Authors & affiliations: Tiffany Shi1, Louis Matuschek1, Celine S. Lages1, Ramesh Kudira1, Mary Mullen1, Alvaro Ortiz2, Kyoung-Jin Lee2,Douglas Zook2, Brandee Wagner2, Alexander Miethke1
1Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
2Metacrine, Inc., San Diego, CA, USA
Background: FXR agonists are potent therapeutic regulators of liver metabolic function. Their role in the treatment of inflammatory cholestatic liver disease is less defined. Here, we use Mdr2-/- mice, a model of defective canalicular excretion of phospholipids which leads to biliary precipitation of bile acids, cholangiocyte injury, sterile inflammation and fibrosis.
Methods: 45-day-old mdr2-/- female mice underwent oral gavage for 7 days with 30 mg/kg/day of M345, systemic FXR agonist with sustained activity, or 100 mg/kg/day M379 a transient, intestinal FXR agonist, both derivatives of fexaramine, or with vehicle (corn oil) in controls. Serum liver biochemistries and bile acid levels were determined by colorimetric assays and liver, intestinal and hepatic mononuclear cell (MNC) gene expression (8hr after the last oral dose) was quantitated by Taqman-based qPCR. Splenic MNC isolated from mdr2-/- mice were cultured with 0.1uM M345 or DMSO control for 16 hours with LPS stimulation and qPCR was performed.
Results: Compared to vehicle control mice, M345-treated mice showed improved weight gain (+4.8 vs -0.9 g compared to baseline; p=0.00), lower serum liver biochemistries (ALT 298 vs 981 IU/L; p=0.01, ALP 149 vs 197 IU/L; p= 0.03, TB 1.2 vs 6.8 mg/dL; p= 0.01) and decreased serum bile acid levels (282 vs 923 umol/L; p=0.03). In contrast, M379-treated mice showed continued weight loss and no significant change in serum liver biochemistries or serum bile acid levels. While both M345 and M379 significantly induced intestinal Shp and Fgf15 gene expression and reduced hepatic mRNA expression of Cyp8b1 and liver bile acid levels, only M345 treatment reduced whole liver Tnfα (down-89%; p=0.00) and IL1β (down-79%; p=0.02) as well as hepatic MNC Tnfα (down-69%; p=0.05) and IL1β (down-96%; p=0.03) mRNA expression when compared to controls. In vitro experiments also showed a decrease in Tnfα and IL1β mRNA expression in LPS stimulated splenic MNCs from mdr2-/-treated with M345 when compared to DMSO controls.
Conclusion: While both the sustained, systemic and transient, intestinal FXR agonists down-regulate transcription of key enzymes of de novo bile acid synthesis and subsequently liver bile acid concentration, only the sustained, systemic FXR agonist M345 represses Tnfα and IL1β expression in hepatic MNC, which is associated with attenuation of the mdr2-/- phenotype. Cell culture experiments suggest direct effects of M345 on pro-inflammatory cytokine production by MNCs.