Biochemical and physiological studies on the safety and efficacy of blood products and their interactions with the vascu

1. Laboratory of Biochemistry and Vascular Biology Biochemistry of Blood Substitutes Abdu I. Alayash, Ph.D. (PI) Yiping Jia, Ph.D. Physical Protein Chemistry Andrew Shrake, Ph.D. (PI) (Ewa Marszal, Ph.D.) Vascular Biology Felice D’Agnillo, Ph.D. (PI) Research • Biochemical and physiological studies on the safety and efficacy of blood products and their interactions with the vascular system • Albumin, PPF, Dextrans, HESs, A1PI, • r-Apolipoprotein A1, C1 Esterase Inhibitor, Butyrylcholinesterase, Hb and PFC-based blood substitutes, and heme-based products • Develop policy and guidance documents Regulatory Review

2. Laboratory of Biochemistry and Vascular Biology Biochemistry of Blood Substitutes Abdu Alayash, Ph.D. Yiping Jia, Ph.D. BPAC March ‘06

3. a PFC b b 2,3 DPG PFC a PFC Oxygen Therapeutics, “Blood Substitutes” RBC PFC Emulsion Tetramer Conjugated Tetramer Polymer Encapsulated Hb

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

5. HBOCs: Redox Challenges Outside RBCs! • Free Hb is inherently toxic : generates ROS, and reacts with ROS & Þ RNS (i.e. NO) vascular “ injury ” • Nature of chemical modification: Þ Non - site specificity conformational & heme instability ROS = Reactive Oxygen; RNS = Reactive Nitrogen Species

6. O-R-PolyHbA0 (HbA0 cross-linked and polymerized with O-raffinose)Hallmarks of Functional Abnormality • Non-sigmoidal oxygen equilibrium curve • Non-saturating • Non-cooperative (Hill coefficient = 1.0 vs. 2.5) • pH insensitivity Biochemistry (2002)

7. O-R-PolyHbA0Identification of the Origin of Altered Function O2 O2 Locked (T) State O2 O2 O2 O2 Tense (T) Oxy O2 O2 O2 Normal Conformational Change O2 O2 Tense (T) Deoxy Relaxed (R) Oxy Tetragonal Heme Fe Rhombic Heme Fe (1) Heme Destabilization O-R-PolyHbA0 HbA0 (2) Protein Destabilization (locked T state) Biochemistry (2002), Biochemical J. (2004)

8. O-R-PolyHbA0:Actual Chemical Modification (MALDI-MS) • Non-uniform O-raffinose oxidation products • Non-specific cross-links • Modified cysteines Destabilization of the protein! Proteins (2005)

9. Research Significance Our studies on the complex chemical and recombinant modifications associated with HBOCs provide an invaluable pre-clinical tool in predicting product stability, functionality and potential toxicity.

10. Laboratory of Biochemistry and Vascular Biology Physical Protein Chemistry Andrew Shrake, Ph.D. (Ewa Marszal, Ph.D.) BPAC March ‘06

11. a1-PI Projects ● Investigation of the structure of a1-PI polymer ● Characterization of differences in isoelectric focusing behavior of licensed a1-PI products ● Development of WHO a1-PI reference standard ● Expression of human a1-PI in E. coli and A. niger ● Assay development (ELISA and potency assay)

12. Pathogenesis of a1-PI deficiency Lungs Emphysema Liver Disease a1-PI Neutrophil elastase Neutrophils Bone marrow Adapted from Crystal et al., 1997

13. Structure of a1-PI Polymer • Relevance of investigation of the structure of a1-PI polymer • protein polymers form in vivo mechanisms underlying conformational diseases prevention • protein polymers are present in products safety issue related to long-time use

14. The structure of a1-PI A sheet C sheet RCL C232 PDB 1QLP

15. Loop-A sheet model ofa1-PI polymer Nature Cell Biol. 2, E207 (2000)

16. Head-to-head model of a1-PI polymer head-to-tail head-to-head A b-sheet C232 Marszal et al. JBC 2003 Listed as one of the models of a1-PI polymers in a review “A protein family under “stress” – Serpin stability, folding, and misfolding” by Devlin & Bottomley, Frontiers in Bioscience 2005

17. Research Significance • Expertise in plasma-derived product characterization, comparability, safety, and follow-on biologics • Impact on potential new treatment modalities

18. Laboratory of Biochemistry and Vascular Biology Vascular Biology Felice D’Agnillo, Ph.D. BPAC March ‘06

19. Vascular Endothelial Responses to Biologics and Pathogens • Hemoglobin-based oxygen carriers • Counterterrorism – Anthrax • Blood-derived products

20. H2O2 Fe2+ Fe4+ Fe3+ H2O2 Medium Redox active aaHb Redox Active Hb Induces Endothelial Cytotoxicity D’Agnillo, Am J Physiol. 287, 2004.

21. Vascular Endothelial Responses to Anthrax Toxin ●Hemorrhages ● Vasculitis ● Vascular leakage

22. * * * * Anthrax Lethal Toxin Induces Endothelial Barrier Dysfunction Warfel et al., Am J Pathol. 166, 2005. 120 100 80 TEER (% relative to control) 60 Medium 40 LT 20 0 0 20 40 60 80 Time (h)

23. Anthrax Lethal Toxin Alters Endothelial Adherens Junctions Phase F-actin/nuclei VE-cadherin Medium LT

24. Research Significance Contribute to the safety and efficacy evaluation of current and anticipated blood-derived biologics by examining preclinical in vitro and in vivo models and relevant vascular biomarkers