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