Saul J. Karpen, M.D., Ph.D.

The Re-vival of Bile Acid 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

Cholic acid CA Primary HO Chenodeoxycholic acid Primary Key bile acids HO Bacterial Modification H CDCA 7 H OH OH 3 7 OH Deoxycholic acid DCA HO H OH COOH COOH COOH The enterohepatic circulation of bile acids 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 Ursodeoxycholic acid ASBT Non-human, Rx UDCA HO H OH COOH 7β

Key Components of the Enterohepatic Circulation of Bile Acids Biliary Bile acids Certain bacteria metabolize bile acids and change active biliary components Fecal Bile acids CA + CDCA: 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 Nuclear Receptors CAR VDR PXR LXR FXR MAPK SHP FGF19 PI3K JNK p38 MAPK ERK1/2 Cell Signaling PGC1α PKC TRAIL FAS AP1 TGR5 S1PR2 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 ASBT inhibitors TGR5 activators See Ghosh. Abst 14, Thursday 10:30

Hepatic FXR targeting to improve adaptation to BA retention BA import NTCP ntcp RXR RAR bsep RXR FXR BA export BSEP RXRα FXR ost α & β shp RXR FXR SHP BA sinusoidal export OST α/β RXR FXR cyp7a LRH-1 RXR LXR BA synthesis Mouse Liver ChIP-SEQ: FXR: 7800 sites Thomas Pharm Res 2013

FXR Deficiency Neonatal Cholestasis/Liver Failure NR1H4: ARG176* 2 Families: Presentation at birth 6 w of age Direct Bilirubin Coagulopathy Mild ALT & AST Low GGT 2 died (5 weeks, 8 months) 2 transplanted (4 & 22 months) BSEP RXR Absent BSEP expression FXR New disease discovered through exome sequencing. Nature Commun. 2016

FXR Agonists 6-α-ethyl-CDCA Cholic Acid Chenodeoxycholic acid Deoxycholic acid GW4064 (Obeticholic acid) EC 50 : 20 µm 4-10 µm 19-50 µm 99 nm 37-80 nm Fexaramine 25 nm Fexarine 38 nm Fexarene 36 nm AGN31 2 µm (also RXR) Z-Guggulsterone IC 50 : 10 µm FXR Antagonists Lithocholic Acid 10-30 µm Stigmasterol 10 µm Makishima Science 1999 Parks Science 1999 Wang Mol Cell 1999 Urizar Science 2002 Yu JBC 2002 Pellicciari J Med Chem 2002 Hawkins JCI 2002 Dussault JBC 2003 Downes Mol Cell 2003 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 BA synthesis Cholic acid is an FXR agonist 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 x See Foster Abst. 22, 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 * : p < 0.01 compared to Placebo Plans for Phase 3 Study # : p < 0.025 comparing 1500 to 500 mg

ASBT inhibitors: FXR & LXR Modulators

ASBT

ASBT inhibitors in clinical trials Clinical trials.gov A4250 Elobixibat; A3309 Maralixibat; SHP625 Diseases: Alagille Syndrome PFIC1 PFIC2 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 5-D Itch Scale Serum BA 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 50 th -74 th 1.32 (1.2-1.5) 75 th -84 th 1.76 (1.5-2.0) 85 th -94 th 2.25 (2.0-2.6) > 95 th 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 ASBT GLP1 Cholestasis Glucose Metabolism 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 HFD-induced hepatic steatosis Fexaramine (Intestinal FXR agonist) improves HFD-induced hepatic steatosis Intestinal FXR Antagonism improves NASH in mice J Clin Invest. 2015;125:386 402. Intestinal FXR Agonism improves NASH in mice Nat Med. 2015;21:159 165.

NAFLD & NASH: FXR Agonism or FXR Antagonism Both work Why?

Intact Enterohepatic BA Recirculation Interrupted Enterohepatic BA Recirculation BA Pool size Ileal ASBT Inhibition Liver BA Synthesis Cholesterol Ileum BA Uptake FXR-FGF15/19 signaling ASBT Hypothesis: Ileal ASBT inhibition will improve the hepatic and whole body response to HFD in mice ASBT Colon BA s Microbial BA metab. TGR5 signaling

Study Design & Endpoints Weeks. 0 4 8 12 16.... Weekly Body Weights Weekly Caloric & fluid intake Chow Week 15 GTT, ITT HFD HFD ASBTi Mice: HFD: ASBTi [SC-435] x 16wk Male, C57BL6J, 4-6 weeks, n=7-16/group ALIOS (45% fat; 0.2% cholesterol), + Added Sugars in the Drinking Water ASBTi: 0.006% SC-435, 10 mg/kg/day Rao A et al. Sci Transl Med 2016; 357: ra122. 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.

Relative gene expression 4 3 2 1 0 Cyp7a1 a a b Chow HFD HFD ASBTi SC-435 Inhibits Ileal ASBT Function Liver 4 Cyp8b1 3 2 1 0 a Chow a b HFD HFD ASBTi Relative gene expression 20 15 10 5 0 Ilbp a Chow Colon a b HFD HFD ASBTi Relative gene expression 3 2 1 0 Fgf15 b a Chow c HFD HFD ASBTi Ileum Shp a Chow a HFD b HFD ASBTi ASBT Fecal Bile Acids (µm/24 hr) a HFD Feces b HFD ASBTi

ASBTi Restores Glucose Tolerance GTT ITT Glucose (mg/dl) * * * * Chow HFD HFD + ASBTi * * * Chow HFD HFD + ASBTi * Significantly different from HFD + Asbti GTT AUC ITT AUC a a,b b a a b Time (mins) Chow HFD HFD ASBTi

ASBTi Improves Hepatic NAFLD Activity & Steatosis Scores Chow HFD HFD + ASBTi NAFLD Activity Score (NAS) a b c (µg/mg liver) Triglycerides a b a (µg/mg liver) Cholesteryl Esters a b a (pmol/mg liver tissue) Total Bile Acids Chow HFD HFD ASBTi Chow HFD HFD ASBTi Chow HFD HFD ASBTi Chow HFD HFD ASBTi

ASBTi Markedly Alters Hepatic BA Composition Bile Acid (pmol/mg tissue) FXR Agonist * * TCA TCDCA TDCA TLCA CA * * FXR Antagonist HFD HFD + Asbti * * * * * α-tmca β-tmca ω-tmca THDCA TUDCA α-mca β-mca 17% FXR Agonist 58% 42% 83% FXR Antagonist HFD HFD + Asbti

Hypothesized Mechanisms of Action of ASBTi in Liver HFD ASBTi Insulin resistance Hyperglycemia Hyperinsulinemia ASBTi SREBP-1c LXR Hepatic cholesterol ChREBP FXR Hepatic BA Composition Lipogenic genes TG Hepatic Steatosis ASBTi Ileal ASBT inhibition Markedly Alters Hepatic BA Composition ASBT FXR Agonist TCA TCDCA TDCA FXR Antagonist TMCA s THDCA TUDCA 58% 42% HFD 17% 83% 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 Paul Dawson, PhD Astrid Kosters, PhD Anuradha Rao, PhD Angelica Amanso, PhD JP Berauer, MD Gina Ramirez Anya Mezina,MD MSCR Courtney Ferrebee Jamie Mells, PhD Kim Pachura Jianing Li, PhD Grace Wynn 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 Wujuan Zhang, PhD Funding (NIH) R01 DK056239 R01 DK047987 Philanthropies: Alpard Foundation Spain Fund Moss Fund