(19)
(11)EP 3 173 081 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
25.09.2019 Bulletin 2019/39

(21)Application number: 15197046.4

(22)Date of filing:  30.11.2015
(51)Int. Cl.: 
A61K 31/343  (2006.01)
A61P 35/04  (2006.01)
A61P 35/00  (2006.01)

(54)

DESETHYLAMIODARONE FOR USE IN CANCER TREATMENT

DESETHYLAMIODARON ZUR VERWENDUNG IN DER KREBSBEHANDLUNG

DÉSÉTHYLAMIODARONE POUR UTILISATION DANS LE TRAITEMENT DU CANCER


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(43)Date of publication of application:
31.05.2017 Bulletin 2017/22

(73)Proprietor: Pécsi Tudományegyetem
7622 Pécs (HU)

(72)Inventors:
  • Bognár, Zita
    7831 Pellérd (HU)
  • Sümegi, Balázs
    7634 Pécs (HU)
  • Ifj. Gallyas, Ferenc
    7633 Pécs (HU)
  • Tóth, Kálmán
    7634 Pécs (HU)

(74)Representative: Lengyel, Zsolt 
Danubia Patent & Law Office LLC Bajcsy-Zsilinszky út 16
1051 Budapest
1051 Budapest (HU)


(56)References cited: : 
WO-A2-2004/105696
  
  • VAN DER GRAAF ET AL: "IN VITRO AND IN VIVO MODULATION OF MULTI-DRUG RESISTANCE WITH AMIODARONE", INT. J . CANCER, vol. 48, 1 January 1991 (1991-01-01), pages 616-622, XP055268384,
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

Background of invention



[0001] Desethylamiodarone (2-butyl-3-benzofuranyl)[4-[2-(ethylamino)ethoxy]-3,5-diiodophenyl]-methanone, DEA), the major metabolite of the widely used antiarrhythmic drug amiodarone (AM), also has antiarrhythmic activity, significantly increasing the action potential duration (class III antiarrhythmic effect) and decreasing the maximum rate of depolarization (class I antiarrhythmic effect) at clinically relevant concentrations. (1, 2) Amiodarone is widely used in the treatment of a variety of cardiac diseases. (3, 4, 5) Amiodarone and its main metabolite DEA are both strongly bound to plasma proteins.(6) DEA rapidly accumulates in extra cardiac tissues (especially in the lungs) after amiodarone treatment, sometimes in higher concentrations than amiodarone itself. (7, 8, 9, 10) Tissue concentrations of AM and of DEA are 100 to above 1000 times higher than the corresponding plasma concentrations. (11) Organs that store these drugs are adipose tissue, liver and lung, but also skin, pancreas, myocardium and thyroid gland. Except in the adipose tissue, the tissue concentrations of the metabolite are higher than that of the parent drug following chronic administration of AM. (12) Repetitive exposures of cell cultures to AM and DEA respectively resulted in a cumulative and partially saturable drug uptake. Under all conditions tested DEA accumulation was always higher than that of AM. (13) The mean elimination half-life of DEA is 40 days and varies considerably between individuals. (14) Direct and indirect evidence for intralysosomal localization have been presented for AM and DEA in vivo and in vitro. (15) The therapeutic range of amiodarone has been recommended to be <5.7 µM (16). Indeed, it has been reported that the concentrations of amiodarone and DEA in patient plasma are 1.6-5.3 µM and 1.7-4.5 µM, respectively. (17) Amiodarone was found to activate mainly necrotic cell death pathways, whereas DEA also activated apoptotic pathways. (18) DEA may act synergistically with amiodarone. (19) DEA has greater cytotoxic potency in vitro compared to AM (20), however this toxic effect is further enhanced in the presence of amiodarone. To avoid this enhanced toxic effect, as well as to decrease the likeliness of the side effects we will perform our experiments exclusively with DEA. After administration of amiodarone in therapeutic doses, plasma concentration of DEA is in a range of 1.7-4.5 µM (17), however in the lungs DEA can reach mM concentrations due to its significant accumulation in that organ. (21)

[0002] Modern surgical techniques and new chemotherapeutic approaches have significantly improved the effectiveness of treatment in primary tumors, but metastasis remains the leading cause of death in patients with cancer. (22) In determining the stage, relevant treatment, and prognosis of most solid cancers metastasis is the key factor. Tumor metastasis is a complex process that involves local invasion, intravasation, and survival in the circulation, extravasation, and colonization. The tumor cells must overcome numerous hurdles to successfully colonize in the target organ (23, 24) Lung metastases are identified in 30-55% of all cancer patients, though prevalence varies based on the type of primary cancer. Almost any cancer has the ability to spread to the lungs, but the tumors that most commonly do so include bladder cancer, colon cancer, breast cancer, prostate cancer, sarcoma, Wilms tumor, and neuroblastoma.(23,24) The lungs are the most common site of metastasis in melanoma (22). Although metastasis only occurs in approximately 10% of melanoma patients, it remains the major cause of death (25).

[0003] The present inventors surprisingly found that DEA has significant anti-tumor effect. Although DEA is a well-known metabolite, which accumulates in lungs, and is capable of inducing apoptotic cell death, no such anti-tumor effect has been suggested.

Detailed description of the invention



[0004] The present invention provides a pharmaceutical composition comprising a compound selected from the group consisting of desethylamiodarone (DEA) and pharmaceutically acceptable salts, hydrates and solvates thereof, together with a pharmaceutically acceptable excipient, vehicle and/or carrier, for use in the treatment of cancer, wherein said compound is the sole active ingredient administered. The invention is defined by the claims. Any subject-matter falling outside the scope of the claims is provided for information purposes only. Any references in the description to methods of treatment refer to the compounds and pharmaceutical composition of the present invention for use in a method of treatment of the human or animal body by therapy.

[0005] In another preferred embodiment, the composition is administered together with radiotherapy.

[0006] In another disclosure, the composition is administered with one or more anticancer therapies. The one or more anticancer therapy is selected from the group consisting of surgery, and chemotherapy. In another disclosure, the composition is administered together with one or more bioactive agents. In another preferred embodiment, the bioactive agents are selected from a group consisting of antibodies, growth factors, hormones, cytokines, anti-hormones, xanthines, interleukins, interferons, and cytotoxic agents.

[0007] In another preferred embodiment, the cytotoxic agent is selected from the group consisting of doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carubicin, nogalamycin, menogaril, pitarubicin, valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluridine, pentostatin, broxuridine, capecitabine, cladribine, decitabine, floxuridine, fludarabine, gougerotin, puromycin, tegafur, tiazofurin, adriamycin, cisplatin, camoplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine methotrexate, flurouracils, etoposide, taxol, taxal analogs, and mitomycin.

[0008] In another preferred embodiment, the composition is administered together with, prior to, or subsequent to the administration of one or more combinations of cytotoxic agents as part of a treatment regimen, wherein the combination of cytotoxic agents is selected from:

A. CHOPP (cyclophosphamide, doxorubicin, vincristine, prednisone, and procarbazine);

B. CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone);

C. COP (cyclophosphamide, vincristine, and prednisone);

D. CAP-BOP (cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine, and prednisone);

E. m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone, and leucovorin);

F. ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, leucovorin, mechloethamine, vincristine, prednisone, and procarbazine);

G. ProMACE-CytaBOM (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide. teucovorin, cytarabine, bleomycin, and vincristine);

H. MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin, and leucovorin);

I. MOPP (mechloethamine, vincristine, prednisone, and procarbazine);

J. ABVD (adriamycin/doxorubicin, bleomycin, vinblastine, and dacarbazine);

K. MOPP (mechloethamine, vincristine, prednisone, and procarbazine) alternating with ABV (adriamycin/doxorubicin, bleomycin, and vinblastine);

L. MOPP (mechloethamine, vincristine, prednisone, and procarbazine) alternating with ABVD (adriamycin/doxorubicin, bleomycin, vinblastine, and dacarbazine);

M. ChIVPP (chlorambucil, vinblastine, procarbazine, and prednisone);

N. IMVP-16 (ifosfamide, methotrexate, and etoposide);

O. MIME (methyl-gag, ifosfamide, methotrexate, and etoposide);

P. DHAP (dexamethasone, high-dose cytarabine, and cisplatin);

Q. ESHAP (etoposide, methylpredisolone, high-dose cytarabine, and cisplatin);

R. CEPP(B) (cyclophosphamide, etoposide, procarbazine, prednisone, and bleomycin); S. CAMP (lomustine, mitoxantrone, cytarabine, and prednisone);

T. CVP-1 (cyclophosphamide, vincristine, and prednisone);

U. ESHOP (etoposide, methylpredisolone, high-dose cytarabine, vincristine and cisplatin);

V. EPOCH (etoposide, vincristine, and doxorubicin for 96 hours with bolus doses of cyclophosphamide and oral prednisone);

W. ICE (ifosfamide, cyclophosphamide, and etoposide);

X. CEPP(B) (cyclophosphamide, etoposide, procarbazine, prednisone, and bleomyan);

Y. CHOP-B (cyclophosphamide, doxorubicin, vincristine, prednisone, and bleomycin); and

Z. P/DOCE (epirubicin or doxorubicin, vincristine, cyclophosphamide, and prednisone).



[0009] In another preferred embodiment, the cancer is cutaneous or intraocular melanoma, mesothelioma, renal cell carcinoma, breast cancer, head and neck cancer, a primary or secondary brain tumor, carcinoma of the cervix, cancer of the urethra, prostate cancer, pancreatic cancer, testicular cancer, hepatobiliary cancer, hepatic duct cancer, biliary duct cancer, colorectal cancer, bladder cancer, ovarian cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, colon cancer, rectal cancer, or cancer of the anal region.

[0010] In a particularly preferred embodiment, the cancer is selected from the group consisting of lung cancer, urinary bladder carcinoma, melanoma, mammary carcinoma, cervical cancer, and the metastasis thereof.

[0011] The prior art has not suggested the use of DEA for the treatment of proliferative disorders. WO2004105696 relates to the treatment of neoplasms, and amiodarone is mentioned as a compound the anti-proliferative effects are enhanced by the disclosed triazole compounds. DEA is mentioned as a related compound to amiodarone, but no treatment is disclosed, especially not for DEA alone, in this document.

[0012] In the current invention, we report the ability of an amiodarone metabolite, desethyamiodarone, to inhibit growth and induce apoptosis in several type of cancer cells, which indicates its potential as an antitumor agent for the treatment of cancer. Desethylamiodarone (DEA) is the main metabolite of the widely used anti-arrhythmic drug amiodarone. DEA also has antiarrhythmic activity in clinical relevant concentration. (1,2) Our previous results showed that DEA caused mitochondrial permeability transition and induced apoptotic cell death.(27,28,29,30) DEA accumulates rapidly in the lungs.(7,8) DEA seemed to act synergistically with amiodarone.(19) We used DEA and not amiodarone for the experiments planned. Using DEA we exactly know the dose which was given and we do not have to worry about this synergic cytotoxicity. Here, we checked whether Desethylamiodarone can affect invasion, migration of cancer cells. First of all, we initially checked whether desethylamiodarone has effect on the viability of T24 (human urinary bladder carcinoma), B16F10 (mice melanoma), 4T1 (mouse mammary carcinoma) cells using MTT assay. As shown in Figure 1-3 Desethylamiodarone had significant cytotoxic activity in very low concentrations. To assess the recovery ability of cells that survived treatment, long-term (7 days) colony formation assay was performed. As shown in Figure 5-8 one week following treatment with desethylamiodarone indicated already a significant decrease in the number and the size of colonies in T24 (human urinary bladder carcinoma), B16F10 (mice melanoma), 4T1 (mouse mammary carcinoma) A549 (human lung adenocarcinoma epithelial), HeLa (human cervix adenocarcinoma) cell lines. Metastasis is fundamental property of malignant cancer cells, and occurs through a series of sequential steps including invasion, intravasation, survival and translocation in the circulation system, extravasation and survival in new organ. Cancer recurrence by metastasis is one of the main causes of mortality in cancer patients and is currently a main target for cancer therapy (31) Furthermore, metastasis causes about 90% of human cancer-associated deaths (32, 33,34). Thus, a number of researchers are investigating molecular mechanisms related to cancer metastasis, and searching drugs which can suppress metastatic potential of cancer. We began to detect the antimetastatic ability of DEA in vivo; thus, we performed experimental lung metastasis tests. The results show that DEA can significantly reduce the number of tumor nodules and decrease lung weight. Therefore, DEA has a significant anti-metastatic effect in vivo. In addition, the number of cancer nodules in the lungs of DEA-treated mice was significantly reduced by injecting B16F10 cells.

[0013] PI3K/Akt pathway plays an important role in cell division, apoptosis and tumourigenesis. PI3K is a lipid second messenger of intracellular signal transduction, and Akt is its main target. Phosphorylated Akt possesses a wide range of biological effects, such as anti-apoptotic and cell survival-promoting activity(35) Consistent activation of the PI3K/Akt pathway has been detected in numerous human tumor cells, including breast cancer, bladder cancer (36,37). This signal pathway promotes tumor survival, progression and metastasis (36,37). Akt regulates several cellular activities, including proliferation, the cell cycle and apoptosis (38). In the current study, we demonstrated that DEA repressed Akt phosphorylation in a concentration and time-dependent manner. DEA is a direct inhibitor and can interfere with the activation of PI3K/Akt. The pro-apoptotic effect of DEA might be due to its ability to induce BCL-2 degradation and enhance the expression of Bax and P53. We therefore determined whether DEA-induced decreased AKT phosphorylation leads to changes in GSK-3β phosphorylation inT24 bladder carcinoma. We found that the decrease in AKT phosphorylation after treatment with DEA new as accompanied by decrease in GSK-3β phosphorylation. PI3K/AKT and MAPK/ERK pathways activation have been regarded as important key players in cancer carcinogenesis (39). However, only limited data on their effects have been published thus far.

[0014] Due to the fact that DEA inhibits the growth of tumor cells, reduce their colony forming potential, induces cell death and significantly increases the high energy irradiation induced cell death, the invention is useful in the treatment of cancer and increases the efficacy of the high energy irradiation in cancer treatment. The molecular mechanisms of desethylamiodarone among other possible effects is the inhibition of cytoprotective PI-3-K-Akt and ERK1/2 pathways which play significant role in cell death and radiosensitization.

[0015] In certain embodiments, DEA reduces Akt activity and ERK1/2 activities contributing to cell death (Fig. 2) and reduces the colony formation (Fig. 5) and so metastases formation of bladder cancer cell, so improves bladder cancer therapy.

[0016] In certain other embodiments, DEA reduces Akt activity and ERK1/2 activities contributing to cell death (Fig. 3) and reduces the colony formation (Fig. 6) and so metastases formation of melanoma cell, so improves metastatic melanoma therapy.

[0017] In certain other embodiments, DEA reduces Akt activity and ERK1/2 activities contributing to cell death (Fig. 4) and reduces the colony formation and so metastases formation of breast cancer cell, so improves breast cancer therapy.

[0018] In certain other embodiments, DEA reduces Akt activity and ERK1/2 activities contributing to cell death and reduces the colony formation (Fig. 7) and so metastases formation of lung cancer cell, so improves lung cancer therapy.

[0019] In certain other embodiments, DEA reduces Akt activity and ERK1/2 activities contributing to cell death and reduces the colony formation (Fig. 8) and so metastases formation of cervix cancer cell, so improves cervix cancer therapy.

[0020] In a preferred embodiment of the invention, DEA may be used in conjunction with radiotherapy. Standard protocols may be adapted easily by the person skilled in the art to combine the DEA therapy with the state of the art radiotherapy regimens.

[0021] In certain other embodiments, DEA through the reduction of Akt activity and ERK1/2 activities, contributing to its radio sensitizing effects (Fig. 9) making a more effective bladder cancer therapy as radiosensitizer.

[0022] In certain other embodiments, DEA through the reduction of Akt activity and ERK1/2 activities contributing to its radio sensitizing effects (Fig. 10) making a more effective lung cancer therapy as radiosensitizer.

[0023] In certain other embodiments, DEA through the reduction of Akt activity and ERK1/2 activities contributing to its radio sensitizing effects (Fig. 11) making a more effective cervix cancer therapy as radiosensitizer.

[0024] In certain other embodiments, DEA provides a suitable adjunct to enhance efficacy of radiotherapy in models of metastatic melanome (B16F10 cell line) by reducing cancer stem cell formation determined from significantly reduced spheroid numbers, and decreased the number of cells with CD133, CD24 and CD44 cancer stem cell markers.

[0025] In certain other embodiments, DEA provides a suitable adjunct to enhance efficacy of radiotherapy in models of metastatic bladder cancer (T24 cell line) by reducing cancer stem cell formation determined from significantly reduced spheroid numbers, and decreased the number of cells with CD133, CD24 and CD44 cancer stem cell markers.

[0026] In certain other embodiments, DEA provides a suitable adjunct to enhance efficacy of radiotherapy in models of breast cancer (4T1 mouse mammary carcinoma cell line) by reducing cancer stem cell formation determined from significantly reduced spheroid numbers, and decreased the number of cells with CD133, CD24 and CD44 cancer stem cell markers.

[0027] In certain other embodiments, DEA provides a suitable adjunct to enhance efficacy of radiotherapy in models of pancreatic tumors (Panc-1 cell line) by reducing cancer stem cell formation determined from significantly reduced spheroid number, and decreased the number of cells with CD13, CD44 and CD24 cancer stem cell markers.

[0028] In a preferred embodiment of the invention, DEA may be used in conjunction with surgery. The attending physicians will prescribe how to combine the DEA therapy with the state of the art surgery protocols.

[0029] In a preferred embodiment of the invention, DEA may be used in conjunction with other chemotherapeutic protocols. Standard protocols may be adapted easily by the person skilled in the art to combine the DEA therapy with the state of the art regimens.

[0030] In certain other disclosures, DEA provides a suitable adjunct to enhance efficacy of a specific chemotherapy regimen (Temozolomide, Nab-paclitaxel, Paclitaxel, Carmustine (also known as BCNU) Cisplatin) in models of metastatic melanome (B16F10 cell line) by reducing cancer stem cell formation determined from significantly reduced spheroid number, and decreased the number of cells with CD133, CD24 and CD44 cancer stem cell markers.

[0031] In certain other disclosures, DEA provides a suitable adjunct to enhance efficacy of chemotherapy ( Temozolomide, Nab-paclitaxel, Paclitaxel, Carmustine (also known as BCNU) Cisplatin) in models of metastatic bladder cancer (T24 cell line) by reducing cancer stem cell formation determined from significantly reduced spheroid number, and decreased the number of cells with CD133, CD24 and CD44 cancer stem cell markers.

[0032] In certain other disclosures, DEA provides a suitable adjunct to enhance efficacy of chemotherapy (Docetaxel, Paclitaxel, Platinum agents) in models of breast cancer (4T1 mouse mammary carcinoma cell line) by reducing cancer stem cell formation determined from significantly reduced spheroid number, and decreased the number of cells with CD133, CD24 and CD44 cancer stem cell markers.

[0033] In certain other disclosures, DEA provides a suitable adjunct to enhance efficacy of chemotherapy (Gemcitabine, 5-fluorouracil (5-FU), Oxaliplatin, Albumin-bound paclitaxel, Cisplatin, Paclitaxel, Docetaxel) in models of pancreas cancer (Panc-1 cell line) by reducing cancer stem cell formation determined from significantly reduced spheroid number, and decreased the number of cells with CD133, CD24 and CD44 cancer stem cell markers.

[0034] The administration of DEA or the pharmaceutical formulations comprising thereof is carried out by conventional means. It is readily within the capabilities of the person skilled in the art to determined the appropriate administration for any given circumstances. As a way of non-limiting examples, DEA may be administered subcutaneously, intraperitoneally, intravenously, intravesically, intraarterially, intramedullarly, intrathecally, transdermally, transcutaneously, intranasally, topically, entereally, intravaginally, sublingually or rectally.

[0035] In a preferred embodiment, DEA may be administered intravenously.

[0036] In another preferred embodiment, DEA may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth.

[0037] Formulation for delivering DEA may include any pharmaceutically acceptable salts, hydrates and solvates thereof, together with a pharmaceutically acceptable excipient, vehicle and/or carrier. The person skilled in the art will have no difficulty to produce suitable formulations using his general knowledge in the field For guidance, see e.g. Remington's Pharmaceutical Sciences (Mack Publishing Company, N.J. 1991). DEA and common pharmaceutical ingredients are available from commercial sources.

[0038] Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids, or powders, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, solid solution, liposome, films, ovules, sprays and liquid formulations.

[0039] Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet. Solid formulations for oral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

[0040] Further, the pharmaceutical formulations of the invention may also be administered directly into the blood stream, into muscle, or into an internal organ.

[0041] For the purposes of the present invention, especially for the administration to human patients, the total daily dose of the compound of the invention is typically in the range from about any of 10 mg/kg to 25 mg/kg to 50 mg/kg to 100 mg to 150 mg/kg to 200 mg to 250 mg/kg or more, depending, of course, on the mode of administration. For example, the compound of the invention may be administered at about 10 mg/kg, 25 mg/kg, 50 mg/kg, 100 mg, 150 mg/kg, 200 mg or 250 mg/kg.

[0042] The total daily dose may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein. Several dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the physician wishes to achieve. The dosing regimen can vary over time.

[0043] These dosages are based on an average human subject having a weight of about 65 kg to 70 kg. The physician will readily be able to determine doses for subjects whose weight falls outside this range. The precise effective amount for a human subject will depend upon the severity of the disease state, the general health of the subject, the age, weight and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgment of the clinician.

Brief description of the figures



[0044] 

Figure 1. Structure of desethylamiodarone.

Figure 2. Effect of Desethylamiodarone on cell death in T24 cells.

Figure 3. Effect of Desethylamiodarone on cell proliferation in B16F10 murine melanoma cells.

Figure 4. Effect of Desethylamiodarone on cell proliferation in 4T1 mouse mammary carcinoma cells.

Figure 5. The effect of treatment with Desethylamiodarone on the colony forming abilities of T24 human bladder cancer cell line.

Figure 6. Inhibition of colony formation in B16F10 cells by desethylamiodarone.

Figure 7. Inhibition of colony formation in A549 cells by desethylamiodarone.

Figure 8. Inhibition of colony formation in Hela cells by desethylamiodarone.

Figure 9. Inhibitory effects of DEA on artificial lung metastasis.

Figure 10. Histopathology analysis of lung metastasis.

Figure 11. The effects of a DEA on the effectiveness of radiotherapy on the colony forming abilities of T24 human bladder cancer cell line.

Figure 12. The effects of a DEA on the effectiveness of radiotherapy on the colony forming abilities of A549 human lung adenocarcinoma epithelial cell line.

Figure 13. The effects of a DEA on the effectiveness of radiotherapy on the colony forming abilities of HeLA human cervix adenocarcinoma cell line.

Figure 14. Effect of desethylamiodarone on Akt, GSK-3β and ERK pathways in T24 cell line.


Example 1: Materials and methods



[0045] Materials. Protease inhibitor cocktail, and all chemicals for cell culture were purchased from Sigma-Aldrich Kft (Budapest, Hungary). Desethylamiodarone (Dea) was a gift from Professor Varro (Department of Pharmacology and Pharmacotherapy, University of Szeged, Szeged, Hungary.)The following antibodies were used: anti-Akt, anti-phospho-Akt, anti-phospho-glycogen synthase kinase-3β (GSK), anti -ERK, anti-Phospho-ERK (Cell Signaling, 1:1000), anti-rabbit IgG (Sigma-Aldrich Kft).

[0046] Cell viability assay. The cells were seeded into 96-well plates at a starting density of 104 cells per well and cultured overnight before DEA was added to the medium for 24h and 48 h. The medium was changed to a fresh one containing 0.5% of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT+) for an additional 3 hours, then the MTT+ reaction was terminated by adding HCl to 10 mM final concentration. The amount of blue formazan dye formed from MTT+ was proportional to the number of live cells, and was determined with an Anthos Labtech 2010 ELISA reader at 550 nm. All experiments were run in at least 4 replicates and repeated 3 times.

[0047] Effect of desethylamiodarone on colony formation. Cells were trypsinized and plated in triplicate into 6-well plates at 500 cells/well. Cells were treated with different concentration of Dea. After 7 days of incubation, cells were washed and stained with Coomassie-blue, and the colonies containing more than 50 cells were counted. The number of colonies were determined and normalized to the number of colonies in controls.

[0048] Mouse model of lung metastasis. To produce experimental metastasis model, male C57BL/6 mice (6 weeks old) were used. B16F10 cells (5 x 105/0.1 ml) were injected into the lateral tail vein of C57BL/6 mice, using a 30G1/2 needle and a 1-mL syringe. Mice were randomly divided into 3 groups of 6 mice each. Each mouse was given a daily intragastric injection of either 100 µl 15% ethanol solution as the vehicle control or 25 mg/kg DEA. Treatment was started 1 day before cell injection and was given every third day, lasted for consecutive 16 days Sixteen days after injection, the animals were weighed and killed. The lungs were removed, rinsed in PBS, and weighed. The lung weight index was calculated as the ratio of lung weight vs body weight. The harvested lungs were fixed in in 4% formalin. Tumor foci on the surfaces of the lungs were counted under a stereomicroscope. Then, the whole lung was embedded in optimum cutting temperature compound (Sakura Finetek, SA), sectioned (12 mm thickness, 5 levels), and stained with hematoxylin and eosin (H&E) according to routine protocols. Histological observations were performed under a microscope (BX51, Olympus, Japan). The percentage of total area of the stained sections occupied by tumor was measured using the Panoramic viewer 1.15.4 (3DHISTECH, Hungary)

[0049] Ethics. Animal experiments were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996), and was approved by the Animal Research Review Committee of the University of Pecs, Medical School.

[0050] Western blot analysis. 106 cells were seeded into regular plates and treated as for the cell viability assay. Cells were harvested at intervals in a chilled lysis buffer containing 0.5 mM sodium-metavanadate, 1 mM ethylenediaminetetraacetic acid (EDTA) and protease inhibitor cocktail in phosphate-buffered saline (PBS). Probes were boiled and subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis then transferred to nitrocellulose membranes. The membranes were blocked in 5% low fat milk for 1.5 h at room temperature then exposed to primary antibodies at 4 °C overnight in blocking solution. Appropriate horseradish peroxidase-conjugated secondary antibodies were used at a 1:5000 dilution (anti-mouse and anti-rabbit IgGs; Sigma-Aldrich, Steinheim, Germany) and visualized by enhanced chemiluminescence (Amersham Biosciences, Piscataway, New Jersey, USA). All experiments were repeated 3 times.

[0051] The effects of a DEA on the effectiveness of radiotherapy. Cells were trypsinized and plated in triplicate into 6-well plates at 500 cells/well. Cells were treated with different concentration of Dea and irradiated with a dose of 1.0 and 2.0 Grays. A telecobalt (Therathron 780 C) external irradiation equipment (average photon energy of 1.25 MeV) will be applied for irradiating cells. After 7 days of incubation, cells were washed and stained with Coomassie-blue, and the colonies containing more than 50 cells were counted. The number of colonies were determined and normalized to the number of colonies in controls.

Example 2. Inhibitory effects of Desethylmaiodarone on Viability and Proliferation on tumor cell lines



[0052] The effect of desethylamiodarone was measured in T24 (human urinary bladder carcinoma), A549 (human lung adenocarcinoma epithelial), B16F10 (mice melanoma), HeLa (human cervix adenocarcinoma), 4T1 (mouse mammary carcinoma) cell lines. Cells were treated with increasing concentrations of desethylamiodarone for 24 and 48 h. Cell growth was assessed by measuring metabolic activity, which reflects viability in the MTT-test in the course of 24h, 48h of continued exposure to desethylamiorarone. The untreated cells served as negative controls. The viability of the cells, was lower in all DEA-treated than in the control group, with the difference being statistically significant. The results showed that DEA significantly inhibited the viability of these tumor cell lines.

Effect of DEA on viability on T24/83 human urinary bladder carcinoma cell line



[0053] T24 cells were exposed for 24h (A), 48h (B) to 5µmol, 10 µM, 15 µM and 15 µM desethylamiodarone (filled bars). Viability of the cells was detected by the MTT+ method. Untreated cells served as controls. Data represent means±S.E.M. of three independent experiments, each running in four replicates. Significantly different from control group at p<0.01.

[0054] The data shown on Fig. 2 provide evidence that DEA can be useful to treat bladder cancer.

Effect of DEA on viability on B16F10 mice melanoma cell line



[0055] B16F10 cells were incubated with the indicated concentrations (2.5, 5, 7.5 or 10µM) of desethylamiodarone for 24 h (A) and 48 h (B) and cell viability was measured by MTT assay (filled bars). Untreated cells served as controls. Data represent means±S.E.M. of three independent experiments, each running in four replicates. Significantly different from control group at p<0.01.

[0056] The data shown on Fig. 3 provide evidence that DEA can be useful to treat melanoma.

Effect of DEA on viability on 4T1 mouse mammary carcinoma cell line



[0057] 4T1 cells were incubated with the indicated concentrations (2.5, 5, 7.5 or 10µM) of desethylamiodarone for 24 h and 48 h and cell viability was measured by MTT assay (filled bars). Untreated cells served as controls. Data represent means±S.E.M. of three independent experiments, each running in four replicates. Significantly different from control group at p<0.05.

[0058] The data shown on Fig. 4 provide evidence that DEA can be useful to treat breast cancer.

Example 3. Effect of desethylamiodarone on colony formation on tumor cell lines



[0059] To assess the recovery ability of cells that survived treatment, long-term (7 days) colony formation assay was performed. One week following treatment with desethylamiodarone at the lowest concentrations (500nM, 1, 2, 2.5 and 3 µM respectively) indicated already a significant decrease in the number and the size of colonies.

Effect of desethylamiodarone on colony formation on T24/83 human urinary bladder carcinoma cell line



[0060] Untreated cells served as controls. The results are mean ± SEM of 3 independent experiments performed in at least quadruplicate, p < 0.01 compared to the corresponding control group.

[0061] The data shown on Fig. 5 provide evidence that DEA can be useful to treat bladder cancer.

Effect of desethylamiodarone on colony formation on B16F10 mice melanoma cell line



[0062] B16F10 cells were incubated with the indicated concentrations of desethylamiodarone (1, 1.5, 2, 2.5 or 3µM for one week and cell colonies were fixed, stained and counted. (A-B) Data are expressed as the mean ± SD of ≥3 experiments; a p<0.01 is considered statistically significant.

[0063] The data shown on Fig. 6 provide evidence that DEA can be useful to treat melanoma.

Effect of desethylamiodarone on colony formation on A549 human lung adenocarcinoma epithelial cell line



[0064] A549 cells were incubated with the indicated concentrations of desethylamiodarone (1, 1.5, 2, 2.5 or 3µM for one week and cell colonies were fixed, stained and counted. (A-B) Data are expressed as the mean ± SD of ≥3 experiments; ap<0.01 is considered statistically significant.

[0065] The data shown on Fig. 7 provide evidence that DEA can be useful to treat lung cancer.

Effect of desethylamiodarone on colony formation on HeLa human cervix adenocarcinoma cell line



[0066] HeLa cells were incubated with the indicated concentrations of desethylamiodarone (1, 1.5, 2, 2.5 or 3µM for one week and cell colonies were fixed, stained and counted. (A-B) Data are expressed as the mean ± SD of ≥3 experiments; ap<0.01 is considered statistically significant

[0067] The data shown on Fig. 8 provide evidence that DEA can be useful to cervix carcinoma.

Example 4. Effect of desethylamiodarone on lung metastasis formation of melanoma on B16F10 mice melanoma cell line


Inhibitory effects of DEA on artificial lung metastasis.



[0068] Murine melanoma B16F10 cells were injected into the lateral tail vein of 6-weeks-old male C57BL/6 mice and divided into two groups (6 mice/group). Intraperitoneal administration of 25 mg/kg DEA or vehicle control was started 24 h after tumor cell inoculation, and repeated every third day. The treatment group received desethylamiodarone injection, while the control group received vehicle control treatment. As shown in Fig. 9, desethylamiodarone treatment substantially inhibited tumor appearance. At the 16th day of the experiment lungs were removed for analysis. Lungs were photographed (Fig. 9 A,B), weighted and lung weight index was calculated as the ratio of lung weight versus body weight of host mice, and was compared between the two groups.(Fig. 9 C) Desethylamiodarone treatment significantly *P < 0.001 inhibited lung weight increase in B16F10 injected mice. Metastatic nodules on lung surface were counted. A dramatically decrease in the number of tumor nodules was observed in the lungs after desethylamiodarone treatment. This observation was confirmed by the statistical analysis of the number of tumor nodules, which showed that the difference between the model group and the desethylamiodarone - treated mice group was significant *P < 0.001. (Fig. 9 D) Accordingly, the lung weight index, and the number of tumor nodules indicated that metastasis was less remarkable for desethylamiodarone treated mice than control group.

[0069] Lung metastasis in C57BL/6 mice induced from B16F10 tail vein injection. 24h after injection of melanoma B16F10 cells, mice in the control group were treated with vehicle (alcohol), mice in the treatment group were injected with desethylamiodarone (25 mg/kg, i.p., every 3rd day.) After 16 day's mice were sacrificed and then analyzed. Effect of desethylamiodarone on lung metastasis formation was photographed .A and B: mouse with B16F10 lung metastasis vehicle treated or DEA treated, respectively. Lung weight was determined and lung weight index was calculated (C). Tumor growth was monitored also by counting the tumor nodules (D). Treatment caused a statistically significant decrease in metastasis (***P < 0.001). Data are presented as the mean ± standard deviation. *P<0.001 compared with control.

[0070] These data provide evidence that DEA can be useful to inhibit melanoma metastasis.

Histopathology analysis of lung metastasis



[0071] Lungs of the metastasis-induced animals were fixed in neutral buffered formalin and stained with hematoxyline and eosine. Lung tissue sections were analyzed histopathological after H & E staining.. Histopathology analysis was made of lung metastasis from mice treated with the vehicle, control (A,C) and of lung metastasis from mice treated with 25mg/kg/3days DEA(B,D). Histopathology of the lung also showed marked reduction in tumor mass in the lungs of Desethylamiodarone-treated animals (Figure 10 A,B,C,D). It showed a difference in tumor nodule pattern distribution, concentration.. Image analysis with the Pannoramic Viewer Imaging System results quantifying percentage coverage of tumor area over lung section area n=6 per group.(E) Data are reported as means±SD. **P < 0.001, unpaired Student t-test, n=6. The percentage of tumor area was determined in comparison to total section area (Figure 10 E). The percentage of tumor area was significantly decreased in HE stained sections *p<0.001. From a structural point of view, it was observed that melanoma cells with poliedric morphology with a great amount of melanin content as cytoplasm granules or in a perinuclear distribution. Additionally, aberrant nodular proliferation in Broncho alveolar regions, characteristic of epithelial melanoma, was observed (Figure 10A,C). After desethylamiodarone treatment (Figure 10 B,D) tumor nodules were decreased and organized in a predominantly in the lung parenchymal distribution, whereas, in control group, the nodules were larger and distributed in all part of the lung. The results are in accordance with those of macroscopic observations. This result indicates that DEA can inhibit in vivo melanoma tumor metastasis.

[0072] These data provide evidence that DEA can be useful to inhibit melanoma metastasis.

Example 5. Sensitization of cancer cells to irradiation.



[0073] Sensitizing cancer cells to irradiation is a major challenge in clinical oncology therefore we used DEA to demonstrate its radio sensitizing effects. To examine the effects of a DEA on the effectiveness of radiotherapy the cells were treated with 1Gy or 2Gy single dose irradiation alone or combined with 500nM-3 µM DEA. DEA treatment significantly increases the effectiveness of Radiotherapy.

The effects of a DEA on the effectiveness of radiotherapy on the colony forming abilities of T24 human bladder cancer cell line.



[0074] The cells were treated with 1Gy or 2Gy single dose irradiation alone or combined with 500nM-3 µM DEA. Untreated cells served as controls. The results are mean ± SEM of 3 independent experiments performed in at least quadruplicate. P < 0.05 compared to the corresponding control group.

[0075] The data shown on Fig. 11 provide evidence that DEA can be useful to sensitize bladder cancer to irradiation

The effects of a DEA on the effectiveness of radiotherapy on the colony forming abilities of A549 human lung adenocarcinoma epithelial cell line.



[0076] The cells were treated with 1Gy or 2Gy single dose irradiation alone or combined with 500nM-3 µM DEA. Untreated cells served as controls. The results are mean ± SEM of 3 independent experiments performed in at least quadruplicate, p < 0.05 compared to the corresponding control group.

[0077] The data shown on Fig. 12 provide evidence that DEA can be useful to sensitize lung cancer to irradiation

[0078] The effects of a DEA on the effectiveness of radiotherapy on the colony forming abilities of HeLA human

cervix adenocarcinoma cell line.



[0079] The cells were treated with 1Gy or 2Gy single dose irradiation alone or combined with 500nM-3 µM DEA. Untreated cells served as controls. The results are mean ± SEM of 3 independent experiments performed in at least quadruplicate, p < 0.05, compared to the corresponding control group.

[0080] The data shown on Fig. 13 provide evidence that DEA can be useful to sensitize cervix cancer to irradiation

Example 6. The Effect of Desethylamiodarone on Key Signaling Pathways.



[0081] To determine the downstream consequences of DEA treatment, we examined the phosphorylation status of AKT (protein kinase B), GSK-3β and p42/44 mitogen-activated protein kinase (MAPK) (ERK1/2) by immunoblotting in T24 (human urinary bladder carcinoma), B16F10 (mice melanoma) cell lines. Cells were cultured in the presence of 5-10-15 µM DEA for 6 hours. The cells were prepared and western blot analysis was carried out to perform both phosphorylated and non-phosphorylated forms of the previously listed proteins. Analyzing the activity of the protein ERK, commonly involved in tumor proliferation, we found a decrease in its phosphorylation after 6 hours of treatment with 5-10-15 µM DEA. Since previous reports have indicated that the PI3K/Akt pathway provides a survival signal to protect cells from apoptosis (26), we determined the effect of DEA treatment on the activity of Akt. We observed that DEA repressed Akt (Ser473) phosphorylation in a concentration-dependent manner (Fig. 12). We extended our observations to evaluate whether AKT downstream target GSK-3β was affected. DEA treatment also decreased the phosphorylation of GSK-3β after 6 hours at a concentration dependent manner. These results indicate that the efficacy of this drug could be related with the modulation of ERK1/2, AKT, and GSK-3β proteins.

Example 7. The Effect of Desethylamiodarone on CSC subpopulation



[0082] The CSC hypothesis is an important concept arising in cancer research. This hypothesis postulates the existence of a subgroup of cancer cells, the CSCs, which has the ability to self-renew and to differentiate into all cell types of the original heterogeneous tumor, thus resembling the function of normal epithelial stem cells.

[0083] To efficiently treat cancer, it is important to target the CSCs, ideally in combination therapies that also target the bulk of the tumor. To examine the effect of DEA on the cancer stem cell populations, we carried out flow cytometry using the cancer stem cell markers (CD44+/ CD24-/low). Desethylamiodarone treatment will be tested for decreasing the CD44+ cell population and decreasing the incidence of CSC cells.

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Claims

1. A pharmaceutical composition comprising a compound selected from the group consisting of desethylamiodarone and pharmaceutically acceptable salts, hydrates and solvates thereof, together with a pharmaceutically acceptable excipient, vehicle and/or carrier, for use in the treatment of cancer, wherein said compound is the sole active ingredient administered.
 
2. The composition for use according to claim 1, wherein the composition is administered together with radiotherapy.
 
3. The composition for use according to claims 1-2, wherein the cancer is cutaneous or intraocular melanoma, mesothelioma, renal cell carcinoma, breast cancer, head and neck cancer, a primary or secondary brain tumor, carcinoma of the cervix, cancer of the urethra, prostate cancer, pancreatic cancer, testicular cancer, hepatobiliary cancer, hepatic duct cancer, biliary duct cancer, colorectal cancer, bladder cancer, ovarian cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, colon cancer, rectal cancer, or cancer of the anal region.
 
4. The composition for use according to claims 1-3, wherein the cancer is selected from the group consisting of lung cancer, urinary bladder carcinoma, melanoma, mammary carcinoma, cervical cancer, and the metastasis thereof.
 


Ansprüche

1. Pharmazeutische Zusammensetzung enthaltend eine Verbindung ausgewählt aus der Gruppe bestehend aus Desethylamiodaron und pharmazeutisch annehmbaren Salzen, Hydraten und Solvaten dieser, zusammen mit einem pharmazeutisch annehmbaren Hilfsstoff, Vehiculum und/oder Trägerstoff, zur Verwendung in der Behandlung von Krebs, worin die Verbindung der einzige verabreichte Wirkstoff ist.
 
2. Die Zusammensetzung zur Verwendung nach Anspruch 1, wobei die Verbindung zusammen mit Radiotherapie verabreicht wird.
 
3. Die Zusammensetzung zur Verwendung nach den Ansprüchen 1-2, wobei der Krebs Haut- oder Augenmelanom, Mesotheliom, Nierenkarzinom, Brustkrebs, Kopf-Hals-Krebs, primärer oder sekundärer Hirntumor, Zervixkarzinom, Harnröhrenkrebs, Prostatakrebs, Bauchspeicheldrüsenkrebs, Hodenkrebs, hepatobiliärer Krebs, Lebergangkrebs, Gallengangkrebs, Darmkrebs, Blasenkrebs, Eierstockkrebs, Lungenkrebs, nicht-kleinzelliger Lungenkrebs, kleinzelliger Lungenkrebs, Kolonkrebs, Enddarmkrebs oder Krebs der Analregion ist.
 
4. Die Zusammensetzung zur Verwendung nach der Ansprüchen 1-3, wobei der Krebs aus der Gruppe enthaltend Lungenkrebs, Harnblasenkarzinom, Melanom, Mammakarzinom, Gebärmutterhalskrebs und ihrer Metastasen ausgewählt ist.
 


Revendications

1. Composition pharmaceutique comprenant un composé choisi dans le groupe constitué par la déséthylamiodarone et ses sels, hydrates et solvates pharmaceutiquement acceptables, conjointement avec un excipient, véhicule et/ou support pharmaceutiquement acceptable, pour utilisation dans le traitement du cancer, où ledit composé est le seul ingrédient actif administré.
 
2. Composition pour utilisation selon la revendication 1, où la composition est administrée conjointement avec une radiothérapie.
 
3. Composition pour utilisation selon les revendications 1 à 2, où le cancer est le mélanome cutané ou intraoculaire, le mésothéliome, le carcinome à cellules rénales, le cancer du sein, le cancer de la tête et du cou, une tumeur primitive ou secondaire du cerveau, le carcinome du col utérin, le cancer de l'urètre, le cancer de la prostate, le cancer du pancréas, le cancer du testicule, le cancer hépatobiliaire, le cancer du canal hépatique, le cancer du canal biliaire, le cancer colorectal, le cancer de la vessie, le cancer de l'ovaire, le cancer du poumon, le cancer du poumon non à petites cellules, le cancer du poumon à petites cellules, le cancer du côlon, le cancer du rectum ou le cancer de la région anale.
 
4. Composition pour utilisation selon les revendications 1 à 3, où le cancer est choisi dans le groupe constitué par le cancer du poumon, le carcinome de la vessie, le mélanome, le carcinome mammaire, le cancer du col de l'utérus et leurs métastases.
 




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REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description