PLN - 74809, a dual α V β 6 /α V β 1 integrin inhibitor, inhibits fibrosis in precision - cut liver tissue from PSC and PBC patients and the Mdr2 knockout mouse S Turner 1 , M Decaris 1 , S Ho 1 , C Chen 1 , G Lee 1 , V Rao 1 , M Marlow 1 , S Martin 1 , T Chen 1 , J Cha 1 , M Munoz 1 , T Hom 1 , K Leftheris 1 , Y Popov 2 and J Schaub 1 1 Pliant Therapeutics, South San Francisco, CA, 2 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA Rationale Integrins α V β 6 and α V β 1 are heterodimeric proteins that bind and activate latent TGF-β. In primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), α V β 6 and α V β 1 integrins are thought to play a role in the development and propagation of fibrosis through TGF-β activation by cholangiocytes and stellate cells, respectively. We have identified an orally available, dual-selective, small-molecule inhibitor of α V β 6 and α V β 1 , PLN-74809, and tested its ability to block fibrosis through inhibition of integrin-mediated TGF-β activation. Methods IHC and ELISA-based assays were used to evaluate expression of α V β 6 and α V β 1 in human tissue samples from PSC and PBC patients. Anti-fibrotic properties of PLN-74809 and a similarly potent, dual-selective compound, PLN-75068, were evaluated in vivo in two mouse models of biliary fibrosis (Mdr2 -/- and DDC diet) and in precision-cut liver tissue slices (PCLivS) prepared from PSC and PBC patient liver explants by IHC, hydroxyproline (OHP) quantitation, and gene expression analysis. Results 1) Integrins α V β 6 and α V β 1 were significantly elevated in fibrotic Mdr2 -/- mice, and in PSC and PBC patient liver tissue 2) PLN-75068 reduced relative hepatic collagen levels and serum ALP levels in DDC diet-injured mice 3) PLN-74809 reduced hepatic TGF-β signaling, relative hepatic collagen levels, and serum ALP levels in Mdr2 -/- mice 4) PLN-74809 reduced collagen gene expression in PCLivS prepared from PSC and PBC patient liver explants Integrins α V β 6 and α V β 1 Activate Latent TGF-β, Resulting in Profibrotic Gene Expression Conclusions Dual inhibition of α V β 6 (on cholangiocytes) and α V β 1 (on fibroblasts) offers a targeted approach for blocking TGF-β signaling in biliary fibrosis Levels of α V β 6 and α V β 1 , two integrins that activate latent TGF-β through binding to LAP, were increased in biliary fibrosis in human and mouse samples Dual inhibition of α V β 6 /α V β 1 with PLN-74809 or PLN-75068 significantly reduced hepatic TGF-β signaling, hepatic collagen deposition, and serum ALP in either the Mdr2 -/- or DDC mouse models of biliary fibrosis Dual inhibition of α V β 6 /α V β 1 with PLN-74809 reduced collagen gene expression in PCLivS prepared from PSC and PBC patient tissue explants PLN-74809 warrants further investigation as a direct anti-fibrotic in PSC and PBC clinical trials References: Peng Z, et al. Hepatology 2016,6(1):217–232 Key abbreviations: ALP, alkaline phosphatase; DDC, 3.5-Diethoxycarbonyl-1.4-dihydrocollidine; ELISA, enzyme-linked immunosorbent assay; IHC, immunohistochemistry; LAP, latency-associated peptide; OCA, obeticholic acid; OHP, hydroxyproline; PBC, primary biliary cirrhosis; PCLivS, precision-cut liver slices; PSC, primary sclerosing cholangitis; QD, once daily Disclosures: All authors, except YP, were employed by Pliant Therapeutics Inc. at the time of their contribution to the studies reported here Control Diet Vehicle 75068 150 mg/kg BID 75068 500 mg/kg BID OCA 30 mg/kg QD 0 100 200 Serum Alkaline Phosphatase U/L ** ** * p = 0.06039 **p<0.01 TGF-β-Activating Integrins α V β 6 and α V β 1 Are Present at Elevated Levels in Liver Tissue with Biliary Fibrosis IHC staining showed α V β 6 expression and SMAD3 phosphorylation in PSC and PBC liver tissue ELISA-based assay showed elevated α V β 1 levels in PSC liver tissue α V β 6 levels were elevated in Mdr2 -/- mouse livers; Itgb6 -/- mice were protected from fibrosis α V β 1 levels were elevated in Mdr2 -/- mouse livers 1 Control PSC 0 5 10 15 Human Integrin α v β 1 pg/µg total protein ** Balb/c Mdr2 -/- 0.0 0.1 0.2 0.3 pg/µg total protein p = 0.0707 Integrin α v β 1 Balb/c Mdr2 -/- 0 2 4 6 pg/µg total protein *** Integrin α v β 6 Dual α V β 6 /α V β 1 Inhibition Reduced Hepatic Collagen Deposition in the DDC Biliary Fibrosis Model Week 2 Week 4 Week 6 Week 8 Tissue Collection Week 0 PLN-75068 oral BID DDC diet Picrosirius red β 6 Integrin 4 weeks of 0.03% DDC feeding induced collagen deposition and integrin α V β 6 expression in the liver Control Diet Vehicle 75068 150mg/kg BID 75068 500mg/kg BID OCA 30mg/kg QD 0.0 0.2 0.4 0.6 Relative Hepatic Collagen (OHP) ug OHP/mg liver * ** p = 0.06253 Dual α V β 6 /α V β 1 inhibition with PLN-75068 significantly and dose-dependently reduced collagen deposition and serum ALP in DDC-injured mice PSC Liver PBC Liver pSMAD3 β 6 Integrin Peng et al., 2016 Mdr2 -/- Mdr2 -/- ;Itgb6 -/- 2 ***p<0.001 *p<0.05; **p<0.01 Dual α V β 6 /α V β 1 Inhibition Reduced Hepatic Collagen Deposition in the Mdr2 -/- Biliary Fibrosis Model 3 Week 3 Week 6 Week 9 Week 12 Tissue Collection Week 0 PLN-74809 oral QD Mdr2 -/- Picrosirius red staining showed a dose-dependent decrease in hepatic collagen deposition with PLN-74809 Vehicle 100 mg/kg 300 mg/kg 1000 mg/kg Picrosirius red PLN-74809 Dual α V β 6 /α V β 1 inhibition with PLN-74809 significantly and dose-dependently reduced SMAD3 phosphorylation and collagen deposition in Mdr2 -/- livers Balb/c Vehicle 100 mg/kg 300 mg/kg 1000 mg/kg 0.0 0.1 0.2 0.3 pSMAD3 (Normalized to total protein) * *** Hepatic pSMAD3 PLN-74809 (QD) *** *p<0.05; **p<0.01; ***p<0.001 Vehicle 100 mg/kg 300 mg/kg 1000 mg/kg 0 200 400 600 Serum Alkaline Phosphatase IU/L PLN-74809 (QD) ** Vehicle 100 mg/kg 300 mg/kg 1000 mg/kg 0 1 2 3 4 Serum Total Bilirubin mg/dL PLN-74809 (QD) Dual α V β 6 /α V β 1 inhibition with PLN-74809 dose-dependently reduced serum markers of biliary injury Vehicle 100 mg/kg 300 mg/kg 1000 mg/kg 0 50 100 150 200 Relative Hepatic Collagen (OHP) µ g of OHP/100 mg liver PLN-74809 (QD) * **p<0.01 Dual α V β 6 /α V β 1 Inhibition Reduced Collagen Expression in PBC/PSC Patient Tissue PCLivS from PBC and PSC patient explants showed a reduction in collagen gene expression with PLN-74809 treatment 4 Day 0 Day 2 Day 0 Day 2 0.0 0.5 1.0 1.5 Viability Relative Viability PBC PSC DMSO 100 nM 1 µ M 10 µ M Alk 5 Inh 0.0 0.5 1.0 COL1A1 Relative expression PLN-74809 **** * p = 0.0897 DMSO 100 nM 1 µ M 10 µ M Alk 5 Inh 0.0 0.5 1.0 COL1A2 Relative expression PLN-74809 * DMSO 100 nM 1 µ M 10 µ M Alk 5 Inh 0.0 0.5 1.0 COL3A1 Relative expression ** PLN-74809 DMSO 100 nM 1 µ M 10 µ M Alk 5 Inh 0.0 0.5 1.0 TGFB1 Relative expression *** * PLN-74809 p = 0.0559 PCLivS were viable for 48 hours in culture Picrosirius red staining for collagen showed extensive fibrosis in PCLivS tissue PSC Liver PBC Liver *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001