converted to the weight of a single substance (non-hydrate).
  • Paclitaxel injection (paclitaxel injection “Sawai”) was purchased from Sawai Pharmaceutical Co., Ltd. and used as paclitaxel.
  • Rat anti-mouse F4 / 80 antigen monoclonal antibody, rabbit anti-iNOS polyclonal antibody, and rabbit anti-nitrotyrosine antibody are AbD Serotec (Oxford, UK), Santa Cruz Biotechnology (Santa Cruz, CA, US), and Millipore, respectively. (Billerica, MA, USA) was used.
  • test substance FK330 was suspended in a 0.5% methylcellulose aqueous solution (0.5% MC) (20 mg / mL) and orally administered.
  • Paclitaxel was prepared at the time of use at 1 mg / mL in physiological saline for injection (Otsuka Pharmaceutical).
  • Cells Human lung cancer-derived Calu-6 (HTB-56) is an American Type Culture Collection (VA, From USA). The cells were cultured under the conditions of 37 ° C. and 5% CO 2 using RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS).
  • Control group untreated FK330 single administration group: FK330 100 mg bid (200 mg / kg / day, Day 0 to Day 6) Paclitaxel alone administration group: Paclitaxel 10 mg / kg / day (Day 0 to Day 5) Combination group: FK330 100 mg / kg / kg bid (200 mg / kg / day, Day 0 to Day 6) + paclitaxel 10 mg / kg / day (Day 0 to Day 5) FK330 was administered by oral gavage (5 mL / kg) twice a day at intervals of 6 to 8 hours. Paclitaxel was administered as an intravenous bolus (10 mL / kg) from the tail vein 1 to 4 hours after administration of FK330.
  • Tissue fixation and frozen block preparation On Day 6, tumor tissues were collected from mice. Infiltration was fixed with 4% paraformaldehyde-phosphate buffer (Wako Pure Chemical Industries, Ltd.) at 4 ° C. for 12 hours. Tissue fixation, 10%, 15% and 20% sucrose / phosphate buffer were used, and sucrose substitution was performed stepwise at 4 ° C. according to a conventional method. After the replacement, the cancer tissue was embedded in an OCT compound (Toronto Research Chemicals, Toronto, Canada), a frozen block was prepared with dry ice / acetone, and stored at ⁇ 80 ° C.
  • OCT compound Toronto Research Chemicals, Toronto, Canada
  • Mouse macrophage staining Primary antibody treatment: incubated with rat anti-mouse F4 / 80 antigen monoclonal antibody (1: 200 dilution) for 60 minutes at room temperature. Secondary antibody treatment: Incubated with biotinylated anti-rat IgG antibody (1: 100 dilution, vector laboratories) for 30 minutes at room temperature. Detection system: Sections were incubated for 30 minutes at room temperature using VECTASTAIN ABC kit (Vector Laboratories). Color development: treated with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Dako) reagent for 1 minute at room temperature.
  • DAB 3,3′-diaminobenzidine tetrahydrochloride
  • iNOS staining Primary antibody treatment: Incubated with rabbit anti-iNOS polyclonal antibody (1: 500 dilution) overnight at 4 ° C. Detection system: Sections were incubated for 30 minutes at room temperature using EnVision (DAKO). Color development: treated with DAB reagent for 1 minute at room temperature. Nitrotyrosine staining: Primary antibody treatment: Incubated with rabbit anti-nitrotyrosine antibody (1: 500 dilution) overnight at 4 ° C. Detection system: Sections were incubated for 30 minutes at room temperature using EnVision (DAKO). Color development: treated with DAB reagent for 1 minute at room temperature.
  • Stained images (200x for F4 / 80 and iNOS images, 400x for nitrotyrosine-stained images) are loaded into a computer via a microscope, and image analysis system equipped with WinROOF (version 5.7) (Mitani Corporation) Analyzed. The value was expressed as a ratio (%) of positive area per total area. Five visual fields were measured for each section, and the average value was taken as the value.
  • FK330 which is an iNOS inhibitor
  • FK330 which is an iNOS inhibitor
  • paclitaxel induced macrophage infiltration into tumor tissue and iNOS protein expression in tumor tissue, and as a result, induced production of NO in tumor.
  • the combined use with FK330 which is an iNOS inhibitor suppressed iNOS-derived NO production induced by paclitaxel.
  • Example 9 Test substance The dose of FK330 was converted to the weight of a single substance (non-hydrate).
  • Paclitaxel was purchased from LC Laboratories (Woburn, MA, USA) and used as paclitaxel.
  • Preparation and administration of test substance FK330 was suspended in a 0.5% aqueous methylcellulose solution (0.5% MC) (20 mg / mL) and orally administered.
  • aqueous methylcellulose solution (0.5% MC) (20 mg / mL) and orally administered.
  • Cells Human hormone resistant prostate cancer-derived PC-3 (CRL-1435) was obtained from the American Type Culture Collection (VA, USA). The cells were cultured under conditions of 37 ° C. and 5% CO 2 using DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells collected using trypsin were suspended in PBS at 6 ⁇ 10 7 cells / mL and mixed with an equal amount of Matrigel TM Basement Membrane Matrix (Becton Dickinson, Bedford, MA, USA).
  • FBS heat-inactivated fetal bovine serum
  • FK330 was administered by oral gavage at 5 mL / kg, and paclitaxel was administered by tail vein at 10 mL / kg. Paclitaxel was administered continuously for 5 days from Day 0 to Day 4, followed by a 25-day rest (1 cycle). In the second cycle of paclitaxel, it was administered continuously from Day 30 for 4 days.
  • Control group untreated FK330 single administration group: FK330 100 mg / kg bid (200 mg / kg / day, continuous administration from the start of the experiment to the end of the experiment)
  • Paclitaxel alone administration group Paclitaxel 10 mg / kg / day was administered continuously for 5 days from Day 0 to Day 4, followed by a 25-day rest period (1 cycle), and paclitaxel was administered continuously for 4 days from Day 30 to Day 33 ( 2 cycles).
  • Combination group FK330 100 mg / kg bid (200 mg / kg / day, continuous administration from the start of the experiment to the end of the experiment) + paclitaxel 10 mg / kg / day (first cycle for 5 consecutive days, second cycle for 4 consecutive days) ).
  • FK330 was administered by oral gavage (5 mL / kg) twice a day at intervals of 6 to 8 hours.
  • Paclitaxel was administered as an intravenous bolus (10 mL / kg) from the tail vein 1 to 4 hours after administration of FK330.
  • Tumor diameter was measured twice a week using body weight and digital calipers. The tumor volume was calculated from the ellipse volume calculation formula (major axis x [minor axis] 2 x 0.5).
  • FIG. 19 shows the results of examining the combined effect of FK330 with paclitaxel in human hormone-resistant prostate cancer.
  • FK330 markedly enhanced the antitumor effect of paclitaxel, and the tumor volume reduction rate relative to the paclitaxel alone group at Day 30 was 63.1% (P ⁇ 0.05), at Day 55 The tumor volume reduction rate for the paclitaxel alone group was 72.1% (P ⁇ 0.001).
  • PC-3 human hormone-resistant prostate cancer
  • Example 10 Test substance The dose of FK330 was converted to the weight of a single substance (non-hydrate). Docetaxel (docetaxel hydrate injection (Taxotere TM intravenous drip infusion)) was purchased from Sanofi-Aventis Co., Ltd. and used as docetaxel. 2) Preparation and administration of test substance FK330 was suspended in a 0.5% aqueous methylcellulose solution (0.5% MC) (20 mg / mL) and orally administered. Docetaxel (40 mg / mL) was adjusted to 20 mg / mL with 95% ethanol and then diluted to 1 mg / mL with physiological saline for injection (Otsuka Pharmaceutical) immediately before administration and administered into the tail vein.
  • Docetaxel docetaxel hydrate injection (Taxotere TM intravenous drip infusion)
  • MC aqueous methylcellulose solution
  • Docetaxel 40 mg / mL was adjusted to 20 mg /
  • Cells Human hormone resistant prostate cancer-derived PC-3 (CRL-1435) was obtained from the American Type Culture Collection (VA, USA). The cells were cultured under conditions of 37 ° C. and 5% CO 2 using DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells collected using trypsin were suspended in PBS at 6 ⁇ 10 7 cells / mL and mixed with an equal amount of Matrigel TM Basement Membrane Matrix (Becton Dickinson, Bedford, MA, USA).
  • FBS heat-inactivated fetal bovine serum
  • FK330 was administered by oral gavage at 5 mL / kg, and docetaxel was administered by tail vein at 10 mL / kg. Docetaxel was administered for 2 days continuously on Day 0 and Day 1 and then was withdrawn for 25 days (1 cycle). In the second cycle of docetaxel, administration was continued for 2 days from Day 27.
  • Control group untreated FK330 single administration group: FK330 100 mg / kg bid (200 mg / kg / day, continuous administration from the start of the experiment to the end of the experiment)
  • Docetaxel alone administration group Docetaxel 10 mg / kg / day was administered continuously for 2 days, Day 0 and Day 1, followed by a 25-day rest period (1 cycle), and docetaxel was administered continuously for 2 days from Day 27 (2 cycles)
  • Combination group FK330 100 mg / kg bid (200 mg / kg / day, continuous administration from the start of the experiment to the end of the experiment) + docetaxel 10 mg / kg / day (continuous administration for 2 days in each cycle)
  • FK330 was administered by oral gavage (5 mL / kg) twice a day at intervals of 6 to 8 hours.
  • Docetaxel was administered as an intravenous bolus (10 mL / kg) from the tail vein 1 to 4 hours after administration of FK330. Tumor diameter was measured twice a week using body weight and digital calipers. The tumor volume was calculated from the ellipse volume calculation formula (major axis x [minor axis] 2 x 0.5).
  • FIG. 20 shows the results of examining the combined use effect of FK330 with docetaxel in human hormone-resistant prostate cancer.
  • PC-3 human prostate cancer
  • PC-3 tumor-bearing mice combined use with FK330 enhanced the antitumor effect of docetaxel by multiple cycles administration.
  • Example 11 Test substance The dose of FK330 was converted to the weight of a single substance (non-hydrate). Docetaxel (docetaxel hydrate injection (Taxotere TM intravenous drip infusion)) was purchased from Sanofi-Aventis Co., Ltd. and used as docetaxel. 2) Preparation and administration of test substance FK330 was suspended in a 0.5% aqueous methylcellulose solution (0.5% MC) (20 mg / mL) and orally administered. Docetaxel (40 mg / mL) was adjusted to 20 mg / mL with 95% ethanol, then diluted to 1.5 mg / mL with physiological saline for injection (Otsuka Pharmaceutical) and administered into the tail vein immediately before administration.
  • Docetaxel (docetaxel hydrate injection (Taxotere TM intravenous drip infusion)) was purchased from Sanofi-Aventis Co., Ltd. and used as docetaxel. 2) Preparation and administration of test
  • HTB-132 Human breast cancer-derived MDA-MB-468 was obtained from the American Type Culture Collection (VA, USA). The cells were cultured under the conditions of 37 ° C. and 5% CO 2 using RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells collected using trypsin were suspended in PBS at 6 ⁇ 10 7 cells / mL and mixed with an equal amount of Matrigel TM Basement Membrane Matrix (Becton Dickinson, Bedford, MA, USA).
  • FBS heat-inactivated fetal bovine serum
  • FK330 was administered by oral gavage at 5 mL / kg, and docetaxel was administered by tail vein at 10 mL / kg. Docetaxel was administered for 36 days after Day 0 administration (1 cycle) and administered again on Day 37 (2 cycles).
  • Docetaxel single administration group After docetaxel 15 mg / kg / day was administered on Day 0, a 36-day withdrawal period was placed (1 cycle), and docetaxel was administered again on Day 37 (2 cycles).
  • Combination group FK330 100 mg / kg bid + docetaxel 15 mg / kg / day.
  • FK330 was administered by oral gavage (5 mL / kg) twice a day at intervals of 6 to 8 hours.
  • Docetaxel was administered as a bolus (10 mL / kg) from the tail vein 1 to 4 hours after administration of FK330.
  • Tumor diameter was measured twice a week using body weight and digital calipers. The tumor volume was calculated from the ellipse volume calculation formula (major axis x [minor axis] 2 x 0.5).
  • FIG. 21 shows the results of examining the combined use effect of FK330 with docetaxel in a human breast cancer-bearing model.
  • FK330 markedly enhanced the antitumor effect of docetaxel, and the tumor volume reduction rate compared to docetaxel alone group at Day37 was 46.6% (P ⁇ 0.05), at Day65 The tumor volume reduction rate for the docetaxel alone group was 76.6% (P ⁇ 0.01).
  • P ⁇ 0.05 The tumor volume reduction rate for the docetaxel alone group was 76.6% (P ⁇ 0.01).
  • Example 12 Test substance The dose of FK330 was converted to the weight of a single substance (non-hydrate).
  • Paclitaxel was purchased from LC Laboratories (Woburn, MA, USA) and used as paclitaxel.
  • Preparation and administration of test substance FK330 was suspended in a 0.5% aqueous methylcellulose solution (0.5% MC) (20 mg / mL) and orally administered. After preparing a 10 mg / mL stock solution in ethanol / Cremophor solution (mixed 1: 1), paclitaxel is diluted to 1 mg / mL with physiological saline for injection (Otsuka Pharmaceutical) immediately before administration. And administered via the tail vein.
  • physiological saline for injection Otsuka Pharmaceutical
  • JCRB0240 Cells Human ovarian cancer-derived MCAS (JCRB0240) was obtained from the Pharmaceutical Research Institute (HSRRB). The cells were cultured under conditions of 37 ° C. and 5% CO 2 using EMEM medium supplemented with 20% heat-inactivated fetal bovine serum (FBS). Cells collected using trypsin were suspended in PBS at 6 ⁇ 10 7 cells / mL and mixed with an equal amount of Matrigel TM Basement Membrane Matrix (Becton Dickinson, Bedford, MA, USA).
  • FBS heat-inactivated fetal bovine serum
  • FK330 was administered by oral gavage at 5 mL / kg, and paclitaxel was administered by tail vein at 10 mL / kg. Paclitaxel was administered continuously for 5 days from Day 0 to Day 4, followed by 24 days of withdrawal (1 cycle). The second cycle of paclitaxel was administered from Day 29 for 5 consecutive days. Administration of paclitaxel in the third cycle after 29 days of withdrawal was continued for 5 consecutive days from Day 63.
  • Control group untreated FK330 single administration group: FK330 100 mg / kg bid (200 mg / kg / day, continuous administration from the start of the experiment to the end of the experiment)
  • Paclitaxel alone administration group Paclitaxel 10 mg / kg / day was administered continuously for 5 days from Day 0 to Day 4, and a 24-day drug holiday was placed (1 cycle). Next, paclitaxel was continuously administered for 5 days from Day 29, followed by a 29-day drug holiday (2 cycles), and paclitaxel was continuously administered for 5 days from Day 63 (3 cycles).
  • Combination group FK330 100 mg / kg bid (200 mg / kg / day, continuous administration from the start of the experiment to the end of the experiment) + paclitaxel 10 mg / kg / day (continuous administration for each cycle for 5 days)
  • FK330 was administered by oral gavage (5 mL / kg) twice a day at intervals of 6 to 8 hours.
  • Paclitaxel was administered as a bolus (10 mL / kg) from the tail vein 1 to 4 hours after administration of FK330.
  • Tumor diameter was measured twice a week using body weight and digital calipers. The tumor volume was calculated from the ellipse volume calculation formula (major axis x [minor axis] 2 x 0.5).
  • FIG. 22 shows the results of examining the combined use effect of FK330 with paclitaxel in human ovarian cancer.
  • FK330 markedly enhanced the antitumor effect of paclitaxel, and the tumor volume reduction rate compared to the paclitaxel alone group at Day 63 was 61.5% (no significant difference), and paclitaxel alone at Day 91 The tumor volume reduction rate for the group was 86.0% (P ⁇ 0.05).
  • MCAS human ovarian cancer
  • Paclitaxel was purchased from LC Laboratories (Woburn, MA, USA) and used as paclitaxel.
  • Docetaxel, doxorubicin, irinotecan and carboplatin purchased from Sigma-Aldrich (St. Louis, MO, USA) were used as docetaxel, doxorubicin, irinotecan and carboplatin, respectively.
  • Gemcitabine purchased from Toronto Research Chemicals (Ontario, Canada) was used as gemcitabine.
  • the cell viability measurement kit (CellTiter-Glo TM Luminescent Cell Viability assay kit) purchased from Promega (Madison, WI, USA) was used. 2) Cells Human lung cancer-derived Calu-6 (HTB-56) was obtained from the American Type Culture Collection (VA, USA). MKN-28 (JCRB0253) was obtained from National Institute of Biomedical Innovation (HSRRB). The cells were cultured under the conditions of 37 ° C. and 5% CO 2 using RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS).
  • FBS heat-inactivated fetal bovine serum
  • Paclitaxel, docetaxel and gemcitabine 0.1 nmol / L to 1 ⁇ mol / L
  • Doxorubicin 1 nmol / L to 10 ⁇ mol / L
  • Irinotecan 1 ⁇ mol / L and 10 ⁇ mol / L
  • Carboplatin 10 ⁇ mol / L and 100 ⁇ mol / L 72 hours after treatment with various chemotherapeutic agents, the cell viability of cancer cells was measured using a commercially available kit (CellTiter-Glo TM luminescent cell viability assay kit) according to the instructions. The luminescence intensity was measured using a luminescence measuring device ARVO (Perkin Elmer, Waltham, MA, USA). The experiment was conducted three times, and the average value was taken as the value. Cell viability was expressed relative to SNAP and chemotherapeutic agent untreated.
  • FIGS. 23 and 24 show the inhibitory action of SNAP on the decrease in cell viability of human lung cancer cell line Calu-6 and human gastric cancer cell line MKN-28 induced by various chemotherapeutic agents.
  • SNAP had no effect on reduced cell viability by gemcitabine, doxorubicin, irinotecan and carboplatin.
  • SNAP significantly suppressed the decrease in cell viability caused by paclitaxel and docetaxel.
  • the NO donor selectively inhibited the cancer cell viability-reducing action of taxane drugs in human lung cancer cell Calu-6 and human gastric cancer cell MKN-28.
  • Example 14 Test substance NO donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) purchased from Cayman Chemical Company (Ann Arbor, MI, USA) was used.
  • Paclitaxel was purchased from LC Laboratories (Woburn, MA, USA) and used as paclitaxel.
  • Docetaxel was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as docetaxel.
  • Cell viability measurement kit CellTiter-Glo TM Luminescent Cell Viability assay kit
  • caspase-3 / 7 activity measurement kit Caspase-Glo TM 3/7 assay kit
  • HTB-56 Cells Human lung cancer-derived Calu-6 (HTB-56) was obtained from the American Type Culture Collection (VA, USA). MKN-28 (JCRB0253) was obtained from National Institute of Biomedical Innovation (HSRRB). The cells were cultured under the conditions of 37 ° C. and 5% CO 2 using RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). 3) Test method Human cancer cells were seeded in a 96-well plate (manufactured by Iwaki Glass Co., Ltd.) at a cell density of 1 ⁇ 10 4 cells / well and cultured overnight.
  • FBS heat-inactivated fetal bovine serum
  • Example 15 Test substance The doses of iNOS inhibitors KD-7040 and GW-274150 were converted to the weight of the simple substance (cation part). Paclitaxel was purchased from LC Laboratories (Woburn, MA, USA) and used as paclitaxel. 2) Preparation and administration of test substance KD-7040 and GW-274150 are suspended in 0.5% aqueous methylcellulose solution (0.5% MC) (KD-7040 is 6 mg / mL, GW-274150 is 20 mg / mL) and administered orally. did.
  • paclitaxel is diluted to 1 mg / mL with physiological saline for injection (Otsuka Pharmaceutical) immediately before administration. And administered via the tail vein.
  • physiological saline for injection Otsuka Pharmaceutical
  • HTB-56 Cells Human lung cancer-derived Calu-6 (HTB-56) was obtained from the American Type Culture Collection (VA, USA). The cells were cultured by the same method as in Example 2, and the cells collected using trypsin were suspended in PBS at 4 ⁇ 10 7 cells / mL, and an equal amount of Matrigel TM Basement Membrane Matrix (Becton Dickinson Co., Bedford , MA, USA).
  • KD-7040 single administration group KD-7040 30 mg / kg bid (60 mg / kg / day, continuous administration from the start of the experiment to the end of the experiment)
  • GW-274150 single administration group GW-274150 100 mg / kg bid (200 mg / kg / day, continuous administration from the start of the experiment to the end of the experiment)
  • Paclitaxel alone administration group Paclitaxel 10 mg / kg / day was administered continuously for 5 days from Day 0 to Day 4, followed by a 19-day drug holiday (1 cycle), and paclitaxel was administered continuously for 5 days from Day 24 to Day 28 ( 2 cycles).
  • Combination group KD-7040 30 mg / kg bid (60 mg / kg / day, continuous administration from the start of the experiment to the end of the experiment) + paclitaxel 10 mg / kg / day (continuous administration for each cycle for 5 days).
  • GW-274150 100 mg / kg bid (200 mg / kg / day, continuous administration from the start of the experiment to the end of the experiment) + paclitaxel 10 mg / kg / day (continuous administration for each cycle for 5 days).
  • KD-7040 and GW-274150 were administered orally by gavage (5 mL / kg) twice a day, generally at intervals of 6 to 8 hours.
  • Paclitaxel was administered as an intravenous bolus (10 mL / kg) from the tail vein 1 to 4 hours after the first administration of each cycle of KD-7040 or GW-274150. Tumor diameter was measured twice a week using body weight and digital calipers. The tumor volume was calculated from the ellipse volume calculation formula (major axis x [minor axis] 2 x 0.5).
  • the pharmaceutical composition for cancer treatment containing a NO production inhibitor as an active ingredient which is administered in combination with the taxane antitumor agent of the present invention, is used in combination with a taxane antitumor agent. Because of its therapeutic effect, it is particularly useful for the treatment of various cancers known to be applied to existing taxane antitumor agents.
  • Applicable cancers are solid cancers and lymphomas in which taxane antitumor agents are used, and in some embodiments, breast cancer, endometrial cancer, ovarian cancer, prostate cancer, lung cancer, stomach (gastric gland) cancer, non-cancerous Small cell lung cancer, head and neck squamous cell carcinoma, esophageal cancer, bladder cancer, melanoma, colon cancer, renal cell carcinoma, non-Hodgkin lymphoma and the like.

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Abstract

Selon l'invention, un procédé de traitement amplifiant les effets d'un agent anti-tumoral à base de taxane sur diverses tumeurs, est obtenu. Après considérations relatives à un procédé d'augmentation de l'action anti-tumorale de l'agent anti-tumoral à base de taxane, il a été vérifié qu'une composition médicamenteuse pour le traitement du cancer comprenant en tant que principe actif un inhibiteur de production de NO, augmente l'action anti-tumorale de l'agent anti-tumoral à base de taxane. Plus précisément, l'invention concerne une composition médicamenteuse pour le traitement du cancer comprenant en tant que principe actif un inhibiteur de production de NO destiné à une administration combinée à l'agent anti-tumoral à base de taxane.
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