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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
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