Combined inhibition of bile salt synthesis and intestinal uptake reduces cholestatic liver damage and colonic bile salts in mice

Abstract

Background & aims: Intestine-restricted inhibitors of the apical sodium-dependent bile acid transporter (ASBT, or ileal bile acid transporter) are approved as treatment for several inheritable forms of cholestasis but are also associated with abdominal complaints and diarrhoea. Furthermore, blocking ASBT as a single therapeutic approach may be less effective in moderate to severe cholestasis. We hypothesised that interventions that lower hepatic bile salt synthesis in addition to intestinal bile salt uptake inhibition provide added therapeutic benefit in the treatment of cholestatic disorders. Here, we test combination therapies of intestinal ASBT inhibition together with obeticholic acid (OCA), cilofexor, and the non-tumorigenic fibroblast growth factor 15 (Fgf15)/fibroblast growth factor 19 (FGF19) analogue aldafermin in a mouse model of cholestasis.

Methods: Wild-type male C57Bl6J/OlaHsd mice were fed a 0.05% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet and received daily oral gavage with 10 mg/kg OCA, 30 mg/kg cilofexor, 10 mg/kg ASBT inhibitor (Linerixibat; ASBTi), or a combination. Alternatively, wild-type male C57Bl6J/OlaHsd mice were injected with adeno-associated virus vector serotype 8 (AAV8) to express aldafermin, to repress bile salt synthesis, or to control AAV8. During a 3-week 0.05% DDC diet, mice received daily oral gavage with 10 mg/kg ASBTi or placebo control.

Results: Combination therapy of OCA, cilofexor, or aldafermin with ASBTi effectively reduced faecal bile salt excretion. Compared with ASBTi monotherapy, aldafermin + ASBTi further lowered plasma bile salt levels. Cilofexor + ASBTi and aldafermin + ASBTi treatment reduced plasma alanine transaminase and aspartate transaminase levels and fibrotic liver immunohistochemistry stainings. The reduction in inflammation and fibrogenesis in mice treated with cilofexor + ASBTi or aldafermin + ASBTi was confirmed by gene expression analysis.

Conclusions: Combining pharmacological intestinal bile salt uptake inhibition with repression of bile salt synthesis may form an effective treatment strategy to reduce liver injury while dampening the ASBTi-induced colonic bile salt load.

Impact and implications: Combined treatment of intestinal ASBT inhibition with repression of bile salt synthesis by farnesoid X receptor agonism (using either obeticholic acid or cilofexor) or by expression of aldafermin ameliorates liver damage in cholestatic mice. In addition, compared with ASBT inhibitor monotherapy, combination treatments lower colonic bile salt load.

Keywords: ASBT; Aldafermin; Alkaline phosphatase; Cholestasis; Cilofexor; Faecal bile salt; Fibroblast growth factor 15/19; IBAT; Intestine-restricted ASBT inhibitors; Liver injury; NGM282; OCA.

Graphical abstract

Fig. 1

OCA + ASBTi and mostly cilofexor + ASBTi reduce plasma liver injury markers. (A) Schematic overview of the experiment. (B) Plasma ALP. (C) Plasma ALT. (D) Plasma AST. (E) Liver weight. (F) Spleen weight. (G) Ileum Fgf15 gene expression relative to Gapdh. Data are shown as mean ± SD, and individual data points represent individual mice. Healthy controls (n = 3) are indicated by the dotted line. Statistical differences were measured using a Kruskal–Wallis one-way ANOVA, ∗p ≤0.05. ALP, alkaline phosphatase; ALT, alanine transaminase; ASBT, apical sodium-dependent bile acid transporter; ASBTi, ASBT inhibitor; AST, aspartate transaminase; DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine; Fgf15, fibroblast growth factor 15; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; OCA, obeticholic acid.

Fig. 2

Improved liver health with cilofexor and cilofexor + ASBTi treatment. (A) H&E, Sirius Red, CK7, PDGFRβ, αSMA, COL1A1, and DESMIN stain of liver tissue; the scale bar represents 50 μm. (B) Ranking of liver injury by individual treatments. αSMA, alpha smooth muscle actin; ASBT, apical sodium-dependent bile acid transporter; ASBTi, ASBT inhibitor; CK7, cytokeratin 7; COL1A1, collagen type 1 alpha 1; OCA, obeticholic acid; PDGFRβ, platelet-derived growth factor receptor beta.

Fig. 3

Cilofexor + ASBTi combination treatment improves liver inflammatory and fibrotic markers. compared with ASBTi monotherapy. Liver mRNA expression of (A) Mcp1, (B) Il-1β, (C) Tnfα, (D) Timp1, (E) Col1a1, and (F) αSma, relative to Gapdh. Data are shown as mean ± SD, and individual data points represent individual mice. Healthy controls (n = 3) are indicated by the dotted line. Statistical differences were measured using a Kruskal–Wallis one-way ANOVA, ∗p ≤0.05. αSma, alpha smooth muscle actin; ASBT, apical sodium-dependent bile acid transporter; ASBTi, ASBT inhibitor; Col1a1, collagen type 1 alpha 1; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; Mcp1, monocyte chemoattractant protein-1; OCA, obeticholic acid; Timp1, TIMP metallopeptidase inhibitor 1; Tnfα, tumour necrosis factor alpha.

Fig. 4

Cilofexor + ASBTi lowered plasma bile salt concentration and hydrophobicity and repressed faecal bile salt excretion. (A) Plasma bile salt concentration. (B) Plasma hydrophobicity index. (C) Plasma bile salt composition. (D) Faecal bile salt excretion. mRNA expression of liver (E) Shp, (F) Cyp7a1, (G) Cyp7b1, and (H) Cyp8b1 relative to Gapdh. Data are shown as mean ± SD, and individual data points represent individual mice. Healthy controls (n = 3) are indicated by the dotted line. Statistical differences were measured using a Kruskal–Wallis one-way ANOVA, ∗p ≤0.05. αMC, α-muricholic acid; ASBT, apical sodium-dependent bile acid transporter; ASBTi, ASBT inhibitor; βMC, β-muricholic acid; BW, body weight; Cyp7a1, cholesterol 7 alpha-hydroxylase; Cyp7b1, cholesterol 7 beta-hydroxylase; Cyp8b1, sterol 12-alpha-hydroxylase; DC, deoxycholic acid; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; OCA, obeticholic acid; Shp, small heterodimer particle (or Nr0b2); TɑMC, taurine-conjugated α-muricholic acid; TβMC, taurine-conjugated β-muricholic acid; TC, taurine-conjugated cholic acid; THDC, taurine-conjugated hyodeoxycholic acid; TLC, taurine-conjugated lithocholic acid.

Fig. 5

Aldafermin AAV elevated plasma FGF19 and improves cholestatic markers. (A) Schematic overview of the experiment. (B) Plasma FGF19 3 weeks after AAV8 injection. Statistical differences were measured using a non-parametric Mann–Whitney U test, ∗p ≤0.05. (C) Plasma ALP. (D) Plasma ALT. (E) Plasma AST. (F) Endpoint bodyweight. (G) Liver weight. (H) Spleen weight. Data are shown as mean ± SD, and individual data points represent individual mice. Statistical differences were measured using a Kruskal–Wallis one-way ANOVA, ∗p ≤0.05. AAV, adeno-associated virus; AAV8, AAV vector serotype 8; ALP, alkaline phosphatase; ALT, alanine transaminase; ASBT, apical sodium-dependent bile acid transporter; ASBTi, ASBT inhibitor; AST, aspartate transaminase; DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine; FGF19, fibroblast growth factor 19.

Fig. 6

Aldafermin ameliorates cholestasis-induced liver injury only in combination with ASBTi. (A) H&E, Sirius Red, CK7, PDGFRβ, αSMA, COL1A1, and DESMIN stain of liver tissue; the scale bar represents 50 μm. Liver mRNA expression of (B) Mcp1, (C) Il-1β, (D) Tnfα, (E) Timp1, and (F) Col1a1 relative to Gapdh. Data are shown as mean ± SD, and individual data points represent individual mice. Statistical differences were measured using a Kruskal–Wallis one-way ANOVA, ∗p ≤0.05. αSMA, alpha smooth muscle actin; ASBT, apical sodium-dependent bile acid transporter; ASBTi, ASBT inhibitor; CK7, cytokeratin 7; COL1A1, collagen type 1 alpha 1; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; Mcp1, monocyte chemoattractant protein-1; Timp1, TIMP metallopeptidase inhibitor 1; Tnfα, tumour necrosis factor alpha; PDGFRβ, platelet-derived growth factor receptor beta.

Fig. 7

Aldafermin repressed not only bile salt synthesis but also bile salt transport and signalling. Gene expression of (A) liver Cyp7a1, (B) liver Shp, (C) liver Slc51b, (D) liver Abcb11, (E) liver Slc10a1, (F) liver Cyp7b1, (G) liver Cyp8b1, (H) liver Abcc2, (I) ileum Fgf15, (J) ileum Fabp6, (K) ileum Slc51a, (L) ileum Slc51b, and (M) ileum Slc10a2. (N) Normalised protein expression of BSEP and NTCP including quantification relative to Na⁺/K⁺ ATPase. Data are shown as mean ± SD, and individual data points represent individual mice. Liver gene expression is corrected for Gapdh and ileum gene expression for Gapdh and Hprt. Statistical differences were measured using a Kruskal–Wallis one-way ANOVA, ∗p ≤0.05. Abcb11, ATP binding cassette subfamily B member 11; Abcc2, ATP-binding cassette sub-family C member 2; ASBT, apical sodium-dependent bile acid transporter; ASBTi, ASBT inhibitor; BSEP, bile salt export pump; Cyp7a1, cholesterol 7 alpha-hydroxylase; Cyp7b1, cholesterol 7 beta-hydroxylase; Cyp8b1, sterol 12-alpha-hydroxylase; Fabp6, fatty acid binding protein 6; Fgf15, fibroblast growth factor 15; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; Hprt, hypoxanthine guanine phosphoribosyltransferase; NTCP, Na⁺-taurocholate cotransporting polypeptide; Shp, small heterodimer particle (or Nr0b2); Slc10a1, solute carrier family 10 member 1; Slc51a, solute carrier family 51 subunit alpha; Slc51b, solute carrier family 51 subunit beta.

Fig. 8

Combination therapy of aldafermin + ASBTi reduced plasma bile salt concentrations and faecal bile salt excretion. (A) Plasma bile salt concentration. (B) Plasma bile salt pool composition. (C) Plasma bile salt hydrophobicity index. (D) Faecal bile salt excretion. Data are shown as mean ± SD, and individual data points represent individual mice. Statistical differences were measured using a Kruskal–Wallis one-way ANOVA, ∗p ≤0.05. ASBT, apical sodium-dependent bile acid transporter; ASBTi, ASBT inhibitor; BW, body weight; CA, cholic acid; DC, deoxycholic acid; GHC, glycine-conjugated hyocholic acid; GHDC, glycine-conjugated hyodeoxycholic acid; HC, hyocholic acid; TɑMC, taurine-conjugated α-muricholic acid; TβMC, taurine-conjugated β-muricholic acid; TC, taurine-conjugated cholic acid; TDC, taurine-conjugated deoxycholic acid; TCDC, taurine-conjugated chenodeoxycholic acid; THDC, taurine-conjugated hyodeoxycholic acid; TLC, taurine-conjugated lithocholic acid; UDC, ursodeoxycholic acid; ΩMC, Ω-muricholic acid.