Aminothiolester compounds or pharmaceutically acceptable salts thereof, for use for the treatment of cancer

Abstract

The present invention relates to the treatment of cancer in a subject, wherein cancer cells of said subject overproduce H.sub.2O.sub.2 and have a level of GSH below 0.5 nmol for 25 000 cells, with aminothiolester compounds or pharmaceutically acceptable salts thereof, in particular with S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate or a pharmaceutically acceptable salt thereof, more particularly with 4-(Dimethylamino)-4-methyl-2-pentynethioic acid S-methyl ester fumarate. It also relates to a method for selecting a subject suffering from a cancer and who will most likely benefit from a treatment with aminothiolester compounds or pharmaceutically acceptable salts thereof, in particular with S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate or a pharmaceutically acceptable salt thereof, more particularly with 4-(Dimethylamino)-4-methyl-2-pentynethioic acid S-methyl ester fumarate.

Claims

1. A method of treating cancer in a subject in need thereof, comprising: measuring levels of H.sub.2O.sub.2 and GSH in cancer cells of the subject; and if the cancer cells of the subject (i) have a level of H.sub.2O.sub.2 of at least 200% compared with a basal level of H.sub.2O.sub.2 in normal cells originating from the same tissue as the cancer cells, and (ii) have a level of GSH below 0.5 nmol for 25,000 cells; then administering to the subject a therapeutically effective amount of a compound of formula (I): ##STR00010## wherein X1 and X2, identical or different, are chosen among a C.sub.1-C.sub.7 alkyl group, a phenyl, a benzyl or X1 and X2 together with the nitrogen atom to which they are linked form a heterocycle; or a pharmaceutical acceptable salt thereof; or if the cancer cells of the subject do not (i) have a level of H.sub.2O.sub.2 of at least 200% compared with a basal level of H.sub.2O.sub.2 in normal cells originating from the same tissue as the cancer cells, and (ii) have a level of GSH below 0.5 nmol for 25,000 cells; then administering to the subject an anticancer agent other than a compound of formula (I).

2. The method according to claim 1, wherein the heterocycle is a piperidine or a morpholine.

3. The method according to claim 1, wherein said H.sub.2O.sub.2 level is determined by quantifying the level of Fluorescence Intensity.

4. The method according to claim 3, wherein said H.sub.2O.sub.2 level is higher than 20000 Relative Fluorescence Intensity.

5. The method according to claim 1, wherein the GSH level is determined by luminescence.

6. The method according to claim 1, wherein the cancer is chosen from bladder cancer, brain tumors, breast cancer, melanoma, multiple myeloma, leukemia, lymphoma, prostate cancer, cervical cancer, stomach cancer, liver cancer, tongue cancer, ovarian cancer, pancreatic cancer, renal cancer, pleuramesothelomia, osteosarcoma, muscle cancer, lung cancer, kidney cancer, head and neck cancer, colon cancer, blood cancer, cancers of the nervous central system and sarcoma.

7. The method according to claim 1, wherein in said compound of formula (I), X1 and X2, identical or different, are chosen among a methyl, a phenyl, a benzyl, at least one of X1 or X2 being a methyl, or X1 and X2 together with the nitrogen atom to which they are linked form a piperidine or a morpholine.

8. The method according to claim 1, wherein the compound of formula (I) is selected from the group consisting of: S-methyl 4-methyl-4-(piperidin-1-yl)pent-2-ynethioate; S-methyl 4-[benzyl(methyl)amino]-4-methylpent-2-ynethioate; S-methyl 4-methyl-4-[methyl(phenyl)amino]pent-2-ynethioate; S-methyl 4-methyl-4-(morpholin-4-yl)pent-2-ynethioate; and S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate.

9. The method according to claim 1, wherein said compound of formula (I) is the S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate or a pharmaceutical acceptable salt thereof.

10. The method according to claim 9, wherein said pharmaceutical acceptable salt is 4-(Dimethylamino)-4-methyl-2-pentynethioic acid S-methyl ester fumarate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0154] The present invention will be further illustrated by the following figures and examples.

[0155] FIG. 1: efficacy of S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate (viability assay (resazurin, XTT or MTT) on various cancer cell lines

[0156] FIG. 2: H.sub.2O.sub.2/S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate IC.sub.50 correlation: relationship between IC.sub.50 of S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate in μM and the endogenous H.sub.2O.sub.2 activity levels in cancer cells

[0157] FIG. 3: H.sub.2O.sub.2/S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate IC.sub.50 correlation: Determination of a cut-off

[0158] FIG. 4: H.sub.2O.sub.2/S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate IC.sub.50 correlation: Studies by tissue origin

[0159] FIG. 5: Comparison of the H.sub.2O.sub.2 level of melanoma cells (cancer cells) and of melanocytes cells (corresponding normal cells)

[0160] FIG. 6: S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate effect in HL60 cancers cells that overproduce H.sub.2O.sub.2 and have or not a level of GSH below 0.5 nmol for 25 000 cells

[0161] FIG. 7: Inhibition of S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate efficacy by enhancing the level of GSH in cells having an overproduction of a H.sub.2O.sub.2

[0162] FIG. 8: S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate efficacy in Colo357 cells having an overproduction of a H.sub.2O.sub.2 and a level of GSH above 5 nmol for 25000 cells

[0163] FIG. 9: Total GSH level in nmol per 25000 cells observed in sensitive and resistant cells

[0164] FIG. 10: Quantification of MDA and HNE adducts in S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate sensitive cells (HL-60, NT2/D1) (A) and S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate resistant cells (MSC) treated with S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate 5 or 10 μmol.Math.L.sup.−1 during 24 hours (B)

[0165] Note: in all the figures mentioned above: DIMATE is given for S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate.

DETAILED DESCRIPTION

Example 1

[0166] Material and Methods

[0167] Cell Lines

[0168] A panel of 52 human tumor cells representing 10 tissue types has been selected to cover a broad set of different oncogenes and according to their response to different standard chemotherapeutics. Cells were obtained from the American Type Culture Collection (ATCC), the European Collection of Cell Cultures (ECACC) and from primary culture of cancer cells derived from patients' tumors (Research Institute of Vall d'Hebron (VHIR), Barcelone, Spain; Vall d'Hebron Institute of Oncology (VHIO), Barcelone, Spain; Oncotest, Freiburg, Germany; Oncodesign, Dijon, France; Universitá Degli Studi di Palermo, Oncology and Surgical Sciences, Palermo, Italy; and Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Barcelona, Spain (See Table 2 for description of the tumor cell panel).

TABLE-US-00002 TABLE 2 Tissue Characteristics Cells Type of cell line Bladder UM-UC-3 established cell line Bladder HT-1197 established cell line Bladder LB831-BLC established cell line Bladder RT112 established cell line Bladder T24 established cell line Blood HL-60 established cell line Blood OCI-AML2 established cell line Blood Raji established cell line Blood U-937 established cell line Blood Kasumi-1 established cell line Blood MOLM-14 established cell line Blood KG-1 established cell line Blood K-562/imatinib established cell line Blood THP-1 established cell line Brain CGL-1 established cell line Brain SK-N-AS established cell line Brain U-87 established cell line Brain BrGI2 established cell line Brain BrGI3 established cell line Brain BrGI6 established cell line Brain BrA1 established cell line Brain CGL-9 established cell line Breast SK-BR-3 established cell line Breast BT-20 established cell line Breast BT-474C established cell line Breast MCF-7 established cell line Breast MDA-MB-231 established cell line Breast MDA-MB-468 established cell line Breast BA-pt1102 established cell line Breast MBC-pt1106 established cell line Breast BA-pt1201 established cell line Breast BA-pt1202 established cell line Breast BA-pt1205 established cell line Cervix Hela established cell line Colon LoVo established cell line Colon SW620 established cell line Colon COLO-205 established cell line Colon HCT-15 established cell line Colon CRA07 established cell line Colon CRA11 established cell line Colon CRA13 established cell line Head & Neck LB1617-HNSCC established cell line Head & Neck Fadu established cell line Head & Neck KB established cell line Kidney Caki-1 established cell line Kidney A-498 established cell line Liver Hep G2 established cell line Liver Hep 3B2.1-7 established cell line Lung A-549 established cell line Lung A-549 established cell line Lung H-522 established cell line Lung PC-9 established cell line Lung HCC4006 established cell line Lung HCC2935 established cell line Lung H1975 established cell line Lung H1650 established cell line Lung H820  established cell line Lung H2935 established cell line Lung HCC4006 established cell line Lung HCC827  established cell line Lung H1299 established cell line Lung Hop62 established cell line Lung H522 established cell line Lung H23  established cell line Lung H460 established cell line Lung H441 established cell line Melanoma SKMEL-103 established cell line Melanoma SKMEL-147 established cell line Melanoma MLMN-9  established cell line Melanoma MLMN-10 established cell line Melanoma UACC-903 established cell line Melanoma SKMEL-28 established cell line Melanoma MBrM12 established cell line Muscle A-673 established cell line Normal HMVEC established cell line Normal HUV-EC-C established cell line Normal MRC-5 established cell line Normal HSC CD34+ established cell line Normal MSG established cell line Osteosarcoma OS-0411 established cell line Ovary PD-OVC-17 established cell line Ovary A2780 established cell line Ovary A2780/Cis established cell line Ovary IGROV-1 established cell line Ovary SK-OV-3 established cell line Ovary OVCAR-3 established cell line Ovary OVCAR-4 established cell line Ovary PD-OVC-11 established cell line Ovary PD-OVC-02 established cell line Ovary PD-OVC-05 established cell line Pancreas Capan-1 established cell line Pancreas Capan-2 established cell line Ovary PD-OVC-17 established cell line Pancreas MIA PaCa-2 established cell line Prostate 22Rv1 established cell line Prostate LNCap established cell line Prostate PC3 established cell line Prostate DU145 established cell line Stomach KATO III established cell line Bladder 1036 PDC Bladder 1218 PDC Bladder 1228 PDC Bladder 1258 PDC Bladder 1352 PDC Bladder 439 PDC Central Nervous 498 PDC System Cervix 1729 PDC Cervix 1783 PDC Cervix 2025 PDC Cervix 280 PDC Cervix 742 PDC Cervix 94 PDC Cervix 975 PDC Gastric Asian 3013 PDC Asian Gastric Asian 3044 PDC Asian Gastric Asian 3052 PDC Gastric Caucasian 1172 PDC Gastric Caucasian 214 PDC Gastric Caucasian 251 PDC Head and Neck; Caucasian 1842 PDC Lung Adeno 1041 PDC Lung Adeno 1584 PDC Lung Adeno 1647 PDC Lung Adeno 289 PDC Lung Adeno 526 PDC Lung Adeno 623 PDC Lung Adeno 629 PDC Lung Adeno 629 PDC Lung Adeno 923 PDC Lung Adeno 983 PDC Lung Epidermoid 1422 PDC Lung Epidermoid 397 PDC Lung Large Cell 1072 PDC Lung Large Cell 1121 PDC Lung Large Cell 1674 PDC Lung Large Cell 430 PDC Lung Large Cell 529 PDC Breast 1162 PDC Breast 1322 PDC Breast 1384 PDC Breast 583 PDC Breast 713 PDC Breast MX1 established cell line Melanoma 1765 PDC Melanoma 1792 PDC Melanoma 274 PDC Melanoma 276 PDC Melanoma 462 PDC Melanoma 520 PDC Melanoma 622 PDC Melanoma 672 PDC Ovarian 1023 PDC Ovarian 1353 PDC Ovarian 1544 PDC Ovarian 899 PDC Pancreas 1872 PDC Pancreas 1900 PDC Pancreas 1986 PDC Pancreas 2033 PDC Pancreas 2082 PDC Pancreas 2116 PDC Pancreas 546 PDC Prostate DU-145 established cell line Prostate MRI-H-1579 established cell line Prostate PC-3M established cell line Pleurameso- 1752 PDC thelioma Pleurameso- 541 PDC thelioma Renal 1114 PDC Renal 1183 PDC Renal 1393 PDC Renal 486 PDC Renal 616 PDC Sarcoma 1937 PDC Colon  CXF 1297 PDC Colon CXF 243 PDC Colon CXF 280 PDC Colon CXF 647 PDC Colon CXF 676 PDC Gastric Asian GXA 3011 PDC Gastric Asian GXA 3023 PDC Gastric Caucasian GXF 97  PDC Head & Neck Caucasian HNXF 536 PDC Head & Neck Caucasian HNXF 908 PDC Lung Adeno LXFA 400 PDC Lung Adeno LXFA 586 PDC Breast MAXF 449 PDC Breast MAXF 508 PDC Breast MAXF 583 PDC Breast MAXF 713 PDC Breast MAXF MX1 PDC Melanoma MEXF 1829 PDC Melanoma MEXF 989  PDC Ovary OVXF 1023 PDC Ovary OVXF 1353 PDC Ovary OVXF 1544 PDC Ovary OVXF 899  PDC Pancreas PAXF 1998 PDC Pancreas PAXF 2005 PDC Pancreas PAXF 2045 PDC Pancreas PAXF 2046 PDC Pancreas PAXF 2053 PDC Pancreas PAXF 2059 PDC Pancreas PAXF 2094 PDC Pancreas PAXF 546  PDC Renal RXF 1220 PDC Renal RXF 1781 PDC Renal RXF 631  PDC Skin SXFS 117 PDC
All cells were cultivated in appropriate media according to supplier recommendations.

[0169] Treatment with S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate

[0170] Cells were seeded into 96-well cell culture plates at concentrations required to ensure approximately 80% confluence in control (untreated cells) at the end of experiment (0.5×10.sup.4-5×10.sup.4 cell/well). The sensitivity towards S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate was determined using different concentrations of the drug (from 0.01 to 100 μM). After 48 hours, the growth-inhibitory effect of the drug was analyzed using Alamar blue, according to manufactures instructions. The Alamar Blue assay was used based on earlier observations: similar results were found with Alamar blue, tetrazolium reduction (XTT assay). To ensure good data quality and to minimize impact of pipetting errors, each particular drug concentration was assessed based on mean fluorescence intensity from 8 separate wells. The drug response was quantified by the half maximal inhibitory concentration (IC.sub.50) for each particular cell line, and determined by non-linear regression analysis of log-dose/response curves. Cut-off value for resistance to S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate was determined statistically (>2 S.D. above the IC.sub.50 geometric mean). The in-vitro threshold value for hypersensitivity to the drug has been defined as <IC.sub.50 geometric mean.

[0171] 3D Clonogenic Assay, 96 Well Format

[0172] The clonogenic assay test was carried out in 96 well plate format. For each test a frozen aliquot of tumor cells prepared from tumor xenografts was thawed and assay plates were prepared as follows: each test well contains 3 layers of equal volume, 2 layers of semi-solid medium (bottom and top layer), and one layer of medium supernatant, with or without test compound. The bottom layer consists of 0.05 mL/well Iscove's Modified Dulbecco's Medium (Invitrogen), supplemented with 20% (v/v) fetal calf serum (Sigma), 0.01% (w/v) gentamicin (Invitrogen) and 0.75% (w/v) agar (BD Biosciences). Tumor cells were added to 0.05 mL of the same culture medium supplemented with 0.4% (w/v) agar and plated onto the bottom layer. After 24 h test compounds were added after serial dilution in DMSO and transfer in cell culture medium, and left on the cells for the duration of the experiment (continuous exposure, 0.05 mL drug overlay). Every dish includes six untreated control wells and drug-treated groups in duplicate at 10 concentrations. Cultures were incubated at 37° C. and 7.5% CO.sub.2 in a humidified atmosphere for 8 to 13 days and monitored closely for colony growth using an inverted microscope. Within this period, ex vivo tumor growth leads to the formation of colonies with a diameter superior to 50 μm. At the time of maximum colony formation, counts were performed with an automatic image analysis system. 24 h prior to evaluation, vital colonies were stained with a sterile aqueous solution of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (1 mg/mL, 100 μL/well). Sigmoidal concentration-response curves were fitted to the data points obtained for each tumor model using 4 parameter non-linear curve fit (Oncotest Warehouse Software). IC.sub.50 values were reported as relative IC.sub.50 values, being the concentration of test compound that give a response (inhibition of colony formation/viability) half way between the top and bottom plateau of the sigmoidal concentration-response curve (inflection point of the curve), or as absolute IC.sub.50 values, being the concentration of test compound at the intersection of the concentration-response curves with T/C=50%. For calculation of mean IC.sub.50 values the geometric mean was used. Results were presented as mean graph plots or heat maps (individual IC.sub.50 values relative to the geometric mean IC.sub.50 value) over all tumor models as tested.

[0173] Measurement of H.sub.2O.sub.2 Production

[0174] Intracellular H.sub.2O.sub.2 production in live cells was measured using Total ROS/superoxide detection kit (Enzo life science), following the manufacturer's instructions. The assay uses specific H.sub.2O.sub.2/RNS probes that upon reaction with H.sub.2O.sub.2 and RNS species were oxidized rapidly to highly fluorescent compounds. Fluorescence intensity is proportional to the total H.sub.2O.sub.2/RNS levels within the sample. The experimental tests were performed using untreated cells, or cells treated with S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate (1-5 uM), vehicle HEPES, N-acetyl-L-cysteine (NAC) (10 mM). The day before the analysis, cells were seeded in 96-well black/clear bottom plates at a density of 2×10.sup.4 in culture media according to cell suppliers instructions, supplemented with FBS (10% v/v), 10 units of penicillin/mi and 100 mg of streptomycin. Cells were kept at 37° C. in a humid atmosphere of 95% air: 5% CO.sub.2 overnight. On the day of the analysis, cells media was replaced by 100 μL of phenol free-media supplemented as above, without any treatment, with S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate for 6 hours, vehicle HEPES for 6 hours, or with NAC for 20 min. After the incubation period, wells were loaded with 100 μL of H.sub.2O.sub.2 Detection Solution, containing the respective treatment agent or controls and incubated for 60 min at 37° C. in the dark. Several wells were left without cells for the background fluorescence control measurements. Plates were read using fluorescence microplate reader (ex=560 nm, Em=600). Data were expressed as the change in arbitrary fluorescence units produced from equal amounts of cells and normalized to total protein input.

[0175] Measurement of GSH Production

[0176] Cells were seeded in 96-well black walled plate (5×10.sup.4 cells/mL) and incubated overnight for attachment. The cells were treated either with vehicle, DMSO, BCNU 100 μM, S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate 20 μM, for 24 hours. The total glutathione was measured according to the instructions of the kit (GSH-Glo™, Promega) and results were obtained for 25 000 cells.

[0177] Quantitative Determination of HNE-Protein and MDA/Protein Adducts by ELISA

[0178] The formation of HNE-adducts and MDA-adducts was quantified with the Oxiselect HNE Adduct Elisa kit (Cell Biolabs, San Diego, Calif.) and the OxiSelect MDA Adduct ELISA Kit (Cell Biolabs), respectively. Briefly, after a treatment with S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate during 24 hours, cells were lysed by sonication in reducing SDS Sample Buffer. Homogenates were diluted to 10 μg protein/mL and adsorbed in 96-well protein binding plates by incubation at 37° C. for at least 2 hours. Wells were washed twice with PBS and incubated for an additional 2 hours at room temperature on an orbital shaker. Following three washes in PBS, 100 μL of anti-HNE antibody or anti-MDA antibody were added to the wells and incubated for 1 hour at room temperature. Subsequently, goat anti-rabbit secondary antibody-HRP conjugate (diluted 1/1000 with the assay diluent) was added and incubation continued for 1 hour. Wells were washed five times in PBS and HRP-substrate was added. Reaction was stopped with an acidic solution, and absorbance read on a microplate reader at 450 nm. The level of HNE-adducts and of MDA-adducts was determined by comparison with a standard curve prepared from HNE-BSA and MDA-BSA standards supplied by the manufacturer.

[0179] Induction of Resistance to S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate Treated Cells.

[0180] To revert S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate-induced cell death, drug sensitive cells HL-60 were exposed to S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate (10 μM) in the presence of the reduced glutathione analog Glutathione Monoethyl Ester (GSH-MEE) at concentrations 10 mM or S-methyl 4-(dimethylamino)-4-methylpent ynethioate in combination. HL-60 were seeded, into 96-wells plate, at a concentration of 5×10.sup.4 cells/well in 100 μL of complemented medium supplemented with Glutathione Monoethyl Ester (GSH-MEE) 10 mM. After 90 minutes incubation, cells were washed with PBS and cells were incubated using different concentrations of the drug (from 0.01 to 100 μM). After 24 h and 48 h cell viability was measure using the Rezasurin assay as described above.

[0181] Statistical Analysis

[0182] Values are expressed as mean±SD or frequencies and proportions. Differences between groups were determined by unpaired t test, Chi-square, Fisher's exact test or ANOVA, where appropriate. P<0.05 was considered statistically significant. Analysis was performed using GraphPad prism version 5.0 (GraphPad software, San Diego Calif. USA) and JMP software version 12.01 (SAS Institute Inc. North Carolina USA).

[0183] Results

[0184] The results obtained are shown in FIGS. 1 to 10.

[0185] Efficacy of S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate In Vitro

[0186] As shown in FIG. 1, S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate is effective in a wide variety of cancer cell lines which overproduce H.sub.2O.sub.2 (Viability assay-resazurin, XTT or MTT as explained above).

[0187] H.sub.2O.sub.2/S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate IC.sub.50 Correlation

[0188] FIG. 2 shows that a correlation is observed between high H.sub.2O.sub.2 activity and low IC.sub.50 (monitoring by Total ROS/superoxide detection kit as mentioned above).

[0189] In addition, FIG. 3 shows that by separating the cells in two parts with a cut-off (20 000 Relative Fluorescence Intensity), a distinction of S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate sensitivity to H.sub.2O.sub.2 high from H.sub.2O.sub.2 low is revealed (Pvalue<0.05).

[0190] In FIG. 4, the correlation between high H.sub.2O.sub.2 activity and low IC.sub.50 is illustrated by tissue origin.

[0191] In addition, to control the selectivity of the S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate toward cancer cells, the compound according to the invention was also tested on normal cells by using the same method as the one described above. The results show that the compound according to the invention has no effect on normal cells as shown in Table 3 below.

TABLE-US-00003 TABLE 3 S-methyl 4-(dimethylamino)-4-methylpent- 2-ynethioate in normal human primary cells Cell line Description IC50 (uM) hMCSs- Human mesenchymal stem cells from >50 BM bone marrow huvec Human umbilical vein endothelial cells >50 BEAS-2B Human bronchial epithelial cells >50 HPF Human Pulmonary fibroblasts >100 NHEM Normal Human epidermal melanocytes >100

[0192] In addition, other compounds according to the invention were tested by following the above mentioned material and method on the cell line DU145 (prostatic cancer line) and the cell line HL60 (leukemia) mentioned in Table 2.

[0193] The results regarding the efficacy of those compounds are provided in the Table 4 below.

TABLE-US-00004 TABLE 4 IC.sub.50 IC.sub.50 IUPAC Name Formula (DU145) (HL-60) S-methyl 4-methyl-4- (piperidin-1-yl)pent-2- ynethioate [00005] Formula Weight: 225.4   Formula: C.sub.12H.sub.19NOS  22 μM  7.211 μM S-methyl 4- [benzyl(methyl)amino]-4- methylpent-2-ynethioate [00006] Formula Weight: 261.4   Formula: C.sub.15H.sub.19NOS 1.4 μM 10.408 μM S-methyl 4-methyl-4- [methyl(phenyl)amino] pent-2-ynethioate [00007] Formula Weight: 247.4   Formula: C.sub.14H.sub.17NOS 12.5 μM    24.8 μM S-methyl 4-methyl-4- (morpholin-4-yl)pent-2- ynethioate [00008] Formula Weight: 227.3   Formula: C.sub.11H.sub.17NO.sub.2S 9.3 μM     30 μM S-methyl 4- (dimethylamino)-4- methylpent-2-ynethioate [00009] Formula Weight: 185.28   Fumarate Salt: 7.2 μM    7.6 μM 301.2   Formula: C.sub.9H.sub.15NOS
Correlation Between H.sub.2O.sub.2 Level and GSH Level

[0194] The FIG. 6 shows S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate effect in HL60 cancers cells that overproduce H.sub.2O.sub.2 and have or not a level of GSH below 0.5 nmol for 25 000 cells.

[0195] In particular, FIG. 6(A) shows that the H.sub.2O.sub.2 activity level in Relative Fluorescence Unit (RFU) was increased after treatment with S-methyl 4-(dimethylamino)-4-methylpent ynethioate 5 μM for 24 hours. However, no H.sub.2O.sub.2 increase was observed using a treatment of S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate in addition with 2.5 mM N-acetyl-Cysteine (NAC). In FIG. 6(B), the amount of GSH level was significantly increased in HL-60 cells treated with 2.5 mM NAC and the cells were thus overproducing H.sub.2O.sub.2 and have a level of GSH above 0.5 nmol for 25 000 cells.

[0196] FIG. 6 (C) shows the viability of HL-60 cells treated with S-methyl 4-(dimethylamino)methylpent-2-ynethioate (5, 10 μmol.Math.L-1) and/or 2.5 mM NAC, and that the inhibition of efficacy of S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate was significantly observed in HL-60 cells treated with NAC.

[0197] In addition, FIG. 7 shows that after a pre-treatment of 10 mM GSH-MEE, a Glutathione reduced ethyl ester (GSH-MEE) being a membrane/lipid permeable derivative of GSH that may be used to partially supplement the GSH supply, treatment of HL-60 cells using GSH-MEE inhibits the cytotoxic effect of S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate. By adding more GSH in cells, the status of cells became: overproduction H.sub.2O.sub.2 and level of GSH above 0.5 nmol for 25 000 cells, and S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate is no more effective.

[0198] Furthermore, to confirm these results, FIG. 8 shows that (A) the treatment of Colo357 cells with 100 μM carmustine (BCNU) allows a 50% decrease of the GSH level and to change the status of cells in: overproduction H.sub.2O.sub.2 and level of GSH below 0.5 nmol for 25 000 cells and that as a consequence (B) S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate was effective on these cells, decreasing the viability of 50%.

[0199] In FIG. 9, the total GSH level in nmol per 25000 cells is observed in sensitive and resistant cells. A significative difference (***, p-value<0.001) was observed. Using a threshold of 0.5 nmol per 25000 cells for distinction of GSH high from GSH low cells, 100% of sensitive cells are GSH low and 100% of resistant cells are GSH high.

[0200] FIG. 10 shows the quantification of MDA and HNE adducts in S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate sensitive cells (HL-60, NT2/D1) (A) and S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate resistant cells (MSC) treated with S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate 5 or 10 μmol.Math.L.sup.−1 during 24 hours (B). For MDA-adducts, in sensitive S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate cells, the threshold is above 100 ng per μg of total protein, and in resistant cells, the threshold is below this threshold. For HNE-adducts, the same observation can be made with a threshold of 1 μg per μg of total protein.

Example 2

[0201] The H.sub.2O.sub.2 level from a melanoma cells sample is compared to a control value to determine if these cells would be eligible to a treatment with the compounds according to the invention. The H.sub.2O.sub.2 level from human melanoma cells (obtained as mentioned in example 1) is thus measured as well as the H.sub.2O.sub.2 level from the corresponding normal cells, here melanocytes (Normal human epithelial melanocytes from juvenile foreskin, Promocell (NHEM, pool) cultivated with Melanocyte growth medium M2 of Promocell).

[0202] The two samples are subjected to determination of the H.sub.2O.sub.2 level using the Total ROS/superoxide detection kit (Enzo life science). Relative Fluorescence Intensities of both samples are measured on Appliskan fluorescence microplate reader (Thermo Scientific) (Ex/Em=488/520 nm and Ex/Em=550/610 nm).

[0203] As can be seen from FIG. 5, the H.sub.2O.sub.2 level of the cancer cells sample exceeds the H.sub.2O.sub.2 level of the control sample (i.e. normal cells) by 280%.

[0204] These cancer cells are therefore eligible to treatment with the compounds according to the invention, and in particular with 4-(Dimethylamino)-4-methyl-2-pentynethioic acid S-methyl ester fumarate, if the level of GSH is at the same time below 5 nmol for 25000 cells.