1. Overview of Hemoglobin-based Oxygen Carriers (HBOCs) Abdu I. Alayash, Ph.D. Laboratory of Biochemistry and Vascular Biology, DH, OBRR, CBER, FDA BPAC Meeting, December 14, 2006

2. Oxygen Therapeutics, “Blood Substitutes”     2,3 DPG PFC RBCPFC Emulsion TetramerConjugated Tetramer PolymerEncapsulated Hb

3. HBOC Associated Pre-Clinical and Clinical Side Effects Vasoactivity/hypertension Gastrointestinal side effects Pancreatic and liver enzyme elevation Oxidative stress Cardiac involvement Proinflammatory responses Neurotoxicity (Mackenzie C.F. and Bucci E. Hosp. Med. 65:582, 2004)

4. HBOCs: Challenges Outside RBCs (A) Nature of chemical modification non-site specificity  conformational & heme instability (B) Vascular/oxidative effects of free Hb a) consumes NO  vasoconstriction/hypertension b) generates/reacts with ROS  vascular “injury” ROS = Reactive Oxygen Species

5. Oxyglobin® (HBOC-301): Biochemical and Physiological Properties of an FDA Approved HBOC for Veterinary Medicine

6. Buehler et al., Anal Chem 77:3466, 2005 Molecular Size Distribution of HBOC-301 and its Fractions (min)

7. Oxygen Equilibrium Curves (OECs) for HBOC-301 and its Fractions Buehler et al., Anal Chem 77:3466, 2005

8. HBOCs: Challenges Outside RBCs (A) Nature of chemical modification non-site specificity  conformational & heme instability (B) Vascular/oxidative effects of free Hb a) consumes NO  vasoconstriction/hypertension b) generates/reacts with ROS  vascular “injury” ROS = Reactive Oxygen Species

9. Cell-free Hb and The Vasculature (Alayash, A., Nature Rev. Drug Disc. 3:152-159, 2004)

10. HBOC-301 Associated Hypertension after 50% Exchange Transfusion

11. Baldwin et al., AJP 283:H1292, 2002; Baldwin et al, AJP 284:H81, 2003; and Baldwin et al., JAP 96:893, 2004 CNmetHb HBOC Associated Oxidative Vascular Injury HBOC-301 O-R-PolyHbA 0  -DBBF-Hb CNmetHb

12. HBOCs: General Summary Most HBOCs are vasoactive, in part due to the scavenging of vascular NO. The proximity of HBOCs or its fractions to NO vascular sources may determine the degree of response. HBOCs that can withstand initial NO/oxidative insult due to heme stability and/or in the presence of endogenous reductants may deliver O 2.