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. 2020 Nov;40(11):2860-2876.
doi: 10.1111/liv.14643.

Dual _targeting of hepatic fibrosis and atherogenesis by icosabutate, an engineered eicosapentaenoic acid derivative

Geurt Stokman  1 , Anita M van den Hoek  1 , Ditte Denker Thorbekk  2 , Elsbet J Pieterman  1 , Sanne Skovgård Veidal  2 , Brittany Basta  3 , Marta Iruarrizaga-Lejarreta  4 , José W van der Hoorn  1 , Lars Verschuren  5 , Jimmy F P Berbée  6 , Patrick C N Rensen  6 , Tore Skjaeret  7 , Cristina Alonso  3 , Michael Feigh  2 , John J P Kastelein  8 , Scott L Friedman  3 , Hans M G Princen  1 , David A Fraser  7
Affiliations

Dual _targeting of hepatic fibrosis and atherogenesis by icosabutate, an engineered eicosapentaenoic acid derivative

Geurt Stokman et al. Liver Int. 2020 Nov.

Abstract

Background & aims: While fibrosis stage predicts liver-associated mortality, cardiovascular disease (CVD) is still the major overall cause of mortality in patients with NASH. Novel NASH drugs should thus ideally reduce both liver fibrosis and CVD. Icosabutate is a semi-synthetic, liver-_targeted eicosapentaenoic acid (EPA) derivative in clinical development for NASH. The primary aims of the current studies were to establish both the anti-fibrotic and anti-atherogenic efficacy of icosabutate in conjunction with changes in lipotoxic and atherogenic lipids in liver and plasma respectively.

Methods: The effects of icosabutate on fibrosis progression and lipotoxicity were investigated in amylin liver NASH (AMLN) diet (high fat, cholesterol and fructose) fed ob/ob mice with biopsy-confirmed steatohepatitis and fibrosis and compared with the activity of obeticholic acid. APOE*3Leiden.CETP mice, a translational model for hyperlipidaemia and atherosclerosis, were used to evaluate the mechanisms underlying the lipid-lowering effect of icosabutate and its effect on atherosclerosis.

Results: In AMLN ob/ob mice, icosabutate significantly reduced hepatic fibrosis and myofibroblast content in association with downregulation of the arachidonic acid cascade and a reduction in both hepatic oxidised phospholipids and apoptosis. In APOE*3Leiden.CETP mice, icosabutate reduced plasma cholesterol and TAG levels via increased hepatic uptake, upregulated hepatic lipid metabolism and downregulated inflammation pathways, and effectively decreased atherosclerosis development.

Conclusions: Icosabutate, a structurally engineered EPA derivative, effectively attenuates both hepatic fibrosis and atherogenesis and offers an attractive therapeutic approach to both liver- and CV-related morbidity and mortality in NASH patients.

Keywords: NASH; apoptosis; arachidonic acid; atherosclerosis; lipotoxicity; oxidised phospholipids.

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Conflict of interest statement

Northsea Therapeutics BV acquired the commercial rights for icosabutate. JK and SF are paid consultants and have stock options in Northsea Therapeutics BV. DF and TS are employees of NorthSea Therapeutics BV but had no role in data collection. SF acts as a paid consultant for the following companies: 89 Bio, Amgen, Axcella Health, Blade Therapeutics, Bristol Myers Squibb, Can‐Fite Biopharma, ChemomAb, Escient Pharmaceuticals, Forbion, Galmed, Gordian Biotechnology, Glycotest, Glympse Bio, In sitro, Morphic Therapeutics, Novartis, Ono Pharmaceuticals, Scholar Rock, Surrozen. All other authors have no conflict of interest to declare.

Figures

FIGURE 1
FIGURE 1
Both icosabutate and OCA reduce liver fat, but only icosabutate reduces liver enzymes and liver inflammation in AMLN ob/ob mice. Effects of treatment on terminal bodyweight (A), liver weight (B) and liver lipids as measured by % steatosis (C) or hepatic triacylglycerol content (D), plasma ALT (E) and hepatic galectin‐3 (F). Values represent mean ± SEM for 12 mice per group. (G) Representative histological photomicrographs of liver cross sections stained with H&E or anti‐Galectin 3, magnification 20x. *P < .05, **P < .01, ***P < .001 vs vehicle
FIGURE 2
FIGURE 2
Icosabutate prevents hepatic collagen deposition in AMLN ob/ob mice. Liver col1A1 content as measured by either percent area (A) or total content (B). Change in liver col1A1 content between baseline and post‐treatment (C). Histological photomicrographs of liver cross sections stained with anti‐col1A1 pre‐ vs post‐treatment (D), magnification 20×. Liver hydroxyproline (HYP) content (E‐F). Values represent mean ± SEM for 12 mice per group. *P < .05, **P < .01, ***P < .001 vs vehicle
FIGURE 3
FIGURE 3
Icosabutate reduces the hepatic content of activated stellate cells in AMLN ob/ob mice and proliferation of human stellate cells. Liver α‐SMA content as measured by percent area (A) and total content (B). Change in liver α‐SMA content between baseline and post‐treatment (C). Histological photomicrographs of liver cross sections stained with anti‐α‐SMA (a marker of activated stellate cells) pre‐ vs post‐treatment (D), magnification 20×. Values represent mean ± SEM for 12 mice per group. *P < .05, **P < .01, ***P < .001 vs vehicle. LX‐2 cell viability (E) and proliferative responses (F). Results are presented as normalised mean values ± SEM of 5 (2 for OA) independent experiments performed in triplicate. **P < .005, ***P < .0001 vs vehicle
FIGURE 4
FIGURE 4
Icosabutate reduces hepatic NASH‐associated lipotoxic lipids species, oxidative stress and oxPLs in AMLN ob/ob mice. Post‐treatment hepatic concentrations of FFAs (A), DAG (B), ceramides (C), bile acids (D) HETEs (comprising 11(R)‐, 12‐ and 15(S) isomers) (E), AA‐containing PC (F), oxidised glutathione (G), reduced/oxidised glutathione ratio (H) and oxPLs (comprising PC‐AA‐OH and LPC‐LA‐OH) (I). Values represent mean ± SEM for 12 mice per group. *P < .05, **P < .01, ***P < .001 vs vehicle
FIGURE 5
FIGURE 5
Icosabutate preferentially _targets highly unsaturated hepatic TAG, DAG and reduces ceramides in AMLN ob/ob mice. Influence of the number of carbons and double bond content in the decrement of TAG (A). Change in specific DAG (B) and ceramide (C) species. Colour code represents the transformed ratio between means of the groups: green sections denote metabolites that were reduced (negative log2 fold‐changes) and red sections denote increased metabolites (positive log2 fold‐changes). For TAG the y axis denotes the number of carbons and the x axis the number of double bonds. Data are presented as mean ± SEM, *P < .05, **P < .01, ***P < .001 vs vehicle
FIGURE 6
FIGURE 6
Icosabutate reduces hepatic apoptosis in AMLN ob/ob mice. Effects of treatments on the number of apoptotic cells numbers at study termination as measured by TUNEL (A). Representative images of cross sections of liver stained with TUNEL at termination (magnification 20×, scale bar = 50 μm) (B). Values represent mean ± SEM for 12 mice per group. *P < .05, **P < .01 vs vehicle
FIGURE 7
FIGURE 7
Icosabutate reduces plasma lipids in association with increased hepatic clearance and upregulated hepatic LDL‐R expression in APOE*3Leiden.CETP mice. Total plasma TAG (A), plasma cholesterol (B) and HDL cholesterol (C). (D) Hepatic VLDL‐TAG production expressed as production rate per hour (mM/h). (E) Hepatic VLDL clearance was determined by measuring glycerol tri[3H]oleate‐derived activity and (F) [14C]cholesteryl oleate in plasma and expressed as plasma half‐life. Activity of (G) hepatic lipase and (H) lipoprotein lipase was determined in post‐heparin plasma as the rate of free fatty acid (FFA) release from VLDL‐TAG. (I) Protein expression of LDL‐R in liver tissue was determined by Western blotting. Data were normalised vs controls. (J) Liver free cholesterol, cholesteryl esters and triacylglycerol contents. (K) Excretion of bile acids and neutral sterols in faecal samples. Data are presented as mean ± SEM, *P ≤ .05, **P ≤ .01, ***P ≤ .001
FIGURE 8
FIGURE 8
Icosabutate reduces atherosclerotic plaque formation in APOE*3Leiden.CETP mice. Representative images of the aortic root (A). (B) Total cholesterol exposure. (C) Total lesion area per cross section of the aortic root. (D) Number of lesions per cross section. (E) Percentage of undiseased aortic root segments and pathohistological scoring of lesions expressed as percentage of total number of lesions. Severity was classified as type I‐II: initial lesion/fatty streak, type III: intermediate lesion, type IV‐V: (fibro)atheroma lesion. Data are presented as mean ± SEM, *P ≤ .05, **P ≤ .01, ***P ≤ .001
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