Blockade of Inflammation

Inhibition of Hepatic Fibrosis

Prevention of Liver Fat Accumulation

Potent bifunctional inhibition of intracellular uric acid production blocks fructose-induced lipogenesis, lipid peroxidation, and markers of inflammation in HepG2 liver cellsLaura Gabriela Sanchez-Lozada1, Fernando E. García-Arroyo1, J. Gabriel Juárez-Rojas1, Guillermo Gonzaga1, and Raymond P. Warrell, Jr. 2

Dept. of Nephrology, INC Ignacio Chavez, Mexico City, Mexico; 2Acquist Therapeutics, Inc., Westfield, NJ USA

Background: A prototype drug markedly reduced serum uric acid (UA) in human subjects, frequently < 1.0 mg/dL. We have

developed potent bifunctional derivatives that inhibit xanthine oxidase (XO) and URAT1, along with uricase. In vivo, fructose

induces NAFLD that can progress to NASH. Both steatosis and inflammation can be blocked by hypouricemic drugs, but only in

models with elevated UA. Uricase inhibition has precluded in vivo efficacy assessment of these new agents. However, in vitro

incubation of hepatocytes with fructose increases both intracellular (IC) UA and triglycerides (TGs). We evaluated whether

metabolic effects were modulated in vitro using new bifunctional drugs.

Methods: Control HepG2 cells were cultured in DMEM w/low glucose w/o phenol red for 48-72 h and changed every 24 h. Test

cells were cultured with added fructose (25mM), plus new or standard drugs (RLBN1001, RLBN11127, RLBN1133, allopurinol

[AP], and probenecid [PBN]) (100mM). IC and extracellular (EC) UA and triglycerides (TGs) were by measured by enzymatic kits

(Sekisui). Lipid content was confirmed by Red Oil O and colorimetry (Cayman). 4-hydroynonenal (4HNE) and micro-CRP were

assessed by colorimetry and ELISA, respectively (Cayman). Experiments were conducted three times, each in triplicate.

Prolipogenic enzyme expression was evaluated by WB.

Results: Fructose sharply increased IC UA and TGs with confirmed steatosis at 48 hours (see chart). All drugs significantly

blocked UA production and significantly reduced IC TGs, 4HNE and CRP. Allopurinol and RLBN1127 suppressed the UA increase by

95% and > 98%, respectively, but both RLBN1127 and RLBN1133 were superior to AP in blocking TG production (P < 0.0005).

Over-expression of fatty acid synthase, acetyl CoA carboxylase and ATP citrate lyase were blocked by all drugs (data not shown).

Conclusion: These in vitro data confirm in vivo results that suppression of fructose-induced increases in UA with hypouricemic

drugs significantly reduce TGs and markers of lipid peroxidation and inflammation in hepatocytes. While XO over-activity is

believed a primary stimulus of UA production, probenecid – nominally a “pure” uricosuric that inhibits URAT1 – also significantly

suppressed UA/TG content, albeit less than XOIs. These results suggest that bifunctional activity may confer advantages over

monofunctional targeting. RLBN1127 is expected to enter clinical trials in biomarker-targeted inflammatory diseases.

BACKGROUND

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1. Uric acid (UA) is a mediator of lipogenesis and a trigger for inflammasomeactivation

2. 30-40% of NASH patients exhibit elevated serum uric acid and may requireurate-lowering therapy (ULT) to treat or prevent gout

3. ULT drugs favorably alter both upstream and downstream biomarkers ofNASH in human hepatocytes

4. ULT drugs renormalized fructose-induced reduction of AMPK activity andexpression in vitro

5. Beneficial effects of ULT on NASH parameters are observed in vitro (humanhepatocytes) and in vivo (rodent NAFLD/NASH models with elevated UA)

6. Available xanthine oxidase inhibitors, allopurinol and febuxostat, havesafety issues (i.e., worsening LFTs in pts with NASH1 and increased CV andall-cause mortality,2 respectively) that suggest unsuitability for NASH pts

7. Elevated sUA may describe a biomarker-defined population responsive toULT, and specific clinical studies are needed in NAFLD/NASH.

Dose (mg/m2/day) Initial Value Lowest Value

100 6.2 (4.2–6.9) 1.0 (0.8–1.8)

150† 5.4 (3.1–8.1) 1.2 (0.6–3.9)

150 6.2 (3.4–6.2) 1.2 (0.6–3.9)

200 5.8 (3.7–10.5) 0.8 (0.7–1.3)

250 5.8 (4.4–7.8) 1.2 (0.8–1.3)

750 4.7 (3.6–9.0) 1.1 (0.4–1.3)

1000 5.3 (3.8–6.9) 1.2 (0.7–2.5)

1500 4.4 (3.1–7.3) 1.2 (0.7–1.7)

Mean (SD) 5.5 (3.6–7.4) 1.2 (0.6–1.8)

• 50 patients < 5 days

• 15-fold dose range

• 80% mean reduction

• Minimal effective dose

is below starting dose

Fig. 1: RLBN1001 induced marked reductions In serum uric acid in human subjects

In clinical testing of a novel small molecule (RLBN1001), we unexpectedly

observed an off-target effect of exceptionally potent lowering of uric acid

in human subjects (Fig. 1). We developed derivatives of this prototype

and confirmed that these drugs are highly potent bifunctional inhibitors of

both xanthine oxidase and URAT1.

RLBN1127 inhibits uricase, which converts UA to allantoin. Uricase is

expressed but inactive in humans, and fully active in most species. Due

to uricase inhibition, RLBN1127 could not be tested in vivo using

standard models. We explored potential applications in gout and NASH.

Since others have established efficacy of urate-lowering therapy (ULT) in

certain NASH models (Figs 2 and 3), we evaluated ULT in vitro using

fructose-incubated human hepatocytes.

FBXControl Diet

ControlDietFBX FBX

10

20

30

40

1

2

3

P<0.014

0 0

P<0.05

FebuxostatControl

Alanine Transaminase (IU/ml)

Uric Acid

(µMx103)

0

2

3

Uric Acid(µMx103)

P<0.05

4

Control Diet

N.S.

FBX

1

FBX

10

20

30

Alanine Transaminase

(IU/ml)

40

Control Diet

50

FBXFBX0

N.S.

MCD+ControlMCD+

Febuxostat

Sirius Red (collagen)

H&E Stain

Diet: methionine/choline-deficient; 8 weeksDiet: High fructose/high trans fatty acids; 8 weeks

Fig 2: UA Reduction Prevents Inflammation and Fibrosis in NASH

Febuxostat: XO inhibition only; model with elevated UA

Fig. 3: No ULT Effect in NASH not Associated with Elevated UA

No benefit in models with normal uric acid

Adapted from Nakatsu Y et al.: Am J Physiol Gastrointest Liver Physiol 309: G42-51, 2015

P=0.0005

P<0.0001P=0.0001

P=0.0001

RLBN1127 decreases uric acid > 98% RLBN1127 decreases triglycerides > 50%7

00

µg/

mg ➤

Fig. 4: RLBN1127 Lowers Uric Acid and Reduces Liver Triglycerides as a Class Effect in Human Liver Cells

Methods: HepG2 cells were incubated in fructose at a concentration pre-

determined as causing the greatest increase in intracellular triglycerides. Test

agents and controls were added at a uniform concentration of 100 uM.

C F F+ A P F+ RL BN

1 0 0 1

F+ RL BN

1 0 0 1 .1

F+ RL BN

1 1 2 7

F+ RL BN

1 1 3 3

0 .0

0 .5

1 .0

1 .5

2 .0

A c e ty l C o A c a rb o x y la s e e x p re s s io n

Arb

itra

ry

un

its

-A c t in , 4 2 k D a

A c C o A C b x , 2 6 0 k D a

C F + R L B N

1 001

F + A PF F + R L B N

1 0 01 .1

F + R L B N

1 127

F + R L B N

1 133

C F F+ A P F+ RL BN

1 0 0 1

F+ RL BN

1 0 0 1 .1

F+ RL BN

1 1 2 7

F+ RL BN

1 1 3 3

0 .0

0 .2

0 .4

0 .6

0 .8

F a tty A c id S y n th a s e e x p re s s io n

Arb

itra

ry

un

its

-A c t in , 4 2 k D a

F A S , 2 7 3 k D a

C F + R L B N

1 001

F + A PF F + R L B N

1 0 01 .1

F + R L B N

1 127

F + R L B N

1 133

C F F+ A P F+ RL BN

1 0 0 1

F+ RL BN

1 0 0 1 .1

F+ RL BN

1 1 2 7

F+ RL BN

1 1 3 3

0 .0

0 .5

1 .0

1 .5

2 .0

A T P C itr a te L y a s e e x p re s s io n

Arb

itra

ry

un

its

-A c t in , 4 2 k D a

A T P C it L y a , 1 2 1 k D a

C F + R L B N

1 001

F + A PF F + R L B N

1 0 01 .1

F + R L B N

1 127

F + R L B N

1 133

Acetyl CoA Carboxylase Expression ATP Citrate Lyase Expression

Fatty Acid Synthase Expression

#

* * * **

#

* * * * *

#

* * * * *

Fig. 5: ULT as a Class Effect Favorably Alters Lipogenic Parameters in Human Liver Cells

C F F+ A P F+ RL BN

1 0 0 1

F+ RL BN

1 0 0 1 .1

F+ RL BN

1 1 2 7

F+ RL BN

1 1 3 3

0

1

2

3

4

5

A M P K e x p re s s io n

Arb

itra

ry

u

nits

-A c t in , 4 2 k D a

A M P K , 6 0 k D a

C F + R L B N

1 001

F + A PF F + R L B N

1 0 01 .1

F + R L B N

1 127

F + R L B N

1 133

C F F+ A P F+ RL BN

1 0 0 1

F+ RL BN

1 0 0 1 .1

F+ RL BN

1 1 2 7

F+ RL BN

1 1 3 3

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

A M P K / P h o s p h o -A M P K (T h r 1 7 2 ) ra t io

Arb

itra

ry

u

nits

p -A M P K , 6 2 k D a

C F + R L B N

1 001

F + A PF F + R L B N

1 0 01 .1

F + R L B N

1 127

F + R L B N

1 133

A M P K , 6 0 k D a

* * * # * * * * ***

#

1400

1500

0

300

200

1200

(C-r

eact

ive

pro

tein

)(p

g/m

l in

cu

ltu

re m

ediu

m) (4

-Hyd

roxy N

on

enal)

(nm

/mg p

rotein

)

100

1300

RLBN1127 (100mM)

RLBN1001 (100mM)

Allopurinol (100mM)

#

Probenecid(100mM)

Control

Fructose (25mM)

0.06

0.04

0.02

0

#

#

* *

**

*

**

*

+ Fructose (25mM)

4HNECRP

Fig. 7: Uric Acid Reduction lowers downstream markers of inflammation

Fig. 6: Urate-Lowering Drugs Restore AMPKα Expression and Activity

AP = Allopurinol; RLBN1001 = Prototype; RLBN1001.1 = Probenecid; RLBN1127 = Clinical lead; RLBN1133 = Analog# = P < 0.0001, Fructose vs. control; * = P < 0.0001, Drugs vs. fructose

CONCLUSIONS

Ref:rences:

1. Lee JS et al.: Liver safety of febuxostat compared with allopurinol in gout patients with fatty liver disease. Proc Am Coll Rheumatol (Abstract 2234), 2018.

2. White WB et al.: Cardiovascular safety of febuxostat or allopurinol in patients with gout. N Engl J Med 2018;378:1200-10.

ABSTRACT

OBJECTIVES