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