A Medium-Chain Fatty Acid Analogue Prevents Intestinal Failure-Associated Liver Disease in Preterm Yorkshire Piglets

Abstract

Background & aims: At least 20%-30% of patients with intestinal failure receiving long-term parenteral nutrition will develop intestinal failure-associated liver disease (IFALD), for which there are few therapeutic options. SEFA-6179 is a first-in-class structurally engineered medium-chain fatty acid analogue that acts through GPR84, PPARα, and PPARγ agonism. We hypothesized that SEFA-6179 would prevent biochemical and histologic liver injury in a preterm piglet model of IFALD.

Methods: Preterm Yorkshire piglets were delivered by cesarean section, and parenteral nutrition was provided for 14 days via implanted central venous catheters. Animals were treated with either medium-chain triglyceride vehicle control or SEFA-6179.

Results: Compared to medium-chain triglyceride vehicle at day of life 15, SEFA-6179 prevented biochemical cholestasis (direct bilirubin: 1.9 vs <0.2 mg/dL, P = .01; total bilirubin: 2.7 vs 0.4 mg/dL, P = .02; gamma glutamyl transferase: 172 vs 30 U/L, P = .01). SEFA-6179 also prevented steatosis (45.6 vs 13.9 mg triglycerides/g liver tissue, P = .009), reduced bile duct proliferation (1.6% vs 0.5% area cytokeratin 7 positive, P = .009), and reduced fibrosis assessed by a masked pathologist (median Ishak score: 3 vs 1, P = 0.007). RNA sequencing of liver tissue demonstrated that SEFA-6179 broadly impacted inflammatory, metabolic, and fibrotic pathways, consistent with its in vitro receptor activity (GPR84/PPARα/PPARγ agonist).

Conclusions: In a preterm piglet model of IFALD, SEFA-6179 treatment prevented biochemical cholestasis and steatosis and reduced bile duct proliferation and fibrosis. SEFA-6179 is a promising first-in-class therapy for the prevention and treatment of IFALD that will be investigated in an upcoming phase II clinical trial.

Keywords: Animal; Cholestasis; Intestinal Failure; Liver Diseases/Drug Therapy; Models; Parenteral Nutrition.

Fig. 1.. Study design.

Figure created with Biorender.com.

Fig. 2.. In vitro, SEFA-6179 is a GPR84 partial agonist, PPARα partial agonist, and PPARγ full agonist.

Chemical structure of SEFA-6179 (A) is shown in comparison to decanoic acid (B). GPR84 activity of SEFA6179 was determined using a Hit Hunter^® cAMP assay with Embelin acting as a positive control (C, D). PPARα activity of SEFA-6179 was determined using PathHunter^® nuclear hormone receptor cell lines with GW7647 as a positive control (E, F). PPARγ activity of SEFA-6179 was determined using PathHunter^® nuclear hormone receptor cell lines with troglitazone as a positive control (G, H).

Fig. 3.. Nutritional outcomes in preterm piglets receiving 14 days of parenteral nutrition.

Piglets receiving medium chain triglyceride (MCT) vehicle received similar parenteral nutrition (A) and gained similar weight (B) as piglets receiving SEFA-6179 (median with interquartile range). Compared to MCT, piglets receiving SEFA-6179 had higher plasma albumin at day of life 8 and 15 (C, boxplot with range). Comparisons of weight gain assessed using rank-based analysis of covariance (rANCOVA); Comparison of parenteral nutrition volume assessed by mean area under the curve and exact Wilcoxon rank sum. Comparisons of plasma albumin were done with exact Wilcoxon rank-sum tests. * P < 0.05; ** P<0.01.

Fig. 4.. SEFA-6179 prevents biochemical cholestasis and increases serum adiponectin.

Piglets received daily orogastric gavage of medium chain triglyceride (MCT) vehicle or SEFA-6179. Serum adiponectin was assessed at DOL 15. Boxplots with range. Comparisons between MCT and SEFA-6179 piglets at DOL 1, 8, and 15 were performed with exact Wilcoxon rank-sum tests. *P < 0.05 **P < 0.01

Fig. 5.. Medium chain triglyceride (MCT) piglets show steatosis, bile duct proliferation, and fibrosis on representative liver histology, which is reduced by SEFA-6179 treatment.

Formalin-fixed paraffin-embedded liver sections were stained with hematoxylin & eosin (H&E, A-B). Frozen liver sections were stained with oil red O (C-D). Immunohistochemical staining (DAB, brown) of formalin-fixed paraffin-embedded liver sections was performed for cytokeratin 7 (E-F) and αSMA (G-H). Formalin-fixed paraffin-embedded liver sections were stained with Masson’s trichrome (I-J). MCT piglets demonstrate steatosis (A, asterisks; C), extensive bile pigment (A, arrows), bile duct proliferation (E), extensive αSMA staining (G), and fibrosis indicated by bridging portal-portal and portal-central vein fibrosis (I, arrows). SEFA-6179 piglets demonstrate no steatosis (D), mild bile pigment (B, arrows), mild bile duct proliferation (B, asterisks; F), reduced αSMA staining (H), and minimal fibrotic portal expansion (J, arrows). Magnification: A– 400X; B – 200x; C – 200nm reference bars. D – 1mm reference bars. E – 100X.

Fig. 6.. SEFA-6179 reduces liver steatosis, bile duct proliferation, hepatic stellate cell activation, and Ishak fibrosis score.

SEFA-6179 piglets, compared to MCT piglets, demonstrated decreased oil red O staining (A) and liver triglyceride content (B) consistent with minimal hepatosteatosis, decreased cytokeratin 7 staining (C) consistent with minimal bile duct proliferation, decreased αSMA staining (D) consistent with decreased hepatic stellate cell activation, and decreased Ishak fibrosis score (E) assessed by a masked pathologist. Boxplots with range. Comparisons (A-D) made with exact Wilcoxon rank-sum tests. The mean Ishak score is presented as a diamond. Ishak fibrosis scores were compared with Fisher’s exact test. * P < 0.05 ** P < 0.01.

Fig. 7.. RNA-Seq demonstrates enrichment of fatty acid oxidation, retinal metabolism, and PPAR signaling from SEFA-6179 treatment.

RNA-Seq was performed on the Illumina NovaSeq 6000 platform and differentially expressed genes were identified (A). Gene ontogeny analysis (B) demonstrated biological process (BP), cellular component (CC), and molecular function (MF) categories of commonly enriched genes, including fatty acid oxidation, lipid oxidation, and cellular adhesion. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (C) demonstrated key enriched pathways including retinol metabolism and PPAR signaling. Key canonical pathways determined using Ingenuity Pathway Analysis (D) include increased oxidative phosphorylation and fatty acid β-oxidation, with downregulated granzyme A signaling and LPS/IL-1 Mediated Inhibition of RXR Function.