biochemical and physiological studies on the safety and efficacy of blood products and their...

• 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

Laboratory of Biochemistry and Vascular Biology

Physical Protein ChemistryAndrew Shrake, Ph.D. (PI)

(Ewa Marszal, Ph.D.)

Vascular BiologyFelice D’Agnillo, Ph.D. (PI)

Biochemistry of Blood SubstitutesAbdu I. Alayash, Ph.D. (PI)

Yiping Jia, Ph.D.

Biochemistry of Blood Substitutes

Abdu Alayash, Ph.D.Yiping Jia, Ph.D.

Laboratory of Biochemistry andVascular Biology

BPAC March ‘06

Oxygen Therapeutics, “Blood Substitutes”

2,3 DPGPFC

PFC

PFC

RBC PFC Emulsion

Tetramer Conjugated Tetramer

Polymer Encapsulated Hb

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

ROS = Reactive Oxygen; RNS = Reactive Nitrogen Species

HBOCs: Redox Challenges Outside RBCs!

• Free Hb is inherently toxic:

generates ROS, and reacts with ROS & RNS (i.e. NO) vascular “injury”

• Nature of chemical modification: Non-site specificity conformational & heme instability

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

log PO2

-0.5 0.0 0.5 1.0 1.5 2.0

Sa

tura

tio

n

0.00

0.25

0.50

0.75

1.00

BloodO-R-PolyHbA0

HbA0

Biochemistry (2002)

Biochemistry (2002), Biochemical J. (2004)

O-R-PolyHbA0

Identification of the Origin of Altered Function

(1) Heme Destabilization

(2) Protein Destabilization (locked T state)

O2 O2

O2

O2 O2

O2 O2

Tense (T) Deoxy

Tense (T) Oxy

Locked (T) State

O2 O2

O2 O2

Relaxed (R) Oxy

Normal Conformational Change

Tetragonal Heme Fe

HbA0

Rhombic Heme Fe

O-R-PolyHbA0

O-R-PolyHbA0:Actual Chemical Modification

(MALDI-MS)

Proteins (2005)

•Non-uniform O-raffinose oxidation products

•Non-specific cross-links•Modified cysteines

Destabilization of the protein!

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.

Physical Protein Chemistry

Andrew Shrake, Ph.D.(Ewa Marszal, Ph.D.)

Laboratory of Biochemistry andVascular Biology

BPAC March ‘06

1-PI Projects

● Investigation of the structure of 1-PI polymer

● Characterization of differences in isoelectric focusing behavior of licensed 1-PI products

● Development of WHO 1-PI reference standard

● Expression of human 1-PI in E. coli and A. niger

● Assay development (ELISA and potency assay)

LungsEmphysema

Liver Disease

1-PI

Neutrophil elastase

Neutrophils

Bone marrow

Pathogenesis of 1-PI deficiency

Adapted from Crystal et al., 1997

Structure of 1-PI Polymer

Relevance of investigation of the structure of 1-PI polymer

protein polymers form in vivo

mechanisms underlying conformational diseasesprevention

protein polymers are present in products

safety issue related to long-time use

A sheet

C sheet

RCL

C232

The structure of 1-PI

PDB 1QLP

Loop-A sheet model of 1-PI polymer

Nature Cell Biol. 2, E207 (2000)

C232

A -sheet

Head-to-head model of 1-PI polymer

Marszal et al. JBC 2003

Listed as one of the models of 1-PI polymers in a review “A protein family under “stress” – Serpin stability, folding, and misfolding”by Devlin & Bottomley, Frontiers in Bioscience 2005

head-to-tailhead-to-head

Research Significance

• Expertise in plasma-derived product characterization, comparability, safety, and follow-on biologics

• Impact on potential new treatment modalities

Vascular Biology

Felice D’Agnillo, Ph.D.

Laboratory of Biochemistry andVascular Biology

BPAC March ‘06

Vascular Endothelial Responses to Biologics and Pathogens

• Hemoglobin-based oxygen carriers

• Counterterrorism – Anthrax

• Blood-derived products

Redox Active Hb Induces Endothelial Cytotoxicity

Medium

Redox activeHb

H2O2

Fe2+ Fe4+ Fe3+

H2O2

D’Agnillo, Am J Physiol. 287, 2004.

● Hemorrhages

● Vasculitis

● Vascular leakage

Vascular Endothelial Responsesto Anthrax Toxin

Anthrax Lethal Toxin Induces Endothelial Barrier Dysfunction

Time (h)

*

*

*

*

TE

ER

(% r

ela

tiv

e t

o c

on

tro

l)

0

20

40

60

80

100

120

0 20 40 60 80

Medium

LT

Warfel et al., Am J Pathol. 166, 2005.

LT

Medium

Phase F-actin/nuclei VE-cadherin

Anthrax Lethal Toxin Alters Endothelial Adherens Junctions

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