Saul J. Karpen, M.D., Ph.D.Raymond F. Schinazi Distinguished Biomedical Chair

Professor of PediatricsHepDart Kona, Hawaii

December 6, 2017

The Re-vival of Bile Acid Based Therapeutics for Children and Adults

FXRRXRα

Disclosures

• Consultant– Intercept Pharmaceuticals– Retrophin– Albireo– Regulus

• NIH NIDDK Consortia– ChiLDReN (14 NA sites children with biliary atresia & other cholestatic diseases)

– NASH CRN (8 US sites adults & children with NASH)

Topics

• Bile acid (BA) primer: FXR, TGR5, ASBT, NTCP

– Including the microbiome …

• BA Roles & Targets in Cholestasis– FXR activators– ASBT (SLC10A2) & NTCP (SLC10A1) inhibitors– Cholic Acid (CA), UDCA & NorUDCA – FGF19 analogues– Potentiators & Chaperones

• BA Roles & Targets in NAFLD– FXR activators & inhibitors– ASBT inhibitors– TGR5 activators

• Cholesterol Bile acids (~ 14 enzymes)• > 95 % efficient• Specific BA transporters in Liver & Intestine• Each BA circulates 8-10 times a day• Luminal bacterial modifications• BA flux is relevant for:

• Feedback regulation of BA synthesis

• Bile flow

• Absorption of Fats & Vitamins ADEK

• Metabolism

ASBT

COOH

OH

OHH

HO

Cholic acidCA

HO

COOH

OHH

Chenodeoxycholic acid

CDCA

3 7

7

The enterohepatic circulation of bile acids

COOH

HO

OH

H

Deoxycholic acid

DCABacterialModification

HO

COOH

OHH

Ursodeoxycholic acid

UDCA

7β

Non-human,Rx

Primary

Primary

Key bile acids

Key Components of the Enterohepatic Circulation of Bile Acids

Fickert & Wagner J Hepatol 2017 Ridlon J Lipid Res 2006

Certain bacteria metabolize bile

acids and change active biliary components

CA + CDCA:70% of BAs in bile4% of BAs in feces

Biliary Bile acids

Fecal Bile acids

LXRFXR

MAPK

PI3K

PKC

TGR5

AP1FASTRAIL

PGC1α

CAR

VDR

PXR

CellSignaling

Apoptosis

NuclearReceptors

JNKp38MAPK

ERK1/2

SHPFGF19

S1PR2

Multiple Molecular Roles for Bile Acids

Bile acid based approaches

Trauner M, Fuchs CD, Halilbasic E, Paumgartner G. New therapeutic concepts … . Hepatology. 2017

Topics

• Bile acid (BA) primer: FXR, TGR5, ASBT, NTCP

– Including the microbiome …

• BA Roles & Targets in Cholestasis– FXR activators– ASBT (SLC10A2) & NTCP (SLC10A1) inhibitors– Cholic Acid (CA), UDCA & NorUDCA – FGF19 analogues– Potentiators & Chaperones

• BA Roles & Targets in NAFLD– FXR activators & inhibitors– ASBT inhibitors– TGR5 activators

See Ghosh. Abst 14, Thursday 10:30

NTCP

⬇BA import

BSEP

ost α & β

cyp7aLRH-1

shpSHPRXR FXR

bsep

OST α/β

RXR FXR

RXR LXR

RXR RAR

RXR FXR

ntcp

⬇BA synthesis

Hepatic FXR targeting to improve adaptation to BA retention

RXRα FXR

⬆BA export

⬆BA sinusoidal export

Thomas Pharm Res 2013

Mouse Liver ChIP-SEQ:• FXR: 7800 sites

FXR Deficiency Neonatal Cholestasis/Liver Failure

Nature Commun. 2016

2 Families:• Presentation at birth6 w of age

• ⬆ Direct Bilirubin • Coagulopathy• Mild ⬆ ALT & AST• Low GGT

• 2 died (5 weeks, 8 months)• 2 transplanted (4 & 22 months)

NR1H4: ARG176*

Absent BSEP expression

BSEPRXR FXR

New disease discovered

through exome sequencing.

Cholic Acid Chenodeoxycholic acid

Lithocholic AcidZ-Guggulsterone

FXR Agonists

FXR Antagonists

GW4064EC50: 4-10 µM 37-80 nM≈ 20 µM

IC50:

Deoxycholic acid19-50 µM

6-α-ethyl-CDCA (Obeticholic acid)99 nM

≈ 10 µM

Makishima Science 1999Parks Science 1999Wang Mol Cell 1999Urizar Science 2002Yu JBC 2002Pellicciari J Med Chem 2002Hawkins JCI 2002Dussault JBC 2003Downes Mol Cell 2003Carter Ped Res 2007

≈ 10-30 µMStigmasterol≈ 10 µM

Fexaramine25 nM

Fexarine38 nM

Fexarene36 nM

AGN312 µM

(also RXR)

Cholic Acid Therapy in BA Synthesis Defects (3 β HSD Deficiency): ⬇ hepatotoxic atypical bile acid intermediates

Adapted from Setchell KD. Adolf Windaus Prize Lecture 2004. In: Paumgartner G et al, eds. Bile Acid Biology and Its Therapeutic Implications. Proceedings of the Falk Symposium 141 (XVIII Internationale Bile Acid Meeting) held in Stockholm, Sweden, June 18-19, 2004. Netherlands: Springer; 2005:1-15.

Atypical Bile Acids(URINE)

cyp7aLRH-1

shpSHPRXR FXR

RXR LXR

⬇BA synthesisCholic acid is an FXR agonist ↓ CYP7A (↓ BA synthesis)

NEJM 2016: NCT01473524

93% Female

56 ±10 y

73 ±13 kgAlk Phos 324 ± 174 Serum BA 48±68

93% on UDCAPruritus Scores: Itch, PBC-40, 5-D Itch

OCA

NTCP Inhibition: Blocking Bile Acid Return to Hepatocytes

Karpen: adapted from: Liver Diseases in ChildrenSlijepcevic D, et al Hepatology. 2017; online 5.12.2017

• Myrcludex B: Potent NTCP inhibitor• Peptide derived from aa 2-47 of pre-S1 HBV sequence• Inhibits HBV (& HDV) entry into hepatocytes

• Note: NTCP (SLC10A1) S267F variant in 10% of Chinese resists HBV infection.

Li & Urban J Hepatology 2016

Myrcludex B in Human Volunteers

x

xx

xx

See Foster Abst. 22, CRV431

Fickert P. J Hepatol. 2017

NorUDCA for PSC: 12 week Phase 2 Trial

38 Sites12 European countries

159 Participants:• 40 UDCA naïve • 58 UDCA responders• 55 UDCA non-responders

Exclusions:• < 18 or > 80 y• Concurrent immunosuppressive meds• Recent endoscopic Rx• T Bili > 3, etc…

NorUDCA for PSC: Change in Alk Phos as Primary Endpoint

* : p < 0.01 compared to Placebo

# : p < 0.025 comparing 1500 to 500 mg

Plans for Phase 3 Study

ASBT inhibitors:

FXR & LXR Modulators

ASBT

A4250 Maralixibat; SHP625Elobixibat; A3309

Volixibat; SHP626 GSK2330672

ASBT inhibitors in clinical trials

Clinical trials.gov

Diseases:Alagille SyndromePFIC1PFIC2Biliary Atresia

PBCPSC

NASH

Constipation

ASBT inhibition in the Abcb4-/- mouse: 2 mouse models of PFIC3

Miethke, Hepatology 2015

ASBTi fecal BA loss & & altered Liver BAs

Baghdasaryan, Journal of Hepatology, 2016

ASBTi Fibrosis & Inflammation

Lancet 2017: NCT01899703

11 pts

10 pts

19 F/ 2 M 52 ±10 y 73 ±13 kgAlk Phos 264 ± 174 IU/LSerum BA 48±68 µMPruritus Scores: Itch, PBC-40, 5-D Itch

Dose escalation: 45 mg po BID Days 1-390 mg po BID Days 4-14

ASBT Inhibitors for Pruritus

5-D Itch ScaleSerum BA

C4

PBC: 14 days of ASBT inhibition pruritus & serum BA levels

Note:ASBTi serum UDCA & DCA

No change in liver indices

Topics

• Bile acid (BA) primer: FXR, TGR5, ASBT, NTCP

– Including the microbiome …

• BA Toxicity & Targets in Cholestasis– FXR activators– ASBT (SLC10A2) & NTCP (SLC10A1) inhibitors– Cholic Acid (CA), UDCA & NorUDCA – FGF19 analogues– Potentiators & Chaperones

• BA Roles & Targets in NAFLD– FXR activators & inhibitors– ASBT inhibitors– TGR5 activators

NHANES data: 1988 to 2010 Prevalence of NAFLD among U.S. Adolescents

0

2

4

6

8

10

12

1988-94 1999-02 2003-06 2007-10

% S

uspe

cted

NA

FLD

ALT >25.8 U/L for boys; >22.1 U/L for girls

Welsh, Karpen, Vos, J Ped 2013Source: Dr. Miriam Vos

Obese (& Normal) Adolescents Adults with CV Disease

NEJM 2016

• Israeli Army Recruits• 1967-2010• Mean Age: 17 yo• 60% Male BMI %ile CV Mortality

Hazard Ratio • 50th-74th 1.32 (1.2-1.5)• 75th-84th 1.76 (1.5-2.0)• 85th-94th 2.25 (2.0-2.6)• > 95th 3.46 (2.9-4.1)

J Hepatol. 2016;65:363–368.

BMI > 25 is a risk factor for the development of

liver disease

Roles for bile acid signaling in addressing NAFLD & NASH

FXRTGR5ASBTGLP1

CholestasisGlucose MetabolismFat MetabolismCholesterol KineticsInsulin Resistance

LiverIntestineVisceral FatMuscle

OCA

J Clin Invest. 2015;125:386–402.

Intestinal FXR Antagonism improves NASH in mice

Intestinal FXR Agonism improves NASH in mice

Essential, but seemingly contradictory effects of FXR & BA signaling in NAFLD

Intestinal FXR ko protects against HFD-induced hepatic steatosis

Nat Med. 2015;21:159–165.

Fexaramine (Intestinal FXR agonist) improves HFD-induced hepatic steatosis

NAFLD & NASH:

FXR Agonism or FXR Antagonism

Both work Why?

Intact Enterohepatic BA Recirculation

ASBT

Ileal ASBTInhibition

ASBT

Interrupted EnterohepaticBA Recirculation

Ileal ASBT inhibition will improve the hepatic

and whole body response to HFD in

mice

Hypothesis:

ColonBA’sMicrobial BA metab.TGR5 signaling

BA Pool size

LiverBA Synthesis Cholesterol

IleumBA UptakeFXR-FGF15/19 signaling

ALIOS (45% fat; 0.2% cholesterol), + Added Sugars in the Drinking Water

Chow

HFD

ASBTi [SC-435] x 16wk

HFDASBTi

HFD:

ASBTi: 0.006% SC-435, 10 mg/kg/day

Mice: Male, C57BL6J, 4-6 weeks, n=7-16/group

4 8 12 16. . . ..0Weeks • Weekly Body Weights

• Weekly Caloric & fluid intake

• Week 15 GTT, ITT

• Week 16• Serum Liver Indices• Feces Bile Acids• Ileum RNA• Colon RNA• Liver Histology

• Lipids & Bile Acids

• RNA-Seq• RNA & Protein• Hydroxyproline

• Statistics: Mean ± SD• ANOVA

Tetri LH. Am J Physiol GI 2008 Nov;295(5):G987–95.

Mells JE J Nutr Biochem. 2015 Mar;26(3):285–92.

Study Design & Endpoints

Rao A et al. Sci Transl Med 2016; 357: ra122.

Liver

0

1

2

3

4

Chow HFD HFDASBTi

a

b

a

Cyp7a1

0

1

2

3

4 Cyp8b1

a

b

a

Chow HFD HFDASBTi

Rel

ativ

e ge

ne e

xpre

ssio

n

Colon

0

5

1 0

1 5

2 0 Ilbp

a a

b

Chow HFD HFDASBTi

Rel

ativ

e ge

ne e

xpre

ssio

n

ASBT

Feca

l Bile

Aci

ds (µ

M/2

4 hr

) Feces

HFD

a

b

0

1

2

3Fgf15

a

b

c

Chow HFD HFDASBTi

Ileum

Rel

ativ

e ge

ne e

xpre

ssio

n

HFDASBTi

Chow HFD

Shp

ab

a

SC-435 Inhibits Ileal ASBT Function

HFDASBTi

Glu

cose

(mg/

dL)

Time (mins)

Chow

HFD + ASBTiHFD

*

ITT

AUC

a,bb* * *

a

**

*

Chow

HFD + ASBTiHFD

GTT

AU

C a

b

a

* Significantly different from HFD + Asbti

GTT

ITT

ASBTi Restores Glucose Tolerance

Chow HFD HFDASBTi

Chow HFD HFD + ASBTi

NAFLD Activity Score (NAS)

Chow HFD HFDASBTi

a

b

c

ASBTi Improves Hepatic NAFLD Activity & Steatosis Scores

Triglycerides Cholesteryl Esters(µ

g/m

g liv

er)

(µg/

mg

liver

)

a

b

a

a

b

a

Chow HFD HFDASBTi

Chow HFD HFDASBTi

Chow HFD HFDASBTi

(pm

ol/m

g liv

er ti

ssue

)

Total Bile Acids

FXR AntagonistFXR Agonist

HFD

58% 42%

HFD + Asbti

83%

17%

Bile

Aci

d (p

mol

/mg

tissu

e)

* * * * *α-TMCA β-TMCA ω-TMCA THDCA TUDCA α-MCA β-MCA

FXR Antagonist

HFDHFD + Asbti

TCA TCDCA TDCA TLCA CA

*

*

*

FXR Agonist

*

ASBTi Markedly Alters Hepatic BA Composition

HFD

Insulin resistance

Hyperglycemia Hyperinsulinemia

ChREBP

Lipogenic genes TG

SREBP-1c LXR Hepatic cholesterol

FXR Hepatic BA Composition

ASBTi

ASBTi

ASBTi

Hepatic Steatosis

ASBT

Hypothesized Mechanisms of Action of ASBTi in Liver

FXR Antagonist

FXR Agonist

HFD

58% 42%

HFD + Asbti

83%

17%

Ileal ASBT inhibition Markedly Alters Hepatic BA Composition

TMCA’s THDCA TUDCA

TCA TCDCA TDCA

Summary: Re-vival of Bile Acid Biology & New Therapeutics• Bile acid (BA) biology: FXR, TGR5, ASBT, NTCP

– Gut-Liver-Microbe-Gene Axis involves BAs

– Differential effects in Ileum, Colon, Liver, Fat, …

– Individual BA’s have distinct functional properties

• Cholestasis– Roles for FXR Agonism, ASBT inhibition, norUDCA

– ? NTCP Inhibition

• NAFLD– Complex roles for FXR Agonism & Antagonism– Roles for ASBT inhibition (Await human study: Shire, SHP626: NCT02787)– ? NTCP inhibition (CRV231)

Saul Karpen, MD, PhDPaul Dawson, PhDAstrid Kosters, PhDAnuradha Rao, PhDAngelica Amanso, PhDJP Berauer, MDGina Ramirez

Funding (NIH)• R01 DK056239 • R01 DK047987• Philanthropies: • Alpard Foundation• Spain Fund• Moss Fund

Anya Mezina,MD MSCRCourtney FerrebeeJamie Mells, PhDKim PachuraJianing Li, PhDGrace WynnPrabhu Shankar, MD

Emory University (Saul-Paul Lab)

Hong Yin, MD (Pathology)

Dean Jones, PhD (Metabolomics)Sophia BantonShuzhao Li

Hao Wu, PhD (School of Public Health)

Emory University

Brad Keller, PhD (Lumena/Shire)

Ken Setchell, PhD

Wujuan Zhang, PhD

Cincinnati Children’s