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Sunrise Free Radical School. Redox, Kinetic, and Biological Necessities to Create an Effective Metalloenzyme-mimetic James D. Crapo, M.D. Free Radical-Mediated Pathologies. Normal Metabolism, Aging Chemical Hyperoxia Ischemia-Reperfusion Inflammation Autoimmune Cancer. Antioxidants.

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slide1

Sunrise Free Radical School

Redox, Kinetic, and Biological Necessities to Create an Effective Metalloenzyme-mimetic

James D. Crapo, M.D.

free radical mediated pathologies
Free Radical-Mediated Pathologies
  • Normal Metabolism, Aging
  • Chemical
  • Hyperoxia
  • Ischemia-Reperfusion
  • Inflammation
  • Autoimmune
  • Cancer
antioxidants
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

slide4

+

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

slide7

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

metalloporphyrin antioxidant mimetics efficacy of first generation tbap
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

slide10

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

metals
Metals
  • Manganese
  • Iron
  • Copper
  • Cobalt
  • Nickel
slide13

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

slide14

+

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

slide15

N

+

N

N

+

N

+

Mn

N

+

N

N

+

N

MN TE-2-PyP

Molecular Formula:

C48H56N8Cl5Mn

Molecular Weight:

977

5Clˉ

slide16

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

slide18

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

slide19

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

slide20

Antioxidant Properties of Metalloenzyme Mimetics

  • Attenuate O2- Mediated Injury
  • Attenuate H2O2 Mediated Injury
  • Prevent Formation of Lipid Peroxides
  • Scavenge ONOO-
pharmacokinetics
Pharmacokinetics
  • Route
  • Uptake
  • Distribution
  • Half Life
    • Plasma
    • Tissue
mouse plasma concentrations of mntm 2 5 ip iv bolus
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

slide23
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

slide24
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

pharmacokinetics of mntm 2 5 ip in rats 24 mg kg sc
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

toxicity
Toxicity
  • MTD
  • Organ specific
  • Mutagenicity
  • Cardiovascular
mechanisms
Mechanisms
  • Antioxidant - targeted
cuzn sod concentrations in hepatocyte organelles
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

distribution of cuzn sod molecules in hepatocyte organelles
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

slide43

800

700

600

500

400

Units/g lung

300

200

100

0

CuZn SOD

Mn SOD

EC-SOD

Human

SOD Total Activity in Lung

slide44

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

slide45

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

slide46

EC-SOD

600

500

400

Units/g wet weight

300

200

100

0

Liver

Kidney

Heart

Brain

Lung

Human

estimated aoe in 70 kg human
Estimated AOE in 70 Kg Human
  • CuZn SOD – 10-20 gm
  • Mn SOD 5-10 gm
  • EC-SOD 1-2 gm
mechanisms1
Mechanisms
  • Antioxidant - targeted
  • NFκB inhibition
slide49
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
nf b pathway
NF-κB Pathway

Adapted from: www.emdbiosciences.com/html/CBC/NFKB_NFkappaB_IKB_IKK_Pathway_Products.htm

nf b pathway1
NF-κB Pathway

ub

proteosome

Adapted from: www.emdbiosciences.com/html/CBC/NFKB_NFkappaB_IKB_IKK_Pathway_Products.htm

slide52

p65

Reducing conditions in the nucleus facilitate DNA-binding

p50

p65

p50

Cys62

Cys62

SH

Inhibition by MnTDE

Cell Free System using purified p50

Catalytic antioxidants display oxidoreductase activity to oxidize redox-sensitive transcription factors to prevent DNA-binding

Free Radic Biol Med 36(2):233-247, 2004

what does an antioxidant mimetic do
What Does an Antioxidant Mimetic Do?
  • Redox chemistry
  • Mediate signal transduction
  • Control inflammatory response via regulation of transcription factor(s)