Redox, Kinetic, and Biological Redox, Kinetic, and Biological

Necessities to Create an Effective Necessities to Create an Effective

Metalloenzyme-mimeticMetalloenzyme-mimetic

James D. Crapo, M.D.James D. Crapo, M.D.

Sunrise Free Radical Sunrise Free Radical SchoolSchool

Free Radical-Mediated PathologiesFree Radical-Mediated Pathologies

Normal Metabolism, AgingNormal Metabolism, Aging

ChemicalChemical

HyperoxiaHyperoxia

Ischemia-ReperfusionIschemia-Reperfusion

InflammationInflammation

AutoimmuneAutoimmune

CancerCancer

AntioxidantsAntioxidants

TocopherolTocopherol

Non EnzymaticNon Enzymatic 11

EfficiencyEfficiency

EnzymaticEnzymatic1,000 – 10,0001,000 – 10,000

AscorbateAscorbateββ Carotene CaroteneNACNAC

Superoxide DismutasesSuperoxide Dismutases

CatalaseCatalase

MimeticsMimetics 1,000 – 10,0001,000 – 10,000Metalloenzyme Metalloenzyme MimeticsMimetics

Several Classes of Several Classes of Catalytic AntioxidantsCatalytic Antioxidants

Salen MimeticSalen Mimetic[EUK-134][EUK-134]

N

O

OCH3

N

O

CH3O

Mn+

Macrocylic MimeticMacrocylic Mimetic[M-40403][M-40403]

N

NN

NMn

N

meso-Porphyrin Mimeticmeso-Porphyrin Mimetic

N

N

N

N

N

NN

N

N N

NN

Mn+

+

+

+

+

Metalloenzyme MimeticMetalloenzyme Mimetic

+0.94+0.94+0.94+0.94

OO22--.. + 2H + 2H++ + e + e-- H H22OO22

OO22--.. O O22 + e + e--

-0.33-0.33

MnSODMnSODCuZnSODCuZnSOD

+0.3+0.3

E E 1/2 1/2 (NHE)(NHE)

2O2O22--.. + 2H + 2H++ H H22OO22

The Redox Potentials for the Half Reactions of the The Redox Potentials for the Half Reactions of the Dismutation of Superoxide and Superoxide DismutasesDismutation of Superoxide and Superoxide Dismutases

Mn TBAPMn TBAP

N

N

N

N

CO2H

HO2C CO2H

CO2H

Mn+

Metalloporphyrin Antioxidant MimeticsMetalloporphyrin Antioxidant MimeticsEfficacy of First Generation - TBAPEfficacy of First Generation - TBAP

LungLung

Paraquat InjuryParaquat Injury

Carregeenin InflammationCarregeenin Inflammation

Bleomycin-Induced FibrosisBleomycin-Induced Fibrosis

CNSCNS

Kainate-Induced SeizuresKainate-Induced Seizures

Cerebral VasoconstrictionCerebral Vasoconstriction

Spinal Cord InjurySpinal Cord Injury

LiverLiver

Ischemia-ReperfusionIschemia-Reperfusion

SteatosisSteatosis

Acetaminophine InjuryAcetaminophine Injury

Fas-Mediated Acute InjuryFas-Mediated Acute Injury

CardiovascularCardiovascular

CardiomyopathyCardiomyopathy

Zymoson-Induced ShockZymoson-Induced Shock

JointJoint

Carregeenin Paw EdemaCarregeenin Paw Edema

Development of an Antioxidant MimeticDevelopment of an Antioxidant Mimetic

Modify side chainsModify side chains

Modify chargeModify charge

Modify redox potentialModify redox potential

Alter backboneAlter backbone

Change metalChange metal

N

N

N

N

RR

Mn+

RR RR

RR

MetalsMetals

ManganeseManganese IronIron CopperCopper CobaltCobalt NickelNickel

Mn TM-4-PyPMn TM-4-PyP

N

N

N

N

N

N

N

N

CH3

CH3

CH3

H3C Mn

AEOL-10110

+

+

+

+

+

+0.94+0.94

OO22--.. + 2H + 2H++ + e + e-- H H22OO22

OO22--.. O O22 + e + e--

-0.33-0.33

MnSODMnSODCuZnSODCuZnSOD

+0.3+0.3

MnTBAPMnTBAP

-0.23-0.23

MnTMPyPMnTMPyP

+0.06+0.06

E E 1/2 1/2 (NHE)(NHE)

2O2O22--.. + 2H + 2H++ H H22OO22

The Redox Potentials for the Half Reactions of the The Redox Potentials for the Half Reactions of the Dismutation of Superoxide and Superoxide DismutasesDismutation of Superoxide and Superoxide Dismutases

MnMnNN

NN

NN

NN

RR 22

RR 33

RR 11

RR 44

225225

336336

10,64810,648

RR11

,,22

,,33

,,44

== NN CC HH 33

++

RR11

,,22

,,33

,,44

==

NN

CC HH33

++

RR11

,,22

,,33

,,44

==

NN

HH33

CC

++

The “Ortho Effect”The “Ortho Effect”

SOD Activity SOD Activity units/mgunits/mg

MN TE-2-PyPMN TE-2-PyP

Molecular Formula:Molecular Formula: C48H56N8Cl5MnC48H56N8Cl5Mn

Molecular Weight:Molecular Weight:977977

5Clˉ5Clˉ

N

N

N

N

N

N

N

NMn+

+

+

+

+

Antioxidant ActivitiesAntioxidant Activities

LipidLipid ONOOONOOSODSOD peroxidationperoxidation scavengerscavenger

CatalaseCatalase(U/mg)(U/mg) IC50 (IC50 (M)M) (M(M-1-1SS-1-1) ) % %

activityactivity

CuZn SODCuZn SOD 5,1005,100 1515 —— — —

Mn TBAPMn TBAP 179179 2929 3.0x103.0x1055 0.420.42

Mn TM-4-PyPMn TM-4-PyP 550550 1616 1.8x101.8x1088 0.450.45

MnTE-2-PyPMnTE-2-PyP 8,5008,500 11 1.0x10 1.0x1077 1.411.41

N

N

N

N

N

NN

N

N N

NN

Mn+

Aeol-10150

+

+

+

+

MnTM-2,5-IPMnTM-2,5-IP

Antioxidant ActivitiesAntioxidant Activities

LipidLipid ONOOONOOSODSOD peroxidationperoxidation scavengerscavenger

CatalaseCatalase(U/mg)(U/mg) IC50 (IC50 (M)M) (M(M-1-1SS-1-1) ) % %

activityactivity

CuZn SODCuZn SOD 5,1005,100 1515 —— — —

Mn TBAPMn TBAP 179179 2929 3.0x103.0x1055 0.420.42

Mn TM-4-PyPMn TM-4-PyP 550550 1616 1.8x101.8x1088 0.450.45

MnTE-2-PyPMnTE-2-PyP 8,5008,500 11 1.0x10 1.0x1077 1.411.41

MnTM-2,5-IPMnTM-2,5-IP 14,80014,800 11 1.0x10 1.0x1066 1.671.67

+0.94+0.94

OO22--.. + 2H + 2H++ + e + e-- H H22OO22

OO22--.. O O22 + e + e--

-0.33-0.33

MnSODMnSODCuZnSODCuZnSOD

+0.3+0.3

MnTBAPMnTBAP

-0.23-0.23

MnTMPyPMnTMPyP

+0.06+0.06

E E 1/2 1/2 (NHE)(NHE)

2O2O22--.. + 2H + 2H++ H H22OO22

The Redox Potentials for the Half Reactions of the The Redox Potentials for the Half Reactions of the Dismutation of Superoxide and Superoxide DismutasesDismutation of Superoxide and Superoxide Dismutases

MnTM-2,5-IPMnTM-2,5-IPMnTE-2-PyPMnTE-2-PyP

+0.23+0.23 +0.33+0.33

Attenuate OAttenuate O22- - Mediated InjuryMediated Injury

Attenuate HAttenuate H22OO22 Mediated Injury Mediated Injury

Prevent Formation of Lipid PeroxidesPrevent Formation of Lipid Peroxides

Scavenge ONOOScavenge ONOO--

Antioxidant Properties of Antioxidant Properties of Metalloenzyme MimeticsMetalloenzyme Mimetics

PharmacokineticsPharmacokinetics

RouteRoute UptakeUptake DistributionDistribution Half LifeHalf Life

» PlasmaPlasma» TissueTissue

Mouse Plasma Concentrations ofMouse Plasma Concentrations ofMnTM-2,5-IP (iv bolus)MnTM-2,5-IP (iv bolus)

0 1 2 3 4 5 6 711

1010

100100

10001000

1000010000

100000100000

1mg/kg1mg/kg

3mg/kg3mg/kg

10mg/kg10mg/kg

30mg/kg30mg/kg

TT1/21/2 ~44 minutes~44 minutes

Hours

MnT

M-2

,5-I

PM

nTM

-2,5

-IP

(ng/

ml)

(ng/

ml)

MnTM-2,5-IP Steady-State From Mini-Osmotic PumpMnTM-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) (1.12 mg/kg loading dose followed by 1.8 mg/kg/hr infusion for 24 hours)

00 44 88 1212 1616 2020 242411

100100

1000010000

10000001000000

serumserum 2,0002,000

brainbrain 9090

heartheart 1,0001,000

liverliver 16,30016,300

lunglung 1,6001,600

kidneykidney 34,10034,100

Steady-StateSteady-State(ng/g or ng/ml)(ng/g or ng/ml)

HoursHours

Mn

TM

-2,5

-IP

Mn

TM

-2,5

-IP

(ng/

g o

r n

g/m

l)(n

g/g

or

ng

/ml)

MnTM-2,5-IP Clearance from Mini-Osmotic PumpMnTM-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)(1.12 mg/kg loading dose followed by 1.8 mg/kg/hr infusion for 24 hours)

00 2424 4848 7272 969611

100100

1000010000

10000001000000

serumserum 88

brainbrain 4949

heartheart 9696

liverliver 136136

lunglung 8383

kidneykidney 140140

estimated half-life (hrs)estimated half-life (hrs)

HoursHours

Mn

TM

-2,5

-IP

Mn

TM

-2,5

-IP

(ng/

g o

r n

g/m

l)(n

g/g

or

ng

/ml)

Pharmacokinetics of MnTM-2,5-IP in RatsPharmacokinetics of MnTM-2,5-IP in Rats(24 mg/kg, SC)(24 mg/kg, SC)

00 22 44 66 88 1010 1212 141410001000

1000010000

100000100000 TT1/21/2 ~ 3 hrs~ 3 hrsTime to peak ~ 6 hrsTime to peak ~ 6 hrsEffective dosing interval ~ 9 hrsEffective dosing interval ~ 9 hrs

HoursHours

Pla

sma

MnT

M-2

,5-I

PP

lasm

a M

nTM

-2,5

-IP

(ng/

ml)

(ng/

ml)

ToxicityToxicity

MTDMTD Organ specificOrgan specific MutagenicityMutagenicity CardiovascularCardiovascular

MnTM-2,5-IPMnTM-2,5-IPToxicology Toxicology

Species Route Endpoint Value Cynomolgus

monkey IV (24 h infusion) MTD 96 mg/kg - day

Cynomolgus monkey

IV (1 week continuous

infusion) MTD 72 mg/kg - day

Mouse IP (bolus) LD50 240 mg/kg

Mouse

IV (7 day

continuous infusion)

Female - ovarian cysts at 50 and 100 mg/kg - day Male - no adverse effects at 100 mg/kg - day

Rat IV, IP, oral Hypotension Route-dependent

Rat ICV Serotonin Syndrome

>9000 ng 4500 ng NOEL

MechanismsMechanisms

Antioxidant - targetedAntioxidant - targeted

EC-SOD in Large Elastic Pulmonary ArteryEC-SOD in Large Elastic Pulmonary Artery

50 μ

EC-SOD in Muscular Pulmonary ArteryEC-SOD in Muscular Pulmonary Artery

50 μ

EC-SOD in Small ArterioleEC-SOD in Small Arteriole

Immunolocalization of EC-SODImmunolocalization of EC-SOD

CuZn SOD Concentrations in CuZn SOD Concentrations in Hepatocyte OrganellesHepatocyte Organelles

OrganellesOrganelles mg SOD/cmmg SOD/cm33

NucleusNucleus 0.71 0.71 ± 0.06± 0.06

Cytoplasmic MatrixCytoplasmic Matrix 1.36 1.36 ± 0.30± 0.30

MitochondriaMitochondria 0.21 0.21 ± 0.01± 0.01

RERRER 0 0

SERSER 0.02 0.02 ± 0.01± 0.01

Golgi ApparatusGolgi Apparatus 0 0

LysosomesLysosomes 5.81 5.81 ± 1.55± 1.55

PeroxisomesPeroxisomes 0.27 0.27 ± 0.08± 0.08

Distribution of CuZn SOD Molecules in Distribution of CuZn SOD Molecules in Hepatocyte OrganellesHepatocyte Organelles

OrganellesOrganelles # # μμmm33

NucleusNucleus 13,300 13,300

Cytoplasmic MatrixCytoplasmic Matrix 25,500 25,500

MitochondriaMitochondria 3,900 3,900

SERSER 400 400

LysosomesLysosomes 108,900 108,900

PeroxisomesPeroxisomes 5,000 5,000

SOD Total Activity in LungSOD Total Activity in Lung

00

100100

200200

300300

400400

500500

600600

700700

800800

CuZn SODCuZn SOD Mn SODMn SOD EC-SODEC-SOD

HumanHuman

Un

its/

g l

un

gU

nit

s/g

lu

ng

SOD Activities in Specific CompartmentsSOD Activities in Specific Compartments

00

55

1010

1515

2020

2525

3030

3535

4040

4545

5050

CuZn SODCuZn SODin Cellsin Cells

MnSOD inMnSOD inMitochondriaMitochondria

EC-SOD inEC-SOD inInterstitiumInterstitium

Un

its/

cmU

nit

s/cm

33 ti

ssu

e t

issu

e

CuZn SODCuZn SOD

00100010002000200030003000400040005000500060006000700070008000800090009000

LiverLiver KidneyKidney BrainBrain HeartHeart LungLung

Un

its/

g w

et

wei

gh

tU

nit

s/g

we

t w

eig

ht

HumanHuman

Mn SODMn SOD

00

500500

10001000

15001500

20002000

25002500

LiverLiver KidneyKidney HeartHeart BrainBrain LungLungHumanHuman

Un

its/

g w

et

wei

gh

tU

nit

s/g

we

t w

eig

ht

A B

EC-SODEC-SOD

00

100100

200200

300300

400400

500500

600600

LiverLiver KidneyKidney HeartHeart BrainBrain LungLung

HumanHuman

Un

its

/g w

et

we

igh

tU

nit

s/g

we

t w

eig

ht

Estimated AOE in 70 Kg HumanEstimated AOE in 70 Kg Human

CuZn SOD – 10-20 gmCuZn SOD – 10-20 gm

Mn SOD 5-10 gmMn SOD 5-10 gm

EC-SOD 1-2 gmEC-SOD 1-2 gm

MechanismsMechanisms

Antioxidant - targetedAntioxidant - targeted

NFNFκκB inhibitionB inhibition

NF-NF-κκBB

Nuclear Factor-Kappa BNuclear Factor-Kappa B First discovered as an enhancer of First discovered as an enhancer of

B cells B cells (Sen & Baltimore 1986, Cell)(Sen & Baltimore 1986, Cell)

Ubiquitous transcription factor Ubiquitous transcription factor Shown to be involved in cancer, Shown to be involved in cancer,

immune response, redox regulation, immune response, redox regulation, apoptosisapoptosis

NF-NF-κκB B PathwayPathway

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

ub

proteosome

NF-NF-κκB B PathwayPathway

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

Catalytic antioxidants display oxidoreductase Catalytic antioxidants display oxidoreductase activity to oxidize redox-sensitive transcription activity to oxidize redox-sensitive transcription

factors to prevent DNA-bindingfactors to prevent DNA-binding

p50p65

Cys62

Reducing conditions in Reducing conditions in the nucleus facilitate the nucleus facilitate DNA-bindingDNA-binding p50p65

Cys62

SH

Cell Free System using purified p50Cell Free System using purified p50

Inhibition by MnTDE

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

What Does an Antioxidant Mimetic Do?What Does an Antioxidant Mimetic Do?

Redox chemistryRedox chemistry

Mediate signal transductionMediate signal transduction

Control inflammatory response via Control inflammatory response via

regulation of transcription factor(s)regulation of transcription factor(s)