Inhibitors and combinations for treating diseases

Abstract

The present disclosure relates to inhibitors, compounds, and combinations for inhibiting ferroptosis and uses thereof for treating disease.

Description

[0001] INHIBITORS AND COMBINATIONS FOR TREATING DISEASES

[0002] TECHNOLOGICAL FIELD

[0003] The present disclosure relates to compounds, inhibitors, combinations and compositions for treating diseases.

[0004] BACKGROUND ART

[0005] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0006] - International patent application, publication No. WO17027976

[0007] - Xu J, Zhou F, Wang X and Mo C (2023) Role of ferroptosis in pregnancy related diseases and its therapeutic potential. Front. Cell Dev. Biol. 11:1083838.

[0008] Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

[0009] BACKGROUND

[0010] Ferroptosis is a form of programmed cell death characterized by disbalanced redox homeostasis and has been implicated in multiple human diseases (11, 13, 48, 49). Preeclampsia is a systemic disorder of pregnancy characterized by hypertension and proteinuria that develops after 20 weeks of gestation in an estimated 3- 8% of all pregnancies (5, 16-18).

[0011] WO17027976 relates to a method of measuring level of early predictive marker of pre-eclampsia (PE).

[0012] Xu J. et al describes mechanisms and advances of ferroptosis and the pathogenic role of ferroptosis in pregnancy related diseases and the potential medicines for its treatment.

[0013] GENERAL DESCRIPTION

[0014] In accordance with some aspects, the present disclosure provides a combination comprising at least two small molecule compounds (SMC) selected from the group consisting of:

[0015] 2,2',2",2"'-((4,8-di(piperidin-l-yl)pyrimido[5,4-d]pyrimidine-2,6- diyl)bis(azanetriyl))tetraethanol (designated herein as SMC 7),

[0016] (RS)-N,N-Dimethyl-l-(10H-phenothiazin-10-yl)propan-2-amine (designated herein as

[0017] SMC 2),

[0018] (±)-[3-(9H-carbazol-4-yloxy)-2-hydroxypropyl][2-(2-methoxyphenoxy)ethyl]amine

[0019] (designated herein as SMC 3), (RS)-2[(3,3-Diphenylpropyl)(methyl)amino]-l,l-dimethylethyl methyl 2,6-dimethyl-4-

[0020] (3-nitrophenyl)-l,4-dihydropyridine-3,5-dicarboxylate (designated herein as SMC 4),

[0021] 4-[(7-chloroquinolin-4-yl)amino]-2-[(diethylamino)methyl]phenol (designated herein as

[0022] SMC 5), and

[0023] (2R)-2,5,7,8-Tetramethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]-3,4-dihydro-2H-l- benzopyran-6-ol (designated herein as SMC 60)

[0024] In accordance with some other aspects, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the combination as described herein, and wherein the composition optionally further comprises at least one pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s.

[0025] In accordance with some further aspects, the present disclosure provides a method for (i) mitigating (reducing / inhibiting) ferroptosis-induced death in trophoblasts, (ii) reducing sFLT-1 secretion from placental tissue, (iii) curbing (reducing / inhibiting) excessive sFLT-1 triggered due to ferroptotic death or (iv) any combination thereof, the method comprising the step of contacting the cell with an effective amount of the combination as described herein.

[0026] In accordance with yet some aspects, the present disclosure provides a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with increased sFLT-1 secretion and / or cell ferroptosis in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of the combination as described herein.

[0027] In accordance with yet some other aspects, the present disclosure provides a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying preeclampsia (PE) in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of the combination as described herein.

[0028] In accordance with yet some further aspects, the present disclosure provides a use of a combination as described herein in the preparation of a composition for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with increased sFLT-1 secretion and / or cell ferroptosis in a subject in need thereof.

[0029] In accordance with yet some further aspects, the present disclosure provides a use of a combination as described herein in the preparation of a composition for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of preeclampsia in a subject in need thereof.

[0030] In accordance with some aspects, the present disclosure provides a ferroptosis inhibitor for use in a method for inhibiting secretion of sFLT-1 from a cell.

[0031] In accordance with some other aspects, the present disclosure provides a ferroptosis inhibitor for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease associated with increased sFLT- 1 secretion in a subject in need thereof.

[0032] In accordance with some further aspects, the present disclosure provides a ferroptosis inhibitor for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of preeclampsia in a subject in need thereof.

[0033] In accordance with some further aspects, the present disclosure provides a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with increased sFLT-1 secretion and / or cell ferroptosis in a subject in need thereof, said method comprises administering to said subject a therapeutically effective amount of at least one ferroptosis inhibitor.

[0034] In accordance with some further aspects, the present disclosure provides a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying preeclampsia in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of at least one ferroptosis inhibitor.

[0035] In accordance with some further aspects, the present disclosure provides a use of at least one ferroptosis inhibitor in the preparation of a composition for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with increased sFLT-1 secretion and / or cell ferroptosis in a subject in need thereof.

[0036] In accordance with some further aspects, the present disclosure provides a use of at least one ferroptosis inhibitor in the preparation of a composition for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying preeclampsia in a subject in need thereof.

[0037] In accordance with some further aspects, the present disclosure provides a screening method for identifying at least one modulator of sFLT-1 secretion, the method comprising the steps of:

[0038] (a) determining in at least one cell contacted with a candidate compound: at least one of: (i) ferroptosis; (ii) lipid peroxide accumulation; (iii) glutathione depletion;

[0039] (b) determining that the candidate compound is capable of at least one of: (i) inhibiting ferroptosis; (ii) inhibiting lipid peroxide accumulation; (iii) inhibiting glutathione depletion.

[0040] EMBODIMENTS

[0041] Some embodiments of this disclosure will now be described in the following numbered paragraph. The following description intends to add on the above general description and not limit it in any manner.

[0042] 1. A combination comprising at least two compounds selected from Table 2 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0043] 2. A combination comprising at least two compounds selected from Table 3 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof. 3. A combination comprising at least two compounds selected from the group consisting of Compound 1 to Compound 56 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0044] 4. A combination comprising at least two compounds selected from Table 4 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0045] 5. A combination comprising at least two compounds selected from the group consisting of Compound 1 to Compound 32 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0046] 6. A combination comprising at least two compounds selected from Table 5 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0047] 7. A combination comprising at least two compounds selected from the group consisting of Compound 1 to Compound 5 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0048] 8. A combination comprising at least two small molecule compounds (SMC) selected from the group consisting of: SMC 1, SMC 2, SMC 3, SMC 4, SMC 5 and SMC 60 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0049] 9. The combination according to any one of Embodiments 1 to 8, comprising one or more of (i) SMC 1 and SMC 2, (ii) SMC 1 and SMC 5, (iii) SMC 1 and SMC 60, (iv) SMC 2 and SMC 5.

[0050] 10. The combination according to Embodiment 9, comprising one or more of (i) SMC 1 and SMC 2.

[0051] 11. A pharmaceutical composition comprising a therapeutically effective amount of the combination according to any one of Embodiments 1 to 10, and wherein the composition optionally further comprises at least one pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s.

[0052] 12. The combination according to any one of Embodiments 1 to 10 and / or the pharmaceutical composition according to Embodiment 11, for use in inhibiting ferroptosis in a cell.

[0053] 13. The combination according to any one of Embodiments 1 to 10 and / or the pharmaceutical composition according to Embodiment 11, for use in reducing sFLT-1 secretion from a cell.

[0054] 14. A method for inhibiting ferroptosis in a cell, the method comprising the step of contacting the cell with an effective amount of the combination according to any one of Embodiments 1 to 10 thereby inhibit ferroptosis.

[0055] 15. A method for reducing sFLT-1 secretion from a cell, the method comprising the step of contacting the cell with an effective amount of the combination according to any one of Embodiments 1 to 10 to thereby reduce sFLT-1 secretion.

[0056] 16. The combination for use and / or the pharmaceutical composition for use according to any one of Embodiments 12 and 13 and / or the method according to Embodiment 14 or 15, wherein the cell is a trophoblast cell, an endothelia cell, or a cancer cell.

[0057] 17. The combination for use and / or the pharmaceutical composition for use according to any one of Embodiments 12 and 13 and / or the method according to Embodiment 14 or 15, wherein the cell is a trophoblast cell.

[0058] 18. The combination for use and / or the pharmaceutical composition for use according to any one of Embodiments 12 and 13 and / or the method according to Embodiment 14 or 15, wherein the cell is of a subject suffering from a disorder or condition associated with cell ferroptosis.

[0059] 19. The combination for use and / or the pharmaceutical composition for use according to any one of Embodiments 12 and 13 and / or the method according to Embodiment 14 or 15, wherein the cell is of a subject suffering from a disorder or condition associated with increased sFLT-1 secretion.

[0060] 20. The combination for use and / or the pharmaceutical composition for use and / or the method according to Embodiment 18 or 19, wherein the disorder or condition is associated with vascular dysfunction and / or abnormal angiogenesis.

[0061] 21. The combination for use and / or the pharmaceutical composition for use and / or the method according to Embodiment 18 or 19, wherein the disorder or condition is an excitotoxic disorder. 22. The combination for use and / or the pharmaceutical composition for use and / or the method according to Embodiment 18 or 19, wherein the disorder or condition is a neurodegenerative disease, a cardiovascular disease, cancer, a kidney disease, a liver disease, a respiratory disease, an inflammatory disease, an autoimmune disease, a skin disease, a gastrointestinal disease or any combination thereof.

[0062] 23. The combination for use and / or the pharmaceutical composition for use and / or the method according to Embodiment 18 or 19, wherein the disorder or condition is preeclampsia.

[0063] 24. The combination according to any one of Embodiments 1 to 10, for use in at least one of (i) mitigating ferroptosis-induced death in trophoblasts, (ii) reducing sFLT-1 secretion from placental tissue, (iii) curbing excessive sFLT-1 triggered due to ferroptosis death or (iv) any combination thereof.

[0064] 25. A method for at least one of (i) reducing ferroptosis-induced death in a trophoblast cell, (ii) reducing sFLT-1 secretion from placental tissue, (iii) curbing (reducing / inhibiting) excessive sFLT-1 triggered due to ferroptosis death or (iv) any combination thereof, the method comprising the step of contacting the cell with an effective amount of the combination according to any one of Embodiments 1 to 10 to thereby inhibit ferroptosis and / or reduce sFLT-1 secretion.

[0065] 26. The combination for use according to Embodiment 24 and / or the method according to Embodiment 25, wherein the cell is a trophoblast cell.

[0066] 27. The combination for use and / or the method according to Embodiment 26, wherein the trophoblast cell is obtained from subject suffering from preeclampsia.

[0067] 28. The combination for use according to any one of Embodiments 1 to 10, for use in inhibiting placental ferroptosis in a subject in need thereof.

[0068] 29. A method for inhibiting placental ferroptosis in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of the combination according to any one of Embodiments 1 to 10.

[0069] 30. The combination according to any one of Embodiments 1 to 10, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with cell ferroptosis in a subject in need thereof. 31. The combination according to any one of Embodiments 1 to 10, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with increased sFLT-1 secretion in a subject in need thereof.

[0070] 32. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with cell ferroptosis in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of the combination according to any one of Embodiments 1 to 10.

[0071] 33. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with increased sFLT-1 secretion in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of the combination according to any one of Embodiments 1 to 10.

[0072] 34. The combination for use according to Embodiment 30 or 31 and / or the method according to Embodiment 32 or 33, wherein the disorder or condition is associated with vascular dysfunction and / or abnormal angiogenesis.

[0073] 35. The combination for use according to Embodiment 30 or 31 and / or the method according to Embodiment 32 or 33, wherein the disorder or condition is an excitotoxic disorder.

[0074] 36. The combination for use according to Embodiment 30 or 31 and / or the method according to Embodiment 32 or 33, wherein the disorder or condition is a neurodegenerative disease, a cardiovascular disease, cancer, a kidney disease, a liver disease, a respiratory disease, an inflammatory disease, an autoimmune disease, a skin disease, a gastrointestinal disease or any combination thereof.

[0075] 37. The combination for use according to Embodiment 30 or 31 and / or the method according to Embodiment 32 or 33, wherein the disorder or condition is preeclampsia.

[0076] 38. The combination according to any one of Embodiments 1 to 10, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of preeclampsia in a subject in need thereof.

[0077] 39. A method of treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a neurodegenerative disease, a cardiovascular disease, cancer, a kidney disease, a liver disease, a respiratory disease, an inflammatory disease, an autoimmune disease, a skin disease, a gastrointestinal disease or any combination thereof, in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of the combination according to any one of Embodiments 1 to 10.

[0078] 40. A method of treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of preeclampsia in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of the combination according to any one of Embodiments 1 to 10.

[0079] 41. Use of a combination according to any one of Embodiments 1 to 10 in the preparation of a composition for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with increased sFLT- 1 secretion and / or cell ferroptosis in a subject in need thereof.

[0080] 42. At least one ferroptosis inhibitor for use in a method for inhibiting secretion of sFLT-1 from a cell.

[0081] 43. The ferroptosis inhibitor for use according to Embodiment 42, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease associated with increased sFLT-1 secretion in a subject in need thereof.

[0082] 44. A method of inhibiting secretion of sFLT-1 from a cell, the method comprising contacting the cell with an effective amount of at least one ferroptosis inhibitor thereby inhibit secretion of sFLT-1.

[0083] 45. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with increased sFLT-1 secretion and / or cell ferroptosis in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of at least one ferroptosis inhibitor.

[0084] 46. The ferroptosis inhibitor for use according to Embodiment 42 or 43 and / or the method according to Embodiment 44 or 45, wherein the cell is a trophoblast cell, an endothelia cell, or a cancer cell.

[0085] 47. The ferroptosis inhibitor for use according to Embodiment 42 or 43 and / or the method according to Embodiment 44 or 45, wherein the cell is a trophoblast cell. 48. The ferroptosis inhibitor for use according to Embodiment 42 or 43 and / or the method according to Embodiment 44 or 45, wherein the cell is of a subject suffering from a disorder or condition associated with cell ferroptosis.

[0086] 49. The ferroptosis inhibitor for use according to Embodiment 42 or 43 and / or the method according to Embodiment 44 or 45, wherein the cell is of a subject suffering from a disorder or condition associated with increased sFLT-1 secretion.

[0087] 50. The ferroptosis inhibitor for use according to Embodiment 42 or 43 and / or the method according to Embodiment 44 or 45, wherein the disorder or condition is associated with vascular dysfunction and / or abnormal angiogenesis.

[0088] 51. The ferroptosis inhibitor for use according to Embodiment 42 or 43 and / or the method according to Embodiment 44 or 45, wherein the disorder or condition is an excitotoxic disorder.

[0089] 52. The ferroptosis inhibitor for use according to Embodiment 42 or 43 and / or the method according to Embodiment 44 or 45, wherein the disorder or condition is a neurodegenerative disease, a cardiovascular disease, cancer, a kidney disease, a liver disease, a respiratory disease, an inflammatory disease, an autoimmune disease, a skin disease, a gastrointestinal disease or any combination thereof.

[0090] 53. The ferroptosis inhibitor for use according to Embodiment 42 or 43 and / or the method according to Embodiment 44 or 45, wherein the disorder or condition is preeclampsia.

[0091] 54. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the ferroptosis inhibitor is at least one compound selected from Table 2 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0092] 55. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the at least one ferroptosis inhibitor comprises at least two compounds selected from Table 2 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0093] 56. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the ferroptosis inhibitor is at least one compound selected from Table 3 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0094] 57. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the at least one ferroptosis inhibitor comprises at least two compounds selected from Table 3 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0095] 58. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the at least one ferroptosis inhibitor is selected from the group consisting of Compound 1 to Compound 56 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0096] 59. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the at least one ferroptosis inhibitor comprises a combination of two compounds, selected from the group consisting of Compound 1 to Compound 56 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0097] 60. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the ferroptosis inhibitor is at least one compound selected from Table 4 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0098] 61. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the at least one ferroptosis inhibitor comprises at least two compounds selected from Table 4 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0099] 62. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the at least one ferroptosis inhibitor is selected from the group consisting of Compound 1 to Compound 32 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0100] 63. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the at least one ferroptosis inhibitor comprises a combination of two compounds, selected from the group consisting of Compound 1 to Compound 32 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof. 64. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the ferroptosis inhibitor is at least one compound selected from Table 5 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0101] 65. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the at least one ferroptosis inhibitor comprises at least two compounds selected from Table 53 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0102] 66. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the at least one ferroptosis inhibitor is selected from the group consisting of Compound 1 to Compound 5 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0103] 67. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the at least one ferroptosis inhibitor comprises a combination of two compounds, selected from the group consisting of Compound 1 to Compound 5 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0104] 68. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the at least one ferroptosis inhibitor is at least one of SMC 1, SMC 2, SMC 3, SMC 4, SMC 5, SMC 60 or any combination thereof.

[0105] 69. The ferroptosis inhibitor for use and / or the method according to any one of Embodiments 42 to 53, wherein the at least one ferroptosis inhibitor comprises a combination selected from (i) SMC 1 and SMC 2, (ii) SMC 1 and SMC 5, (iii) SMC 2 and SMC 5.

[0106] 70. A screening method for identifying at least one modulator of sFLT-1 secretion, the method comprising the steps of:

[0107] (a) determining in at least one cell contacted with a candidate compound: at least one of: (i) ferroptosis; (ii) lipid peroxide accumulation; (iii) glutathione depletion;

[0108] (b) determining that the candidate compound is capable of at least one of: (i) inhibiting ferroptosis; (ii) inhibiting lipid peroxide accumulation; (iii) inhibiting glutathione depletion. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] 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:

[0110] Figures. 1A-1C are graphs showing three phosphatidylethanolamine (PEA) species in preeclampsia patients (“Early PE') and in controls “Early Confy Figure 1A - PEA (36:4), Figure IB - PEA (38:4), and Figure 1C - PEA (38:5).

[0111] Figures 2A-2E show the effect of RSL-3 and induction of ferroptosis; Figure 2A is a graph showing RSL-3 induces a dose response in LDH release in BeWo cells, Figure 2B is a graph showing Fer-1 inhibition of RSL-3 induced LDH release in placental explants obtained from normal pregnancies, Figures 2C-2E are images of control untreated BeWo cells, cells treated with RSL-3, and cells treated with RSL-3 and Fer-1, respectively.

[0112] Figures 3A-3C show soluble fms-like tyrosine kinase-1 (sFLT-1); Figure 3A is a graph showing RSL-3 induction of ferroptosis and Fer-1 on sFLT-1 secretion in placental explants obtained from normal pregnancies; Figures 3B and 3C are graphs showing the effect of Fer-1 and deferoxamine (DFO) on sFLT-1 secretion in ex -vivo preeclamptic placental explants.

[0113] Figures 4A-4C are graphs showing the effect of different compounds on LDH release; Figure 4A is a graph showing the effect of different compounds on LDH release in human trophoblast line BeWo, Figure 4B is a graph showing the effect of different compounds on LDH release in BeWo cells silencing GPX4, Figure 4C is a graph showing the effect of different compounds on LDH release in human placental explant culture exposed to RSL3; deferoxamine (DFO), a-Tocopherol (a-TOC), mitoquinone mesylate (MitoQ), mitochondria (mito)-TEMPO (mTEMPO), Esomeprazole (ESO), Metformin (MET), Pravastatin (PRA).

[0114] Figures 5A-5E relate to characterization and development of a high-throughput screening (HTS) of different compounds, Figures 5A and SB are graphs showing cell viability at different conditions, Figure SC is a schematic representation of the screening of ferroptosis inhibitors at a concentration of 1 pM; Figure 5D is a schematic example of drug selection; Figure 5E shows activity of known cell death inhibitors in inhibiting ferroptosis.

[0115] Figures 6A- 6M show validation of HTS results, Figure 6A illustrates the doseresponse relationship of each drug for inhibiting ferroptosis in BeWo cells; Figure 6B is a graph the effect of candidates with ML 162 or with RSL-3 on LDH release, Figures 6C-6J are images showing cell morphology in BeWo cells, control cells (Figure 6C), cells with RSL3 (Figure 6D), cells with RSL3 and Fer (Figure 6E), cells with RSL3 and DPY (Figure 6F), cells with RSL3 and PMZ (Figure 6G), cells with RSL3 and CRVD (Figure 6H), cells with RSL3 and AMDQ (Figure 61), cells with RSL3 and LRC (Figure 6J), Figure 6K is a graph showing the effect of the compounds on LDH in BeWo cells in which GPX4 was silenced using shRNA, Figure 6L is a graph showing the effect of the compounds on LDH in BeWo cells in ex-vivo normal human placental tissue, Figure 6M is a graph showing the effect of staurosporine-induced apoptosis as compared to RSL-3 induced ferroptosis effect.

[0116] Figures 7A 7E- are graphs show effect of different drugs on cell viability, Figure 7A shows cell viability by Fer-1, Dipyridamole, Sildenafil, Theophylline, and Triflusal; Figure 7B shows cell viability by Fer-1, Promethazine, Terfenadine, Loratadine, and Desloratadine, Figure 7C shows B0DIPY-C11 fluorescence by different compounds, Figures 7D and 7E show intracellular glutathione levels by different compounds.

[0117] Figures 8A-8D are graphs showing validation of HTS results on sFLT-1 secretion, Figures 8A and 8B in ex vivo placental explant model with tissue from normal term pregnancies Figures 8C and 8D in ex vivo preeclamptic placental explants.

[0118] Figures 9A-9J are graphs showing synergistic effect of various combinations in BeWo cells, Figures 9A and 9B combination of Dipyridamole and a-Tocopherol; ZIP score: 17.30, Figure 9C combination of Amodiaquine and dipyridamole, Figure 9D combination of Carvedilol and Dipyridamole, Figure 9E combination of Lercanidipine and dipyridamole, Figure 9F combination of Promethazine and Dipyridamole, Figure 9G combination of Amodiaquine and Promethazine, Figure 9H combination of Carvedilol and Promethazine, Figure 91 combination of Esomeprazole and Promethazine, Figure 9J combination of Lercanidipine and Promethazine.

[0119] Figures 10A-10B are graphs showing synergistic effect of combination of Promethazine (PMZ) and Dipyridamole (DPY) in placental explants, Figure 10A effect of the combination on LDH level, Figure 10B effect of the combination on sFLT 1 secretion.

[0120] DETAILED DESCRIPTION OF EMBODIMENTS

[0121] The present disclosure is based on the identification that trophoblasts are particularly sensitive to ferroptosis and that ferroptosis in these cells leads to excessive secretion of soluble fms-like tyrosine kinase- 1 (sFLT-1) from placental tissue. This establishes a novel and unique link between ferroptosis and sFLT-1.

[0122] As shown in Example 1 below, placentas from early-onset preeclampsia show elevated accumulation of hydroperoxy- phosphatidylethanolamine (PEA) (designated as HOO-PEA) levels, indicative of increased placental ferroptosis.

[0123] In addition, as shown in Example 2 below, ferroptosis was associated with significantly elevated sFLT-1 secretion in both trophoblast cells and human placental ex- vivo explants. Inhibiting ferroptosis reduced or mitigated the secretion of sFLT-1.

[0124] Since sFLT-1 plays a pivotal role in the onset of preeclampsia, the findings presented in this application offer a potential therapeutic avenue of treating pregnancy related diseases, such as preeclampsia.

[0125] Accordingly, it was suggested that anti-ferroptosis compounds may be used for reducing secretion of sFLT-1 from cells, including, inter alia, trophoblast cells.

[0126] Hence, in accordance with some aspects, the present disclosure provides an inhibitor of ferroptosis for use in a method for inhibiting secretion of sFLT-1 from a cell.

[0127] In the following text, when referring to the ferroptosis inhibitor it is to be understood as also referring to compounds, combinations, synergistic combinations, pharmaceutical compositions, methods, kits and uses, disclosed herein. Thus, whenever providing a feature with reference to the ferroptosis inhibitor, it is to be understood as defining the same feature with respect to the compounds, combinations, synergistic combinations, pharmaceutical compositions, methods, kits and uses, mutatis mutandis.

[0128] As used herein ferroptosis refers to a form of regulated programmed cell death characterized by iron overload, accumulation of lipid peroxidation, and disruption of antioxidant system. As shown herein, a novel connection was observed between ferroptosis and sFLT- 1. sFlt-1 or sVEGFR-1 as used herein refers to a tyrosine kinase protein with antiangiogenic properties. sFlt-1 is a truncated form of the VEGF receptor Flt-1; it contains an extracellular domain identical to that of Flt-1 and lacks both the transmembrane and intercellular domains present in Flt-1. sFlt-1 sequence may be provided by NCBI Reference Sequence: NP_001153392.1. sFlt-1 may have the following sequence:

[0129] The reduction in sFLT-1 secretion is not limited to a specific cell and is applicable to any cell that secrets sFLT-1, which secretion can be inhibited by at least one ferroptosis inhibitor.

[0130] In some examples, the cell is a trophoblast cell, an endothelia cell, or a cancer cell.

[0131] In some examples, the cell is from placental tissue.

[0132] In some examples, the cell is of a subject suffering from a disorder or condition associated with cell ferroptosis.

[0133] In some examples, the cell is of a subject suffering from a disorder or condition associated with increased sFLT-1 secretion.

[0134] The present disclosure is not limited to a specific disorder or condition. In some examples, the disorder or condition is associated with vascular dysfunction and / or abnormal angiogenesis.

[0135] In some examples, the disorder or condition is an excitotoxic disorder.

[0136] In some examples, the disorder or condition is a neurodegenerative disease, a cardiovascular disease, cancer, a kidney disease, a liver disease, a respiratory disease, an inflammatory disease, an autoimmune disease, a skin disease, a gastrointestinal disease or any combination thereof.

[0137] In some examples, the disorder or condition is preeclampsia.

[0138] In accordance with some aspects, it is provided a ferroptosis inhibitor for use in reducing (inhibiting) secretion of sFLT-1 from placental tissue.

[0139] The ability to reduce sFLT-1 secretion was suggested to be relevant in treatment of various disorders.

[0140] In accordance with some aspects, the present disclosure provides a ferroptosis inhibitor for use in a method of treating a neurodegenerative disease, a cardiovascular disease, cancer, a kidney disease, a liver disease, a respiratory disease, an inflammatory disease, an autoimmune disease, a skin disease, a gastrointestinal disease or any combination thereof.

[0141] Further, the present disclosure provides a ferroptosis inhibitor for use in treating pregnancy related disease.

[0142] In some examples, the ferroptosis inhibitor is for use in treating preeclampsia.

[0143] As shown in Example 3 below, compounds which were reported to inhibit / attenuate preeclampsia also inhibited ferroptosis. This finding supports the connection between ferroptosis and preeclampsia. In addition, the fact that the tested compounds inhibited ferroptosis at concentrations which were similar or even lower than the previously reported to attenuate sFLT-1 secretion in animal model suggests the importance of ferroptosis in treating preeclampsia.

[0144] Hence, given the potential involvement of placental ferroptosis in preeclampsia pathophysiology, compounds which would inhibit placental ferroptosis, may offer a novel insight into therapeutic efficacy of preeclampsia. As described and shown herein, compounds that inhibit ferroptosis (i.e. ferroptosis inhibitor), reduced or mitigated the secretion of sFLT-1.

[0145] The termferroptosis inhibitor as used herein refers to a compound, agent or a drug that partially or totally blocks stimulation, decreases, prevents, delays activation, inactivates, desensitizes, downregulates the activity or dampens ferroptosis. The inhibitor may comprise a synthetic inhibitor or natural inhibitor.

[0146] The present disclosure is not limited to a specific ferroptosis inhibitor.

[0147] In some examples, the ferroptosis inhibitor may be a nucleic acid modulator, a protein modulator, peptidomimetic modulator, a compound, a small molecule compound (SMC), or any combination thereof.

[0148] In some examples, the ferroptosis inhibitor may be at least one of an aptamer, an antisense RNA, a single-stranded RNA (ssRNA), a double- stranded RNA (dsRNA).

[0149] In some examples, the ferroptosis inhibitor may be one or more of a polypeptide, an antibody or fragment thereof.

[0150] In some examples, the ferroptosis inhibitor may be a SMC.

[0151] A small molecule in the context of the present disclosure refers to a low molecular weight organic compound, having a molecular weight lower than 900 Daltons.

[0152] The ferroptosis inhibitor may be identified in some examples by high throughput screening (HTS) methods. For example, the ferroptosis inhibitor may be identified by the HTS assay described herein and shown schematically in Figure 5D and detailed in Example 4.

[0153] As shown in Example 4 below, HTS was applied to identify inhibitors of trophoblast ferroptosis, i.e. to identify anti-ferroptosis compounds.

[0154] As shown in Table 2, 287 compounds were found to inhibit BeWo ferroptosis (cut off >70% protection). In some examples, the ferroptosis inhibitor is at least one compound selected from the list of compounds of Table 2.

[0155] It should be noted that when referring to compound or inhibitor (one or more, two or more, combinations) selected from / provided in / detailed in one or more of Table 2, Table 3, Table 4, Table 5, each compound or inhibitor represents an embodiment of this disclosure. Among the 287 compounds, 56 compounds were identified as known drugs. Table 3 provides a list of 56 compounds that were found to inhibit BeWo ferroptosis and are known drugs.

[0156] In some examples, the ferroptosis inhibitor is at least one compound selected from the list of compounds of Table 3.

[0157] Among the 56 known drugs, 32 compounds were identified as being safe for use during pregnancy. Table 4 provides a list of 32 compounds that were found to inhibit BeWo ferroptosis and are known drugs and safe for use during pregnancy.

[0158] In some examples, the ferroptosis inhibitor is at least one compound selected from the list of compounds of Table 4.

[0159] The compounds identified in the HTS may be used alone or as combinations, i.e. two or more compounds (such as synergistic combination). These inhibitors and combinations thereof (such as synergistic combination) were suggested as promising new interventions for managing various disorders, for example, preeclampsia.

[0160] As shown in Example 8, combination comprising two compounds, including inhibitors identified by the HTS assay were effective in reducing ferroptosis and sFLT-1 secretion.

[0161] Hence, in accordance with some aspects, the present disclosure provides a combination comprising two or more compounds identified by the HTS described herein.

[0162] In accordance with some aspects, the present disclosure provides a combination comprising at least two compounds selected from Table 2.

[0163] In accordance with some other aspects, the present disclosure provides a combination comprising at least two compounds selected from Table 3.

[0164] Hence, the present disclosure provides a combination comprising at least two compounds selected from the group consisting of:

[0165]

[0166] or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0167] In accordance with some other aspects, the present disclosure provides a combination comprising at least two compounds selected from compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, compound 12, compound 13, compound 14, compound 15, compound 16, compound 17, compound 18, compound 19, compound 20, compound 21, compound 22, compound 23, compound 24, compound 25, compound 26, compound 27, compound 28, compound 29, compound 30, compound 31, compound 32, compound 33, compound 34, compound 35, compound 36, compound 37, compound 38, compound 39, compound 40, compound 41, compound 42, compound 43, compound 44, compound 45, compound 46, compound 47, compound 48, compound 49, compound 50, compound 51, compound 52, compound 53, compound 54, compound 55, compound 56 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0168] In accordance with some other aspects, the present disclosure provides a combination comprising at least two compounds selected from Table 4.

[0169] In accordance with some other aspects, the present disclosure provides a combination comprising at least two compounds selected from compound 1 , compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, compound 12, compound 13, compound 14, compound 15, compound 16, compound 17, compound 18, compound 19, compound 20, compound 21, compound 22, compound 23, compound 24, compound 25, compound 26, compound 27, compound 28, compound 29, compound 30, compound 31, compound 32.

[0170] As shown in Example 5, after analyzing the EC50 of various compounds, five compounds that exhibited high anti-ferroptotic activity were selected for further analysis.

[0171] Hence, the present disclosure provides a combination comprising at least two small molecule compounds (SMCs) selected from the group consisting of: SMC 1,

[0172] SMC 2 SMC 3, SMC 4, SMC 5.

[0173] SMC 1 is known as Dipyridamole; having CAS Registry Number® 58-32-2 and IUPAC name 2,2',2",2"'-((4,8-di(piperidin-l-yl)pyrimido[5,4-d]pyrimidine-2,6- diyl)bis(azanetriyl))tetraethanol. Dipyridamole is represented by the following chemical structure denoted as Formula (I):

[0174] Dipyridamole is denoted herein at times in short as DPY.

[0175] SMC 2 is known as Promethazine; having CAS Registry Number® 60-87-7 and IUPAC name (RS)-N,N -Dimethyl- 1 -( 1 OH -phenothiazin- 10-yl)propan-2-amine.

[0176] Promethazine is represented by the following chemical structure denoted as Formula (II):

[0177] Promethazine is denoted herein at times in short as PMZ.

[0178] SMC 3 is known as Carvedilol; having CAS Registry Number® 72956-09-3 and

[0179] IUPAC name (±)-[3-(9H-carbazol-4-yloxy)-2-hydroxypropyl][2-(2- methoxyphenoxy)ethyl] amine.

[0180] Carvedilol is represented by the following chemical structure denoted as

[0181] Formula (III):

[0182]

[0183] Carvedilol is denoted herein at times in short as CRVD.

[0184] SMC 4 is known as Lercanidipine; having CAS Registry Number® 100427-26-7 and IUPAC name (RS)-2[(3,3-Diphenylpropyl)(methyl)amino]-l,l-dimethylethyl methyl 2,6-dimethyl-4-(3-nitrophenyl)- 1 ,4-dihydropyridine-3,5-dicarboxylate.

[0185] Lercanidipine is represented by the following chemical structure denoted as Formula (IV):

[0186] Lercanidipine is denoted herein at times in short as LRC.

[0187] SMC 5 is known as Amodiaquine; having CAS Registry Number® 86-42-0 and IUPAC name 4- [(7 -chloroquinolin-4-yl)amino] -2- [(diethylamino)methyl]phenol.

[0188] Amodiaquine is represented by the following chemical structure denoted as Formula (V): Amodiaquine is denoted herein at times in short as AMDQ.

[0189] In some examples the compound is:

[0190] (2R)-2,5,7,8-Tetramethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]-3,4-dihydro-2H-l- benzopyran-6-ol (designated herein as SMC 60) or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0191] SMC 60 is known as a-Tocopherol; having CAS Registry Number® 59-02-9 and IUPAC name (2R)-2,5,7,8-Tetramethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]-3,4- dihydro-2H- 1 -benzopyran-6-ol.

[0192] In accordance with some aspects, the present disclosure provides a combination comprising at least two small molecule compounds (SMCs) selected from the group consisting of SMC 1, SMC 2, SMC 3, SMC 4, SMC 5 and SMC 60.

[0193] It should be noted that in accordance with the present disclosure, when referring to a compound and / or a small molecule compound (e.g. any one of SMC1, SMC2, SMC3, SMC4, SMC5 or SMC60), it may encompasses one or more of the following as well as any combinations thereof: a solvate, a hydrate, a stereoisomer, a pharmaceutically acceptable prodrug, a pharmaceutically active metabolite, a pharmaceutically acceptable salt, a crystalline form, an amorphous form, a physiologically functional analogue, a physiologically functional derivative thereof or a combination thereof.

[0194] It should be further noted that a solvate, a hydrate, a stereoisomer, a pharmaceutically acceptable prodrug, a pharmaceutically active metabolite, a pharmaceutically acceptable salt, a crystalline form, an amorphous form, a physiologically functional analogue, a physiologically functional derivative in the context of the present disclosure are considered to have similar biological or physiological activity as the small molecule to which they relate or any small molecule related thereof, for example, inhibiting ferroptosis and / or inhibiting sFLT-1 secretion / production.

[0195] In some examples, the combination comprises SMC1 and SMC2. In some examples, the combination comprises SMC1 and SMC3. In some examples, the combination comprises SMC1 and SMC4. In some examples, the combination comprises SMC1 and SMC5. In some examples, the combination comprises SMC1 and SMC60.

[0196] In some examples, the combination comprises SMC2 and SMC3. In some examples, the combination comprises SMC2 and SMC4. In some examples, the combination comprises SMC2 and SMC5. In some examples, the combination comprises SMC2 and SMC60.

[0197] In some examples, the combination comprises SMC3 and SMC4. In some examples, the combination comprises SMC3 and SMC5. In some examples, the combination comprises SMC3 and SMC60.

[0198] In some examples, the combination comprises SMC4 and SMC5. In some examples, the combination comprises SMC4 and SMC60.

[0199] In some examples, the combination comprises SMC5 and SMC60.

[0200] In some examples, the combination consists of SMC1 and SMC2. In some examples, the combination consists of SMC1 and SMC3. In some examples, the combination consists of SMC1 and SMC4. In some examples, the combination consists of SMC1 and SMC5. In some examples, the combination consists of SMC1 and SMC60.

[0201] In some examples, the combination consists of SMC2 and SMC3. In some examples, the combination consists of SMC2 and SMC4. In some examples, the combination consists of SMC2 and SMC5. In some examples, the combination consists of SMC2 and SMC60.

[0202] In some examples, the combination consists of SMC3 and SMC4. In some examples, the combination consists of SMC3 and SMC5. In some examples, the combination consists of SMC3 and SMC60.

[0203] In some examples, the combination consists of SMC4 and SMC5. In some examples, the combination consists of SMC4 and SMC60.

[0204] In some examples, the combination consists of SMC5 and SMC60.

[0205] As shown in the Examples, the compounds and / or SMCs of the present disclosure, either alone or in combinations were shown to inhibit RSL3-induced LDH release from cells. In other words, the ferroptosis inhibitors, either alone or in combinations, inhibited RSL3-induced LDH release from cells. As shown in Example 8, combination of compounds were effective in reducing LDH level and sFLT 1 secretion.

[0206] Hence, in accordance with some aspects, the present disclosure provides one or more compounds for use in inhibiting ferroptosis in a cell and / or reducing sFLT-1 production / secretion from a cell, wherein the one or more compounds is selected from the compounds detailed in Table 2 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof. In some examples, the combination for use in inhibiting ferroptosis in a cell and / or reducing sFET-1 production / secretion from a cell comprises two or more compounds detailed in Table 2 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0207] The terms “inhibition”, “reduction”, “suppression” or “attenuation” as referred to herein, relate to the retardation, restraining or reduction of activity, such as EDH release or sFET-1 production, by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%. The “inhibition”, “reduction”, “suppression” or “attenuation” may be compared for example to a value of for example EDH release or sFLT-1 production measured in the absence of one or more of the SMCs described herein.

[0208] In accordance with some other aspects, the present disclosure provides one or more compounds for use in inhibiting ferroptosis in a cell and / or reducing sFLT-1 production / secretion from a cell, wherein the one or more compounds is selected from the compounds detailed in Table 3 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0209] In some examples, the combination for use in inhibiting ferroptosis in a cell and / or reducing sFLT-1 production / secretion from a cell comprises two or more compounds detailed in Table 3 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0210] In some examples, the combination for use in inhibiting ferroptosis in a cell, and / or reducing sFLT-1 production / secretion from a cell comprises at least two compounds selected from compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, compound 12, compound 13, compound 14, compound 15, compound 16, compound 17, compound 18, compound 19, compound 20, compound 21, compound 22, compound 23, compound 24, compound 25, compound 26, compound 27, compound 28, compound 29, compound 30, compound 31, compound 32, compound 33, compound 34, compound 35, compound 36, compound 37, compound 38, compound #9, compound 40, compound 41, compound 42, compound 43, compound 44, compound 45, compound 46, compound #47, compound 48, compound 49, compound 50, compound 51, compound 52, compound 53, compound 54, compound 55, compound 56, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0211] In accordance with some other aspects, the present disclosure provides one or more compounds for use in inhibiting ferroptosis in a cell and / or reducing sFLT-1 production / secretion from a cell, wherein the one or more compounds is selected from the compounds detailed in Table 4 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0212] In some examples, the combination for use in inhibiting ferroptosis in a cell and / or reducing sFLT-1 production / secretion from a cell comprises two or more compounds detailed in Table 4 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0213] In some examples, the combination for use in inhibiting ferroptosis in a cell and / or reducing sFLT-1 production / secretion from a cell, comprises at least two compounds selected from compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, compound 12, compound 13, compound 14, compound 15, compound 16, compound 17, compound 18, compound 19, compound 20, compound 21, compound 22, compound 23, compound 24, compound 25, compound 26, compound 27, compound 28, compound 29, compound 30, compound 31, compound 32, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof. In accordance with some other aspects, the present disclosure provides one or more compounds for use in inhibiting ferroptosis in a cell and / or reducing sFLT-1 production / secretion from a cell, wherein the one or more compounds is selected from the compounds detailed in Table 5 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0214] In some examples, the combination for use in inhibiting ferroptosis in a cell and / or reducing sFLT-1 production / secretion from a cell comprises two or more compounds detailed in Table 5 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0215] In some examples, the combination for use in inhibiting ferroptosis in a cell and / or reducing sFLT-1 production / secretion from a cell, comprises at least two compounds selected from SMC1, SMC2, SMC3, SMC4, SMC5 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0216] In accordance with some other aspects, the present disclosure provides one or more compounds for use in inhibiting ferroptosis in a cell and / or reducing sFLT-1 production / secretion from a cell, wherein the one or more SMC is selected from the group consisting of SMC1, SMC2, SMC3, SMC4, SMC5 and SMC60 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0217] Hence, in accordance with some aspects, the present disclosure provides one or more SMCs for use in inhibiting ferroptosis in a cell and / or reducing sFLT-1 production / secretion from a cell, wherein the one or more SMC is selected from the group consisting of SMC1, SMC2, SMC3, SMC4, SMC5 and SMC60 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0218] In accordance with some aspects, the present disclosure provides a combination for use in inhibiting ferroptosis in a cell and / or reducing sFLT-1 production / secretion from a cell, the combination comprising at least two small molecule compounds (SMC) selected from the group consisting of SMC1, SMC2, SMC3, SMC4, SMC5 and SMC60 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof. In accordance with some aspects, the present disclosure provides one or more compounds detailed in Table 2, Table 3, Table 4 or Table 5 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof for use in suppressing pro-ferroptotic lipid signal in a cell.

[0219] In some examples, the combination for use in suppressing pro-ferroptotic lipid signal in a cell, wherein the combination comprises two or more compounds detailed in Table 3 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0220] In some examples, the combination for use in suppressing pro-ferroptotic lipid signal in a cell, wherein the combination comprises two or more compounds detailed in Table 4 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0221] In some examples, the combination for use in suppressing pro-ferroptotic lipid signal in a cell, wherein the combination comprises two or more compounds detailed in Table 5 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0222] In some examples, the combination for use in suppressing pro-ferroptotic lipid signal in a cell, wherein the combination comprises two or more compound SMCs, wherein the SMC is selected from the group consisting of SMC1, SMC2, SMC3, SMC4, SMC5 and SMC60 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0223] As noted herein, the present disclosure is not limited to a specific cell and may be applicable to a variety of cells, for example, cells forming the placenta.

[0224] In some examples, the cell treated by the combinations used in accordance with the present disclosure, may be a trophoblast cell, an endothelia cell, or a cancer cell.

[0225] Trophoblast cell as used herein refers to a specialized type of cell that plays a role in the development, and function of the placenta during pregnancy. Trophoblast cells are initially derived from the fertilized egg, specifically from the blastocyst stage of embryo development and contribute to the formation of the outer layer of the blastocyst. An endothelial cell as used herein refers to cells line the interior surface of blood vessels, lymphatic vessels, and the heart. They form a single layer, known as the endothelium, which serves as a barrier between the blood and the rest of the vessel wall. Endothelial cells are involved in various functions, including regulating blood flow, vascular tone, and permeability, as well as mediating inflammation and blood clotting.

[0226] Cancer cell as used herein refers to abnormal cells that have undergone genetic mutations leading to uncontrolled growth and division. Unlike normal cells, cancer cells do not respond to the body's regulatory mechanisms that control cell proliferation and apoptosis (programmed cell death). As a result, they can form tumors, invade surrounding tissues, and spread to other parts of the body (metastasis). Cancer cells often exhibit changes in their metabolism, signaling pathways, and interactions with the immune system, allowing them to survive and thrive in conditions that would be unfavorable for normal cells.

[0227] In some examples, the cell treated by the compound, inhibitor or combinations used in accordance with the present disclosure, may be a trophoblast cell and / or a primary trophoblast cell.

[0228] Placental explants as used herein refer to tissue samples that are isolated directly from human placental tissue, obtained following term or early preterm (preeclampsia) delivery.

[0229] In some examples, the cell treated by one or more of the SMCs or any of the combinations may be obtained from a placental explant.

[0230] In some examples, the cell treated by one or more of the SMCs or any of the combinations is of a subject suffering from a disorder or condition associated with increased sFLT-1 secretion.

[0231] In some examples, the disorder or condition is associated with vascular dysfunction and / or abnormal angiogenesis.

[0232] In some examples, the disorder or condition is an excitotoxic disorder.

[0233] An excitotoxic disorder as used herein refers to a neurological condition that arises from excitotoxicity, in which neurons are damaged and killed due to excessive stimulation by neurotransmitters such as glutamate and similar substances.

[0234] In some other examples, the cell treated one or more of the SMCs or any of by the combinations is of a subject suffering from a disease (disorder) or condition associated with cell ferroptosis, at times increased ferroptosis.

[0235] In some examples, the disorder or condition is a neurodegenerative disease, a cardiovascular disease, cancer, a kidney disease, a liver disease, a respiratory disease, an inflammatory disease, an autoimmune disease, a skin disease, a gastrointestinal disease or any combination thereof.

[0236] In some examples, the disease associated with cell ferroptosis is one or more cancer, neurodegenerative diseases, ischemia / reperfusion injury, or autoimmune disease.

[0237] In some other embodiments, the cell is obtained from a subject suffering from one or more of cancer, neurodegenerative diseases, ischemia / reperfusion injury, or autoimmune disease, or who is predicted to suffer from one or more of cancer, neurodegenerative diseases, ischemia / reperfusion injury or who has been diagnosed to potentially develop one or more of cancer, neurodegenerative diseases, ischemia / reperfusion injury.

[0238] As shown in Example 2 below, a concentration-dependent increase in cell death (as indicated by LDH release to the medium) in response to RSL3 in BeWo cells. In addition, Example 2 shows that Ferrostatin -1 (Fer-1 or Fer a potent and selective ferroptosis inhibitor which acts as a radical scavenger and 15E0X inhibitor) inhibited EDH release placental explants obtained from normal pregnancies. These results show a dose-dependent activation of ferroptosis using RSE3 (as indicated by an increase by EDH release), and an inhibition of this increase by Fer-1 (200 nM).

[0239] Interestingly, as also shown in Example 2, placental ferroptosis induced by RLS- 3 in placental explants obtained from normal pregnancies, was associated with a significant increase in medium sFLT-1 levels and this increase was blocked by addition of Fer-1 (that inhibits ferroptosis).

[0240] Hence, based on the results in Example 2 demonstrating that ferroptosis elevates placental sFlt-1 release in placental explants, a direct association between placental ferroptosis and sFlt-1 levels was suggested.

[0241] These findings highlight the potential of targeting ferroptosis as a novel strategy for treating or preventing preeclampsia. In other words, it is suggested that anti- ferroptosis compounds either used alone or in combination as described herein that reduce / inhibit sFLT-1 secretion may be considered as candidates for treatment of preeclampsia.

[0242] As shown in Example 5, the identified compound (ferroptosis inhibitor) inhibited ferroptosis cell death induced by RSL3, in normal-term placental explants culture.

[0243] In some embodiments, the one or more compound or SMCs or the combination is for use in at least one of (i) mitigating (reducing / inhibiting) ferroptosis-induced death in trophoblasts, (ii) reducing sFLT-1 secretion from placental tissue, (iii) curbing (reducing / inhibiting) excessive sFLT-1 triggered due to ferroptotic death, or (iv) any combination thereof.

[0244] Mitigating as used herein refers to the process of reducing or lessening the severity or impact of cell death, specifically ferroptosis.

[0245] In some examples, the disease is associated with placenta dysfunction.

[0246] In some examples, the disease is a pregnancy related disease.

[0247] In some examples, the pregnancy related disease is one or more of spontaneous abortion, preeclampsia, gestational diabetes mellitus (GDM), intrahepatic cholestasis of pregnancy (ICP), spontaneous preterm birth or any combination thereof.

[0248] In some examples, the disorder or condition is preeclampsia.

[0249] Interestingly, and as shown in Example 7, drugs identified by the HTS as ferroptosis inhibitors reduced sFET-1 release from placental explants of preeclamptic patients, supporting the importance of inhibiting ferroptosis and offering new avenues for preeclampsia treatment.

[0250] In some other embodiments, the cell is obtained from a subject suffering from placenta dysfunction, or who is predicted to suffer from placenta dysfunction, or who has been diagnosed to potentially develop placenta dysfunction.

[0251] In some other embodiments, the cell is obtained from a subject suffering from a pregnancy related disease, or who is predicted to suffer from a pregnancy related disease, or who has been diagnosed to potentially develop a pregnancy related disease.

[0252] In some other embodiments, the cell is obtained from a subject suffering from pre- eclampsia or who is predicted to suffer from preeclampsia or who has been diagnosed to potentially develop preeclampsia.

[0253] In some aspects, the one or more of the SMCs or any of the combinations of the present disclosure is for use in a method for inhibiting ferroptosis in a subject in need thereof.

[0254] In some aspects, the one or more of the SMCs or any of the combinations of the present disclosure is for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of disease associated with ferroptosis in a subject in need thereof.

[0255] In some aspects, the one or more of the SMCs or any of the combinations of the present disclosure is for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of one or more cancer, neurodegenerative diseases, ischemia / reperfusion injury, or autoimmune disease.

[0256] In some aspects, the one or more of the SMCs or any of the combinations of the present disclosure is for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of placenta dysfunction.

[0257] In some aspects, the one or more of the SMCs or any of the combinations of the present disclosure is for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pregnancy related disease.

[0258] In some aspects, the one or more of the SMCs or any of the combinations of the present disclosure is for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of one or more of spontaneous abortion, preeclampsia, GDM, ICP, spontaneous preterm birth or any combination thereof.

[0259] In some aspects, the one or more of the SMCs or any of the combinations of the present disclosure is for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of pre-eclampsia.

[0260] As shown in the Examples below, various combinations of the SMCs had a synergistic effect in inhibiting trophoblast ferroptosis. This inhibition was superior compared to the effect of each one of the SMCs when tested as single compound. As also shown in the Examples below, this effect was observed in a broad range of concentrations and concentration ratios.

[0261] Hence, it was suggested that the SMCs (drugs) identified in the present disclosure can interact synergistically to inhibit ferroptosis.

[0262] Thus, the present disclosure provides a synergistic combination comprising at least two SMCs selected from the group consisting of SMC1, SMC2, SMC3, SMC4, SMC5 and SMC60.

[0263] The synergistic combination may be applicable for uses as described herein.

[0264] A synergistic combination as used herein refers to a quantity of a combination comprising at least two SMCs as described herein that is statistically significantly more effective / active than the additive effects of the at least two SMCs when used individually (e.g. not in combination).

[0265] Synergism between at least two of the SMCs described herein may be based on the results obtained from a synergy model. In some examples, synergy can be calculated by HAS model. In some examples, synergy can be calculated by Loewe model. In some examples, synergy can be calculated by Bliss model. In some examples, synergy can be calculated by ZIP model.

[0266] The synergistic effect of the SMCs can be determined by the effect on LDH secretion using any known assay known in the art as described in the Examples below. In some embodiments, the synergistic effect of the SMCs on LDH secretion is determined as a synergistic combination using a Loewe additivity model method. In some embodiments, the synergistic effect of the SMCs on LDH secretion is determined as a synergistic combination using a HAS model. In some embodiments, the synergistic effect of the SMCs on LDH secretion is determined as a synergistic combination using a ZIP model.

[0267] In accordance with some aspects, it is provided a synergistic combination comprising SMC1 and SMC2.

[0268] In accordance with some aspects, it is provided a synergistic combination comprising SMC1 and SMC3.

[0269] In accordance with some aspects, it is provided a synergistic combination comprising SMC1 and SMC4.

[0270] In accordance with some aspects, it is provided a synergistic combination comprising SMC1 and SMC5.

[0271] In accordance with some aspects, it is provided a synergistic combination comprising SMC1 and SMC60.

[0272] In accordance with some aspects, it is provided a synergistic combination comprising SMC2 and SMC3.

[0273] In accordance with some aspects, it is provided a synergistic combination comprising SMC2 and SMC4.

[0274] In accordance with some aspects, it is provided a synergistic combination comprising SMC2 and SMC5.

[0275] In accordance with some aspects, it is provided a synergistic combination comprising SMC2 and SMC60.

[0276] In accordance with some aspects, it is provided a synergistic combination comprising SMC3 and SMC4.

[0277] In accordance with some aspects, it is provided a synergistic combination comprising SMC3 and SMC5.

[0278] In accordance with some aspects, it is provided a synergistic combination comprising SMC3 and SMC6.

[0279] In accordance with some aspects, it is provided a synergistic combination comprising SMC4 and SMC5.

[0280] In accordance with some aspects, it is provided a synergistic combination comprising SMC4 and SMC60.

[0281] In accordance with some aspects, it is provided a synergistic combination comprising SMC5 and SMC60.

[0282] At least one of the at least two SMCs may be present in the combination in a concentration capable of providing a synergistic effect. In other words, and in accordance with some other embodiments, at least one of the at least two SMCs and at times at lest two, is present in the combination in a synergistic amount.

[0283] A “synergistic amount” for a component in a combination (e.g. SMC1 and SMC60) is defined as an amount (e.g. concentration) providing a synergistic effect.

[0284] As detailed herein and shown in the Examples, the synergistic amount may be provided by a range of concentrations for one or more of the at least two SMCs, such that various combinations of the at least two SMCs provide a synergistic effect. It should be noted that when referring to a combination comprising a range of SMCs concentrations, it encompasses all possible combinations comprising individual concentration of each SMCs.

[0285] Hence, in accordance with the present disclosure, the synergistic amount may be a range of concentrations or may be a single concentration.

[0286] In accordance with some embodiments that can be considered as aspects of the present disclosure, it is provided a combination comprising at least two SMCs, wherein each one of the at least two SMCs is present in the combination in a synergistic amount.

[0287] As appreciated, each one of the at least two SMCs may have the same synergistic amount (either a range of concentrations or a single concentration as shown herein) or a different synergistic amount (either a range of concentrations or a single concentration as shown herein). Additionally, or alternatively, in case the synergistic amount is a range of concentrations, the ranges may partially overlap.

[0288] In accordance with some aspects, it is provided a synergistic combination comprising SMC1 and SMC2, at a ratio of at least 0.05, at times between about 0.08 and about 40, at times between about 0.1 and about 30.

[0289] In accordance with some aspects, it is provided a synergistic combination comprising SMC1 and SMC3 at a ratio of at least 0.05, at times between about 0.08 and about 40, at times between about 0.1 and about 30.

[0290] In accordance with some aspects, it is provided a synergistic combination comprising SMC1 and SMC4 at a ratio of at least 0.001, at times between about 0.002 and about 40, at times between about 0.003 and about 30.

[0291] In accordance with some aspects, it is provided a synergistic combination comprising SMC1 and SMC5 at a ratio of at least 0.05, at times between about 0.08 and about 40, at times between about 0.1 and about 30.

[0292] In accordance with some aspects, it is provided a synergistic combination comprising SMC1 and SMC60 at a ratio of at least 0.001, at times between about 0.005 and about 5, at times between about 0.01 and about 3, at times between about 0.02 and about 2.

[0293] In accordance with some aspects, it is provided a synergistic combination comprising SMC2 and SMC3 at a ratio of at least 0.05, at times between about 0.08 and about 40, at times between about 0.1 and about 30.

[0294] In accordance with some aspects, it is provided a synergistic combination comprising SMC2 and SMC4 at a ratio of at least 0.001, at times between about 0.002 and about 40, at times between about 0.003 and about 30.

[0295] In accordance with some aspects, it is provided a synergistic combination comprising SMC2 and SMC5 at a ratio of at least 0.05, at times between about 0.08 and about 40, at times between about 0.1 and about 30.

[0296] In accordance with some aspects, it is provided a synergistic combination comprising SMC2 and SMC60.

[0297] In accordance with some aspects, it is provided a synergistic combination comprising SMC3 and SMC4.

[0298] In accordance with some aspects, it is provided a synergistic combination comprising SMC3 and SMC5.

[0299] In accordance with some aspects, it is provided a synergistic combination comprising SMC3 and SMC6.

[0300] In accordance with some aspects, it is provided a synergistic combination comprising SMC4 and SMC5.

[0301] In accordance with some aspects, it is provided a synergistic combination comprising SMC4 and SMC60.

[0302] In accordance with some aspects, it is provided a synergistic combination comprising SMC5 and SMC60.

[0303] As suggested, the combinations of the present disclosure and / or pharmaceutical compositions comprising the same, may be used in various methods as described herein.

[0304] According with some aspects of the present disclosure, it is provided a method for mitigating ferroptosis-induced death in trophoblasts cell of a subject in need thereof. The method comprises the step of contacting the cell with an effective amount of the SMCs or the combinations described herein.

[0305] According with other aspects of the present disclosure, it is provided a method for reducing sFLT-1 secretion from placental tissue of a subject in need thereof. This method involves contacting the placental tissue with an effective amount of the SMCs or the combinations described herein.

[0306] According with further aspects of the present disclosure, it is provided a method for curbing excessive sFLT-1 triggered due to ferroptosis death. The method entails contacting a cell of a subject in need thereof with an effective amount of the SMCs or the combinations described herein.

[0307] According with yet other aspects of the present disclosure, it is provided a method for utilizing the SMCs or any combination of the above methods in treating conditions related to ferroptosis death.

[0308] The term “contacting” means to bring, put, incubates or mix together. As such, a first item is contacted with a second item when the two items are brought or put together, e.g., by touching them to each other or combining them. In the context of the present invention, the term "contacting" includes all measures or steps which allow interaction between the at least two SMCs and a cell, specifically a cell characterized by ferroptosis. In some examples, the cell is a trophoblast cell and / or placental explant.

[0309] The methods of the invention may be in vitro methods, ex vivo methods and / or in vivo methods.

[0310] In some embodiments, the method is an in vitro method.

[0311] In some other embodiments, the method is an in in vivo method.

[0312] In some embodiments, the method is an ex vivo method.

[0313] In some examples, the method is for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of one or more cancer, neurodegenerative diseases, ischemia / reperfusion injury, or autoimmune disease in a subject in need thereof. In some examples, the method is for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease associated with placenta dysfunction in a subject in need thereof.

[0314] In some examples, the method is for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a pregnancy related disease in a subject in need thereof.

[0315] In some examples, the method is for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of one or more of spontaneous abortion, preeclampsia, gestational diabetes mellitus (GDM), intrahepatic cholestasis of pregnancy (ICP), spontaneous preterm birth in a subject in need thereof.

[0316] In some examples, the method is for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of pre-eclampsia in a subject in need thereof.

[0317] According to some aspects of the present disclosure it is provided a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with increased sFLT-1 secretion in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of one or more SMC or a combination comprising at least two SMCs, the SMC is selected from the group consisting of SMC 1, SMC 2, SMC 3, SMC 4, SMC 5 and SMC 60 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0318] According to a further aspect of the present disclosure it is provided a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with ferroptosis in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of one or more SMC or a combination comprising at least two SMCs, the SMC is selected from the group consisting of SMC 1, SMC 2, SMC 3, SMC 4, SMC 5 and SMC 60 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0319] According to another aspect of the present disclosure it is provided a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of one or more of cancer, neurodegenerative diseases, ischemia / reperfusion injury, or autoimmune disease in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of one or more SMC or a combination comprising at least two SMCs, the SMC is selected from the group consisting of SMC 1 , SMC 2, SMC 3, SMC 4, SMC 5 and SMC 60 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0320] As used herein cancer refers to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non-solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors.

[0321] In general, the SMCs and combinations comprising the same and methods of the present invention may be applicable for the treatment of a patient suffering from any one of non-solid and solid tumors.

[0322] As used herein "autoimmune disease or disorder" refers to a condition associated with dysfunction of the immune system of a subject, either through activation or suppression of the immune system or that can be treated, prevented or diagnosed by targeting a certain component of the immune response in a subject, such as the adaptive or innate immune response. Such disorder may be any one of psoriasis or rheumatoid arthritis.

[0323] According to another aspect of the present disclosure it is provided a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease associated with placenta dysfunction in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of one or more SMC or a combination comprising at least two SMCs, the SMC is selected from the group consisting of SMC 1, SMC 2, SMC 3, SMC 4, SMC 5 and SMC 60 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0324] Placenta dysfunction also known as placental dysfunction, refers to abnormalities or impairments in the structure or function of the placenta.

[0325] According to another aspect of the present disclosure it is provided a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a pregnancy related disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more SMC or a combination comprising at least two SMCs, the SMC being selected from the group consisting of SMC 1, SMC 2, SMC 3, SMC 4, SMC 5 and SMC 60 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0326] According to another aspect of the present disclosure it is provided a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of one or more of spontaneous abortion, pre-eclampsia, GDM, ICP, spontaneous preterm birth in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of one or more SMC or a combination comprising at least two SMCs, the SMC being selected from the group consisting of SMC 1, SMC 2, SMC 3, SMC 4, SMC 5 and SMC 60 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0327] According to another aspect of the present disclosure it is provided a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of pre-eclampsia in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of one or more SMC or a combination comprising at least two SMCs, the SMC is selected from the group consisting of SMC 1 , SMC 2, SMC 3, SMC 4, SMC 5 and SMC 60 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

[0328] In some examples, the subject to be treated by the combinations and methods described herein is a female subject.

[0329] In some examples, the subject is a pregnant woman.

[0330] In accordance with the present invention, the subject to be treated with a combination, as disclosed herein, is one which has been found or determined to possibly benefit from the treatment with the combination and specifically the synergistic combination, and for whom a therapeutic protocol has been tailored, specifying amounts of the one or more SMCs, at times at least two SMCs, such that the amount of one component is adjusted to or determined based on the amount of the other component or to any one other protocol parameter which may, inter alia, depend on subject health and personal factors as well as on time of administration, sequence and administration regimen. As noted herein above, the combination of the at least two SMCs can be present in a single composition. Hence, the present disclosure provides in accordance with some aspects, a pharmaceutical composition comprising a therapeutically effective amount of the combinations of the present disclosure, and optionally further comprises at least one pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s.

[0331] Additionally or alternatively, each one of the at least two SMCs is present in a different composition.

[0332] In some embodiments, the pharmaceutical composition comprises at least one pharmaceutically acceptable excipient or carrier.

[0333] As used herein “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.

[0334] The pharmaceutical composition of the invention can be administered and dosed by the methods of the invention, in accordance with good medical practice. The combinations, compositions used in the methods and kits of the invention, described herein after, may be adapted for administration by systemic, parenteral, intraperitoneal, transdermal, oral (including buccal or sublingual), rectal, topical (including buccal or sublingual), vaginal, intranasal and any other appropriate routes. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).

[0335] The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central blood system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, intra-placental, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

[0336] Regardless of the route of administration selected, the at least two SMCs may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

[0337] The pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0338] Pharmaceutical compositions used to treat subjects in need thereof according to the invention generally comprise a buffering agent, an agent who adjusts the osmolarity thereof, and optionally, one or more pharmaceutically acceptable carriers, excipients and / or additives as known in the art. Supplementary active ingredients can also be incorporated into the compositions. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0339] Local administration to the area in need of treatment may be achieved by, for example, local infusion during surgery, topical application, direct injection into the specific organ, etc.

[0340] Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, solid solution, liposome, films, ovules, sprays or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0341] It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

[0342] It should be understood that methods of the invention making use of administration of the at least two SMCs in "combination" (at times denoted "together"') refers to a multiple’s treatment schedules.

[0343] In accordance with some embodiments, combination denotes a regimen involving administration of the at least two SMCs by the methods of the invention in a single treatment cycle. As may be appreciated, administration of the combination requires an assessment of various parameters, inter alia, treatment schedule, predetermined ratio of the at least two SMCs (dosing), number of treatment cycles, and others. Assessment of the combination may be carried out before onset of treatment.

[0344] The at least two SMCs described herein can be administered and dosed by the methods of the invention, in accordance with good medical practice, such as systemically, for example by parenteral, e.g. intravenous, intraperitoneal or intramuscular injection.

[0345] In some embodiments, the administration of at least two SMCs by the methods of the invention may be by the same or different modes of administration and at the same or different frequency, i.e. at the same or different time points.

[0346] In accordance with some embodiments, administration of the at least two SMCs by the methods of the invention is at different dosage forms. In other words, at least two SMCs are not present in the same composition.

[0347] As detailed above, various modes of administration are known in the art, including, but are not limiting to, topical administration, enteral administration, parental administration. For example by injection (e.g., using a subcutaneous, intramuscular, intravenous, intraperitoneal, or intradermal injection), infusion (e.g. intraperitoneal), transdermal, transmucosal, intranasal administration, by inhalation or sublingually.

[0348] In some embodiments, at least two SMCs are administered by the methods of the invention in different routes.

[0349] In some embodiments, each of at least two SMCs is administered by the methods of the invention separately, i.e. not within the same composition / dosage form.

[0350] In some examples, the at least two SMCs are formulated separately, i.e., in two different pharmaceutical compositions. In some examples, the two pharmaceutical compositions can be designed and / or dosed for sequential administration. In some other examples, the two pharmaceutical compositions can be designed and / or dosed for concomitant administration.

[0351] In some other embodiments, the at least two SMCs are administered by the methods of the invention simultaneously.

[0352] As noted above and in accordance with some embodiments, the at least two SMCs are formulated in the same composition. In some embodiments, the at least two SMCs are administered in a single unit dose; namely in a unit which is suitable for administration to the subject as detailed herein below. The unit dose can contain an effective amount- a prescribed quantity of the at least two SMCs sufficient to produce a therapeutic effect.

[0353] Finally, disclosed herein is a package or kit comprising a combination comprising at least two SMCs; and instructions for use of a combination for treating a disease as described herein.

[0354] In some examples, one SMC of the at least two SMCs in the kit is preferably contained separately from the at least one additional SMC. The first and the additional SMCs are as defined hereinabove with respect to the at least two SMCs for use in the disclosed combination.

[0355] In some embodiments, the different reservoirs are different syringes or different formulation containers comprising the actives in solid or liquid or solution forms.

[0356] In some embodiments, the kit comprise a container means for containing separate compositions; such as a divided bottle or a divided foil packet.

[0357] In some embodiments, the kit includes directions for the administration of the separate components. It should be understood that such a kit is advantageous when the at least two SMCs are preferably administered in different dosage forms, are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.

[0358] It should be appreciated that each of the multiple components of the kit may be administered simultaneously.

[0359] Alternatively, each of the multiple dosage forms may be administered sequentially in either order. Further, the kits described herein can include a composition as described, or in separate multiple dosage unit forms, as an already prepared dosage form ready for administration or, alternatively, can include the composition as described as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid oral dosage form.

[0360] When the kit includes a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid dosage form (e.g., for oral administration), the kit may optionally include a reconstituting solvent.

[0361] In some embodiments, the kit comprises the at least two SMCs in the same composition, e.g., the pharmaceutical composition of the present disclosure.

[0362] In some embodiments, the kit comprising instructions for the at least two SMCs in a combination for use in administration in a subject in need thereof according with the methods descried herein.

[0363] In accordance with some further aspects, the present disclosure provides use of at least two SMCs in the preparation of a pharmaceutical composition, or a combination as described herein.

[0364] As noted herein, the present disclosure relates to combinations comprising at least two SMC or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, prodrug, metabolite, derivative, functional analogue, or physiologically functional derivative thereof.

[0365] The term "pharmaceutically acceptable salt" refers to salts derived from organic and inorganic acids of the SMC described herein. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, hydrochloride, bromide, hydrobromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, camphorsulfonate, napthalenesulfonate, propionate, succinate, fumarate, maleate, malonate, mandelate, malate, phthalate, and pamoate. The term “pharmaceutically acceptable salt” as used herein also refers to a salt of a compound described herein having an acidic functional group, such as a carboxylic acid functional group, and a base. Exemplary bases include, but are not limited to, hydroxide of alkali metals including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, organic amines such as unsubstituted or hydroxyl-substituted mono-, di-, or trialkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(Ci-C6)-alkylamine), such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D- glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids such as arginine, lysine, and the like. The term “pharmaceutically acceptable salt” also includes hydrates of a salt of a compound described herein.

[0366] The term “solvate” refers to an aggregate of a compound with one or more solvent molecules, such as hydrate, alcoholate (aggregate or adduct with alcohol), and the like.

[0367] The term “hydrate” refers to a compound formed by the addition of water. The hydrates may be obtained by any known method in the art by dissolving the compounds in water and recrystallizing them to incorporate water into the crystalline structure.

[0368] The term “stereoisomer” as used herein encompasses an "enantiomer", the enantiomer refers to a compound that is superposable with respect to its counterpart only by a complete inversion / reflection (mirror image) of each other. The small molecule in accordance with the present disclosure encompass any enantiomers (i.e., R or S).

[0369] The term “prodrug” or “pharmaceutically acceptable prodrug” as used herein refers to a compound that may be converted under physiological conditions to the specified compound or to a pharmaceutically acceptable salt of such compound. Prodrugs may be useful for facilitating the administration of a parent drug.

[0370] The term “metabolite” or “pharmaceutically acceptable metabolite” as used herein refers to a compound that is formed under physiological conditions to of degrading and eliminating the compounds. Oxidative metabolite may an example.

[0371] The term "physiologically functional derivative” used herein relates to any physiologically acceptable derivative of a compound as described herein. The physiologically functional derivatives also include prodrugs of the compounds of the invention. As noted herein, such prodrugs may be metabolized in vivo to a compound of the invention. These pro-drugs may or may not be active themselves and are also an object of the present invention.

[0372] The term "derivative" in accordance with the SMC of the present invention also encompasses chemically modified SMC derived from a parent compound of the invention that differs from the parent compound by one or more elements, substituents and / or functional groups such that the derivative has the same or similar biological properties / activities as the parent compound.

[0373] The term "functional analog" used herein relates to any chemical entity that exhibits at least an activity in at least one of the above uses and / or methods, the activity of the analog can be the same or similar to that of the SMC to which it is analogous (herein the "parent SMI”). For the sake of clarity, when referring to a "parent SMI" it is to be understood to refer to an SMC that has been experimentally shown to be potentially effective when tested in a combination as disclosed herein. The potential effect can be determined based on the above description in in vitro, ex vivo or in vivo models.

[0374] In some examples, the functional analog is a chemical derivative of the parent SMI, i.e., having at least one modification (e.g., deletion, substitution, insertion of a chemical group, isomerization etc.) with respect to the parent SMC and yet it has an effect as the parent SMC. The effect may not need to be to the same extent as that of the parent SMI and can be statistically greater, smaller, the same (as long as statistically, there is an inhibition). In some examples, the parent compound may be one or more of SMC 1 , SMC 2, SMC 3, SMC 4, SMC 5 and SMC 60.

[0375] In some examples, the functional analog may be structurally significantly different from the parent SMC (i.e., would not be considered a chemical derivative) and yet exhibit an effect as the parent SMC.

[0376] A crystalline and / or amorphous form of the SMCs described herein include, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms of the compounds, as well as mixtures thereof.

[0377] The term "effective amount" or “therapeutically effective dose” as used herein is that determined by such considerations as are known to the man of skill in the art. The amount must be sufficient to treat, prevent or delay the onset of disease as described herein. As known, the "effective amount" for purposes herein may be determined by such considerations as known in the art. The amount must be effective to achieve the desired therapeutic effect, depending, inter alia, on the type and severity of the disease to be treated and the treatment regime. As generally known, the effective amount depends on a variety of factors including for example the distribution profile within the body, a variety of pharmacological parameters such as half-life in the body, on undesired side effects, if any, on factors such as age and gender, and others.

[0378] The effective amount and / or therapeutically effective dose of the at least two SMCs in accordance with some embodiments is a synergistic amount.

[0379] It should be noted that determination of an effective amount, therapeutically effective amount or synergistic amount or any of these amounts is well within the skills of a person skilled in the art as described herein and specifically in view of the disclosure herein, including the Examples provided below. Any combination or pharmaceutically composition used in the invention, for example in the methods of the invention, the effective amount, therapeutically effective amount or synergistic amount or dose can be estimated from in vitro and cell culture assays described herein. For example, a dose can be formulated in animal models to achieve a required concentration or titer. Such information can be used to accurately determine useful doses in humans.

[0380] As appreciated, doses determined in the animal model such as mouse model can be converted for the treatment other species such as human and other animals diagnosed with the disease, using conversion Tables known to the skilled in the art.

[0381] Hence, as appreciated by those skilled in the art, the in vitro data shown herein from cell culture assays can be used with animal studies to formulate a range of dosage for use in human.

[0382] In the context of the present disclosure, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0383] The term “treatment or prevention” as used herein, refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, a disease or condition as described herein.

[0384] It is understood that the interchangeably used terms "associated", “linked” and "related", when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder condition or pathology causes the second disease, disorder, condition or pathology.

[0385] The present invention relates to the treatment of subjects, or patients, in need thereof. By “patient” or “subject in need” it is meant any organism who may be affected by the above-mentioned conditions, and to whom the treatment methods herein described are desired, including humans, domestic and non-domestic mammals such as canine and feline subjects, bovine, simian, equine and murine subjects, rodents, domestic birds, aquaculture, fish and exotic aquarium fish. It should be appreciated that the treated subject may be also any reptile or zoo animal. More specifically, the methods and compositions of the invention are intended for mammals. By “mammalian subject” is meant any mammal for which the proposed therapy is desired, including human, equine, canine, and feline subjects, most specifically humans. It should be noted that specifically in cases of non-human subjects, the method of the invention may be performed using administration via injection, drinking water, feed, spraying, oral gavage and directly into the digestive tract of subjects in need thereof. It should be further noted that particularly in case of human subject, administering of the compositions of the invention to the patient includes both self-administration and administration to the patient by another person.

[0386] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. As used herein the term "about" refers to ± 10 %.

[0387] It should be noted that various embodiments of this invention may be presented in a range format. The description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range inclusive the end points values. For example, description of a range such as from 1 to 6 or between 1 and 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6.

[0388] It should be noted that the term “at least one” refers to one or more of the following and encompasses any one of 1, 2, 3, or more of the specified information.

[0389] It is to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[0390] Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0391] It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0392] The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

[0393] It should be noted that the various embodiments and examples detailed herein in connection with various aspects of the invention may be applicable to one or more aspects disclosed herein. It should be further noted that any embodiment described herein, for example, related to components of the microbial consortium, may be applied separately or in various combinations. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. The phrases “in another embodiment” or any refence made to embodiment as used herein do not necessarily refer to different embodiment, although it may. Thus, various embodiments of the invention can be combined (from the same or from different aspects) without departing from the scope of the invention. NON-LIMITING EXAMPLES

[0394] Materials and Methods:

[0395] Placental Sample Collection :

[0396] The study utilized placental biopsy samples collected from two centers. Samples for phospholipidomics analysis were obtained from the Steve N. Caritis Magee Obstetric Maternal and Infant (MOMI) Database and Biobank at Magee-Womens Research Institute. All other experiments were conducted using samples collected and preserved at Hadassah Hospital in Israel.

[0397] This study was approved by The Hadassah Hospital Medical Center Institutional Review Board, following the principles of the Helsinki Declaration. All patients provided written informed consent for placental biopsy collection.

[0398] Early-onset preeclampsia was defined as cases of preeclampsia manifesting before 34 weeks of gestation, according to the guidelines of the American College of Obstetricians and Gynecologists, with clinical evaluations most recent to the participants’ delivery. Unless otherwise specified, experiments were conducted on normal placentas from women who delivered at term without any illnesses or complications during pregnancy or delivery.

[0399] Biopsies (2-3 cm) were taken within 30 minutes post-delivery from a mid-region between the cord insertion and placental margin. These biopsies were used for placental explants preparation or snap-frozen in liquid nitrogen for subsequent phospholipidomics analysis.

[0400] Cell culture

[0401] The BeWo trophoblast cells (ATCC, CCL- 98) were cultured and maintained in F12K Kaighn’s modified medium (ATCC) supplemented with 10% Fetal bovine serum (Gibco) and antibiotics and maintained at conditions of 37°C, 21% O2 and 5% CO2 (F- 12K-C).

[0402] Activation, inhibition, and quantification of ferroptosis in culture

[0403] For ferroptosis induction experiments, 80,000 cells / well were seeded in a 24-well plate using 10% serum medium. After 24 hours, the cells were exposed to RSL3 (0.4 pM 1S-3R-RSL3, SML2234, Merck) or ML162 (0.25 pM, Merck), with or without Fer-1 (positive control for ferroptosis inhibition, 0.5 pM, HY-100579, MCE). Induction of ferroptosis in BeWo cells was performed using 1% serum medium.

[0404] Drug treatment, kinetics, and synergistic assays

[0405] To evaluate the effect of selected drugs on ferroptosis, the following drugs were added 30 minutes after RSL3: Dipyridamole (5 pM, D9766, Merck); Promethazine - hydrochloride (5 pM, P-4651, Merck); Amodiaquine (5 pM, S5948, Selleckchem); Lercanidipine (5 pM, S5861, Selleckchem); Carvedilol (5 pM, C3993, Merck); Esomeprazole magnesium (100 pM, HY-B1446, MCE); Z-VAD-FMK (20 pM, HY- 16658B, MCE); DFO (300 pM, S5742, Selleckchem); MitoQ (5 pM, S8978, Selleckchem); and MitoTEMPO (100 nM, SML0737, Merck).

[0406] In experiments targeting ferroptosis induction via GPX4 knockdown (shRNA), cells were maintained in a 0.5 pM Fer-1 -containing medium to avoid premature activation of ferroptosis in cells lacking GPX4. To activate ferroptosis, the medium was washed with PBS and then replaced with Fer-1 -free medium. For apoptosis induction, cells were exposed to Staurosporine (1 pM, S1412, Selleckchem) instead of RSE3.

[0407] ELISA analysis

[0408] Concentrations of sFlt-1 was measured in conditioned cell culture media using commercially available EEISA kit (R&D systems), as described elsewhere.

[0409] Analysis of combined synergistic effect

[0410] Synergistic assays were performed in cell cultures by dose-response curve with different titers of drugs and drugs combinations. For synergism analysis, the web application "SynergyFinder" was used, and synergism was determined by the Zero Interaction Potency (ZIP) model. ZIP model is a reference model for evaluating the effect of combination of two drugs by comparing the change in the potency of the dose -response curves between individual drugs and their combinations.

[0411] Placental explants (ex-vivo) experiments

[0412] Biopsies for placental explant preparation (from normal or early preeclamptic patients) were thoroughly washed in ice-cold PBS, dissected into small fragments (1-2 mm), and four pieces were transferred to each well in a 24-well plate. The explants were cultured in DMEM with 10% FBS and 1% antibiotic-antimycotic, maintained at 5% CO2 and 8% 02 (mimicking the oxygenation state after 14 weeks of gestation), with fresh medium changed every 24 hours.

[0413] Activation of ferroptosis: Explants from normal pregnancies were cultured for 72 hours. On the fourth day, they were washed in PBS and transferred to F-12K medium with 1% FBS and 1% antibiotic-antimycotic. They were incubated for 24 hours with different concentrations of RSL3 with or without additional compounds.

[0414] Inhibition of spontaneous tissue ferroptosis: Explants from preeclamptic pregnancies were cultured for 48 hours. Fer-1, DFO, or DMSO (control) were added on the third day. On the fourth day, the explants were washed once in PBS and transferred to F-12K medium containing 1% FBS and 1% antibiotic-antimycotic. They were then incubated for another 24 hours with Fer-1, DFO, or DMSO.

[0415] At the end of the experiments, supernatant samples were collected, and placental pieces from each well were dried and weighed. EDH and sFET-1 release was calculated relative to the dry weight of the placental tissue (e.g., EDH measurement / dry weight).

[0416] Cell death assay

[0417] Cell death was measured using a colorimetric assay to quantify lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells (Roche, 11644793001). The assay was performed according to the manufacturer’s instructions. Sample OD was measured at 540 nm using the BIOTEK ELx8O8 Microplate Reader. sFlt-1 maturement

[0418] Cell culture supernatants were collected as described above, to analyze sFlt-1 release (R&D Systems, DY321B), and an enzyme-linked immunosorbent assay was performed, according to the manufacturer’s instructions.

[0419] Lipid peroxidation assay

[0420] Lipid peroxidation of BeWo cells was assessed using the fluorescent probe BODIPY (581 / 591) Cl l (Invitrogen, D3861). Briefly, following trypsinization, the cells washed twice using PBS xl in a conical 15mL tube. The pellet then resuspended in ImL of PBS xl containing final concentration of luM of BODIPY (581 / 591) Cl l and incubated for 30 minutes at 370C and 21% 02. Afterward, lOmL of F-12K-C was added and sent to another 10 minutes of incubation. Following the second incubation the cells were centrifuged at 1500 rpm for 5 minutes, resuspended in F-12K-C and 30,000 cells were seeded in a 48-well dish and incubated overnight at 370C, 21% 02 and 5% CO2. The desired treatments were applied during the next 24 hours and then subjected to flow cytometry following another 24 hr.

[0421] GSH / GSSG assay

[0422] The concentrations of total and oxidized glutathione (GSH and GSSG, respectively) in cultured BeWo cells were determined using the Colorimetric GSH Assay Kit (ab239709, Abeam), following the manufacturer's protocol.

[0423] Lentivirus and shRNA-Mediated Silencing

[0424] To establish stable silencing of GPX4 in BeWo cells, we employed MISSION shRNA plasmids (Sigma- Aldrich). We utilized lentiviral vectors pLKO.l, which were engineered to express either non-targeting shRNA (SHC016, Sigma- Aldrich) or shRNA specifically targeting human GPX4 (TRCN0000046251 , labeled as shGPX4, Sigma- Aldrich). These vectors were co-transfected with pMD2.G and psPAX2 plasmids (Addgene 12259 and 12260, respectively) into HEK293T cells for viral packaging, using TransIT-Lenti transfection reagent (Minis) for the process. Between 48 and 72 hours post-transfection, the resulting viral particles were collected from the culture supernatant via filtration using a 0.45-pm syringe filter, concentrated through centrifugation at 18,000 rpm for 2 hours, and preserved at -80°C. These prepared viral vectors, containing either the non-specific or GPX4-targeted shRNA, were then introduced into BeWo cells, facilitated by the addition of polybrene (8 pg / mL, Sigma- Aldrich) for one hour. Selection of successfully transduced cells was performed after 48 hours using puromycin (2 pg / mL, Sigma- Aldrich). Additionally, to mitigate any spontaneous ferroptosis, Ferrostatin-1 (0.5 pM) was incorporated into the cell culture.

[0425] High-throughput screening (HTS) procedure and CDP / CTG multiplex luminescence assay

[0426] Screen and hit validation experiments were performed using the HTS equipment: Echo555 Acoustic transfer system (Labcyte, Germany), Combi MultiDrop (Thermo Fischer Scientific), Washer Dispenser II (GNF, San Diego, CA, USA), Luminescence signals were measured by luminescence module of PheraStar FS plate reader (BMG Labtech, Ortenberg, Germany). Chemical libraries containing an overall 6520 compounds were purchased from Selleck Chemicals Bioactive, LOP AC (Sigma), Spectrum Collection (MicroSource) and Prestwick. All of these were screened by their ability to inhibit ferroptosis in BeWo cells, where cell viability was measured by CellTiter-Glo® (CTG) Luminescent Cell Viability Assay (G7572, Promega).

[0427] Briefly, 4000 cells / well were seeded for 24 hr in 50pl of growth medium into a white / white 384-well TC plate (Greiner, #781080) before treatments with the compounds or controls were applied followed by the addition of RSL3 (0.4 pM 1S-3R-RSL3, SML2234, Merck). After an additional 24 hr cells were subjected to CTG assay.

[0428] Phospholipidomics

[0429] Lipids were extracted using a modified Folch procedure under a nitrogen atmosphere, and lipid phosphorus was determined by a micromethod as previously described in Beharier et al. Liquid chromatography / electrospray ionization-mass spectrometry (LC / ESI-MS) analysis of lipids was conducted using a Dionex HPLC system coupled to an Orbitrap Fusion Lumos mass spectrometer. Phospholipids were separated on a normal phase column with gradient solvents, and data were acquired in data-dependent MS2 targeted-MS3 mode. All procedures followed the protocol detailed in Beharier et al.

[0430] Statistical Analysis

[0431] Statistical analyses for each experiment are detailed in the corresponding figure legends. Data are expressed as mean ± S.E.M. All analyses and visualizations were conducted using GraphPad Prism 7 software. Group comparisons were assessed using a two-tailed Student’s t-test or one-way ANOVA, followed by Dunnett’s or Bonferroni post hoc tests where appropriate. For placental explant experiments involving two conditions from specific donors, matched-pair analysis was employed. Statistical significance was defined as a P value of less than 0.05 Results

[0432] Example 1: Early-onset Preeclampsia Placentas Exhibit Ferroptosis

[0433] Quantitative redox phospholipidomics studies of placenta samples from early preeclampsia and gestational age-matched controls (preterm control) were performed.

[0434] As can be seen in Figures 1A-1C, the level of three phosphatidylethanolamine (PEA) molecules; PEA (36:4), PEA (38:4), and PEA (38:5), were significantly higher in placenta samples from preeclampsia women who experienced an early delivery (“Early PE”) versus placenta samples from women who experienced an early delivery but were not diagnosed with preeclampsia (“Early Cont”).

[0435] Based on these results, it was suggested that placental ferroptosis is abundant in preeclampsia.

[0436] Example 2: Placental ferroptosis positively regulates sflt-1 release

[0437] Ferroptosis was induced by using RAS-selective lethal 3 (RSL3), a selective Glutathione peroxidase 4 (GPX4) inhibitor.

[0438] A concentration-dependent increase in cell death (as indicated by LDH release to the medium) in response to RSL3 was observed in BeWo cells (Figure 2A)

[0439] As can be seen from Figure 2B, Ferrostatin -1 (Fer-1 or Fer a potent and selective ferroptosis inhibitor which acts as a radical scavenger and 15L0X inhibitor) inhibited LDH release placental explants obtained from normal pregnancies. As can be seen, in the presence of 0.8uM RSL and luM Fer, LDH release was inhibited.

[0440] As can be seen from the images in Figures 2C-2E, treatment of cells with RSL3 induced cell death (Figure 2D) which was inhibited upon addition of Fer-1 (Figure 2E).

[0441] These results show a dose-dependent activation of ferroptosis using RSL3 (as indicated by an increase by LDH release), and an inhibition of this increase by Fer-1 (200 nM).

[0442] Interestingly, placental ferroptosis induced by RLS-3 in placental explants obtained from normal pregnancies, was associated with a significant increase in medium sFLT-1 levels (Figure 3A) and this increase was blocked by addition of Fer-1 (that inhibits ferroptosis). sFLT-1 plays a significant role in the pathophysiology of preeclampsia and elevated placental sFLT-1 release is characteristic of preeclampsia (22-24).

[0443] Hence, based on the results in Figure 3A demonstrating that ferroptosis elevates placental sFlt-1 release in placental explants, a direct association between placental ferroptosis and sFlt-1 levels was suggested.

[0444] As can be seen in Figures 3B and 3C, Fer-1 and deferoxamine (DFO), an iron chelator that blocks ferroptosis, markedly lowered sFLT-1 secretion in preeclamptic placental explants.

[0445] These findings highlight the potential of targeting ferroptosis as a novel strategy for treating or preventing preeclampsia.

[0446] In other words, it is suggested that anti-ferroptosis compounds that reduce / inhibit sFLT-1 secretion may be considered as candidates for treatment of preeclampsia.

[0447] Example 3: Antioxidant drugs that attenuate rodent preeclampsia-like phenotypes inhibit trophoblast ferroptosis

[0448] Esomeprazole (ESO), Pravastatin (PRA), Metformin (MET), mitoquinone mesylate (MitoQ), and mitochondria (mito)-TEMPO (mTEMPO), were previously shown to inhibit / attenuate preeclampsia (in animal models of the disease), i.e. alleviate preeclampsia-like symptoms in vivo, and lower sFlt-1 secretion from the placenta. Each one of these compound acts via distinct mechanism of action.

[0449] In order to verify if these compounds, which were known to inhibit / attenuate preeclampsia, may also affect ferroptosis, ferroptosis was induced in different tested systems.

[0450] Figure 4A shows the effect of the tested drugs on RSL-3 induced ferroptosis in human trophoblast line, BeWo. As can be seen in Figure 4A, Fer, deferoxamine (DFO), a-Tocopherol (a-TOC), MitoQ, mTEMPO, and ESO inhibited RSL3-induced ferroptosis.

[0451] PRA and MET, which were reported to inhibit / attenuate preeclampsia had no effect on LDH release (Figure 4A).

[0452] A similar inhibition of LDH release was observed in BeWo cells in which ferroptosis was triggered by silencing GPX4 (Figure 4B). In addition, inhibition of LDH release was observed when ferroptosis was induced using RSL3 in placental explant from healthy pregnancy (Figure 4C).

[0453] As can be seen in Figures 4A and 4B, a-Tocopherol impedes ferroptosis in BeWo cells, however, this effect was not observed in human placental tissue (Figure 4C). The results show that ferroptosis cell death induced by RSL3, was reduced by compounds previously known to alleviate preeclampsia.

[0454] Interestingly, the concentrations in which ESO, MitoQ, and mTEMPO inhibited ferroptosis in Figures 4A were similar or even lower than the previously reported to attenuate sFLT-1 secretion in animal model (31, 32).

[0455] Hence, taken together these results show that some of the compounds which were reported to inhibit / attenuate preeclampsia also inhibit ferroptosis. This finding supports the connection between ferroptosis and preeclampsia. In addition, the fact that the tested compounds inhibited ferroptosis at concentrations which were previously shown to attenuate sFLT-1 secretion in animal model suggests the importance of ferroptosis in treating preeclampsia.

[0456] Hence, given the potential involvement of placental ferroptosis in preeclampsia pathophysiology, compounds which would inhibit placental ferroptosis, may offer a novel insight into therapeutic efficacy of preeclampsia.

[0457] Example 4: High-Throughput Screening for Drugs that Inhibit Trophoblast Ferroptosis.

[0458] To identify drugs that mimic Fer-1 activity, and can be safely used in human pregnancy, a HTS system was used. This system allows unbiased screening of a large library of drugs and compounds.

[0459] The CellTiter-Glo (CTG) viability assay, which measures intracellular ATP levels as an indicator of viability, detects ferroptosis and was used to monitor the extent of cell death in our HTS screening. Notably, the CTG assay detected a significant drop in ATP levels even in a sublethal dose of RSL3 (Figure 5A).

[0460] Increased sensitivity to RSL3 was noted in an assay medium composed of 1% fetal bovine serum (FBS), compared to media with 10% FBS (Figure SB).

[0461] Therefore, for the screening assays, a medium containing 1% FBS was used and ferroptosis was induced using 400 nM of RSL3.

[0462] Given the objective to identify drugs with potential clinical applicability, the screening protocol was tuned to detect ferroptosis inhibitors at a concentration of 1 pM (Figure 5C).

[0463] Using the RSL-induced cell death system, a total of 6520 compounds including drugs and hormones were screened. Figure 5D shows a schematic example of the screening protocol.

[0464] Table 1 shows a summary of the tested compounds.

[0465] Table 1: Summary of tested compounds sources

[0466] Among the compounds that were found to inhibit ferroptosis, several ferroptosis inhibitors were detected, while inhibitors of pyroptosis, apoptosis, and necroptosis did not rescue cells (Figure 5E).

[0467] A functionally coherent clustering among the modulatory compounds was detected that target known pathways that are implicated in ferroptosis regulation. These insights collectively confirm the ability of HTS to identify potent inhibitors of trophoblast ferroptosis.

[0468] From the tested compounds, 287 compounds were selected based on the % of ferroptosis inhibition.

[0469] Table 2 shows the list of 287 compounds that were found to inhibit BeWo ferroptosis (cut off >70% protection). Table 2: List of compounds that were found to inhibit BeWo ferroptosis

[0470]

[0471]

[0472]

[0473] Among the 287 compounds that were found to inhibit BeWo ferroptosis 56 are known drugs shown in Table 3.

[0474] Table 3: List of approved drugs that were found to inhibit BeWo ferroptosis

[0475]

[0476]

[0477]

[0478]

[0479] Example 5: Screen for Drugs that Inhibit Ferroptosis

[0480] The drug library was screened using the HTS described in Example 4 for ferroptosis-inhibitor medications that are potentially safe for use during pregnancy, based on available safety information. Among the 56 known drugs, 32 drugs are classified as FDA categories A-C for pregnancy treatment and are shown in Table 4. Details are as provided in Table 3.

[0481] Table 4: List of drugs are classified as FDA categories A-C for pregnancy treatment that were found to inhibit BeWo ferroptosis (compound # is as identified in Table 3)

[0482] After analyzing the ECso of various drugs, five drugs that exhibited high anti- ferroptotic activity were selected for further analysis.

[0483] These drugs included Dipyridamole (DPY) (denoted as compound #1 or SMC #1), Promethazine (PMZ) (denoted as compound #2 or SMC #2), Carvedilol (CRVD) (denoted as compound #3 or SMC #3), Lercanidipine (LRC) (denoted as compound #4 or SMC #4)and Amodiaquine (AMDQ) (denoted as compound #5 or SMC #5).

[0484] Table 5 shows the anti-ferroptotic bioactivity (EC50) of the selected drugs as determined in the cell viability assay of the HTS.

[0485] Table 5: EC50 values of the selected SMC

[0486] Figure 6A illustrates the dose-response relationship of each drug for inhibiting ferroptosis in BeWo cells.

[0487] The effective concentration ranges required for ferroptosis prevention as determined from Figure 6A are provided in Table 6.

[0488] Table 6: EC50 values of ferroptosis prevention

[0489] As can be seen from Table 6, Promethzaine and Dipyridamole were the most effective in inhibiting RSL3-induced ferroptosis at concentrations of 0.106 and 0.146 pM, respectively.

[0490] Cytotoxicity assays and cell morphology evaluations demonstrated that all positive candidates effectively prevented RSL3- and ML162-induced cell death (detected by LDH release), regardless of their diverse pharmacologic activities.

[0491] Specifically, as can be seen from Figure 6B, the small molecules compounds that were found in the HTS, reduced LDH release when tested in the presence of each one of the GPX4 inhibitors, ML162 and RSL-3. These results in BeWo cells indicated that the validity of the HTS assay and the compatibility of the compounds obtained by this assay. Figures 6C-6J demonstrated that the small molecules compounds prevented cell death.

[0492] In addition, the small molecules compounds inhibited ferroptosis induced by silencing of GPX4 using shRNA as can be see in Figure 6K.

[0493] The in vitro results were verified also in ex-vivo normal human placental tissue. As can be seen in Figs. 6L, LDH release induced by RSL3 was blocked by Fer-1 as well as by the SMCs obtained by the HTS assay.

[0494] Interestingly, the small molecules compounds did not protect against staurosporine-induced apoptosis (Figure 6M) suggesting that the cell death was not apoptosis.

[0495] These results indicated that the SMCs obtained by the HTS assay inhibit ferroptotic cell death induced by RSL3 in normal-term placental explants culture.

[0496] Intriguingly, Esomeprazole, which was previously identified as a potential novel drug for preeclampsia, was found to inhibit trophoblast ferroptosis, supporting the present hypothesis. Nevertheless, Esomeprazole concentrations that inhibit ferroptosis (lOOpM) were within an order of magnitude higher than conventional clinical treatment, and therefore Esomeprazole was not selected for further testing.

[0497] Further, these findings indicate that the protective effect of the drugs varies between two-dimensional cell cultures and three-dimensional, multicellular native placental tissues. In these experimental settings, it was found that dipyridamole and promethazine were particularly effective in protect the tissue at the lowest concentrations (Figure 6L).

[0498] Example 6: Assessment of the mechanism of action of Dipyridamole and Promethazine

[0499] Dipyridamole is a phosphodiesterase (PDE) inhibitor, mostly used as an anticoagulant due to its inhibitory effect on platelet activation and aggregation. As can be seen in Figure 7A, Sildenafil, Theophylline, and Triflusal that also block PDE, did not show any anti-ferroptotic activity.

[0500] Promethazine is a non-selective histamine receptor blocker. As can be seen in Figure 7B, Terfenadine, Loratadine, and Desloratadine, which also block these receptors, did not block trophoblast ferroptosis. These data suggest that the anti-ferroptosis effect of Dipyridamole and Promethazine, was not mediated through their known pharmacologic mechanism.

[0501] As can be seen in Figure 7C, an increase in lipid peroxidation in the cells exposed to RSL3 was observed, which was confirmed by the elevated B0DIPY-C11 fluorescence. When RSL3 was co-administered with either of Dipyridamole and Promethazine or Fer- 1, there was a significant reduction in B0DIPY-C11 fluorescence, indicating a decrease in lipid peroxide accumulation.

[0502] As shown in Figures 7D and 7E, while exposure to RSL3 led to a decrease in intracellular glutathione levels, co-treatment with Dipyridamole or Promethazine or Fer- 1 attenuated intracellular glutathione.

[0503] This observation suggests that Dipyridamole and Promethazine effectively counteract both lipid peroxidation and the consequent glutathione depletion.

[0504] Dipyridamole and Promethazine radical- scavenging potentials was tested using the Diphenyl- 1-picrylhydrazyl (DPPH) assay. Promethazine was effective in free-radical scavenging, that is less effective than vitamin E, while Dipyridamole did not exhibit direct radical-scavenging activity (data not shown).

[0505] Additionally, electron paramagnetic resonance (EPR) spectrometry was used to investigate the Fe-binding capacities of these drugs, focusing on their ability to suppress ascorbate radical formation, a hallmark of iron chelation. The results showed no detectable Fe-binding activity among the compounds, suggesting their anti-ferroptotic action is not mediated through iron chelation (data not shown).

[0506] Together, our findings demonstrate that the anti-ferroptotic Dipyridamole and Promethazine effectively mitigate lipid peroxidation and preserve glutathione levels, without affecting Fe-binding activity, underscoring their therapeutic potential against ferroptosis-driven pathologies.

[0507] Example 7: Dipyridamole and Promethazine Decrease sFlt-1 Secretion From Preeclamptic Placental Explants.

[0508] The therapeutic potential of Dipyridamole and Promethazine was tested in an ex vivo placental explant model with tissue from normal term pregnancies and from early preterm preeclamptic placentas. Figures 8A and 8B show results of the therapeutic potential of Dipyridamole and Promethazine in ex vivo placental explant model with tissue from normal term pregnancies treated with RSL3 to induce ferroptosis.

[0509] As can be seen in Figure 8A, Dipyridamole and Promethazine, like Fer-1, effectively inhibited ferroptosis induced by RSL3. In addition, as can be seen in Figure 8B, placental sFLT-1 secretion was blocked by Fer-1 as well as by Dipyridamole, and Promethazine, both identified in the HTS assay.

[0510] Hence, these results suggested that the compounds identified in the HTS assay inhibit ferroptotic cell death, reduce sFLT-1 secretion and can be used for treating preeclampsia.

[0511] The effect of Dipyridamole and Promethazine was studied in early preterm preeclamptic placentas.

[0512] Table 7 provides the clinical information on the women participating in this study. All women were diagnosed with early preeclampsia and delivered a baby by cesarean delivery.

[0513] Table 7: clinical information of women with early preeclampsia

[0514] Figures 8C and 8D show results of the therapeutic potential of Dipyridamole and Promethazine in early preterm preeclamptic placentas. Importantly, as can be seen in Figure 8C, in early preterm preeclamptic placentas, a decrease in LDH levels was observed in the presence of Fer-1 as well as by Dipyridamole, and Promethazine. These results suggest reduced tissue damage.

[0515] In addition, as can be seen Figure 8D, in early preterm preeclamptic placentas, sFLT-1 secretion was significantly attenuated by Fer-1 as well as Dipyridamole and Promethazine.

[0516] This highlights the potential of Dipyridamole and Promethazine as novel treatments for mitigating placental injury and sFLT-1 secretion in preeclampsia.

[0517] Example 8: Synergism assay

[0518] Example 8 A: Synergism assay - in vitro assay

[0519] Synergistic combinations of the six identified small molecules compounds from the HTS assay were tested.

[0520] Fig. 9A shows synergy data for combinations of Dipyridamole and a-Tocopherol, and Fig. 9B shows the Zero interaction potency (ZIP) model with the synergy score.

[0521] Fig. 9C shows synergy for combinations of Amodiaquine and dipyridamole, Fig. 9D shows synergy data for combinations of Carvedilol and dipyridamole, Fig. 9E shows synergy data for combinations of Lercanidipine and Dipyridamole, Fig. 9F shows synergy data for combinations of Promethazine and Dipyridamole, Fig. 9G shows synergy data for combinations of Amodiaquine and Promethazine, Fig. 9H shows synergy data for combinations of Carvedilol and Promethazine, Fig. 91 shows synergy data for combinations of Esomeprazole and Promethazine, Fig. 9J shows synergy data for combinations of Lercanidipine and Promethazine. Table 8 shows the ZIP score of these combinations. Table 8: ZIP score of drug combinations

[0522] As can be seen from Table 8, combinations comprising Dipyridamole - Promethazine, Promethazine - Amodiaquine, Dipyridamole - Amodiaquine and Dipyridamole and a-Tocopherol showed synergistic effect with the combination of Dipyridamole and a-Tocopherol showing the most significant synergistic effect.

[0523] Example 8B: Synergism assay - Placental explants

[0524] Biopsies for placental explant preparation (from normal patients) were thoroughly washed in ice-cold PBS, dissected into small fragments (1-2 mm), and four pieces were transferred to each well in a 24-well plate. The explants were cultured in DMEM with 10% FBS and 1% antibiotic-antimycotic, maintained at 5% CO2 and 8% 02 (mimicking the oxygenation state after 14 weeks of gestation), with fresh medium changed every 24 hours.

[0525] Activation and inhibition of ferroptosis: Explants from normal pregnancies were cultured for 72 hours. On the fourth day, they were washed in PBS and transferred to F- 12K medium with 1% FBS and 1% antibiotic-antimycotic. They were incubated for 24 hours with 0.8 uM of RSL3 with combinations of different concentrations of DPY and PMZ. At the end of the experiment, supernatant samples were collected, and placental pieces from each well were dried and weighed. LDH and sFLT-1 release was calculated relative to the dry weight of the placental tissue (e.g., LDH measurement / dry weight).

[0526] Figs. 10A and 10B show synergy data for combinations of Promethazine (PMZ) and Dipyridamole (DPY) in placental explants in which ferroptosis was induced by RSL3.

[0527] Figure 10A shows the synergistic effect of the combination on LDH level. As can be seen, the combination of PMZ and DPY induced a synergistic effect. The ZIP score of this combination was 15.78 indicating that the combination has a synergistic effect in reducing LDH level.

[0528] Figure 10B shows the synergistic effect of the combination on sFLT 1 secretion. As can be seen, the combination of PMZ and DPY induced a synergistic effect and that the effect is a negative synergistic effect.

[0529] The negative synergism suggests that PMZ and DPY interact such that the overall effect is diminished, producing a result that is lower / reduced than what each agent would achieve on its own. The ZIP score of this combination was -45.73 indicating that the combination has a synergistic effect in reducing sFLT 1 secretion.

[0530] Example 9: Characterization of drug transfer via the human placenta using the ex-vivo dually perfused cotyledon model

[0531] The ex-vivo dually human-perfused cotyledon model is used for the evaluation of placental clearance and the transfer of various endogenous compounds, pharmaceutical drugs under dynamic, near-physiological conditions. This ex vivo model, contains independent maternal and fetal perfusion circuits that individually maintain stable pH, temperature, flow rates, and perfusion pressure during the dual-perfusion process. The model takes advantage of the full complexity of the intact human term placental tissue and generally shows a good correlation with in vivo data. This model is used to characterize drug transfer via the human placenta. Placentas are obtained from cesarean deliveries will be used in order to evaluate drug transplacental transfer. Cotyledons will be cannulated and dually perfused. Drugs maternal-to fetal transfer will be evaluated by repeat sampling of maternal and fetal circuits. Drugs samples will be analyzed for drug concentrations (HPLC) in order to calculate maternal-fetal transport rate. Following isolation of human placental cotyledons, fetal arteryvein pair will be cannulated using an internal pressure monitor (series 50 IP-2; Philips, Ontario, Canada). The maternal flow will be established with blunt tipped needles inserted into the maternal tissue at the fetal pair site. Circuits will be closed, and perfused in a designated chamber for a 4 hours experiment. Perfusates will be equilibrated with a pre-humidified gas mixture of 95% 02 and 5% CO2 in the maternal reservoir and 95% N2 and 5% CO2 in the fetal reservoir. Drugs (in a concentration similar to that obtained in the standard treatment) and antipyrine (50pg / mL) will be added to the maternal circulation in the absence or the presence of the organic anion transporting polypeptide (OATPs), inhibitor rifampin (lOpg / mL), or the P-glycoprotein inhibitor valspodar (2pg / mL). Placental viability will be ensured by monitoring feto-placental pressure, pH, fetal reservoir volume, glucose and lactate concentrations, and the contraction response to lOOnM angiotensin II at the end of the experiment.

[0532] Example 10: Efficacy and safety of SMCs in animal models of PE.

[0533] The efficacy and safety of the SMCs is evaluated in two mouse models of PE: (i) reduced uterine perfusion pressure (RUPP) model in pregnant mice and (ii) uninephrectomized mouse model in pregnant outbred mice. End points evaluated will be a) plasma sFLT-1 levels and blood pressures during third trimester b) birth weight and litter sizes c) lipid markers of ferroptosis in placental samples.

[0534] RUPP model: on gestational day 13.5, mice will be anesthetized using 3% isoflurane. An abdominal incision will be made and the uterus containing the placental- fetal unit exteriorized. The arterial (Ovarian and Uterine) branches on both sides will be partially ligated with 6-0 silk suture. The ligation will be accomplished by blunt dissection, followed by a single suture tie around the artery with a monofilament thread alongside the vessel to maintain some (-50%) vessel patency.

[0535] Sham mice will be operated in a similar fashion, but without ligation. For the RUPP mice, blood pressures will be monitored via telemetry procedure. Uninephrectomy model of PE: Female CD1 mice weighing 20-25 grams (Charles River) will be randomly assigned to receive either Sham surgery or unilateral nephrectomy (UNx). At the time of nephrectomy procedure, these mice will also receive radiotelemetry probes suitable for chronic hemodynamic monitoring of blood pressure and heart rate.

Claims

CLAIMS:

1. A combination comprising at least two small molecule compounds (SMC) selected from the group consisting of:2,2',2",2"'-((4,8-di(piperidin-l-yl)pyrimido[5,4-d]pyrimidine-2,6- diyl)bis(azanetriyl))tetraethanol (designated herein as SMC / ),(RS)-N,N-Dimethyl-l-(10H-phenothiazin-10-yl)propan-2-amine (designated herein asSMC 2),(±)-[3-(9H-carbazol-4-yloxy)-2-hydroxypropyl][2-(2-methoxyphenoxy)ethyl]amine(designated herein as SMC 3),(RS)-2[(3,3-Diphenylpropyl)(methyl)amino]-l, 1 -dimethylethyl methyl 2,6-dimethyl-4-(3-nitrophenyl)-l,4-dihydropyridine-3,5-dicarboxylate (designated herein as SMC 4),4-[(7-chloroquinolin-4-yl)amino]-2-[(diethylamino)methyl]phenol (designated herein asSMC 5), and(2R)-2,5,7,8-Tetramethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]-3,4-dihydro-2H-l- benzopyran-6-ol (designated herein as SMC 60) or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

2. The combination according to claim 1 , comprising one or more of (i) SMC 1 and SMC 2, (ii) SMC 1 and SMC 5, (iii) SMC 1 and SMC 60, (iv) SMC 2 and SMC 5.

3. The combination according to claim 1 or 2 for use in inhibiting ferroptosis in a cell and / or reducing sFLT-1 secretion from a cell.

4. The combination for use according to claim 3, wherein said cell is a trophoblast cell, an endothelia cell, or a cancer cell.

5. The combination for use according to claim 4, wherein said cell is of a subject suffering from a disorder or condition associated with cell ferroptosis and / or increased sFLT-1 secretion.

6. The combination for use according to claim 5, wherein said disorder or condition is associated with vascular dysfunction and / or abnormal angiogenesis.

7. The combination for use according to claim 5, wherein said disorder or condition is an excitotoxic disorder.

8. The combination for use according to claim 5, wherein said disorder or condition is a neurodegenerative disease, a cardiovascular disease, cancer, a kidney disease, a liver disease, a respiratory disease, an inflammatory disease, an autoimmune disease, a skin disease, a gastrointestinal disease or any combination thereof.

9. The combination for use according to claim 5, wherein said disorder or condition is preeclampsia.

10. The combination according to claim 1 or 2 for use in at least one of (i) mitigating ferroptosis-induced death in trophoblasts, (ii) reducing sFLT-1 secretion from placental tissue, (iii) curbing excessive sFLT-1 triggered due to ferroptosis death or (iv) any combination thereof.

11. The combination for use according to claim 10, wherein said cell is a trophoblast cell.

12. The combination for use according to claim 11 , wherein said trophoblast cell is obtained from subject suffering from preeclampsia.

13. The combination for use according to any one of claims 3 to 12, for use in inhibiting placental ferroptosis in a subject in need thereof.

14. The combination of claim 1 or 2, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with cell ferroptosis and / or increased sFLT-1 secretion in a subject in need thereof.

15. The combination for use according to claim 14, wherein said disorder or conditionis associated with vascular dysfunction and / or abnormal angiogenesis.

16. The combination for use according to claim 14, wherein said disorder or condition is an excitotoxic disorder.

17. The combination for use according to claim 14, wherein said disorder or condition is a neurodegenerative disease, a cardiovascular disease, cancer, a kidney disease, a liver disease, a respiratory disease, an inflammatory disease, an autoimmune disease, a skin disease, a gastrointestinal disease or any combination thereof.

18. The combination for use according to claim 14, wherein said disorder or condition is preeclampsia.

19. The combination of claim 1 or 2, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of preeclampsia in a subject in need thereof.

20. A pharmaceutical composition comprising a therapeutically effective amount of the combination according to claim 1 or 2, and wherein said composition optionally further comprises at least one pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s.

21. A method for inhibiting ferroptosis in a cell and / or reducing sFLT-1 secretion from a cell, said method comprising the step of contacting said cell with an effective amount of the combination according to claim 1 or 2 to thereby inhibit ferroptosis and / or reduce sFLT-1 secretion.

22. A method for at least one of (i) reducing ferroptosis-induced death in a trophoblast cell, (ii) reducing sFLT-1 secretion from placental tissue, (iii) curbing (reducing / inhibiting) excessive sFLT-1 triggered due to ferroptosis death or (iv) any combination thereof, said method comprising the step of contacting said cell with an effective amount of the combination according to claim 1 or 2 to thereby inhibit ferroptosis and / or reduce sFLT-1 secretion.

23. The method according to claim 22, wherein said cell is obtained from placental explant.

24. The method according to claim 22 or 23, wherein said cell is of a subject sufferingfrom preeclampsia.

25. The method according to any one of claims 22 to 24, for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of preeclampsia in a subject in need thereof.

26. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with increased sFLT-1 secretion and / or cell ferroptosis in a subject in need thereof, said method comprises administering to said subject a therapeutically effective amount of the combination according to claim 1 or 2.

27. The method according to claim 26, wherein said disorder or condition is preeclampsia.

28. Use of a combination according to claim 1 or 2 in the preparation of a composition for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with increased sFLT-1 secretion and / or cell ferroptosis in a subject in need thereof.

29. A ferroptosis inhibitor for use in a method for inhibiting secretion of sFLT-1 from a cell.

30. The ferroptosis inhibitor for use according to claim 29, wherein said cell is a trophoblast cell, an endothelia cell, or a cancer cell.

31. The ferroptosis inhibitor for use according to claim 30, wherein said cell is a trophoblast cell.

32. The ferroptosis inhibitor for use according to claim 29, wherein said cell is of a subject suffering from a disorder or condition associated with cell ferroptosis and / or increased sFLT-1 secretion.

33. The ferroptosis inhibitor for use according to 32, wherein said disorder or condition is associated with vascular dysfunction and / or abnormal angiogenesis.

34. The ferroptosis inhibitor for use according to claim 32, wherein said disorder or condition is an excitotoxic disorder.

35. The ferroptosis inhibitor for use according to claim 32, wherein said disorder or condition is a neurodegenerative disease, a cardiovascular disease, cancer, a kidney disease, a liver disease, a respiratory disease, an inflammatory disease, an autoimmune disease, a skin disease, a gastrointestinal disease or any combination thereof.

36. The ferroptosis inhibitor for use according to claim 32, wherein said disorder or condition is preeclampsia.

37. The ferroptosis inhibitor for use according to any one of claims 29 to 36, being one or more compound presented in Table 4.

38. The ferroptosis inhibitor for use according to any one of claims 29 to 37, being one or more compound presented in Table 5.

39. The ferroptosis inhibitor for use according to any one of claims 29 to 38, being at least one of SMC 1, SMC 2, SMC 3, SMC 4, SMC 5 or any combination thereof.

40. The ferroptosis inhibitor for use according to any one of claims 29 to 39, comprising one or more combination (i) SMC 1 and SMC 2, (ii) SMC 1 and SMC 5, (iii) SMC 2 and SMC 5.

41. The ferroptosis inhibitor for use according to any one of claims 29 to 40, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease associated with increased sFLT-1 secretion in a subject in need thereof.

42. The ferroptosis inhibitor for use according to any one of claims 29 to 40, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of preeclampsia in a subject in need thereof.

43. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with increased sFLT-1 secretion and / or cell ferroptosis in a subject in need thereof, said method comprises administering to said subject a therapeutically effective amount of at least one ferroptosis inhibitor.

44. The method according to claim 43, wherein said disorder or condition is associated with vascular dysfunction and abnormal angiogenesis.

45. The method according to claim 43, wherein said disorder or condition is an excitotoxic disorder.

46. The method according to claim 43, wherein said disorder or condition is a neurodegenerative disease, a cardiovascular disease, cancer, a kidney disease, a liver disease, a respiratory disease, an inflammatory disease, an autoimmune disease, a skin disease, a gastrointestinal disease or any combination thereof.

47. The method according to claim 43, wherein said disorder or condition is preeclampsia.

48. The method according to any one of claims 43 to 47, wherein said ferroptosis inhibitor is one or more compound presented in Table 4.

49. The method according to any one of claims 43 to 47, wherein said ferroptosis inhibitor is one or more compound presented in Table 5.

50. The method according to any one of claims 43 to 49, wherein said ferroptosis inhibitor is at least one of SMC 1, SMC 2, SMC 3, SMC 4, SMC 5 or any combination thereof.

51. The method according to any one of claims 43 to 50, wherein said ferroptosis inhibitor is one or more combination (i) SMC 1 and SMC 2, (ii) SMC 1 and SMC 5, (iii) SMC 2 and SMC 5.

52. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying preeclampsia in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of at least one ferroptosis inhibitor.

53. Use of at least one ferroptosis inhibitor in the preparation of a composition for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disorder or condition associated with increased sFLT-1 secretion and / or cell ferroptosis in a subject in need thereof.

54. Use of at least one ferroptosis inhibitor in the preparation of a composition for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying preeclampsia in a subject in need thereof.

55. A screening method for identifying at least one modulator of sFLT-1 secretion, the method comprising the steps of:(a) determining in at least one cell contacted with a candidate compound: at least one of: (i) ferroptosis; (ii) lipid peroxide accumulation; (iii) glutathione depletion;(b) determining that the candidate compound is capable of at least one of: (i) inhibiting ferroptosis; (ii) inhibiting lipid peroxide accumulation; (iii) inhibiting glutathione depletion.

Images