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

2. 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: , 2005)

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

4. 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

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

6. 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

7. 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.

8. 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)

9. 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.

10. No body weight and liver weight changes with C9

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

12. Fibrosis area is reduced with Oral C9

13. Intellectual Property Portfolio
Lisofylline Analogs And Methods For Use (Expires 2028)
US 8,481,580
US 8,987,321
Canada 2,618,970
Japan EP
(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 (Germany, France, UK, Ireland, Switzerland)

14. Preliminary ADME data

15. 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

16. 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

17. Metabolic Stability/CYP Inhibition New Analogs