Shawn M. Talbott, PhDCNS, LDN, FACSM, FAIS, FACN

Chief Science Officer

NRF2 REVOLUTION What is Nrf2 and

Why should I care? Mark Gordon, MD, FACC

W H AT I S O X I D AT I V E S T R E S S ?

• Oxidative stress, is a disturbance in the balance between the production of reactive oxygen species (free radicals) and antioxidant defenses,

O X I D AT I V E S T R E S S

N R F 2

• Messenger Protein

• Activates genes

• Redox balance

• Delicate balance

• Master Regulator

T H E K E A P 1 / N R F 2 PAT H W AY

Nrf 2 activator

Nrf 2 activator

N R F 2 A C T I O N S :

• Increases the production of a variety of proteins that:

• Reduce oxidative stress (anti-oxidant enzymes) (SOD, Catalase, Glutathione)

• Have anti-inflammatory action (downregulates NFKB)

• Detoxify toxic molecules

D E T O X I F I C AT I O N :

D E T O X I F I C AT I O N :

G L U TAT H I O N E :

• Dramatically increased with Nrf 2 activation

• Single most important detoxification enzyme

• Acts as anti oxidant

• Regulates Nitric Oxide cycle - (blood vessel health)

• Involved in DNA repair, protein synthesis, enzyme activation

• Every organ system is affected by Glutathione

N R F 2 I S P U L S AT I L E I N T H E N O R M A L H E A LT H Y S TAT E

N O T… P E D A L T O T H E M E TA L ! ! !

– S P E A K E R AT A N T I - A G I N G C O N F E R E N C E , A P R I L 2 0 1 4

“Nrf 2 is a revolution in science,…and is the most important anti-aging pathway in the human body.”

EXPLORE YOUR LIMITS!

ACTIVATE YOUR NRF2 AND

Shawn M. Talbott, PhDCNS, LDN, FACSM, FAIS, FACN

Chief Science Officer

Managing Cellular Stress

NRF2

Lamin-B1

YOUNG OLD

Nrf2 Declines with Age and Induces Oxidative Stress

YOU

NG

O

LD

Nrf2 0

0.5

1

1.5

Young Old

Den

sity

Uni

ts

*

0.000

0.250

0.500

YOUNG OLDNrf2

/ARE

Bin

ding

Act

ivity

(A45

0)*

WT (C57/Bl6) Mouse

0

10

20

30

40

50

Young OldRO

S (E

PR-C

MH

Sig

nals

) nm

ol/m

in/m

g pr

otei

n p<0.05

p<0.05

Increased oxidative stress

Bacopa

Silymarin

O

OH

OH

OH

OH

OH O

O

OH

OH

OH

Epigallocatechin  gallate  EGCG

HOCH2

OHO

OO

C H3

C H3

C H3

C H3O

Withaferin  A

OO

OH OH

OOCH3 CH3

H

Curcumin

Nrf2 = a powerful “master regulator” of antioxidant enzymes and survival genes

0 5 0 1 0 0 1 5 0 2 0 0 2 5 00

1 0

2 0

3 0

4 0

A R E re p o r te r a s s a y

u g /m L

Fo

ld I

nd

uc

tio

n

A sh w a g h a n d a

B la c k P e p p e r

M ilk T h is tle

T u m e r ic

G re e n T e a

B a c o p a

P ro ta n d im U S

P ro ta n d im J a p a n

All five ingredients together produced an 18-fold increase in the expression of this antioxidant gene. !Protandim works 18 times more effectively than the sum of its parts.

Fold

Indu

ctio

n H

eme

Oxy

gena

se m

RN

A

Alcohol extract from 30 ug protandim or individual components per ml medium

0

2

4

6

8

10

12

14

16

18

20

Ashwag

antha

Bacopa

Green Te

a

Silymari

n

Curcumin

Protandim

Control

SYNERGY = Action greater than the sum of the parts

First Patent Issued: July 10, 2007 Second Patent Issued: July 10, 2008 Third Patent Issued: August 25, 2009 Fourth Patent Issued: April 12, 2011

 Patented  Technology

010

2030

4050

6070

80

%  In

crease  in  Catalase

0 30 120

Days  on  Protandim

*

05

101520253035404550

0 30 120

Days  on  Protandim

%  increase  SOD  

*

After 120 days… SOD increased by 34% Catalase increased by 54%

0

0.5

1

1.5

2

2.5

3

3.5

4

0 20 40 60 80 100

AGE

TBAR

S  (m

icromolar)

Before  Supplementation

R 2  =  0.238

Oxidative  Stress

R 2  =  0.003After  Supplementation  30  d

Vit E and Vit C

No supplements

After 30 days… !“Remarkably, this age-dependent increase in TBARS was almost completely abolished by Protandim treatment (Fig. 1D), with an overall average reduction of the oxidative stress marker by 40%.”

Nutragenomics: Modifying gene expression with natural products

0

20

40

60

80

100

120

140

0 20 40 60 80Protandim µg/ml

Fold

Indu

ctio

n

0

20

40

60

80

100

120

140

0 5 10Sulforaphane µM

Fold

Indu

ctio

n

0

20

40

60

80

100

120

140

0 0.1 0.2 0.3 0.4 0.5 0.6Bardoxolone-Me µM

Fold

Indu

ctio

n

0

20

40

60

80

100

120

140

0 50 100BG12 (Dimethylfumarate) µM

Fold

Indu

ctio

n

FImax = 135

Cmax = 48 µg/ml

FImax = 67

Cmax = 0.3 µM

FImax = 21

Cmax = 6 µM

FImax = 55

Cmax = 60 µM

A B

DC

Protandim 135 Bardoxolone Me 67 Dimethylfumarate 55 Sulforaphane 21

Agent Fold Induction

A side-by-side comparison of Nrf2 activating potencies

Of the 19 genes, 16 (84%) are modulated by Protandim in the opposing direction of the disease process

19  genes  associated  with  atherosclerosis  are  regulated  by  Protandim

Of the 28 genes, 25 (89%) are modulated by Protandim in the opposing direction of the disease process

28  genes  associated  with  colon  cancer  are  regulated  by  Protandim

Genes  1-­‐33

66  genes  associated  with  Alzheimer’s  are  regulated  by  Protandim

Genes  34-­‐66

Of the 66 genes, 43 (65%) are modulated by Protandim in the opposing direction of the disease process

66  genes  associated  with  Alzheimer’s  are  regulated  by  Protandim

Original Contribution

Nrf2 activation: A potential strategy for the prevention of acutemountain sickness

Christina Lisk a, Joe McCord b, Swapan Bose b, Tim Sullivan b, Zoe Loomis a,Eva Nozik-Grayck c, Thies Schroeder d, Karyn Hamilton e, David C. Irwin a,n

a Division of Cardiology, Cardiovascular Pulmonary Research Group, School of Medicine, Aurora, CO 80045, USAb Pulmonary Division, University of Colorado at Denver, Anschutz Medical Campus, Aurora, CO 80045, USAc Department of Pediatrics, Cardiovascular Pulmonary Research Group, School of Medicine, Aurora, CO 80045, USAd Department of Radiation Oncology, Duke University, Durham, NC 27710, USAe Exercise and Sports Science, Colorado State University, Fort Collins, CO 80523, USA

a r t i c l e i n f o

Article history:Received 22 October 2012Received in revised form9 April 2013Accepted 16 May 2013Available online 27 May 2013

Keywords:Nrf2High altitude sicknessCerebralVascular leakCalcium channel blockersCarbon anhydrase inhibitorsEndothelin receptor antagonistFree radicals

a b s t r a c t

Reactive oxygen species (ROS) formed during acute high altitude exposure contribute to cerebral vascularleak and development of acute mountain sickness (AMS). Nuclear factor (erythroid-derived 2)-relatedfactor 2 (Nrf2) is a transcription factor that regulates expression of greater than 90% of antioxidant genes,but prophylactic treatment with Nrf2 activators has not yet been tested as an AMS therapy. Wehypothesized that prophylactic activation of the antioxidant genome with Nrf2 activators wouldattenuate high-altitude-induced ROS formation and cerebral vascular leak and that some drugs currentlyused to treat AMS symptoms have an additional trait of Nrf2 activation. Drugs commonly used to treatAMS were screened with a luciferase reporter cell system for their effectiveness to activate Nrf2, as wellas being tested for their ability to decrease high altitude cerebral vascular leak in vivo. Compounds thatshowed favorable results for Nrf2 activation from our screen and attenuated high altitude cerebralvascular leak in vivo were further tested in brain microvascular endothelial cells (BMECs) to determine ifthey attenuated hypoxia-induced ROS production and monolayer permeability. Of nine drugs tested,with the exception of dexamethasone, only drugs that showed the ability to activate Nrf2 (Protandim,methazolamide, nifedipine, amlodipine, ambrisentan, and sitaxentan) decreased high-altitude-inducedcerebral vascular leak in vivo. In vitro, Nrf2 activation in BMECs before 24 h hypoxia exposure attenuatedhypoxic-induced hydrogen peroxide production and permeability. Prophylactic Nrf2 activation iseffective at reducing brain vascular leak from acute high altitude exposures. Compared to acetazolamide,methazolamide may offer better protection against AMS. Nifedipine, in addition to its knownvasodilatory activities in the lung and protection against high altitude pulmonary edema, may provideprotection against brain vascular leak as well.

& 2013 Elsevier Inc. All rights reserved.

Acute mountain sickness (AMS) is a well-described syndromethat affects 60% of unacclimated individuals ascending to altitudesabove 8000 ft [1–4] with the prevalence increasing to 75% ofindividuals ascending to 12,000 ft [1–4]. The most commonsymptoms of AMS are headache, nausea, and fatigue [3]; however,in rare cases high altitude illness can progress to the life-threatening conditions of high altitude cerebral (HACE) [3] orpulmonary edema (HAPE) [4]. The decreased barometric pressureand subsequent reduction of available oxygen are the primarycausal factors of AMS, but the exact mechanism(s) by whichhypoxia induces AMS is unclear. Recently, it has been suggestedthat hypoxia-induced cerebral vascular leak and subsequent

astrocyte swelling in the trigeminal areas play a key role in thedevelopment of AMS [1]. This has led some investigators tohypothesize that hypoxia triggers increased production of reactiveoxygen species (ROS) in the brain, which are subsequentlyresponsible for endothelial cell barrier dysfunction, increasedcerebral vascular permeability, and astrocyte swelling [1,5–8].

An innate defense mechanism of the body against increasedoxidative stress is the activation of the nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) transcription factor. Nrf2 isresponsible for regulating the gene expression of phase II detox-ification enzymes and antioxidant proteins through an enhancersequence known as the antioxidant-responsive element (ARE) [9].Importantly, the ARE is a promoter element common to nearly allof the antioxidant enzymes, including peroxiredoxins, thioredoxins,catalase, glutathione peroxidase, and heme oxygenase-1 [9–11].Hence, Nrf2 has been termed “the master regulator” of the ARE-

Contents lists available at SciVerse ScienceDirect

journal homepage: www.elsevier.com/locate/freeradbiomed

Free Radical Biology and Medicine

0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.freeradbiomed.2013.05.024

n Corresponding author. Fax: +1 303 724 3693.E-mail address: David.Irwin@UCDenver.edu (D.C. Irwin).

Free Radical Biology and Medicine 63 (2013) 264–273

• Funded  by  DARPA  (Dept  of  Defense  Advanced  Research  Projects  Agency)  !

• Hypoxic  high  altitude  environment  can  induce  leaky  blood  vessels  in  lungs/brain  !

• “…Nrf2  activation  either  by  Protandim  or  from  ‘off-­‐target’  effects  of  other  compounds  before  high  altitude  or  hypoxia  exposure  decreased  cerebral  vascular  leak  in  vivo…”

“Protandim-mediated HO-1 induction involved

the presence of ARE sites in the HO-1 promoter and nuclear translocalization of the

transcription factor Nrf2”

“Overall, induction of antioxidant enzymes by

Protandim may serve as a practical and potent

approach for cancer prevention”

“Our results suggest that suppression of p53

and induction of MnSOD may play an

important role in the tumor

suppressive activity of

Protandim.”

After 6 months on Protandim: !

• TBARS reduced 48% !

• Osteopontin reduced 57% !

• PON1 increased 35% !

• MRI signal reduced 38%

“[Protandim] prevented fibrosis and capillary loss

and preserved [heart] function”

“Protandim blocks IH and reduces cellular proliferation to that of freshly isolated HSV. !Protandim treatment increased the activity of SOD, HO-1, and catalase 3-, 7-, and 12-fold, respectively, and decreased the levels of superoxide and the lipid peroxidation product 4-HNE.”

Don’t Take Antioxidants Make Antioxidants