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

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Plas

ma

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Plasma Triglycerides

Gemcabene

n.s.p < 0.001

p < 0.001

p < 0.001p < 0.05

Plasma Triglycerides Plasma CRP

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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

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apoC

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36B

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SE

M

Apolipoprotein C-III

Gemcabene

n.s.p < 0.001

n.s.

p < 0.01p < 0.0001

Apolipoprotein C-III (ApoC-III)

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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)

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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)

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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)

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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

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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

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(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

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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

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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

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1.5

CR

P m

RN

A / 3

6B4

mR

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± SE

M

CRP

Gemcabene

n.s.

p < 0.0001p < 0.0001

n.s.

n.s.Ve

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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