Electrophilic Signaling Mediators
Pleiotropic Inhibitors of Metabolic and Inflammatory Disorders
Bruce Freeman, PhD
UPMC/Irwin Fridovich Professor and Chair
Dept Pharmacology and Chemical Biology
University of Pittsburgh School of Medicine
Pharmacology and Chemical Biology Mission:
Apply basic chemical principles to the understanding of cell
signaling and the creation of new therapeutic strategies
Nitric Oxide, Fatty Acids - Cell Signaling
Nobel Prize, 1998
Organonitrates
PD-5 inhibitors
Nobel Prize, 1988
Aspirin
NSAIDS
Steroids
Nitric Oxide and Nitrite Produce Nitrogen Dioxide (.NO2)
This Nitrates Unsaturated Fatty Acids
.NO
+ O2
.-
ONOO.NO
CO2
NO2 + MPO
NO2
.NO
-
H+
+ O2
NO2-
H 2 O2
.NO
HNO2
NO2
2
.NO
.NO
ONO. .OH
ONO. CO3.-
ONOOCO2
H 2 O2
+ MPO
-
H+
ONOO-
2
Nature, Nat Chem Biol, Nat Med, Science, PNAS, JCI
R
2
R
Nitric Oxide and Nitrite Produce Nitrogen Dioxide (.NO2)
This Nitrates Unsaturated Fatty Acids
.NO
+ O2
.-
ONOO.NO
CO2
NO2 + MPO
ONO. .OH
ONO. CO3.-
ONOOCO2
H 2 O2
+ MPO
-
H+
ONOO-
NO2-
15NO
2-LA
(MRM 325.3/47)
H 2 O2
.NO
2
14NO
NO2
.NO
-
H+
+ O2
.NO
HNO2
.NO
(MRM 324.3/46)
2
2
NO2
R
Science, Nature, Nat Chem Biol, Nat Med, PNAS, JCI
2-LA
R
NO2
R
O
R
Nitro-FA
Keto-FA
R
R
• Electrophilic lipids provide a linkage between metabolic and
inflammatory status and adaptive signaling responses
• Organisms have evolved key regulatory proteins and enzymes
containing functionally-significant, electrophile-reactive
nucleophilic amino acids - cysteine
• These sentinel proteins sense metabolic state and the
inflammatory milieu, thus orchestrating adaptive responses
Complexa (Pittsburgh-Based) Drug Development Strategy
Administer synthetic homologs of electrophilic signaling mediators to
increase endogenous levels and promote salutary responses
Electrophilic Fatty Acids Regulate Gene Expression
Human Coronary Artery Endothelium
10-NO2-octadec-9-enoic acid
CXA-10
330 genes
93 genes
~425 genes total
Inflammation
Oxidant defense
Metabolism
DNA, protein repair
Conjugation, excretion
J Biol Chem
Other Natural, Near-Natural Electrophiles
Dimethylfumarate (BG-12, Tecfidera - Biogen-Idec)
•
•
•
•
Oral drug for psoriasis, FDA-approved 2012 for MS
MOA – Nrf2 activation, rapidly metabolized
Not detectable in plasma after administration
Recommended dosing = 240 mg, 3x/d
Sulforaphane (broccoli sprouts, florets)
•
•
•
Orally bioavailable, anti-cancer, anti-inflammatory
MOA – Nrf2 activation
Long track record of safety
Contrast
Contrast Induced
Induced Nephropathy
Nephropathy
J JAm
2008;51(15):1419-1428 McCullough
McCullough
AmColl
CollCardiol.
Cardiol. 2008;51(15):1419-1428
8
Rationale for CXA-10 in CI-AKI
CXA-10
Nrf2, HSF-1 activation, NFkB inhibition
Increase HO-1, CuZn and Mn SOD,
catalase, eNOS, GSH synthesis
GSH transferase/reductase,
Inhibit ATII/sEH signaling
Decrease iNOS, cytokine, ANP, TGFb,
PDGF signaling, MMP and NAD(P)H
oxidases, fetal gene expression, collagen
Inhibit oxidative inflammatory reactions/byproducts
Enhance detoxification/repair reactions
Reduce fibroblast, smooth muscle cell proliferation
Improve vascular
function
Limit inflammatory responses, adverse remodeling
Broader and more potent activity than NAC, Fe chelators
IMPROVED RENAL FUNCTION, DECREASED MORTALITY
Role of Heme Oxygenase-1 in AKI Prevention
Rationale for Use of CXA-10
Anupam Agarwal, MD
Chief, Renal Division
UAB School of Medicine
Recent Interim Chair of Medicine
and Dean of the UAB School of
Medicine
Past President, ASN (2011-2)
10
Nrf2-Regulated Genes in Renal Protection
•
Transcriptional regulatory factor Nrf2 is protective during
ischemic/nephrotoxic AKI in mice [Liu et al, Kid Int (2009), JHU SOM]
– Nrf2 deficiency in knock-out mice enhances susceptibility to both
ischemic/nephrotoxic AKI
•
Sulforaphane and other isothiocyanates in cruciferous vegetables
(broccoli) limits renal fibrosis [Oh et al, FRBM (2011), Kyungpook
SOM,Korea]
– Mediates Nrf2-dependent induction of phase II detoxifying enzymes including
HO-1
– Protective effect on acute renal fibrosis due to Nrf2-mediated and antioxidantindependent inhibition of TGF-β/Smad signaling
– TGF-β/Smad signaling down-regulated by CXA-10, current data suggests
broader anti-fibrotic effects that need to be tested
11
Preservation of kidney function during sepsis
• Sepsis increased creatinine levels due to impaired renal function
• CXA-10 re-established normal plasma creatinine and urea levels
• CXA-10 decreased all measured parameters of inflammation
CXA-10 Protects Against Ischemic Kidney Injury
Nitro-oleic acid protects the mouse kidney from ischemia and reperfusion injury
Am J Physiol (Renal) 295:942-949, 2008 (T Yang, Univ Utah SOM):
CXA-10 administered 50 min and q 6H x 24 hours after release of clamp
Delayed administration of CXA-10 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 also in histopathology
o
Decreased leucocyte recruitment
No Rx
No Protection
+ CXA-10
Protection
Kidney, 24 hr post-IR
CXA-10 Attenuates Nephrotoxin-Induced AKI
Nitro-Oleic Acid Protects against Adriamycin-Induced Nephropathy in Mice
T. Yang, Am J Physiol Renal Physiol, (2013)
• Attenuates ADR-induced, podocyte loss,
glomerulosclerosis and tubulointerstitial
fibrosis.
• Suppresses indices of oxidative stress
including plasma and urinary TBARS and
renal expression of NAD(P)H oxidase
p47phox and gp91phox
•Suppresses ADR induced inflammation
including renal expression of TNF-α, IL1β and MCP-1
Renal Protection by CXA-10 in Ischemia Induced AKI
(Contracted by Complexa to UAB O’Brien Kidney Center - A. Agarwal)
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
(measured by HPLC-MS)
* p<0.01
Basal
24
48
Time (hr)
72
Renal Protection by CXA-10 in I/ Induced AKI
UAB O’Brien Kidney Center - A. Agarwal
Veh + Sham
CXA 12.5+ Sham
Veh + I/R
CXA12.5 + I/R
Outer Medulla
Cortex
A
B
Percentage of Tubules (%)
50
40
Veh + IR
CXA12.5 + IR
*
30
20
10
0
Necrotic Tubules
16
#
*
BB Loss
Casts
Histological evaluation (12.5 mg/kg) following I/R injury *p<0.01, p<0.05); analysis
CKD Model
Uni-nephrectomised mice high fat/high salt diet,
deoxycorticosterone acetate Rx
At initiation of Rx, the model is
hypertensive with glomerular
injury, podocytopathy (loss of
renal filtration barrier)
Urine/blood collection
Study Design
Age (week)
6
Unx
8
Start diet,
DOCA
10
11
CXA-10
DOCA
2.5 mg/kg reimplant
PO, QD
12
14
Tissue
collection
Endpoints
Normal
Damage (Effacement)
•
•
•
•
•
.
Serum and urine analysis
Histological assessment and quantitation of podocyte
number and endothelial loss
Immunohistochemistry – inflammatory, fibrotic markers
qRT-PCR for gene expression
Drug PK and PD
CXA-10 (2.5 mg/kg PO) Protects Against Renal Injury
Legend:
Ctrl
CXA-10 2.5mpk ,* p<0.05; **p<0.01 vs. untreated
DOCA
16
CXA-10 reduced urinary albumin excretion
3000
CXA-10 reduced renal/cardiac hypertrophy (wk 4)
Weight ratio (mg/g)
2000
1500
*
**
1000
500
12
10
8
6
4
2
0
1
2
3
Week of Treatment
3000
4
Kidney/body ratio
CXA-10 reduction in urinary albumin excretion paralleled nephrin
60
excretion, indicating podocyte protection (wk 4)
2500
2000
1500
1000
500
0
0
*
Nephrinuria (ng/24 h)
0
Albuminuria (ug/24 h)
Albumin µg/24 Hours
14
2500
50
40
30
20
10
0
**
Heart/body ratio
Electrophilic Anti-inflammatory/Metabolic Mediators
• Naturally occurring in humans, fish, plants, insects
• Levels increase during digestion, metabolic and inflammatory stress
• Mediate salutary pleiotropic metabolic and inflammatory signaling
• Link metabolic/inflammatory status with adaptive gene expression
• Proven as viable drug candidates