Bruce A Freeman, Ph.D. Irwin Fridovich Professor and Chair Dept. of Pharmacology and Chemical...
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Transcript of Bruce A Freeman, Ph.D. Irwin Fridovich Professor and Chair Dept. of Pharmacology and Chemical...
Bruce A Freeman, Ph.D.Irwin Fridovich Professor and Chair
Dept. of Pharmacology and Chemical BiologyUniversity of Pittsburgh School of Medicine
Apply basic chemical principles in the understanding of cell signaling and the creation of new therapeutic strategies
Formation and Signaling Actions of Electrophilic Fatty Acids
Nitric Oxide and Nitrite Produce Nitrogen Dioxide (.NO2) nitrates biomolecules
forms electrophilic products
H+.NO + O2
.- ONOO- ONO. .OH
NO2- + MPO .NO2
CO2ONOO- ONOOCO2 ONO. CO3
.-
H2O2
NO2-
H+
HNO2 .NO2
.NO + MPO NO2-
H2O2
.NO + O2 .NO2
NO2
R R
Science, Nature, PNAS, J Clin Invest, J Biol Chem
Oral Bacteria
Deoxy-HbXOR
Peroxidases Nitrite-NO reaction
Activate sGCInhibit cytochrome oxidase
Protein modificationNO-like bioactivity
Fatty acid nitrationProtein nitration
NO3-
NO2-
NO2. NO. SNO
Acidification Acidification
Lundberg, Weitzberg
MammalianNOx Cycle
Normal Gastric Conditions
40 42 44 46 480
2.0103
4.0103
6.0103
8.0103
1.0104 Basal
Time (min)
Inte
nsi
ty,
cps
40 42 44 46 480
1.0105
2.0105
3.0105
4.0105 Basal +Diet
Time (min)
Inte
nsi
ty,
cps
Human Plasma NO2-CLA Human Urine NO2-CLA
NO2-Fatty Acid Generation (Humans)1 gm/d CLA, 8 oz/d beet root juice x 3d
37 38 39 400
5.0102
1.0103
1.5103
Basal
Time (min)
Inte
nsi
ty,
cps
37 38 39 400
5.0102
1.0103
1.5103 Basal +Diet
Time (min)
Inte
nsi
ty,
cps
NO2-Fatty Acid Generation (Humans) [15N]O2
- (nitrite, 20 mg)
[15N]O3- (nitrate, 1 gm)
18:2, 3 gm @ 0, 48 hr
0 24 48 72 hr
15NOx 15NOxpause, wash-out 6 time points6 time points
MeasurementsBlood pressure, heart rateNox species – urine, bloodPBMC gene expressionPlatelet functionMitochondrial function
FDA IND, IRB PRO11120134 – Freeman, Gladwin
Plasma NO2-cLA Levels
[14N]
[15N]
Nitrite Nitrate
A Key Reactivity of NO2-FA and Keto-FARapid, Reversible Michael Addition Reaction
Cysteine Reaction Rate
Species K (M-1
s-1)
NO
0
H2O2
~1
15d-PGJ2 <1
NO2-oleate 190 NO2-linoleate 240 Keto-DPA, DHA ~100
N+O
O-
R'
H
H
RS-N+
O-
O-
R'
H
H
N+O-
O-
R'
H
HH+
N+O-
O-
R'
H
H H
RS
RS
RS
Electrophilic Fatty Acids Regulate Gene ExpressionHuman Vascular Endothelium
10-NO2-octadec-9-enoic acid
330 genes
93 genes
~425 genes total
43 genes Nrf2-regulated
InflammationOxidant defenseMetabolismDNA, protein repair Conjugation, excretion
PNAS, J Biol Chem
• Generated endogenously by both enzymatic and random redox reactions during digestion, metabolism and inflammation
• Contribute to the beneficial actions of dietary unsaturated fatty acids and NO3
- + NO2- (Mediterranean/Japanese diets)
• Signal via Michael addition of susceptible proteins: pleitropictranscription factors, pro-inflammatory enzymes
• Established metabolic pathways regulate electrophilic lipid levels
COI Disclosure: Complexa, Inc.Drug Development Strategy
Administer homologs of electrophilic lipid signaling mediators to increase endogenous concentrations and promote beneficial
metabolic and inflammatory signaling responses
Nitro-FAKeto-FA
O
R R
NO2
R R
Dimethylfumarate (BG-12, Tecfidera - Biogen-Idec)
• Oral drug for multiple sclerosis, FDA approval - 2012• MOA – Nrf2 activation, rapidly metabolized• Not detectable in plasma after administration• Recommended dosing = 240 mg, 3 x day
Comparable Natural, Near-Natural Electrophiles
Sulforaphane (broccoli sprouts, florets)
• Orally bioavailable, anti-cancer, anti-inflammatory • MOA – Nrf2 activation• Long track record of safety
**
*
Study #1Pre-clinical Disease Proof-of-Concept
CXA-10 Protects Against Ischemic Kidney Injury (Treatment)
Nitro-oleic acid protects the mouse kidney from ischemia and reperfusion injuryAm J Physiol (Renal) 295:942-949, 2008 (T Yang, Univ Utah School of Medicine)
Kidney, 24 hr post-IR
CXA-10 Administration:
Resulted in significant renal protection:o Decreased plasma urea and
creatinine, preservation of urine output
o Decreased plasma and organ indices of inflammation
o Evidenced in histopathologyo Decreased leucocyte recruitment
+ CXA-10Protection
No RxNo Protection
CXA-10 administered 50 min and q 6H x 24 hours after ischemia
Basal 24 48 72 Time (hr)
I/R = Bilateral renal artery blocking with small clamps, 35min
* p<0.01
Study #2Confirmatory Kidney IR PK/PD Study (Prophylaxis)
(UAB O’Brien Kidney Center, Anupam Agarwal, MD)
• Rat I/R n=6; sham n=3; CXA-10 12.5 mg/kg
• Dosed 1 hr prior to ischemic insult• >45% reduction in serum
creatinine
• CXA-10 administered to mice via chronic 4-week oral dosing, exerted significant renal-protective effects:
• Significantly reduced albuminuria, fibrotic gene expression, urinary nephrin and MCP-1 excretion
• Inhibited gene expression of pro-inflammatory cytokines, ECM and profibrotic factor, PAI-1
• Reduction in cardiac hypertrophy (not seen with control)• Positive impact on cholesterol metabolism
• MOA - anti-inflammatory, antioxidant and anti-fibrotic effects.
Study 4Evaluation of CXA-10 in the DOCA Salt Model of
Chronic Renal Injury
Histologically, untreated mice developed tubular damage and interstitial fibrosis. CXA-10 improved overall tubular appearance Representative picrosirius red stained sections (x200)
Control Untreated CXA-10
Electrophilic Fatty AcidsAnti-inflammatory Mediators, Drug Candidates
• Naturally occurring in humans, fish, plants, insects• Levels increase during metabolic and inflammatory stress, also
from diets rich in polyunsaturated fatty acids and NO2-/NO3
-
• Mediate salutary pleitropic metabolic and inflammatory signaling• Link between metabolic/inflammatory status and adaptive, tissue-
protective gene expression and enzymatic activities
-20
-15
-10
-5
0
0 1.25 2.5 5 10 20 (mg/kg)
DSy
stol
ic B
P de
crea
se(m
mH
g)
OA-NO2
OA
*
*
*
Mechanisms
Up• eNOS expression• HO-1 expression
Down• Epoxide hydrolase• Nox expression• Nox catalytic activity
OA-NO2 Limits Angiotensin II-induced Hypertension
Circ Res, PNAS
p< 0.05
020406080
100120140
Baseline
Angiotensin II
Syst
olic
Blo
od P
ress
ure
(mm
Hg)
OA OA-NO2 OA OA-NO2
Remaining Challenge – ID of total pool of lipid electrophiles
Metabolic Fate of Fatty Acid Nitroalkenes
NO2
R R
Protein adduction
GSH reactionMDRP export
b-OxidationEsterification
Complex lipids
Excretion
Glucuronidation
Nitroalkene ReductionProstaglandin reductase
(PtGR1)Signaling (PTM)Keap1/Nrf2
PPARg, HSF-1NFkB p65, PTPs
Mito: Site II-70 kDaXOR, sEH