This study investigated whether BAR704, a potent and selective FXR agonist, protects against liver fibrosis in a mouse model. Mice were administered carbon tetrachloride to induce liver fibrosis over 8 weeks. Treatment with BAR704 reversed biochemical and histological signs of fibrosis, reducing markers of liver damage and collagen deposition. BAR704 also decreased expression of profibrogenic genes and normalized levels of genes related to endothelial function. The results suggest that selective FXR agonists like BAR704 may be an attractive treatment for liver fibrosis.

1. BAR704, a potent and selective FXR agonist protects against liver fibrosis
Adriana Carino*, Sabrina Cipriani # , Michele Biagioli*, Silvia Marchianò*, Angela Zampella † and Stefano Fiorucci*
*Dipartimento di Scienze Chirurgiche e Biomediche, Università degli studi di Perugia Nuova Facoltà di Medicina e Chirurgia, Sant’Andrea delle Fratte, Perugia, Italy
#Dipartimento di Medicina, Università degli studi di Perugia Nuova Facoltà di Medicina e Chirurgia, Sant’Andrea delle Fratte, Perugia, Italy
†Dipartimento di Farmacia, Università di Napoli Federico II, Napoli, Italy
Background. FXR and GPBAR1, two bile acid-activated receptors, represent a new opportunities for the pharmacological treatment of liver and metabolic disorders. Even if dual FXR/GPBAR1 agonists are considered a novel opportunity in the treatment of these diseases,
selective targeting of one of these receptors represents an attractive therapeutic approach for a wide range of disorders in which dual modulation associates with severe side effects. BAR704 is a potent and selective FXR agonist, showing an EC50 of 950 nM in transactivation
assay on HepG2 cells. Administrated to intact mice at the dose of 15 mg/kg, this agent significantly increased the expression of OSTα, SHP and BSEP, three canonical FXR target genes. Further, exposure to this agent results in a marked reduction of total and single
unconjugated bile acids, as well as the reduction blood concentrations of 7α -hydroxy-4-cholesten-3-one, an intermediate in the synthesis of primary bile acids.
Aim of the study. To investigate whether BAR704 protects against development of liver fibrosis and cirrhosis in rodent model of chronic liver damage.
Methods. Liver fibrosis was induced by carbon tetrachloride (CCl4) administration. For this purpose C57BL6 mice were administered i.p. 500 µl/Kg body weight of CCl4 twice a week for 8 weeks. CCL4 mice were randomized to receive BAR704 (15 mg/Kg daily by gavage) or
vehicle (distilled water). At the end of the treatment Portal perfusion pressure was evaluated. The animals were sacrificed and blood and liver collected for plasma AST, Albumin and Bilirubin measurement, liver histopathology analysis (H/E and Sirius Red) and for evaluation of
fibrosis markers genes expression (RT-PCR) .
Results. Treating mice with CCl4 resulted in an increased AST and bilirubin plasma levels, and in a marked reduction of albumin plasma levels. These biochemical features were supported by development of severe fibrosis as demonstrated by liver histology
and by the increased expression of pro-fibrogenetic genes (including Colα1, αSMA). Treating mice with BAR704 reversed this pattern and protected against liver fibrosis development. BAR704 reduced AST and Bilirubin plasma levels, increased Albumin plasma
levels and reduced ECM deposition as measured by assessing the fibrosis score and expression of pro-fibrogenetic genes. Exposure to BAR704 also reduced portal pressure and this effect was likely linked to reduction of collagen deposition but also involved
reshaping of additional genes such as CSE, CBS, ET-1 and Caveolin 1.
Conclusions. This study shows that BAR704 a selective FXR agonist exerts benefiicial effects on liver fibrosis. This study suggest that the use of FXR selective agonists might represent an attractive strategy for the treatment of fibrosis in liver disease.
Figure 3: Total RNA extracted from liver was used to evaluate by RT-PCR the relative mRNA expression of (A)
marker genes of fibrosis, (B) Nuclear Receptor genes and (C) genes involved in endothelial function. Values are
normalized to GAPDH and are expressed relative to those of not treated animals (CTRL) which are arbitrarily
settled to 1. The relative mRNA expression is expressed as 2(-ΔΔCt). Results are the mean ± SE of 6–12 mice per
group. *p < 0.05 vs CTRL mice. #p<0.05 versus CCl4 alone (Figure 4, A-C)
Figure 1: (A) Molecular structure of BAR704. (B) Transactivation assay on FXR ligand
binding domain. HepG2 cells were transiently transfected with pSG5-FXR, pSG5-RXR,
p(hsp27)TKLUC vectors and with pGL4.70, pGL4.70 (Promega), a vector encoding the
human Renilla gene. 24 hours post-transfection cells were stimulated with 10 µM of
BAR704, CDCA or 6E-CDCA, used as positive controls. Results are expressed as the
mean ± standard error (*p<0.05 vs not treated cells (NT). (C) Concentration−response
curves for BAR704. HepG2 cells were transiently transfected as described before, and
then stimulated with increasing concentration of BAR704 (0.1, 1 and 10 µM). (Figure 1, A-
C).
Figure 2: Liver fibrosis was induced by carbon tetrachloride (CCl4) administration (i.p. 500 μL/Kg body weight, twice a week for 8
weeks). CCl4 mice were randomized to receive BAR704 (15 mg/Kg daily by gavage) or vehicle (distilled water). Data shown are
plasmatic levels of (A) portal pressure, (B) AST, (C) Albumin and (D) Bilirubin. (E) Hematoxylin and eosin (H&E) staining. (F) Sirius
red staining. (G) Image J quantification of Sirius red staining. Data are mean ± SE of 6–12 mice per group. *p < 0.05 versus CTRL
mice. # p< 0.05 versus CCl4. (Figure 2, A-G)
0
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10-7 10-6 10-5 10-4
EC50: 950nM
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Col11 SMA TGF TNF IL1
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mRNArelativeexpressiontoGAPDH
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mRNArelativeexpressiontoGAPDH
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mRNArelativeexpressiontoGAPDH
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HepG2 FXR-RXR
NT CDCA 6E-CDCA BAR704
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