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)