1. FONT Phase II Clinical Trial PI: Howard Trachtman, M.D. Co-PI: Debbie Gipson, M.S., M.D. Study Coordinator: Suzanne Vento, RN Study Coordinator: Emily Herreshoff

2. Justification for Novel FSGS Therapy Poor Survial for Resistant FSGS Presler/Gipson, 2005Cattran, JASN, 2003A a. Pediatric FSGSb. Adult FSGS CR 12 12 8 5 5 PR 20 18 13 11 9 NR 28 25 20 10 9

3. Specific Aims 1: To evaluate two novel therapies for resistant FSGS -- anti-TNF-α antibody and galactose against standard therapy 2: To identify one or more novel agents as candidates for future study in a Phase III randomized clinical trial 3: To develop a network of sites to foster the performance of clinical trials for this disease and other glomerular disorders

4. Eligible Patients with Resistant FSGS RANDOMIZATION Galactose N=17 Standard Rx N=17 Adalimumab N=17 Pause for Efficacy Review YES, Continue recruitment to N=42* NO, Discontinue study arm ≥2/17 with Up/c<50% baseline and stable GFRe FONT II REVISED PROJECT

5. TNF-  Inflammatory cytokine Produced by circulating or infiltrating mononuclear cells, macrophages, and kidney mesangial cells Postulated mechanisms –recruitment of leukocytes to the site of glomerular injury –induction of cytokines and growth factors –generation of oxygen radicals resulting in increased glomerular endothelial cell permeability to albumin –direct cytotoxicity to glomerular mesangial and epithelial cells, and induction of apoptosis Increases in TNF- α and TNF- α mRNA described in FSGS

6. TNF  levels from cultured PBMC increased in nephrotic syndrome Bakr, 2003 TNF-alpha (pg/ml) P=0.015 P=0.006 P=0.002 2500 2000 1500 1000 500 0 FSGSMCGNDMP-SRMCNS-SR

7. TNF  levels low during remission of nephrotic syndrome GroupN PBMC TNF  (pg/ml) Median IQR FSGS111,9551,786-2060 MCNS-SS21 1915-26 MCNS-SR 5 3424-82.5 Remission11 5.54.9-6.0 Control10 5.54.8-6.0 Bakr, 2003

8. Phase I Adalimumab N=10 (4M:6F) Age 16.8±9.0 yr GFRe 105±50 ml/min/1.73 m 2 Dose 24 mg/m 2 (max: 40 mg) sc q 14 d Up/c 15.9±10.4

9. Adalimumab: PK The half life, area under the curve, and clearance were increased in proteinuric FSGS compared with patients with rheumatoid arthritis FSGS steady state RA Tmax (hr)34.2±9.8*130±55 Cmax (μg/mL)12.8±8.313.7±2.7 T1/2 (hr)273±402389±71 AUC2019±1693*3622±587 Cl/F53.2±43.3*11.2±2.0

10. Adalimumab Safety 9 AEs –1 probably related: injection site reaction –No discontinuation due to AE Relationship of T 1/2 to Up/c and albumin TSQM (effectiveness, side effects, convenience, global satisfaction) –Scores: 61, 92, 71, 59

11. Phase I: Long-term follow-up Adalimumab (n=10) Rosiglitazone (n=11) Follow-up (months) 16.15.718.310.2 50% decline in Up/c 43 Progression to ESKD 14 Stabilization in GFRe (Δ/month) 71%56%

12. Galactose Arm Rationale Role of FSGS permeability factor Effect of galactose on FSGS permeability factor

13. Circulating permeability factor(s) in FSGS Permeability activity: In vitro glomerular albumin permeability (P alb ) predicts recurrence. Evidence: Early post-transplant recurrence of proteinuria Proteinuria after injection of FSGS plasma Clinical benefit of plasmapheresis (PP) 1 st & 2 nd 1 PP rx IV Galactose P alb of patient specimens Savin VJ, et al. Transl Res 2008

14. Permeability is increased most by serum from FSGS patients with severe disease or recurrence after transplantation P alb 1 1 1 1. Savin, NEJM, 1996 2. Butcher, ASN, 1998 3. McCarthy, ASN, 1997 2 3

15. More rapid progression to kidney failure is seen in patients with high activity (Pudur) 0 P alb < 0.5 P alb  0.5 Time from diagnosis to ESRD, years Survival distribution Function 5 years 10 15.25.50.75 1.0 Low activity, about 40% good function after 5 years High activity, only about 25% good function after 5 years

16. Permeability activity is decreased by galactose Galactose taken twice daily in water. Activity decreased at 2 weeks and remained low. P alb Pre-treatment 6 weeks 1 week 2 weeks 4 weeks 4 weeks after stopping galactose Savin VJ, et al. Transl Res 2008

17. Galactose and Potential Mechanisms of Action Permeability factor may gain access to podocyte by sugar binding. Galactose may block the binding between factor and podocyte. Factor-galactose complex may be removed by specific receptors in liver or other tissues. FSPF galactose Galactose receptors FSPF-galactose complex FSPF FSPF-galactose complex galactose FSPF FSPF Podocytes Filtration slits Savin VJ, et al. Transl Res 2008

18. Cardiotrophin-like cytokine (CLC-1) is present in plasma of a patient with recurrent FSGS We used galactose to purify active proteins from a patient with FSGS Proteins were separated on a gel. A band of low molecular weight protein was present. CLC-1 was the single cytokine identified in this material by mass spectrometry. Patient plasma Proteins of interest Savin VJ, ASN 2008 (abstract)

19. Rat glomeruli were treated with CLC-1 in various concentrations. CLC-1 increased permeability as does FSGS serum. Synthesis of nephrin, the main protein of the slit- junction, is decreased. P alb, CLC-1 * * ** * P< 0.01 vs C CLC-1 increases permeability and decreases expression of nephrin, the main junction protein Savin VJ, ASN 2008 (abstract)

20. Antibody to CLC-1 prevents permeability caused by patient serum or by CLC-1 itself Anti-CLC-1 mAb vs. without mAb: Fisher exact test, p < 0.02; paired t-test, p<0.005. Savin VJ, ASN 2008

21. Summary of current work about circulating factor in FSGS Permeability activity of plasma is highest in patients with severe disease. Galactose binds the active protein and can be used for purification or for therapy. CLC-1 is a candidate for the FSGS permeability factor due to – presence in FSGS plasma and – same effects of FSGS plasma in vitro

22. Galactose: NDT 2009;24:2938-40 48 year old man, onset of NS 2004 Resistant to prednisone, cyclophosphamide, cyclosporine, and plasmapheresis Elevated Palb Galactose 10 g BID started Feb 2007 After 7 months: –Palb 0.54→0.09 –Proteinuria 4.5→0.6 g/24 hours Galactose Rx through 1/09, proteinuria 1.1 g/24 h, stable GFR, off cyclosporine

23. Galactose: Safety IND #77,091 23 patients Duration of therapy: 28 days No SAE directly related to drug AE: abdominal pain

24. Galactose: Dosing Dispensed by Aptuit –Powder –500 g in plastic container Dose: 0.2 g/kg/dose BID (max: 15 g BID) Dissolve in 15-30 ml water Administer 15 minutes before breakfast and dinner

25. Study Summary Visit week/year Study Visit Blood and Urine Tests Quality of Life Pregnancy Screening Week -4 xx x Week 0 xxxx Week 2 xx Week 8 xx x Week 16 xx x Week 26 xxx Month 7 xx Month 9 xx Month 12 xxx Month 18 xx Year 2 x* x * May be phone follow up

26. FONT Study Network 15 study sites Boston Children’s Carolinas Medical Center Children’s Mercy Kansas City Cincinnati Children’s Cohen Children’s Medical Center Emory University Mayo Clinic Miami Children’s MUSC Nationwide Children’s Oregon Stollery Texas Tech El Paso University of Kansas University of Michigan

27. Contact Information Study Website: www.fonttrial.orgwww.fonttrial.org Email: font2@umich.edufont2@umich.edu Phone: 1-855-4-FONT II (1-855-436-6844)