Treatment response of murine sclerosing cholangitis to systemic versus intestinal FXR agonists segregates with their effects on hepatic pro-inflammatory cytokine production

Oral presentation at AASLD – The Liver Meeting, November 2018

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

Abstract:

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.

Treatment response of murine sclerosing cholangitis to systemic versus intestinal FXR agonists segregates with their effects on hepatic pro-inflammatory cytokine production

Oral presentation at XXV International Bile Acid Meeting: Bile Acids in Health and Disease, July 2018

Tiffany Shi1, Celine S. Lages1, Ramesh Kudira1, Louis Matuschek1, Mary Mullen1, Alvaro Ortiz2, Kyoung-Jin Lee2, Douglas Zook2, Brandee Wagner2, Alexander Miethke1

1. Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
2. Metacrine, Inc., San Diego, CA, USA

Introduction: 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 systemic FXR agonist M345 or 100 mg/kg/day of intestinal FXR agonist M379, 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 and intestinal gene expression (8hr after the last oral dose of FXR agonist) was quantitated by Taqman-based qPCR.

Results: Compared to vehicle control mice, M345-treated mice displayed 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 when compared to controls. While both M345- and M379 significantly induced intestinal Shp and Fgf15 gene expression and reduced hepatic mRNA expression of Cyp8b1 (down 91% vs 79%; p=0.22) and liver bile acid levels (down 38% vs 29%; p=0.36), only M345-treatment reduced hepatic TNFα mRNA expression when compared to controls (down-89%; p=0.00).

Discussion / Conclusion: While both systemic and intestinal FXR agonists down-regulate transcription of key enzymes of de novo bile acid synthesis and subsequently liver bile acid concentration, only the systemic FXR agonist M345 represses hepatic TNFα expression, which is associated with attenuation of the sclerosing cholangitis phenotype.

M480, an oral FXR agonist, is superior to Cyclosporine A and equally efficacious as anti-IL12/23 in the adoptive T-cell transfer model of IBD colitis

Oral presentation at Digestive Disease Week, June 2018

Xueqing Liu1, Harry Dedman1, Robert O’Connell2, Alison Bendele2, Connor Ohlsen2, Steve Govek1, Johnny Nagasawa1, Karensa Douglas1, Angelica Milik1, Nhin Lu1, Jing Qian1, Alvaro Ortiz1, Kyoung-Jin Lee1, Nicholas Smith1, Brandee Wagner1, Ken Song1

1. Metacrine, San Diego, CA
2. Bolder BioPath, Inc., Boulder, CO

Introduction: Inflammatory bowel disease (IBD) is a debilitating disease in which most effective therapies are injectable biologics with immunosuppressive side effects. Targeting the farnesoid X receptor (FXR), a nuclear hormone receptor activated by bile acids, represents a novel approach. FXR is a ligand-activated transcription factor highly expressed in the liver and gastrointestinal tract. Previous studies with FXR agonists have shown prophylactic efficacy in DSS and TNBS chemically induced IBD models. Unlike DSS and TNBS models, the adoptive T-cell transfer model is thought to be more representative of human IBD. Therefore, we examined the efficacy of M480, a potent non-bile acid FXR agonist, in therapeutic treatment mode in adoptive T-cell transfer colitis.

Methods: Colitis was induced by transplanting purified CD4+CD45RBhi T-cells to recipient C.B-17 SCID mice. Body weights of all mice were monitored throughout study duration. Treatments with vehicle (n=10), M480 (10 and 30 mg/kg p.o., n=10), anti-IL-12p40 (neutralizing both IL-12 and IL-23, 0.5mg/mouse, i.p., n=5) and cyclosporine A (CsA, 50 mg/kg p.o., n=10) were administered starting 21 days post T-cell transfer and maintained for 4 weeks. Terminal endpoints included colon weight to length ratio (W/L), colon histopathology and gene expression.  Histopathology analysis included inflammation, erosion, gland loss and hyperplasia, each with a score of 0-5, and cumulative sum score of 0-20.

Results: CD4+CD45RBhi T-cell transfer led to a 12% (p<0.05) reduction in body weight from baseline, which was reversed by M480 (10 mg/kg), anti-IL-12p40 and CsA. A marker of colitis, colon W/L, was increased 2.8 fold (p<0.05) in the vehicle group, relative to control mice without T-cell transfer. As compared to vehicle, M480 treated mice showed 41% and 38% reduction in colon W/L at 10 mg/kg and 30 mg/kg respectively (p<0.01).  Treatment with anti-IL-12/23 and CsA showed 52% and 34% improvement in colon W/L, respectively. The vehicle treated mice averaged histopathology scores of 4, 3 and 2 for inflammation, hyperplasia and gland loss, respectively, with little or no erosion, and an average histopathology sum score of 10. M480 at 10 and 30 mg/kg significantly reduced the sum score by 71% (p<0.01) and 74% (p<0.01), respectively, comparable to 78% reduction by anti-IL-12/23 (p<0.01).  Both M480 and anti-IL-12/23 showed similar improvement across all histopathology endpoints. While CsA improved the colon W/L it failed to show significant improvement in histopathology parameters.

Conclusion:  M480, a non-bile acid FXR agonist, is efficacious in reducing colitis in the adoptive T-cell transfer model with efficacy superior to CsA and comparable to anti-IL-12/23 treatment.  M480 represents a novel class of oral agents that may offer an alternative treatment for IBD.