NOVEL DOPAMINE PRECURSORS

Abstract

The invention disclosed herein concerns a novel class of compounds suitable for the treatment of neurodegenerative diseases, such as Parkinson’s Disease.

Claims

1. A compound of the general formula (I): ##STR00009## wherein R is a C.sub.1-C.sub.5alkyl; and n is zero or 1, excluding a compound wherein n is 0 and R is methyl.

2. The compound according to claim 1, wherein n is 1.

3. The compound according to claim 1, wherein the C.sub.1-C.sub.5alkyl is selected from methyl, ethyl, propyl, butyl and pentyl.

4. The compound according to claim 1, wherein the C.sub.1-C.sub.5alkyl is selected from methyl, n-butyl, iso-propyl, tert-butyl and n-pentyl.

5. The compound according to claim 1, wherein the C.sub.1-C.sub.5alkyl is methyl.

6. The compound according to claim 1, wherein n is 1 and R is methyl.

7. A L-dopa precursor of dopamine having a structure according to claim 1.

8. An inhibitor of oxidative induced inflammatory mitogen activated protein kinases (MAPK) pathway, the inhibitor having a structure according to claim 1.

9. The inhibitor according to claim 8, wherein the MAPK is JNK and P38.sup.MAPK.

10. A composition comprising a compound according to claim 1.

11. The composition according to claim 10, being a pharmaceutical composition.

12. The composition according to claim 11, being adapted for oral administration, administration by an aerosol, administration by inhalation, nasal administration, parenteral administration, subcutaneous administration, transdermal administration, intradermal administration, intravenous administration, intramuscular administration, buccal administration, intraperitoneal administration, rectal administration or vaginal administration.

13. The composition according to claim 12, being suitable for oral administration.

14. The composition according to claim 11, for use in protecting cells from oxidative stress.

15. Use of a compound according to claim 1 in an in vivo method of reducing or reversing oxidative stress, or an inflammatory state of a human or animal cell.

16. Use of a compound according to claim 1, in treating a neurodegenerative disease or disorder, or a disease or disorder characterized by or associated with reduced levels of brain dopamine.

17. A method of reducing or reversing oxidative stress, or an inflammatory state of a human or animal cell, the method comprising treating a subject with a compound of the formula (I): ##STR00010## wherein R is a C.sub.1-C.sub.5alkyl; and n is zero or 1.

18. The method according to claim 17, for treating a disease or disorder characterized by or associated with reduced levels of brain dopamine.

19. A method of treating a neurodegenerative disease or disorder, or a disease or disorder characterized by or associated with reduced levels of brain dopamine, the method comprising administering a compound to a subject suffering therefrom or having disposition to suffering therefrom, wherein the compound is of the general formula (I): ##STR00011## wherein R is a C.sub.1-C.sub.5alkyl; and n is zero or 1.

20. The method according to claim 19, wherein the disease or disorder is caused by damage to the central nervous system.

21. The method according to claim 19, wherein the disease or disorder is characterized by progressive dysfunction, degeneration and death of neurons optionally synaptically interconnected.

22. The method according to claim 19, wherein the disease or disorder is associated or based on oxidative stress, or an inflammatory state of a human or animal cell.

23. The method according to claim 22, wherein the disease or disorder is associated with reduced levels of brain dopamine.

24. The method according to claim 19, wherein the neurodegenerative diseases and disorders is selected from Huntington’s disease, spinocerebellar ataxias, Parkinson’s disease, secondary parkinsonism, morbus Alzheimer, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), amyotrophic lateral sclerosis (ALS), Shy Drager syndrome, dopamine-responsive dystonia, cystic fibrosis, familial amyloidotic polyneuropathy, spongiform encephalopathies, dementia with Lewy body disease (LBD), akinesia, bradykinesia, hypokinesia, frontotemporal dementia with Parkinsonism, spinocerebellar ataxias, spinal and bulbar muscular atrophy, hereditary dentatorubral-pallidoluysian atrophy, familial British dementia, familial Danish dementia, prion disease, mild brain trauma mTBI, atherosclerosis, and allergic airway disease.

25. The method according to claim 19, wherein the disease or disorder is Parkinson’s disease or dopamine-responsive dystonia.

26. The method according to claim 19, wherein the compound of claim 1 is a compound wherein R is a C1-C5alkyl and n is zero or 1.

27. The method according to claim 19, wherein the compound of claim 1 is a compound wherein n is 1.

28. The method according to claim 19, wherein the compound of claim 1 is a compound wherein n is zero.

29. The method according to claim 19, wherein the compound of claim 1 is a compound wherein the C1-C5alkyl is selected from methyl, ethyl, propyl, butyl and pentyl.

30. The method according to claim 19, wherein the compound of claim 1 is a compound wherein the C1-C5alkyl is selected from methyl, n-butyl, iso-propyl, tert-butyl and n-pentyl.

31. The method according to claim 19, wherein the compound of claim 1 is a compound wherein the C1-C5alkyl is methyl.

32. The method according to claim 19, wherein the compound of claim 1 is a compound wherein n is zero or 1 and R is methyl.

33. The method according to any one of claims 17 to 32, wherein the compound is: ##STR00012## or ##STR00013## .

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0075] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0076] FIG. 1 demonstrates the ability of compound SD-444 to rescue human neuroblastoma cells (SH-SY5Y) from auranofin- (AuF) induced cell death. Viability of cells pre-treated with 5 .Math.M AuF for 30 min, washed and later exposed to increasing concentrations of SD-444, was determined 24 h later. Data is displayed as mean ± S.E.M.

[0077] FIG. 2 demonstrates the ability of SD-444 to protect human neuroblastoma cells (SH-SY5Y) from oxidative stress induced inflammation, as shown by inhibition- induced phosphorylation of p38.sup.MAPK. Cells were treated with 10 .Math.M AuF with or without SD-444. Phosphorylation was determined by western blot analysis. The amount of each band was quantitated by densitometry and plotted with a linear regression program normalized with β catenin (b-cat).

[0078] FIG. 3 demonstrates the ability of SD-444 to protect cells from oxidative stress by inhibition-induced phosphorylation of JNK. SH-SY5Y cells were treated with 10 .Math.M AuF with or without SD-444. Phosphorylation was determined by western blot analysis. The amount of each band was quantitated by densitometry and plotted with a linear regression program normalized to total JNK.

[0079] FIG. 4 demonstrates the reversal of morphological changes in PC 12 cells by oxidative stress, by utilizing SD-444. PC12 cells were treated with AuF with or without SD-444. Morphological changes were visualized 4 hr later (magnification x200).

[0080] FIG. 5 demonstrates the ability of SDA-341 to protect human neuroblastoma cells (SH-SY5Y) from oxidative stress induced inflammation, as shown by inhibition-induced phosphorylation of ERK1/2. SH-SY5Y cells were treated with 10 .Math.M AuF with or without SDA-341. Phosphorylation was determined by western blot analysis. The amount of each band was quantitated by densitometry and plotted with a linear regression program normalized to beta-catenin.

[0081] FIGS. 6A-C demonstrate the ability of SDA-341 to protect PC12 cells from oxidative stress induced inflammation, as shown by inhibiting auranofin-induced phosphorylation of ERK1/2. Cells were treated with 10 .Math.M AuF with or without SDA-341. Phosphorylation was determined by western blot analysis (FIG. 6A). The amount of each band was quantitated by densitometry (FIG. 6B) and plotted with a linear regression program normalized to total ERK2 (FIG. 6C).

[0082] FIGS. 7A-C demonstrate the reversal of morphological changes in PC12 cells by oxidative stress, by utilizing SDA. (FIG. 7A) Control untreated PC12 cells (FIG. 7B). PC12 cells were treated with AuF (2 .Math.M; 30 min) lose their morphology and become rounded, as compared to untreated cells. (FIG. 7C) AuF-treated (2 .Math.M; 30 min) cells incubated with 150 .Math.M SDA showed normal morphology (magnification X100 and X200). Cells were treated with AuF (2 .Math.M) for 30 min, washed and then SDA was added at 25 .Math.M or 100 .Math.M. Cells were visualized 4 hr later (magnification, 100 × and 400 x).

[0083] FIG. 8 summarizes animals body weights of rats treated with rotenone, a pesticide that by exerting mitochondrial stress mimics PD disease characteristics, and is widely used as a model of PD in rats and mice. Body weight of rats treated with rotenone with SD or SDA, and naive rats.

[0084] FIG. 9 provides rearing behavior test results in rats treated with rotenone alone, or in the presence of either SD or SDA and naive rats. Rats treated with rotenone (3.0 mg/kg), with or without SD-444 (33 mg/kg) or SDA-341 (33 mg/kg). Rearing behavior in rats treated with rotenone alone, or in the presence of either SD or SDA and naive rats treated with rotenone with SD or SDA, and naive rats is shown at days 4, 8, and 10.

[0085] FIG. 10 provides rotarod results in rats treated with rotenone alone, or in the presence of either SD or SDA and naive rats. Rats treated with rotenone (3.0 mg/kg), with or without SD-444 (33 mg/kg) or SDA-341 (33 mg/kg). Rotarod behavior was tested in rats treated with rotenone alone, or in the presence of either SD or SDA and naive rats treated with rotenone with SD or SDA, and naive rats is shown at days 4, 8, and 10.

[0086] FIG. 11 provides rat beam walk test results in rats treated with rotenone alone, or in the presence of either SD or SDA and naive rats. Rats treated with rotenone (3.0 mg/kg), with or without SD-444 (33 mg/kg) or SDA-341 (33 mg/kg). Walk beam behavior was tested in rats treated with rotenone alone, or in the presence of either SD or SDA and naive rats treated with rotenone with SD or SDA, and naive rats is shown at days 4, 8, and 10.

DETAILED DESCRIPTION OF EMBODIMENTS

Results

Synthesis

[0087] Acetyle-Cys-2,3 dihydroxyphenylalanin-Cys- amide (SD-444) was prepared by standard peptide synthesis procedure.

[0088] SD-444 was tested for protecting neuronal cells from activating apoptotic signaling:

[0089] A) The anti-apoptotic activity of SD-444 was tested on human neuronal cells SH-SY5Y. The cells were challenged by auranofin (AuF) that induces cellular stress by selectively blocking the thioredoxin reductase activity.

[0090] SH-SY5Y cells were plated on 96-well plates and treated with AuF in different concentrations for 30 min. Then the cells were washed with PBS and treated as indicated. Twenty-four hours later, the cells were fixed with glutaraldehyde in final concentration of 0.5% for 10 min. Cells were washed 3 times with DDW dried overnight, and washed once with borate buffer (0.1 M, pH 8.5). The fixed cells were stained with 200 .Math.l of 1% methylene blue dissolved in borate buffer for 1 h. After extensive washing and drying, the color was extracted with 200 .Math.l of 0.1 M HCl for 1 h at 37° C. and absorbance was read in spectrophotometer at 630 nm.

[0091] When SD-444 was applied to the cells, the AuF effect was partially reversed and neuronal cell-viability was partially restored (FIG. 1).

[0092] B) The ability of SD-444 to prevent apoptosis was monitored and the molecular mechanism through which it exerts protection of the cells was identified to be the ASK-MAPK pathway.

[0093] In the assay, twenty to thirty micrograms of protein samples were loaded on 10-12% SDS-PAGE gels. The proteins were then transferred electrophoretically to nitrocellulose (Whatman, Germany). The blots were blocked by incubation for 1 h at RT in TBS-T (25 mM Tris-HCl pH 7.4, 0.9% NaCl and 0.02% Tween-20) with 4% Difco skim milk (BD, USA), and incubated over-night at 4° C. with the primary antibody: p-p38.sup.MAPK (Thr180/Tyr182), rabbit mAb; p38, rabbit Ab.

[0094] As shown in FIG. 2 and FIG. 3, stress induced by AuF in the neuronal SH-SY5Y cells led to the activation of cell-death pathways through the activation of MAPK p38 and JNK. In cells treated with SDA-444 there was a significant reduction in p38 activation already at 30 .Math.M (FIG. 2) and at 10 .Math.M in JNK (FIG. 3).

[0095] C) Phase microscopy studies: The ability of SD-444 to rescue cells from oxidative stress was tested by exposing the PC12 to 2 .Math.M auranofin for 30 min, and after washing incubated with 250 .Math.M SD-444 at 37° C. for additional 4 hrs.

[0096] As shown in FIG. 4, cells that were incubated with AuF and then with SD-444 showed similar morphology to un-treated cells, as opposed to cells that were exposed to auranofin, which looked rounded, losing normal morphology.

[0097] Acetyle-Cys-2,3 dihydroxyphenylalanine-amide (SDA-341) was synthesized, purified, and chemically analyzed. SDA-341 was prepared by the conventional standard liquid-phase method.

[0098] Activity of SDA-341

[0099] SDA-341 was tested for protecting neuronal cells from activating apoptotic signaling:

[0100] A) The ability of SDA-341 to prevent apoptosis was monitored and the molecular mechanism through which it exerts protection of the cells was identified to be the ASK-MAPK pathway.

[0101] In the assay, twenty to thirty micrograms of protein samples were loaded on 10-12% SDS-PAGE gels. The proteins were then transferred electrophoretically to nitrocellulose (Whatman, Germany). The blots were blocked by incubation for 1h at RT in TBS-T (25 mM Tris-HCl pH 7.4, 0.9% NaCl and 0.02% Tween-20) with 4% Difco skim milk (BD, USA), and incubated over-night at 4° C. with the primary antibody: p-JNK.sup.MAPK, and β-catenin.

[0102] As shown in FIG. 5 stress induced by AuF in the neuronal SH-SY5Y cells leads to the activation of cell-death pathways through the activation of MAPK JNK. In cells treated with SDA-341 EC.sub.50 of 100 .Math.M in reduction of JNK activation.

[0103] The ability of SDA-341 to lower AuF induced activation of ERK1/2 in PC12 cells with the corresponding anti ERK1/2 and ERK2 antibodies was also tested, as shown in FIG. 6. Calculated EC.sub.50=80.Math.M.

[0104] B) Phase microscopy studies: The ability of SDA to rescue the cells from oxidative stress was tested by exposing the PC12 to auranofin 2 .Math.M for 30 min with or without 150 .Math.M SDA. AuF was washed after 30 min and 150 .Math.M SDA was added and allowed to incubate at 37° C. for additional 4 hrs.

[0105] Phase microscopy showed the morphology of the cells after 4 hrs as shown in FIG. 7. PC12 cells incubated with SDA displayed a similar morphology to un-treated cells, as opposed to cells that were exposed to AuF, which looked rounded loosing normal morphology.

Animal Study: Purpose

[0106] The aim of this study was to examine the efficacy of two new compounds, SuperDopa (SD; 444) and Superdopamide (SDA; 341) in protecting neuronal pathways in vivo, using a Rotenone -induced model of Parkinson’s disease (PD) in rats. The study was performed with Sprague Dawley rats (Tables 1 and 2).

TABLE-US-00001 Test System Species/Strain: Sprague Dawley rats Source: Envigo Sex: Males Total No. of Animals: 20 Age: 7-9 weeks of age at treatment administration. Body Weight: 292-315 g at study initiation. Weight variation of animals at the time of treatment initiation should not exceed ± 20% of the required weight. Acclimation period: 7 days. Animals Health: Only animals in good health acclimatized to laboratory conditions for 7 days were used in the study. Animals with any evidence of disease or physical abnormalities were not selected for study.

TABLE-US-00002 Utilities and environmental control Animal Housing: Housing: Animal handling was performed according to guidelines of the National Institute of Health (NIH) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animals are housed in cages polysulphone (3/cage) measuring425×266×185mm, with stainless steel top grill facilitating pelleted food and drinking water in plastic bottle; bedding: steam sterilized clean paddy husk (Harlan, Sani-chip, Cat#: 7090A) was be used and bedding material was changed along with the cage at least twice a week. Environment: Automatically controlled environmental conditions were set to maintain temperature at 22 -/+2° C. with a relative humidity (RH) of 55 -/+ 15%, a 12:12 hour light:dark cycle and 10-30 air changes/hr in the study room. Identification: Animals were given a unique animal identification number. This number also appeared on a cage card, visible on the front of each cage. The cage card also contained the study number, route of administration and all other relevant details as to treatment group and dose level. Diet and water: Animals are provided ad libitum a commercial rodent diet (Harlan Teklad TRM Rat/Mouse Diet cat #: 2018SC), sterilized. Animals had free access to acidified autoclaved drinking water (pH 3.5) obtained from the municipality supply.

Group and Experimental Design

[0107] Animals were divided into 4 groups as indicated in Table 3:

[0108] 1-SD (Rotenone + SD); 2 - SDA (Rotenone + SDA); 3 - Control (Rotenone); 4 -Naive. The experimental groups were comprised of 6 animals for treated groups (1-3) and 2 animals in naive group (4). Rotenone was administrated 3.0 mg/kg intraperitoneally (IP) once a day in the morning (days 1-9). SD 33 mg/kg and SDA 33 mg/kg were administrated intraperitoneally once a day in the afternoon (days 1-9).

[0109] Rearing behavior, rotarod and beam walk tests were performed before initiation of treatment (day 0) as well as on days 4, 8 and 10 of the experiment.

[0110] Animals weight was measured before initiation of treatment (day 0), on days 4,7, 9 during the experiment and on termination day 11. On day 11, animals were sacrificed and brains were harvested for further histopathological analysis.

TABLE-US-00003 Animal groups Group number Number of animals Material and Route of administration 1-SD n=6 (#13-18) Rotenone (3 mg/kg) + SD (33 mg/kg) IP 2 - SDA n=6 (#7-12) Rotenone (3 mg/kg) +SDA (33 mg/kg) IP 3 - Control n=6 (#1-6) Rotenone (3 mg/kg) IP 4 - Naïve n=2 (#19-20) None None

EXPERIMENTAL PROCEDURES

Groups’ Allocation

[0111] On the last day of acclimation period, animals were allocated into treatment groups (3 rats in a cage) based on their body weight, while the average body weight was be similar in all treatment groups.

Body Weight Monitoring

[0112] All animals were weighed before dosing, weight measurements are presented in Table 4 and FIG. 8.

TABLE-US-00004 Animals body weight Day 0 4 7 9 11 21/08/18 25/8/18 28/08/18 30/08/18 01/09/18 Weight (gr) Weight (gr) Weight (gr) Weight (gr) Weight (gr) Group rat 1-SD 13 307 296 281 285 278 14 294 290 286 280 284 15 315 310 305 298 283 16 300 299 294 286 274 17 313 305 301 297 290 18 300 299 298 295 290 Average 304.83 299.83 294.17 290.17 283.17 Std 7.51 6.36 8.35 6.82 5.84 T-test 1-SD vs Control 0.068308 0.005668 0.003981 0.000042 0.001039 2-SDA 7 292 284 274 275 272 8 307 293 283 272 261 9 307 299 287 267 269 10 305 294 283 274 266 11 315 299 288 281 283 12 300 296 291 284 277 Average 304.33 294.17 284.33 275.50 271.33 Std 7.06 5.08 5.41 5.62 7.18 T-test 2-SDA vs Control 0.693888 0.166815 0.865534 0.176523 0.071247 3 - Control 1 294 290 283 269 252 2 306 286 279 263 250 3 308 291 284 274 263 4 295 290 286 255 261 5 305 292 290 282 274 6 308 293 287 270 268 Average 302.67 290.33 284.83 268.83 261.33 Std 5.88 2.21 3.44 8.43 8.40 4 -Naïve 19 305 313 325 334 346 20 310 315 319 326 332 Average 307.50 314.00 322.00 330.00 339.00 Std 2.50 1.00 3.00 4.00 7.00

Drug Administration

[0113] IP administration: Rotenone was administrated to groups 1-3. Rotenone was injected intraperitoneally at a dose 3.0 mg/kg once a day in the morning (days 1-9). SD and SDA was administrated both at a dose of 33 mg/kg and were injected intraperitoneally once a day in the afternoon (days 1-9). Group number 4 was untreated, and remained as a naive group.

Rat Rearing Behavior Test

[0114] Animals were placed in a clear glass cylinder (40 cm high and 20 cm diameter) and number of rears in 2 min was observed. Rear was considered as animals raised their hands above the shoulder and made contact with the wall of cylinder with their forelimb. The results of rat rearing behavior test are presented in Table 5 and FIG. 9.

TABLE-US-00005 Rearing behavior (cylinder) test results: Day 0 4 8 10 21/08/18 25/08/18 29/08/18 31/08/18 Cylinder cylinder cylinder cylinder Group rat rise and touch rise and touch rise and touch rise and touch 1 -SD 13 8 9 10 13 14 18 12 18 17 15 9 13 15 9 16 13 15 12 15 17 13 13 10 8 18 13 22 9 16 Average 12.33 14.00 12.33 13.00 Std 3.25 4.00 3.20 3.42 T-test 1-SD vs Control 0.583 0.047 0.007 0.002 2-SDA 7 18 14 15 11 8 9 12 19 14 9 12 14 8 9 10 12 11 12 10 11 18 15 16 14 12 8 10 19 17 Average 12.83 12.67 14.83 12.50 Std 3.93 1.80 3.89 2.75 T-test 2-SDA vs Control 0.838 0.136 0.054 0.002 3 - Control 1 8 9 6 0 2 18 13 1 4 3 13 14 8 1 4 15 6 0 0 5 8 10 8 8 6 12 10 20 9 average 12.33 10.33 7.17 3.67 Std 3.59 2.62 6.54 3.68 4 - Naïve 19 14 15 18 25 20 18 18 14 16 average 16.00 16.50 16.00 20.50 Std 2.00 1.50 2.00 4.50

Rotarod Behavior Assay

[0115] The test was used to evaluate motor coordination and balance. Apparatus was set to accelerate from 4 to 40 rpm in 300 s, and animals from same cage are placed in separate lanes on rod initially rotating at 4 rpm. Rotarod test results are presented in Table 6 and FIG. 10.

TABLE-US-00006 Rotarod results day 0 4 8 10 21/08/18 25/08/18 29/08/18 31/08/18 R.R (sec) R.R (sec) R.R (sec) R.R (sec) group rat 1-SD 13 280 268 251 255 14 288 300 282 258 15 300 300 267 266 16 291 247 254 224 17 267 255 237 192 18 274 280 249 248 average 283.33 275.00 256.67 240.50 std 10.98 20.45 14.34 25.32 T-test 1-SD vs Control 0.076115 0.000486 0.0000002 0.000116 2-SDA 7 278 298 281 270 8 300 274 249 195 9 269 263 234 123 10 290 300 277 260 11 300 300 268 243 12 274 283 289 271 average 285.17 286.33 266.33 227.00 std 12.25 14.24 19.11 53.18 T-test 2-SDA vs Control 0.708284 0.013093 0.000103 0.000357 3 -Control 1 282 250 78 0 2 280 189 105 12 3 300 230 132 108 4 276 230 76 0 5 289 292 192 88 6 300 202 122 114 average 287.83 232.17 117.50 53.67 std 9.42 33.37 39.20 50.44 4 -Naïve 19 270 300 300 300 20 294 285 282 282 average 282.00 292.50 291.00 291.00 std 12.00 7.50 9.00 9.00

Rat Beam Walk Test

[0116] Animals were gently placed on 1 m long narrow aluminum beam facing one of the ends and allowed to walk to the end of the beam. The results of rat beam walk test are presented in Table 7 and FIG. 11.

TABLE-US-00007 Rat beam walk test results: Day 0 4 8 10 21/08/18 25/08/18 29/08/18 31/08/18 beam beam beam beam Group rat end time(sec) end time(sec) end time(sec) end time(sec) 1 - SD 13 19 13 22 23 14 5 15 18 17 15 8 6 16 12 16 10 38 21 27 17 16 18 32 14 18 10 31 23 43 average 11.33 20.17 22.00 22.67 Std 4.75 10.95 5.07 10.43 T-test 1-SD vs Control 0.089 0.030 0.004 0.031 2-SDA 7 7 12 16 22 8 3 32 23 17 9 10 14 52 34 10 8 21 18 10 11 12 10 19 21 12 17 16 26 18 average 9.50 17.50 25.67 20.33 Std 4.35 7.34 12.23 7.23 T-test 2-SDA vs Control 0.511 0.138 0.152 0.026 3 -Control 1 12 100 120 120 2 8 25 33 52 3 13 10 9 26 4 19 44 86 120 5 10 36 47 70 6 6 27 32 40 average 11.33 40.33 54.50 71.33 Std 4.15 28.65 37.37 36.85 4 -Naïve 19 20 10 18 9 20 10 27 22 16 Average 15.00 18.50 20.00 12.50 Std 5.00 8.50 2.00 3.50

Study Termination

[0117] Animals was euthanized by CO.sub.2. Blood was collected and serum was separated. Organs (brains), 20 samples, from 20 rats, were harvested and fixed in 2.5% PFA. Brains were dissected to obtain sections from the Substantia Nigra Pars compacta (SNC) and the striatum (ST) using a rat brain matrix. After the dissection in a standard position per brain sections were put in an embedding cassette.

Study Results

[0118] In vitro- Studies in tissue culture showed that SD and SDA protect human neuroblastoma SH-SY5Y cells from oxidative stress induced by selectively inhibiting thioredoxin reductase by auranofin (AuF). AuF triggers activation of MAPKs pathway through the phosphorylation JNK and p38. The two compounds SD-444 and SDA-341 inhibit JNK and p38 phosphorylation and thereby inhibitg the apoptotic pathway. Preventing apoptosis was accompanied by increasing cell-viability shown in phase microscopy.

[0119] In vivo - Parkinsonian features, such as loss of dopaminergic neurons in the substantia nigra and motor impairment are demonstrated by exposure of rats to rotenone. Rotenone exerts mitochondrial stress and is widely used as a model for PD.

[0120] Using the rotenone rat model, both SD-444 and SDA-341 when administered intraperitoneally, appeared to rescue motor activity in the three motor tests the rotarod, the cylinder, and the walk-beam tests.

[0121] The goal of Walk-beam test is to evaluate motor balance and to show the ability of the rat to stay upright and walk across an elevated narrow beam to a safe platform. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod. As shown both SD and SDA were very effective in this test, reversing the rotenone induced imbalance.

[0122] The Cylinder test is designed to evaluate locomotor asymmetry in rodent models of CNS disorders like the rotenone. It can be used to evaluate novel chemical entities for their effect on motor performance. Here we have shown that SD and SDA were very effective in maintaining locomotactivity in Rotenone-treated rats.

[0123] The rotarod test motor coordination has been assessed also by the rotarod-test that is based on a rotating rod with forced motor activity. The assay that evaluates balance, grip strength, and motor coordination, showed that SD and SDA significantly reversed motor dysregulation mediated by rotenone. Both compounds significantly improved balance, grip strength, and motor coordination.

[0124] In summary, our studies showed that SD and SDA increase viability of neuronal cells in vitro and inhibit the MAPK apoptotic pathway. Both SD and SDA appeared to be effective anti-apoptotic reagents, manifested by inhibiting the AuF-induced MAPKs phosphorylation reversing the AuF oxidative stress effects.

[0125] In vivo, they effectively improved motor performance, reversing the rotenone impaired motor performance, which is induced by mitochondrial stress. Rescue activity was shown in three motor tests. Hence, SD and SDA could potentially become effective in treating neurodegenerative diseases and neurodegenerative related-disorders.