Desmethylanethole trithione derivatives for the treatment of diseases linked to mitochondrial reactive oxygen species (ROS) production

Abstract

The present invention relates to desmethylanethole trithione (AOX) and derivatives thereof, especially derivatives of formula (I), for the prevention and treatment of diseases whose initiation and/or evolution relates to the production and effects of reactive oxygen species (ROS) of mitochondrial origin, ##STR00001##

Claims

1. A compound of formula (I′): ##STR00058## or a pharmaceutically acceptable tautomer, salt or solvate thereof, wherein: X represents S, O or NHOH; Y represents CH, C or N; R.sup.1, R.sup.4 and R.sup.5 each independently represent hydrogen, hydroxy, halo, amino, alkylsulfonyl, aminosulfonyl, cyano, nitro, carboxy, aryl, alkoxy, haloalkyl, alkylamino, am inoalkyl, nitrooxyalkyl or carboxyalkyl; R.sup.2′ and R.sup.3′ together with the carbon atoms to which they are attached form a 5-membered heteroaryl moiety wherein —R.sup.3′—R.sup.2′— represents -A-CR.sup.6=B— or —B=CR.sup.6-A—; wherein: A represents O, S or NR′; wherein R.sup.7 represents hydrogen, C1—C8 alkyl or alkyloxycarbonyl; B represents CH or N; and R.sup.6 represents hydrogen, hydroxy, halo, amino, alkylsulfonyl, aminosulfonyl, cyano, nitro, carboxy, aryl, alkoxy, haloalkyl, alkylamino, aminoalkyl, nitrooxyalkyl or carboxyalkyl.

2. The compound according to claim 1, of formula (IIa), (IIb), (IIIa) or (IIIb): ##STR00059## or a pharmaceutically acceptable tautomer, salt or solvate thereof, wherein X, Y, A, B, R.sup.1, R.sup.4, R.sup.5 and R.sup.6 are as defined in claim 1.

3. The compound according to claim 1, of formula (IIa-1), (IIa-2), (IIIa-1) or (IIIa-2) ##STR00060## or a pharmaceutically acceptable tautomer, salt or solvate thereof, wherein X, A, B, R.sup.1, R.sup.4, R.sup.5 and R.sup.6 are as defined in claim 1.

4. The compound according to claim 1, of formula (IIa-1a), (IIa-1b), (IIa-1c), (IIa-1d), (IIa-1e), (IIa-2a), (IIa-2b), (IIa-2c), (IIa-2d), (IIa-2e), (IIIa-1a), (IIIa-1b), (IIIa-1c), (IIIa-1d), (IIIa-1e), (IIIa-2a), (IIIa-2b), (IIIa-2c), (IIIa-2d) or (IIIa-2e) ##STR00061## ##STR00062## ##STR00063## or a pharmaceutically acceptable tautomer, salt or solvate thereof, wherein X, R.sup.1, R.sup.4, R.sup.5, R.sup.6 and R.sup.7 are as defined in claim 1.

5. The compound according to claim 1, being of formula (IIb-1), (IIb-2), (IIb-3), (IIb-4), (IIb-5), (IIIb-1), (IIIb-2), (IIIb-3), (IIIb-4) or (IIIb-5) ##STR00064## or a pharmaceutically acceptable tautomer, salt or solvate thereof, wherein X, R.sup.1, R.sup.4, R.sup.5, R.sup.6 and R.sup.7 are as defined in claim 1.

6. The compound according to claim 1, wherein X is S or O; and/or Y is CH or N.

7. The compound according to claim 1, wherein X is S; and/or Y is CH.

8. The compound according to claim 1, said compound being selected from the group consisting of: 5-(2-hydroxybenzo[d]oxazol-5-yl)-3H-1,2-dithiole-3-thione; 5-(2-hydroxybenzo[d]thiazol-6-yl)-3H-1,2-dithiole-3-thione; 5-(benzofuran-5-yl)-3H-1,2-dithiole-3-thione; and methyl 5-(3-thioxo-3H-1,2-dithiol-5-yl)-1H-indole-1-carboxylate.

9. A pharmaceutical composition comprising a compound according to claim 1, or a pharmaceutically acceptable tautomer, salt or solvate thereof, and at least one pharmaceutically acceptable excipient.

10. A medicament comprising a compound according to claim 1, or a pharmaceutically acceptable tautomer, salt or solvate thereof.

11. A process for manufacturing a compound of Formula (IIa-1) according to claim 3 or a pharmaceutically acceptable tautomer, salt or solvate thereof, characterized in that it comprises: a) cyclizing a compound of formula (C) ##STR00065## wherein A, B, R.sup.1, R.sup.4, R.sup.5 and R.sup.6 are as defined in claim 3; with a sulfur-based reagent, in the presence of a siloxane; to obtain a compound of formula (IIa-1′) ##STR00066## or a pharmaceutically acceptable tautomer, salt or solvate thereof, wherein A, B, R.sup.1, R.sup.4, R.sup.5 and R.sup.6 are as defined in claim 3; and optionally: b1) compound of formula (IIa-1′) can react with an oxidant; to obtain a compound of formula (IIa-1″) ##STR00067## or a pharmaceutically acceptable tautomer, salt or solvate thereof, wherein A, B, R.sup.1, R.sup.4, R.sup.5 and R.sup.6 are as defined in claim 3; or b2) compound of formula (IIa-1′) can react with hydroxylamine NH.sub.2OH—HCl; in the presence of a base; to obtain a compound of formula (IIa-1′″) ##STR00068## or a pharmaceutically acceptable tautomer, salt or solvate thereof, wherein A, B, R.sup.1, R.sup.4, R.sup.5 and R.sup.6 are as defined in claim 3.

12. The process according to claim 11, wherein the base is sodium acetate (AcONa) and/or the oxidant is mercury acetate Hg(OAc).sub.2.

13. A method for treating free oxygen radicals-related diseases in a subject in need thereof, comprising administering to said subject an inhibitor of production of reactive oxygen species (ROS), wherein said inhibitor is a compound of formula (I′): ##STR00069## or a pharmaceutically acceptable tautomer, salt or solvate thereof wherein: X represents S, O, or NHOH; Y represents CH, C, or N; R.sup.1, R.sup.4, and R.sup.5 each independently represent hydrogen, hydroxy, halo, amino, alkylsulfonyl, aminosulfonyl, cyano, nitro, carboxy, aryl, alkoxy, haloalkyl, alkylamino, am inoalkyl, nitrooxyalkyl or carboxyalkyl; R.sup.2′ and R.sup.3′ together with the carbon atoms to which they are attached form a 5-membered heteroaryl moiety wherein —R.sup.3′—R.sup.2′— represents -A-CR.sup.6=B— or —B=CR.sup.6-A—; wherein: A represents O, S, or NR.sup.7′; wherein R.sup.7 represents hydrogen, C1-C8 alkyl or alkyloxycarbonyl; B represents CH or N; and R.sup.6 represents hydrogen, hydroxy, halo, amino, alkylsulfonyl, aminosulfonyl, cyano, nitro, carboxy, aryl, alkoxy, haloalkyl, alkylamino, aminoalkyl, nitrooxyalkyl or carboxyalkyl; wherein said free oxygen radicals-related diseases are selected from the group consisting of cardiovascular diseases, aging diseases, auto-immune diseases, progeroid syndromes, Parkinsonian syndromes, neurological diseases, ischemic and reperfusion injuries, infectious diseases, muscles diseases and lung, kidney and liver diseases.

14. The method according to claim 13, wherein said compound is selected from: 5-(2-hydroxybenzo[d]oxazol-5-yl)-3H-1, 2-dithiole-3-thione; 5-(2-hydroxybenzo[d]thiazol-6-yl)-3H-1,2-dithiole-3-thione; 5-(benzofuran-5-yl)-3H-1,2-dithiole-3-thione; and methyl 5-(3-thioxo-3H-1,2-dithiol-5-yl)-1H-indole-1-carboxylate.

15. The method according to claim 13, wherein said compound is of formula (II) or (III): ##STR00070## or a pharmaceutically acceptable tautomer, salt or solvate thereof, wherein X, Y, R.sup.1, R.sup.4, R.sup.5, R.sup.6, A and B are as defined in claim 13.

16. The method according to claim 13, wherein said compound inhibits mitochondrial production of ROS.

17. The method according to claim 16, wherein the compound inhibits mitochondrial production of ROS at site I.sub.Q of complex I of mitochondria.

18. The method according to claim 13, wherein said cardiovascular diseases are selected from the group comprising myocardial infarction, ischemia-reperfusion injury, heart failure, thrombosis and embolism, cardiopulmonary diseases, cardiac toxicity of anthracyclines, cardiac toxicity of anti-cancer drugs, cardiac toxicity of antiviral drugs, cardiac toxicity of quinolones, ischemia, stroke, cardiac fibrillation, pulmonary arterial hypertension, heart attack, hypertension and cardiomyopathies.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the absence of effect of AOL (5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione) on mitochondrial respiration. Panel A: After incubation in the presence of AOL (20 μM in this example), isolated mitochondria from rat heart were oxidizing glutamate+malate (GM) as substrate. Phosphorylation was triggered by adenosine diphosphate (ADP) and stopped by atractyloside (ATR), a specific inhibitor of adenine translocator. Panels B to D: The classical study of mitochondrial oxidative phosphorylation in the presence of various respiratory substrates was carried out in the presence of increasing concentrations of AOL (from 5 to 80 μM). There were no statistical differences in mitochondrial respiration under the different energetic states after addition of AOL. Oxidation rate after ADP addition reflects the adenosine triphosphate (ATP) synthesis activity of isolated mitochondria.

(2) FIG. 2 presents the main sites of oxygen radicals' production by isolated mitochondria in the presence of substrates of both complexes I and III in the presence of ATR (state 4) in order to obtain maximal mitochondrial ROS production. As previously stated, mitochondrial ROS production is highly dependent on mitochondrial activity and conditions. Although AOL has been tested under numerous conditions, for the sake of clarity, we chose to present here only the most demonstrative results. The presence of all the substrates (i.e., glutamate, malate and succinate), giving the electrons to the whole chain, is the closest to in situ conditions in the cell. Under these conditions of substrates, we evaluated the effect of the presence of AOL on ROS production by the complete chain under ATR (inhibition of phosphorylation: maximum production), and by the complex I (inhibited by rotenone) and complex II (inhibited by Antimycin A). Colors refer to FIG. 3.

(3) FIG. 3 presents the effect of AOL (5 to 80 μM) on ROS/H.sub.2O.sub.2 production by isolated mitochondria in the presence of substrates of complexes I and II in the presence of ATR (state 4), effect of rotenone, antimycin A and myxothiazol. In the absence of specific inhibitors of the complexes, ROS production is at maximum and mainly comes from reverse electron transport at site I.sub.Q (see FIG. 4). After addition of rotenone, which specifically reverse electron transport by inhibiting I.sub.Q, production decreases and occurs almost entirely at site III.sub.QO. The subsequent addition of Antimycin A, which blocks the transfer of the electrons to oxygen, increases ROS production at site III.sub.QO and finally myxothiazol blocks ROS production at site III.sub.QO (see FIG. 2 for details).

(4) FIG. 4 is a scheme presenting the site of action of AOL on mitochondria ROS production and the sites where AOL has no or little action.

(5) FIG. 5 is a histogram showing the effect of 10 and 20 μM AOL on glucose stimulated insulin secretion (GSIS) in isolated islets from male C57Bl/6J mice. Combination of three experiments is displayed. Islets from two mice for each experiment; five islets each well; four to six wells each condition. Insulin secretion data were normalized to 11 mM Glc-Veh group, which was considered 100%. *p<0.05, **p<0.01 and ***p<0.001 versus 3 mM Glc-Veh; #p<0.05, ##p<0.01 and ###p<0.001 versus 11 mM Glc-Veh; One-way ANOVA and Bonferroni's post-hoc test.

(6) FIG. 6 is a histogram showing the fat mass determined after 3 weeks of treatment. Fat mass is expressed in grams (g). Data are expressed as mean±SEM.

(7) FIG. 7 is a histogram showing the lean mass determined after 3 weeks of treatment. Lean mass is expressed in grams (g). Data are expressed as mean±SEM.

(8) FIG. 8 is a graph showing the effect of chronic treatment with AOL (5 mg/kg and 10 mg/kg) on glucose responses during an insulin tolerance test (ITT). The graph represents changes in blood glucose levels during an ITT. Data are expressed as mean±SEM.

(9) FIG. 9 is a histogram showing the effect of AOL on blood glucose levels. After five weeks of treatment, blood glucose was measured in 2-h fasted mice. Data are expressed as mean±SEM.

(10) FIG. 10 is a histogram showing the neuroprotective effect of AOL (5 mg/kg bid for 11 days) on TH-positive cell counts in the SN in MPTP-treated mice. Data expressed as mean±SEM (n=10-11) and analyzed using one-way repeated measures ANOVA followed by Dunnett's multiple comparison test. **P<0.01; ***P<0.001 c.f. MPTP+vehicle.

(11) FIG. 11 is a graph showing the effect of AOL on the recovery of heart contractility during the reperfusion phase following ischemia. Data are expressed as mean±SEM for control (black) and AOL treated (grey) for 6 independent experiments.

(12) FIG. 12 is a graph showing the effect of AOL on the infarct size of the slices of ischemic hearts. At the end of the reperfusion period, hearts were stained by triphenyltetrazolium chloride (TTC). Living tissue appears red, while damaged tissue appears white.

(13) FIG. 13 is a set of two graphs showing the effect of AOL treatment on pulmonary arterial pressure and heart remodeling. Panel A: effect of AOL (hatched columns) on the mean pulmonary arterial pressure (mPAP) measured in normoxic rats (N, white columns), chronic hypoxic rats (CH, light grey columns) and monocrotaline-treated rats (MCT, dark grey columns). Panel B: right ventricular hypertrophy expressed as the Fulton index (i.e., ratio of right ventricle weight (RV) to left ventricle plus septum weight (LV+S)). n is the number of rats. *, ** and *** indicate a significant difference for P<0.05, 0.01 and 0.0001 respectively versus N. ### indicates a significant difference for P<0.05 versus CH. † and †† indicate a significant difference for P<0.05 and 0.01 respectively versus N+AOL. ‡ indicates a significant difference for P<0.05 versus MCT.

(14) FIG. 14 is a set of graphs showing the effect of AOL on pulmonary arteries (PA) remodeling. The effect of AOL (hatched columns) on PA remodeling was assessed by measuring the percentage of PA medial thickness in normoxic rats (N, white columns), chronic hypoxic rats (CH, light grey columns) and monocrotaline-treated rats (MCT, dark grey columns). Intraacinar arteries observed to estimate PA remodeling were separated into three groups with different cross-sectional diameters (Panel A: under 50 μm; Panel B: between 50 to 100 μm; Panel C: between 100 to 150 μm). n is the number of vessels. *, ** and *** indicate a significant difference for P<0.05, 0.01 and 0.0001 respectively versus N. ## and ### indicate a significant difference for P<0.01 and 0.0001 versus CH. a significant difference for P<0.05 and 0.01 respectively versus N+AOL. ‡ indicates a significant difference for P<0.05 versus MCT.

(15) FIG. 15 is a set of graphs showing the effect of AOL on the thickness of the outer nuclear layer (ONL) of the retina, in progressive light-induced retinal degeneration. Panel A: effect of vehicle and AOL on “untransferred” animals. Animals were bred under cyclic low-intensity lighting and received injections of vehicle or AOL three times a day for 7 days. Fifteen days after the end of the treatment, histological analysis of the retina was carried out. Data are expressed as mean±SEM thickness of the ONL, for untreated animals (light grey, square dots), vehicle-treated animals (dark grey, triangle dots) and AOL-treated animals (black, round dots), in μm, from the optical nerve and every 0.39 mm in the superior and inferior poles of the optic disc. Panel B: effect of vehicle and AOL on “transferred” animals. Animals were bred under cyclic low-intensity lighting and transferred to cyclic high-intensity lightning for 7 days, during which they received injections of vehicle or AOL three times a day. At the end of the treatment, animals were transferred back under cyclic low-intensity lighting conditions, and histological analysis of the retina was carried out fifteen days later. Data are expressed as mean±SEM thickness of the ONL, for untreated animals (light grey, square dots), vehicle-treated animals (dark grey, triangle dots) and AOL-treated animals (black, round dots), in μm, from the optical nerve and every 0.39 mm in the superior and inferior poles of the optic disc.

(16) FIG. 16 is a set of graphs showing a lifespan SOD2-KO experiment on four groups of mice (WT-KOL: wild-type mice treated with vehicle; WT-AOL: wild-type mice treated with AOL; KO-KOL: SOD2-KO mice treated with vehicle; KO-AOL: SOD2-KO mice treated with AOL). Data are expressed as mean values. Panel A: evolution of mice body weight, in grams over time, in days. Panel B: baseline-corrected of mice body weight, as a percentage of weight gain over time, in days. Panel C: survival proportion among the KO-KOL and KO-AOL groups, in percentage over time, in days.

(17) FIG. 17 is a graph showing the succinate dehydrogenase (SDH) activity in heart, among five groups of mice (WT-KOL: wild-type mice treated with vehicle; WT-AOL: wild-type mice treated with AOL; KO-KOL: SOD2-KO mice treated with vehicle; KO-AOL: SOD2-KO mice treated with AOL; WT: wild-type untreated mice). The optical density of SDH reaction sampled from heart sections was measured with the image processing software Image Analyst MKII (Akos). This density was expressed in the form of mean grey level where mean grey level=sum of grey/number of pixels measured. Data are expressed as mean values of the measured optical density.

(18) FIG. 18 is a set of graphs showing Oil Red O staining of liver slices from WT and SOD2-KO mice, treated or not with AOL. Histogram represent the average size of lipids droplets (Panel A), the droplet density (droplets number/liver area) (Panel B) and the total lipid area (average size×droplets number) (Panel C).

(19) FIG. 19 shows the effect of AOX on mitochondrial oxidation. A typical experiment showing mitochondrial respiration under various oxidative phosphorylation conditions in the presence of AOX is shown in FIG. 19A. AOX has been added to mitochondria for a 5-minute-incubation before oxidative phosphorylation assay. Oxygen consumption (dark grey trace) starts after substrate addition (GM: Glutamate-Malate) and is activated by ADP (phosphorylation state). Phosphorylation is stopped by ATR (atractyloside) and low residual respiration reflects mitochondrial inner membrane integrity. FIG. 19B presents the evolution of mitochondrial oxidation rates during the different respiratory states described above (Succ/ROT: GM substrates, succinate and rotenone) in the presence of increasing concentrations of AOX in DMSO.

(20) FIG. 20 shows the effect of AOX on mitochondrial ATP phosphorylation. Phosphorylation rate has been measured simultaneously with oxidation rate as previously described in Gouspillou et al. (2014. Aging Cell. 13(1):39-48), and the effects of increasing concentrations of AOX have been reported. Results are expressed as a relative percentage to the control of the pH unit/sec evolution.

(21) FIG. 21 shows the effect of increasing AOX concentrations on oxygen radicals production by isolated mitochondria in the presence of substrates of both complexes I and III (glutamate, malate and succinate) in the presence of ATR, therefore under conditions of major production by complex I. The measurement of ROS produced by mitochondria was carried out by using the classical peroxidase-Amplex Red system which measures the appearance of H.sub.2O.sub.2 by the oxidation of Amplex Red giving fluorescent resorufin (see FIG. 22B).

(22) FIG. 22 shows the absence of effect of AOX on non-mitochondrial ROS in an in vitro assay, in comparison with AOL and Oltipraz. Non-mitochondrial ROS/H.sub.2O.sub.2 were obtained by using commercially available NAD(P)H oxidase in the presence of reduced NAD(P)H and again measured using the classical peroxidase-Amplex Red system (FIG. 22B). The effect of increased concentrations of the different molecules tested is presented in FIG. 22A. Straight horizontal lines represent the averaged percentage of ROS production across all concentrations tested, for AOL (99.2%) and of AOX (109.9%).

(23) FIG. 23 shows the effect of AOX on the viability of carcinoma cell lines A549 and H460, expressed in % to the control (0 μM AOX). Cells were incubated with increasing doses, from 0 to 500 μM AOX, and cytotoxicity was assessed by sulforhodamine B (SRB) assay (FIG. 23A). Results are also expressed as the log of AOX concentration (FIG. 23B).

(24) FIG. 24 shows the absence of effect of AOX and AOL on the respiration of H460 cells, as assed by oxygraphic approach.

(25) FIG. 25 shows the absence of effect of AOX on metastatic activity of carcinoma cells, using the Transwell test. Briefly, H460 carcinoma cells were placed on the upper layer of a cell permeable membrane, and following an incubation period, the cells that have migrated through the membrane are stained and counted. FIG. 25A shows photographs of the dyed cells in the lower compartment for the different conditions (control, 5 μM AOX, 10 μM AOX and 10 μM NAC [N-acetyl cysteine]). FIG. 25B is an histogram showing the results of FIG. 25A.

(26) FIG. 26 shows the contraction of intrapulmonary arteries induced by serotonin (5HT) or endothelin (ET-1) in presence of AOL (FIG. 26A) or AOX (FIG. 26B) at different concentrations. 2-way ANOVA: * p<0.05; ** p<0.01; *** p<0.001.

(27) FIG. 27 shows the proliferation of pulmonary arterial smooth muscle cells (PASMC) after incubation in 10% fetal calf serum (FCS), 0.2% FCS and 0.2% FCS+100 μM serotonin (5HT), in presence or in absence of AOL.

(28) FIG. 28 shows the effect of AOL, AOX and Oltipraz (0 to 80 μM) on ROS/H.sub.2O.sub.2 production by sites I.sub.Q and III.sub.QO of isolated mitochondria, when individually targeted using the combination of succinate (energy substrate of respiratory complex 2) and known inhibitors of respiratory chain, namely for site I.sub.Q, 10 mM succinate alone and for site III.sub.Qouter, 10 mM succinate, 4 μM rotenone and 2.5 μM antimycin A. FIG. 28A shows the effect of the three molecules on site I.sub.Q and FIG. 28B shows the effect on site III.sub.QO.

(29) FIG. 29 shows the effect of AOX analogs (Cp1; Cp2; Cp3; Cp4; Cp5; Cp6a; Cp8; Cp9a) (from 0 to 25 μM) on ROS/H.sub.2O.sub.2 production by sites I.sub.Q and III.sub.QO of isolated mitochondria, when individually targeted using the combination of succinate (energy substrate of respiratory complex 2) and known inhibitors of respiratory chain, namely for site I.sub.Q, 10 mM succinate alone and for site III.sub.Qouter, 10 mM succinate, 4 μM rotenone and 2.5 μM antimycin A. FIG. 29A shows the effect of (upper panel) Cp1, Cp2, Cp3, Cp4, (lower panel) Cp5, Cp6a, Cp8 and Cp9a on site I.sub.Q and FIG. 29B shows the effect of Cp5, Cp6a and Cp9a on site III.sub.QO.

EXAMPLES

(30) The present invention is further illustrated by the following examples.

Chemistry Examples

(31) General Experimental Procedures

(32) Reagents and Solvents

(33) All reagents of synthetic grade and solvents were used as supplied. The solvents used in reactions were dried, distilled if required in accordance with the state of the art. Some solvents were commercially available as dry and were used as such.

(34) Reaction Conditions

(35) When dry conditions were required, glassware was oven dried and reactions were carried out under a nitrogen atmosphere. Room temperature (r.t.) refers to 20-25° C. Reaction temperatures of −78° C. were obtained using solid C02 and acetone. For those at 0° C., an ice bath was employed and where heat was required an oil bath with contact thermometer was used.

(36) Reactions were monitored by TLC. TLC was carried out using Merck, DC Kieselgel 60 F.sub.254 plates UV254 pre-coated aluminum sheets with silica gel and fluorescent indicator. Indicators used included ethanolic phosphomolybdic acid solution

(37) Flash Chromatography

(38) Silica gel, MN Kieselgel 60, 15-40 microns grade from Macherey-Nagel was used in the purification of crude products by flash column chromatography. The samples were either applied directly to the top of the silica/solvent column or applied as dry silica gel slurry.

(39) Automated Flash Chromatography

(40) Teledyne Isco Combiflash Companion™ purification system Crude samples were dissolved in a small amount of suitable solvent and applied to RediSep® prepacked columns. The column was placed within the Teledyne Isco Combiflash Companion® purification system and automated purification was carried out using a solvent gradient program. The system was used either with the automated fraction collection facility where compounds were detected by UV or by collecting all fractions.

(41) Nuclear Magnetic Resonance Spectroscopy (NMR)

(42) NMR was recorded on Bruker UltraShield instruments operating at 400 MHz (.sup.1H), and 100 MHz (.sup.13C). Calibration was carried out using the residual solvent shift from the deuterated solvent. When CDCl.sub.3 was employed as the solvent, calibration was carried out on this solvent signal at 7.26 (.sup.1H) and 77.16 (.sup.13C). When aromatics were present in the sample to be analyzed, Me.sub.4Si was added to CDCl.sub.3 and the spectra were calibrated at 0.0 (1H). When D20 was used, the water signal was designated as the internal reference at 4.79 ppm (1H). For CD.sub.3OD, the internal reference was designated at 3.31 ppm (.sup.1H) and 49.0 ppm (.sup.13C NMR). For (CD.sub.3).sub.2SO the internal reference was designated at 2.50 ppm (.sup.1H) and 39.5 ppm (.sup.13C). For .sup.19F, CFCl.sub.3 as an external reference was used. Chemical shifts are reported in parts per million (ppm) and coupling constants are given in Hertz (Hz). The abbreviations for the multiplicity of the proton and carbon signals are as follows: s singlet, d doublet, dd doublet of doublet, dt doublet of triplets, ddt doublet of doublet of triplets, t triplet, tt triplet of triplets, q, quintet, m multiplet.

(43) Mass Spectroscopy (MS) and Liquid Chromatography Coupled to Tandem Mass Spectrometry (LC-MS) Analysis

(44) Mass analysis was carried out on a Waters 3100 Mass detector, Waters Alliance 2695. 1 μg sensitivity with either ESI or APCI. 10,000 Da/sec scan speeds up to 2,000 Da for full compatibility with seconds wide fast LC peaks. Dual orthogonal sampling ionization with ZSpray™ source. Multiple detection strategies available with supported Tunable UV (TUV), Photodiode Array (PDA), and Evaporative Light Scattering (ELS) optical detectors. Or mass analysis was carried out on UPLC/MS: Xevo G2 Qtof The Xevo G2 QT of mass spectrometer, with UPLC®/MSE and QuanT of technology.

(45) Melting Point Analyses

(46) Melting points were measured on a STUART SMP3 instrument.

(47) High Performance Liquid Chromatography (HPLC)

(48) HPLC-DAD analyses were carried out on a Waters analytical HPLC system equipped with suitable analytical column, Empower software, Waters Delta 600 Multisolvent delivery system and a photodiode array detector (Waters 2996) and/or refractometer, a system controller (Waters 600), and a Rheodyne injector 7725i with a 20 μL sample loop was used.

(49) Synthesis of Intermediate Compounds (C)

(50) General procedure A. A 100 mL round bottom flask fitted with magnetic stirrer is charged with appropriate commercial arylethanone and dimethylcarbonate. Sodium hydride (60% in mineral oil) is added slowly with stirring and the whole is refluxed overnight. The mixture is poured into water, acidified with HCl (2M) and extracted with ethyl acetate. The organic layer is washed with water (50 mL) and saturated brine solution (50 mL). The organic layer is dried over anhydrous Na.sub.2SO.sub.4 and the solvent is removed under reduced pressure. The crude material is purified by silica gel column chromatography using suitable solvent to give the corresponding methyl 3-oxo-3-arylpropanoate.

(51) Intermediates 7, 11 and 13 were synthesized using general alkylation procedure A.

Intermediate 7: methyl 5-(3-methoxy-3-oxopropanoyl)-1H-indole-1-carboxylate

(52) ##STR00046##

(53) The reaction was carried out according to the general alkylation procedure A, using 6 as arylethanone (1.03 g, 6.47 mmol), dimethylcarbonate (12 mL) and sodium hydride (2.5 g, 64.7 mmol). After workup and purification, compound 7 was obtained (m=1.4 g, 78%). .sup.1H NMR (400 MHz, DMSO-D.sub.6) δ(ppm) 8.30 (d, J=1.4 Hz, 1H), 7.78 (dd, J=8.7, 1.7 Hz, 1), 7.55 (d, J=8.7 Hz, 1H), 7.48 (d, J=3.1 Hz, 1H), 6.64 (dd, J=3.1, 0.7 Hz, 1H), 4.23 (s, 2H), 3.84 (s, 3H), 3.66 (s, 3H).

Intermediate 11: methyl 3-(2-hydroxybenzo[d]oxazol-5-yl)-3-oxopropanoate

(54) ##STR00047##

(55) The reaction was carried out according to the general alkylation procedure A, using 10 as arylethanone (1.9 g, 10.7 mmol), dimethylcarbonate (24 mL) and sodium hydride (4.75 g, 118 mmol). After workup and purification, compound 11 was obtained (m=580 mg, 23%). .sup.1H NMR (400 MHz, DMSO-D.sub.6) δ(ppm) 11.94 (s, 2H), 7.77 (dd, J=8.4, 1.8 Hz, 2H), 7.58 (d, J=1.7 Hz, 2H), 7.44 (d, J=8.4 Hz, 2H), 4.23 (s, 3H), 3.64 (s, 6H).

Intermediate 13: methyl 3-(benzofuran-5-yl)-3-oxopropanoate

(56) ##STR00048##

(57) The reaction was carried out according to the general alkylation procedure A, using 12 as arylethanone (1.45 g, 9.06 mmol), dimethylcarbonate (25 mL) and sodium hydride (2.17 g, 90.6 mmol). After workup and purification, compound 13 was obtained (m=1.58 g, 80%). .sup.1H-NMR (300 MHz, CD.sub.3OD): δ(ppm) 8.35 (d, J=1.8 Hz, 1H), 7.98-8.01 (m, 1H), 7.89 (d, J=2.1 Hz, 1H), 7.61 (d, J=8.7 Hz, 1H), 7.00-7.01 (m, 1H), 4.17 (s, 2H), 3.65 (s, 3H).

Intermediate 9: methyl 3-(2-hydroxybenzo[d]thiazol-6-yl)-3-oxopropanoate

(58) ##STR00049##

(59) A 100 mL round bottom flask fitted with magnetic stirrer was charged with Sodium hydride (60% in mineral oil) (1 g, 48 mmol), dimethylcarbonate (4.2 mL) and tetrahydrofuran (30 mL). The commercial arylethanone 8 (1.94 g, 10 mmol) in tetrahydrofuran (30 mL) was added slowly with stirring and the whole was refluxed for 72 h. The mixture was slowly poured into water, acidified with saturated ammonium chloride (50 mL) and extracted with ethyl acetate. The organic layer was washed with water (50 mL) and dried over anhydrous Na.sub.2SO.sub.4. After evaporation under reduce pressure, the crude material was purified by crystallization in ethyl acetate to give the corresponding methyl 3-oxo-3-arylpropanoate 9 (m=1.89 g, 75%). .sup.1H NMR (400 MHz, DMSO-D.sub.6) δ(ppm) 12.33 (s, 1H), 8.24 (d, J=1.7 Hz, 1H), 7.89 (dd, J=8.4, 1.8 Hz, 1H), 7.22 (d, J=8.4 Hz, 1H), 4.17 (s, 2H), 3.65 (s, 3H).

(60) Synthesis of compound Cp1

(61) ##STR00050##

(62) Step 1. Cp1-methoxy: 5-(4-methoxyphenyl)-3H-1,2-dithiol-3-on. Mercury (II) acetate (4 g, 12.5 mmol) was added to a solution of commercial dithione 1 (1 g, 4.16 mmol) in mixture of acetic acid (v=25 mL) and chloroform (v=80 mL). The reaction mixture was stirred at room temperature overnight. The whole was filtrated and evaporated. The obtained solid was chromatographed on silica gel (petroleum ether:acetone=90:10) to give a yellow solid Cp1-methoxy (800 mg, 85% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) δ(ppm) 7.59 (d, J=8.9 Hz, 2H), 6.98 (d, J=8.9 Hz, 2H), 6.77 (s, 1H), 3.88 (s, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ(ppm) 194.29, 170.13, 162.52, 128.08, 125.08, 116.30, 114.75, 55.60.

(63) Step 2. Cp1: 5-(4-hydroxyphenyl)-3H-1,2-dithiol-3-one. A mixture of methyl aryl ether Cp1-methoxy (410 mg, 1.83 mmol) and pyridine hydrochloride (630 mg, 5.5 mmol) were placed in a round bottom flask and subjected to microwave irradiation at 250 W for 5 minutes. After complete conversion, the reaction mixture was passed through a column chromatography (dichloromethane:methanol=97:3) to give the dithione Cp1 (m=70 mg, 10% yield). .sup.1H NMR (400 MHz, Acetone-D.sub.6) δ(ppm) 9.31 (s, 1H), 7.80-7.66 (m, 2H), 7.05-7.00 (m, 2H), 6.98 (s, 1H). .sup.13C NMR (101 MHz, Acetone-D.sub.6) δ(ppm) 192.95, 170.48, 161.08, 160.96, 128.34, 123.94, 116.25, 116.16, 115.17.

Synthesis of Compound Cp2

(64) ##STR00051##

(65) Cp2: 5-(4-hydroxyphenyl)-3H-1,2-dithiol-3-one oxime. Hydroxylamine hydrochloride (140 mg, 2 mmol) was added to a solution of commercial dithione 2 (224 mg, 1 mmol) and sodium acetate (165 mg, 2 mmol) in ethanol (v=5 mL). The reaction mixture was stirred at room temperature overnight and then concentrated under reduce pressure. The obtained solid was chromatographed on silica gel (dichloromethane) to give the dithione Cp2 (m=51 mg, 32%) as a red solid. .sup.1H NMR (400 MHz, DMSO-D.sub.6) δ(ppm) 11.55 (s, 1H), 10.08 (s, 1H), 7.51 (d, J=8.7 Hz, 2H), 7.07 (s, 1H), 6.84 (d, J=8.7 Hz, 2H). .sup.13C NMR (101 MHz, DMSO-D.sub.6) δ(ppm) 161.99, 159.83, 153.46, 128.36, 123.71, 116.30, 112.77.

Synthesis of Compound Cp3

(66) ##STR00052##

(67) Ref.: MacDonald & McKinnon, 1967. Can. J. Chem. 45(11):1225-1229

(68) Step 1. Cp3-methoxy: 5-(4-methoxyphenyl)-3H-1,2,4-dithiazole-3-thione. The commercial isothiocyanate 3 (3 g, 15.5 mol) and phosphorus pentasulfide (6 g, 13.5 mmol) in carbon disulfide (18 mL) were placed in a round bottom flask and subjected to microwave irradiation at 65 W for 15 minutes. After complete conversion, the solution was filtered and evaporated under reduced pressure. The oily residue was treated with ethanol (approximately 30 mL) and cooled to 0° C. The crude dithiones were filtered off and recrystallized from (dichloromethane:ethanol=1:1) to give a yellow solid Cp3-methoxy (m=220 mg, 7% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) δ(ppm) 8.12 (d, J=8.9 Hz, 2H), 7.26 (s, 1H), 7.00 (d, J=9.0 Hz, 3H), 3.92 (s, 3H). .sup.13C NMR (101 MHz, DMSO-D.sub.6) δ(ppm) 185.35, 160.71, 126.65, 119.04, 110.23, 51.10.

(69) Step 2. Cp3: 5-(4-hydroxyphenyl)-3H-1,2,4-dithiazole-3-thione. A mixture of methyl aryl ether Cp3-methoxy (300 mg, 1.25 mmol) and dichloromethane (v=6 mL) were placed in a round bottom flask and cooled to 0° C. Boron tribromide, 1M in dichloromethane (v=6 mL, 6 mmol) was added slowly and the whole was stirred overnight. After complete conversion the reaction mixture was poured into water to give a suspension. The solid was filtered off and washed with water. Precipitation in dichloromethane gives the desired phenol Cp3 (m=270 mg, 95%). .sup.1H NMR (400 MHz, DMSO-D.sub.6) δ(ppm) 11.09 (s, 1H), 8.07 (d, J=8.8 Hz, 2H), 6.97 (d, J=8.8 Hz, 2H). .sup.13C NMR (101 MHz, DMSO-D.sub.6) δ(ppm) 214.93, 191.66, 165.12, 132.15, 122.22, 117.20.

Synthesis of Compound Cp4

(70) ##STR00053##

(71) Ref.: Brown et al., 2014. Bioorg Med Chem Lett. 24(24):5829-5831

(72) Step 1. Cp4-methoxy: 4-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione. The mixture of methyl 2-(4-methoxyphenyl)-3-oxopropanoate 4 (3.79 g, 19.52 mmol), Lawesson's reagent (7.89 g, 19.52 mmol), and sulfur (313 mg, 9.59 mmol) in 250 mL of toluene were heated to reflux for 270 minutes. When the reaction was completed the mixture was filtered and the filtrate was concentrated. Purification by column chromatography (petroleum ether:acetone=10:1) allowed to a red solid. The obtained solid was washed with ether and crystalized in acetone to give Cp4-methoxy (1.56 g, 33% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) δ(ppm) 8.37 (s, 1H), 7.50 (d, J=8.8 Hz, 2H), 6.97 (d, J=8.8 Hz, 2H), 3.84 (s, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ(ppm) 214.12, 160.10, 153.04, 149.08, 130.28, 125.43, 113.88, 55.35.

(73) Step 2. Cp4: 4-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione. A mixture of methyl aryl ether Cp4-methoxy (171 mg, 0.71 mmol) and pyridine hydrochloride (264 mg, 0.86 mmol) were placed in a round bottom flask and subjected to microwave irradiation at 250 W for 5 minutes. After complete conversion the reaction mixture was passed through a column chromatography (dichloromethane:methanol=97: 3) to give the dithione Cp4 (m=51 mg, 32%). H NMR (400 MHz, Acetone-D.sub.6) δ(ppm) 8.94 (s, 1H), 8.67 (s, 1H), 7.50 (d, J=8.6 Hz, 1H), 6.91 (d, J=8.6 Hz, 1H).

Synthesis of Compound Cp5

(74) ##STR00054##

(75) General procedure B. Phosphorus pentasulfide (0.7 mmol), sulfur (1 mmol), hexamethyldisiloxane HMDO (3 mmol) are heated in xylene (2.5 mL) at 145° C. for 5 minutes. The appropriate methyl 3-oxo-3-arylpropanoate is added by portions and the reaction mixture is refluxed for 1 h where the reaction is finished. Subsequently, the crude thiones are filtered off and the filtrates are concentrated. Purification by column chromatography and crystallizations allow to give the corresponding aryldithione.

(76) Cp5: 5-(2-hydroxybenzo[d]oxazol-5-yl)-3H-1,2-dithiole-3-thione. The synthesis of compound Cp5 was carried out according to the general thionation procedure B, using intermediate 11 (580 mg, 2.47 mmol), P.sub.4S.sub.10 (658 mg, 1.53 mmol), sulfur (79 mg, 2.55 mmol), HMDO (0.76 mL, 7.65 mmol), and xylene (5 ml). After workup, rapid purification on silica gel (THF) and crystallization in ethanol and acetone, Cp5 (m=70 mg) was obtained as a red-dark solid. .sup.1H NMR (400 MHz, DMSO-D.sub.6) δ(ppm) 12.07 (s, 1H), 7.84 (s, 1H), 7.66 (dd, J=8.4, 2.0 Hz, 1H), 7.59 (d, J=1.8 Hz, 1H), 7.45 (d, J=8.4 Hz, 1H). .sup.13C NMR (101 MHz, DMSO-D.sub.6) δ(ppm) 215.73, 173.92, 154.61, 146.65, 136.06, 132.07, 127.48, 122.31, 110.94, 108.69.

Synthesis of Compound Cp6a

(77) ##STR00055##

(78) CP6a: 5-(2-hydroxybenzo[d]thiazol-6-yl)-3H-1,2-dithiole-3-thione. The reaction was carried out according to the general thionation procedure Busing intermediate 9 (700 mg, 2.55 mmol), P.sub.4S.sub.10 (680 mg, 1.53 mmol), sulfur (81.5 mg, 2.55 mmol), HMDO (1.63 mL, 7.65 mmol), and xylene (6 mL). After workup, rapid purification on silica gel (THF) and further purification by reverse phase on C18 (acetonitrile: water gradient) Cp6a (m=6.7 mg) was obtained as red solid. .sup.1H NMR (400 MHz, DMSO-D.sub.6) δ(ppm) 8.26 (d, J=1.9 Hz, 1H), 7.84 (dd, J=8.4, 1.9 Hz, 1H), 7.77 (s, 1H), 7.22 (d, J=8.4 Hz, 1H). .sup.3C NMR (101 MHz, DMSO-D.sub.6) δ(ppm) 173.85, 170.59, 144.13, 140.39, 135.24, 126.37, 125.48, 122.20, 112.62.

Synthesis of Compound Cp8

(79) ##STR00056##

(80) Cp8: 5-(benzofuran-5-yl)-3H-1,2-dithiole-3-thione. The reaction was carried out according to the general thionation procedure B, using intermediate 12 (1.58 g, 7.25 mmol), P.sub.4S.sub.10 (1.93 g, 4.35 mmol), sulfur (232 mg, 7.25 mmol), HMDO (0.76 mL, 21.7 mmol), and xylene (15 mL). After workup, rapid purification on silica gel (dichloromethane) and crystallization in ethanol and acetone, Cp8 (m=890 mg) was obtain as a yellow solid. H NMR (400 MHz, DMSO-D.sub.6) δ(ppm) 8.26 (d, J=1.7 Hz, 1H), 8.14 (d, J=2.2 Hz, 1H), 7.86-7.81 (m, 3H), 7.75 (d, J=8.7 Hz, 1H), 7.06 (dd, J=2.2, 0.9 Hz, 1H). .sup.13C NMR (101 MHz, DMSO-D.sub.6) δ(ppm) 215.64, 174.84, 156.73, 148.51, 135.82, 128.82, 126.87, 124.25, 121.26, 113.04, 107.68.

Synthesis of Compound Cp9a

(81) ##STR00057##

(82) Cp9a: methyl 5-(3-thioxo-3H-1,2-dithiol-5-yl)-1H-indole-1-carboxylate. The reaction was carried out according to the general thionation procedure Busing intermediate 7 (700 mg, 2.55 mmol), P.sub.4S.sub.10 (680 mg, 1.53 mmol), sulfur (81.5 mg, 2.55 mmol), HMDO (1.63 mL, 7.65 mmol), and xylene (6 mL). After workup, rapid purification on silica gel (THF) and crystallization in ethanol and acetone, we have obtained Cp9a (m=55 mg) as a red-dark solid. .sup.1H NMR (400 MHz, DMSO-D.sub.6) δ(ppm) 8.20 (d, J=1.6 Hz, 1H), 7.81 (s, 1H), 7.69 (dd, J=8.7, 1.8 Hz, 1H), 7.59 (d, J=8.7 Hz, 1H), 7.49 (d, J=3.1 Hz, 1H), 6.59 (d, J=3.1 Hz, 1H), 3.84 (s, 3H). .sup.13C NMR (101 MHz, DMSO-D.sub.6) δ(ppm) 176.78, 138.90, 134.27, 132.59, 128.85, 122.84, 120.72, 111.52, 102.57, 34.12, 33.24, 31.09.

BIOLOGY EXAMPLES

Example 1: AOL does not Affect Mitochondrial Oxidative Phosphorylation

(83) Material and Methods

(84) Animal Procedures and Ethics Statement

(85) All experiments described were carried out in agreement with the National and European Research Council Guide for the care and use of laboratory animals. P. Diolez has a valid license to conduct experiments on animals by the Service Vétérinaire de la Santé et de la Protection Animale of the Ministère de l'agriculture et de la Forêt, France (Mar. 17, 1999, license number 3308010).

(86) Materials

(87) All the chemicals were reagent grade, purchased from Sigma Chemical (St. Louis, Mo.), except for sucrose and NADH oxidase (that were obtained from Merck (Darmstadt, Germany)). The trithio-AnethOL compound (AOL, corresponding to 5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione) was a gift from the private company GMPO (Paris, France). 15 mM stock solution was prepared in DMSO, and kept in darkness at 0° C. for only few days.

(88) Isolation of Mitochondria

(89) Male Wistar rats (250-325 g; obtained from Janvier Labs, Le Genest-Saint-Isle, France) were killed by stunning and cervical dislocation, and the heart was quickly removed and washed in cold isolation medium containing 100 mM sucrose, 180 mM KCl, 50 mM Tris, 5 mM MgCl.sub.2, 10 mM EDTA, and 0.1% (w/v) defatted BSA (pH 7.2).

(90) Isolation of heart mitochondria was performed in a cold chamber. Before homogenization, hearts (about 1.5 g) were minced with scissors and treated for 5 minutes in 5 mL of the same medium supplemented with protease (2 mg of bacterial proteinase type XXIV per mL of isolation buffer) with stirring. The tissue suspension was poured into a 50-mL glass Potter homogenizer, diluted with 20 mL of isolation buffer, then homogenized for 3 minutes using a motorized Teflon pestle. The homogenate was filtered through bolting cloth (Sefar Nitex) to remove debris, and centrifuged at 8,000 g for 10 minutes. The resulting pellet was rinsed with 5 mL of isolation buffer, resuspended in 25 mL of the same buffer, then subjected to low speed centrifugation (400 g) for 8 minutes. The resulting supernatant was centrifuged twice at 7,000 g for 15 minutes to yield a washed mitochondrial pellet that was gently resuspended in 150 μL of isolation buffer. Protein concentration was determined by the Bradford method (Sigma, kit #B6916) using BSA as standard. Mitochondria were kept on ice at a final concentration of 40-50 mg/mL for less than 5 hours.

(91) Mitochondrial Respiration

(92) Oxygen consumption rates of heart mitochondria (0.1 mg/mL), incubated in the absence or presence of AOL at increasing doses (from 0 to 80 μM final concentration), were recorded polarographically under constant stirring at 25° C. using a high-resolution oximeter (Oxygraph-2K, Oroboros Instruments, Austria). The respiration medium consisted in 140 mM sucrose, 100 mM KCl, 1 mM EGTA, 20 mM MgCl.sub.2, 10 mM KH.sub.2PO.sub.4, and 1 g/L (w/v) BSA essentially fatty acid free (pH 7.2).

(93) Mitochondrial ROS/H.sub.2O.sub.2 Production

(94) Rates of ROS/H.sub.2O.sub.2 production from heart mitochondria were assessed through the oxidation of the colorless, non-fluorescent indicator Amplex Red in the presence of exogenous horseradish peroxidase (HRP, EC 1.11.1.7, Sigma). H.sub.2O.sub.2 reacts with Amplex Red in a 1:1 stoichiometry, yielding the fluorescent compound resorufin (excitation: 560 nm; emission: 585 nm) which is stable once formed. Fluorescence was measured continuously with a spectro-fluorometer equipped with temperature control and stirring (SAFAS Xenius, Monaco). Isolated mitochondria (0.1 mg/mL) were incubated in the same experimental buffer than previously, supplemented with 15 μM Amplex Red and 10 μg/mL HRP. Glutamate (5 mM)/malate (2.5 mM) together with succinate (5 mM) were used as complex I and complex II substrates, respectively. Experiments were conducted under non-phosphorylating conditions in the presence of 15 μM atractyloside, i.e., under state IV conditions where mitochondrial membrane is maximal. Afterwards, rotenone (1.5 μM), antimycin A (2 μM), and myxothiazol (0.2 μM) were sequentially added to inhibit the redox centers within the electron transfer chain (FIG. 2), namely sites I.sub.Q, I.sub.F (with rotenone), III.sub.Qi (with antimycin A) and III.sub.QO (with myxothiazol). Assay was finally calibrated with known amounts of H.sub.2O.sub.2 (steps of 300 nM), in the presence of all relevant compounds, including AOL. The control test of the absence of effect of AOL on the Amplex Red assay itself and NAD(P)H oxidase ROS/H.sub.2O.sub.2 production was carried out in the absence of cardiac mitochondria and the presence of NAD(P)H oxidase (EC 1.6.3.3, 5 mU/mL, Sigma) and NADH (100 μM) solutions.

(95) Results

(96) We first verified (FIG. 1) that the AOL compound did not affect oxidative phosphorylation directly on isolated mitochondria from rat heart. This has been carried out by using the now classical oxygraph method. Mitochondria were first incubated with various AOL concentrations (5 to 80 μM) then respiratory substrate was added (substrate state, black curve), followed by a saturating ADP concentration to get the maximal oxidative phosphorylation rate (grey curve), and finally the addition of atractyloside (ATR) which inhibits the ADP/ATP translocator and gives the mitochondrial leak rate under non-phosphorylating conditions (FIG. 1A). The other panels of FIG. 1B-D presents the results obtained with different respiratory substrate combinations: glutamate+malate which feed electrons to complex I, succinate (+ rotenone) for complex II and glutamate+malate+succinate to feed electrons to both complexes. This last substrate combination has been chosen since it most closely resembles to in vivo conditions where Krebs cycle functions and both succinate and NADH are oxidized by respiratory chain. The results indicate that statistically no differences were observed in the presence of AOL for the large range of concentrations tested (FIG. 1), demonstrating under these conditions the absence of effect of AOL on mitochondrial oxidative phosphorylation—i.e., both on respiratory chain activity and ATP synthesis—as well as on mitochondrial inner membrane integrity (leak rate, after ATR addition). This last result indicates that AOL does not affect oxidative phosphorylation yield. Together, all these results confirm the absence of any harmful effect of AOL, documented by the use of this drug for human health for a long time.

Example 2: AOL Inhibits Superoxide/H.SUB.2.O.SUB.2 .Production by Mitochondria

(97) As previously stated, mitochondrial ROS production is highly dependent on mitochondrial activity and conditions. Although we tested the effects of AOL on ROS production by mitochondria under numerous conditions, we chose to present here, for the sake of clarity, only the most demonstrative results of the very specific effects of AOL. As already discussed, the presence of the substrate combination (i.e., glutamate, malate and succinate) (FIG. 2A) giving the electrons to the whole respiratory chain, is the most representative of in situ conditions in the cell where the metabolism is active. Furthermore, maximal mitochondrial ROS/H.sub.2O.sub.2 production does not occur under conditions of high mitochondrial phosphorylation but under conditions of high reduction of electron transporters, i.e., low or no phosphorylation. These conditions are fulfilled in the presence of ATR (inhibition of ATP/ADP translocator by ATR, (FIG. 1) and we could effectively verify that the addition of ATR under conditions of saturating ADP triggered the production of ROS which was at the detection limit under maximal phosphorylating conditions (results not shown). Under these conditions, ROS are produced at different sites of the respiratory chain (Orr et al., 2013. Free Radic. Biol. Med. 65:1047-1059; Quinlan et al., 2013. Redox Biol. 1:304-312) (FIG. 2). The main sites of production are located at complexes I and III, where large changes in potential energy of electrons occur (Balaban et al., 2005. Cell. 120(4):483-495; Goncalves et al., 2015. J. Biol. Chem. 290(1):209-27), which also allow proton pumping at these sites.

(98) We designed a series of inhibitor titrations in order to decipher the action of AOL on ROS production by the whole respiratory chain under conditions of maximal ROS production (FIG. 2E). In the absence of specific inhibitors of the complexes, ROS production is at maximum and mainly comes from reverse electron transport at site I.sub.Q (FIG. 2A). It is crucial to note that ROS produced by complex I, either by site I.sub.Q (quinone site) or site I.sub.f (flavin site), are delivered to the inner—matrix—side of the inner mitochondrial membrane. After addition of rotenone, a classical inhibitor of complex I which specifically binds to I.sub.Q, the ROS production decreases strongly and occurs almost entirely at site III.sub.QO and a remaining production at site I.sub.f due to the presence of complex I substrates and NADH production which are not inhibited by rotenone (FIG. 2B). The subsequent addition of antimycin A, an inhibitor of the electron transfer to cytochrome c, causes an increase in the reduced over oxidized quinone ratio, which is still reduced by complex II activity, and therefore a concomitant increase in ROS production at site III.sub.QO (FIG. 1C). Finally, the addition of myxothiazol, an inhibitor of complex III site III.sub.QO, abolishes complex III ROS production and the remaining very low production may be ascribed to the flavin site of complex I, for which we have no known inhibitor (Goncalves et al., 2015. J. Biol. Chem. 290(1):209-27).

(99) FIG. 3 illustrates the effect of the presence of increasing concentrations of AOL (from 5 to 80 μM) on ROS/H.sub.2O.sub.2 production measured under the different conditions defined in FIG. 2. It clearly appears from the results presented in this figure that AOL only affects the ROS production measured in the absence of inhibitor, by approximately 80%, while no statistical differences were observed with this range of AOL concentrations on ROS/H.sub.2O.sub.2 measured under the other conditions. As can be seen on FIG. 2, this specific condition (only ATR present) is the only condition in our assay where ROS are produced by complex I (site I.sub.Q). When rotenone is added to the assay, ROS/H.sub.2O.sub.2 production appears insensitive to AOL, even at high concentrations, whatever the site being involved. The clear absence of effect on several sites of mitochondrial ROS production is not only surprising but also asks interesting questions about the very mechanism of action of AOL on mitochondria. Indeed, these results rule out the basic hypothesis of the mode of action of AOL described in the previous papers and at the basis of the patent for its therapeutic use. These results effectively demonstrate that AOL is not a radical scavenger; otherwise its action would be independent of the origin of the ROS. However, since AOL clearly strongly decreases ROS production by complex I at site I.sub.Q, and only that site, we have the evidence that AOL specifically inhibits the formation of ROS at this site.

(100) Although the mechanism has still to be investigated, evidence is presented here that AOL compound specifically interferes with mitochondrial complex I and selectively inhibits superoxide production from the ubiquinone-binding site of complex I (site I.sub.Q) with no effects on superoxide production from other sites or on oxidative phosphorylation. To our knowledge, there is only one compound with comparable properties that has recently been described, the N-cyclohexyl-4-(4-nitrophenoxy) benzenesulfonamide (Orr et al., 2013. Free Radic. Biol. Med. 65:1047-1059). Like AOL, this compound does not modify the activity of complex I as a component of the respiratory chain and oxidative phosphorylation.

(101) The specificity of AOL was further tested in vitro using the peroxidase-Amplex Red system utilized for the measurement of ROS/H.sub.2O.sub.2 by mitochondria, which in fact measures the appearance of H.sub.2O.sub.2 by the oxidation of Amplex Red to the fluorescent resorufin (see FIG. 4). In the absence of mitochondria and by adding instead a H.sub.2O.sub.2-producing system to the measuring system, it was possible to test the effect of AOL on this system. This has been carried out by using commercial NAD(P)H oxidase which produces H.sub.2O.sub.2 in the presence of added NAD(P)H and measuring the reduction of Amplex Red to resorufin (FIG. 4). We did not observe any inhibition of the fluorescence under these conditions, which exclude any effect of AOL on the NAD(P)H oxidase or on the peroxidase activity (results not shown). These results confirm that AOL does not interfere either with the measurement system or directly interact with H.sub.2O.sub.2. Interestingly, these results also demonstrate that AOL does not inhibit the ROS/H.sub.2O.sub.2 production by the NADP(H) oxidase, which is one—if not the—major non-mitochondrial ROS/H.sub.2O.sub.2 producer in the cells. The scheme on FIG. 4 recapitulates the different information on the mode of action of AOL on ROS/H.sub.2O.sub.2 production by mitochondria and NAD(P)H oxidase and stresses the very high specificity demonstrated here. These results are in striking contrast with previous assertions on the putative effect of AOL as radical scavenger.

(102) When tested on isolated mitochondria from rat heart, AOL effectively decreases mitochondrial ROS/H.sub.2O.sub.2 production (in isolated mitochondria, H.sub.2O.sub.2 is produced from the reduction of ROS by mitochondrial superoxide dismutase). However, the results presented here clearly demonstrate that AOL does not act as a simple antioxidant or radical scavenger. While antioxidants are general ROS/H.sub.2O.sub.2 scavengers, AOL presents a complete selectivity towards the formation of ROS by site I.sub.Q in complex I, which demonstrates that AOL does not simply interact with superoxide radicals but specifically prevents their formation in complex L In that respect, AOL therefore appears as a member of a brand-new class of oxidative stress protectants, whose only one member has been described very recently (Orr et al., 2013. Free Radic. Biol. Med. 65:1047-1059). Whereas antioxidants generally do not interfere directly with electron transport and scavenge ROS and/or H.sub.2O.sub.2 downstream from production and therefore can never fully suppress the effect of ROS (Orr et al., 2013. Free Radic. Biol. Med. 65:1047-1059), AOL may act differently by preventing ROS formation and thus being more active to protect mitochondria from their own ROS.

(103) Data presented here go further and demonstrate that AOL is a specific inhibitor of ROS formation at site I.sub.Q of complex I of mitochondrial respiratory chain. Further experiments are however required to ascertain that AOL has completely no effect on other mitochondrial sites, but this does not preclude the above conclusions. We also show here some evidences that AOL may only interact with mitochondria without affecting oxygen radicals' formation in cytosol, and therefore would not affect intracellular signalisation.

(104) Inhibition of complex I activity by rotenone or the neurotoxin MPP.sup.+ has been linked to parkinsonism in both rodents and humans, suggesting a link between dysfunctional complex I, ROS production, and neurodegeneration (Langston et al., 1983. Science. 219(4587):979-980; Betarbet et al., 2000. Nat. Neurosci. 3(12):1301-1306). In contrast, comparative analyses show an inverse relationship between maximal superoxide/H.sub.2O.sub.2 production from site I.sub.Q, but not site I.sub.F, and maximum life span across diverse vertebrate species (Lambert et al., 2007. Aging Cell. 6(5):607-618; Lambert et al., 2010. Aging Cell. 9(1):78-91). Therefore, selective modulators of superoxide/1202 production from site I.sub.Q or site I.sub.F would offer unique opportunities to probe the putative role of mitochondrial ROS production in normal and pathological processes (Orr et al., 2013. Free Radic. Biol. Med. 65:1047-1059). There are also some speculations, even controversial, that site III.sub.Q—not affected by AOL—play an important role in cellular signalling during hypoxia.

(105) In conclusion, it appears that AOL properties may represent a breakthrough in the search for specific modulators of ROS/H.sub.2O.sub.2 production in cells. This is a current important issue in research, and AOL has an enormous advantage toward newly discovered molecules since it is already authorized for human use. AOL acts upstream from ROS production, therefore insuring higher protection than classical antioxidants; AOL acts specifically on mitochondrial ROS production; AOL ensures mitochondrial protection, crucial for numerous diseases, especially cardiac ones; AOL does not interfere with cell signalisation; AOL acts specifically on site I.sub.Q in complex I, which is the main mitochondrial site and may be implicated in important diseases, including Parkinson's disease and cardiac fibrillation. AOL may represent the first member of a new class of “protectants” that specifically prevent ROS production inside mitochondria, and may therefore be used for mitochondrial protection during various oxidative stress and therefore prevent diseases, with very little side effects on crucial cellular ROS signalling.

Example 3: Effect of AOL in a Cardiovascular Disease: Diabetes

(106) Effect of the Compound AOL on Glucose-Stimulated Insulin Secretion (GSIS) in Mouse Pancreatic Islets

(107) The aim of the study was to investigate the ability of the compound AOL in modulating glucose-stimulated insulin secretion (GSIS) in isolated pancreatic islets from mice.

(108) Material and Methods

(109) Experiments were conducted in strict compliance with the European Union recommendations (2010/63/EU) and were approved by the French Ministry of Agriculture and Fisheries (authorization no 3309004) and the local ethical committee of the University of Bordeaux. Maximal efforts were made to reduce the suffering and the number of animals used.

(110) Three independent experiments were carried out and, for each of them, two mice were sacrificed and islets isolated according to the procedure further described below.

(111) Pancreatic islets were isolated using the collagenase digestion method. Briefly, pancreas was inflated with Hanks solution containing 0.33 mg/mL of collagenase (Sigma-Aldrich), 5.6 mM glucose and 1% bovine serum albumin, pH 7.35, removed and kept at 37° C. for 6-9 minutes. After tissue digestion and exocrine removal by three consecutive washes, the islets were manually collected, under a binocular magnifier. Islets were left recovering from digestion by culturing for 20-24 hours in RPMI-1640 medium containing 11 mM glucose (Invitrogen, CA, USA) and supplemented with 2 mM glutamine, 200 IU/mL penicillin, 200 μg/mL streptomycin and 8% fetal bovine serum stripped with charcoal-dextran (Invitrogen).

(112) For each static GSIS experiment, islets from two mice were first incubated for 2 hours at 37° C. in 3 mL Krebs-bicarbonate buffer solution (in mM): 14 NaCl, 0.45 KCl, 0.25 CaCl.sub.2, 0.1 MgCl.sub.2, 2 HEPES and 3 glucose, equilibrated with a mixture of 95% 02:5% C02, pH 7.4. Then, groups of five size-matched islets were transferred to 24-well plate wells with 0.5 mL fresh buffer containing either one of the following stimulus: 3 mM glucose (Glc) and 11 mM glucose plus vehicle (0.4% DMSO in Krebs-bicarbonate buffer), or 11 mM glucose plus the diluted drug to be tested (10 μM or 20 μM of AOL in vehicle), and further incubated for 1 hour. Six different wells were used for each experimental condition. At the end of the incubation, bovine albumin was added to each well to a final concentration of 1%, and the plate was put at 4° C. for 15 minutes to stop insulin secretion. Next, the media was collected and stored at −20° C. for subsequent measurement of insulin content by ELISA (kit from Mercodia, Uppsala, Sweden), according to the manufacturer's instructions. Insulin secretion in each well was calculated as ng of insulin per islet and per hour of incubation, and then expressed as percentage of insulin secretion in 11 mM glucose vehicle group, which was considered 100%.

(113) Description of the experimental groups is shown in Table 1.

(114) TABLE-US-00001 TABLE 1 11 mM 11 mM Group 3 mM 11 mM Glc + AOL Glc + AOL abbreviation Glc-Veh Glc-Veh 10 μM 20 μM Group Group treated Group treated Group treated Group treated definition with vehicle with vehicle with AOL with AOL and 3 mM and 11 mM 10 μM and 20 μM and glucose glucose 11 mM 11 mM glucose glucose Number of 4-6 5-6 4-6 6 wells

(115) Results

(116) Individual insulin secretion values obtained in each of the three experiments were combined and averaged. These are expressed as the relative percentage of insulin secretion, normalized to the 11-mM glucose vehicle group (FIG. 5).

(117) Combined analysis of data shows that AOL enhanced GSIS at both 10 and 20 μM, showing a similar potency, with a GSIS increase ranging around 65-75% as compared to the 11 mM glucose vehicle group (One-way ANOVA; Bonferroni's post-test). For statistical analysis see Table 2.

(118) TABLE-US-00002 TABLE 2 One-way Sum of Degrees of Mean of ANOVA squares freedom squares F p Experiment Treatment 50630 3 16880 18.36 <0.0001 #1 Residual 16540 18 919.0 Experiment Treatment 44790 3 14930 21.46 <0.0001 #2 Residual 12530 18 695.9 Experiment Treatment 193700 3 64570 35.16 <0.0001 #3 Residual 33060 18 1837 Combined Treatment 244100 3 81360 33.13 <0.0001 experiments Residual 152200 62 2455

Conclusion

(119) The study demonstrates that AOL, at the doses tested (10 and 20 μM), enhances GSIS and significantly stimulates insulin secretion in vitro, in isolated pancreatic islets from mice.

(120) Thus, these findings suggest that AOL might be particularly useful in pathological conditions in which insulin secretion is deficient, such as diabetes, including type 1 diabetes, type 2 diabetes and other types of diabetes such as MODY (Maturity Onset Diabetes of the Young).

(121) Effects of a Chronic Treatment with AOL on Food Intake, Body Weight and Glucose Metabolism in Diet-Induced Obese Mice Material and Methods

(122) The aim of the study was to determine whether the compound AOL, administered daily at the doses of 5 mg/kg and 10 mg/kg for up to five weeks by intraperitoneal (i.p.) daily administration in diet-induced obese (DIO) mice fed with a high-fat diet (HFD), modifies food intake, body weight, adiposity and glucose metabolism.

(123) Mice were fed ad libitum with a HFD (60% of calories from fat, mostly lard) for twelve weeks before the pharmacological study begun. Animals received AOL or its vehicle by intraperitoneal (i.p.) administration and were maintained on HFD for the length of the study. Food intake and body weight were measured daily and recorded for up to three consecutive weeks.

(124) For appropriate distribution of the mice in the different experimental groups before the start of the pharmacological study, we evaluated their body composition in vivo using an Echo MRI 900 (EchoMedical Systems, Houston, Tex., USA) (see also Cardinal et al., 2014. Mol. Metab. 3(7):705-16; Cardinal et al., 2015. Endocrinology. 156(2):411-8). Daily food intake and body weight measurements were obtained using a balance (model TP1502, Denver Instruments).

(125) Thirty 7-weeks-old male C57/B16J mice arrived to the laboratory on 25 Feb. 2016 and underwent a first in vivo body composition analysis (Echo MRI 900, EchoMRI Systems) after 1 week of adaptation to the experimental housing room. After this first MRI analysis, animals were fed a high-fat diet (HFD) ad libitum for a period of twelve weeks. Thereafter, they underwent a second MRI analysis and were distributed into 3 experimental groups of equivalent body weight and body composition.

(126) Once the pharmacological treatment started (day 1), food intake (FI) and body weight (BW) were measured daily before the dark phase in animals housed in their home cage. Spillage of food was checked daily. The food consumed was calculated by subtracting the food left in the hoppers from the initial pre-weighted amount. FI and BW were measured for three consecutive weeks. Afterwards animals underwent a third MRI analysis in order to observe potential effects of the treatment onto the body composition (changes in fat and lean mass), followed by a glucose tolerance test (GTT) and an insulin tolerance test (ITT). Mice received daily i.p. administration of AOL or its vehicle for a total length of five weeks, until they were sacrificed.

(127) A nuclear echo magnetic resonance imaging whole-body composition analyzer (Echo MRI 900; EchoMedical Systems) was used to repeatedly assess body fat and lean mass in conscious mice.

(128) GTT and ITT are routinely used to assess dynamic modulation of glucose metabolism respectively during a glucose challenge and an insulin challenge. They give information on the presence of glucose intolerance and possible resistance to the action of the hormone insulin.

(129) Animals were injected i.p. with 1.5 g/kg of D-Glucose (Sigma-Aldrich) for the GTT or with 0.5 U/kg of insulin (Humulin, Lilly, France) for the ITT. For the GTT and the ITT, animals were fasted overnight. The tests were conducted the following morning. Blood samples were taken from the tail vein at different time points (0, 15, 30, 60, 90 and 120 minutes after the i.p. administration of glucose or insulin) and glucose concentration was measured using glucose sticks (OneTouch Vita, Lifescan France, Issy les Moulineaux, France).

(130) At sacrifice, blood samples were collected, blood glucose was rapidly assessed using glucose sticks and blood samples were then centrifuged at 3000 rpm for 15 minutes. The obtained plasma was stored at −80° C. for subsequent measurement of insulin, which was carried out by performing an ELISA (kit from Mercodia, Uppsala, Sweden), according to the manufacturer's instructions.

(131) HOMA-IR index, which gives information about the presence of insulin resistance, was calculated using the formula (Glucose mmol/L×Insulin mU/L)/22.5.

(132) Statistical analyses were carried out using GraphPad Prism Software (San Diego, Calif., USA). Repeated measurements two-way ANOVA were carried out to analyze the effects of the treatment factor, the time factor and their interaction on food intake, body weight, GTT and ITT. One-way ANOVA was carried out to compare the effect of the treatment factor on cumulative food intake, body composition, AUC of GTT and ITT, and circulating glucose, insulin and HOMA-IR at time of sacrifice. When ANOVA results were significant (p<0.05), the Tukey post-hoc test was performed to allow adequate multiple comparisons among the groups. Data are expressed as mean±SEM. Graphs were generated using GraphPad Prism software.

(133) Results

(134) The treatment did not have a significant effect on body weight or on the percentage (%) of change of the body weight calculated from day 1 in which body weight was measured before the first administration of AOL.

(135) Chronic administration of AOL after three weeks tended to reduce fat mass (p=0.13, FIG. 6), whilst it did not have any effect on lean mass (FIG. 7). The mean±SEM values are represented in FIG. 6 and FIG. 7 and statistical analysis are shown in Table 3 and Table 4, respectively.

(136) TABLE-US-00003 TABLE 3 Statistical analysis of data represented in FIG. 6. One-way Sum of Degrees of Mean of ANOVA squares freedom squares F p Treatment 18.50 2 9.248 2.151 0.1366 Residual 111.8 26 4.299

(137) TABLE-US-00004 TABLE 4 Statistical analysis of data represented in FIG. 7. One-way Sum of Degrees of Mean of ANOVA squares freedom squares F p Treatment 2.773 2 1.387 1.256 0.3014 Residual 28.70 26 1.104

(138) AOL at the dose of 10 mg/kg significantly blunted the action of insulin on circulating glucose levels during an ITT (FIG. 8), suggesting the presence of insulin resistance. Accordingly, a treatment effect was also found when analyzing the AUC (AUC veh: 12812.50±750.35, AUC AOL 5 mg/kg: 15006.56±1139.69, AUC AOL 10 mg/kg: 18168.33±1562.90, one-way ANOVA F(2, 23)=5.186, p=0.0138), with the AOL 10 mg/kg group having an AUC significantly higher than the vehicle group (Tukey post-hoc, p=0.0107). The mean±SEM values are represented in FIG. 8 and statistical analysis are shown in Table 5.

(139) TABLE-US-00005 TABLE 5 Post-hoc analysis on the treatment factor for data in FIG. 8. The numbers in the Tukey Post-hoc analysis table represent the p values. Values in bold correspond to significant (p < 0.05) results. Tukey post-hoc Vehicle AOL 5 mg/kg AOL 10 mg/kg Vehicle 0.532016 0.023546 OP 5 mg/kg 0.532016 0.232976 OP 10 mg/kg 0.023546 0.232976

(140) At time of sacrifice, after five weeks of treatment, blood glucose levels were measured in 2-hour fasted mice.

(141) AOL tended to decrease blood glucose levels (FIG. 9) and statistical analysis in Table 6.

(142) TABLE-US-00006 TABLE 6 Statistical analysis of data represented in FIG. 9. One-way Sum of Degrees of Mean of ANOVA squares freedom squares F p Treatment 3972 2 1986 2.586 0.0980 Residual 16898 22 768.1

Conclusion

(143) In diet-induced obese animals, chronic daily administration of AOL tended to decrease body weight and food intake in DIO mice (data not shown). Accordingly, this was associated with a trend to decrease fat mass and basal blood glucose levels.

(144) Overall, these data suggest that AOL might have some beneficial effects in a model of dietary obesity.

Example 4: Effect of AOL in a Neurologic Disease: Parkinson Disease

(145) In this study, the potential neuroprotective effects of AOL were assessed by counting the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN) in the sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Mice were treated with AOL (5 mg/kg; i.p.) or vehicle for 11 consecutive days. MPTP (20 mg/kg; i.p.) or saline was administered on treatment days 4-8. All mice were killed on day 12 following final administration of treatment.

(146) Sub-chronic MPTP administration in C57/bl6 mice induces degeneration of nigrostriatal dopaminergic neurons, which leads to reduced number of TH-positive neurons in the SN which was, on this occasion, a reduction of 39%.

(147) Material and Methods

(148) For vehicle conditions, the test item was dissolved in 0.5% DMSO/0.95% Tween 20 in saline while AOL was administered intraperitoneally (i.p.) at the dose of 5 mg/kg. The volume of administration was 10 mL/kg.

(149) C57bl/6 male mice (Janvier) weighing 22-28 g were housed in a temperature-controlled room under a 12-hour light/dark cycle with free access to food and water. In order to tentatively achieve final numbers of n=10 per group, n=12 per group were used to account for possible losses in the course of the experiment. To produce neurodegeneration of dopaminergic neurons in the substantia nigra, mice were treated with MPTP hydrochloride (20 mg/kg i.p. once daily for five consecutive days).

(150) Mice were humanely euthanized by cervical dislocation after the last administration.

(151) The caudal half of the brain (containing the substantia nigra) was placed in paraformaldehyde (4% in 0.1 M Phosphate Buffer Saline (PBS) pH 7.4) for 5 days and then transferred to 20% sucrose (20% in 0.1 M PBS) for cryoprotection. The tissue was then frozen in cold isopentane (at −50° C.±2° C.).

(152) The striata were dissected out, weighed and snap frozen separately in dry ice (at −70° C.±10° C.). Tissues samples are stored at −70° C. (±10° C.) for an optional HPLC analysis of dopamine and its metabolites. If this option is not taken, the striata will be destroyed.

(153) Coronal serial sections of the entire mesencephalon were cut on a cryostat at 50 μm intervals. Sections were collected free-floating in well-plates containing cryoprotectant solution, which were then stored at −20° C. until the day of TH immunohistochemical processing.

(154) TH immunohistochemistry was performed as follows on every fourth section. Tissue sections were taken from the −20° C. freezer, left to adjust to room temperature, and then rinsed in PBS solution. Endogenous peroxidase was inhibited by incubating in PBS containing 0.3% H.sub.2O.sub.2 for 10 minutes. Following this, sections were washed in PBS, incubated in PBS 4% normal horse serum (NHS) and 0.3% Triton X-100 for 30 minutes, for the blockade of non-specific antigenic sites. Sections were then incubated overnight at room temperature in antibody dilutant+primary antibody for tyrosine hydroxylase (TH) (anti-TH affinity isolated antibody, Sigma T8700) at a dilution of 1/10,000. Sections were then rinsed thoroughly in PBS and incubated for 30 minutes in ImmPRESS Ig peroxidase polymer detection reagent (Vector MP7401). Following this, sections were thoroughly washed with PBS. Immunological staining was then revealed with 3,3′-Diaminobenzidine (DAB)/Tris/H.sub.2O.sub.2 kit (Vector SK4100). After one minute, revelation was stopped with several PBS washes. Sections were mounted and counterstained with 0.1% cresyl violet.

(155) Unbiased stereological analysis was used to estimate the number of TH-immunopositive (TH+) neurons (Mercator, Explora Nova, La Rochelle, France). The boundaries of the SN were determined by examining the size and shape of the different TH+ neuronal groups. The volume was calculated by using the formula: V=ΣS td; where ΣS is the sum of surface areas, t is the average section thickness and d is the number of slices between two consecutive sections measured. One in every 4 sections was used; optical dissectors were distributed using a systematic sampling scheme. Dissectors (50 μm length, 40 μm width) were separated from each other by 150 μm (x) and 120 μm (y). The following formula was used to estimate the number of TH+ neurons: N=V(SN) (ΣQ−/ΣV(dis)); where N is the estimation of cell number, V is the volume of the SN, ΣQ− is the number of cells counted in the dissectors, and ΣV(dis) is the total volume of all the dissectors. Mean estimated number of neurons and SEM was then calculated for each group.

(156) All statistical analyses were performed using Graphpad prism version 7. All data are presented as mean±the standard error of the mean (SEM). The effect of AOL was analyzed with a one-way ANOVA followed by Dunnett's multiple comparisons post-hoc analysis. A P value of less than 0.05 was considered significant.

(157) Results

(158) There was a significant effect of treatment on the number of TH+ cells in the SN (F2,29=10.94, p<0.001, FIG. 10). The number of TH+ cells in the SN was reduced by 39% (p<0.001) in MPTP-treated compared to vehicle-treated animals. Following administration of AOL, the number of TH+ cells in the SN was increased by 44% (p<0.01) compared to vehicle in the MPTP-treated mice.

Conclusion

(159) AOL treatment for 11 consecutive days, at a dose of 5 mg/kg, has a significant neuroprotective effect compared to vehicle, in preventing the MPTP-induced reduction in TH+ cells in the SN, resulting in 44% more cells surviving in the SN with the administration of AOL.

(160) These data suggest that AOL treatment can protect the dopaminergic neurons in the substantia nigra from MPTP intoxication.

Example 5: Effect of AOL in Cardiovascular Disease: Ischemia-Reperfusion Injury

(161) The present study aims at evaluating the capacity for AOL to protect perfused rat heart from the damages occurring after global ischemia and reperfusion.

(162) The consequences of 30 minutes' global ischemia followed by 120 minutes' reperfusion (FIG. 11) on contractility and tissue viability were studied on isolated perfused rat heart pretreated or not (control vehicle) with 10 μM AOL.

(163) Material and Methods

(164) All procedures conformed to the UK Animals (Scientific Procedures) Act 1986 and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23. revised 1996). Male Wistar rats (250-300 g) were anesthetized by 3% isoflurane, heparinized and euthanized by a lethal IP injection of pentobarbital (130 mg/kg). Hearts (˜0.95 g of fresh weight) were rapidly harvested and placed into ice cold Krebs-Henseleit buffer containing (in mmol/L): NaCl 118, NaHCO.sub.3 25, KCl 4.8, KH.sub.2PO.sub.4 1.2, MgSO.sub.4 1.2, glucose 11 and CaCl.sub.2 1.8; gassed with 95% O.sub.2/5% CO.sub.2 at 37° C. (pH 7.4). Langendorff heart perfusions were performed (Garlid et al., 2006. Am. J. Physiol. Heart Circ. Physiol. 291(1):H152-60) and contractility was assessed by continuous measurement of the rate pressure product (RPP) thanks to a balloon placed in the left ventricle and connected to a pressure transducer. Hearts were perfused in a constant flow mode (12 mL/min). After 10 minutes for stabilization followed by 10 minutes of treatment with the vehicle (Control) or 10 μM AOL solution, global normothermic ischemia was induced by halting perfusion flow for 30 minutes while immersing the heart in perfusion buffer at 37° C. At the end of the reperfusion period, hearts were stained to assess infarct size, or freeze-clamped using liquid-nitrogen cooled tongues. In the latter case, hearts were grinded under liquid nitrogen, and stored at −80° C. for further analyses.

(165) At the end of the reperfusion period, hearts were stained with triphenyltetrazolium chloride (TTC): hearts were perfused for 7 minutes at 13 mL/min with a 12% (w/v) TTC solution in order to get a 1% final concentration in the heart. Hearts were then detached from the cannula and incubated for an additional 4 minutes at 37° C. before being sliced perpendicular to the longitudinal axis into 6 slices. The slices were then treated in 4% (w/v) formalin solution overnight at 4° C. and weighed, before both sides of each slice were photographed. The surface of the necrotic and at-risk areas of each side were determined on each photography by planimetry (AlphaEase v5.5), and infarct size was expressed as a percentage of the total cross-sectional area of the heart, since total heart was subjected to ischemia.

(166) Data from 6 independent preparations are expressed as means±SEM. The n number in each group being smaller than 20, the distribution was considered as non-normal and consequently a non-parametric Mann-Whitney test (SPSS statistics 17.0) was performed to compare the two groups. Results were considered statistically significant if the p-value was below 0.05.

(167) Results

(168) FIG. 11 presents the evolution of the RPP—considered here as a surrogate of heart contractility—during the critical phase of the reperfusion following ischemia. Clearly, AOL improves the contractility and after an identical evolution as compared to the control hearts treated with AOL, showed an improvement of contractility which was about three times higher than control hearts after 2 hours of reperfusion. At this time, hearts were prepared for TTC staining to assess tissue viability. The higher contractile activity for AOL hearts was confirmed by TTC staining, and photographs of the slices of treated and non-treated hearts (data not shown) clearly show that AOL induced an important protection of cardiac tissue. This protection has been analyzed more thoroughly and the results are presented in FIG. 12. The infarct size—damaged tissue—is expressed as percentage of the total surface for each independent experiment together with the mean value for AOL-treated and non-treated hearts. Results clearly show that AOL highly significantly protects cardiac tissue from ischemia/reperfusion damages. In fact, about 50% of infarcted tissue was rescued by pre-treatment with AOL (FIG. 12).

Conclusion

(169) These results extend, under ex vivo (living organ) conditions, the role of AOL as an inhibitor of mitochondrial ROS production, most probably at the level of complex I.

(170) They also evidence the therapeutic interest of AOL for tissue protection against ischemia/reperfusion damages, not only in heart but also in any tissue subjected to ischemia.

Example 6: Effect of AOL in a Cardiovascular Disease: Pulmonary Hypertension

(171) The present study aims at studying the role of mitochondria in the pulmonary vasculature physiology and providing a new alternative treatment of pulmonary hypertension. This disease is characterized by increased pulmonary arterial pressure and remodeling of pulmonary arteries (PA), leading to increased pulmonary vascular resistance, hypertrophy of the right ventricle, right heart failure and ultimately, death.

(172) Pulmonary hypertension can be divided into five groups, among which the group 1 corresponds to pulmonary arterial hypertension. As for group 3, it includes pulmonary hypertension due to lung diseases (such as chronic obstructive pulmonary disorder) and/or alveolar hypoxemia.

(173) To address the issue of the effect of AOL, two different rat models were used: a hypoxia model and a monocrotaline-induced model, that share pathophysiological characteristics with group 3 and group 1 pulmonary hypertension, respectively.

(174) Material and Methods

(175) Male Wistar rats (300-400 g) were separated into 3 groups and used 4 weeks later: the first group (control or normoxic rats—N rats) was housed in ambient room air; the second group (chronic hypoxic rats—CH rats) was exposed to chronic hypoxia for 3 weeks in a hypobaric chamber (50 kPa); and the third group (MCT rats) was injected with a single intraperitoneal dose of monocrotaline at a dose of 60 mg/kg. MCT (Sigma, St Quentin Fallavier, France) was dissolved in an equal volume of HCl (1 M) and NaOH (1 M).

(176) In each group, some animals were treated with AOL (Sulfarlem, EG Labo Eurogenerics. Crushed tablets mixed with food, fed ad libitum) and some other animals were untreated. Eaten food was weighed every day to estimate the AOL dose administered. 10 mg/kg/day was thus administered during the 3 weeks of experiment for the second and third groups.

(177) For each condition, 7 to 10 rats were used. All animal care and experimental procedures complied with the recommendations of the Federation of European Laboratory Animals Science Association, and were approved by the local ethics committee (Comité d'éthique régional d'Aquitaine—referenced 50110016-A).

(178) Pulmonary hypertension was assessed by measuring both the mean pulmonary arterial pressure (mPAP) and right ventricle hypertrophy. To measure PAP, N, CH and MCT rats were anesthetized with pentobarbital sodium (Centravet) by intraperitoneal injection (60 mg/kg) and mPAP was measured, in closed-chest rats, through a catheter inserted in the right jugular vein, then through the right atria and the right ventricle into the pulmonary artery, and attached to a Baxter Uniflow gauge pressure transducer. Right ventricle hypertrophy was estimated by the ratio of right ventricle (RV) to left ventricle plus septum (LV+S) weight (Fulton index).

(179) Pulmonary arteries (PA) remodeling was assessed by measuring the percentage of the PA medial thickness from sections of paraffin-embedded lung. Lung sections were first stained with hematoxylin and eosin (VWR) according to common histological procedure. On each section, three groups of 10 intracinar arteries with different cross-sectional diameters were observed to evaluate medial wall thickness (namely cross-sectional diameters under 50 μm, between 50 to 100 μm and between 100 to 150 μm).

(180) Results

(181) Results are expressed as mean±SEM of n independent observations. All data were analyzed using a non-parametric test for unpaired samples (Mann-Whitney test). FIG. 13 shows the effect of AOL on pulmonary arterial pressure (FIG. 13A) and heart remodeling (FIG. 13B). n indicates the number of rats for mPAP and Fulton index measurements. All bar graphs and statistics were performed with Graphpad PRISM software (v6, Graphpad Software). P<0.05 were considered significant. As seen, AOL had no significant effect on the control group (N rats). However, mean pulmonary arterial pressure was decreased in MCT rats treated with AOL, and even more significantly in CH rats treated with AOL. AOL treatment had however no effect on the Fulton index.

(182) FIG. 14 shows the effect of AOL on pulmonary arteries remodeling. n indicates the number of vessels analyzed for % of medial thickness measurement. All bar graphs and statistics were performed with Graphpad PRISM software (v6, Graphpad Software). P<0.05 were considered significant. AOL shows a significant effect in CH rats, in which pulmonary arteries diameter was reduced by 30%.

Conclusion

(183) AOL treatment, at an oral dose of 10 mg/kg/day, has a significant effect in the prevention and/or treatment of pulmonary hypertension in vivo, in particular in group 3 pulmonary hypertension. Results indeed show a significant improvement of clinical symptomatology.

(184) These data suggest that mitochondria play a major role in the pulmonary vasculature physiology, and extend the use of AOL to the treatment of pulmonary hypertension.

Example 7: Effect of AOL in Aging Disease and Progeroid Syndromes: Macular Degeneration

(185) The present study aims at evaluating the capacity for AOL to protect retina against progressive degeneration.

(186) Material and Methods

(187) Rats bred under cyclic low-intensity lighting were transferred to cyclic high-intensity lighting for one week and divided into 3 groups (non-treated animals, vehicle-treated animals and AOL-treated animals). Treated animals received injections of vehicle or AOL at a dose of 6 mg/kg/day, three times a day for the 7 days of the transfer (30 minutes before light-ON; at 01.00 μm; at 09.00 μm). After one week, animals were transferred in the dark (D0).

(188) A control group (“untransferred”) was not transferred to cyclic high-intensity lighting but received the same treatment as described above: injections of vehicle or AOL three times a day for 7 days, followed by a transfer in the dark (D0).

(189) On the day following the transfer in the dark (D1), a first electroretinography is performed. It measures the electrophysiological signal which is generated by the retina, in response to a light stimulation. It is typically characterized by two waves, namely a-wave and b-wave. a-wave represents the initial corneal-negative deflection, derived from the cones and rods of the outer photoreceptor layers. It reflects the hyperpolarization of the photoreceptors due to closure of sodium ion channels in the outer-segment membrane. b-wave represents the corneal-positive deflection, derived from the inner retina (predominantly Muller and ON-bipolar cells). Analysis of the electroretinogram consists in measuring the amplitude and/or latency of these waves, as a function of the intensity of the light stimulation. a-wave amplitude, for a given light stimulation intensity, depends on the number of photoreceptors; whereas amplitude of b-wave, for a given light stimulation intensity and a given number of photoreceptors, indicates the signal transmission efficiency.

(190) After D1's electroretinography, animals were transferred back under cyclic low-intensity lighting conditions, and a second electroretinography is performed at D15.

(191) Animals were then sacrificed for histological analysis. The thickness of the various layers of the retina, in particular the thickness of the outer nuclear layer (ONL) and inner nuclear layer (INL) were measured (in μm, from the optical nerve and every 0.39 mm in the superior and inferior poles of the optic disc).

(192) Results

(193) Histological analysis is reported on FIG. 15. It shows that, in the control group (“untransferred”), treatment with AOL has no effect on ONL's thickness (FIG. 15A). This suggests that AOL does not have a toxic effect on retina's photoreceptors.

(194) On the contrary, transfer in cyclic high-intensity lighting conditions (“transferred”) induces a significant decrease (by half in some areas) of the ONL, in non-treated animals. AOL however tends to protect the ONL against light-induced damages. Histological analysis has indeed shown a significant increase of the thickness of the ONL in AOL-treated/cyclic high-intensity lighting-exposed animals (FIG. 15B).

Conclusion

(195) AOL treatment has a significant protective effect against light-induced damages on the retina. In particular, the thickness of the retina was shown to be preserved as compared to non-treated animals, after prolonged cyclic high-intensity lighting exposure.

Example 8: Effect of AOL in Diseases Related to Mitochondrial Dysfunctions

(196) The present study aims at testing the effect of AOL in vivo, in a model of oxidative phosphorylation dysfunction.

(197) Material and Methods

(198) Mice deficient in mitochondrial Mn-Superoxide Dismutase (Sod2-KO) on a CD1-background were used. This genetic alteration leads to an adverse phenotype and the death of animals at an average of 8 days old. Mitochondrial superoxide dismutase is a free radical scavenging enzyme which transforms superoxide (highly reactive) into hydrogen peroxide (less reactive), that could then cross mitochondrial membranes and be detoxified by matrix and cytosolic anti-oxidant systems. The aim of this study was to test if AOL could rescue the Sod2-KO phenotype through its activity on I.sub.Q superoxide production.

(199) After birth, pups were genotyped (3 day-old) and the litter size was reduced to 6 pups per cage. Animals were then treated (AOL in Kolliphor®—5 mg/kg) or not (Kolliphor® only, noted KOL below). The choice of the dosage was mainly driven by the solubility limit of the compound (2.8 mM in Kolliphor®) and the maximum injectable volume in pups (6 to 7 μL per gram body mass). Two studies were conducted on two different generations from the same parents: lifespan; and succinate dehydrogenase activity in heart (SDH) and Oil Red O staining in liver. Animals were weighed and injected (intra-peritoneal) daily.

(200) Succinate dehydrogenase activity is a marker of superoxide in the mitochondrial matrix. Thus, a lack of SOD2 is associated with a decrease of SDH activity in heart. The aim of this experiment was to test whether or not AOL could restore SDH activity in KO mice.

(201) Oil Red O staining is a marker of lipid that has been shown to accumulate in Sod2-KO liver. However, the direct link between superoxide/hydrogen peroxide production and liver lipid accumulation is not established. The aim of the study was to test the potency of AOL to prevent liver lipid accumulation in Sod2-KO mice.

(202) Results

(203) Lifespan

(204) 4 groups were constituted:

(205) 1—WT-KOL (n=7), a group of wild-type mice treated with the vehicle only;

(206) 2—WT-AOL (n=17), a group of wild-type mice treated with AOL;

(207) 3—KO-KOL (n=2), a group of Sod2-KO mice treated with the vehicle only;

(208) 4—KO-AOL (n=4), a group of Sod2-KO mice treated with AOL.

(209) Animals were injected once a day from 3 days old until their death.

(210) FIG. 16A and FIG. 16B show the evolution of body weight (A) and percentage of initial body weight. These results show that body weight and body weight gain were lower in KO-mice than in WT-mice. Treatment with AOL however tends to alleviate this effect as seen from day 8 to 12, suggesting a potential beneficial effect of the compound.

(211) FIG. 16C shows the survival proportion of Sod2-KO mice whether they were treated with AOL or not. As expected in view of the above results, both median lifespan and maximal lifespan were slightly improved by AOL treatment in KO mice, with AOL-treated mice living up to 2 days longer as compared to untreated mice, supporting a beneficial effect of AOL.

(212) SDH activity in heart & Oil Red O staining

(213) 5 groups were constituted:

(214) 1—WT-non-injected (n=6), a group of untreated wild-type mice;

(215) 2—WT-KOL (n=6), a group of wild-type mice treated with the vehicle only;

(216) 3—WT-AOL (n=6), a group of wild-type mice treated with AOL;

(217) 4—KO-KOL (n=4), a group of Sod2-KO mice treated with the vehicle only;

(218) 5—KO-AOL (n=6), a group of Sod2-KO mice treated with AOL.

(219) In this study, animals were treated daily (5 mg/kg) from day 3 to day 5. Heart and liver were harvested at day 6.

(220) As expected, SDH activity tended to decrease (not significant) in KO compared to WT animals. However, AOL showed only a very slight increase in SDH activity in KO mice, but could not restore SDH activity to the levels of WT mice (FIG. 17).

(221) FIG. 18 shows lipid droplets average size (Panel A), density (Panel B) and area (Panel C). Untreated KO mice exhibited a high lipid content phenotype compared to WT animals. In AOL-treated KO mice however, lipid droplets density decreased as compared to untreated animals. More importantly, these results also show that AOL treatment was able to restore the total lipid area in KO mice, consistent with on-target suppression of mitochondrial superoxide production in vivo in Sod2-KO mice.

Conclusion

(222) In vivo studies show encouraging results. Although AOL treatment could not fully counteract the effects of SOD2 depletion in mice, results show that lifespan could still be extended by a couple of day as compared to untreated KO animals, together with an alleviation of the decrease in body weight gain. This suggests a potential effect of AOL.

(223) AOL bioavailability is known to be very short. Thus, treatment with higher doses might lead to improve AOL effects in these experiments. However, constitutive KO remains a high adverse phenotype to rescue with only one very specific treatment and may require synergic action with other drugs.

(224) In vivo, results also showed that AOL could restore lipid content and/or prevent lipid accumulation in liver of Sod2-KO mice.

Example 9: AOX Affects Mitochondrial Oxidative Phosphorylation at High Concentrations (Over 20 μM)

(225) Material and Methods

(226) Animal Procedures and Ethics Statement

(227) All experiments described were carried out in agreement with the National and European Research Council Guide for the care and use of laboratory animals. P. Diolez has a valid license to conduct experiments on animals by the Service Vétérinaire de la Santé et de la Protection Animale of the Ministère de l'agriculture et de la Forêt, France (Mar. 17, 1999, license number 3308010).

(228) Materials

(229) All the chemicals were reagent grade, purchased from Sigma Chemical (St. Louis, Mo.), except for sucrose and NADH oxidase (that were obtained from Merck (Darmstadt, Germany)). AOL and AOX were gift from OP2 (Bordeaux, France). 15 mM stock solutions were prepared in DMSO, and kept in darkness at 0° C. for only few days.

(230) Isolation of Heart Mitochondria

(231) Male Wistar rats (250-325 g; obtained from Janvier Labs, Le Genest-Saint-Isle, France) were killed by stunning and cervical dislocation, and the heart was quickly removed and washed in cold isolation medium containing 100 mM sucrose, 180 mM KCl, 50 mM Tris, 5 mM MgCl.sub.2, 10 mM EDTA, and 0.1% (w/v) defatted BSA (pH 7.2).

(232) Isolation of heart mitochondria was performed in a cold chamber. Before homogenization, hearts (about 1.5 g) were minced with scissors and treated for 5 minutes in 5 mL of the same medium supplemented with protease (2 mg of bacterial proteinase type XXIV per mL of isolation buffer) with stirring. The tissue suspension was poured into a 50-mL glass Potter homogenizer, diluted with 20 mL of isolation buffer, then homogenized for 3 minutes using a motorized Teflon pestle. The homogenate was filtered through bolting cloth (Sefar Nitex) to remove debris, and centrifuged at 8,000 g for 10 minutes. The resulting pellet was rinsed with 5 mL of isolation buffer, resuspended in 25 ml of the same buffer, then subjected to low speed centrifugation (400 g) for 8 minutes. The resulting supernatant was centrifuged twice at 7,000 g for 15 minutes to yield a washed mitochondrial pellet that was gently resuspended in 150 μL of isolation buffer. Protein concentration was determined by the Bradford method (Sigma, kit # B6916) using BSA as standard. Mitochondria were kept on ice at a final concentration of 40-50 mg/mL for less than 5 hours.

(233) Mitochondrial Oxygen Consumption and ATP Synthesis

(234) Oxygen consumption rates of heart mitochondria (0.1 mg/mL), incubated in the absence or presence of AOX at increasing doses (from 0 to 100 μM final concentration), were recorded polarographically under constant stirring at 25° C. using a high-resolution oximeter (Oxygraph-2K, Oroboros Instruments, Austria). The respiration medium consisted in 140 mM sucrose, 100 mM KCl, 1 mM EGTA, 20 mM MgCl.sub.2, 10 mM KH.sub.2PO.sub.4, and 1 g/L (w/v) BSA essentially fatty acid free (pH 7.2).

(235) ATP synthesis was measured under the same conditions using a high sensitivity pH electrode (Metrohm) as previously described (Gouspillou et al., 2014. Aging Cell. 13(1):39-48).

(236) Mitochondrial ROS/H.sub.2O.sub.2 Production

(237) Rates of ROS/H.sub.2O.sub.2 production from heart mitochondria were assessed through the oxidation of the colorless, non-fluorescent indicator Amplex Red in the presence of exogenous horseradish peroxidase (HRP, EC 1.11.1.7, Sigma). H.sub.2O.sub.2 reacts with Amplex Red in a 1:1 stoichiometry, yielding the fluorescent compound resorufin (excitation: 560 nm; emission: 585 nm) which is stable once formed. Fluorescence was measured continuously with a spectro-fluorometer equipped with temperature control and stirring (SAFAS Xenius, Monaco).

(238) Isolated mitochondria (0.1 mg/mL) were incubated in the same experimental buffer than previously, supplemented with 15 μM Amplex Red and 10 μg/mL HRP. Glutamate (5 mM)/malate (2.5 mM) together with succinate (5 mM) were used as complex I and complex II substrates, respectively. Experiments were conducted under non-phosphorylating conditions in the presence of 15 μM atractyloside. Afterwards, rotenone (1.5 μM), antimycin A (2 μM), and myxothiazol (0.2 μM) were sequentially added to inhibit the redox centers within the electron transfer chain (see FIG. 2), namely sites I.sub.Q, I.sub.F (with rotenone), III.sub.Qi (with antimycin A) and III.sub.QO (with myxothiazol). Assay was finally calibrated with known amounts of H.sub.2O.sub.2 (steps of 300 nM), in the presence of all relevant compounds, including AOX. The control test of the absence of effect of AOL, AOX and Oltipraz on the Amplex Red assay itself and NAD(P)H oxidase ROS/1202 production was carried out in the absence of cardiac mitochondria and the presence of NAD(P)H oxidase (EC 1.6.3.3, 5 mU/mL, Sigma) and NADH (100 μM) solutions.

(239) Results

(240) We first tested if AOX compound affects oxidative phosphorylation directly on isolated mitochondria from rat heart. This has been carried out by using the now classical oxygraph method (FIG. 19A). Mitochondria were first incubated with various AOX concentrations (20 to 100 μM), then respiratory substrate was added (substrate state, black curve), followed by a saturating ADP concentration to get the maximal oxidative phosphorylation rate (slope of oxygen consumption, grey curve), and finally the addition of atractyloside (ATR) which inhibits the ADP/ATP translocator and gives the mitochondrial leak rate under non-phosphorylating conditions (FIG. 19A). FIG. 19B presents the results obtained with succinate (+ rotenone) to feed electrons to respiratory chain. This substrate has been chosen since it most closely reflects respiratory chain regulation. The results indicate that under “substrate” state and ATR state (inner membrane proton leak rate), AOX induced an increase for concentrations up to 50 μM followed by a decrease for concentrations over 50 μM. These data suggest an uncoupling effect of AOX on oxidative phosphorylation for concentrations higher than 20 μM and a concomitant inhibition of oxidation rate. Data demonstrate under these conditions, the effect of high concentrations of AOX (higher than 20 μM) on mitochondrial oxidative phosphorylation—i.e., on both respiratory chain activity and ATP synthesis—as well as on mitochondrial inner membrane integrity (leak rate measured after ATR addition).

(241) FIG. 20 presents the effects of AOX on the phosphorylation rate (ATP synthesis rate) of isolated rat heart mitochondria. Results confirm that concentrations lower than 20 μM do not modify ATP synthesis by isolated mitochondria. However, higher concentrations do effectively decrease phosphorylation rate and completely abolish it at 60 μM.

Example 10: AOX Inhibits Superoxide/H.SUB.2.O.SUB.2 .Production by Mitochondria

(242) As previously stated, mitochondrial ROS production is highly dependent on mitochondrial activity and conditions. Although we tested the effects of AOX on ROS production by mitochondria under numerous conditions, we chose to present here, for the sake of clarity, only the most demonstrative results of the very specific effects of AOX. The presence of the complete substrate combination (i.e., glutamate, malate and succinate) (FIG. 2A), giving the electrons to the whole respiratory chain, is the most representative of in situ conditions in the cell where metabolism is active. Furthermore, maximal mitochondrial ROS/H.sub.2O.sub.2 production does not occur under conditions of high mitochondrial phosphorylation but under conditions of high reduction of electron transporters, i.e., low or no phosphorylation. These conditions are fulfilled in the presence of ATR and we could effectively verify that the addition of ATR under conditions of saturating ADP triggered the production of ROS which was at the detection limit under maximal phosphorylating conditions (results not shown). Under these conditions, ROS are produced at different sites of the respiratory chain (Orr et al., 2013. Free Radic. Biol. Med. 65:1047-1059; Quinlan et al., 2013. Redox Biol. 1:304-312) (FIG. 2).

(243) We designed a series of inhibitor titrations in order to decipher the action of AOX on ROS production by the whole respiratory chain under conditions of maximal ROS production (FIG. 2E). In the presence of substrate combination and the absence of specific inhibitors of the complexes, ROS production is at maximum and mainly comes from reverse electron transport at site I.sub.Q (FIG. 2A). After addition of rotenone, a classical inhibitor of complex I which specifically binds to I.sub.Q catalytic site, the ROS production decreases strongly and occurs almost entirely at site III.sub.QO (FIG. 2B). Therefore, the decrease in ROS production (measured by the Amplex Red method, FIG. 22) after rotenone addition represents the activity of ROS production by complex I.

(244) FIG. 21 illustrates the effect of increasing concentrations of AOX (from 2.5 to 20 μM) on ROS/H.sub.2O.sub.2 production measured under these conditions. It clearly appears from the results presented in this figure that AOX strongly inhibits the ROS production by complex I, at concentrations lower than required with AOL (cf. FIG. 3 for comparison). Indeed, AOX concentrations as low as 2.5 μM showed an inhibitory effect on the ROS production by site I.sub.Q, with an estimated IC.sub.50 of about 9.5 μM (Minimum: −72.5272±68.64; Maximum: 554.045±19.73; IC.sub.50: 9.46768±1.018; Hill coefficient: 2.61579±0.5706).

Example 11: AOX does not Scavenge Non-Mitochondrial Superoxide/H.SUB.2.O.SUB.2 .Production by NAD(P)H Oxidase

(245) The mechanism of action of AOX was further tested in vitro using the peroxidase-Amplex Red system utilized for the measurement of ROS/H.sub.2O.sub.2 by mitochondria, which in fact measures the appearance of H.sub.2O.sub.2 by the oxidation of Amplex Red to the fluorescent resorufin (FIG. 22). In the absence of mitochondria and by adding instead a H.sub.2O.sub.2-producing system to the assay, it was possible to test the effect of AOX on this non-mitochondrial superoxide/H.sub.2O.sub.2 production. This has been carried out by using commercial NAD(P)H oxidase which produces H.sub.2O.sub.2 in the presence of added NAD(P)H and measuring the reduction of Amplex Red to resorufin. We compared the effects of increasing concentrations (10 to 100 μM) of AOX to those of AOL and Oltipraz (FIG. 22A). Results presented show that while both AOL and AOX had no global effect on ROS measurement under these conditions, Oltipraz constantly decreased the amount of ROS measured by the assay. These results therefore demonstrate that Oltipraz either inhibits NAD(P)H oxidase or acts as a moderate (poor) radical scavenger and binds superoxide/H.sub.2O.sub.2 which are not available for peroxidase assay, but in all circumstances, inhibits non-mitochondrial (mimicking cytosolic) superoxide/H.sub.2O.sub.2 production. They also exclude any effect of AOX and AOL on the NAD(P)H oxidase or on the peroxidase activity. These results confirm that AOX and AOL do not interfere either with the measurement system or directly interact with H.sub.2O.sub.2. Interestingly, these results also demonstrate that AOX and AOL do not inhibit the ROS/H.sub.2O.sub.2 production by the NADP(H) oxidase, which is one—if not the—major non-mitochondrial ROS/H.sub.2O.sub.2 producer in the cells. The scheme on FIG. 22B recapitulates the different information on the mode of action of AOX and AOL on ROS/H.sub.2O.sub.2 production by mitochondria and NAD(P)H oxidase.

(246) When tested on isolated mitochondria from rat heart, AOX effectively decreases mitochondrial ROS/H.sub.2O.sub.2 production (in isolated mitochondria, H.sub.2O.sub.2 is produced from the reduction of ROS by mitochondrial superoxide dismutase). However, the results presented here clearly demonstrate that AOX does not act as an antioxidant or radical scavenger. While antioxidants are general ROS/H.sub.2O.sub.2 scavengers, AOX presents a selectivity towards the formation of ROS by site I in complex I, which demonstrates that AOX does not simply interact with superoxide radicals but specifically prevents their formation in complex I. In that respect, AOX therefore appears as a member of a brand-new class of oxidative stress protectants, by preventing ROS formation and thus being more active to protect mitochondria from their own ROS. We also show here some evidences that AOX may only interact with mitochondria without affecting oxygen radicals' formation in cytosol, and therefore would not affect intracellular signalization.

(247) Inhibition of complex I activity by rotenone or the neurotoxin MPP.sup.+ has been linked to parkinsonism in both rodents and humans, suggesting a link between dysfunctional complex I, ROS production, and neurodegeneration (Langston et al., 1983. Science. 219(4587):979-980; Betarbet et al., 2000. Nat. Neurosci. 3(12):1301-1306). In contrast, comparative analyses show an inverse relationship between maximal superoxide/H.sub.2O.sub.2 production from site I.sub.Q, but not site I.sub.F, and maximum life span across diverse vertebrate species (Lambert et al., 2007. Aging Cell, 6(5):607-618; Lambert A J et al., 2010. Aging Cell, 9(1):78-91). Therefore, selective modulators of superoxide/H.sub.2O.sub.2 production from site I.sub.Q or site I.sub.F would offer unique opportunities to probe the putative role of mitochondrial ROS production in normal and pathological processes (Orr et al., 2013. Free Radic. Biol. Med. 65:1047-1059).

(248) These results effectively demonstrate that AOX is not a radical scavenger; otherwise its effects on ROS measurements would be independent of the origin of the superoxide/H.sub.2O.sub.2 origin: mitochondrial or non-mitochondrial. Although the mechanism has still to be investigated, evidence is presented here that AOX compound specifically interferes with mitochondrial complex I and selectively inhibits superoxide production from the ubiquinone-binding site of complex I (site I.sub.Q).

(249) In conclusion, it appears that AOX properties may represent a breakthrough in the search for specific mitochondria-targeted modulators of ROS/H.sub.2O.sub.2 production in cells. AOX acts upstream from ROS production, therefore insuring higher protection than classical antioxidants.

Example 12: AOX Specifically Inhibits Mitochondrial ROS Production but not Cytosolic ROS Production

(250) Material and Methods

(251) Human non-small cell carcinoma cell line H460 and A549 were obtained from ATCC. Cells were cultured in growth medium consisting of DMEM (GIBCO), 10% FBS (GIBCO) and 100 units of penicillin and 100 μg/mL streptomycin (GIBCO). The cells were maintained in either 75-cm.sup.2 T-flasks or 175-cm.sup.2 T-flasks (BD Biosciences) in an incubator (model 3100 series, Forma Scientific, Marietta, Ohio) controlled at 37° C., 95% humidity, and 5% CO.sub.2. Cell culture medium was refreshed every other day. Every 2 to 3 days, H460 and A549 cultures were detached from the flasks using a 0.25% trypsin solution (Gibco) and subcultured. All cultures were maintained at 80-90% confluence at the time of subculture.

(252) Cytotoxicity screening was carried out by sulforhodamine B colorimetric assay. A549 and H460 cells (2.Math.10.sup.3) were seeded in a 96-well plate, and after adherence, cells were incubated with 5 to 500 μM of AOL or AOX or control dimethyl sulfoxide (DMSO). Cytotoxicity was evaluated after 48 hours treatment sulforhodamine B (SRB) assay, according to Vichai (Vichai et al., 2006. Nat. Protoc. 1(3):1112-6).

(253) Cell migration assays were performed in Transwells (Corning Inc., 8.0-μm pore size). For migration assay, 2.Math.10.sup.4 cells in serum-free medium were added to the upper wells. Media containing 10% FBS were added to the lower wells. Cells that migrated through the filter after 16 hours were stained with 0.2% crystal violet and counted using the software Image J.

(254) Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc.). The results are expressed as mean±SEM values for n independent experiments. Comparisons between groups were done by one-way ANOVA and a posteriori Dunnett's test. When appropriate, unpaired Student's t-tests or Mann-Whitney's test were employed. Differences of p<0.05 were considered to be significant.

(255) Results

(256) Absence of Cytotoxicity of AOX on Cultured Cells

(257) Cytotoxicity of AOX was performed on two carcinoma cell lines (H460 and A459) for a large range of concentrations (0 to 500 μM). Results are presented in FIG. 23.

(258) Positively no significant toxicity could be observed for concentrations lower than 100 μM, and cell viability only decreased abruptly for higher concentrations of AOX (FIG. 23A). By plotting these results on a semi-logarithmic plot (FIG. 23B), we could calculate the IC.sub.50 for high dose AOX toxicity as 134.8±0.3 μM. These results demonstrate that AOX has no harmful effect on cultured cells, even for high concentrations (100 μM).

(259) Absence of Effect of AOX on Carcinoma Cells Respiration

(260) The effects of AOX (0 to 40 μM) were assessed on carcinoma cells using the classical oxygraphic approach and compared to the effects of sister molecule AOL.

(261) Oxygen consumption of H460 resting cells has been measured continuously and increasing concentrations of AOL and AOX (0 to 40 μM) have been injected to the preparation (FIG. 24). It can be seen than there has been no change in the rate of oxygen consumption by H460 cells either with AOX or with AOL, indicating the absence of intracellular perturbation of mitochondrial activity and cell energetic metabolism. These results confirm the absence of harmful effect of AOL and AOX on cell functions.

(262) Effect of AOX on Carcinoma Cells Metastatic Activity

(263) The Transwell test of cell migration has been utilized for this assay and the migration of H460 carcinoma cells has been measured in the presence of two different concentrations of AOX (5 and 10 μM) and compared to the effect of 100 μM N-acetyl cystein (NAC) as a positive control of cytosolic ROS production (FIG. 25).

(264) FIG. 25A presents the photographs of dyed cells in the lower compartment for the different assay conditions. The results are presented in the histogram (FIG. 25B) and demonstrate the absence of induction of metastatic activity by AOX since the number of cells crossing the membrane are equivalent for the control and AOX conditions. By contrast, in the presence of NAC—a typical cell permeable radical scavenger targeting cytosolic ROS—, we observed a huge increase in metastatic activity.

(265) These results clearly demonstrate that the action of AOX as inhibitor of mitochondrial ROS production does not interfere with cytosolic ROS production.

Conclusion

(266) When directly applied to isolated mitochondria, AOX has some adverse effects on mitochondrial function for high concentrations (above 20 μM), probably due to the protonability of the molecule, which could act as a weak acid and perturb mitochondrial membrane potential. However, we could observe that these conditions are not attained in intact cells where even high concentrations do not trigger mitochondrial dysfunction.

(267) These results demonstrate thus that AOX has no toxic effect on cultured cell for concentrations over 100 μM, i.e., well over potential circulating concentrations under therapeutic treatment. This has been confirmed on the absence of effect of AOX under these conditions on cell respiration and energetic metabolism.

(268) The absence of effect of AOX on the metastatic activity of carcinoma cells by contrast to N-acetyl cystein (NAC), which increased this activity, confirms the specificity of AOX for mitochondrial ROS inhibition and the absence of effect of AOX on cytosolic ROS production.

Example 13: Effect of AOX in a Cardiovascular Disease: Pulmonary Hypertension

(269) The present study aims at providing a new alternative treatment of pulmonary hypertension. This disease is characterized by increased pulmonary arterial pressure and remodeling of pulmonary arteries (PA), leading to increased pulmonary vascular resistance, hypertrophy of the right ventricle, right heart failure and ultimately, death.

Comparative Effects of AOX and AOL on Intrapulmonary Arteries Contractility

(270) To address the issue of the effect of AOX, isometric tension measurements were carried out on intrapulmonary arteries, upon agonist-induced contractions using serotonin or endothelin, in absence and in presence of AOL or AOX.

(271) Material and Methods

(272) Intrapulmonary arteries of the first order (IPA1) were divided into short tubular segments with an external diameter of 1.5-2 mm and used for isometric contraction measurement. Arterial rings were mounted in isolated organ bath systems, containing Krebs solution (containing 118.4 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO.sub.4, 25 mM NaHCO.sub.3, 1.2 mM KH.sub.2PO.sub.4, 2.5 mM CaCl.sub.2) and 11.1 mM D-glucose, pH 7.4 adjusted with NaOH) at 37° C. and bubbled continuously with 15% O.sub.2/5% CO.sub.2. An initial load of 0.8 to 1 g was applied to arterial rings. Tissues were allowed to equilibrate for 1 hour in Kreb's solution and washed out every 15 minutes. A high KCl solution (80 mM) was applied in order to obtain a reference contraction used to normalize subsequent contractile responses.

(273) Contractile responses to different agonists were then tested by constructing a cumulative concentration-response curve (CCRC) to serotonin (5HT, 10.sup.−4 to 10.sup.−8 μM) or endothelin (ET-1, 10.sup.−7 to 10.sup.−10 M). When indicated, AOL or AOX were preincubated during 30 minutes, and then CCRC to agonist was performed in the presence of the drug. High potassium solutions were obtained by substituting an equimolar amount of KCl for NaCl from Kreb's solution. Endothelial function was tested on each ring by examining the relaxation induced by 10 μM carbamylcholine on 0.3 μM Phe-induced preconstricted pulmonary arterial rings. Passive and active mechanical properties were assessed using transducer systems, coupled to IOX software (EMKA Technologies, Paris, France) in order to facilitate data acquisition and analysis.

(274) Results

(275) The contraction was dependent of the concentration of serotonin (5HT) or endothelin (ET-1) with maximal contractions measured with 100 μM 5HT and 0.1 μM ET-1.

(276) AOL could relax contractions induced with up to 5.Math.10.sup.−5 M of serotonin, but had no effect on endothelin-induced contractions (FIG. 26A).

(277) The same pattern of response was observed on rings incubated in a bath containing AOX at 10 μM (FIG. 26B). Using higher concentrations of AOX however, contractions induced with 10.sup.−4 M of serotonin could be relaxed.

Conclusion

(278) These data further confirm that mitochondria play a major role in the pulmonary vasculature physiology, and suggest a greater efficacy of AOX than AOL for the lessening of pulmonary artery contractility and therefore, for the prevention and/or treatment of pulmonary hypertension.

(279) Effect of AOL on Pulmonary Arterial Smooth Muscle Cells Proliferation

(280) Material and Methods

(281) Detection of pulmonary arterial smooth muscle cells (PASMC) proliferation was assayed by using a colorimetric immunoassay kit (Roche Applied Science, Indianapolis, Ind., USA) based on the measurement of 5-bromo-2′-deoxy-uridine (BrdU) incorporation during DNA synthesis.

(282) Isolated PASMC were seeded in culture medium supplemented with 10% fetal calf serum (FCS) at a density of 5.Math.10.sup.3 cells/well/100 μL in a 96-well culture plate. The culture plate was placed in a humidified incubator at 37° C. under 5% CO.sub.2 in air. After 48 hours, cells were subjected to 48 hours of growth arrest in serum-free culture medium supplemented with 1% insulin-transferrin-selenium. At the end of this period, PASMC were incubated for 24 hours in culture medium containing either: 0.2% FCS (control condition); 0.2% FCS+10, 20, 60 or 100 μM AOL; 0.2% FCS+100 μM 5HT (serotonin); 0.2% FCS+100 μM 5HT+10, 20, 60 or 100 μM AOL; 10% FCS; 10% FCS+10, 20, 60 or 100 μM AOL.

(283) Each condition was tested in triplicate. 10 μL of BrdU (100 μM) was then added to the media and cells were incubated for an additional 2 hours at 37° C. DNA synthesis was then assayed using the colorimetric method, according to the manufacturer's instructions. Newly synthesized BrdU DNA was determined by measuring absorbance of the samples in an ELISA reader (Spectrostar/nano; BMG Labtech, Champigny-sur-Marne, France) at a wavelength of 380 nm with a reference wavelength of 490 nm.

(284) Results

(285) As reported in FIG. 27, AOL successfully inhibited the proliferation induced by a high concentration of FCS (10%) or in more physiological conditions, i.e., in presence of 0.2% of FCS and 100 μM 5HT.

Conclusion

(286) These data suggest that AOL has a potential effect in the prevention and/or treatment of pulmonary hypertension.

Example 14: Effect of AOX in a Cardiovascular Disease: Cardiac Toxicity of Anthracyclines

(287) The present study aims at evaluating the effect of AOX in a model of cardiac toxicity of anthracyclines. This was assessed by administering anthracycline-derived anti-cancer drugs, together with AOX, to 10-week-old rats for 14 to 17 days.

(288) Material and Methods

(289) The studies were performed on Sprague-Dawley rats aged 10 weeks and different treatments were administered intraperitoneally for 14 to 17 days, before collection of the heart for analysis. To respect the “three Rs principle” in animal experimentation, the number of group tested was limited as much as possible, in particular by focusing the experiments on one anthracycline molecule only, namely Doxorubicine, and by comparing the effect of AOX to one alternative protective molecule only, namely Dexrazoxane.

(290) At the end of the experiment, the heart of the rats treated will be removed and cardiac function will be studied exhaustively after perfusion of these hearts in a Langendhorf system to determine the function cardiac affected by doxorubicin and whether AOX treatment is efficient.

(291) The study comprises 5 different groups for 8 rats each: 1—Control group. Rats received the vehicle only, composed of 5% DMSO+95% NaCl 0.9%, twice a day (morning and evening) for 17 days; 2—Doxo group. Rats received Doxorubicine at a dose of 3 mg/kg (i.p.), every two days (morning), from day 3, for 14 days. Rats received vehicle only for every other injection; 3—Dexra group. Rats were treated with Dexrazoxane (reference protecting agent) at a dose of 30 mg/kg i.p. simultaneously with Doxorubicin at a dose of 3 mg/kg i.p. (according to the dosage ratio recommended by the French Regional Health Agency “ARS” in 2011), every two days, from day 3, for 14 days. Rats received vehicle only for every other injection; 4—AOX group. Rats were treated with AOX and Doxorubicin: 4 mg/kg i.p. of AOX, mornings and evenings, for 72 hours preceding the first injection of Doxorubicin; on the days of Doxorubicin injection (based on the Doxo group): 4 mg/kg i.p. AOX together with the Doxorubicin injection, followed 90 minutes later by a second injection of AOX at a dose of 4 mg/kg i.p.; on the days without Doxorubicin injection: 4 mg/kg i.p. of AOX, mornings and evenings. 5-AOX/Carv/Enal group. Rats were treated similarly than rats from the AOX group here above. AOX injections were supplemented with a classical treatment for cardiac insufficiency (Carvediol, a β-blocker, at a dose of 1 mg/kg, and Enalapril, a vasodilator, at a dose of 0.5 mg/kg).

Example 15: Effect of AOX in an Autoimmune Disease: Scleroderma

(292) The present study aims at testing the effect of AOX on fibroblasts from patients with scleroderma. Scleroderma is a chronic systemic autoimmune disease characterized by an increased synthesis of collagen, damages to small blood vessels, activation of T lymphocytes and production of altered connective tissues.

(293) Material and Methods

(294) Fibroblasts from both a healthy donor and a patient with scleroderma are cultured in flasks, in complete DMEM medium (10% FCS, 1% antibiotic). After 6-hour adhesion, cells are deprived of serum overnight.

(295) AOX is extemporaneously prepared. AOX was weighed and dissolved in DMSO at 5 mg/mL. This stock solution was further diluted to 10 and 5 mM final, in DMSO. AOX was further diluted in complete DMEM medium, to reach final concentrations of 40, 20 and 10 μM.

(296) Cultured cells were contacted with AOX at 40, 20 and 10 μM. Control cells were contacted with complete DMEM medium, supplemented with DMSO (0.2%) and N-acetylcysteine (3 mM). Cells are incubated under normoxic conditions (37° C., 20% 02) and under hypoxia (37° C., 1% O.sub.2) for 6 or 24 hours.

(297) For MMP-1, MIP and MCP secretion analysis, the culture supernatant is harvested, aliquoted and stored at −20° C. for dosage. MMP-1 is quantified by ELISA (Abcam), according to the manufacturer's instructions. MCP-1 and MIP-la concentrations are quantified by CBA (Cytometric Bead Array, Biolegend).

(298) For MMP1, collagen and CCl2 expression analysis, cells are detached with trypsin and washed with PBS. The cell pellet is then resuspended in lysis buffer, and RNA extraction is carried out according to the manufacturer's instructions (Nucleospin RNA Plus, Macherey Nagel). 1 μg of RNA is retro-transcribed (GoScript, Promega), then diluted 10-fold before SYBR Green qPCR (SYBR qPCR Premix Ex Taq, Takara) in a BioRad CFX384 PCR machine. Primers for MMP-1, CollA2 and CCl2 are used to measure genes of interest; primers for Ppia, RPLP0 and EEF1A1 are used to measure reference genes.

Example 16: AOX Inhibits Selectively ROS Production at Site I.SUB.Q .Contrary to Oltipraz

(299) Material and Methods

(300) After freshly isolating mitochondria from rat heart, then measuring protein quantity of the mitochondrial suspension, H.sub.2O.sub.2 production in response to the exogenous addition of increasing concentrations of AOL, AOX and Oltipraz was measured fluorimetrically with Amplex Red and horseradish peroxidase (HRP).

(301) The two major sites of mitochondrial ROS/H.sub.2O.sub.2 production were targeted individually using the combination of succinate (energy substrate of respiratory complex 2) and known inhibitors of respiratory chain, namely for site I.sub.Q, 10 mM succinate alone (CCCP as positive control) and for site III.sub.Qouter, 10 mM succinate, 4 μM rotenone and 2.5 μM antimycin A (myxothiazol as positive control). These “reaction” solutions were designed to generate maximal rates of ROS/H.sub.2O.sub.2 production predominantly from a single site within the chain (Quinlan et al. 2013. Redox Biol. 1:304-12).

(302) For measurement, a working solution of 50 μM Amplex red reagent and 20 μg/ml HRP was mixed to the reaction buffer into the corresponding wells of a 96-well plate (Greiner 96 F-bottom), in the absence or presence of increasing concentrations (from 0 to 80 μM) of AOL, AOX or Oltipraz. Addition of mitochondria to a final concentration of 0.125 mg/mL per well initiated the assay. The appropriate total volume per microplate well was 200 μL.

(303) Plates were incubated in the dark at room temperature for 20-25 minutes. A double orbital shaking (100 rpm-3 seconds) was applied before reading the endpoint fluorescence values on a CLARIOstar plate reader (BMG LABTECH GmbH, Germany). In fact, H.sub.2O.sub.2 reacts with Amplex Red in a 1:1 stoichiometry, yielding the fluorescent compound resorufin that was analyzed using the following optic settings (excitation wavelength at 546-20 nm; emission at 600-40 nm; gain: 750).

(304) Also, an H.sub.2O.sub.2 standard curve, with concentrations ranging from 0 to 5 μM, was prepared in the experimental buffer that consisted in (in mM): 140 sucrose, 100 KCl, 1 EGTA, 20 MgCl.sub.2, 10 KH.sub.2PO.sub.4, and 1 g/L (w/v) BSA essentially fatty acid free (pH 7.2).

(305) Results

(306) FIG. 28 illustrates the effect of increasing concentrations of AOL, AOX and Oltipraz (from 0 to 80 μM) on ROS/H.sub.2O.sub.2 production measured under these conditions.

(307) It clearly appears from the results of FIG. 28A that AOL and AOX strongly inhibit the ROS production by complex I, at concentrations as low as 2.5 μM. On the contrary, Oltipraz, at the same concentrations, and up to 80 μM, showed only a poor effect on the inhibition of ROS production in site I.sub.Q.

(308) FIG. 28B further shows that none of the three compounds have an effect on the inhibition of ROS production by complex III.

(309) Together, these results clearly demonstrate that AOL and AOX are site IQ-selective inhibitors of mitochondrial ROS production, contrary to Oltipraz.

Example 17: AOX Analogs Inhibit Superoxide/H.SUB.2.O.SUB.2 .Production by Mitochondria

(310) Material and Methods

(311) The same protocol as described above in Example 16 was used to measure ROS/H.sub.2O.sub.2 production in response to the exogenous addition of various AOX analogs. Namely, a working solution of 50 μM Amplex red reagent and 20 μg/mL HRP was mixed to the reaction buffer into the corresponding wells of a 96-well plate (Greiner 96 F-bottom), in the absence or presence of increasing concentrations (from 1 to 25 μM) of eight compounds (Cp1; Cp2; Cp3; Cp4; Cp5; Cp6a; Cp8; Cp9a). Addition of mitochondria to a final concentration of 0.125 mg/mL per well initiated the assay. The appropriate total volume per microplate well was 200 μL.

(312) Results

(313) FIGS. 29A and 29B illustrate the effect of increasing concentrations of AOX analogs (from 2.5 to 25 μM) on ROS/H.sub.2O.sub.2 production measured under these conditions.

(314) It clearly appears from the results presented in these figures that AOX analogs inhibits the ROS production by complex I, selectively with respect to complex III.