Redox, Kinetic, and Biological Necessities to Create an Effective Metalloenzyme-mimetic James D. Crapo, M.D.

1. Sunrise Free Radical School Redox, Kinetic, and Biological Necessities to Create an Effective Metalloenzyme-mimetic James D. Crapo, M.D.

2. Free Radical-Mediated Pathologies • Normal Metabolism, Aging • Chemical • Hyperoxia • Ischemia-Reperfusion • Inflammation • Autoimmune • Cancer

3. Antioxidants Efficiency a Tocopherol Non Enzymatic Ascorbate 1 β Carotene NAC Enzymatic Superoxide Dismutases Catalase 1,000 – 10,000 Metalloenzyme Mimetics 1,000 – 10,000 Mimetics

4. + N N N N N + Mn N N N N N N O O + M n Mn + + N N N N N N C H O O C H 3 3 Salen Mimetic Macrocylic Mimetic [EUK-134] [M-40403] N N + meso-Porphyrin Mimetic Several Classes of Catalytic Antioxidants

5. Metalloenzyme Mimetic

7. The Redox Potentials for the Half Reactions of the Dismutation of Superoxide and Superoxide Dismutases E 1/2 (NHE) -0.33 +0.94 +0.3 O2-. O2 + e- O2-. + 2H+ + e- H2O2 MnSOD CuZnSOD 2O2-. + 2H+ H2O2

8. Mn TBAP

9. Metalloporphyrin Antioxidant MimeticsEfficacy of First Generation - TBAP Cardiovascular Cardiomyopathy Zymoson-Induced Shock Joint Carregeenin Paw Edema Lung Paraquat Injury Carregeenin Inflammation Bleomycin-Induced Fibrosis CNS Kainate-Induced Seizures Cerebral Vasoconstriction Spinal Cord Injury Liver Ischemia-Reperfusion Steatosis Acetaminophine Injury Fas-Mediated Acute Injury

10. R N N + Mn R R N N R Development of an Antioxidant Mimetic Modify side chains Modify charge Modify redox potential Alter backbone Change metal Aeol-10150

11. Metals • Manganese • Iron • Copper • Cobalt • Nickel

12. Mn TM-4-PyP

13. The Redox Potentials for the Half Reactions of the Dismutation of Superoxide and Superoxide Dismutases E 1/2 (NHE) -0.33 +0.94 -0.23 +0.06 +0.3 O2-. O2 + e- O2-. + 2H+ + e- H2O2 MnTMPyP MnSOD CuZnSOD MnTBAP 2O2-. + 2H+ H2O2

14. + R , , , = N C H 1 2 3 4 3 R R 2 1 N R , , , = 1 2 3 4 N Mn N N + C H 3 N R R 3 4 R , , , = 1 2 3 4 N + H C 3 The “Ortho Effect” SOD Activity units/mg 225 336 10,648

15. N + N N + N + Mn N + N N + N MN TE-2-PyP Molecular Formula: C48H56N8Cl5Mn Molecular Weight: 977 5Clˉ

16. Antioxidant Activities Lipid ONOO SOD peroxidation scavenger Catalase (U/mg) IC50 (M) (M-1S-1) % activity CuZn SOD 5,100 15 — — Mn TBAP 179 29 3.0x105 0.42 Mn TM-4-PyP 550 16 1.8x108 0.45 MnTE-2-PyP 8,500 1 1.0x107 1.41

17. MnTM-2,5-IP

18. Antioxidant Activities Lipid ONOO SOD peroxidation scavenger Catalase (U/mg) IC50 (M) (M-1S-1) % activity CuZn SOD 5,100 15 — — Mn TBAP 179 29 3.0x105 0.42 Mn TM-4-PyP 550 16 1.8x108 0.45 MnTE-2-PyP 8,500 1 1.0x107 1.41 MnTM-2,5-IP 14,800 1 1.0x106 1.67

19. The Redox Potentials for the Half Reactions of the Dismutation of Superoxide and Superoxide Dismutases E 1/2 (NHE) MnTE-2-PyP MnTM-2,5-IP -0.33 +0.94 -0.23 +0.06 +0.3 +0.23 +0.33 O2-. O2 + e- O2-. + 2H+ + e- H2O2 MnTMPyP MnSOD CuZnSOD MnTBAP 2O2-. + 2H+ H2O2

20. Antioxidant Properties of Metalloenzyme Mimetics • Attenuate O2- Mediated Injury • Attenuate H2O2 Mediated Injury • Prevent Formation of Lipid Peroxides • Scavenge ONOO-

21. Pharmacokinetics • Route • Uptake • Distribution • Half Life • Plasma • Tissue

22. Mouse Plasma Concentrations ofMnTM-2,5-IP (iv bolus) 100000 T ~44 minutes 1/2 10000 1000 MnTM-2,5-IP (ng/ml) 30mg/kg 100 10mg/kg 3mg/kg 10 1mg/kg 1 0 1 2 3 4 5 6 7 Hours

23. MnTM-2,5-IP Steady-State From Mini-Osmotic Pump (1.12 mg/kg loading dose followed by 1.8 mg/kg/hr infusion for 24 hours) Steady-State 1000000 (ng/g or ng/ml) kidney 34,100 liver 16,300 10000 MnTM-2,5-IP serum 2,000 (ng/g or ng/ml) lung 1,600 heart 1,000 100 brain 90 1 0 4 8 12 16 20 24 Hours

24. MnTM-2,5-IP Clearance from Mini-Osmotic Pump(1.12 mg/kg loading dose followed by 1.8 mg/kg/hr infusion for 24 hours) 1000000 estimated half-life (hrs) kidney 140 10000 liver 136 MnTM-2,5-IP (ng/g or ng/ml) lung 83 heart 96 100 brain 49 serum 8 1 0 24 48 72 96 Hours

25. Pharmacokinetics of MnTM-2,5-IP in Rats(24 mg/kg, SC) T ~ 3 hrs 100000 1/2 Time to peak ~ 6 hrs Effective dosing interval ~ 9 hrs Plasma MnTM-2,5-IP (ng/ml) 10000 1000 0 2 4 6 8 10 12 14 Hours

26. Toxicity • MTD • Organ specific • Mutagenicity • Cardiovascular

27. MnTM-2,5-IPToxicology

28. Mechanisms • Antioxidant - targeted

35. EC-SOD in Large Elastic Pulmonary Artery 50 μ

36. EC-SOD in Muscular Pulmonary Artery 50 μ

37. EC-SOD in Small Arteriole

38. Immunolocalization of EC-SOD

41. CuZn SOD Concentrations in Hepatocyte Organelles Organelles mg SOD/cm3 Nucleus 0.71 ± 0.06 Cytoplasmic Matrix 1.36 ± 0.30 Mitochondria 0.21 ± 0.01 RER 0 SER 0.02 ± 0.01 Golgi Apparatus 0 Lysosomes 5.81 ± 1.55 Peroxisomes 0.27 ± 0.08

42. Distribution of CuZn SOD Molecules in Hepatocyte Organelles Organelles # μm3 Nucleus 13,300 Cytoplasmic Matrix 25,500 Mitochondria 3,900 SER 400 Lysosomes 108,900 Peroxisomes 5,000

43. 800 700 600 500 400 Units/g lung 300 200 100 0 CuZn SOD Mn SOD EC-SOD Human SOD Total Activity in Lung

44. 50 45 40 35 30 Units/cm3 tissue 25 20 15 10 5 0 CuZn SODin Cells MnSOD inMitochondria EC-SOD inInterstitium SOD Activities in Specific Compartments

45. 2500 9000 8000 2000 7000 6000 1500 Units/g wet weight 5000 Units/g wet weight 4000 1000 3000 500 2000 1000 0 0 Liver Kidney Heart Brain Lung Liver Kidney Brain Heart Lung Human Human A B CuZn SOD Mn SOD

46. EC-SOD 600 500 400 Units/g wet weight 300 200 100 0 Liver Kidney Heart Brain Lung Human

47. Estimated AOE in 70 Kg Human • CuZn SOD – 10-20 gm • Mn SOD 5-10 gm • EC-SOD 1-2 gm

48. Mechanisms • Antioxidant - targeted • NFκB inhibition

49. NF-κB • Nuclear Factor-Kappa B • First discovered as an enhancer of B cells (Sen & Baltimore 1986, Cell) • Ubiquitous transcription factor • Shown to be involved in cancer, immune response, redox regulation, apoptosis

50. NF-κB Pathway Adapted from: www.emdbiosciences.com/html/CBC/NFKB_NFkappaB_IKB_IKK_Pathway_Products.htm