The Re-vival of Based Therapeutics for Children and Adults

RXRα FXR

Saul J. Karpen, M.D., Ph.D. Raymond F. Schinazi Distinguished Biomedical Chair Professor of Pediatrics HepDart Kona, Hawaii December 6, 2017 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 Key bile acids The enterohepatic circulation of bile acids Cholic acid CA OH COOH  Primary • Cholesterol Bile acids (~ 14 enzymes)

7 • > 95 % efficient HO OH H • Specific BA transporters in Liver & Intestine 3 7 • Each BA circulates 8-10 times a day COOH CDCA • Luminal bacterial modifications Primary

HO OH H • BA flux is relevant for: • Feedback regulation of BA synthesis Deoxycholic acid OH • Bile flow DCA COOH Bacterial • Absorption of Fats & Vitamins ADEK Modification • HO Metabolism H

Ursodeoxycholic acid ASBT

COOH UDCA Non-human,

HO OH Rx H 7β Key Components of the Enterohepatic Circulation of Bile Acids Biliary Bile acids

Certain bacteria metabolize bile acids and change active biliary components

CA + CDCA: Fecal Bile acids 70% of BAs in bile 4% of BAs in feces

Fickert & Wagner J Hepatol 2017 Ridlon J Lipid Res 2006 Multiple Molecular Roles for Bile Acids SHP FGF19 LXR JNK Nuclear PXR FXR p38MAPK Receptors ERK1/2 VDR MAPK Cell CAR PI3K Signaling

PGC1α PKC

TRAIL TGR5 S1PR2 FAS AP1

Apoptosis 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 See Ghosh. Abst 14, – ASBT inhibitors Thursday 10:30 – TGR5 activators Hepatic FXR targeting to improve adaptation to BA retention

BA import

⬇ BA export NTCP ntcp bsep RXR RAR RXR FXR ⬆ BSEP RXRα FXR shp RXR FXR SHP ost α & β RXR FXR cyp7a LRH-1 RXR LXR BA sinusoidal export Mouse Liver ChIP-SEQ: ⬆ BA synthesis • FXR: 7800 sites OST α/β ⬇

Thomas Pharm Res 2013 FXR Deficiency  Neonatal Cholestasis/Liver Failure

NR1H4: ARG176*

New disease Absent BSEP expression 2 Families: discovered  • Presentation at birth 6 w of age BSEP through exome • Direct Bilirubin RXR FXR sequencing. • Coagulopathy ⬆ • Mild ALT & AST • Low GGT ⬆ • 2 died (5 weeks, 8 months) • 2 transplanted (4 & 22 months)

Nature Commun. 2016 FXR Agonists

6-α-ethyl-CDCA Cholic Acid Chenodeoxycholic acid Deoxycholic acid GW4064 ≈ µ µ µ () EC50: 20 M 4-10 M 19-50 M 99 nM 37-80 nM

AGN31 Fexaramine Fexarine Fexarene 2 µM 25 nM 38 nM 36 nM (also RXR)

Makishima Science 1999 FXR Antagonists Parks Science 1999 Wang Mol Cell 1999 Urizar Science 2002 Yu JBC 2002 Pellicciari J Med Chem 2002 Hawkins JCI 2002 Z- Lithocholic Acid Stigmasterol Dussault JBC 2003 ≈ µ ≈ µ ≈ µ Downes Mol Cell 2003 IC50: 10 M 10-30 M 10 M Carter Ped Res 2007 Cholic Acid Therapy in BA Synthesis Defects (3 β HSD Deficiency):  hepatotoxic atypical bile acid intermediates Atypical Bile Acids (URINE)⬇

shp RXR FXR SHP

cyp7a LRH-1 RXR LXR Cholic acid is an FXR agonist BA synthesis  ↓ CYP7A (↓ BA synthesis) ⬇

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. OCA

93% Female

56 ±10 y

73 ±13 kg Alk Phos 324 ± 174 Serum BA 48±68

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

NEJM 2016: NCT01473524 NTCP Inhibition: Blocking Bile Acid Return to Hepatocytes See Foster Abst. 22, x CRV431 x x

Myrcludex B in Human Volunteers x x

• 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.

Karpen: adapted from: Liver Diseases in Children Slijepcevic D, et al Hepatology. 2017; online 5.12.2017 Li & Urban J Hepatology 2016 NorUDCA for PSC: 12 week Phase 2 Trial

38 Sites 12 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… Fickert P. J Hepatol. 2017 NorUDCA for PSC: Change in Alk Phos as Primary Endpoint

Plans for Phase 3

* : p < 0.01 compared to Placebo Study

# : p < 0.025 comparing 1500 to 500 mg ASBT inhibitors:

FXR & LXR Modulators ASBT ASBT inhibitors in clinical trials

Clinical trials.gov

Diseases: Alagille Syndrome PFIC1 PFIC2 A4250 Elobixibat; A3309 Maralixibat; SHP625 Biliary Atresia

PBC PSC

NASH

Volixibat; SHP626 GSK2330672 Constipation ASBT inhibition in the Abcb4-/- mouse: 2 mouse models of PFIC3 ASBTi  fecal BA loss &  & altered Liver BAs

ASBTi   Fibrosis & Inflammation

Miethke, Hepatology 2015 Baghdasaryan, Journal of Hepatology, 2016 ASBT Inhibitors for Pruritus

19 F/ 2 M 52 ±10 y 73 ±13 kg Alk Phos 264 ± 174 IU/L Serum BA 48±68 µM Pruritus Scores: Itch, PBC-40, 5-D Itch

11 pts

10 pts

Dose escalation: 45 mg po BID Days 1-3 90 mg po BID Days 4-14

Lancet 2017: NCT01899703 PBC: 14 days of ASBT inhibition   pruritus & serum BA levels

Serum BA 5-D Itch Scale

C4

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 12

10

8

6

4

2 % Suspected NAFLD

0 1988-94 1999-02 2003-06 2007-10 ALT >25.8 U/L for boys; >22.1 U/L for girls

Source: Dr. Miriam Vos Welsh, Karpen, Vos, J Ped 2013 Obese (& Normal) Adolescents  Adults with CV Disease

• 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) NEJM 2016 BMI > 25 is a risk factor for the development of liver disease

J Hepatol. 2016;65:363–368. Roles for bile acid signaling in addressing NAFLD & NASH

FXR TGR5 Cholestasis ASBT Glucose Metabolism GLP1 Fat Metabolism Cholesterol Kinetics Insulin Resistance Liver Intestine Visceral Fat Muscle OCA

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

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

Intestinal FXR Antagonism Intestinal FXR Agonism improves NASH in mice improves NASH in mice

J Clin Invest. 2015;125:386–402. Nat Med. 2015;21:159–165. NAFLD & NASH:

FXR Agonism or FXR Antagonism

Both work  Why? Intact Enterohepatic Interrupted Enterohepatic BA Recirculation BA Recirculation

 BA Pool size

Ileal ASBT Liver Inhibition BA Synthesis  Cholesterol

Ileum BA Uptake FXR-FGF15/19 signaling Hypothesis: ASBT ASBT Ileal ASBT inhibition will improve the hepatic Colon and whole body BA’s response to HFD in Microbial BA metab. TGR5 signaling mice Study Design & Endpoints

Weeks • Weekly Body Weights 12 .0 .4 8. . 16. • Weekly Caloric & fluid intake

Chow • Week 15 GTT, ITT

• Week 16 HFD • Serum Liver Indices • Feces Bile Acids HFD • Ileum RNA ASBTi • Colon RNA ASBTi [SC-435] x 16wk • Liver Histology • Lipids & Bile Acids Mice: Male, C57BL6J, 4-6 weeks, n=7-16/group • RNA-Seq • RNA & Protein • Hydroxyproline HFD: ALIOS (45% fat; 0.2% cholesterol), + Added Sugars in the Drinking Water • Statistics: Mean ± SD • ANOVA

ASBTi: 0.006% SC-435, 10 mg/kg/day Tetri LH. Am J Physiol GI 2008 Nov;295(5):G987–95. Mells JE J Nutr Biochem. 2015 Mar;26(3):285–92. Rao A et al. Sci Transl Med 2016; 357: ra122. SC-435 Inhibits Ileal ASBT Function Liver Colon 4 Cyp7a1 4 Cyp8b1 b 20 Ilbp b b 3 3 15

2 2 a a 10 a a

1 1 5 a

Relative gene expression gene Relative a Relative gene expression gene Relative 0 0 0 Chow HFD HFD Chow HFD HFD Chow HFD HFD ASBTi ASBTi ASBTi

Fgf15 Ileum Shp

3 ) Feces

b a hr b

ASBT M/24

2 µ

a

1 a a b c Fecal Bile Acids ( Acids Bile Fecal Relative gene expression gene Relative 0 Chow HFD HFD Chow HFD HFD HFD HFD ASBTi ASBTi ASBTi GTT ITT

Glucose (mg/dL) Chow HFD HFD + Chow HFD HFD + ASBTi * * Significantly different from HFD +Asbti ASBTi ASBTi Time (mins) * * Restores GlucoseRestores Tolerance * * * *

ITT AUC GTT AUC Chow a,b a HFD b a ASBTi HFD a b ASBTi Improves Hepatic NAFLD Activity & Steatosis Scores Chow HFD HFD + ASBTi

NAFLD Activity Score Triglycerides Cholesteryl Esters Total Bile Acids (NAS) b b b

c a g/mg liver) g/mg g/mg liver) g/mg µ µ ( ( a a a a tissue) liver (pmol/mg

Chow HFD HFD Chow HFD HFD Chow HFD HFD Chow HFD HFD ASBTi ASBTi ASBTi ASBTi ASBTi Markedly Alters Hepatic BA Composition

FXR Agonist FXR Antagonist /mg tissue)

pmol * HFD HFD + Asbti Bile Acid ( Acid Bile * * * * * * * * TCA TCDCA TDCA TLCA CA α-TMCA β-TMCA ω-TMCA THDCA TUDCA α-MCA β-MCA

17% 58% 42% 83% FXR Agonist FXR Antagonist

HFD HFD + Asbti Hypothesized Mechanisms of Action of ASBTi in Liver

HFD

ASBTi Insulin resistance

ASBTi Hyperglycemia Hyperinsulinemia

Hepatic LXR SREBP-1c cholesterol

ChREBP FXR Hepatic BA Composition

Hepatic Steatosis Lipogenic genes TG ASBTi

Ileal ASBT inhibition Markedly Alters Hepatic BA Composition ASBT

FXR Agonist 17% TCA TCDCA TDCA 58% 42% 83% FXR Antagonist TMCA’s THDCA TUDCA HFD HFD + Asbti 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) Emory University (Saul-Paul Lab)

Saul Karpen, MD, PhD Anya Mezina,MD MSCR Paul Dawson, PhD Courtney Ferrebee Astrid Kosters, PhD Jamie Mells, PhD Anuradha Rao, PhD Kim Pachura Angelica Amanso, PhD Jianing Li, PhD JP Berauer, MD Grace Wynn Gina Ramirez Prabhu Shankar, MD

Emory University Hong Yin, MD (Pathology)

Dean Jones, PhD (Metabolomics) Sophia Banton Shuzhao Li

Hao Wu, PhD (School of Public Health)

Brad Keller, PhD (Lumena/Shire)

Cincinnati Children’s

Ken Setchell, PhD Funding (NIH) R01 DK056239 Wujuan Zhang, PhD • • R01 DK047987 • Philanthropies: • Alpard Foundation • Spain Fund • Moss Fund