Plasma total BA Feces total bile acids plasma FGF19 d ... · DCA 18.60 3.99 TDCA 4.75 2.16 GDCA...
1
FXR (NR1H4) is a bile acid-activated nuclear receptor which controls several steps in bile acid synthesis and conjugation. Beyond this primary function, FXR exerts strong effects on cholesterol and lipid metabolism, and on liver steatosis, inflammation and fibrotisation. Recently, a phase IIb trial using Obeticholic Acid (OCA) as the first clinical stage FXR agonist has demonstrated improvements in all histopathological parameters underlying Non-alcoholic Steato-hepatitis (NASH). However, OCA demonstrated side effects such as worsening the HDL/LDL ratio or pruritus. This prompted a quest for novel, synthetic and more selective FXR agonists with an improved therapeutic profile. PX20606 is one such an FXR agonist wich we developed from early stage drug discovery up to phase I clinical studies. In order to monitor PX20606-mediated FXR activation in these human volunteers, it was essential to qualify pharmacodynamic biomarkers from blood or feces that would allow to extrapolate from PX20606 mediated changes in FXR target gene expression in animals to the human situation without the need for biopsies. Here, we present data on changes in FXR target gene expression along with their corresponding changes in plasma and fecal bile acid profiles, plasma FGF-19 levels from mouse, monkey and human phase I studies. Human Phase I clinical data Monkey experimental data Mouse experimental data 10 days repeated oral dosing study in mice Male C57BL/6J mice were purchased from Elevage Janvier (Rennes, France) at the age of 8 weeks. Animals were kept on standard rodent chow. Treatment with FXR agonists lasted over 10 days with one dose per day in the morning. PX20606 or OCA were given at the doses indicated by oral gavage in vehicle (0,5% PVP, 0,1% Tween 80 in 20 mM Tris pH 7,4). On the day of sacrification, animals were fasted for 4h before receiving a final gavage. 28 days repeated oral dosing study in monkey Cynomolgus monkey (Macaca fascicularis) (age 2–3 years; body weight 2–3 kg) were maintained on Teklad 2056 NC monkey diet and housed under standard laboratory conditions. For continuous 28 day treatment PX20606 (20mg/kg) was administered once daily by oral gavage after suspension in vehicle (0.5% (w/v) polyvinylpyrrolidone and 0.1% (v/v) Tween 80, in phosphate-buffered saline, pH 7.4). 7 days clinical Phase 1 randomized placebo controlled study The clinical Phase I studies have been registered at ClinicalTrials.gov Identifier NCT01998672 for the multiple ascending dose (MAD) Study. 0.1% polysorbate (Tween 80), 0.5% polyvinyl-pyrrolidone (PVP 30), 1.5% benzyl alcohol, 0.3 ppm denatonium benzoate anhydrous (Bitrex™) in purified water was used as vehicle for oral delivery. The FXR agonist PX20606 was suspended in vehicle solution (named Px-102 in the clinical study) and orally administered in a volume adjusted to the body weight of the subject in the fasted state alongside the groups receiving vehicle solution alone. Seperation of bile acids by HPLC-MS Plasma/lyophilized feces were hydrolyzed with NaOH for 1 hour at 60°C and further diluted with water. SPE purification was accomplished using Chromabond C18 cartridges. The eluate was dried, reconstituted in methanol/water and subsequently subjected to LC-HRMS. The analytes were separated on a Kinetex Phenyl-Hexyl, analytical column (Phenomenex, Germany) and detected by LC-HRMS (Q-Exactive) in the full scan negative mode Expression changes in Cyp7A1 and Cyp8B1 upon FXR agonist treatment changes bile acid pool composition Induction of conjugated bile acids / muricholic acid (TMCA). Reduction of cholic acid CA consistent with Cyp8B1 repression In mice FXR agonist Px-102 potently induces FGF-15 RNA in ileum while inducing SHP and repressing Cyp7A1 & Cyp8B1 RNA expression in the liver. This resulted in a dramatic shift in plasma bile acid (BA) composition and changes in total bile acid pool size in plasma and feces. Px-102 potently induced plasma FGF-19 in monkeys and humans in parallel with significant changes in plasma BA amount and composition. These data demonstrate the utility of individual BA composition and FGF-19 as sensitive biomarkers allowing close to “real- time” monitoring of pharmacological FXR activation during clinical trials. Expression changes in Cyp7A1 cause a reduction of total bile acid production. Repression of TBA pool in plasma could be sustained over 4 weeks of treatment Repression of liver Cyp7A1 expression should cause a reduction of total bile acid production, however the loss of TBA was only evident in feces while plasma TBAs in mice increased upon FXR agonist treatment Bile acid composition after 7 days of PX20606 treatment Upon FXR activation total bile acids were reduced in plasma and feces % bile acid species Placebo PX20606 0.5 mg/kg PX20606 1.5 mg/kg CDCA 6.2 2.9 6.5 TCDCA 4.9 6.0 3.0 GCDCA 39.3 31.4 47.6 CA 3.1 1.5 4.6 TCA 1.0 0.6 0.8 GCA 9.9 4.3 15.7 DCA 10.0 5.3 6.4 TDCA 2.3 6.6 0.9 GDCA 20.2 37.4 13.2 LCA 0.3 0.3 0.3 TLCA 0.4 1.0 0.1 GLCA 2.5 2.6 0.8 FXR controls genes important for bile acid synthesis (SHP PX20606 3.1±0.7 & OCA 2.6±0.7; Cyp7A1 repression PX20606 0.1±0.05 & OCA 0.2±0.04 -> dependent of FGF15/ileum; Cyp8B1 repression PX20606 0.01±0.001 & OCA 0.06±0.02 -> liver specific). Induction of FGF15 expression in the ileum ( PX20606 10.5±2.3, OCA 1.3±0.3) Cyp7A1 mRNA was downregulated 0.06(±0.08) fold by PX20606 while gene expression of FGF19 in liver tissue was induced 6.9 (±1.9) fold. Plasma FGF19 levels (4 h after admin.) were induced up to 4840 (±229) pg/ml after 1 week, 17,920 (±4,957) pg/ml after 2 weeks and 32,667 (±10,585) pg/ml Plasma FGF19 levels (4h after p.o dosing for 7 days) were induced up to 580 (±147) pg/ml at 0.5 mg/kg, 1,105 (±138) pg/ml at 1.0 mg/kg and 1,707, (±409) at 1.5 mg dose of PX20606. bile acid species OH HO OH Optionaly added in liver: determines CA vs.CDCA OH O Conjugation side From mice to men - Plasma and fecal bile acid composition as sensitive markers of pharmacological FXR activation: Results from animal and human phase I studies using the potent and selective FXR agonist PX20606 Eva Hambruch, Sanja Perović-Ottstadt, Desiree Helen Krol, Martin Hornberger, Christoph Steeneck, Olaf Kinzel, Ulrich Deuschle, Manfred Birkel & Claus Kremoser* Phenex Pharmaceuticals AG , Waldhofer Str. 104, 69123 Heidelberg, Germany * = presenting author Dr. Claus Kremoser, email: [email protected], phone: 0049 6221 65282-0 RESULTS BACKGROUND MATERIALS & METHODS CONCLUSIONS REFERENCES Cholesterol CA CDCA Primary bile acid synthesis Cyp8B1 Cyp7A1 liver TCA TCDCA GCDCA Secondary bile acid synthesis Microbiota LCA intestine TDCA, DCA, UDCA Bacs/Baat conjugation TCA GCA CDCA GCA Cholesterol CA CDCA Primary bile acid synthesis Cyp8B1 Cyp7A1 liver TCA TCDCA GCDCA Secondary bile acid synthesis Microbiota LCA intestine TDCA, DCA, UDCA Bacs/Baat conjugation TCA GCA CDCA GCA Cholesterol CA CDCA Primary bile acid synthesis Cyp8B1 Cyp7A1 liver TCA TCDCA,TMCA Secondary bile acid synthesis Microbiota THCA,TMDCA,THDCA,TUDCA intestine TDCA Bacs/Baat conjugation Cyp7A1 Liver Gut FXR FXR FGF15/19 Shp Bile acid pool suppression FGFR4/ ß-klotho Cyp8B1 Change in CA/CDCA ratio FXR‘s control of bile acid homeostasis bile acid Control 10 mg/kg PX20606 CDCA 16.67 15.40 TCDCA 5.23 39.69 GCDCA 3.41 6.60 CA 17.78 2.91 TCA 5.56 1.96 GCA 14.75 0.20 DCA 18.60 3.99 TDCA 4.75 2.16 GDCA 3.97 0.74 LCA 8.17 13.99 TLCA 0.69 8.93 GLCA 0.42 3.42 % bile acid species F Kuipers, T Claudel, E Sturm, B. Staels: The farnesoid X receptor (FXR) as modulator of bile acid metabolism Rev Endocr Metab Disord, 5 (2004), pp. 319–326 G Rizzo, B Renga, A Mencarelli, R Pellicciari, S. Fiorucci: Role of FXR in regulating bile acid homeostasis and relevance for human diseases Curr Drug Targets Immune Endocr Metabol Disord, 5 (2005), pp. 289–303 T Matsubara, F Li, FJ. Gonzalez: FXR signaling in the enterohepatic system; Mol Cell Endocrinol, 368 (2013), pp. 17–29 CONTACT INFORMATION Ratio of conjugated / unconjugated bile acid species Ratio of CDCA / CA- derived bile acid species Ratio of conjugated / unconjugated bile acid species Ratio of CDCA / CA- derived bile acid species Ratio of conjugated / unconjugated bile acid species Ratio of CDCA / CA- derived bile acid species Gene expression analysis in liver tissue after 10 days of oral gavage treatment with PX20606 and Obeticholic acid (OCA) Cyp7A1 gene expression in liver and FGF19 gene expression in ileum after 4 weeks of treatment with FXR agonist PX20606 Plasma FGF19 levels after 7 days of treatment (left: 4 h after administration; right: time course over 7 days) Bile acid species in plasma Bile acid species in plasma Bile acid species in plasma Total bile acids (TBA) in plasma Total bile acids (TBA) in plasma and feces Total bile acids (TBA) in plasma and feces Gene regulation Gene regulation and FGF19 plasma level Gene regulation and FGF19 plasma level Control OCA 30 mg/kg PX20606 30 mg/kg 0 100 200 300 400 Feces total bile acids μmol/L ** ** Control OCA 30 mg/kg PX20606 30 mg/kg 0 5 10 15 20 25 Plasma total BA μmol/L **** ** Control PX20606 20 mg/ kg 0.0 0.5 1.0 1.5 2.0 Cyp7A1 Liver relative fold TBP norm **** Pl ac ebo 0 .5 mg/kg 1 .0 mg/kg 1 .5 mg/kg 0 500 1,000 1,500 2,000 2,500 FGF19 plasma (+4h) FGF19 (pg/ml) *** * PX20606 Placebo 0.5 mg/kg 1.0 mg/kg 1.5 mg/kg 0 1,000 2,000 3,000 Fecal bile acids bile acids(μg/g) PX20606 * ** *** day 1 day 7 day 1 day 7 day 1 day 7 day 1 day 7 0 2 4 6 8 10 plasma total bile acids TBA (μmol/l) placebo PX20606 0.5 mg/kg PX20606 1.0 mg/kg PX20606 1.5 mg/kg * ** FGF19 Induction results in increase in protein levels measured in plasma TBA μmol/l mean (±SEM) Control OCA 30mg/kg PX20606 30mg/kg Plasma 2.8 (±0.8) 15.5 (±1.8) 8.8 (±1.6) Feces 235.2 (±43.2) 79.5 (±6.7) 85.2 (±8.2) TBA μmol/l mean (±SD) Control PX20606 20mg/kg Plasma week1 6.1 (±0.7) 6.7 (±1.4) Plasma week 2 6.7 (±2.2) 3.0 (±0.5) Plasma week 4 10.9 (±6.8) 4.0 (±0.4) TBA (±SEM) placebo PX20606 0.5 mg/kg PX20606 1.0 mg/kg PX20606 1.5 mg/kg feces (mg/g) 2255 (±402) 423 (±61) 379 (±165) 190 (±38) Plasma (μmol/l) day 1 5.5 (±0.9) 6.8 (±1.3) 5.3 (±1.0) 6.6 (±4.7) Plasma (μmol/l) day 7 6.3(±0.8) 3.8 (±0.8) 3.2 (±0.4) 4.3 (±3.4) Bile acid composition after 28 days of treatment Bile acid composition after 10 days of treatment Induction of conjugated bile acids (most prominent taurine conjugates of CDCA and LCA). Reduction of cholic acid CA consistent with Cyp8B1 repression FXR induced changes in Cyp7A1 and Cyp8B1 as well as control over conjugation enzymes (Bacs, Baat) cause shifts in bile acid pool composition Changes in plasma bile acid composition in humans are less pronounced compared to mice and monkeys Evident shift towards CDCA derived BA species. Increase in conjugated BA species developed at 1,5 mg/kg PX20606 for 7 days. PX20606 0 24 48 72 96 120 144 0 500 1,000 1,500 2,000 2,500 FGF19 plasma level hours FGF19 (pg/ml) Placebo 0.5 mg/kg 1.0 mg/kg 1.5 mg/kg PX20606 % bile acid species Control 30 mg/kg OCA 30 mg/kg PX20606 CDCA 0.24 0.06 0.08 TCDCA 0.10 0.06 0.29 CA 7.25 0.51 2.34 TCA 0.12 0.01 0.20 DCA 5.63 0.42 0.69 TDCA 0.10 0.05 0.25 LCA 0.56 0.54 0.55 TLCA 0.07 0.002 0.003 MCA 0.33 8.26 5.05 TMCA 0.09 6.34 2.09 *** * * *** * Mean ± SEM Mean ± SEM Mean ± SEM Mean ± SEM Control OCA 30 mg/kg PX20606 30 mg/kg 0.0 0.5 1.0 1.5 Cyp7A1 Liver relative fold TBP norm *** *** Control OCA 30 mg/kg PX20606 30 mg/kg 0.0 0.5 1.0 1.5 Cyp8B1 Liver relative fold TBP norm **** **** Control OCA 30 mg/kg PX20606 30 mg/kg 0 1 2 3 4 5 6 7 8 FGF15 Ileum relative fold TBP norm * Control OCA 30 mg/kg PX20606 30 mg/kg 0 1 2 3 4 SHP Liver relative fold TBP norm ** ** C ontrol P x 2 0 mg / kg C ontrol P x 2 0 mg / kg C ontrol P x 2 0 mg / kg 0 5 10 15 20 plasma total bile acids TBA (μmol/l) 1st week 2 weeks 4 weeks ns ** * C ontrol P x 2 0 mg / kg C ontrol P x 2 0 mg / kg C ontrol P x 2 0 mg /kg 0 200 400 2,500 5,000 7,500 10,000 20,000 30,000 40,000 50,000 FGF19 (pg/ml) 1st week 2 weeks 4 weeks **** **** **** PX = PX20606 Control PX20606 20 mg/kg 0 2 4 6 8 10 FGF19 Ileum relative fold TBP norm ** PX = PX20606
Transcript of Plasma total BA Feces total bile acids plasma FGF19 d ... · DCA 18.60 3.99 TDCA 4.75 2.16 GDCA...
FXR (NR1H4) is a bile acid-activated nuclear receptor which controls
several steps in bile acid synthesis and conjugation. Beyond this
primary function, FXR exerts strong effects on cholesterol and lipid
metabolism, and on liver steatosis, inflammation and fibrotisation.
Recently, a phase IIb trial using Obeticholic Acid (OCA) as the first
clinical stage FXR agonist has demonstrated improvements in all
histopathological parameters underlying Non-alcoholic Steato-hepatitis
(NASH). However, OCA demonstrated side effects such as worsening
the HDL/LDL ratio or pruritus. This prompted a quest for novel,
synthetic and more selective FXR agonists with an improved
therapeutic profile. PX20606 is one such an FXR agonist wich we
developed from early stage drug discovery up to phase I clinical
studies. In order to monitor PX20606-mediated FXR activation in these
human volunteers, it was essential to qualify pharmacodynamic
biomarkers from blood or feces that would allow to extrapolate from
PX20606 mediated changes in FXR target gene expression in animals
to the human situation without the need for biopsies. Here, we present
data on changes in FXR target gene expression along with their
corresponding changes in plasma and fecal bile acid profiles, plasma
FGF-19 levels from mouse, monkey and human phase I studies.
Human Phase I clinical data Monkey experimental data Mouse experimental data
10 days repeated oral dosing study in mice
Male C57BL/6J mice were purchased from Elevage Janvier (Rennes, France) at the
age of 8 weeks. Animals were kept on standard rodent chow. Treatment with FXR
agonists lasted over 10 days with one dose per day in the morning. PX20606 or OCA
were given at the doses indicated by oral gavage in vehicle (0,5% PVP, 0,1% Tween 80
in 20 mM Tris pH 7,4). On the day of sacrification, animals were fasted for 4h before
receiving a final gavage.
28 days repeated oral dosing study in monkey
Cynomolgus monkey (Macaca fascicularis) (age 2–3 years; body weight 2–3 kg) were
maintained on Teklad 2056 NC monkey diet and housed under standard laboratory
conditions. For continuous 28 day treatment PX20606 (20mg/kg) was administered
once daily by oral gavage after suspension in vehicle (0.5% (w/v) polyvinylpyrrolidone
and 0.1% (v/v) Tween 80, in phosphate-buffered saline, pH 7.4).
7 days clinical Phase 1 randomized placebo controlled study
The clinical Phase I studies have been registered at ClinicalTrials.gov Identifier
NCT01998672 for the multiple ascending dose (MAD) Study. 0.1% polysorbate (Tween
80), 0.5% polyvinyl-pyrrolidone (PVP 30), 1.5% benzyl alcohol, 0.3 ppm denatonium
benzoate anhydrous (Bitrex™) in purified water was used as vehicle for oral delivery.
The FXR agonist PX20606 was suspended in vehicle solution (named Px-102 in the
clinical study) and orally administered in a volume adjusted to the body weight of the
subject in the fasted state alongside the groups receiving vehicle solution alone.
Seperation of bile acids by HPLC-MS
Plasma/lyophilized feces were hydrolyzed with NaOH for 1 hour at 60°C and further
diluted with water. SPE purification was accomplished using Chromabond C18
cartridges. The eluate was dried, reconstituted in methanol/water and subsequently
subjected to LC-HRMS. The analytes were separated on a Kinetex Phenyl-Hexyl,
analytical column (Phenomenex, Germany) and detected by LC-HRMS (Q-Exactive) in
the full scan negative mode
Expression changes in Cyp7A1 and Cyp8B1 upon FXR
agonist treatment changes bile acid pool composition
Induction of conjugated bile acids / muricholic acid (TMCA).
Reduction of cholic acid CA consistent with Cyp8B1
repression
In mice FXR agonist Px-102 potently
induces FGF-15 RNA in ileum while
inducing SHP and repressing Cyp7A1 &
Cyp8B1 RNA expression in the liver. This
resulted in a dramatic shift in plasma bile
acid (BA) composition and changes in total
bile acid pool size in plasma and feces.
Px-102 potently induced plasma FGF-19 in
monkeys and humans in parallel with
significant changes in plasma BA amount
and composition.
These data demonstrate the utility of
individual BA composition and FGF-19 as
sensitive biomarkers allowing close to “real-
time” monitoring of pharmacological FXR
activation during clinical trials.
Expression changes in Cyp7A1 cause a reduction of
total bile acid production. Repression of TBA pool in
plasma could be sustained over 4 weeks of treatment
Repression of liver Cyp7A1 expression should cause
a reduction of total bile acid production, however the
loss of TBA was only evident in feces while plasma
TBAs in mice increased upon FXR agonist treatment
Bile acid composition after 7 days of
PX20606 treatment
Upon FXR activation total bile acids
were reduced in plasma and feces
% bile acid
speciesPlacebo
PX20606
0.5 mg/kg
PX20606
1.5 mg/kg
CDCA 6.2 2.9 6.5
TCDCA 4.9 6.0 3.0
GCDCA 39.3 31.4 47.6
CA 3.1 1.5 4.6
TCA 1.0 0.6 0.8
GCA 9.9 4.3 15.7
DCA 10.0 5.3 6.4
TDCA 2.3 6.6 0.9
GDCA 20.2 37.4 13.2
LCA 0.3 0.3 0.3
TLCA 0.4 1.0 0.1
GLCA 2.5 2.6 0.8
FXR controls genes important for bile acid synthesis (SHP PX20606 3.1±0.7 & OCA 2.6±0.7; Cyp7A1 repression
PX20606 0.1±0.05 & OCA 0.2±0.04 -> dependent of FGF15/ileum; Cyp8B1 repression PX20606 0.01±0.001 &
OCA 0.06±0.02 -> liver specific). Induction of FGF15 expression in the ileum ( PX20606 10.5±2.3, OCA 1.3±0.3)
Cyp7A1 mRNA was downregulated 0.06(±0.08) fold by
PX20606 while gene expression of FGF19 in liver tissue
was induced 6.9 (±1.9) fold.
Plasma FGF19 levels (4 h after admin.) were induced
up to 4840 (±229) pg/ml after 1 week, 17,920 (±4,957)
pg/ml after 2 weeks and 32,667 (±10,585) pg/ml
Plasma FGF19 levels (4h after p.o dosing for 7 days) were induced up to 580 (±147) pg/ml at 0.5 mg/kg, 1,105
(±138) pg/ml at 1.0 mg/kg and 1,707, (±409) at 1.5 mg dose of PX20606.
bile acid species
OH
HO OH
Optionaly added
in liver: determines
CA vs.CDCA OH
O Conjugation
side
From mice to men - Plasma and fecal bile acid composition as sensitive markers of pharmacological FXR activation: Results from animal and human phase I studies using the potent and selective FXR agonist PX20606
Eva Hambruch, Sanja Perović-Ottstadt, Desiree Helen Krol, Martin Hornberger, Christoph Steeneck, Olaf Kinzel, Ulrich Deuschle, Manfred Birkel & Claus Kremoser*
Phenex Pharmaceuticals AG , Waldhofer Str. 104, 69123 Heidelberg, Germany
* = presenting author Dr. Claus Kremoser, email: [email protected],
phone: 0049 6221 65282-0
RESULTS BACKGROUND
MATERIALS & METHODS
CONCLUSIONS
REFERENCES
Cholesterol
CA CDCA
Primary bile acid
synthesis
Cyp8B1
Cyp7A1 liver
TCA
TCDCA
GCDCA
Secondary bile acid
synthesis Microbiota
LCA
intestine
TDCA, DCA,
UDCA
Bacs/Baat
conjugation TCA
GCA
CDCA
GCA
Cholesterol
CA CDCA
Primary bile acid
synthesis
Cyp8B1
Cyp7A1 liver
TCA
TCDCA
GCDCA
Secondary bile acid
synthesis Microbiota
LCA
intestine
TDCA, DCA,
UDCA
Bacs/Baat
conjugation TCA
GCA
CDCA
GCA
Cholesterol
CA CDCA
Primary bile acid
synthesis
Cyp8B1
Cyp7A1 liver
TCA TCDCA,TMCA
Secondary bile acid
synthesis Microbiota
THCA,TMDCA,THDCA,TUDCA
intestine
TDCA
Bacs/Baat
conjugation
Cyp7A1
Liver
Gut
FXR
FXR
FGF15/19
Shp
Bile acid pool
suppression
FGFR4/
ß-klotho Cyp8B1
Change in
CA/CDCA ratio
FXR‘s control of bile acid homeostasis bile acid Control10 mg/kg
PX20606
CDCA 16.67 15.40
TCDCA 5.23 39.69
GCDCA 3.41 6.60
CA 17.78 2.91
TCA 5.56 1.96
GCA 14.75 0.20
DCA 18.60 3.99
TDCA 4.75 2.16
GDCA 3.97 0.74
LCA 8.17 13.99
TLCA 0.69 8.93
GLCA 0.42 3.42
% bile acid
species
F Kuipers, T Claudel, E Sturm, B. Staels: The farnesoid X receptor (FXR) as modulator of bile acid metabolism Rev Endocr Metab Disord, 5 (2004), pp. 319–326
G Rizzo, B Renga, A Mencarelli, R Pellicciari, S. Fiorucci: Role of FXR in regulating bile acid homeostasis and relevance for human diseases Curr Drug Targets Immune Endocr Metabol Disord, 5
(2005), pp. 289–303
T Matsubara, F Li, FJ. Gonzalez: FXR signaling in the enterohepatic system; Mol Cell Endocrinol, 368 (2013), pp. 17–29
CONTACT INFORMATION
Ratio of conjugated /
unconjugated
bile acid species
Ratio of CDCA / CA-
derived bile acid species
Ratio of conjugated /
unconjugated
bile acid species
Ratio of CDCA / CA-
derived bile acid species
Ratio of conjugated /
unconjugated
bile acid species
Ratio of CDCA / CA-
derived bile acid species
Gene expression analysis in liver tissue after 10 days of oral gavage treatment with PX20606
and Obeticholic acid (OCA)
Cyp7A1 gene expression in liver and FGF19 gene
expression in ileum after 4 weeks of treatment with
FXR agonist PX20606
Plasma FGF19 levels after 7 days of treatment (left: 4 h after administration; right: time
course over 7 days)
Bile acid species in plasma Bile acid species in plasma Bile acid species in plasma
Total bile acids (TBA) in plasma Total bile acids (TBA) in plasma and
feces
Total bile acids (TBA) in plasma and feces
Gene regulation Gene regulation and FGF19 plasma
level
Gene regulation and FGF19 plasma
level
Con
trol
OCA 30 m
g/kg
PX20
606 30
mg/
kg
0
100
200
300
400
Feces total bile acids
µm
ol/
L
** **
Con
trol
OCA 30 m
g/kg
PX20
606 30
mg/
kg
0
5
10
15
20
25
Plasma total BA
µm
ol/
L
****
**
Con
trol
PX20
606 20
mg/
kg
0.0
0.5
1.0
1.5
2.0
Cyp7A1 Liver
rela
tive f
old
TB
P n
orm
****
Place
bo
0.5
mg/
kg
1.0 m
g/kg
1.5 m
g/kg
0
500
1,000
1,500
2,000
2,500
FGF19 plasma (+4h)
FG
F19 (
pg
/ml)
***
*
PX20606
Place
bo
0.5
mg/
kg
1.0 m
g/kg
1.5 m
g/kg
0
1,000
2,000
3,000
Fecal bile acids
bile a
cid
s(µ
g/g
)
PX20606
* *****
day 1
day 7
day 1
day 7
day 1
day 7
day 1
day 7
0
2
4
6
8
10
plasma total bile acids
TB
A (
µm
ol/l)
placebo PX206060.5 mg/kg
PX206061.0 mg/kg
PX206061.5 mg/kg
***
FGF19 Induction results in increase
in protein levels measured in plasma
TBA µmol/l
mean (±SEM)Control
OCA
30mg/kg
PX20606
30mg/kg
Plasma 2.8 (±0.8) 15.5 (±1.8) 8.8 (±1.6)
Feces 235.2 (±43.2) 79.5 (±6.7) 85.2 (±8.2)
TBA µmol/l
mean (±SD)Control
PX20606
20mg/kg
Plasma week1 6.1 (±0.7) 6.7 (±1.4)
Plasma week 2 6.7 (±2.2) 3.0 (±0.5)
Plasma week 4 10.9 (±6.8) 4.0 (±0.4)
TBA (±SEM) placeboPX20606
0.5 mg/kg
PX20606
1.0 mg/kg
PX20606
1.5 mg/kg
feces
(mg/g) 2255 (±402) 423 (±61) 379 (±165) 190 (±38)
Plasma
(µmol/l)
day 1
5.5 (±0.9) 6.8 (±1.3) 5.3 (±1.0) 6.6 (±4.7)
Plasma
(µmol/l)
day 7
6.3(±0.8) 3.8 (±0.8) 3.2 (±0.4) 4.3 (±3.4)
Bile acid composition after 28 days of
treatment Bile acid composition after 10 days of
treatment
Induction of conjugated bile acids (most prominent taurine conjugates of
CDCA and LCA). Reduction of cholic acid CA consistent with Cyp8B1
repression
FXR induced changes in Cyp7A1 and Cyp8B1 as well as control over
conjugation enzymes (Bacs, Baat) cause shifts in bile acid pool
composition
Changes in plasma bile acid composition in humans are less
pronounced compared to mice and monkeys
Evident shift towards CDCA derived BA species. Increase in
conjugated BA species developed at 1,5 mg/kg PX20606 for
7 days.
PX20606
0 24 48 72 96 120 1440
500
1,000
1,500
2,000
2,500
FGF19 plasma level
hours
FG
F19 (
pg
/ml)
Placebo0.5 mg/kg1.0 mg/kg1.5 mg/kg
PX20606
% bile acid
speciesControl
30 mg/kg
OCA
30 mg/kg
PX20606
CDCA 0.24 0.06 0.08
TCDCA 0.10 0.06 0.29
CA 7.25 0.51 2.34
TCA 0.12 0.01 0.20
DCA 5.63 0.42 0.69
TDCA 0.10 0.05 0.25
LCA 0.56 0.54 0.55
TLCA 0.07 0.002 0.003
MCA 0.33 8.26 5.05
TMCA 0.09 6.34 2.09
***
*
* *** *
Mean ± SEM Mean ± SEM
Mean ± SEM Mean ± SEM
Con
trol
OCA 3
0 m
g/kg
PX20
606
30 m
g/kg
0.0
0.5
1.0
1.5
Cyp7A1 Liver
rela
tiv
e f
old
TB
P n
orm
******
Con
trol
OCA 3
0 m
g/kg
PX20
606
30 m
g/kg
0.0
0.5
1.0
1.5
Cyp8B1 Liver
rela
tiv
e f
old
TB
P n
orm
********
Con
trol
OCA 3
0 m
g/kg
PX20
606
30 m
g/kg
0
1
2
3
4
5
6
7
8FGF15 Ileum
rela
tiv
e f
old
TB
P n
orm *
Con
trol
OCA 3
0 m
g/kg
PX20
606
30 m
g/kg
0
1
2
3
4
SHP Liver
rela
tiv
e f
old
TB
P n
orm
**
**
Con
trol
Px
20 m
g/kg
Con
trol
Px
20 m
g/kg
Con
trol
Px
20 m
g/kg
0
5
10
15
20plasma total bile acids
TB
A (
µm
ol/l)
1st week 2 weeks 4 weeks
ns
***
Con
trol
Px
20 m
g/kg
Con
trol
Px
20 m
g/kg
Con
trol
Px
20 m
g/kg
0
200
400
2,500
5,000
7,50010,000
20,000
30,000
40,000
50,000
plasma FGF19
FG
F19 (
pg
/ml)
1st week 2 weeks 4 weeks
****
****
****
PX = PX20606 Con
trol
PX20
606 20
mg/
kg
0
2
4
6
8
10
FGF19 Ileum
rela
tive f
old
TB
P n
orm **
PX = PX20606
