Bringing Proteomics to the Clinic: From Discovery to Validation November 4, 2007

1. Bringing Proteomics to the Clinic:From Discovery to Validation November 4, 2007 “Biomarkers of Pulmonary Arterial Hypertension” Frank J. Accurso, MD Professor of Pediatrics University of Colorado Disclosures: None Support: NHLBI, NIDDK, NCRR, AHA, Cystic Fibrosis Foundation Acknowledgements – Dunbar Ivy, MD; Steve Abman, MD

2. Biomarkers of Pulmonary Arterial Hypertension (PAH): Overview • PAH in adults and children • PAH and biomarkers • Exploratory protein biomarker studies in PAH in • children • Take home messages: • Improved biomarkers are needed to quickly • “test” new treatment approaches. • 2. Biomarker studies should be incorporated into • clinical trials and investigations in PAH.

3. PAH - definition • Sustained elevation of mean pulmonary arterial • pressure to > 25 mm Hg at rest or >30 mm Hg • with exercise, with a mean pulmonary capillary • and left atrial pressure < 15 mm Hg at rest. • “fixed” (structural) and “reactive” components • Better treatments are needed. Rubin, Chest supplement, 2004

4. Simonneau G, et al. J Am Coll Cardiol , Supplement, 2004. WHO Classification of PH (Venice, 2003) Former Primary Pulmonary Hypertension (PPH) – idiopathic or unknown cause OR Secondary Pulmonary Hypertension (SPH) – all other causes Current WHO (Venice, 2003) 1. Pulmonary arterial hypertension 2. Pulmonary hypertension with left heart failure 3. PH with lung diseases and/or hypoxemia 4. PH due to chronic thrombotic and/or embolic diseases 5. Miscellaneous

5. PAH (WHO Group I) • Idiopathic (IPAH) • Familial (FPAH) • Associated with (APAH) • Collagen vascular disease • Congenital systemic-to-pulmonary shunts • Portal hypertension • Associated with significant venous or capillary involvement • Pulmonary veno-occlusive disease (PVOD) • Pulmonary capillary hemangiomatosis (PCH) • Persistent pulmonary hypertension of the newborn • HIV infection • Drugs/toxins • Other Simonneau G, et al. J Am Coll Cardiol , Supplement, 2004.

6. PAH: Diagnostic Evaluation • Physiologic* • - Lung function • - Exercise testing • Imaging • Sleep study • Right Heart Catheterization • History* • Physical • Blood • EKG • ECHO • * Difficult in young children • ? Biochemical biomarkers of disease

7. Idiopathic PAH: SurvivalUntreated - survival worse in children 100 Adult median survival: 2.8 years (n=194) Pediatric median survival: 0.8 years (n=16) 80 60 68% % Survival 40 48% 34% 20 (n=194) 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 Years of Follow-up D’Alonzo, et al. Ann Internal Med 1991

8. PAH: Acute vasodilator response depends on age Barst et al.

9. PAH: Treatment Approaches • Pathophysiology • - “fixed” – structural, associated with proliferation • - “reactive” – amenable to vasodilator treatment • - treatments based on known pathways of vasoactivity • Calcium channel blockers – vasodilators • Prostacyclin – vasodilator, antiproliferative • Nitric oxide – vasodilator, antiproliferative • Endothelin – vasoconstrictor, proliferative – need antagonist • Vascular reactivity is a favorable prognostic sign

10. Idiopathic PAH in Children: Survival and Treatment Success with Chronic Oral CCB in Acute Responders Event Free Proportion Years after Diagnosis Yung, et al. Circulation 2004

11. Complex, long-term PAH treatment strategies have been developed CHF Trial of Calcium Channel Blocker Yes Acute Vasodilator Response Epoprostenol Nitric Oxide No Incomplete response Trial of : Bosentan Ambrisentan Sildenafil Iloprost Treprostinil Epoprostenol No Yes Epoprostenol Treprostinil Incomplete response Biomarkers would be helpful in adding treatments. Adapted from Rashid A, Ivy D. 2006: Current Paediatrics

12. Biomarkers of Pulmonary Arterial Hypertension (PAH) • PAH in Adults and Children • Biomarkers and PAH • Exploratory Biomarker Studies in PAH in • Children

13. Outcome Measures Clinical efficacy measures: A characteristic or variable that reflects how a patient feels, functions, or survives. Surrogate endpoint:A laboratory measurement or physical sign that is used in therapeutic trials as a substitute for a clinically meaningful endpoint that is a direct measure of how a patient feels, functions, or survives and is expected to predict the effect of therapy. Biomarker: A characteristic that is objectively measured and evaluated as an indicator of normal biologic process, pathogenic process, or pharmacologic response to a therapeutic intervention. Pulmonary Hypertension Review : Snow and Kawut, 2007 ; Hamblett et al, 2007

14. Biomarker Need in PAH Need Current biochemical biomarker Diagnosis - Early identification No - Staging No Prognosis - Rapid progression of disease No - Ongoing structural Injury No - Predict complications No Treatment - Select treatment No - Response to treatment No - Toxicity with treatment No - Clinical Trials a. Stratification No b. Proof of concept No c. Efficacy No

15. Value Added: Proof of Concept

16. The specific goals of the Clinical Proteomics Program are to: (1) design panels of candidate proteins for disease areas (2) develop high throughput analytic methods (3) assess the predictive value of these proteomic measurements using biological specimens and clinical data from existing study populations (4) establish procedures and standards for quality control http://www.mc.vanderbilt.edu/root/vumc.php?site=proteomics

17. Approaches to Protein Biomarkers Single/Few Protein Assay(s) (Hypothesis) Multiplex Protein Assay(s) (Hypothesis/ Discovery) Candidate - Single - Panel Value Added Validation Proteomics (Discovery)

18. Candidate Biomarker Pathways in PAH Candidate Source Natriuretic peptide (BNP) myocytes Troponin T myocytes Uric acid ubiquitous Endothelin-1 endothelium Serotonin platelets Angiotensin system vascular wall NO related products endothelium Cyclic nucleotides vascular wall, smooth muscle Fibrinogen related products fibrin Cytokines, Growth factors variety Acute Phase Reactants liver Aubert, Swiss Med Weekly, 2007

19. Caveat: Different Reagent Sources Serum Il-6 • Standards • Agree • Controls • Agree • Duplicate • Clinical samples • do not agree • A second analytical • method is needed • for validation. Brand Y Brand X

20. Biomarkers of Pulmonary Arterial Hypertension (PAH): Overview • PAH in adults and children • PAH and biomarkers • Exploratory protein biomarker studies in PAH in • children • Take home messages: • Improved biomarkers are needed to quickly • “test” new treatment approaches. • 2. Biomarker studies should be incorporated into • clinical trials and investigations in PAH.

21. Characteristics of biomarkers • Is the outcome measure specific to a process occurring • either early or late in the disease process? • Is the biomarker in the causal pathway of the drug? • • How is the biomarker expected to change in response to the drug? • • How long will it take to see an expected change in response to the drug?