A promising target for NASH Interleukin-12/STAT4: A promising target for NASH University of Virginia Licensing & Ventures Group BIO 2016

Nonalcoholic Steatohepatitis (NASH) Common liver disease in the US 40% patient w/metabolic syndrome have NASH Progression based on the “2 hit” model. Fatty infiltration from visceral obesity Cytokines (TNFα IL-12) → hepatocyte injury and fibrosis 26% NASH patients progress to cirrhosis IL-12 markedly increased in liver in high fat fed models of NASH (Hepatology 42:880-885, 2005)

STAT4 is a major mediator for pro-inflammatory cytokines LOSS of INSULIN SECRETION, INSULIN RESISTANCE, NASH, ATHEROSCLEROSIS

Interleukin-12/STAT4: A Promising Target for NASH IL-12: pro-inflammatory cytokine important for immune responses leading to type 1 diabetes and atherosclerosis Uses JAK/STAT4 signaling system to induce genes linked to chronic inflammatory disorders High glucose, obesity, and diabetes markedly increase IL-12/STAT4 expression in key tissues Reducing activation of IL-12/STAT4 preserves islet function and viability and reduces insulin resistance. IL-12 is made in insulin producing beta cells of the pancreas and directly leads to reduced insulin secretion and cell death Stat4 deletion prevents type 1 diabetes, reduces insulin resistance, and atherosclerosis STAT4 tracks with the severity of NASH

STAT4 expression is increased in human livers with NASH STAT4 staining score NAFLD NASH STAT4 A-SMA DAPI

STAT4 expression is increased in human livers with NASH STAT4 expression is strongly associated with inflammatory infiltrates and possibly with Kupfer cells STAT4 expression is both cytoplasmic and nuclear nuclear cytoplasmic STAT4 A-SMA DAPI NASH with cirrhosis NASH

Novel Drug Development Candidates Focused library based on Lisofylline structure Structure-Activity Relationship elucidated Improved Pharmacokinetic Parameters Screened for: beta cell protection against cytokines Preservation of insulin secretion Designed for oral bioavailability 40 compounds in library passed 200 test in silico screen Lead compound is orally bioavailable and dietary administration hits target and reduces inflammatory genes in mouse models.

In vitro screening system for IL12R inhibitors Human cell line NK-92: Characteristics of activated human NK cells High basal expression of STAT4 Used in assays to test STAT4 phosphorylation in response to different cytokines/growth factors General protocol for screening: - culture NK-92 cells in 96 well plates at 2-3x105 cells/well add human recombinant IL-12 (heterodimer) incubate for 2 hours add candidate inhibitors at different concentrations dose-dependent studies - specificity of inhibitors for IL-12 pathway (verify response upon IL-23 stimulation or blocking antibodies for IL23R and IL12Rβ2 Readout parameter: STAT 4 activation by Y693 phosphorylation Assay: cell-based ELISA (R&D Systems)

Effects of Lead (Compound 9) in “STAM” Model of NASH 3 groups studied all n=6 a. vehicle alone b. C9-IP 15mmol/kg twice a day c. C9 – oral 15mmol/kg twice daily (23.4 mg/kg/day Treatment started at age 5 weeks and continued to age 9 weeks No change in glucose, lipids or body weight Drug was very well tolerated Positive findings were seen with liver and liver to body weight in oral compound group Highly significant (50%) reduction of fibrosis area in oral Compound 9 treated mice.

No body weight and liver weight changes with C9

Representative photographs of Sirius red-stained liver sections during C9 treatment

Fibrosis area is reduced with Oral C9

Intellectual Property Portfolio Lisofylline Analogs And Methods For Use (Expires 2028) US 8,481,580 US 8,987,321 Canada 2,618,970 Japan 5259408 EP 191867 (Germany, France, Denmark, UK, Ireland, Monaco, Netherlands, Sweden, Switzerland) Lisofylline Analogs And Methods For Use (Expires 2027) US 8,871,764 US 9,278,098 Substituted phthalazines for inducing insulin secretion and protecting beta-cells Canada, 2,679,301 EP 2132183 (Germany, France, UK, Ireland, Switzerland)

Preliminary ADME data

CPW9 PK in mouse Animal Adm AUC/ Dose Cmax (µmol/L) tmax (h) t1/2 (h) po 0.544 10.98 0.17 4.1 2 1.158 7.016 3.5 Mean   0.851 8.998 3.8 Matrix   Blood recalculated to plasma Strain/gender C57bl/6/Female Fasted No Dose po (µmol/kg) 20 Formulation po 28% HPBCD

CPW9 female SPRD rat cassette 1 umol/kg iv, 3 umol/kg po (Veh: TEG/DMA/H20) Animal AUC/Dose Vss (L/kg) T1/2 (h) CL (ml/min/kg) Cmax (umol/L) 1 0.74 0.26 0.3 23 3.85 2 0.69 0.33 24 3.17 3 0.88 0.27 0.36 19 4.84 4 0.67 0.30 0.24 25 3.54 Animal AUC/Dose Cmax (umol/L) Tmax (h) T1/2 (h) F (%) 5 (po) 0.57 0.86 0.5 1.8 77 6 (po) 0.75 0.87 1.3 100

Metabolic Stability/CYP Inhibition New Analogs