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Lipid peroxidation Amino acid and protein modifications Glycation products Endogenous DNA damage

Lipid peroxidation Amino acid and protein modifications Glycation products Endogenous DNA damage. HO -mediated (oxidation) NO -mediated (nitration) HOCl -mediated (halogenation). Oxidative stress markers in the skin. Anti-oxidants Vitamin E (Tocopherol) Ascorbic acid Uric acid

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Lipid peroxidation Amino acid and protein modifications Glycation products Endogenous DNA damage

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  1. Lipid peroxidation Amino acid and protein modifications Glycation products Endogenous DNA damage • HO -mediated (oxidation) • NO -mediated (nitration) • HOCl -mediated (halogenation) Oxidative stress markers in the skin

  2. Anti-oxidants Vitamin E (Tocopherol) Ascorbic acid Uric acid Glutathione Thioredoxin SOD 2O2¯ + 2H+ O2 + H2O2 Catalase H2O2 + H2O2 2 H2O + O2 Natural skin defenses

  3. EUK-134 Oxidative stress and skin aging • PRODUCT: EUK-134 compound • DESCRIPTION: SOD / Catalase mimetic • FEATURES AND BENEFITS: • Protect against lipid peroxidation • Superoxide dismutase / Catalase activity • Anti-oxidant activity • Protect against UVB-induced DNA damage • Prevention of sun damage

  4. EUK-134 • 1- Mechanism of action • SOD activity of EUK-134 • Catalase activity of EUK-134 • Self regenerating property • 2- Chain reaction caused by UV exposure (UV damages) • 3- In vitro tests • Prevention of UV-induced DNA damage • Reduction of UV-stimulated p53 production • Improvement of keratinocytes survival • Prevention of UV-induced cell death (sunburn cells) • 4- Clinical trials • Reduction of UV-induced erythema • Prevention of UV-induced lipid peroxidation • 5- Potential applications and formulations

  5. EUK-134 • 1- Mechanism of action • SOD activity of EUK-134 • Catalase activity of EUK-134 • Self regenerating property • 2- Chain reaction caused by UV exposure (UV damages) • 3- In vitro tests • Prevention of UV-induced DNA damage • Reduction of UV-stimulated p53 production • Improvement of keratinocytes survival • Prevention of UV-induced cell death (sunburn cells) • 4- Clinical trials • Reduction of UV-induced erythema • Prevention of UV-induced lipid peroxidation • 5- Potential applications and formulations

  6. EUK-134 Oxidative stress and skin aging • ACTIVITY: Mimics the activity of two natural skin enzymes • - SOD • - Catalase • Superoxide dismutase (SOD): • O2− + H2O → H2O2 • Catalase: • H2O2→ H2O + O2

  7. EUK-134 Oxidative stress and skin aging Mechanism of action Catalase SOD O2− + H2O H2O2 H2O + O2 Skin Damage No Skin Damage

  8. EUK-134 Oxidative stress and skin aging Mechanism of action Catalase SOD O2− + H2O H2O2 H2O + O2 Skin Damage No Skin Damage

  9. EUK-134 Oxidative stress and skin aging Mechanism of action Catalase SOD O2− + H2O H2O2 H2O + O2 Skin Damage No Skin Damage

  10. H O 2 2 Superoxide dismutase activity assay NBT xanthine oxidase O NBT * xanthine 2- SOD Control + SOD Absorbance (NBT*) Time

  11. 0 8 5 0.7 3 2.8 0 0 60 120 EUK-134 is a catalytic superoxide scavenger EUK-134 (nmoles) 0.1 NBT Reduced (nmoles) 1.4 Time (sec) 2O2– + 2H+→ O2 + H2O2

  12. EUK-134 Oxidative stress and skin aging Mechanism of action Catalase SOD O2− + H2O H2O2 H2O + O2 Skin Damage No Skin Damage

  13. 8 7 6 5 4 3 2 1 0 30 40 50 60 70 EUK-134 exhibits catalase activity ) 5 [Oxygen] (M x 10 2 Time (sec) H2O2 + H2O2→ 2 H2O + O2

  14. EUK-134 exhibits catalase activity H2O2 + H2O2→ 2 H2O + O2

  15. Effect of UVA on the in vitro catalase activity of EUK-134

  16. EUK-134 Oxidative stress and skin aging Mechanism of action Catalase SOD O2− + H2O H2O2 H2O + O2 Skin Damage No Skin Damage

  17. EUK-134 Mechanisms of action Self regenerating property Dismutation of O2− Mn(III) + O2−→ Mn(II) + O2 Reduction of Mn (III) to Mn (II) 2H+ +Mn(II) + O2− → Mn(III) + H2O2 Mn (II) oxidized to Mn (III) Scavenging of H2O2 Mn(III) + H2O2 → Mn(V)O2− + H2O Oxidized into oxoMn-salen by H2O2 Mn(V)O2− + H2O2 → Mn(III) + H2O + O2 OxoMn-salen reduced to Mn (III)

  18. EUK-134 Mechanisms of action Self regenerating property Dismutation of O2− Mn(III) + O2−→ Mn(II) + O2 Reduction of Mn (III) to Mn (II) 2H+ +Mn(II) + O2− → Mn(III) + H2O2 Mn (II) oxidized to Mn (III) Scavenging of H2O2 Mn(III) + H2O2 → Mn(V)O2− + H2O Oxidized into oxoMn-salen by H2O2 Mn(V)O2− + H2O2 → Mn(III) + H2O + O2 OxoMn-salen reduced to Mn (III)

  19. EUK-134 Oxidative stress and skin aging Aging Stress UV exposure Pollution H2O, O2 O2- O2 Catalase SOD H2O2 Cell damages Signs of aging

  20. EUK-134 Oxidative stress and skin aging Aging Stress UV exposure Pollution H2O, O2 O2- O2 EUK-134 (Catalase) EUK-134 (SOD) H2O2 EUK-134 possesses the double action SOD–catalase Cell damages Signs of aging

  21. EUK-134 • 1- Mechanism of action • SOD activity of EUK-134 • Catalase activity of EUK-134 • Self regenerating property • 2- Chain reaction caused by UV exposure (UV damages) • 3- In vitro tests • Prevention of UV-induced DNA damage • Reduction of UV-stimulated p53 production • Improvement of keratinocytes survival • Prevention of UV-induced cell death (sunburn cells) • 4- Clinical trials • Reduction of UV-induced erythema • Prevention of UV-induced lipid peroxidation • 5- Potential applications and formulations

  22. Transmission of • damaged DNA • Cell mutation UV-induced damages UV radiation ROS ROS Protein oxidation DNA damage MMP synthesis Lipid peroxidation MMP activation Arachidonic acid ↑ p53 ↑ Photoaging Pro-inflammatory cytokines Inhibition of cyclin-dependent kinase DNA repair Apoptosis

  23. EUK-134 • 1- Mechanism of action • SOD activity of EUK-134 • Catalase activity of EUK-134 • Self regenerating property • 2- Chain reaction caused by UV exposure (UV damages) • 3- In vitro tests • Prevention of UV-induced DNA damage • Reduction of UV-stimulated p53 production • Improvement of keratinocytes survival • Prevention of UV-induced cell death (sunburn cells) • 4- Clinical trials • Reduction of UV-induced erythema • Prevention of UV-induced lipid peroxidation • 5- Potential applications and formulations

  24. EUK-134 • 1- Mechanism of action • SOD activity of EUK-134 • Catalase activity of EUK-134 • Self regenerating property • 2- Chain reaction caused by UV exposure (UV damages) • 3- In vitro tests • Prevention of UV-induced DNA damage • Reduction of UV-stimulated p53 production • Improvement of keratinocytes survival • Prevention of UV-induced cell death (sunburn cells) • 4- Clinical trials • Reduction of UV-induced erythema • Prevention of UV-induced lipid peroxidation • 5- Potential applications and formulations

  25. Transmission of • damaged DNA • Cell mutation UV-induced damages UV radiation ROS EUK-134 ROS Protein oxidation EUK-134 DNA damage Lipid peroxidation MMP synthesis MMP activation Arachidonic acid ↑ p53 ↑ Photoaging Pro-inflammatory cytokines Inhibition of cyclin-dependent kinase (G1) DNA repair Apoptosis

  26. UVB survival of keratinocytes treated with EUK-134 In Vitro Study • HEK incubated in 96-well plates • Incubation in the presence of EUK-134; 100 µM • UVB exposure to 0 or 60 mJ/cm2 • Neutral red assay; absorbance

  27. 67% protection UVB survival of keratinocytes treated with EUK-134 In Vitro Study

  28. EUK-134 • 1- Mechanism of action • SOD activity of EUK-134 • Catalase activity of EUK-134 • Self regenerating property • 2- Chain reaction caused by UV exposure (UV damages) • 3- In vitro tests • Prevention of UV-induced DNA damage • Reduction of UV-stimulated p53 production • Improvement of keratinocytes survival • Prevention of UV-induced cell death (sunburn cells) • 4- Clinical trials • Reduction of UV-induced erythema • Prevention of UV-induced lipid peroxidation • 5- Potential applications and formulations

  29. Transmission of • damaged DNA • Cell mutation Multiple actions of EUK-134 UV radiation ROS ROS Protein oxidation DNA damage MMP synthesis Lipid peroxidation MMP activation EUK-134 p53 ↑ Arachidonic acid ↑ Photoaging Pro-inflammatory cytokines Inhibition of cyclin-dependent kinase DNA repair Apoptosis

  30. Effects of EUK-134 on p53 protein levels In Vitro Study • NHK incubated in the presence of EUK-134 • UVB exposure (310 nm); 12 mJ/cm2 • NHK harvested at 6 hours post-UVB • Western blot analysis • p53 immunodetection (DO-1)

  31. Effects of EUK-134 on p53 protein levels In Vitro Study Fold induction compared to 0 hr 12 mJ/cm², 6h

  32. Effects of EUK-134 on p53 protein levels • Reduction of the universal sensor of cellular damage; p53 • Protection of the membrane oxidative damage • Protection of UV-induced DNA damage

  33. Effects of EUK-134 on p53 protein levels • EUK-134 • of skin cells oxidative damage • of UV-induced DNA damage • p53 level

  34. EUK-134 • 1- Mechanism of action • SOD activity of EUK-134 • Catalase activity of EUK-134 • Self regenerating property • 2- Chain reaction caused by UV exposure (UV damages) • 3- In vitro tests • Prevention of UV-induced DNA damage • Reduction of UV-stimulated p53 production • Improvement of keratinocytes survival • Prevention of UV-induced cell death (sunburn cells) • 4- Clinical trials • Reduction of UV-induced erythema • Prevention of UV-induced lipid peroxidation • 5- Potential applications and formulations

  35. Multiple actions of EUK-134 UV radiation ROS ROS Protein oxidation EUK-134 DNA damage Lipid peroxidation MMP synthesis MMP activation p53 ↑ Arachidonic acid ↑ Pro-inflammatory cytokines Photoaging Inhibition of cyclin-dependent kinase • Transmission of • damaged DNA • Cell mutation DNA repair Apoptosis EUK-134

  36. Effects of EUK-134 on TT dimer removal In Vitro Study • HEK incubated in 100 mm plates • Incubation in the presence of EUK-134; 50 µM • UVB exposure 0 to 8 mJ/cm2 • Cells harvested after 0/24 hour time point • DNA damage / DNA repair • Dot blot assay for TT dimers

  37. 45%↓ % protection from UVB-induced TT dimer formation with EUK-134 treatment In Vitro Study Cells harvested after 0 hour time point (right after UV exposure) to measure DNA damage % protection from UVB-induced TT dimer formation compared to non- treated NHEK UVB (8 mJ/cm2)

  38. 77% % TT dimer removal in UVB-irradiated NHEK In Vitro Study Cells harvested after 24 hour time point (24 hrs after UV exposure) to measure DNA repair % TT dimer removal in NHEK % TT dimer removal compared to 0 hr UVB (8 mJ/cm²)

  39. Effects of EUK-134 on DNA damage and repair • Reduction of UVB-induced TT dimer • Improved the removal of TT dimer • reduction of UVB-induced DNA damage • increase DNA repair capabilities

  40. EUK-134 • 1- Mechanism of action • SOD activity of EUK-134 • Catalase activity of EUK-134 • Self regenerating property • 2- Chain reaction caused by UV exposure (UV damages) • 3- In vitro tests • Prevention of UV-induced DNA damage • Reduction of UV-stimulated p53 production • Improvement of keratinocytes survival • Prevention of UV-induced cell death (sunburn cells) • 4- Clinical trials • Reduction of UV-induced erythema • Prevention of UV-induced lipid peroxidation • 5- Potential applications and formulations

  41. Multiple actions of EUK-134 UV radiation ROS ROS Protein oxidation EUK-134 DNA damage Lipid peroxidation MMP synthesis MMP activation p53 ↑ Arachidonic acid ↑ Photoaging Pro-inflammatory cytokines Inhibition of cyclin-dependent kinase • Transmission of • damaged DNA • Cell mutation DNA repair EUK-134 Apoptosis EUK-134

  42. UVB-induced sunburn cell formation in EUK-134 treated skin equivalents In Vitro Study • Skin equivalents • Pretreatment with 50 µM EUK-134; 6 hrs • UVB exposure • Cell staining with H&E 24 hours after UVB

  43. 61% protection UVB-induced sunburn cell formation in EUK-134 treated skin equivalents In Vitro Study Number of sunburn cells in selected field UVB 100 mJ/cm²

  44. EUK-134 • 1- Mechanism of action • SOD activity of EUK-134 • Catalase activity of EUK-134 • Self regenerating property • 2- Chain reaction caused by UV exposure (UV damages) • 3- In vitro tests • Prevention of UV-induced DNA damage • Reduction of UV-stimulated p53 production • Improvement of keratinocytes survival • Prevention of UV-induced cell death (sunburn cells) • 4- Clinical trials • Reduction of UV-induced erythema • Prevention of UV-induced lipid peroxidation • 5- Potential applications and formulations

  45. Transmission of • damaged DNA • Cell mutation Multiple actions of EUK-134 UV radiation ROS ROS Protein oxidation DNA damage MMP synthesis Lipid peroxidation MMP activation p53 ↑ Arachidonic acid ↑ Photoaging Pro-inflammatory cytokines EUK-134 Inhibition of cyclin-dependent kinase DNA repair Apoptosis

  46. Effects of EUK-134 on UV-induced erythema Clinical Trial • Nine volunteers, skin type I-III • UVB exposure range 280-320 nm • EUK-134 (0.05%) applied 2X/day; 3 days • 2 MED of UVB radiation • Erythema measured after 24 hours

  47. 46% Effects of EUK-134 on UV-induced erythema Clinical Trial Reduction of UV-induced erythema with EUK-134 Percent (%) reduction

  48. Transmission of • damaged DNA • Cell mutation Multiple actions of EUK-134 UV radiation ROS EUK-134 ROS Protein oxidation DNA damage MMP synthesis Lipid peroxidation MMP activation p53 ↑ Arachidonic acid ↑ Photoaging Pro-inflammatory cytokines Inhibition of cyclin-dependent kinase DNA repair Apoptosis

  49. EUK-134 prevents UVA-induced skin lipid peroxidation Clinical Trial • Ten volunteers, skin type II-IV • EUK-134 0.05% applied inner forearm • 1 hour before UVA exposure 1 J/cm2 • right after UVA exposure 1 J/cm2 • Squalene peroxide vs residual squalene (nmol/ml)

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