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WO2005058324A1 - Utilisation de la siramesine dans le traitement du cancer - Google Patents

Utilisation de la siramesine dans le traitement du cancer Download PDF

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Publication number
WO2005058324A1
WO2005058324A1 PCT/DK2004/000885 DK2004000885W WO2005058324A1 WO 2005058324 A1 WO2005058324 A1 WO 2005058324A1 DK 2004000885 W DK2004000885 W DK 2004000885W WO 2005058324 A1 WO2005058324 A1 WO 2005058324A1
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Prior art keywords
cancer
siramesine
pharmaceutically acceptable
treatment
acceptable salt
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PCT/DK2004/000885
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English (en)
Inventor
Christian Thomsen
Marcel Leist
Marja Jaattela
Marie Stampe Andersen
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H. Lundbeck A/S
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Priority to AU2004298722A priority Critical patent/AU2004298722A1/en
Priority to BRPI0416019-3A priority patent/BRPI0416019A/pt
Priority to EA200600987A priority patent/EA011170B1/ru
Priority to EP04803036A priority patent/EP1715864A1/fr
Priority to CA002550611A priority patent/CA2550611A1/fr
Priority to JP2006544219A priority patent/JP2007514666A/ja
Application filed by H. Lundbeck A/S filed Critical H. Lundbeck A/S
Publication of WO2005058324A1 publication Critical patent/WO2005058324A1/fr
Priority to US11/314,796 priority patent/US20060178389A1/en
Priority to IS8391A priority patent/IS8391A/is
Priority to IL175774A priority patent/IL175774A0/en
Priority to NO20063319A priority patent/NO20063319L/no
Priority to US12/426,520 priority patent/US20090203721A1/en

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/438—The ring being spiro-condensed with carbocyclic or heterocyclic ring systems
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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 the use of Siramesine for the preparation of medicaments useful for the treatment of cancer.
  • Tumor cells have often acquired resistance towards classical treatment modalities, such as classical caspase-mediated apoptosis.
  • classical treatment modalities such as classical caspase-mediated apoptosis.
  • novel efficient drugs for the treatment of cancers there is still a large unmet need for novel efficient drugs for the treatment of cancers.
  • sigma2 receptors are upregulated in cells representing many different types of cancers (Bern et al. 1991, Cancer Res. 51, 6558; Vilner et al. 1995, Cancer Res. 55, 408), and furthermore that sigma2 receptor ligands may inhibit cell proliferation and induce apoptosis in tumor cells (Brent & Pang, 1995, Eur. J. Pharmacol. 278, 151; Crawford & Bowen, 2002, Cancer Res. 62, 313). Additionally, it has been shown that sigma 2 ligands may potentiate the activity of antineoplastic drugs (Crawford & Bowen, 2002, Cancer Res. 62, 313). International Patent Publication No.
  • WO 92/22554 describes a series of sigma receptor ligands considered useful for the treatment of a range of psychic and neurological disorders.
  • the structure activity relationship of these compounds has been further investigated by Perregaard, J. et al, J. Med. Chem., 1995, 38, 11, p. 1998-2008.
  • a medicament for the treatment of cancer is provided.
  • Sigma2 receptor ligands have been known to induce apoptosis in cancer cells of different origin.
  • Siramesine of the invention when used alone is more potent in inducing apoptosis in cancer cells than sigma2 active reference compounds such as haloperidol.
  • chemotherapeutic compounds such as etoposide, doxorubicin, staurosporin, vincristine and tamoxifen.
  • the present invention therefore demonstrate that Siramesine may be used for the manufacture of pharmaceutical compositions for the treatment of cancer, and that such compositions may be used in combination with other chemotherapeutic cancer drugs, and/or in combination with radiotherapy.
  • the drugs may be administered simultaneously or sequentially.
  • the present invention relates to the use of Siramesine or a pharmaceutically acceptable salt thereof, together with a chemotherapeutic drug in a synergistic effective dose for the preparation of a pharmaceutical composition as above, which is adapted for simultaneous administration of the active ingredients.
  • a pharmaceutical composition as above, which is adapted for simultaneous administration of the active ingredients.
  • such pharmaceutical compositions may contain the active ingredients within the same unit dosage form, e.g. in the same tablet or capsule.
  • Such unit dosage forms may contain the active ingredients as a homogenous mixture or in separate compartments of the unit dosage form.
  • the present invention relates to the use of Siramesine or a pharmaceutically acceptable salt thereof together with a chemotherapeutic drug in a synergistic effective dose for the preparation of a pharmaceutical composition or kit as above, which is adapted for sequential administration of the active ingredients, i particular, such pharmaceutical compositions may contain the active ingredients in discrete unit dosage forms, e.g. discrete tablets or capsules containing either of the active ingredients. These discrete unit dosage forms may be contained in the same container or package, e.g. a blister pack.
  • kit means a pharmaceutical composition containing each of the active ingredients, but in discrete unit dosage forms.
  • synthetic effective dosage means the dosages of Siramesine and the chemotherapeutic agent at which their combined use provides a synergistic effect, preferably the maximal obtainable synergistic effect.
  • composition or kit of the invention may be adapted for simultaneous administration of the active ingredients or for sequential administration of the active ingredients, as described above.
  • the present invention relates to the novel use of Siramesine having the general formula or pharmaceutically acceptable salts thereof for the preparation of a medicament for the treatment of cancer.
  • the present invention relates to a method for the treatment of cancer comprising administering to an individual in need thereof a pharmaceutically acceptable amount of Siramesine or a pharmaceutically acceptable salt thereof.
  • a further aspect the invention relates to a method for treatment of cancer0 comprising administering Siramesine or a pharmaceutically acceptable salt thereof to an individual to be treated with or undergoing treatment with a chemotherapeutic agent.
  • the compound -[4-[l-(4-fluorophenyl)-lH-indole-3-yl]-5 l-butyl]-spiro[isober ⁇ zo-furan-l(3H) 5 4'-piperidine] may be used as the base of the compound or as a pharmaceutically acceptable acid addition salt thereof or as an anhydrate or hydrate of such salt.
  • the salts of the compound used in the invention are salts formed with non-toxic organic or inorganic acids.
  • organic salts are those with maleic, fumaric, benzoic, ascorbic, succinic, oxalic,0 bis-methylenesalicylic, methanesulfonic, ethane-disulfonic, acetic, propionic, tartaric, salicylic, citric, gluconic, lactic, malic, mandelic, cinnamic, citraconic, aspartic, stearic, palmitic, itaconic, glycolic, p-amino-benzoic, glutamic, benzene sulfonic and theophylline acetic acids, as well as the 8-halotheophyllines, for example 8-bromo- theophylline.
  • Exemplary of such inorganic salts are those with hydrochloric, '.5 hydrobromic, sulfuric, sulfamic, phosphoric and nitric acids.
  • the compound is used as the furnarate or the hydrochloric salt.
  • the fumarate of -[4-[l-(4-fluorophenyl)-lH-indole-3-yl]-l-butyl]-spiro[isobenzo- furan-l(3H),4'-piperidine] can be prepared as described in Perregaard, J. et al, J. Med. Chem., 1995, 38, 11, 1998-2008 (compound 14f) and the base and other pharmaceutically acceptable salts may be obtained there from by standard procedures.
  • the acid addition salts according to the invention may be obtained by treatment of 1 ' - [4- [ 1 -(4-fluorophenyl)- lH-indole-3 -yl] - 1 -butyl] -spiro [isobenzo-furan- 1 (3H),4 ' - piperidine] with the acid in an inert solvent followed by precipitation, isolation and optionally re-crystallisation by known methods and if desired micronisation of the crystalline product by wet or dry milling or another convenient process, or preparation of particles from a solvent-emulsification process.
  • Precipitation of the salt is preferably carried out in an inert solvent, e.g. an inert polar solvent such as an alcohol (e.g. ethanol, 2-propanol and n-propanol).
  • an inert solvent e.g. an inert polar solvent such as an alcohol (e.g. ethanol, 2-propanol and n-propanol).
  • -[4-[l-(4-fluorophenyl)-lH-indole-3-yl]-l-butyl]- spiro[isobenzofuran-l(3H),4'-piperidine] or a pharmaceutically acceptable salt thereof may be administered in any suitable way e.g. orally or parenterally, and it may be presented in any suitable form for such administration, e.g. in the form of tablets, capsules, powders, syrups or solutions or dispersions for injection.
  • the compound of the invention is administered in the form of a solid pharmaceutical entity, suitably as a tablet or a capsule or in the form of a suspension, solution or dispersion for injection.
  • Tablets may thus be prepared by mixing the active ingredients with ordinary adjuvants and/or diluents and subsequently compressing the mixture in a convenient tabletting machine.
  • adjuvants or diluents comprise: corn starch, lactose, talcum, magnesium stearate, gelatine, lactose, gums, and the like. Any other adjuvant or additive such as colourings, aroma, preservatives, etc. may also be used provided that they are compatible with the active ingredients.
  • composition is intended to encompass a product comprising the specified ingredients in the specific amounts, as well as any product which results, directly or indirectly, from combination of the specific ingredients in the specified amounts.
  • the pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc.
  • inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate
  • granulating and disintegrating agents for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid
  • binding agents for example starch, gelatin, polyvinyl-pyrrolidone or acacia
  • lubricating agents for example, magnesium stearate, stearic acid or talc.
  • compositions of the invention may be used for treatment of cancer in mammals, preferably in humans.
  • Siramesine or a salt thereof for use of the invention is most conveniently administered orally in unit dosage forms such as tablets or capsules, containing the active ingredient (calculated as the free form) in an amount from about 0.01 ⁇ g/kg/day to
  • 100 mg/kg/day preferably 0.01 ⁇ g/kg/day to 30mg/kg/day body weight, most preferably 0.5 mg/day/kg to 7.0 mg/day/kg body weight.
  • siramesine When siramesine is combined with other compounds in order to obtain an increased effect, or in order to allow for the use of a subnormal dose of the other compound, to minimize side effects, then subnormal doses of siramesine and/or the other compound may be used for the treatment. Calculation of patient specific doses is routine practice for those skilled in the art.
  • cancers that may be treated by the compounds, compositions and methods of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxorna, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma,
  • cancer includes a cell afflicted by any one of the conditions identified above, and the term “cancer” includes but is not limited to any of the conditions identified above. Any of the above mentioned conditions are to be considered as single embodiments, and the compositions directed to the treatment of each condition may accordingly be claimed individually or be included in the claimed group when the term cancer is used.
  • any of the above cancer indications are mentioned in relation to use of siramesine, a pharmaceutical composition, a kit, a method of treatment it is intended to be an individual embodiment. Accordingly, each of the indications specified above may individually be claimed together with said use of siramesine, pharmaceutical composition, kit, method of treatment and method for the identification of compounds useful for treatment.
  • Murine fibrosarcoma cells WEHI-S, Wn902, Wn912, and WEHI-R4 are normal, vector control, Hsp70 overexpressing, and hTNF resistant cells, respectively.
  • Other cell types tested include: Human breast cancer cell types MCF7-S 1 and MDA-MB- 468, and non-tumorigenic immortalized breast epithelial cells HBL100.
  • MCF7 casp3.1 and -3.3 and neo2 are single cell clones expressing caspase3 and vector.
  • MCF7 pCEP, -Bcl2 WT, -Bcl2 NT, -Bcl2 Acta, and -Bcl2 Cb5 are single cell clones expressing vector, wildtype Bcl2, cytoplasma localized Bcl2, mitochondria localized Bcl2, and ER localized Bcl2 (Maria H ⁇ yer Hansen, Apoptosis Laboratory, Danish Cancer Society).
  • Human neuroblastoma cell line SK-N-MC cells ATCC, USA.
  • human cervix carcinoma cell lines HeLa (kindly provided by Dr. J. Lukas, Danish Cancer Society) and ME180 were tested.
  • HEK293-A prostata cancer cell line PC3, and non-tumorigenic immortalized prostata epithelial cell line PNT1 A were tested as well.
  • Non-transformed NIH3T3 murine fibroblasts were kindly provided by C. Holmberg (University of Copenhagen, Denmark). Fibroblasts were transduced with pBabe- puro mock, -SV40LT, -v-Ha-ras, -c-src as described (Fehrenbacher et al, 2004).
  • Neuropharmacol., 2002, 43, 95-100 cells were cultured as described, harvested in phosphate buffered saline using a cell scraber and centrifuged (1000 x g, 10 min). The resulting pellets were used for [ 3 H]Lu 28-179 binding assays as described in S ⁇ by et al., 2002.
  • Siramesine (Lu-28-179), Siramesine analogs: 28131M, 28134M, 292880, 32160F, 32124C, and 32168F, and haloperidol (H. Lundbeck A/S, Copenhagen, Denmark), human TNF- ⁇ (Strathmann Biotech Gmbh, Germany), thapsigargin, etoposide, doxorubicin, staurosporine, vincristine, tamoxifen (SIGMA- Aldrich, St Louis, MO), concanamycinA (Alexis Biochemicals, San Diego, CA).
  • antioxidants were used: Butylated hydroxyanisole (BHA), ⁇ - tocopherol, ⁇ -tocopherol, glutathione ethyl ester (GSH), N-acetyl-cysteine (NAC)
  • the effect on cell viability was assessed 24-60h after drug addition by removal of 100 ⁇ L media and addition of 25 jiiL MTT solution (1 mg/mL in PBS, sterile filtered). After 3h incubation 37 °C in darkness, the cells were permeabilized using 100 ⁇ L solubilization buffer (20 % SDS in 50 % dimethylformamide solution) and analyzed on spectrophotometer the following day.
  • LDH lactate dehydrogenase
  • Cytotoxicity (% LDH release) LDHi n me dia/ (LDHi n m edia+ LDHi Starting ⁇ ysa t e ) x 100 %
  • Nuclear condensation The cell death mode was assessed by the type of nuclear condensation and performed by hoechst staining (cell permeable Hoechst 33342, SIGMA- Aldrich) of drug-treated cells.
  • Non cell permeable Sytox Green (Molecular Probes) was used to assay loss of membrane integrity and compared with nuclear morphological changes observed with Hoechst.
  • Cells were incubated with 10.000 x dilution of 25 mg/mL hoechst and /or lO.OOOx dilution of 5 mM sytox green for 5 min at 37°C where after type of nuclear condensation and possible co-stain was analyzed using an inverted Olympus microscope LX-70.
  • cytosolic cystein cathepsin enzyme activity To measure cytosolic cystein cathepsin enzyme activity, subconfluent cells seeded in 24-well plates were treated with an extraction buffer (250 mM sucrose, 20 mM HEPES, 10 mM KC1, 1,5 mM MgCl 2 , lmM EDTA, 1 mM EGTA, lmM pefablock; pH 7,5) containing 20 ⁇ g/mL digitonin for 12-15 min on ice. To measure total cellular cystein cathepsin enzyme activity, cells were treated with the above extraction buffer containing 200 ⁇ g/mL digitonin for 12-15 min on ice.
  • an extraction buffer 250 mM sucrose, 20 mM HEPES, 10 mM KC1, 1,5 mM MgCl 2 , lmM EDTA, 1 mM EGTA, lmM pefablock; pH 7,5
  • caspase extraction buffer (0,5 % Triton X-100, 25 mM HEPES, 5 mM Mg 2 Cl, 1 mM EGTA, 1 mM pefablock, pH 7,5) for 20 min on ice.
  • the caspase 3/7-like activity and cystein cathepsin activities were estimated by adding one volume of 20 ⁇ M Ac-DEVD-AFC (Biomol) in caspase reaction buffer (100 mM HEPES, 20 % glycerol, 0,5 mM EDTA, 0,1 % CHAPS, 5 mM DTT, 1 mM pefablock, pH 7,5) or 20 ⁇ M zFR-AFC (Enzyme System Products) in cathepsin reaction buffer (50 mM sodium acetate, 4 mM EDTA, 8 mM DTT, 1 mM pefablock, pH 6,0), respectively.
  • the V max of the liberation of AFC excitation 400 nni, emission 489 nm
  • AO lysomotropic weak base and metachromatic fluorochrome acridine orange
  • Cells were either evaluated using an inverted Olympus microscope IX-70 or detached from the substratum and analyzed by flow cytometry using a FACSort (Becton Dickinson, San Jose, CA) with an argon ion laser with an output wavelength of 488 nm and analyzed using CELLQuest software.
  • FACSort Becton Dickinson, San Jose, CA
  • Lysosomal stability in vitro assay MCF-7 cells seeded in 14 cm plates were loaded with Fe-dextran for 9h, followed by 16 h clearance in culture medium. Subsequently, the cells were washed in PBS and detached from the substratum. The cell pellet was resuspended in SCA buffer (20 mM Hepes KOH, 10 mM KC1, 1,5 mM Mg 2 Cl, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, pH 7,5) and equilibrated on ice for 20 min. The cell pellet was homogenized by 150-200 strokes using a Teflon pestle.
  • proteins were separated by 6-12% SDS-PAGE and transferred to a nitrocellulose membrane.
  • WEHI-R4 cells (5 x 10 6 ) were implanted subcutaneously in the back of immunocompetent female BALB/c mice.
  • Siramesine treatment commenced two days prior to tumor implant. Mice were divided into groups (5-9/group) and administered peroral 200 ⁇ L 1) vehicle (0,5 % methylcellulose 15 in 0,9 % NaCl solution), 2) 100 mg/kg/day Siramesine in suspension (in 0,5 % methylcellulose 15 in 0,9 % NaCl solution) 7 days per week.
  • 50 mg/kg siramesine was administered 7 days/week in combination with a single dose of etoposide (i.p.) up to 30 mg/kg (administered on day one). Tumor volumes were estimated by the use of a caliper. The effect of the drugs on the tumor growth was monitored over 14-21 days before the mice were sacrificed.
  • Siramesine effectively induces programmed cell death in cultured tumor cell lines of various origins, including cell lines originating from tumors of the prostate, breast and cervix.
  • the sensitivity of cells towards Siramesine is increased upon the oncogenic transformation by ras or src, indicating that siramesine has the desired ability of a cancer drug to induce its effects selectively on the cancer cells with only limited effects on normal cells.
  • siramesine when tested against reference sigma ligands such as Haloperidol and pentazocine in WEHI-S and MCF7 cells, siramesine was a significantly more potent inducer of apoptosis than Haloperidol and pentazocine was.
  • the mode of death induced by siramesine is caspase-independent based on the nuclear morphological changes during the death process, the absence of protection by pharmacological caspase inhibitors, and the absence of effector caspase activation Instead, release of lysosomal cathepsins into the cytosol seems to be involved in the execution of the death process.
  • siramesine was well tolerated in vivo and showed an anti-tumorigenic effect in a syngenic tumor xenograft model in BALB/c mice.
  • a combinational effect of siramesine and etoposide was observed in vivo as compared to groups single-treated with siramesine and etoposide respectively.
  • siramesine worked in a synergistic manner together with etoposide, doxorubicin, staurosporin, vincristine and tamoxifen in induction of cell death in WEHI-S cells.

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  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Dermatology (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

L'invention concerne le traitement du cancer. L'invention concerne en particulier des compositions pharmaceutiques contenant de la siramésine, destinées au traitement du cancer. L'invention concerne encore une méthode de traitement consistant à administrer de la siramésine à un patient atteint d'un cancer.
PCT/DK2004/000885 2003-12-19 2004-12-17 Utilisation de la siramesine dans le traitement du cancer WO2005058324A1 (fr)

Priority Applications (11)

Application Number Priority Date Filing Date Title
BRPI0416019-3A BRPI0416019A (pt) 2003-12-19 2004-12-17 uso de siramesina ou de um seu sal farmaceuticamente aceitável, composição farmacêutica, kit, e, método para o tratamento de cáncer
EA200600987A EA011170B1 (ru) 2003-12-19 2004-12-17 Применение сирамезина в лечении злокачественных опухолей
EP04803036A EP1715864A1 (fr) 2003-12-19 2004-12-17 Utilisation de siramesine dans le traitement du cancer
CA002550611A CA2550611A1 (fr) 2003-12-19 2004-12-17 Utilisation de la siramesine dans le traitement du cancer
JP2006544219A JP2007514666A (ja) 2003-12-19 2004-12-17 癌の治療にシラメシンを使用する方法
AU2004298722A AU2004298722A1 (en) 2003-12-19 2004-12-17 Use of siramesine in the treatment of cancer
US11/314,796 US20060178389A1 (en) 2003-12-19 2005-12-21 Use of siramesine in the treatment of cancer
IS8391A IS8391A (is) 2003-12-19 2006-03-30 Notkun á síramesíni við meðhöndlun á krabbameini
IL175774A IL175774A0 (en) 2003-12-19 2006-05-18 Use of siramesine in the treatment of cancer
NO20063319A NO20063319L (no) 2003-12-19 2006-07-18 Anvendelse av siramesin i behandlingen av cancer
US12/426,520 US20090203721A1 (en) 2003-12-19 2009-04-20 Use of siramesine in the treatment of cancer

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US53167003P 2003-12-19 2003-12-19
US60/531,670 2003-12-19
DKPA200301889 2003-12-19
DKPA200301889 2003-12-19

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US11/314,796 Continuation US20060178389A1 (en) 2003-12-19 2005-12-21 Use of siramesine in the treatment of cancer

Publications (1)

Publication Number Publication Date
WO2005058324A1 true WO2005058324A1 (fr) 2005-06-30

Family

ID=34702153

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2004/000885 WO2005058324A1 (fr) 2003-12-19 2004-12-17 Utilisation de la siramesine dans le traitement du cancer

Country Status (8)

Country Link
EP (1) EP1715864A1 (fr)
JP (1) JP2007514666A (fr)
AU (1) AU2004298722A1 (fr)
BR (1) BRPI0416019A (fr)
CA (1) CA2550611A1 (fr)
EA (1) EA011170B1 (fr)
NO (1) NO20063319L (fr)
WO (1) WO2005058324A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002013789A1 (fr) * 2000-08-15 2002-02-21 H. Lundbeck A/S 4omposition pharmaceutique contenant 1'-[4-[1-(4-fluorophenyl)-1h-indole-3-yl] -1-butyl]-spiro[isobenzofuran-1(3h),4'-piperidine]
EP1224930A1 (fr) * 2001-01-22 2002-07-24 Pfizer Products Inc. Combination d'un SRI et d'un ligand du récepteur sigma pour le traitement de la depression

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002013789A1 (fr) * 2000-08-15 2002-02-21 H. Lundbeck A/S 4omposition pharmaceutique contenant 1'-[4-[1-(4-fluorophenyl)-1h-indole-3-yl] -1-butyl]-spiro[isobenzofuran-1(3h),4'-piperidine]
EP1224930A1 (fr) * 2001-01-22 2002-07-24 Pfizer Products Inc. Combination d'un SRI et d'un ligand du récepteur sigma pour le traitement de la depression

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BRENT P J ET AL: "Sigma Binding site ligands inhibit cell proliferation in mammary and colon carcinoma cell lines and melanoma cells in culture", EUROPEAN JOURNAL OF PHARMACOLOGY, vol. 278, no. 2, 1995, pages 151 - 160, XP002325458, ISSN: 0014-2999 *
CRAWFORD KEITH W ET AL: "Sigma-2 receptor agonists activate a novel apoptotic pathway and potentiate antineoplastic drugs in breast tumor cell lines", CANCER RESEARCH, vol. 62, no. 1, 1 January 2002 (2002-01-01), pages 313 - 322, XP002325457, ISSN: 0008-5472 *
PERREGAARD JENS ET AL: "Sigma Ligands with Subnanomolar Affinity and Preference for the omega-2 Binding Site. 1. 3-(omega-Aminoalkyl)-1H-indoles", JOURNAL OF MEDICINAL CHEMISTRY, vol. 38, no. 11, 1995, pages 1998 - 2008, XP002325456, ISSN: 0022-2623 *

Also Published As

Publication number Publication date
BRPI0416019A (pt) 2007-01-02
EA200600987A1 (ru) 2006-10-27
AU2004298722A1 (en) 2005-06-30
NO20063319L (no) 2006-07-18
CA2550611A1 (fr) 2005-06-30
EA011170B1 (ru) 2009-02-27
JP2007514666A (ja) 2007-06-07
EP1715864A1 (fr) 2006-11-02

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