EA011170B1 - Use of siramesine in the treatment of cancer - Google Patents

Abstract

The present invention relates to the treatment of cancer. In particular, the invention provides pharmaceutical compositions comprising siramesine for the treatment of cancer. The invention further provides a method of treatment comprising administering siramesine to a patient suffering from cancer.

Description

FIELD OF THE INVENTION

The present invention relates to the use of siramezin for the preparation of drugs effective for the treatment of malignant tumors.

BACKGROUND OF THE INVENTION

Tumor cells often have acquired resistance to classical therapeutic techniques, such as classical caspase-mediated apoptosis. However, there remains a large unmet need for new effective drugs for the treatment of malignant tumors. It is known that sigma2 receptors are stimulated in cells representing many different types of malignant tumors (Run and others 1991, Sapseg Kek. 51, 6558; UPPSG et al. 1995, Sapseg Kek. 55, 408), and, in addition, ligands of sigma2 receptors can inhibit cell proliferation and induce apoptosis in tumor cells (Bgep! & Raid, 1995, Eig. 1. Pagtaco. 1. 278, 151; SgMogb & Va \\ 'ep. 2002, Sapseg Kek. 62, 313). Additionally, it was shown that sigma2 ligands can potentiate the activity of antitumor drugs (Sga ^ Togb & Bo ^ ep, 2002, Sapseg Kek. 62, 313). International Patent Publication No. XVO 92/22554 describes a number of sigma receptor ligands that are considered effective in treating a number of mental and neurological disorders. The relationship of the structure of these compounds with their activity was also investigated by Reggedaagb, 1. et al., 1. Meb. Svet., 1995, 38, 11, p. 1998-2008.

Among numerous other compounds in νθ 92/22554, 1 '- [4- [1- (4-fluorophenyl) -1H-indol-3-yl] -1-butyl] spiro [isobenzofuran-1 (3H) 4'-piperidine] compound is disclosed

(siramezin), a new use, which is the subject of the present invention.

The inventors have unexpectedly discovered that siramezin has a significantly more potent antitumor activity than sigma2 ligands according to the above link.

SUMMARY OF THE INVENTION

The present invention relates to a medicament for treating a malignant tumor. Sigma2 receptor ligands were known as agents inducing apoptosis in tumor cells of various origins. The inventors have unexpectedly discovered that, according to the invention, siramezin, when used alone, more potently induces apoptosis in tumor cells than sigma2-active compounds according to the above link, such as haloperidol. In addition, the authors observed a significant synergistic effect of siramesin, used in combination with known chemotherapeutic compounds, such as etoposide, doxorubicin, staurosporine, vincristine and tamoxifen. Therefore, the present invention demonstrates that siramezin can be used to produce pharmaceutical compositions for treating a malignant tumor and that such compositions can be used in combination with other chemotherapeutic antitumor drugs and / or in combination with radiation therapy. With the combined use of siramezin and antitumor chemotherapeutic drugs, drugs can be administered simultaneously or sequentially.

In one embodiment, the present invention relates to the use of siramezin or its pharmaceutically acceptable salt together with a chemotherapeutic agent in a synergistic effective dose for the preparation of a pharmaceutical composition as described above, which is adapted for the simultaneous administration of the active ingredients. In particular, such pharmaceutical compositions may contain the active ingredients in the same unit dosage form, for example, in the same tablet or capsule. Such unit dosage forms may contain the active ingredients in the form of a homogeneous mixture or in separate compartments of the unit dosage form.

In another embodiment, the present invention relates to the use of siramezin or a pharmaceutically acceptable salt thereof together with a chemotherapeutic agent in a synergistic effective dose for the preparation of a pharmaceutical composition or kit, as described above, which are adapted for sequential administration of the active ingredients. In particular, such pharmaceutical compositions may contain the active ingredients in separate unit dosage forms, for example, separate tablets or capsules containing any of the active ingredients. These individual unit dosage forms may be contained in the same container or package, for example a blister pack.

As used herein, the term “kit” means a pharmaceutical composition containing each of the active ingredients, but in separate unit dosage forms. As used herein, the term “synergistic effective dosage” refers to dosages of siramezin and chemothera

- 1 011170 songs, in which their combined use provides a synergistic effect, preferably the maximum achievable synergistic effect.

According to the invention, the pharmaceutical composition or kit can be adapted for the simultaneous administration of the active ingredients or for the sequential administration of the active ingredients, as described above.

In particular, the present invention relates to a new use of siramezin having the general formula or its pharmaceutically acceptable salts for the preparation of a medicament for the treatment of a malignant tumor.

In addition, the present invention relates to a method for treating a malignant tumor, comprising administering to an individual in need thereof a pharmaceutically acceptable amount of siramezin or a pharmaceutically acceptable salt thereof.

In another aspect, the invention relates to a method for treating a malignant tumor, comprising administering siramezin or a pharmaceutically acceptable salt thereof to an individual who has been or is undergoing treatment with a chemotherapeutic agent.

According to the invention, the compound 1 '- [4- [1- (4-fluorophenyl) -1H-indol-3-yl] -1-butyl] spiro [isobenzofuran-1 (3H), 4'-piperidine] (siramezin) may be applied as a base or as a pharmaceutically acceptable acid addition salt or as an anhydrate or hydrate of such a salt. Salts of the compounds used in the invention are salts formed with non-toxic organic or inorganic acids. Of these organic salts, salts with maleic, fumaric, benzoic, ascorbic, succinic, oxalic, bismethylene salicylic, methanesulfonic, ethanedisulfonic, acetic, propionic, tartaric, salicylic, citric, gluconic, milk, apple, almond, cinnamon, almond, cinnamon, are used , palmitic, itaconic, glycolic, para-aminobenzoic, glutamic, benzenesulfonic and theophylline acetic acids, as well as 8-halogenophyophyllines, for example 8-bromteophylline. Of these inorganic salts, salts with hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric and nitric acids are used. Preferably, the compound is used in the form of a fumarate or hydrochloride salt.

Fumarate 1 '- [4- [1 - (4-fluorophenyl) -1H-indol-3-yl] -1-butyl] spiro [isobenzofuran-1 (3H), 4'piperidine] can be prepared as described in Retgsdaagb , 1. et al., 1. Meb. Siesch., 1995, 38, 11, 1998-2008 (compound 141), and the base and other pharmaceutically acceptable salts can be obtained there in accordance with standard procedures.

Thus, according to the invention, acid-added salts can be obtained by treating 1 '- [4- [1 - (4-fluorophenyl) -1H-indol-3-yl] -1-butyl] spiro [isobenzofuran-1 (3H) , 4'-piperidine] acid in an inert solvent, followed by precipitation, isolation and, optionally, recrystallization by known methods and, if desired, micronization of the crystalline product by wet or dry grinding, or another convenient process, or by preparing particles from the solvent emulsification process.

Preferably, the precipitation of the salt is carried out in an inert solvent, for example, an inert polar solvent, such as an alcohol (e.g. ethanol, 2-propanol and n-propanol).

According to the invention, 1 '- [4- [1 - (4-fluorophenyl) -1H-indol-3-yl] -1-butyl] -spiro [isobenzofuran1 (3H), 4'-piperidine] or a pharmaceutically acceptable salt thereof may be administered in any suitable way, for example orally or parenterally, and they can be presented in any suitable form for such administration, for example in the form of tablets, capsules, powders, syrups or solutions or dispersions for injection. Preferably and in accordance with the object of the present invention, the compound according to the invention is administered in the form of a solid pharmaceutical object, respectively, in the form of a tablet or capsule, or in the form of a suspension, solution or dispersion for injection.

Methods for preparing solid pharmaceuticals are well known to science. Thus, tablets can be prepared by mixing the active ingredients with conventional adjuvants and / or diluents and subsequently compressing the mixture in a convenient tablet machine. Examples of adjuvants or diluents include: corn starch, lactose, talc, magnesium stearate, gelatin, lactose, resins and the like. Any other adjuvant or additive, such as colorants, aromas, preservatives, etc., may also be used provided that they are compatible with the active ingredients.

As used herein, the term “composition” refers to a product including certain ingredients.

- 2 011,170 diets in certain quantities, as well as any product that directly or indirectly arises from a combination of certain ingredients in certain quantities. Pharmaceutical compositions containing the active ingredient may be in a form suitable for oral administration, for example, in the form of various types of tablets, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral administration may be prepared in accordance with any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweeteners, flavors, colorants and preservatives, to provide pharmaceutically enjoyable and acceptable drugs. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These fillers 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 crossscarmellose, corn starch or alginic acid; binders, for example starch, gelatin, polyvinylpyrrolidone or gum arabic, and lubricants, for example magnesium stearate, stearic acid or talc.

According to the invention, the compositions can be used to treat a malignant tumor in mammals, preferably humans.

Siramezin or its salt for use according to the invention is most conveniently administered orally in unit dosage forms, such as tablets or capsules containing the active ingredient (calculated as free form) in an amount of from about 0.01 to 100 mg / kg / day, preferably from 0 01 to 30 mg / kg / day body weight, most preferably 0.5 to 7.0 mg / day / kg body weight. When siramezin is combined with other compounds to obtain an increased effect or to enable the use of a subnormal dose of another compound to minimize side effects, then subnormal doses of siramesin and / or other compound may be used for treatment. Calculation of specific doses for patients is a common practice for specialists in this field. The pharmaceutical compositions and methods of the present invention are considered particularly effective for the treatment of malignant tumors, including solid tumors such as carcinomas of the skin, breast, brain, cervix, testicular carcinoma, etc. In particular, malignant tumors that can be treated with the compounds, compositions and methods of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Pulmonary: bronchogenic carcinoma (squamous, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulin, glucagon, gastrinoma, carcinoma, lymphocytic carcinoma, carcinoma, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), colon (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); Genitourinary tract: kidneys (adenocarcinoma, Wilms tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate gland (adenocarcinoma, sarcoma), testicle (seminoma, carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, intermediate cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma; Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma y, hepatocellular adenoma, hemangioma; Bones: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrohistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticular cell sarcoma), multiple myeloma, malignant glandular tumor cell (tumor), , chondroblastoma, chondromixofibroma, osteoidostomy and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, deforming osteitis), about membranes (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma diverse, oligodendroglioma, schwann, retinoblastoma, congenital tumors), cerebrospinal neurofibroma, m); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, precancerous cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulomas cell carcinoma, cellulomas of the tumor cell, cell carcinoma of the cell, cellulomas of the cell wall, tumorous cellulomas, , vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma ohm, mixed mesoderm tumor (embryonic rhabdomyosarcoma), fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acute or chronic

- 0101703], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi’s sarcoma, drifts of dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma. Thus, the term “malignant cell,” as provided herein, includes a cell that is affected by any of the conditions identified above, and the term “malignant tumor” includes, but is not limited to, any of the conditions identified above. Any of the conditions mentioned above should be considered as separate embodiments, and compositions aimed at treating each condition, respectively, can be formulated individually or can be included in the group of claims when the term cancer is used. When any of the above indications for the treatment of a malignant tumor is mentioned with respect to the use of siramezin, pharmaceutical composition, kit, treatment method, an individual embodiment is meant. Accordingly, each of the above indications can be individually formulated together with the indicated use of siramezin, pharmaceutical composition, kit, method of treatment and method for identifying compounds effective for treatment.

Experimental procedure

Cell culture

Murine fibrosarcoma cells ^ EN1-B, ^ и902, \ Up912 and \ УEN1-В4 are normal, vector-controlling, highly-producing Ncr70 and resistant to 11ΤΝΕ cells, respectively. Other types of test cells include: human breast tumor cells types MCE7-B1 and MOA-MB-468, and non-oncogenic immortal breast epithelial cells HB100. MCE7 sacr. 3.1 and -3.3 and peo2 are separate clones of cells expressing caspase-3 and vector. MCE7 pCEP, -Bc12 \ UT cells expressing the vector, wild type Bc12, are limited, Bc12 ΝΤ, -Bc12 Ac1a and -Bc12 Cb5 are separate clones, Bc12 cytoplasm bounded by Bc12 mitochondria and bounded by EP. Vs12 (Mala Nouvek Napkep, Aror1ok18 Lebogaogu, Ωαηίδΐι Sapseg Bos1e! Y). The cell line of human neuroblastoma δΚ-Ν-MS cells (ATCC, USA). In addition, the human cervical carcinoma cell lines HeBa (kindly provided by Dr. TEisak, Eash811 Sapseg Bos1e1u) and ME180 were tested. HEK2 93-A, a PC3 prostate cancer cell line, and the non-oncogenic immortal эп1Α prostate epithelial cell line were also tested. Non-transformed MH3T3 murine fibroblasts were kindly provided by C. No1begd (Ipschetkyu! Sorepyadep, Denmark). The fibroblasts were transformed by pBaE-rigo longing, -BU40BT. -v-Na-hack, -s-kgf, as described (Eyepetbaseg et al., 2004). Cells were propagated in EMEM Dytodep, Ra1k1y, UK) supplemented with 10% heat-inactivated calf serum (Vyuodok1 1pb8yte8, Wei Nayetek, Israel), 0.1 mM non-essential amino acids (Chygodep) and antibiotics or PPM1-1640 Dtyodep ) supplemented with 6% heat-inactivated calf serum and antibiotics at 37 ° C in a humidified air atmosphere with 5% CO ₂ . Cells were retested and found negative for mycoplasma by staining yoosyke! (H-33342, Molecularis, Proof, Eidepe, OK).

The binding of [ ³ N] Siramezin to cell membranes or tissue membranes

The presence of siramesin-sensitive binding sites on tissue prepared from cell lines as described above, or tissue from a rodent or human, was demonstrated using the [ ³ H] Lb 28-179 (siramezin) binding test described previously by Bju, L. and others, No. Igorattaso., 2002, 42, 95-100. Briefly, cells were cultured as described, collected in phosphate buffered saline using a scrubber, and centrifuged (1000 x n, 10 min). The resulting granules were used for [ ³ H] Eu 28-179 binding assays, as described by BoLu et al., 2002.

Apoptotic stimuli

The following apoptotic stimuli were tested: Syramezin (Lb 28-179), analogs of siramezin: 28131M, 28134M, 292880, 32160E, 32124C and 32168E and haloperidol (N. Lippbesk A / B, Copenhagen, Denmark), human ΤΝΕ-α (Bjufta Sty, Germany), thapsigargin, etoposide, doxorubicin, staurosporine, vincristine, tamoxifen (BYUMA-A1bysy, San Luis, Missouri), concanamycin A (A1ex1k Vusyetuana, San Diego, California).

Pharmacological Inhibitors and Medicines

The following protease inhibitors were used: / UAE-Gtk, EEEE-Gtk (Vasiett WiebbogG, Switzerland), EEUE-CHO (Nooku81et8, Strasbourg, France), ENP-CHO, / EA-Gtk (Epute Buket Rgoyisk Etetoge, California) CA-074-Me (Rerybek 1n1ethiopa1, Louisville, ΚΥ), APC11138 (Ce1ega ArrNeb Vu8u81et8, Ecokeg Syu, California, USA), Pepstatin A, PE150606 and Ca1Pix I (C1) inhibitor (Ca1bysoka, Ta Voyeppdet Mappie1t), pnefablock (AEB e \ '1 sec. BE) (Rosye Όίadpokysk, ΌΚ). The following antioxidants were used: Butylated hydroxyanisole (BHA), α tocopherol, γ-tocopherol, glutathione ethyl ether (RLS), АС-acetylcysteine SCHAS) (BYUMA-A1bys, San Luis, Missouri). Cells were preincubated with inhibitors and antioxidants 1 hour before the drug.

- 4 011170 natural means. In addition, the effects of the following drugs on the cytotoxicity of siramezin were tested: sigma2 receptor antagonists ΒΌ1047 (TosGE) and AC927 (contribution from \ ν.Βο \ νοη. ΒΐΌ \ νπ of the United States), 3-methyladenine, actinomycin Ό, cyclohexamide and .

Cell death detection Cell viability

Cell viability was measured using the MTT contraction test. The conversion of the tetrazolium salt of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazodium bromide (MTT, 5> 1СМЛ-Л10пс11) to the blue-colored formazan product was evaluated spectrophotometrically by absorption at 570 nm using a microplate reader Uegkatah (Mo1eci1ag Eeyu1se5). Survival was defined as the percentage of untreated cells or cells treated with the inhibitor: Cells were plated on a 96-well plate containing 200 μl of medium, and the next day treated with drugs. The effect on cell viability was assessed 24-60 hours after drug addition by removing 100 μl of medium and adding 25 μl of MTT solution (1 mg / μl in filter sterilized ΡΒ8). After 3 hours of incubation at 37 ° C in the dark, using 100 μl of solubilization buffer (20% 8E8 in a 50% solution of dimethylformamide), the cell permeability was increased, and the next day they were analyzed on a spectrophotometer.

Cell death and cytotoxicity

Cell death and cytotoxicity were assessed using a lactate dehydrogenase (LDH) releasing sample (Cosier, Mappiet, Germany). The destruction of the plasma membrane, releasing cytoplasmic LDH into the culture medium, is a measure of cytotoxicity or cell lysis. The enzymatic activity of LDH, leading to the oxidation of lactate to pyruvate and the reduction of ΝΑΌ ⁺ to ΝΑΌΗ + Η ⁺ , was measured spectrophotometrically by converting the tetrazolium salt (yellow) to the formazan salt (red). Cells were seeded and treated as described for the MTT assay. After analysis, 50 μl of medium was removed and mixed with 50 μl of reaction buffer. The remaining medium was removed and the cells were lysed in 1% Triton X-100 for 20 min at 37 ° C. The LDH content was also measured in the cell lysate and the% LDH release was estimated:

Cytotoxicity (% LDH release) = LDH _In medium / (LDH _in medium + LDH _in lysate) X 100%

Nuclear seal

The form of cell death was assessed by the type of nuclear compaction when staining NoosyCH (permeable to the cell NoosyCH 33342, 5> 1CMA-A1yps11) cells treated with the drug. The cell-impermeable 8u1ox OGeep (Mo1ecioptera Pyoec) was used to assess the loss of membrane integrity, and the results were compared with the morphological changes in the nuclei observed with the use of Nooches. Cells were cultured with a 10,000x dilution of 25 mg / μl NoosyCH and / or 10,000x dilution of 2 mM 8U1Oh DGHEEP for 5 min at 37 ° C, after which the type of nuclear compaction and possible co-staining were analyzed using an inverted O1utrik ΙΧ- microscope 70.

Gel electrophoresis to detect DNA cleavage

Cells were harvested and cultured at 50 ° C. overnight in 120 μl of lysis buffer (100 mM No. C1, 100 mM Tris-HC1 (pH 8.0), 25 mM 0,5, 0.5% 8Ό8 and 100 μg / ml proteinase TO). Samples were precipitated with 6M No. 1S.'1 and centrifuged at 13,000 rpm for 5 min at 4 ° C. Genomic DNA was subsequently precipitated from the supernatant with 2.5 volumes of 96% ethanol. After centrifugation at 20,000 rpm for 10 min at 4 ° C and washing with 70% ethanol, the granules were dissolved in 10 mM Tris-HCl (pH 8.0) and 1 mM ΕΌΤΑ containing 1 μg / ml Vpake Α. Samples were cultured at 37 ° C for 1.5 h and the DNA concentration was estimated by absorbance (A) at 260 nm. DNA electrophoresis (5 μg / lane) was performed on a 1.5% agarose gel and DNA was visualized by staining with ethidium bromide.

Caspase and cathepsin activity

To measure the activity of the cytosolic enzyme cysteine cathepsin, sublimate cells seeded on 24-well plates were treated with extraction buffer (250 mM sucrose, 20 mM ΗΕΡΕ 8, 10 mM KCl, 1.5 mM MdC1 ₂ , 1 mM ΕΌΤΑ, 1 mM ΕΟΤΑ, 1 mM pefablock; pH 7.5) containing 20 mg / ml digitonin for 12-15 minutes on ice. To measure the total activity of the cysteine cathepsin cell enzyme, the cells were treated with the above extraction buffer containing 200 μg / ml digitonin for 12-15 minutes on ice. To analyze 3/7-like activity, the sub-mating cells were treated with caspase extraction buffer (0.5% Triton X-100, 25 mM ΗΕΡΕ8, 5 mM MdC1 ₂ , 1 mM ΕΟΤΑ, 1 mM pefablock, pH 7.5) for 20 minutes on ice. Activity similar to caspase 3/7 activity and cysteine cathepsin activity was evaluated by adding one volume of 20 μM Ls-OHUE-LBS (Vyuto1) to the reaction buffer with caspase (100 mM ΗΕΡΕ8, 20% glycerol, 0.5 mM ΕΌΤΑ, 0.1 % ΟΗΑΡδ, 5 mM ΌΤΤ, 1 mM pefablock, pH 7.5) or 20 μM ζΓΒ-ΑΕΠ ^ put 8y1et ΡΐΌάικΕ) into the reaction buffer with cathepsin (50 mM sodium acetate, 4 mM ΕΌΤΑ, 8 mM ΌΤΤ, 1 mM pefablock, pH 6.0), respectively. V _max allocation ΑΕΠ (excitation 400 nm, emission 489 nm) was analyzed on

- 5 011170 was performed for more than 20 min at 30 ° С using a fluorometer 8resetgate Setip (Mo1eci1ag Ωονίοοδ. Sunnyvale, California).

Lysosomal stability test. cell culture

The cells were exposed to the lysosomotropic weak base and the metachromatic fluorochrome of acridine orange (AO. MoCecilagin Prococcus), which accumulates in acidic chambers. Highly concentrated AO in acidic lysosomes shows red fluorescence, but after relocalization, green fluorescence in the cytoplasm. To control lysosomal integrity, sublimate drug-treated cells were exposed to 0.1-0.5 μg / ml AO for 3 hours at 37 ° C. Cells were evaluated either using an O1utric 1-70 inverted microscope, or separated from the substrate and analyzed by flow cytometry using a PAC8 (VesUp Pückshkoy, San Jose, California) with an argon ion laser with an output wavelength of 488 nm, and analyzed using a software providing SEBE-Oiekk Test of lysosomal stability ίπ Uygo

MCP-7 cells, seeded on 14 cm plates, were loaded with Re-dextran for 9 hours, followed by 16-hour purification in culture medium. Subsequently, the cells were washed in PB8 and separated from the substrate. The cell fraction was resuspended in 8СЛ buffer (20 mM Nerek KOH, 10 mM KC1, 1.5 mM MdC1 ₂ , 1 mM ΕΌΤΆ, 1 mM ECTA, 250 mM sucrose, pH 7.5) and balanced on ice for 20 minutes. The cell fraction was homogenized with 15-200 strokes of a Teflon pestle. After centrifugation for 5 min at 750 d, the supernatant was isolated and the centrifugation step was repeated. The remaining supernatant was transferred to a column in a magnetic field. The lysosomal fraction was washed twice and eluted from the column. The release of lysosomal cathepsins was subsequently measured in 25 μl of a solution of lysosomes in a black antagonist in 96-well plates, as described for measuring cathepsin activity. After 1.5 hours of reaction at 37 ° C, the _maximization of ARS was analyzed for more than 20 min at 30 ° C using a fluorometer with 8 cells of Oet1 (Mo1eciag Eeeuyue, Sunnyvale, California).

Immunoblot analysis and immunofluorescence

Primary antibodies used included murine monoclonal antibodies against cathepsin B (Art. Vekeagius Przobysp, Boston, MA), cathepsin L (Thaikbisjoe Baogoda, Jupec Lexington, ΚΥ), cytochrome c (ΒΌ Ryagtsdedey), and Gydergel , Nkr70 (2H9; kindly provided by Boris Morgulis, St. Petersburg, Russia) and rabbit polyclonal cathepsin Ό (EAKO sogrogayoi, California), and A1P (aor1ok18 1ab, Eash811 Saiseg Vekeagsy). For immunoblot analysis, proteins were separated by 6-12% δΌδ-PAOE and transferred to a nitrocellulose membrane. After incubation of the primary antibodies for 1 h in real time or at 4 ° C during the night, the blot was cultured with secondary goat anti-mouse or anti-rabbit 1dC antibodies conjugated to horseradish peroxidase for 1 h in real time. Subsequent detection of the protein was performed using ECb or ECb1k (Ategkyat Wyckscheisek, Buckinghamshire, England).

In order to visualize cathepsins, A1P and cy1C, the cells were fixed and their permeability in -20 ° С methanol was increased for 10 min at 25 ° С. After blocking with 5% goat serum (in 1% B8A, 0.3% Triton X-100 in PB8) for 20 min, the cells were cultured with primary antibodies (0.25% B8A, 0.1% Triton X-100 in PB8), as indicated, for 1.5 hours. After 1 hour of incubation with secondary antibodies A1ex P1iog-488 or -546-conjugated anti-mouse 1dC or anti-rabbit 1dC (Mo1ci1ag Prgoec) cells were washed three times in 0.05% Tween 20 in PB8 and are attached using the Kit Prgoid AiNGabe (Mo1ci1ag Prgoec). A confocal analysis was carried out using a YeX Ahuuei 100M microscope with L8M 510 software.

Experiments w u1uo

Cells UEN1-P4 (5 x 106) were implanted subcutaneously on the back of immunocompetent female BABB / s mice. Treatment with siramesine began two days before the implantation of the tumor. Mice were divided into groups (5–9 / group), and 200 μl of 1) vehicle (0.5% methylcellulose 15 in 0.9% solution ΜιΟ) was orally administered, 2) 100 mg / kg / day of siramesin in suspension (in 0.5% methylcellulose 15 in a 0.9% solution No. 1C1) 7 days a week. For combination studies of 1 and 1 uo, 50 mg / kg of siramezin was administered for 7 days / week in combination with a separate dose of etoposide (intraperitoneally) to 30 mg / kg (administered on the first day). Tumor volumes were evaluated using caliper. The effect of drugs on tumor growth was monitored for more than 14-21 days before the killing of mice.

results

In a dose-dependent manner, siramezin 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 to siramesin is increased after oncogenic conversion of HAC or kgf, showing that siramesin has the desired antitumor drug efficacy to induce its selective effect on cancer cells with only limited effects on nor

- 6 011170 male cells. In addition, siramezin, when tested against the background of sigma ligands in the cited link, such as haloperidol and pentazocine, in ^ EN1-8 and M8P7 cells, was a significantly more powerful apoptosis inducer than haloperidol and pentazocin. The death mode induced by siramezin is caspase-independent based on morphological changes in the nuclei during the death process, the lack of protection by pharmacological caspase inhibitors and the absence of a caspase activation effector. Instead, the release of lysosomal cathepsins into the cytosol seems to be involved in the death process. This finding is based on immunohistochemical staining of lysosomal cathepsins B and b, as well as the release of cathepsins ίη νίΐτο from purified lysosomes. Also, the use of pharmacological cathepsin inhibitors can reduce the death induced by siramesin. Tumor cells that were protected from most other antitumor drugs by ectopic expression of Bcl-2 were effectively killed by siramezin. While some chemotherapeutic agents activate the p53-dependent death pathway, siramezin does not activate p53. This shows that the death pathway is significantly different from that induced by DNA damaging agents (such as etoposide), a result that confirms the data shows a wide range of indications for the treatment of a malignant tumor for siramezin, since the p53-dependent death pathway is often compromised in malignant tumors. It is also based on the aforementioned observation that siramesin-induced tumor cell death is not inhibited by Bcl-2.

It is important that siramezin was well resistant to ίη νίνο and showed an anti-oncogenic effect in an isogenic tumor xenograft model on BABB / c mice. In addition, the combination effect of siramesin and etoposide was observed ίη νίνο in comparison with groups treated separately with siramesin and etoposide, respectively. In addition, siramezin acts synergistically with etoposide, doxorubicin, staurosporin, vincristine and tamoxifen in the induction of cell death in ^ EH1-8 cells. These results show that siramezin is a new antitumor drug that is especially effective in comparison with other reference sigmaligands and which can be used alone or in combination with conventional chemotherapeutic agents for treating malignant tumors.

Claims (10)

CLAIM 1. The use of Siramesin or its pharmaceutically acceptable salt for the preparation of a pharmaceutical composition for use in the treatment of a malignant tumor. 2. The use of Siramesin or its pharmaceutically acceptable salt for the preparation of a pharmaceutical composition intended to increase and / or provide an enhanced effect of a chemotherapeutic agent in the treatment of a malignant tumor. 3. The use of Siramesine or its pharmaceutically acceptable salt for the preparation of a pharmaceutical composition for use in combination with a chemotherapeutic agent in the treatment of a malignant tumor. 4. The use of Siramesin or its pharmaceutically acceptable salt for the preparation of a pharmaceutical composition for use in the treatment of a malignant tumor, where the pharmaceutical composition further comprises another chemotherapeutic agent. 5. The use according to any one of claims 1 to 4, wherein the malignant tumor is selected from lung cancer, prostate cancer, breast cancer, glioma, neuroblastoma, melanoma, leukemia, bone marrow tumor or skin cancer. 6. The use according to any one of claims 2 to 4, wherein the chemotherapeutic compound is selected from etoposide, doxorubicin, staurosporine, vincristine and tamoxifen or their pharmaceutically acceptable salts. 7. The use according to any one of claims 1 to 6, wherein said cancer treatment is combined with radiation therapy. 8. The use according to any one of claims 1 to 7, where siramesine is used as fumarate salt or hydrochloride. 9. A method of treating a malignant tumor, comprising administering to the patient an effective amount of siramesine or a pharmaceutically acceptable salt thereof. 10. The method of claim 9, wherein the malignant tumor is selected from lung cancer, prostate cancer, breast cancer, glioma, neuroblastoma, melanoma, leukemia, bone marrow tumor, or skin cancer.