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ABSTRACT
GEMCABENE REGULATES VLDL-REMNANT TRAFFICKING AND INFLAMMATION GENES WITH POTENTIAL IMPACT ON CARDIOVASCULAR DISEASE Daniela C. Oniciu, Rai Ajit K. Srivastava, and Charles L. Bisgaier
Gemphire Therapeutics Inc., 17199 N. Laurel Park, Suite 401, Livonia, MI 48152, USA
INTRODUCTION
Introduction: Hypertriglyceridemia and inflammation are associated with atherosclerosis and CVD. Gemcabene, a small molecule in Phase 2b development for dyslipidemia beneficially affects plasma and hepatic lipids in rodents. In rodents and humans, it reduces VLDL-C, LDL-C, Apo-B, triglycerides (TG) and C-Reactive Protein (CRP). We now describe correlations of gemcabene’s effects on markers of lipid metabolism and inflammation with plasma TG and CRP in a Type I diabetic mouse model. Method: Forty-eight two-day old neonatal C57BL/6 male mice, randomized to 6 groups of 8 animals each, were initially administered a single dose of vehicle (Group 1) or streptozotocin (Groups 2-6). At 4 weeks of age animals were allowed ad libitum chow (Group 1) or a high-fat high-caloric diet (Groups 2-6) until completion of the experiment. Beginning at 6 weeks of age, mice were orally administered either water vehicle (Groups 1 and 2), gemcabene at 30, 100 or 300 mg/kg (Groups 3-5), or the positive comparator telmisartan at 10 mg/kg (Group 6) daily. All groups were sacrificed at week 9. Results: Gemcabene suppressed expression of hepatic genes ApoC-III, angiopoietin-like protein 3 (ANGPTL3), ANGPTL4, Sulfatase-2 (SULF2), lipoprotein lipase (LPL) (lipid trafficking genes), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), chemokine (C-X-C motif) ligand 1 (CXCL1/KC) and CRP (inflammatory genes). Gemcabene did not affect stearoyl-CoA desaturase-1 (SCD1) and ANGPTL8. Conclusions: Gemcabene intervention showed that each lipid trafficking gene was significantly correlated with reduced plasma TG levels, suggesting a coordinated response for TG lowering. Also, the hepatic downregulation by gemcabene of the inflammatory genes IL-6, TNF-α, CXCL1/KC, and CRP were highly correlated with the plasma CRP reduction. These results suggest that gemcabene may have pleiotropic mechanisms that may reduce atherosclerosis progression and cardiovascular risk.
• Patients treated with optimal statin therapy and even PCSK9 inhibitors exhibit considerable residual risk for ASCVD events.1-3
Residual ASCVD risk may occur, in part, because these medications lower only slightly the plasma levels of cholesterol- (C) and triglyceride- (TG) rich remnant Apo-B-lipoproteins (C-TRLs).4-6
• The atherometabolic syndrome and type 2 diabetes (T2DM) increase plasma levels of C-TRLs, owing largely to a defect in hepatic clearance.7 It has also been reported that obesity and T2DM cause hepatic overexpression of sulfatase-2 (SULF2),8,9 and that SULF2 inhibits hepatic disposal of C-TRLs in human T2DM dyslipoproteinemia.10
• Gemcabene reduces VLDL-C, LDL-C, Apo-B, TG and CRP in rodents and humans.11-17 Also, gemcabene-treated STAMTM mice (a rodent model of type I diabetes) showed reduction in plasma TG levels, and downregulation of the SULF2 and apoC-III mRNA expression, with no effect on the low-density lipoprotein receptor (LDLr) mRNA.18 Correlations of gemcabene-induced TG reduction and SULF2 mRNA downregulation in type I diabetic mice are directly proportional,19 suggesting that gemcabene may enhance clearance of C-TRLs in diabetic mice via downregulation of hepatic SULF2 and independent of LDLr mRNA, which would constitute an unprecedented regulation of atherogenic remnant lipoproteins by a small molecule.
• We show herein that the reduction in plasma CRP levels by gemcabene intervention in STAMTM mice was significantly correlated with the downregulation of inflammatory genes: IL-6, TNF-α, CXCL1/KC, and CRP, while lipid trafficking and/or insulin sensitivity genes APOC-III, SULF2, LPL, ANGPTL3 and ANGPTL4 mRNAs were significantly correlated with reductions in plasma TG levels.
Abstract # XVIII P4. 005 ISA Toronto, 2018
EFFECT OF GEMCABENE ON TGs, LDL-C AND CRP IN HUMANS: REDUCTION OF ASCVD RISK
InflammatoryMarkersAtherogenicMarkers
Placebo-corrected Placebo-corrected
Median Percent Change from Baseline at Week 12 in Patients with Hypercholesterolemia on Maximum Tolerated Statin Therapy (ROYAL-1 Study)
-20%
-13%
-9%
-1%
-20%
-15%
-10%
-5%
0%
LSM
ean%Cha
ngefrom
BaselinetoW
eek12
n=28 n=27 n=25n=25
*p<0.05^p=0.065*
^
LDL-CGEM600mg GEM600mgPBO PBO
BackgroundModerate-IntensityStatins
BackgroundHigh-IntensityStatins
-53%
-33%
-6% -6%
-55%
-50%
-45%
-40%
-35%
-30%
-25%
-20%
-15%
-10%
-5%
0%
Med
ian%Cha
ngefrom
BaselinetoW
eek12
n=26 n=26 n=25n=25GEM600mg GEM600mgPBO PBO
BackgroundModerate-IntensityStatins
BackgroundHigh-IntensityStatins
hsCRP
*p<0.05*
*
Gemcabene May Enhance Clearance of C-TRLs in Diabetic Mice via Down-Regulation of Hepatic SULF2 and Independent of LDLr
GEMCABENE MECHANISM OF ACTION
Study Design for Gemcabene Assessment in the STAMTM Murine Model of NASH
Vehi
cle in
Nor
mal
Vehi
cle
in N
ASH
30m
g/kg
100
mg/
kg
300
mg/
kg
Telm
isarta
n 10
mg/
kg
0
500
1000
1500
Plas
ma
Trig
lycer
ides
(mg/
dL) ±
SEM
Plasma Triglycerides
Gemcabene
n.s.p < 0.001
p < 0.001
p < 0.001p < 0.05
Plasma Triglycerides Plasma CRP
Vehi
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al
Vehi
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100
mg/
kg
300
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kg
Telm
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tan
10m
g/kg
0
1
2
3
4
Plas
ma
CR
P (µ
g/m
L) ±
SEM
Plasma CRP
Gemcabene
n.s.
p < 0.0001p < 0.0001
n.s.
p < 0.001
Effect of Gemcabene on Plasma Markers in STAMTM Mice
RESULTS
Veh
icle
in N
orm
al
Veh
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in N
AS
H
30m
g/kg
100
mg/
kg
300
mg/
kg
Telm
isar
tan
10m
g/kg
0.0
0.5
1.0
1.5
apoC
3 m
RN
A /
36B
4 m
RN
A ±
SE
M
Apolipoprotein C-III
Gemcabene
n.s.p < 0.001
n.s.
p < 0.01p < 0.0001
Apolipoprotein C-III (ApoC-III)
Vehi
cle
in N
orm
al
Vehi
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in N
ASH
30m
g/kg
100
mg/
kg
300
mg/
kg
Telm
isar
tan
10m
g/kg
0
2
4
6
8
Sulf2
mR
NA
/ 36B
4 m
RN
A ±
SEM
Sulfatase 2
Gemcabene
n.s.
p < 0.001p < 0.05
p < 0.0001
n.s.Sulfatase 2 (SULF2)
Vehi
cle
in N
orm
al
Vehi
cle
in N
ASH
30m
g/kg
100
mg/
kg
300
mg/
kg
Telm
isar
tan
10m
g/kg
0
2
4
6
8
10
LPL
mR
NA
/ 36B
4 m
RN
A ±
SEM
Lipoprotein Lipase
Gemcabene
p < 0.05
n.s.
p < 0.0001
p < 0.0001p < 0.001
Lipoprotein Lipase (LPL)
Vehi
cle
in N
orm
al
Vehi
cle
in N
ASH
30m
g/kg
100
mg/
kg
300
mg/
kg
Telm
isar
tan
10m
g/kg
0.0
0.5
1.0
1.5
ANG
PTL3
mR
NA
/ 36B
4 m
RN
A ±
SEM
ANGPTL3
Gemcabene
n.s.
n.s.
p < 0.05p < 0.05
p < 0.0001
Angiopoietin 3 (ANGPTL3)
Vehi
cle
in N
orm
al
Vehi
cle
in N
ASH
30m
g/kg
100
mg/
kg
300
mg/
kg
Telm
isar
tan
10m
g/kg
0
1
2
3
4
ANG
PTL4
mR
NA
/ 36B
4 m
RN
A ±
SEM
ANGPTL4
Gemcabene
n.s.
p < 0.0001p < 0.0001
p < 0.0001
p < 0.05
Vehi
cle
in N
orm
al
Vehi
cle
in N
ASH
30m
g/kg
100
mg/
kg
300
mg/
kg
Telm
isar
tan
10m
g/kg
0.0
0.5
1.0
1.5
2.0
2.5
ANG
PTL8
mR
NA
/ 36B
4 m
RN
A ±
SEM
ANGPTL8
Gemcabene
n.s.p < 0.0001
n.s.n.s.
n.s.
0.0 0.2 0.4 0.6 0.8 1.00
500
1000
1500
ApoC-III mRNA/36B4mRNA
Plas
ma
Trig
lycer
ides
(mg/
dL)
Hepatic ApoC-III mRNA vs Plasma TG without Normal
R = 0.57P Value = 0.0007
NASH NASH + GEM30 NASH + GEM100 NASH + GEM300
0.0 0.2 0.4 0.6 0.8 1.00
500
1000
1500
ApoC-III mRNA/36B4mRNA
Pla
sma
Trig
lyce
rid
es
(mg
/dL
)
Hepatic ApoC-III mRNA vs Plasma TG without Normal
R = 0.57P Value = 0.0007
NASH NASH + GEM30 NASH + GEM100 NASH + GEM300
Hepatic ApoC-III mRNA vs Plasma TGs
0 2 4 6 80
500
1000
1500
Sulfatase-2 mRNA/36B4mRNA
Pla
sma
Trig
lyce
ride
s (m
g/d
L)
Hepatic Sulf2 mRNA vs Plasma Triglycerides without Normal
R = 0.52P Value = 0.0022
Hepatic SULF2 mRNA vs Plasma TGs Hepatic LPL mRNA vs Plasma TG w/o Normal
0 2 4 6 8 100
500
1000
1500
LPL mRNA/36B4mRNA
Pla
sma
Trig
lyce
rides
(m
g/dL
)
R = 0.6P Value = 0.0006
Hepatic LPL mRNA vs Plasma TGs Hepatic ANGPTL 3 vs Plasma TG w/o Normal
0.0 0.5 1.0 1.50
500
1000
1500
ANGPTL3 mRNA/36B4mRNA
Pla
sma
Trig
lyce
rides
(m
g/dL
)
R = 0.53P Value = 0.0017
Hepatic ANGPTL 4 vs Plasma TG w/o Normal
0 1 2 3 40
500
1000
1500
ANGPTL4 mRNA/36B4mRNA
Pla
sma
Trig
lyce
rides
(m
g/dL
)
R = 0.57P Value = 0.0007
Hepatic ANGPTL3 mRNA vs Plasma TGs Hepatic ANGPTL4 mRNA vs Plasma TGs
Vehi
cle
in N
orm
al
Veh
icle
in N
AS
H
30m
g/kg
100
mg/
kg
300
mg/
kg
Telm
isar
tan
10m
g/kg
0
5
10
15
20
IL-6
mR
NA
/ 36
B4
mR
NA
± S
EM
Interleukin 6
Gemcabene
p < 0.05
p < 0.05
p < 0.05n.s.
p < 0.05
Vehi
cle
in N
orm
al
Vehi
cle
in N
ASH
30m
g/kg
100
mg/
kg
300
mg/
kg
Telm
isar
tan
10m
g/kg
0.0
0.5
1.0
1.5
CR
P m
RN
A / 3
6B4
mR
NA
± SE
M
CRP
Gemcabene
n.s.
p < 0.0001p < 0.0001
n.s.
n.s.Ve
hicl
e in
Nor
mal
Vehi
cle
in N
ASH
30m
g/kg
100
mg/
kg
300
mg/
kg
Telm
isar
tan
10m
g/kg
0
2
4
6
8
TNFα
mR
NA
/ 36B
4 m
RN
A ±
SEM
Tumor Necrosis Factor α
Gemcabene
n.s.
n.s.
p < 0.05p < 0.05
p < 0.0001
Interleukin (IL) 6 Tumor Necrosis Factor (TNF) α C-Reactive Protein (CRP) mRNA
IL6 mRNA vs Plasma CRP w/o Normal
0 5 10 15 200
1
2
3
4
5
IL-6 mRNA/36B4mRNA
Pla
sma
CR
P (µg
/mL)
R = 0.59P Value = 0.0003
TNFa mRNA vs Plasma CRP w/o Normal
0 2 4 6 80
1
2
3
4
TNFα mRNA/36B4 mRNA
Pla
sma
CR
P (µg
/mL)
R = 0.59P Value = <0.0004
Hepatic CRP mRNA vs Plasma CRP w/o Normal
0.0 0.5 1.0 1.50
1
2
3
4
CRP mRNA/36B4 mRNA
Plas
ma
CR
P (µ
g/m
L)R = 0.81P Value = <0.0001
IL6 mRNA vs Plasma CRP TNFα mRNA vs Plasma CRP Hepatic CRP mRNA vs Plasma CRP
1. Ridker PM, et al. N Engl J Med. 2008;359:2195-207. 2. Robinson JG, et al. N Engl J Med. 2015;372:1489-99. 3. Sabatine MS, et al. N Engl J Med. 2017;376:1713-1722. 4. Deedwania PC, et al. Am J Cardiol. 2005;95:360-6. 5. Blom DJ, et al. N Engl J Med. 2014;370:1809-19. 6. Reyes-Soffer G, et al. Circulation. 2017;135:352-362. 7. Taskinen MR and Borén J. Atherosclerosis. 2015;239:483-95. 8. Chen K, Williams KJ, et al. Hepatology. 2010;52:1957-1967. 9. Hassing HC, Stroes ESG, Williams KJ, et al. Hepatology. 2012;55:1746-1753. 10. Hassing HC, et al. Obesity (Silver Spring). 2014;22:1309-1316.
11. Bisgaier CL, et al. J Lipid Res. 1998;39(1):17-30. 12. Bisgaier CL, Newton RS. US 8,557,835, 2009. 13. Bays HE, et al. Am J Cardiol. 2003;92(5):538-43. 14. Stein E, Bisgaier C, et al. J Clin Lipidol. 2016;10(5):1212-22. 15. Bakker-Arkema, R. Bisgaier, C. J Amer Coll Cardiology 2017, 69(11) Sup: 1863 16. Gemphire Therapeutics, Clinicaltrials.gov identifier NCT02722408 17. Gemphire Therapeutics, Clinicaltrials.gov identifier NCT02634151 18. Oniciu DC, Hashiguchi T, Shibazaki Y, Bisgaier C. PLoS ONE 13(5): e0194568. 19. Bisgaier, C. Oniciu, D.C. Williams, J.K. Circulation 2017, 136, Sup 1, A19177
REFERENCES
Effect of Gemcabene on Hepatic Lipid Regulating Genes in STAMTM Mice
Effect of Gemcabene on Hepatic Inflammatory Regulating Genes in STAMTM Mice
RESULTS
CONCLUSIONS
METHODS AND PLASMA CHANGES IN TGs AND CRP
Oral Dose (mg/kg/day)
Two-Week Gemcabene Treatment Reduces LDL-C in LDL-Receptor Deficient Mice, a
HoFH Rodent Model COBALT-1: Open-Label, Dose-Escalation
(n=8) Diagnosis by genetic confirmation
(including compound heterozygosity) or clinical criteria:
• Hx of LDL-C >500 mg/dL (12.92 mmol/L) plus • xanthoma < 10 years of age • or evidence of HeFH parents
• If Hx is unavailable, LDL-C >300 mg/dL (7.76 mmol/L) on maximally tolerated lipid-lowering drug therapy
• PrimaryEndpoint:
• Changeand%changeinLDL-C• SecondaryEndpoints:
• Changeand%changeinhsCRP,ApoB,otherlipidsandlipoproteins
• Safetyandtolerability
Gemcabene 300 mg (4 weeks)
Gemcabene 600 mg (4 weeks)
Gemcabene 900 mg (4 weeks)
COBALT-1: Open-Label, Dose-Escalation (n=8)
Diagnosis by genetic confirmation (including compound heterozygosity) or
clinical criteria: • History of LDL-C >500 mg/dL (12.92 mmol/L) plus
• xanthoma < 10 years of age • or evidence of HeFH parents
• If history is unavailable, LDL-C >300 mg/dL (7.76 mmol/L) on maximally tolerated lipid-lowering drug therapy
Cobalt-1 Trial Design: Familial Hypercholesterolemia
Cobalt-1 (HoFH Patients) (LDL Receptor Deficient)
Cobalt-1 (HeFH Patients)
• In the STAMTM murine model of NASH, gemcabene reduced plasma levels of triglycerides and CRP.
• Gemcabene regulated the mRNA expression levels of multiple genes that modulate plasma triglyceride levels. With respect to lipid
trafficking genes, gemcabene down-regulated the hepatic mRNA expression levels of ApoC-III, SULF2, ANGPTL3 and ANGPTL4,
and up-regulated that of LPL. Gemcabene intervention showed that each lipid trafficking gene was significantly correlated with
reduced plasma TG levels, and suggests a coordinated response for TG lowering.
• Gemcabene regulated the hepatic mRNA expression levels of multiple genes that are associated with inflammation: IL-6, TNF-α
and CRP, and each of these were highly correlated with plasma CRP reduction.
• These results suggest that gemcabene’s pleiotropic mechanisms may have beneficial effects on residual ASCVD risk and NASH.
Angiopoietin 4 (ANGPTL4) Angiopoietin 8 (ANGPTL8)
LDLreceptor
SR-BI(ChaperoneforSDC1)
Syndecan-1HSPG
Basal membrane
Fenestrated endothelium
Hepatic sinusoid
Space of Disse
Hepatic parenchymal cell
t 1/2~1
h
t 1/2~
10m
in
Endocytosis
C-TRL remnant
Direct receptor- mediated uptake
Healthy Hepatic Uptake of C-TRL
SULF2
LDLreceptor
SR-BI(ChaperoneforSDC1)
Syndecan-1HSPG
Basal membrane
Fenestrated endothelium
Hepatic sinusoid
Space of Disse
Hepatic parenchymal cell
t 1/2~
10m
in
Endocytosis
C-TRL remnant
Direct receptor- mediated uptake
Elevated Sulf2 Blocks Syndecan-1 and Impairs Hepatic Uptake of C-TRL
LDLreceptor
SR-BI(ChaperoneforSDC1)
Syndecan-1HSPG
Basal membrane
Fenestrated endothelium
Hepatic sinusoid
Space of Disse
Hepatic parenchymal cell
t 1/2~
1h
t 1/2~
10m
in
Endocytosis
C-TRL remnant
Direct receptor- mediated uptake
Healthy Hepatic Uptake of C-TRL
SULF2
LDLreceptor
SR-BI(ChaperoneforSDC1)
Syndecan-1HSPG
Basal membrane
Fenestrated endothelium
Hepatic sinusoid
Space of Disse
Hepatic parenchymal cell
t 1/2~
10m
in
Endocytosis
C-TRL remnant
Direct receptor- mediated uptake
Elevated Sulf2 Blocks Syndecan-1 and Impairs Hepatic Uptake of C-TRL
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