ACR Meeting November, 2012
Metabolex Inc. Hayward, CA
Arhalofenate is a Novel Dual-Acting Agent with Uricosuric and Anti-Inflammatory Properties Yun-Jung Choi, Vanina Larroca, Annette Lucman, Vic Vicena, Noe Abarca, Tim Rantz, Brian E. Lavan and Charles A. McWherter
Disclosures
• All Authors, Including Presenting Author, Have the Following Financial Relationships to Disclose: – All authors are current employees of Metabolex, Inc.
4
Key Features of Gout: Hyperuricemia Causes Urate
Crystal Deposits That Produce Gout Flares
Hyperuricemia
↑ Serum uric
acid (sUA)
Mono sodium urate
(MSU) crystal
deposits
Inflammatory
response
↑ IL-1β
Painful gout
flare
Therapeutic
targets
Joint
erosion
5
Arhalofenate Acid has Two Activities in a Single Molecule
Arhalofenate Arhalofenate acid
100%
Bioconversion
Blocks renal tubular urate re-absorption – URAT1, OAT4 and OAT10
Blocks MSU production of pro-IL-1β mRNA – PPAR-γ trans-repression†
† Gregoire et al, Mol. Endocrinol. 2009 23:975-988
OAT10
GLUT9a
OAT4 URAT1
Urate
Urate
Urate
Basolateral Side (Blood)
Apical Side (Urine)
Thiazide diuretics Salicylates
Pyrazinoate Nicotinate
Lactate
Proximal Tubule
Epithelial Cell
GLUT9b
Urate Glucose
Renal Urate Transporters Implicated in the Re-absorption of
Urate in Proximal Tubules
7
Arhalofenate Acid Blocks 14C-Uric Acid Uptake by
Human Renal Transporters
10 -6 10 -5 10 -4 10 -3
0
20
40
60
80
100
120
Arhalofenate Acid (M)
[14C
] U
rate
Up
take (
%C
on
tro
l)
10 -8 10 -7 10 -6 10 -5 10 -4 10 -3
0
20
40
60
80
100
120
Arhalofenate Acid (M)
[14C
] U
rate
Up
take (
%C
on
tro
l)
10 -6 10 -5 10 -4 10 -3
0
20
40
60
80
100
Arhalofenate Acid (M)
[14C
] U
rate
Up
take (
%C
on
tro
l)
Arhalofenate acid concentration
in human urine was 116 ± 23 µM
after dosing at 600 mg
URAT1 OAT4
OAT10
8
Comparison of Arhalofenate Acid with Other Uricosuric Agents
IC50 (μM) for 14C-Uric Acid Uptake
cDNA encoding human renal transporters were transiently transfected into human
embryonic kidney cells. Compound effects on 14C-uric acid uptake was assessed in
comparison to mock transfected parental cells
IC50 (µM) URAT-1 OAT4 OAT10
Arhalofenate acid 92 3 53
Lesinurad 50 4 50
Probenecid 750 30 1000
Benzbromarone 0.3 1.5 3.8
9
MSU Crystal Triggered Inflammation in Joints
Arhalofenate selectively blocks the first step in IL-1β cascade
Macrophage
Neutrophil
Neutrophil
MSU Crystals
Interleukin 1b CXCL1/CXCL2
NALP3 Inflammasome NF-κB genes
Arhalofenate Acid
Neutrophil Activation and Degranulation
Rilonacept/ Canakinumab
Colchicine
Colchicine
Inflammation/ Acute Flare
Prednisone/ NSAIDS
Joint Space
10
Murine Air Pouch Model of MSU Induced Inflammation
• Drugs administered
‒ Arhalofenate 250 mg/kg
‒ Dexamethosone 20 mg/kg
‒ Colchicine 2 mg/kg
• Neutrophil, macrophage and total cell numbers in exudate
measured by FACS analysis
Dose 1 Collect Exudate
6 day Air Pouch Established
Dose 2
24 hrs
Dose 3
24 hrs
MSU
30 min 4 hrs
• Cytokines measured in exudate
by ELISA assay
‒ IL-1b
‒ CXCL1
‒ IL-6
‒ MCP-1
‒ CXCL2
Arhalofenate Potently Inhibits MSU Crystal Induced IL-1β
Production in the Murine Air Pouch Model
+ MSU
0
50
100
150
I L 1
- b (
p g
/ m L
)
No MSU
Vehicle
Arhalo
Dexa
Colchicine
****
**** ****
ns
*
****
Arhalofenate Also Blocks Production of Other NF-κB Inflammatory
Mediators in the Murine Air Pouch Model
No MSU MSU Arhalo Dexa Colchicine
C X
C L
- 1 (
p g
/ m L
)
0
200
400
600 CXCL1
****
*
**
0
100
200
300
400
500
I L - 6
( p
g / m
L )
IL-6
** ***
M C
P - 1
( p
g / m
L )
0
200
400
600
800
1000
**** ****
*
MCP-1
C X
C L
- 2 (
p g
/ m L
)
0
50
100
150
200 CXCL2
***
**
Total Cells Neutrophils Macrophages
Arhalofenate Potently Inhibits MSU Crystal Induced
Inflammatory Cell Production in the Murine Air Pouch Model
(CD45+) (Ly-6B.2+) (F4/80+)
C e
l l n
u m
b e
r ( x
1 0
3 )
0
200
400
600
800
1000
*** *** ** **
C e
l l n
u m
b e
r ( x
1 0
3 )
0
200
400
600
800
1000
****
****
**** ****
No MSU MSU Arhalo Dexa Colchicine
C e
l l n
u m
b e r
( x 1
0 3 )
0
200
400
600
800
1000
14
A Model for Triggering of Gouty Inflammation via
Macrophage Toll-Like Receptors
Macrophage
MSU Crystals
Interleukin 1b
Interleukin 18 CXCL1 CXCL2/IL-8 IL-6 MCP-1 TNF-α + others
NFκB genes: e.g. Pro-IL-1β
TLR2
LPS
Gout attack Monocytes Mast cells Neutrophils
Resolution TGF-β1
cf Liu-Bryan et al, Arth Rheum: 2005: 52:2936 Martinon. et al, Nature 2006: 440: 237
TLR4 FFA
NALP3 Inflammasome Activation
15
MSU and LPS Synergize in Producing IL-1β
Measure IL-1 b
MSU or LPS or LPS + MSU
Culture 2 days 24 hours
cf Petska. et al, Tox. Sci. 2006: 92: 445 Giamarellos-Bourboulis, et al, Ann Rheum Dis 2009: 68:273
0
200
400
600
800
1000
I L 1 - b
( p
g / m
L )
16
Arhalofenate Acid Suppresses IL-1b in Isolated Mouse Macrophages
Comparison to Dexamethasone and Colchicine
Compound Treatment
MSU + LPS Measure Secreted IL-1 b
Culture 2 days 2 hours 24 hours
Arhalofenate Acid
0
200
400
600
800
1000
I L 1
- b (
p g
/ m L
)
****
0
200
400
600
800
1000
I L 1 - b
( p
g / m
L )
Dexamethasone
MSU + LPS MSU + LPS + Dexamethasone
**** 0
200
400
600
800
1000
I L 1
- b (
p g
/ m L
)
Colchicine
MSU + LPS +Arhalofenate Acid MSU + LPS + Colchicine
Arhalofenate Acid Inhibits IL-1β Release in Isolated
Macrophages by Reducing Levels of Intracellular Pro-IL-1β
Intracellular
Pro IL-1β
Secreted IL-1β
Arhalofenate LPS or MSU or LPS + MSU
Measure Secreted and
Intracellular IL-1 b
Culture 2 days 2 hours 24 hours
MSU
LPS
Arhalo +
+
+
+
+
+
+ +
+
0
200
400
600
800
1000
1200 I L
1 - b
( p
g / m
L )
**** ****
****
****
Arhalofenate Acid Inhibits MSU Induced IL-1 b Release in Mouse
Macrophages Through Suppression of Gene Expression
LPS Remove LPS Arhalofenate Acid
Measure secreted IL-1b
and Pro-IL-1b mRNA
Macrophage Culture 2 days 4 hours 2 hours
MSU
18 hours
+ MSU + MSU
Secreted mRNA
0
10
20
30
40
50
I L - 1
b m
R N
A
F o
l d C
h a n
g e
****
****
**
I L 1 - b
( p
g / m
L )
0
10000
20000
30000
**
****
****
****
19
Conclusions
• Arhalofenate acid inhibits uptake by renal uric acid transporters
• Arhalofenate potently blocks MSU crystal-induced inflammation in a mice
– IL-1b and other NF-κB-dependent inflammatory mediators
– Significantly reduces neutrophil influx
• Arhalofenate acid show potent anti-inflammatory action in macrophages
– Suppresses both IL-1b mRNA and protein
• Arhalofenate has a unique dual mode of action to address the key needs
of gout patients:
– Inhibits uric acid uptake
– Suppresses MSU driven inflammation
20
Acknowledgements
• In vivo Pharmacology
– Noe Abarca
– Vanina Barreiro
– Annette Lucman
– Vic Vicena
• In vitro Biology
– Yun-Jung Choi
– Brian Lavan
– Tim Rantz
• Chemistry
– Xin Chen
– Fang Ma
• Stanford Shared FACS Facility
– Marty Bigos
– Bianca Gomez