+ NO 2 - University of Pittsburgh

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Formation and Signaling Actions of
Electrophilic Fatty Acids
Apply basic chemical principles in the understanding of cell signaling
and the creation of new therapeutic strategies
Bruce A Freeman, Ph.D.
Irwin Fridovich Professor and Chair
Dept. of Pharmacology and Chemical Biology
University of Pittsburgh School of Medicine
Nitric Oxide and Nitrite Produce Nitrogen Dioxide (.NO2)
nitrates biomolecules
forms electrophilic products
NO2
R
.NO
+ O2
.-
ONOO.NO
CO2
NO2 + MPO
NO2
.NO
-
H+
+ O2
NO2-
H 2 O2
.NO
2
.NO
HNO2
.NO
ONO. .OH
ONO. CO3.-
ONOOCO2
H 2 O2
+ MPO
-
H+
ONOO-
R
2
2
Science, Nature, PNAS, J Clin Invest, J Biol Chem
Mammalian
NOx Cycle
NO3Oral Bacteria
NO2Peroxidases
Acidification
Deoxy-Hb
XOR
Nitrite-NO reaction
NO2.
NO.
Fatty acid nitration
Protein nitration
Activate sGC
Inhibit cytochrome oxidase
Acidification
SNO
Protein modification
NO-like bioactivity
Lundberg, Weitzberg
NO2-Fatty Acid Generation (Humans)
1 gm/d CLA, 8 oz/d beet root juice x 3d
Normal Gastric Conditions
Human Plasma NO2-CLA
Basal
1.510 3
+Diet
8.010 35
3.010
Intensity, cps
Intensity, cps
4.010 45
1.0
Human Urine NO2-CLA
6.010
2.010 5
4.010 3
3
1.010 5
2.010 3
0
Basal
+Diet
Basal
1.010 3
5.010 2
0
40
42
44
Time (min)
46
48
37
38
39
Time (min)
40
NO2-Fatty Acid Generation (Humans)
[15N]O2- (nitrite, 20 mg)
[15N]O3- (nitrate, 1 gm)
18:2, 3 gm @ 0, 48 hr
15NOx
15NOx
6 time points
0
pause, wash-out
24
6 time points
48
72 hr
Measurements
Blood pressure, heart rate
Nox species – urine, blood
PBMC gene expression
Platelet function
Mitochondrial function
FDA IND, IRB PRO11120134 – Freeman, Gladwin
Plasma NO2-cLA Levels
Nitrite
Nitrate
[14N]
[15N]
A Key Reactivity of NO2-FA and Keto-FA
Rapid, Reversible Michael Addition Reaction
H
RS-
R'
H
N+
O-
H
O
R'
RS
N+
H
O
-
O-
H+
H
R'
RS
H
N+
H
O-
O-
H
R'
N
RS
H
+
O-
O-
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
Electrophilic Fatty Acids Regulate Gene Expression
Human Vascular Endothelium
10-NO2-octadec-9-enoic acid
330 genes
93 genes
~425 genes total
43 genes Nrf2-regulated
Inflammation
Oxidant defense
Metabolism
DNA, protein repair
Conjugation, excretion
PNAS, J Biol Chem
NO2
R
R
O
Nitro-FA
Keto-FA
R
R
• 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: pleitropic
transcription 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
Comparable Natural, Near-Natural Electrophiles
*
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
*
•
•
•
Sulforaphane (broccoli sprouts, florets)
Orally bioavailable, anti-cancer, anti-inflammatory
MOA – Nrf2 activation
Long track record of safety
Study #1
Pre-clinical Disease Proof-of-Concept
CXA-10 Protects Against Ischemic Kidney Injury (Treatment)
Nitro-oleic acid protects the mouse kidney from ischemia and reperfusion injury
Am J Physiol (Renal) 295:942-949, 2008 (T Yang, Univ Utah School of Medicine)
CXA-10 administered 50 min and q 6H x 24 hours after ischemia
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 histopathology
o
Decreased leucocyte recruitment
No Rx
No Protection
+ CXA-10
Protection
Kidney, 24 hr post-IR
Study #2
Confirmatory Kidney IR PK/PD Study (Prophylaxis)
(UAB O’Brien Kidney Center, Anupam Agarwal, MD)
I/R = Bilateral renal artery blocking with small clamps, 35min
• 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
* p<0.01
Basal
24
48
Time (hr)
72
Study 4
Evaluation of CXA-10 in the DOCA Salt Model of
Chronic Renal Injury
• 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.
Control
Untreated
CXA-10
Histologically, untreated mice developed tubular damage and interstitial fibrosis. CXA-10 improved overall tubular
appearance Representative picrosirius red stained sections (x200)
Electrophilic Fatty Acids
Anti-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, tissueprotective gene expression and enzymatic activities
OA-NO2 Limits Angiotensin II-induced Hypertension
p< 0.05
140
Systolic Blood Pressure
(mmHg)
120
100
80
60
Mechanisms
40
20
0
Baseline
OA
OA-NO2
OA
OA-NO2
Up
• eNOS expression
• HO-1 expression
Angiotensin II
Systolic BP decrease
(mmHg)
0
1.25
2.5
5
10
20 (mg/kg)
0
-5
Down
• Epoxide hydrolase
• Nox expression
• Nox catalytic activity
*
-10
-15
-20
OA
OA-NO2
*
*
Circ Res, PNAS
Remaining Challenge – ID of total pool of lipid electrophiles
Metabolic Fate of Fatty Acid Nitroalkenes
Protein adduction
Esterification
Complex lipids
b-Oxidation
NO2
Glucuronidation
R
R
Excretion
Signaling (PTM)
Keap1/Nrf2
PPARg, HSF-1
NFkB p65, PTPs
Mito: Site II-70 kDa
XOR, sEH
GSH reaction
MDRP export
Nitroalkene Reduction
Prostaglandin reductase
(PtGR1)
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