Prooxidative chain-transfer agents for use in the treatment of malignant tumour or infectious diseases

Abstract

Prooxidative chain-transfer agents for use in the treatment of a malignant tumour disease, or infectious disease. The prooxidative chain-transfer agents are selected from lipophilic thiols, lipophilic trithiocarbonates, lipophilic, aromatic dithioesters, and lipophilic, aromatic thiols. The compounds amplify the prooxidative activity at the target site and are therefore highly efficient and specific for their targets.

Claims

1. A prooxidative chain-transfer agent selected from the group consisting of (1) lipophilic thiols comprising the general structure (I) ##STR00009## wherein R.sub.1-R.sub.4 is selected from the group consisting of hydrogen, hydroxyl, a substituted or unsubstituted (C1-C24) alkyl, (C1-C24) hydroxyalkyl, (C1-C24) alkyloxy-(C1-C24) alkyl, (C1-C24) alkylsulfo-(C1-C24) alkyl, (C1-C24) alkylcarboxy-(C1-C24) alkyl, (C1-C24) alkylamino-(C1-C24)alkyl, (C1-C24) alkylamino-(C1-C24) alkylamino, (C1-C24) alkylamino-C1-C24) alkylamino-(C1-C24) alkylamino, a substituted or unsubstituted (C1-C24) aminoalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted arylamino-(C1-C24) alkyl, (C1-C24) haloalkyl, C2-C24 alkenyl, C2-C24 alkynyl; (2) lipophilic, aromatic thiols comprising the general structure (IV) ##STR00010## wherein R.sub.1 is selected from the group consisting of a substituted or unsubstituted (C6-C24) alkyl, (C6-C24) hydroxyalkyl, (C6-C24) alkyloxy, (C6-C24) alkylsulfo, (C6-C24) alkyloxy-(C6-C24) alkyl, (C6-C24) alkylsulfo-(C6-C24) alkyl, (C6-C24) alkylcarboxy-(C6-C24) alkyl, (C6-C24) alkylamino-(C6-C24) alkyl, (C6-C24) alkylamino-(C6-C24) alkylamino, (C6-C24) alkylamino-(C6-C24) alkylamino-(C6-C24) alkylamino, a substituted or unsubstituted (C6-C24) aminoalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted arylamino-(C6-C24) alkyl, (C6-C24) haloalkyl, (C6-C24) alkenyl, (C6-C24) alkynyl; (3) lipophilic trithiocarbonates comprising the general structure (II) ##STR00011## wherein R1 is selected from the group consisting of a substituted or unsubstituted (C6-C24) alkyl, (C6-C24) hydroxyalkyl, (C6-C24) alkyloxy, (C6-C24) alkylsulfo, (C6-C24) alkyloxy-(C6-C24) alkyl, (C6-C24) alkylsulfo-(C6-C24) alkyl, (C6-C24) alkylcarboxy-(C6-C24) alkyl, (C6-C24) alkylamino-(C6-C24) alkyl, (C6-C24) alkylamino-(C6-C24) alkylamino, (C6-C24) alkylamino-(C6-C24) alkylamino-(C6-C24) alkylamino, a substituted or unsubstituted (C6-C24) aminoalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted arylamino-(C6-C24) alkyl, (C6-C24) haloalkyl, C6-C24 alkenyl, C6-C24 alkynyl, and R.sub.2 refers to a hydrogen or a methyl group having one, two, or three substituents, which may be the same or different, each replacing a hydrogen atom (4) lipophilic, aromatic dithioesters comprising the general structure (III) ##STR00012## wherein R.sub.1 is selected from the group consisting of a substituted or unsubstituted (C6-C24) alkyl, (C6-C24) hydroxyalkyl, (C6-C24) alkyloxy, (C6-C24) alkylsulfo, (C6-C24) alkyloxy-(C6-C24) alkyl, (C6-C24) alkylsulfo-(C6-C24) alkyl, (C6-C24) alkylcarboxy-(C6-C24) alkyl, (C6-C24) alkylamino-(C6-C24) alkyl, (C6-C24) alkylamino-(C6-C24) alkylamino, (C6-C24) alkylamino-(C6-C24) alkylamino-(C6-C24) alkylamino, a substituted or unsubstituted (C6-C24) aminoalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted arylamino-(C6-C24) alkyl, (C6-C24) haloalkyl, (C6-C24) alkenyl, (C6-C24) alkynyl, and R.sub.2 refers to a hydrogen or a methyl group having one, two, or three substituents, which may be the same or different, each replacing a hydrogen atom. for use in the treatment or prevention of a malignant tumour disease, or infectious parasitic disease in humans or animals.

2. The prooxidative chain-transfer agent according to claim 1, wherein in the general structure (I) of the lipophilic thiols the hydrophobic group is branched, substituted or unsubstituted.

3. The prooxidative chain-transfer agent according to claim 1, wherein the lipophilic thiol comprising the general structure (I) is selected from the group consisting of n-octylthiol, t-octylthiol, n-dodecylthiol, t-dodecylthiol, n-hexadecylthiol, or n-octadecylthiol.

4. The prooxidative chain-transfer agent according to claim 1, wherein said agent builds thiyl radicals or sulfur radicals in the cell membrane.

5. The prooxidative chain-transfer agent according to claim 1, wherein the carbon atoms within the general structure (I) are part of an aromatic ring system, preferably part of a benzene ring.

6. The prooxidative chain-transfer agent according to claim 1, wherein in the general structure (II) of the lipophilic trithiocarbonates the substitutent is selected from the group consisting of a halogen, hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protected carboxy, cyan, methylsulfonylamino, alkoxy, alkyl, aryl, arylalkyl, acyloxy, or haloalkyl.

7. The prooxidative chain-transfer agent according to claim 1, wherein in the general structure (III) of the lipophilic, aromatic dithioesters, the substituent is selected from the group consisting of a halogen, hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protected carboxy, cyan, methylsulfonylamino, alkoxy, alkyl, aryl, arylalkyl, acyloxy, or haloalkyl.

8. The prooxidative chain-transfer agent according to claim 1, wherein the lipophilic trithiocarbonate comprising the general structure (II) is selected from the group consisting of S-octyl-S′[dimethyl-cyanomethyl]-trithiocarbonate, S-octyl-S′[methyl-hydroxypropyl-cyanomethyl]-trithiocarbonate, S-octyl-S′[methyl-carboxyethyl-cyanomethyl]-trithiocarbonate, S-dodecyl-S′[dimethyl-cyanomethyl]-trithiocarbonate, S-dodecyl-S′[methyl-hydroxypropyl-cyanomethyl]-trithiocarbonate, or S-dodecyl-S′[methyl-carboxyethyl-cyanomethyl]-trithiocarbonate.

9. The prooxidative chain-transfer agent according to claim 1, wherein the lipophilic, aromatic dithioester comprising the general structure (III) is selected from the group consisting of S-[dimethyl-cyanomethyl]-dodecylbenzodithioate, S-[methyl-hydroxypropyl-cyanomethyl]-dodecylbenzodithioate, S-[methyl-carboxyethyl-cyanomethyl]-dodecylbenzodithioate, S-[dimethyl-cyanomethyl]-dodecanoxy-benzodithioate, S-[methyl-hydroxypropyl-cyanomethyl]-dodecanoxy-benzodithioate, or S-[methyl-carboxyethyl-cyanomethyl]-dodecanoxy-benzodithioate.

10. The prooxidative chain-transfer agent according to claim 1, wherein the lipophilic, aromatic thiol comprising the general structure (IV) is selected from the group consisting of 4-dodecylthiophenol, O-dodecyl-4-hydroxythiophenol, S-dodecyl-1,4-benzenedithiol, N-dodecyl-4-aminothiophenol, 4-octadecylthiophenol, O-octadecyl-4-hydroxythiophenol, S-octadecyl-1,4-benzenedithiol, or N-octadecyl-4-aminothiophenol.

11. The prooxidative chain-transfer agent according to claim 11, wherein the infectious parasitic disease is caused by a parasite selected from Acanthamoeba, Anisakis, Ascaris lumbricoides, Botfly, Balantidium coli, Bedbug, Brugia spp., Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania spp., Linguatula serrata, Liver fluke, Loa spp., Onchocerca spp., Paragonimus—lung fluke, Pinworm, Plasmodium spp., Schistosoma spp., Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma gondii, Trypanosoma spp., Whipworm, or Wuchereria bancrofti.

12. The prooxidative chain-transfer agent according to claim 1, wherein the infectious parasitic disease is selected from the group consisting of filariasis, lymphatic filariasis, elephantiasis, chocercosis, malaria, leishmaniasis, trypanosomiasis.

13. The prooxidative chain-transfer agent according to claim 1, wherein the malignant tumour disease is selected from the group consisting of breast cancer, leukemia, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma or head and neck cancer.

14. A prooxidative chain-transfer agent according to claim 1 for use as a medicament.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0079] FIG. 1. Prooxidative activity of chemical Chain-Transfer Agents (CTAs) in biological membranes.

[0080] Native lipid membranes from rat brain were examined on the extent of lipid peroxidation (as malondialdehyde) after treatment with the indicated concentrations of n-dodecylthiol (12-SH).

[0081] Graph (a) shows the absence of any effect in the absence of any radical initiation (“dead membranes”); in graph (b), a low level of free radical initiator (here: iron/ascorbate) was added, as generally found in living cells depending on the cell type. The added amount of initiator alone did not yet have any measurable oxidizing effect (control in (b)); however, its hidden effect was massively amplified by the concomitant addition of a Chain-Transfer Agent.

[0082] The data demonstrate the biochemical activity of the inventive compounds in biological membranes. As such they seem to act as initiator-dependent prooxidants.

[0083] FIG. 2 (FIGS. 2A to 2B). Cytotoxic activity of Chain-Transfer Agents (CTAs) in cultivated HT22 cells and under hypoxic conditions.

[0084] The data confirm the basic toxicity of the compounds pursuant to the invention in living (tumour) cells. It is also shown that despite the postulated pro-oxidative mechanism of action, this toxicity is not dependent on the oxygen partial pressure within the (patho)physiological range of 1% to 20% oxygen, which is relevant for its potential use in hypoxic tumours.

[0085] FIG. 2A. Cytotoxic activity of Chain-Transfer Agents (CTAs) in cultivated HT22 cells.

[0086] HT22 cells were treated for 3 days with the indicated concentrations of n-octylthiol (a) or n-dodecylthiol (b, c). The dotted line indicates the survival of control cultures treated with vehicle.

[0087] FIG. 2B. Cytotoxic activity of Chain-Transfer Agents (CTAs) in cultivated HT22 cells under hypoxic conditions.

[0088] HT22 cells were treated for 1 day (a) or 3 days (b) with the indicated concentrations of n-dodecylthiol under normal oxygen conditions (20% O.sub.2) or under highly hypoxic conditions (1% O.sub.2). Such hypoxic conditions may prevail in solid tumours and can complicate treatment. However, the effectiveness of the chain-transfer agents was not compromised by hypoxic conditions.

[0089] FIG. 3 (FIGS. 3A to 3D) exemplifies the activities of Chain-Transfer Agents (CTAs) in cultivated diploid human fibroblasts.

[0090] The data recapitulate the data from HT22 cells in normal fibroblasts. Representatives of lipophilic thiols falling under the general structure (I) and lipophilic trithiocarbonates falling under the general structure (II) have been used for experimentation. Furthermore, various analyses of the basic mechanism of action of the Chain-Transfer Agents in living cells were conducted. This includes analyses in regard of cellular lipid, protein and DNA damage as well as the physiological response of the cell to this damage. The observed types of damage (e.g. an induction of DNA double-strand breaks) indicate a particular efficacy of Chain-Transfer Agents in tumour cells.

[0091] FIG. 3A. Activities of Chain-Transfer Agents (CTAs) in cultivated diploid human fibroblasts. Cytotoxicity of lipophilic thiols.

[0092] Primary human fibroblasts under cell division-stimulating culture conditions (medium with 10% fetal calf serum) were treated for 3 days with the indicated concentrations of n-decylthiol (a), n-dodecylthiol (b), or n-tetradecylthiol (c).

[0093] FIG. 3B. Activities of Chain-Transfer Agents (CTAs) in cultivated diploid human fibroblasts. Cytotoxicity of lipophilic trithiocarbonates (TTC).

[0094] Primary human fibroblasts under cell division-stimulating culture conditions (medium with 10% fetal calf serum) were incubated for 3 days with the indicated concentrations of S-dodecyl-S′-cyanomethyl trithiocarbonate (D-CM-TTC) (a), S-dodecyl-S′-[dimethyl-cyanomethyl] trithiocarbonate (D-DMCM-TTC) (b), or S-dodecyl-S′-[methyl-hydroxypropyl cyanomethyl] trithiocarbonate (D-MHCM-TTC) (c). The dotted line indicates the survival of control cultures treated with vehicle.

[0095] FIG. 3C. Activities of Chain-Transfer Agents (CTAs) in cultivated diploid human fibroblasts. Biochemical effects.

[0096] Primary human fibroblasts under cell division-stimulating culture conditions (medium with 10% fetal calf serum) were treated with 100 μM (a) and 500 μM (b) n-dodecylthiol (12-SH), respectively, over the indicated time period. Graph (a) shows the concentration of released lipid peroxidation marker 8-isoprostane, graph (b) shows the amount of polyubiquitinated proteins in the cell, which are a marker of protein damage. Both pro-oxidative markers increased significantly within a few hours.

[0097] FIG. 3D. Activities of Chain-Transfer Agents (CTAs) in cultivated diploid human fibroblasts. Genotoxic and pro-apoptotic effects.

[0098] Primary human fibroblasts under cell division-stimulating culture conditions (medium with 10% fetal calf serum) were treated with 500 μM n-dodecylthiol (12-SH) for the indicated time. Panel (a) shows the protein expression of Ser-140 phosphorylated histone 2AX, a marker for DNA double strand breaks and general laboratory proxy for DNA damage. Graph (b) shows the expression of caspase 3 and its active fragment “cleaved” caspase 3. Cleaved caspase 3 is the most important executing caspase in the apoptosis cascade.

[0099] FIG. 4. Prooxidative toxicity of chemical Chain-Transfer Agents (CTAs) in nematodes in vivo.

[0100] Nematodes (C. elegans) were treated with 0.5 mM n-dodecylthiol in the minimum diet supplemented with varying amounts of coliform bacteria as adjunct feed. At the times indicated, the number of worms killed (a) was counted. Panel (b) shows the concentration of the lipid peroxidation marker 8-isoprostane in homogenates of worms treated with 0.1 mM or 0.5 mM n-dodecylthiol (12-SH). This biochemical marker of toxicity increased to a constant maximum after only 6 h, which then led to death after a few days (a).

[0101] The data confirm the general cytotoxic activity of the substances according to the invention in vivo, after oral administration. Also, their pro-oxidative mechanism of action in vivo is evidenced biochemically.

[0102] FIG. 5 (FIGS. 5A to 5B). Ultrastructural effects of Chain-Transfer Agents (CTAs) in insects on different organs in vivo.

[0103] The data demonstrate the effectiveness of chain-transfer agents in vivo in a second, higher organism, in fruit flies (Drosophila). They also show directly the predicted mitochondrial specificity of the cytotoxic effect of these agents.

[0104] FIG. 5A. Ultrastructural effects of Chain-Transfer Agents (CTAs) in insects in vivo. Muscle mitochondria.

[0105] Fruit flies were treated with 1 mM n-dodecylthiol in the feed. The two electron micrographs show intramitochondrial, spiral multilamellar lesions. In tumour cells, such lesions would be expected to lead to apoptosis. The myofibrillar structure, on the other hand, is intact. The black line corresponds to approximately 1 μm.

[0106] FIG. 5B. Ultrastructural effects of Chain-Transfer Agents (CTAs) in insects in vivo. Nervous system and eye.

[0107] Fruit flies were treated with 1 mM n-dodecylthiol in the feed. The electron micrographs show spiral multilamellar lesions of mitochondrial origin in the nervous system (a, b) and in photoreceptor cells (c). Other damage phenotypes are the vacuolization and electron-dense aggregation in (b) as well as the bright lipofuscin accumulation in (c). The black line corresponds to approximately 1 μm.

[0108] FIG. 6. Cytotoxic activity of Chain-Transfer Agents (CTAs) in cultivated Hela cervical carcinoma cells. These cells are primary tumor cells from a donor with cervical carcinoma induced by papillomavirus infection.

[0109] FIG. 7. Cytotoxic activity of Chain-Transfer Agents (CTAs) in cultivated MCF7 breast cancer cells. These cells are primary tumor cells from a donor with invasive breast carcinoma.

[0110] FIG. 8. Cytotoxic activity of Chain-Transfer Agents (CTAs) in cultivated SY5Y neuroblastoma cells. These cells are a subclone of authentic human neuroblastoma cells derived from a bone marrow biopsy conducted in a patient with neuroblastoma.

MODES FOR CARRYING OUT THE INVENTION

[0111] Material and Methods:

[0112] Prooxidative Activity of Chemical Chain-Transfer Agents (CTAs) in Biological Membranes (FIG. 1).

[0113] Native biological membranes from adult rat brain were prepared by differential centrifugation as described (Moosmann and Behl, Proc Nati Acad Sci USA 96:8867-8872, 1999). Samples containing 0.5 mg/ml total protein (as per bicinchoninic acid assay from Pierce, Rockford, Ill., USA) were solubilized by brief sonication in PBS (phosphate-buffered saline) and administered with the indicated concentrations of n-dodecyl thiol (12-SH; from Sigma-Aldrich, St. Louis, Mo., USA) dissolved in ethanol (final concentration: 0.1%). Subsequently, 10 μM Fe.sup.2+/200 μM ascorbate were added as radical-initiating mix; controls received vehicle (water). After the indicated time, the reaction was stopped by adding 2.5 volumes of 5% trichloroacetic acid in 1 M acetic acid, followed by centrifugation (10,000 g for 10 min). Subsequently, thiobarbituric acid-reactive substances (TBARS) as marker of lipid peroxidation were quantified fluorimetrically as detailed before (Hajieva et al., J Neurochem 110:118-132, 2009).

[0114] Cytotoxic Activity of Chain-Transfer Agents (CTAs) in Cultivated HT22 Cells (FIG. 2).

[0115] (FIG. 2A) HT22 are a widely used, immortalized neuronal cell line that has been generated by transforming mouse neuronal tissue with the oncogenic SV40 T-antigen (Morimoto and Koshland, Neuron 5:875-880, 1990). HT22 cells were cultivated in high-glucose DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% heat-inactivated FCS (fetal calf serum), 1 mM pyruvate, 100 mg/L streptomycin and 100 U/mL penicillin. For routine culture, cells were grown in 100 mm dishes at 37° C. in a humidified atmosphere containing 5% CO.sub.2. They were passaged three times per week on reaching approximately 90% confluence and splitted 1:10. All cell culture reagents were from Invitrogen, Carlsbad, Calif., USA.

[0116] For cell survival experiments, HT22 cells were seeded into 96-well plates at a density of ˜5000 cells per well in 0.1 mL medium. After 24 h cultivation, the cells were administered with the indicated concentrations of the tested compounds (n-octyl thiol (8-SH) or n-dodecyl thiol (12-SH)) dissolved in ethanol (final concentration: 1%). After 1 day or 3 days of incubation as indicated, cell survival was analyzed by colorimetric MTT reduction tests (MTT is 3-(4,5-dimethylthiazol-2-yl-)-2,5 diphenyltetrazolium bromide) which were performed exactly as described (Hajieva et al., J Neurochem 110:118-132, 2009).

[0117] (FIG. 2B.) Experiments under hypoxic conditions were conducted accordingly, with the only difference that the 1 day- or 3 day-incubation after n-dodecyl thiol administration was done in a separate incubator that had been set to contain only 1% oxygen (through the controlled addition of nitrogen). The fraction of CO.sub.2 was maintained at 5%.

[0118] Activities of Chain-Transfer Agents (CTAs) in Cultivated Diploid Human Fibroblasts (FIG. 3).

[0119] (FIG. 3A) Normal human diploid fibroblasts were cultivated under standard conditions stimulating cell division, i.e. with 10% serum in the medium. Specifically, the cells were grown in high-glucose DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% heat-inactivated FCS (fetal calf serum), 1× non-essential amino acid (NEAA) supplement, 1 mM pyruvate, 100 mg/L streptomycin and 100 U/mL penicillin. For cultivation, the cells were maintained at 37° C. in a humidified atmosphere containing 5% CO.sub.2. The cells were generally passaged on reaching approximately 90% confluence and were seeded, after counting of the PDLs (population doublings), in new dishes at a density of 10.sup.6 cells per dish in 100 mm dishes, or at a density of 10.sup.4 cells per well in 96-well plates. Cells were used for experiments at PDL 25-30. All cell culture reagents were from Invitrogen, Carlsbad, Calif., USA.

[0120] For cell survival experiments, cells cultivated in 96-well plates for 24 h were administered with the indicated concentrations of the tested compounds (n-decyl thiol (10-SH), n-dodecyl thiol (12-SH), n-tetradecyl thiol (14-SH)) dissolved in ethanol (final concentration: 1%). After 3 days of incubation, cell survival was analyzed by colorimetric MTT reduction tests (MTT is 3-(4,5-dimethylthiazol-2-yl-)-2,5 diphenyltetrazolium bromide) which were performed exactly as described (Hajieva et al., J Neurochem 110:118-132, 2009).

[0121] (FIG. 3B) Cells cultivated in 96-well plates for 24 h were administered with the indicated concentrations of the tested compounds (S-dodecyl-S′-cyanomethyl-trithiocarbonate (D-CM-TTC), S-dodecyl-S′-[dimethyl-cyanomethyl]-trithiocarbonate (D-DMCM-TTC), or S-dodecyl-S′-[methyl-hydroxypropyl-cyanomethyl]-trithiocarbonate (D-MHCM-TTC)) dissolved in ethanol (final concentration: 1%). After 3 days of incubation, cell survival was analyzed by colorimetric MTT reduction tests as above.

[0122] (FIG. 3C) 8-Isoprostanes. Cells cultivated in 96-well plates for 24 h were administered with 100 μM n-dodecyl thiol (12-SH) for the indicated time before removing the cell culture supernatant for 8-isoprostane analysis. 8-Isoprostane analysis was achieved by a commercial enzyme immunoassay (Cayman Chemicals, Ann Arbor, Mich., USA), which was conducted as detailed in the manufacturers instructions.

[0123] Protein polyubiquitination. Cells cultivated in 100 mm dishes for 24 h were administered with 500 μM n-dodecyl thiol (12-SH) for the indicated time before harvesting of the cells in lysis buffer (50 mM Tris-HCl, pH 7.4, 10% sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM Na.sub.3VO.sub.4, 1 mM NaF, 1× protease inhibitor cocktail from Sigma-Aldrich, St. Louis, Mo., USA) followed by brief sonication and denaturation at 95° C. for 2 min. For the specific analysis of protein ubiquitination, Western immunoblotting was performed as described (Hajieva et al., J Neurochem 110:118-132, 2009). In brief, equal amounts of total protein (as per bicinchoninic acid assay from Pierce, Rockford, Ill., USA) were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes by electroblotting. Blocking of was carried out by incubation with Tris-buffered saline/Tween-20 (TBST) containing 2% fat-free dry milk for 60 min at 20° C., followed by incubation with the specific primary antibodies at 4° C. overnight. The primary antibodies were: Rabbit anti-polyubiquitin antibody (dilution 1:2000; from Agilent Dako, Santa Clara, Calif., USA); mouse anti-α-tubulin antibody (dilution 1:1000; from Sigma-Aldrich, St. Louis, Mo., USA); both diluted in TBST. The next day, the membranes were treated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (1:5000; from Jackson Immunoresearch, West Grove, Pa., USA) for 90 min at 20° C. Subsequently, the membranes were washed 3×15 min with TBST. Immunoreactive bands were developed using commercial peroxidase substrate kits (Enhanced Chemiluminescence Plus from Amersham Pharmacia Biotech, Piscataway, N.J., USA), and scanned with a digital chemiluminescent imaging system. Densitometric quantification was performed using automated image analysis software.

[0124] (FIG. 3D) Phospho-H2AX expression and caspase 3 expression. Cells cultivated in 100 mm dishes for 24 h were administered with 500 μM n-dodecyl thiol (12-SH) for the indicated time before harvesting of the cells in lysis buffer and processing for Western blotting as above (Hajieva et al., J Neurochem 110:118-132, 2009). The primary antibodies were: Rabbit anti-phospho-histone H2AX (Ser139) antibody (dilution 1:1000; from Cell Signaling, Cambridge, UK); rabbit anti-caspase 3 antibody (dilution 1:1000; from Cell Signaling, Cambridge, UK); mouse anti-α-tubulin antibody (dilution 1:1000; from Sigma-Aldrich, St. Louis, Mo., USA); all diluted in TBST.

[0125] Prooxidative Toxicity of Chemical Chain-Transfer Agents (CTAs) in Nematodes In Vivo (FIG. 4).

[0126] Caenorhabditis elegans N2 Bristol strain animals were expanded and cultivated at 20° C. on nematode growth medium (NGM) plates in the presence of Escherichia coli strain HB101 as food source following standard protocols (Mocko et al., Neurobiol Dis 40:120-129, 2010). For the toxicity experiments, synchronized adult worms aged 2 days were maintained in liquid culture medium (S-Basal medium supplemented with 5 mg/L cholesterol, 100 mg/L streptomycin, 100 mg/L fluorodeoxyuridine (FUDR)) to which varying amounts of Escherichia coli were added (measured as optical density (OD) at 600 nm wavelength) as described (Mair et al., PLoS One 4:e4535, 2009). Worms distributed in 48-well plates were administered with 500 μM n-dodecyl thiol (12-SH) dissolved in ethanol (final concentration: 1%) and analyzed, after the indicated time, for survival by visual inspection and mechanical stimulation (nose-touch assay) as detailed (Mocko et al., Neurobiol Dis 40:120-129, 2010).

[0127] 8-Isoprostanes. Synchronized, 4-day-old adult worms distributed in cell culture flasks were administered with the indicated concentration of n-dodecyl thiol (12-SH) dissolved in ethanol (final concentration: 1%) and cultivated for 6 h or 48 has indicated. The worms were collected by centrifugation at 1200 g, washed twice with S-Basal medium, and once with DMEM medium containing 100 μM butylated hydroxytoluene (BHT). The worms were then homogenized in DMEM/BHT by sonication (3×20 s at 30 kHz on ice). Equal amounts of protein of the resulting homogenate (as per bicinchoninic acid assay from Pierce, Rockford, Ill., USA) were probed for the presence of 8-isoprostane by a commercial enzyme immunoassay (Cayman Chemicals, Ann Arbor, Mich., USA) following the manufacturer's instructions.

[0128] Ultrastructural Effects of Chain-Transfer Agents (CTAs) in Insects In Vivo (FIG. 5).

[0129] Male Drosophila melanogaster (strain Oregon-R) were maintained at 25° C. in plastic vials covered with air-permeable lids and received standard food (50 g/L refined household sugar, 50 g/L baker's yeast, and 20 g/L agar powder). The medium was boiled under stirring, adding the following supplements at approximately 70° C.: 3 g/L methylparabene (dissolved in ethanol, final concentration: 0.15%), 3 mL/L propionic acid and the appropriate amount of n-dodecyl thiol (12-SH) dissolved in ethanol (final concentration: 0.1%). Synchronized male flies were transferred into new vials and scored for survival every other day. Treatment started on day 2 of adulthood.

[0130] Electron microscopy. Flies harvested after 50 days of treatment were cryofixed by plunge freezing, cut into head, thorax and abdomen before chemical fixation for 90 min with 3% glutaraldehyde and 3% formaldehyde in PBS. The tissues were washed, fixed with 2% OsO.sub.4, washed again, dehydrated with rising concentrations of ethanol, transitionally stabilized with propylene oxide and embedded into epoxy resin essentially as described (Bozzola and Russel, Electron Microscopy: Jones and Bartlett Publishers, Inc., Sudbury, M A, 1999). The polymerized blocks were mounted, trimmed, and sectioned in an ultramicrotome before transfer onto electron microscopy grids. Images were acquired under standard conditions in a Tecnai Transmission Electron Microscope (FEI Company, Hillsboro, Oreg., USA).

[0131] Tumour Therapy

[0132] Data for the anti-cancer effect are shown for HT22 cells (see FIG. 2). To test the cytotoxic activity of Chain-Transfer Agents (CTAs) in cultivated human cells, Hela cervical carcinoma cells cells were treated for 3 days with the indicated concentrations of n-dodecylthiol (a), n-tetradecylthiol (b), or n-octadecylthiol (c) as conducted in the experiments, the results of which are presented in FIG. 6. The dotted line in FIG. 6 indicates the survival of control cultures treated with vehicle.

[0133] The cytotoxic activity of Chain-Transfer Agents (CTAs) in cultivated MCF7 breast cancer cells was analysed in the experiments conducted in FIG. 7. MCF7 cells were treated for 3 days with the indicated concentrations of n-dodecylthiol (a), n-tetradecylthiol (b), or n-octadecylthiol (c). The dotted line indicates the survival of control cultures treated with vehicle.

[0134] The cytotoxic activity of Chain-Transfer Agents (CTAs) in cultivated SY5Y neuroblastoma cells was analysed in the experiments conducted in FIG. 8. SY5Y cells were treated for 3 days with the indicated concentrations of n-dodecylthiol (a), n-tetradecylthiol (b), or n-octadecylthiol (c). The dotted line indicates the survival of control cultures treated with vehicle.

Non-Patent Literature

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