Combination comprising an aminothiolester compound or a pharmaceutically acceptable salt thereof and a compound able to increase the H2O2 level in cancer cells of a subject

Abstract

The present invention relates to a combination comprising an aminothiolester compound or a pharmaceutically acceptable salt thereof, in particular the S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate or a pharmaceutically acceptable salt thereof, and more particularly the 4-(Dimethylamino)-4-methyl-2-pentynethioic acid S-methyl ester fumarate, and a compound able to increase H.sub.2O.sub.2 level in cancer cells of a subject, in particular for use for the treatment of cancer in a subject, wherein cancer cells of said subject do not overproduce H.sub.2O.sub.2 in comparison to a control value and have a level of GSH below 5 nmol for 25000 cells.

Claims

1. A method of treating cancer in a subject in need thereof, comprising administering a combination comprising a compound of formula (I): ##STR00012## 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 an heterocycle; or a pharmaceutical acceptable salt thereof; and a compound able to increase the H.sub.2O.sub.2 level in cancer cells of the subject, wherein the treatment is therapeutic, and wherein cancer cells of said subject: do not overproduce H.sub.2O.sub.2 in comparison to a control value, and have a level of GSH below 0.5 nmol for 25 000 cells.

2. The method according to claim 1, wherein said method comprises measuring the H.sub.2O.sub.2 level and the GSH level in cancer cells of said subject, and wherein said H.sub.2O.sub.2 level is determined by quantifying the level of Fluorescence Intensity.

3. The method according to claim 1, further comprising the step of measuring a MDA-adducts level and/or a HNE-adducts level in a cancer cell sample of said subject after an in vitro treatment with the compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein cancer cells of said subject after said treatment have MDA-adducts level above 75 ng per ng of total protein and/or a HNE-adducts level above 1 g per g of total protein.

4. A method for selecting a subject suffering from a cancer and who will most likely benefit from a treatment with a combination comprising a compound of formula (I): ##STR00013## 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 an heterocycle; or a pharmaceutical acceptable salt thereof; and a compound able to increase the H.sub.2O.sub.2 level in cancer cells of a subject, wherein said method comprises: a) measuring the H.sub.2O.sub.2 level in a cancer cells sample of said subject; b) comparing the resulting level of step a. with a control value; and c) measuring the GSH level in a cancer cells sample of said subject; wherein: a H.sub.2O.sub.2 level of said cancer cells sample of said subject not higher than the control value, and a GSH level of said cancer cells sample of said subject below 5 nmol for 25000 cells, indicates that the subject is likely to benefit from a treatment with a combination comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof and a compound able to increase the H.sub.2O.sub.2 level in cancer cells of a subject.

5. The method according to claim 4, wherein said method comprises: a) measuring the H.sub.2O.sub.2 level in a cancer cells sample of said subject; b) comparing the resulting level of step a. with a control value; d) measuring the GSH level in a cancer cells sample of said subject; and e) measuring the MDA-adducts and/or HNE-adducts level after an in vitro treatment with a compound of formula (I) in a cancer cells sample of said subject; wherein: a H.sub.2O.sub.2 level of said cancer cells sample of said subject not higher than the control value, a GSH level of said cancer cells sample of said subject below 5 nmol for 25000 cells, and a MDA-adducts level above 75 ng per g of total protein and/or a HNE-adducts level above 1 g per g of protein, indicates that the subject is likely to benefit from a treatment with a combination comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof and a compound able to increase the H.sub.2O.sub.2 level in cancer cells of a subject.

6. The method according to claim 4, wherein a H.sub.2O.sub.2 level not higher than 20000 Relative Fluorescence Intensity, indicates that the subject is likely to benefit from a treatment with a combination comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof and a compound able to increase the H.sub.2O.sub.2 level in cancer cells of a subject.

7. The method according to claim 3, wherein the GSH level is determined by luminescence; the MDA-adducts level and/or the HNE-adducts level is determined by immuno-monitoring; or the cancer is chosen from leukemia, lymphomas, blood cancer, breast cancer, lung cancer, melanomas, colon cancer, pancreas cancer, ovarian cancer, osteosarcoma, brain cancer, bladder cancer and gastric cancer.

8. The method according to claim 1, wherein the compound of formula (I) is selected from the group consisting of: a compound in which X1 and X2 are identical or different, and 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; S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate or a pharmaceutical acceptable salt thereof; and 4-(Dimethyl amino)-4-methyl-2-pentynethioic acid S-methyl ester fumarate.

9. The method according to 8, wherein the GSH level is determined by luminescence; the MDA-adducts level and/or the HNE-adducts level is determined by immuno-monitoring; or the cancer is chosen from leukemia, lymphomas, blood cancer, breast cancer, lung cancer, melanomas, colon cancer, pancreas cancer, ovarian cancer, osteosarcoma, brain cancer, bladder cancer and gastric cancer.

10. The method according to claim 4, wherein the compound of formula (I) is selected from the group consisting of: a compound in which X1 and X2 are identical or different, and 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; S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate or a pharmaceutical acceptable salt thereof; and 4-(Dim ethyl amino)-4-methyl-2-pentynethioic acid S-methyl ester fumarate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will be further illustrated by the following figures and examples.

FIGURES

(2) FIG. 1: 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 M and the endogenous H.sub.2O.sub.2 activity levels in cancer cells

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

(4) FIG. 3: H.sub.2O.sub.2/S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate IC.sub.50 correlation: Studies by tissue origin

(5) FIGS. 4 to 6: H.sub.2O.sub.2/S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate IC.sub.50 correlation: induction of sensitivity to S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate by increasing H.sub.2O.sub.2 activity (PCN=pyocyanin) on three cancer cells lines: THP-1, HCC827 and Hop62

(6) FIG. 7: H.sub.2O.sub.2/S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate IC.sub.50 correlation: synergic effect of S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate and H.sub.2O.sub.2 inducer drugs: Arsenic trioxide (As2O3) (ATO) 1 M and Doxorubicine (Doxo) 200 nM

(7) FIG. 8: Combination treatment using S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate and H.sub.2O.sub.2 inducing agent, Cisplatin (CPPD) or Doxorubicin (Doxo), in a context wherein cancer cells Colo357 have a level of GSH higher than 5 nmol for 25000 cells

(8) FIG. 9: Total GSH level in nmol per 25000 cells observed in sensitive and resistant cells

(9) 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 mol.Math.L.sup.1 during 24 hours (B)

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

DETAILED DESCRIPTION MATERIAL AND METHODS

(11) Cell Lines

(12) 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; Universit, Degli Studi di Palermo, Oncology and Surgical Sciences, Palermo, Italy; and Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Barcelona, Spain (See Table 3 for description of the tumor cell panel). All cells were cultivated in appropriate media according to supplier recommendations.

(13) TABLE-US-00003 TABLE 3 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 BrGl2 established cell line Brain BrGl3 established cell line Brain BrGl6 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- established cell line HNSCC 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 MSC 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 System 498 PDC 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 Pleuramesothelioma 1752 PDC Pleuramesothelioma 541 PDC 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

(14) Enhancement of sensitivity of cell line to S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate has been done using selected cell line due to their resistance to S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate. Lung cancer cell lines (HCC827, Hop62) and leukemia cell lines (THP-1) were obtained from the American Type Culture Collection (ATCC), the European Collection of Cell Cultures (ECACC). Cells were cultivated in appropriate media according to supplier recommendations.

(15) Cell Viability Assay, 96 Well Format

(16) 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 (0.510.sup.4-510.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 M). After 48 hours, the growth-inhibitory effect of the drug was analyzed using Rezasurin, according to manufactures instruction. 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.

(17) Enhancement of Sensitivity of Cells to S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate by Increasing the Level of H.sub.2O.sub.2

(18) In a different experiment, cells showing resistance to S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate (THP-1, HCC827, and Hop62) were challenged with H.sub.2O.sub.2 inducer. Cells were seeded as described above and incubated with S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate (from 0.01 to 100 M) alone or S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate in combination with H.sub.2O.sub.2 inducer Pyocyanin (40 M). After 48 h of incubation, cell viability was measure using the Rezasurin assay as described above.

(19) Chemotherapeutic agents that are known to induce H.sub.2O.sub.2 in cells were challenged in association with S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate to treat S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate resistant cells (THP-1). Cells were seeded as described above and incubate S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate (5, 10, 15, 30 M) alone or with other drugs used in chemotherapy and known as H.sub.2O.sub.2 inducer like 2-Metoxyestradiol (2-ME 100 M), Arsenic Trioxide (ATO 1 M), Daunorubicine (40 nM), Doxorubicine (20 nM), Etoposide (500 nM), Mitoxanthrone (50 nM), Parthenolide (100 nM) and Piperlongumine (2 M). After 48 hours of incubation, cell viability was measured using Rezasurin Assay as described above.

(20) Measurement of H.sub.2O.sub.2 Production

(21) 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 are 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, H.sub.2O.sub.2 inducer pyocyanin (PCN, 500 uM) or H.sub.2O.sub.2 inhibitor 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 210.sup.4 in culture media according to cell suppliers instructions, supplemented with FBS (10% v/v), 10 units of penicillin/ml 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 L 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 the different control agents PCN and NAC for 20 min. After the incubation period, wells were loaded with 100 L 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 Appliskan fluorescence microplate reader (Thermo Scientific) (ex=560 nm, Em=600). Data are expressed as the change in arbitrary fluorescence units produced from equal amount of cells and normalized to total protein input.

(22) Statistical Analysis

(23) Values are expressed as meanSD 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).

(24) Measurement of GSH Production

(25) Cells were seeded in 96-well black walled plate (510.sup.4 cells/mL) and incubated overnight for attachment. The cells were treated either with vehicle, DMSO, BCNU 100 M, S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate 20 M, 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.

(26) Quantitative Determination of FINE-Protein and MDA/Protein Adducts by ELISA

(27) 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 g 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 L 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.

(28) Results

(29) The results obtained are shown in FIGS. 1 to 10.

(30) FIG. 1 shows that a correlation is observed between the H.sub.2O.sub.2 activity and the IC.sub.50 (monitoring by Total ROS/superoxide detection kit as mentioned above).

(31) In addition, FIG. 2 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 sensibility of H.sub.2O.sub.2 high from H.sub.2O.sub.2 low is revealed (Pvalue<0.05).

(32) In FIG. 3, the correlation between H.sub.2O.sub.2 activity and IC.sub.50 is illustrated by tissue origin.

(33) Furthermore, FIGS. 4 to 6 and the table 4 below show induction of sensibility to S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate using pre-treatment with pyocianin in a context wherein the cancer cells have a level of GSH below 5 nmol for 25000 cells.

(34) TABLE-US-00004 TABLE 4 abs.IC.sub.50 (M) THP-1 HCC827 Hop62 DIMATE 46.0 24.98 39.0 DIMATE + PCN 10.59 4.92 8.98 (40 M) DIMATE: S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate PCN: pyocianin

(35) Induction of sensibility to S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate was demonstrated using pre-treatment with pyocianin 40 M. Cell exposure to pyocianin causes two-fold increase in H.sub.2O.sub.2 levels without affecting cell viability.

(36) FIG. 7 shows a synergic effect with S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate and a compound able to increase the H.sub.2O.sub.2 level in cancer cells even in cells resistant to those compounds separately.

(37) Furthermore, synergistic results were also shown with the combined treatment by S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate and the chemotherapeutic agents known to induce H.sub.2O.sub.2 on S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate THP-1 resistant cells, as shown in Table 5 below.

(38) TABLE-US-00005 TABLE 5 IC50 (M) P value DIMATE + Etoposide 25.22 P < 0.001 DIMATE + Mitoxanthrone 22.67 P < 0.001 DIMATE + Daunorubicine 12.5 P < 0.001 DIMATE + Parthnolide 14.4 P < 0.001 DIMATE + Piperlongumine 27.6 P < 0.001 DIMATE + Doxorubicine 21.83 P < 0.001 DIMATE + ATO 14.9 P < 0.001 DIMATE + 2-ME <5 P < 0.001 DIMATE 46 DIMATE: S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate

(39) FIG. 8 relates to the combination treatment using S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate and H.sub.2O.sub.2 inducing agent, Cisplatin (CPPD) or Doxorubicin (Doxo), in a context wherein the cancer cells Colo357 have a level of GSH higher than 5 nmol for 25000 cells (contrary to what has been shown before).

(40) It shows in FIG. 8(A) that the treatment using CPPD at 20 mol.Math.L1 and Doxo at 25 mol.Math.L1 increase significantly the H.sub.2O.sub.2 level in Colo357 cell line (respectively 200% and 10 000%) but that, as shown in FIG. 8 (B) no effect was however observed on viability.

(41) FIGS. 9 and 10 illustrate how the thresholds regarding GSH, MDA-adducts and HNE-adducts have been determined.

(42) 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.

(43) FIG. 10 shows the quantification of MDA and HNE adduct in S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate sensitive cells (HL-60, NT2/D1) (A) and 5-methyl_4-(dimethylamino)-4-methylpent-2-ynethioate resistant cells (MSC) treated with S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate 5 or 10 mol.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 g 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 g per g of total protein.