Daniel Freilich, MD, CDR, MC, USN RESUS Sponsor Lead Investigator Naval Medical Research Center, Silver Spring, MD

1. A pivotal, randomized, controlled, and single-blinded trial of the hemoglobin-based oxygen carrier (HBOC), bovine polymerized hemoglobin (HBOC-201), for the prehospital resuscitation of patients with severe hemorrhagic shock (HS) Daniel Freilich, MD, CDR, MC, USN RESUS Sponsor Lead Investigator Naval Medical Research Center, Silver Spring, MD Specialty: Internal Medicine, Infectious Diseases Research interests: hemorrhagic shock, malaria, bioterrorism agents

2. Outline of NMRC/RESUSAdvisory Board presentations • Introduction: Daniel Freilich, MD, CDR, MC, USN • Importance of RESUS to the Navy and Marines Corps: John Mateczun, MD, RADM, MC, USN • Hemorrhagic shock: pathophysiology, clinical presentation, and treatment: Lewis Kaplan, MD • Overview of RESUS study: Richard Dutton, MD • Preclinical HBOC-201 HS studies: Susan Stern, MD • Synopsis of HBOC-201 clinical studies: Gerson Greenburg, MD, PhD • RESUS IND Clinical Hold issues: Daniel Freilich, MD, CDR, MC, USN • Concluding remarks—importance of RESUS to civilian EMS community: Joseph Acker, EMT-P, MPH • Concluding remarks—importance of RESUS to civilian trauma community: Lewis Kaplan, MD

3. Hemorrhagic Shock Lewis J. Kaplan, MD, FACS, FCCM, FCCP Associate Professor Of Surgery Yale University School of Medicine Section of Trauma, Surgical Critical Care and Emergency General Surgery Director, SICU and Surgical Critical Care Fellowship

4. OBJECTIVES • Hemorrhagic shock (HS) • Pathophysiology • Clinically relevant indicators of HS • Acute therapeutic goals • Resuscitation paradigm construction • Outcome benefit

5. HEMORRHAGIC SHOCK • Results from acute blood loss • Trauma (blunt and penetrating) • Emergency general surgery • Elective surgery + complications • Spine reconstruction • Cesarean section • Hepatic transplantation • Interventional Radiology procedures

6. PATHOPHYSIOLOGY • Acute blood loss • Reduced red cell mass • Reduced plasma volume • Reduced tissue oxygen delivery • Impaired balance of tissue oxygen utilization and supply • Demand >>> supply

7. PATHOPHYSIOLOGY • Unbalanced utilization and delivery • Anaerobic metabolism • Systemic lactic acidosis • Cellular hypoxia • Compounded by central shunting of effective circulating volume • protect cardio-pulmonary and cerebral oxygen delivery

8. SHOCK CLASSES: ATLS 2004

9. CLINICAL CORRELATES:Standard Field Criteria PHTLS 2004 • Discoverable by all EMS providers • Tachycardia (HR > 100 bpm) • Hypotension (SBP < 90 mm Hg) • Cool extremities (central shunting and peripheral vasoconstriction) • Multiple manifestations (i.e. unobtainable SaO2) • As shock progresses . . . • Disordered mentation • Obtundation • Loss of consciousness

10. INTERVENTION OPPORTUNITY • Intervene during the evolution of hemorrhagic shock to avoid severe sequelae • Survival directly related to rapidity of hemorrhage control • “Golden hour” • Time to definitive hemorrhage control • Difficult areas: military, rural civilian trauma

11. UNIVERSAL THERAPY (HS):Trauma Bay • ABCDE – ATLS driven • Airway control + 100% O2 • Resuscitation (IVF + PRBC’s) • Laboratory investigation • Initial radiologic survey • Focused Assessment by Sonography for Trauma (FAST)

12. Vital signs Core temperature Peripheral temp Urine output ABG, arterial lactate Derived indices Anion gap Corrected anion gap Unmeasured ions Response to PVE/PRBC Vital signs Cardiac performance CVP/PA Catheter Volume responsive cardiac output ScvO2 / SvO2 Trans-esophageal echocardiography End systolic elastance SHOCK IDENTIFIERS

13. INTERVENTION CHALLENGES: Field and Transport • Lack of definitive control, plasma, RBC • US standard of care = crystalloid solutions • Immune activating • Dilute clotting factors, RBC mass • Induce hyperchloremic acidosis • Compounds lactic acidosis • At best 1/3 remains intravascular • Large volume resuscitation • Potential hemorrhage acceleration

14. FLUIDS FOR SMALL VOLUME RESUSCITATION • Hextend • 6% HES in balanced salt solution • Special Forces, US civilian centers • Hypertonic saline • Single dose due to electrolyte abnormalities • Potential benefit in TBI only • Neither augments oxygen carrying capacity nor RBC mass

15. OPTIMAL FLUIDS FOR SMALL VOLUME RESUSCITATION • Ideal fluid • Small volume • Repeatedly doseable • Free of electrolyte abnormalities • Augments oxygen carrying capacity • Targets reversing the pathophysiology of hemorrhagic shock • Hemoglobin-based oxygen carrier

16. SURVIVAL AND LACTATE CLEARANCE AFTER TRAUMA Abramson et al., J Trauma, 1993; 35:584-9

17. RESUS: In-hospital Trauma CareAssumes resuscitation is ongoing . . . Hemorrhagic Shock BleedingControlled Ongoing Bleeding IVF +PRBC/FFP/Plt OR + IR Hemorrhage Control Hemodynamic Assessment Volume Depletion CVP Volume Replete Persistent: Abnormal VS? Oliguria? High LA/acidosis? Dx Dilemma? PA Catheter Volume Overload Echocardiography

18. RESUS ALGORITHM:PVE and Component Therapy VolumeDepletion Volume Replete Volume Overload Adequate Cardiac Performance? IVF / Colloid PRBC/FFP Consider Diuresis or Afterload Reduction Hemodynamic Reassessment Laboratory reevaluation Hgb 8-10 g/dl (acute) No Yes Hemodynamic Reassessment Laboratory reevaluation Hgb 7-8 g/dl (stable) Consider pressor Goals: Cardiac Performance Optimization (CO, SvO2,LA)

19. HEMORRHAGIC SHOCK, MSOF, AND MORTALITY RISK Death from refractory shock Kaplan L, et al. Curr Op Crit Care, 1999; 5(6):458-463

20. CONCLUSION • Hemorrhagic shock • Known pathophysiology • Targeted intervention • Survival is enhanced with early hemorrhage control and resuscitation • HBOC-201 is a directed PREHOSPITAL intervention to ameliorate HS pathophysiology and offers a sound approach to enhancing survival and minimizing morbidity

21. RESUS Restore Effective Survival in Shock Richard P. Dutton, MD MBA Chief, Trauma Anesthesiology R Adams Cowley Shock Trauma Center University of Maryland Medical Center

22. Objective To compare HBOC-201 with lactated Ringers solution for prehospital resuscitation of patients with severe hemorrhagic shock.

23. Trial Design • Part I (Phase 2b trial): 50 subjects • Part II (Phase 3 trial): 1,108 subjects

24. Inclusion Criteria • Adults 18 to < 70 years old • Injury with suspected bleeding • SBP < 90 mmHg • Revised Trauma Score (RTS) 1 to < 5 • Planned transport to study hospital • IV access secured

25. Revised Trauma Score • Blood pressure • Respiratory rate • Glasgow Coma Scale (GCS) score

26. “Blood transfusion available” exclusion criterion • Intent • To exclude subjects with short transportation delay • Who have access to blood transfusions shortly • Who have insufficient time to potentially benefit from HBOC-201 • Guideline (majority of RESUS subjects) • Expected < 10-15 minutes to hospital arrival  “blood transfusion available” • Expected > 10-15 minutes to hospital arrival  “blood transfusion unavailable” • Exception for critical patients (minority of RESUS subjects) • May enroll critical patients with expected < 10-15 minutes to hospital arrival. • Patients with severely unstable vital signs • Patients not expected to survive to hospital arrival • Per EMS judgment

27. Exclusions • Penetrating brain injury • Paralysis • Known pregnancy • Burns • Cardiac arrest • Allergy • Known opposition to prehospital research • Transport time to study hospital <10-15 min

28. Pre-Hospital Procedures • Screening • Pre-enrollment disclosure or informed consent (when feasible) • Enrollment and randomization • Trial product infusion over 10 minutes • 500 ml HBOC-201 • 1,000 ml LR • Re-infusion if: • SBP < 90 mm Hg, or • SBP 90-99 mm Hg and HR > 100 bpm. • Infusion stopped if: • SBP > 120 mm Hg • Maximum HBOC-201 dosage: 6 units

29. RESUSEMS interventions ATLS ABCDEs: circulation (C) Diagnose HS requiring fluid resuscitation Screen pt Review inclusion criteria (including SPB < 90 mm Hg) and exclusion criteria Inclusion/exclusion criteria not met DO NOT ENROLL Inclusion/exclusion criteria met CONTINUE ENROLLMENT/CONSENT MINORITY OF CASES Patient CONSCIOUS or LAR/family member AVAILABLE Provide IC or Pre-ED (if feasible) MAJORITY OF CASES Patient UNCONSCIOUS, CONFUSED, OR DISORIENTED LAR/family member UNAVAILABLE If do NOT agree to participate DO NOT ENROLL Patient or LAR/family member agree to participate ENROLL PATIENT using PreED or IC ENROLL PATIENT using EIC Open pre-randomization envelope Infuse entire HBOC-201 or LR dose ASSESS CTM RE-INFUSION CRITERIA Be alert for side effects CTM re-infusion criteria met SBP < 90 mm Hg Or SBP 91-99 mm Hg and HR >/= 100 bpm CTM re-infusion criteria NOT met SBP >/= 100 mm Hg Or SBP 91-99 mm Hg and HR < 100 bpm Inadequate Resuscitation Persistent other signs of HS Adequate Resuscitation No persistent other signs of HS Repeat CTM re-infusion cycle Asses and re-infuse CTM PRN Until maximum dose Admin standard IV fluids Re-evaluate NO fluid therapy Re-evaluate Complete CRF Complete CRF Complete CRF

30. In-Hospital Procedures • Finish incomplete trial product infusion • Routine initial care (ATLS) • “Best practice” continuing care • Ongoing informed consent / disclosure • Continued data collection • Surveillance for adverse events

31. RESUS In-Hospital Trauma Care Guidelines • Fluid resuscitation • Prior to hemorrhage control target SBP of 90 mmHg, then • Following hemorrhage control target normal perfusion • Blood composition • Hgb 8-10 g/dL during early resuscitation (active bleeding) • Hgb 7-8 g/dL when hemodynamically stable • Platelets and clotting factors as indicated • Inotropic and vasoconstrictive medications • Guided by advanced monitoring (PA, TEE, SvO2) • Titrated to cardiac output • Traumatic brain injury: per Brain Trauma Foundation guidelines

32. Primary Outcomes • Reduced 28 day mortality • Safe and tolerable

33. Other Outcomes • Key Clinical Parameters: 1. Hemodynamics 11. Hemostasis 2. Tissue oxygenation (lactate) 12. Blood transfusions 3. Renal function 13. Fluid requirements 4. O2 content 14. Immune activation 5. Organ function 15. Neurocognitive function 6. Trauma scores 16. Neurophysiology 7. Infectious complications 17. Safety labs 8. Abdominal complications 18. Clinical safety 9. Length of stay (LOS) 19. Disposition 10. Ventilator support 20. Survival (prehospital) • Composite Surrogate Score (CSS)

34. Consent • Exception from informed consent per 21CFR50.24 • Community consultation & disclosure: • media public service announcements • house mailing brochures • town hall discussions • church meetings • health fairs • Ongoing consent process: • Pre-enrollment disclosure • Post-enrollment discussion • Option to withdraw

37. Pre-enrollment disclosure script 1. You appear to have severe bleeding, are in shock, and need treatment with fluids. 2. As part of a research study, we are testing a new fluid called HBOC-201 that study doctors believe may improve your chance of surviving. 3. This research study is approved by (hospital name)’s committee for the protection of human subjects. 4. There are risks and HBOC-201 could be harmful, but study doctors believe that the benefits outweigh the risks. 5. Unless you object, you will be included in the study and will get HBOC-201 or regular IV fluids. All other medical care will be standard. 6. If you do not want to be in the study, you will get standard medical care. 7. Because treatment needs to start right away, you must tell us immediately if you do NOT want to be in the study.

38. Data Monitoring Committee • Will review the study for efficacy and safety • Planned interim analyses in accordance with 21CFR50.24 • Analysis after accrual of: • 50 patients (5%) • 222 patients (20%) • 554 patients (50%) • 1,108 patients (100%)

39. Stopping Criteria: Efficacy • Absolute: Significantly decreased 28-day relative risk of mortality • Intent-to-treat [ITT] analysis • Appropriate boundary modification (O’Brien-Fleming)

40. Stopping Criteria: Safety • Absolute: Increased risk of death, disability or birth defects in the HBOC-201 group • Relative: Increased serious adverse events, in the absence of a survival benefit • Relative: Worsening of key surrogate measurements, in the absence of a survival benefit

41. RESUS Summary • A pivotal trial of HBOC-201 for prehospital resuscitation of severe hemorrhagic shock • Comparison to the current standard of care • Does not alter in-house trauma treatment • Requires exception from informed consent • Powered to demonstrate a 15% reduction in the relative risk of death

42. Pre-Clinical Data: HBOC-201 Swine Hemorrhagic Shock Studies Susan A. Stern, MD Associate Professor Associate Chair for Education Department of Emergency Medicine University of Michigan Ann Arbor, Michigan

43. Preclinical Hemorrhagic Shock (HS) Studies 22 Trauma Related HS Studies: • 12 Controlled Hemorrhage Models • Fixed volume model- • 40-50% blood volume removed • Concomitant soft tissue injury (muscle crush) • Moderate to severe hemorrhagic insult • Fixed pressure model- • Animal bled to a target MAP = 30-40 mmHg • Mild to moderate hemorrhagic insult • 6 Uncontrolled Hemorrhage Models • Liver crush/laceration • Arterial laceration • 4 Combined HS & Traumatic Brain Injury (TBI)

44. Study Methodologies • Animal preparation • Swine were anesthetized and fasted • One study in which animals were dehydrated • Study design • Simulated prehospital phase - short (30 min) to long (8 hrs) • Simulated hospital phase - hours to days • Fluid resuscitation regimen • Bolus or continuous, target MAP and HR • HBOC-201 total dose: 4-313 ml/kg (7-522% of EBV ) • HBOC-201 infusion rate: 0.5-10 ml/kg/min • COMPARABILITY TORESUS • RESUS individual dose: 7-21 ml/kg • RESUS infusion rate: 0.7 ml/kg/min (50 ml/min)

45. Data • Survival • Hemodynamics • Tissue oxygenation (direct and indirect) • Blood loss • Organ function

46. Survival

47. HBOC-201 Control Survival was Significantly Improved with HBOC-201 100 88 90 80 70 p = 0.004 60 53 50 40 30 103/117 59/112 20 10 0

48. HBOC-201 Control Fluid Less Severe More Severe * * * * * *p < 0.01

49. Hemodynamics

50. = simulated hospital arrival = baseline for HBOC-201 HBOC-201 HEX/LR Mean Arterial Pressure was Significantly Improved with HBOC-201