Novel Treatment Targets for Novel Treatment Targets for Non Alcoholic SteatohepatitisNon Alcoholic Steatohepatitis

Natalie J. Török, MDProfessor

Gastroenterology and HepatologyUC Davis, Northern California VA,

Mather

Cirrhosis MortalityCirrhosis Mortality

Cirrhosis MortalityCirrhosis Mortality

Mortality by underlying and multiple cause, )

Economic BurdenEconomic Burden

• Direct cost (medications, hospitalizations): $2.5 billion

• Indirect cost (loss of productivity): $10.6 billion*

*Ruhl et al: The Burden of Digestive Diseases in the United States. Washington, DC: NIH publication; 2008. pp. 137–143. US Dept. of Health and Human Services, NIH, and NIDDK. 09-6443.

Obesity Epidemic in the U.S.Obesity Epidemic in the U.S.

http://www.cdc.gov/diabetes/statistics.

NAFLD WorldwideNAFLD Worldwide

NAFLD/NASHNAFLD/NASH

• The expected prevalence of NASH in the USA, in 2025 could be > 25 million people.

• This prevalence would exceed by 10-fold the current US prevalence of HCV.

Non Alcoholic Fatty Liver Non Alcoholic Fatty Liver Disease (NAFLD)Disease (NAFLD)

NAFLDSteatosis(macrovesicular)

Non Alcoholic Steatohepatitis(NASH):

Steatosis,Inflammation, Ballooning,+ Mallory hyaline+ Fibrosis

Cirrhosis

Inflammation,Fibrosis StageIV+ Steatosis

Spectrum of disease

?

HCC

Predictors of fibrosis in NASHPredictors of fibrosis in NASH

Independent predictors:– Age– Obesity – Diabetes– AST/ALT>1

Angulo et al, Hepatology, 30, 1356, 1999

Treatment of NASH Treatment of NASH

• Lifestyle modifications

• Surgery (Roux-en Y gastrojejunostomy)

• Medications– Protection of hepatocellular function– Targeting the microbiome– Anti fibrotics

Weight LossWeight Loss

• In obese patients, weight loss remains the main therapy.

• Loss of 5–10% of body weight decreases liver fat by 40–80%.

• Weight loss pills: don’t work (no effect on histology).

The Role of Bariatric SurgeryThe Role of Bariatric Surgery

• Roux-en-Y gastric bypass

• Significant improvement in histology after gastrojejunostomy

• Possible to perform in Child A cirrhotics (rule out significant portal hypertension)

Medical Treatment of NASHMedical Treatment of NASHLipid-lowering

  Gemfibrozil 46 600 mg/day 4 weeks Yes NP

  Clofibrate 16 2 g/day 12 months No No

Insulin-sensitizing

 * Troglitazone 400 mg/day 3 -6 months Yes Yes

  Metformin 500 mg t.d.s. 4 months Yes NP

Hepatoprotective

  *UDCA 24 13 -15 mg/kg/day 12 months Yes Yes

  UDCA 24 250 mg t.d.s. 12 months Yes No

  UDCA + diet 24 10 mg/kg/day 6 months Yes NP

  UDCA + diet 31 10 mg/kg/day 6 months Yes NP

  N-Acetylcysteine 11 1 g/day 3 months Yes NP

  Betaine  8 20 g/day 12 months Yes Yes

  Vitamin E 11* 400 -1200 IU/day 4 -10 months Yes NP

   * Tocopherol 22 300 mg/day 12 months Yes Yes   

Transam. Histology

Obeticholic AcidObeticholic Acid• Farnesoid X Receptor (FXR) agonism

(obeticholic acid): improvement of NASH activity score (NAS) and ALT*

• NASH resolution in 22%

• Improvement in fibrosis in the diabetic subpopulation

• Increase in LDL cholesterol

*Neuschwander-Tetri BA et al: Lancet. 2015;385:956–65.

The Role of the MicrobiomeThe Role of the Microbiome

Bacterial 16S DNABacterial 16S DNALPSLPSPeptidoglycanPeptidoglycan

TLRs

HepatocyteInjury

HSC Activation

Macrophage Activation

ROSROSTNFTNFααIL-1IL-1ββIL-6IL-6

Permeability

Probiotics and synbiotics Probiotics and synbiotics

• Improvement of transaminases

• ? Improvement in inflammation or fibrosis*

* Ferolla et al: World J Hepatol. 2015 Mar 27;7(3)

Targeting Fibrosis-PathogenesisTargeting Fibrosis-Pathogenesis

Quiescent HSCSEC

Active HSC

NormalLiver

Fibrosis

Macrophage

NADPH Oxidases as Core Inducers of NADPH Oxidases as Core Inducers of Stellate Cell ActivationStellate Cell Activation

Phagocytosis

Leptin** NOXs

Angiotensin II*

PDGF***

LPS

AGEs##

**De Minicis et al: Hepatology, 2008 Dec;48(6):2016-26***Adachi et al: Hepatology. 2005 Jun;41(6):1272-81# Becker et al: Cell Physiol Biochem. 2007;19(1-4):89-98.##Jiang et al: Gastro, 2010, Hepatology, 2013 Oct;58(4):1339-48, Bettaieb; Gastro, 2015.

*Bataller R et al: J Clin Invest. 2003 Nov;112(9):1383-94

EtOHBile acid#

HCV

Altenhofer et al: Cellular and Molecular Life Sciences Vol. 69 Issue 14, 2012

NADPH Oxidases (NOXs)NADPH Oxidases (NOXs)

Duox1Duox2

Phagocytic NOX:HSCKupffer cells (liver macrophages)

HSCHepatocytes

HepatocytesHSC,SECs

NADPH + 2O2 ↔ NADP+ + 2O2- + H+

NADPH oxidase 4NADPH oxidase 4

NOX4

p2

2

O2 .-

H2O2.-

O .-

NADPH NADPH+

• Expressed both in hepatocytes and activated stellate cells• Transcriptionally regulated• Produces H2O2

NOX4 is induced by TGFNOX4 is induced by TGFββ in Stellate in Stellate Cells Cells

NO

X4/

Arb

p (

fold

)N

OX

4/A

rbp

(fo

ld)

Day1Day1 Day 8Day 8

NTNT TGF-βTGF-β

NO

X4/

Arb

p (

fold

)N

OX

4/A

rbp

(fo

ld)

DN Smad3DN Smad3

****

****

**

Empty vectorEmpty vector

+TGF-β+TGF-β

Wt Smad3Wt Smad3

*p<0.05, **p<0.01

*

NOX4NOX4

?

Coll.

0

2

4

6

8

10

0

2

4

6

0

1

2

3

4

5

Collagen1A1/β-actin

αSMA/β-Actin

Nox4/β-Actin

*

*

*

8days 1day1day 8days

Nox4-/-WT

****

##

NOX4 induces Fibrogenic TranscriptsNOX4 induces Fibrogenic Transcripts

*p<0.05, #p<0.01,**p<0.001

Jiang et al: Free Radic Biol Med. 2012 Jul 15;53(2):289-96.

Collagen Collagen αα I (2) I (2)

Scr si

NOX4 si

Scr si+cata

NOX4 si+cata

Scr si

NOX4 si

FL

MT

Non si

Scr+cata

No si

*P<0.005*P<0.005

**

Luciferase Intensity (fold)

NOX4 in NOX4 in hepatocyteshepatocytes??

Biopsy samples from patients

Animal Models:Hepatocyte-specific NOX4 deletionChow dietFast food diet

Studies on insulin sensitivity

In vitro Studies

NOX4

?

Stress signaling

Insulin sensitivity

NOX4 Expression is Increased in NASHNOX4 Expression is Increased in NASH

HealthyControl

NASHNO

X4/

B2M

(fo

ld) *

control NASH

Simplesteatosis

*

Chow FFD

NOX4

GAPDH 38kD

67kD

**N

OX

4/G

AP

DH

(f

old)

Chow FFD

*

Chow FFD

NO

X4/

Arb

p(f

old)

NO

X4/

Arb

p(f

old)

Bettaieb et al: Gastroenterology. 2015 Apr 14,

E-cadherin+NOX4

NL

NASH

CK19+NOX4αSMA+NOX4

NOX4 is Induced in Hepatocytes and NOX4 is Induced in Hepatocytes and Stellate Cells in Patients with NASHStellate Cells in Patients with NASH

Oxidative Radicals and Lipid Peroxidation are Oxidative Radicals and Lipid Peroxidation are Decreased Decreased

in the in the NOX4NOX4hepKOhepKO Mice MiceTo address the specific role(s) of NOX4 in hepatocytes: NOX4floxp X Alb-Cre → NOX4hepKO

• Fast food diet• Chow/CSAA/CDAA diet

WTNOX4hepKO

WTNOX4hepKO

AL

T (

U/L

)A

LT

(U

/L)

Chow FFD

**

WTNOX4hepKO

Lu

cig

enin

In

ten

sit

y(f

old)

Chow FFD

**

MD

A (

fold

)

Chow FFD

**

*p<0.05*p<0.05

Inflammation and Apoptosis are Decreased in Inflammation and Apoptosis are Decreased in the NOX4the NOX4hepKOhepKO Mice Mice

***

MC

P1

/AC

T(f

old)

****

TN

Fα

/AC

T(f

old)

Chow FFD Chow FFD

***

WTWT NOX4hepKONOX4hepKO

Act

ive

casp

3 p

osi

tive

cel

ls(f

old)

Act

ive

casp

3 p

osi

tive

cel

ls(f

old)

NOX4hepKONOX4hepKO

wt

*P<0.05**P<0.01***p<0.001

Fibrosis is Decreased in the NOX4Fibrosis is Decreased in the NOX4hepKOhepKO

MiceMice O

H-P

rolin

e (m

g/gm

live

r)O

H-P

rolin

e (m

g/gm

live

r) ***

Chow FFD

WTNOX4hepKO

ChowChow FFDFFD

WT

WT

NO

X4h

epK

ON

OX

4hep

KO

**

**

***T

GF

β/A

CT

(fol

d)T

GF

β/A

CT

(fol

d)α

SM

A/A

CT

(fol

d)α

SM

A/A

CT

(fol

d)

Chow FFD

Pro

colla

gen

α1(

I)/A

CT

(fol

d)

WTNOX4hepKO

*

*P<0.05**P<0.01

Steatosis is Unchanged in the NOX4Steatosis is Unchanged in the NOX4hepKOhepKO Mice on the Mice on the FFDFFD

ChowChow FFDFFD

WT

WT

NO

X4h

epK

ON

OX

4hep

KO

Chow FFD

* nsns

TG

Co

nte

nt

(fol

d)

*

Chow FFD

* **

SR

EB

P1c

(fol

d)

Chow FFD

*

FA

S/A

CT

(fol

d)

PP

AR

γ/A

CT

(fol

d)

*

CD

36/A

CT

(fol

d)

Chow FFD Chow FFD

WTNOX4hepKO

***

WTNOX4hepKO

NOX4hepKO FFDWT FFD

*P<0.05**P<0.01

The Role of NOX4 in Hepatocyte The Role of NOX4 in Hepatocyte Stress SignalingStress Signaling

PKR

eIF2αJNK1

H2O2

NO

X4

O2

p

pp

FA

?PERKp

Insulin signaling

apoptosis

?

0

1

2

3

BSA Pal

pGL-hNOX4-Luc

Lu

cife

rase

ac

tivi

ty

(fol

d)

Ctrl A

d

+BSACtrl

Ad

+Pal

dnSmad3

+Pal

*

* *

Ctrl A

d

Ctrl A

d

+TGFβ

dnSmad3

+TGFβ

* *

NOX4 is induced by Palmitate but not by OleateNOX4 is induced by Palmitate but not by Oleate

Lu

cife

rase

ac

tivi

ty

(fol

d)

NT BSA OA

pGL-hNOX4-Luc

WT NOX4hepKO

Chow

pPKR

pPERK

PERK

PKR

pEiF2α

EiF2α

CHOP

Tub

FFD Chow FFD

pJNK1/2

JNK1/2

Active Casp. 3

NOX4NOX4hepKOhepKO Mice on the FFD are Protected from ER- Mice on the FFD are Protected from ER-stress, stress,

JNK Activation and ApoptosisJNK Activation and Apoptosis

NOX4NOX4hepKOhepKO Mice on the FFD are Protected from ER- Mice on the FFD are Protected from ER-stress, stress,

JNK Activation and ApoptosisJNK Activation and Apoptosis

The PKR/PERK phosphatase PP1c The PKR/PERK phosphatase PP1c activity is Decreased by NOX4/Hactivity is Decreased by NOX4/H22OO22

PP

1c

Ph

osp

hat

ase

act

ivit

y

NTBSAH 2

O 2Pal

GSH+P

al NTBSAPal

**

* WTNOX4-/-

p<0.05

Insulin Sensitivity is Improved in the Insulin Sensitivity is Improved in the

NOX4NOX4hepKOhepKO Mice Mice

0

200

400

600

3030 6060 9090 12012000

Blo

od

Glu

cose

(m

g/dL

) *

0

50

100

150

3030 6060 909000% o

f B

asal

Blo

od

G

luco

se

WTWT

NOX4hepKONOX4hepKO*

* ** *A

UC

(fo

ld)

WT NOX4hepKO

*

WT NOX4hepKO

AU

C (

fold

)

GTT

ITT

WTWT

NOX4hepKONOX4hepKO

(min)(min)

*

(min)(min)

*P<0.05**p<0.01*P<0.05**p<0.01

NOX4 in NASHNOX4 in NASH

PKR

eIF2αJNK1

Caspase-3

H2O2

NO

X4

O2

p

pp

CHOP

Apoptosis

FA

NASH fibrosis

PP1c

p

p

IR

IRS-1

Akt

PERKp

ER stress ER stress signalingsignaling

Stress Stress signalingsignaling

Insulin Insulin resistanceresistance

NOX inhibitionNOX inhibition

• Prolonged NOX2 inhibition may cause increase in infections.

• NOX4 inhibition:– Anti fibrogenic– Anti apoptotic– GKT137831, member of the

pyrazolopyridine dione family, inhibits NOX1/4, orally available (GenKyoTex, Geneva).

ControlControl GKT137831 GKT137831

****R

OS

Pro

du

cti

on

(f

old

)T

arg

et/H

ou

seke

ep

ing

(fo

ld)

##

****

******

CollagenCollagen

αSMAαSMA

TGFβ1TGFβ1

ControlControl GKT137831 GKT137831

GKT137831 Reduces ROS production GKT137831 Reduces ROS production and activation of HSC and activation of HSC in vitroin vitro

*p=0.01, **p<0.01, ***p<0.001

Ap

op

tosi

s R

ate

(%)

Wt control

FasL GKT137831

FasL

**

GKT137831 Reduces Hepatocyte GKT137831 Reduces Hepatocyte ApoptosisApoptosis

*p<0.05

Inhibition of NOX4 Improves Inhibition of NOX4 Improves SteatohepatitisSteatohepatitis

AL

T (

U/L

)A

LT

(U

/L)

Chowveh

FFDveh

* *

Chowveh

FFDveh

FFDinh

TN

Fα

/Arb

p(f

old)

TN

Fα

/Arb

p(f

old)

*** *** ** **

Chowveh

FFDveh

FFDinh

*

Chowveh

FFDveh

FFDinh

IL1β

/Arb

p(f

old)

IL1β

/Arb

p(f

old)

FFDinh

MC

P1/

Arb

p(f

old)

MC

P1/

Arb

p(f

old)

NOX4 inhibitor 60 mg/kg, or vehicle:daily gavage from week 6 of the 12 week FFD

Fibrosis improves after NOX4 Fibrosis improves after NOX4 inhibitioninhibition

Pro

coll

age

nα

1(I)

/Arb

p(f

old)

Chowveh

FFDveh

FFDinh

Chowveh

FFDveh

FFDinh

αS

MA

/Arb

p(f

old)

αS

MA

/Arb

p(f

old)

Chowveh

FFDveh

FFDinh

TG

Fβ

/Arb

p(f

old)

TG

Fβ

/Arb

p(f

old)

** **** ** * ***

Chowveh

FFDveh

FFDinh

0100200300400500600

Glu

cose

(m

g/dL

)G

luco

se (

mg/

dL)

3030 6060 909000 120120 (Min)

050

100150200250300

3030 6060 909000 120120

FFDVehicle

FFDVehicle

FFDinhibitor

FFDinhibitor

* * **

Glu

cose

(m

g/dL

)G

luco

se (

mg/

dL)

GTT

ITT

**

FFDVehicle

FFDVehicle

FFDinhibitor

FFDinhibitor

(Min)

Insulin Sensitivity Improves after Insulin Sensitivity Improves after NOX4 InhibitionNOX4 Inhibition

NOX1/4 Inhibition in NASHNOX1/4 Inhibition in NASH

• Antifibrogenic

• Hepatocyte protective

• Improvement of insulin resistance

• Phase 2 trial in diabetic nephropathy is underway

Potential Future Agents for Potential Future Agents for NASHNASH

• glucagon-like peptide-1 (GLP-1) activator (incretins, Exenatide)-Phase II study-GI side effects.

• Selective cannabinoid receptor (CB) I antagonist-CP 945598

• CB II agonists

• 5HT-2B receptor antagonism, may inhibit fibrosis, enhance regeneration

• 1,25-(OH)2 Vitamin D3

SummarySummaryNovel Novel Approaches in NASH the FutureApproaches in NASH the Future

• NASH-specific approaches

• Fibrosis stage-specific approaches

• Combined treatment strategies

• Individualized treatment

Ahmed Bettaieb, PhD (UC Davis) Ahmed Bettaieb, PhD (UC Davis)

Fawaz Haj, PhD (UC Davis)Fawaz Haj, PhD (UC Davis)

Kathrin SchröderKathrin Schröder (Goethe (Goethe Universitat, Frankfurt am Main)Universitat, Frankfurt am Main)

Ralph Brandes, MD, PhD (Goethe Ralph Brandes, MD, PhD (Goethe Universitat, Frankfurt am Main)Universitat, Frankfurt am Main)

Dave Lambeth, PhDDave Lambeth, PhD(Emory University)(Emory University)

Masato Katsuyama, PhD (Kyoto Masato Katsuyama, PhD (Kyoto Prefectural University of Medicine, Prefectural University of Medicine, Kyoto, Japan)Kyoto, Japan)

Joy X. Jiang, MD, PhDJoy X. Jiang, MD, PhD

Tzu-I Chao, PhDTzu-I Chao, PhD

Sophie Kiss, BScSophie Kiss, BSc

Xiangling Chen, MD, PhDXiangling Chen, MD, PhD

Yu Sasaki, MD, PhDYu Sasaki, MD, PhD

Jijing Tian, PhDJijing Tian, PhD

Hiroo Fukada, MD, PhDHiroo Fukada, MD, PhD

Yannan Xi, BScYannan Xi, BSc

AcknowledgementsAcknowledgements

Blo

od

Glu

cose

(m

g/dL

)(min)(min)

Glu

cose

up

take

(mg/

kg/m

in)

WT NOX4hepKOWT NOX4hepKOC

lam

ped

Insu

lin(u

U/m

l)

GIR

[mg/

(kg.

min

)]

(min)(min)

NOX4hepKONOX4hepKO

WT

WT NOX4hepKO

GIR

(m

g/kg

/min

)

HG

P(m

g/kg

/min

)

WTNOX4hepKO

Basal Clamped

NOX4hepKONOX4hepKO

WT

Hyperinsulinemic-Euglycemic ClampHyperinsulinemic-Euglycemic Clamp