Site Visit Review LBVB: Personal Actions

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Laboratory of Biochemistry and Vascular Biology
Physical Protein Chemistry Andrew Shrake, Ph.D.
(PI) (Ewa Marszal, Ph.D.)
Vascular Biology Felice DAgnillo, Ph.D. (PI)
Biochemistry of Blood Substitutes Abdu I.
Alayash, Ph.D. (PI) Yiping Jia, Ph.D.

• 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

Research
Regulatory Review
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BPAC March 06
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Oxygen Therapeutics, Blood Substitutes
RBC
PFC Emulsion
Tetramer
Conjugated Tetramer
Polymer
Encapsulated Hb
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HBOC Associated Pre-Clinical andClinical Side
Effects(Mackenzie C.F. and Bucci E. Hosp. Med.
65582, 2004)

• Vasoactivity/hypertension
• Gastrointestinal side effects
• Pancreatic and liver enzymes elevation
• Oxidative stress
• Cardiac involvement
• Proinflammatory activity
• Neurotoxicity

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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
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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)
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O-R-PolyHbA0Identification of the Origin of
Altered Function
Tetragonal Heme Fe
Rhombic Heme Fe
(1) Heme Destabilization
O-R-PolyHbA0
HbA0
(2) Protein Destabilization (locked T state)
Biochemistry (2002), Biochemical J. (2004)
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O-R-PolyHbA0Actual Chemical Modification
(MALDI-MS)

• Non-uniform O-raffinose oxidation products
• Non-specific cross-links
• Modified cysteines

Destabilization of the protein!
Proteins (2005)
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Laboratory of Biochemistry and Vascular Biology
Physical Protein Chemistry
Andrew Shrake, Ph.D. (Ewa Marszal, Ph.D.)
BPAC March 06
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• 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)

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

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The structure of a1-PI
A sheet
C sheet RCL
C232
PDB 1QLP
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Loop-A sheet model of a1-PI polymer
Nature Cell Biol. 2, E207 (2000)
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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
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Research Significance

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

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BPAC March 06
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Vascular Endothelial Responses to Biologics and
Pathogens

• Hemoglobin-based oxygen carriers
• Counterterrorism Anthrax
• Blood-derived products

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Redox Active Hb Induces Endothelial Cytotoxicity
DAgnillo, Am J Physiol. 287, 2004.
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Vascular Endothelial Responses to Anthrax Toxin
? Hemorrhages ? Vasculitis ? Vascular leakage
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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
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0
0
20
40
60
80
Time (h)
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Anthrax Lethal Toxin Alters Endothelial Adherens
Junctions
Phase
F-actin/nuclei
VE-cadherin
Medium
LT
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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