1. June 2010 Copeptin in Acute Myocardial Infarction – Background & Clinical Data

2. Vasopressin & Copeptin - FAQs  What is Vasopressin (Copeptin) and where does it come from?  What is the physiological role of Vasopressin?  Why not simply measure Vasopressin?  Is Copeptin produced together with Vasopressin? Do both analytes show the same kinetics?  Which Copeptin levels should be expected in Normals?  What may clinicians ask when you talk about Copeptin (Vasopressin)?  What about the performance of the Copeptin KRYPTOR assay?  Copeptin in early rule out of myocardial infarction

3.  What is Vasopressin (Copeptin) and where does it come from?

4. Structure of Vasopressin O NH 2 NH 2 - O NH 2 -C Arginine-Vasopressin (AVP)  synonym: Vasopressin or antidiuretic hormone (ADH)  peptide hormone  9 amino acids  Disulfide bridge between two cysteine amino acids  C-terminal amidation

5. Synthesis of Vasopressin Figures adapted from: Golenhofen, Basislehrbuch Physiologie, Urban & Fischer; and Morgenthaler NG et al.: Clin Chem 2006 Information: Russel IC and Glover PJ: Critical Care and Resuscitation 2002; Ranger GS: IJCP 2002; Oghlakian G and Klapholz M: Cardiology in Review 2009  Synthesis as a precursor hormone (pre-pro-vasopressin) in the hypothalamus  Cleavage and transport in granules down the axons  Storage in granules in the posterior pituitary  Release into nearby capillaries upon appropriate stimulation

6.  What is the physiological role of Vasopressin?

7. Vasopressin - physiological role AVP: acts via V 2 -receptors in the kidney -> water retention Main role: Regulation of water balance Figure adapted from: Knoers NV N Engl J Med. 2005 May 5;352(18):1847-50 - Increased plasma osmolality - Decreased arterial circulating volume AVP: Synthesis in the Hypothalamus

8. receptorlocationeffect V2kidneywater retention V1 a vascular smooth muscle cells strong vasoconstriction V1 b endocrine cells (e.g. pituitary) regulation of ACTH secretion during stress Vasopressin (AVP) effects Effects of AVP dependent on concentration :  maximal antidiuretic effect: below 15 pg/ml  vasoconstrictor effect at higher concentrations  very little effect on blood pressure at physiological levels! Singh Ranger G, Int J Clin Pract 2002; 56(10):777-782

9. Vasopressin in stress situation ACTH AVP STRESS Cortisol Myocardial infarction

10.  Why not simply measure Vasopressin?

11. Quantification of Vasopressin is difficult Vasopressin Platelets Protease Receptor Only specialized labs measure AVP (time to results several days) Not a single FDA approved AVP assay on the market LIMITED CLINICAL USE Further problem: very unstable ex vivo (even frozen)

12. Morgenthaler NG et al., Clin Chem. 2006 Prohormone processing and assay SignalVasopressin Neurophysin II Neurophysin II Copeptin Signal Peptidase Vasopressin Neurophysin II Neurophysin II Copeptin Vasopressin Vasopressin Prohormone Convertase Copeptin Copeptin Neurophysin II Neurophysin II Copeptin very stable ex vivo Fast assay (KRYPTOR)

13.  Is Copeptin produced together with Vasopressin?  Show both analytes the same kinetics in vivo?

14. r = 0.78 LIA Assay Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9. Jochberger S et al., Schock 2009 31: 132-138 Validation in: Jochberger S et al., Intensive Care Med 2009 35:489-497 Correlation of Vasopressin and Copeptin

15. 97.5 % percentile KRYPTOR: 17.4 pmol/L t 1/2 : few minutes Copeptin – like Vasopressin – is rapidly degraded in vivo Morgenthaler et al. Clin Chem 2006

16.  Which Copeptin levels should be expected in Normals?

17. Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9 Normal distribution Copeptin is not age-related

18. Bhandari SS et al, Clinical Science (2009) 116, 257–263 706 healthy volunteers Significantly higher levels in males Copeptin levels dependent on gender

19. Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9 Copeptin: Influence of exercise 97.5 % pecentile KRYPTOR: 17.4 pmol/L

20.  What may clinicians ask when you talk about Vasopressin / Copeptin?  disturbed Vasopressin / Copeptin secretion and water / salt balance?

21. 38 patients (33 after transphenoidal surgery, 5 without surgery) n = 29 normal posterior pituitary function n = 9 diabetes insipidus centralis Katan et al. JCEM 2007 Diagnosis of diabetes insipidus  Glucose < 2 mmol/l insulin-induced hypoglycemia test

22. 100% sensitivity – 100% specificity Copetin level < 4.75 pmol/L Diagnosis of diabetes insipidus Katan et al. JCEM 2007

23. FAS Kryptor Diagnosis of diabetes insipidus Diabetes Insipidus is no indication for the KRYPTOR Assay! Katan et al. JCEM 2007

24. Hyponatremia  most common fluid and electrolyte disturbance  prevalence: 15-30% of hospitalized patients  variety of disorders causing hyponatremia - treatment varies widely Fenske et al.: J Clin Endocrinol Metab, 2009

25. Assay Performance  What about the performance of the KRYPTOR assay?

26. Copeptin assay parameters Data taken from IFU (instructions for use)

27. Assay Performance  Copeptin in early rule out of myocardial infarction

28. Background  Chest pain patients about 10% of ED consultations  Cardiac Troponin current diagnostic gold standard  Troponin retesting after 6-8 hours necessary due to delayed increase  Rapid and reliable rule out of acute MI already at presentation is a large unmet clinical need

29. Hypothesis rapid and accurate rule out of AMI at initial presentation without Tn retesting after 6 to 8 hours Cardiac Necrosis Troponin Combination of Endogenous Stress Copeptin +

30. Proof of concept study

31.  Consecutive pts with chest pain <12h  Observational study  Serial blood sampling: 0h,1h, 2h, 3h, 6h  Follow up 90d, 360d, 720d  Adjudicated Diagnosis: – 2 independent experts – using all clinical information within 60d FU (History, physical examination, ECG, cTn, chest x-ray, echo, coronary angiography, exercise testing (MPS), CT-scans, endoscopy,....) – Blinded for investigational biomarkers Methods

32. Adjudicated final diagnoses Myocardial Infarction (17%) Unstable Angina (16%) Non-coronary cardiac chest pain (13%) Non-cardiac chest pain (46%) Chest pain of unknown origin (9%) Thereof  STEMI (37%)  NSTEMI (63%)

33. Reichlin et al. J Am Coll Cardiol 2009;54:60-8 Copeptin levels at presentation

34. Reichlin et al. J Am Coll Cardiol 2009;54:60-8 Copeptin and Troponin levels at presentation

35. ROC curves at presentation Reichlin et al. J Am Coll Cardiol 2009;54:60-8

36. 487 pts 314 = 65% (cTnT / Copeptin negative) 173 = 35% (cTnT / Copeptin positive) Rapid rule out of AMI at presentation Reichlin et al. J Am Coll Cardiol 2009;54:60-8

37. 1.Copeptin significantly improves the early diagnosis of AMI (AUC for combination with Troponin T 0.97). 2.The combination of Copeptin and Troponin T allows a rule out of AMI at presentation with a sensitivity of 98.8% and a NPV of 99.7%. 3.The use of Copeptin in conjunction with Troponin T, ECG and clinical findings may obviate the need for prolonged stay in the ED and Troponin retesting after 6 to 8 hours in two-thirds of patients. This change in clinical practice might result in significant medical and economic benefits. Conclusion

38. Paper submitted, confidential Data Validation study Keller et al. J Am Coll Cardiol 2010;55:2096-2106.

39. Methods  1386 patients with suspected acute coronary syndrome  Multicenter approach  Troponin T (4 th generation Roche Diagnostics) used for Gold Standard Diagnosis  Diagnosis NSTEMI: - one value above 0.03 ng/mL ! - and a typical kinetic (rise or fall of at least 20%) Keller et al. J Am Coll Cardiol 2010;55:2096-2106.

40. Baseline characteristics  + 211  + 289 Keller et al. J Am Coll Cardiol 2010;55:2096-2106.

41. Final diagnosis 65% 13% 7% 15% Potential „rule out-portion“: ca. 78% Keller et al. J Am Coll Cardiol 2010;55:2096-2106.

42. Time course of different markers Patients with time of chest pain onset < 2h MI: n=75 NCCP: n=213 Keller et al. J Am Coll Cardiol 2010;55:2096-2106.

43. Paper in preparation for submission, confidential Data AUCs according to time of chest pain onset Diagnostic performance of Copeptin/Troponin T < 3h< 6h< 12hAll Troponin T0.770.80.810.84 Copeptin0.790.78 0.74 Keller et al. J Am Coll Cardiol 2010;55:2096-2106.

44. T=0 Diagnostic performance (1) Best AUC combination Copeptin / Troponin T 0.93  TnT+ Myo: 0.91  TnT + CKMB: 0.88 Keller et al. J Am Coll Cardiol 2010;55:2096-2106. Copeptin + Troponin T

45. Diagnostic performance (2) Troponin T (cut-off: 0.03 ng/mL) Copeptin (cut-off: 13 pmol/L) Combination Sensitivity625888 Specificity977876 Positive predictive value874655 Negative predictive value898595 Keller et al. J Am Coll Cardiol 2010;55:2096-2106.

46. Copeptin and sensitive Troponin Keller et al. J Am Coll Cardiol 2010;55:2096-2106. *Copeptin cut-off 9.8 pmol/l † Copeptin cut-off 13 pmol/l *TnI > 0.04 ng/ml

47. Conclusion Keller et al. J Am Coll Cardiol 2010;55:2096-2106.