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