# Presentation: 718301

## Laboratory of Biochemistry and Vascular Biology

**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**

**Physical Protein Chemistry**
**Andrew Shrake, Ph.D. (PI)**
**(Ewa Marszal, Ph.D.)**

**Vascular Biology**
**Felice D’Agnillo, Ph.D. (PI)**

**Biochemistry of Blood Substitutes**
**Abdu I. Alayash, Ph.D. (PI)**
**Yiping Jia, Ph.D.**

## BPAC March ‘06

## Oxygen Therapeutics, “Blood Substitutes”

**RBC**

**PFC Emulsion**

**Tetramer**

**Conjugated Tetramer**

**Polymer**

**Encapsulated Hb**

**Notes:**
This figure outlines the major classes of products developed as oxygen therapeutics/blood substitutes. There are basically two classes of products that we deal with, fluorochemical and hemoglobin-based products. Fluorocarbon-based products are totally synthetic and are structurally and functionally different from Hb-based products. The focus of our research however, has been on Hb, thus the focus of my presentation will be on Hb-based products.
Hb isolated is from outdated human or in some cases bovine blood and is extensively purified. The purified Hb is then chemically modified. In some cases Hb is expressed in E. Coli. The purpose of chemical modification(s) is to produce a stable, functional tetrameric, conjugated, polymeric or in some cases encapsulated within liposomes. Otherwise, cell free Hb, in a dilute medium outside RBCs will dimerize and would not be appropriate for transfusion. These products are designed to provide oxygen and volume replacement they don’t replace blood.

## HBOC Associated Pre-Clinical andClinical 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

**Notes:**
If you were to review the published literature in terms of known and documented preclinical and clinical adverse events, this what you see: this is a representative list of the most common side effects associated with the infusion of Hbs reported in animals and in humans. Most of these events can be attributed heme-mediated reactivity of the molecule. Vasoactivity/hypertension has been attributed by many to the redox reactions between Hb and Nitric oxide, a vasodilator produced by the vascular system. Removal of NO by Hb lead to vasoconstriction of blood vessels and elevation in blood pressure. Other side effects can also be directly or indirectly attributed to this reaction and to the subsequent hemoglobin redox side reactions.

## HBOCs: Redox Challenges 

- 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

**Notes:**
Based on our research and that of others, we can basically narrow the ORIGIN OF toxicity to a BIOCHEMICALLY two major factors:
First, the Hb is inherently toxic, this largely due to the uncontrolled reactivity of its prosthetic group, the heme. Hb generate oxygen reactive species, if left alone, it will self destruct. It can react with ROS from other cellular sources as well reactive nitrogen species such nitric oxide and its metabolites. These reactions occur even within RBCs, but as you know nature provided reductive mechanisms within..
Second: Chemical modifications employed in production of these products can in some cases enhance these reactions and acceralte Hb destabilization. 

Dr. Jia who will present after me a case-study of one particular product, that we were able to reveal { that both the nature of the modifying agent as well as the non-site specific nature of modification may have led to the destabilization of the protein and ultimately toxicity}.

## Non-sigmoidal oxygen equilibrium curve 
Non-saturating 
Non-cooperative (Hill coefficient = 1.0 vs. 2.5)
pH insensitivity

**Non-sigmoidal oxygen equilibrium curve **
**Non-saturating **
**Non-cooperative (Hill coefficient = 1.0 vs. 2.5)**
**pH insensitivity**

**  ****Biochemistry (2002)**

## O-R-PolyHbA0Identification of the Origin of Altered Function

***O*****-R-PolyHbA****0****Identification of the Origin of Altered Function**

**(1) Heme Destabilization**

** ****(2) Protein Destabilization (locked T state)**

**Tetragonal Heme Fe**

**HbA****0**

**Rhombic Heme Fe**

***O*****-R-PolyHbA****0**

## 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!

## Slide 9

## Laboratory of Biochemistry and

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

- Vascular 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)

## Pathogenesis of 1-PI deficiency

**Lungs**
**Emphysema**
**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 diseases
- 	prevention
-    protein polymers are present in products
- 	safety issue related to long-time use

## The structure of 1-PI

- A sheet

- C sheet
-      RCL

- C232

- The structure of 1-PI

- PDB 1QLP

## Loop-A sheet model of 1-PI polymer

- Loop-A sheet model of 1-PI polymer

- Nature Cell Biol. 2, E207 (2000)

**Notes:**
* The loop-sheet polymer model was proposed about 12 years ago by Lomas and Carroll is widely accepted.
* However, only indirect evidence has been used to support the model.
* The model proposes the insertion of the RCL into the central strands of an A beta-sheet of a neighboring molecule to form a linear polymer, which has been observed in material recoved from inclusion bodies the the livers of deficient patients.
* The loop-sheet model involves a single type of monomer-monomer interaction, loop-A beta-sheet insertion (head-to-tail polymerization).
*  The white surface in the schematic contains the A-beta sheet.

## Head-to-head model of 1-PI polymer

- 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-tail
- head-to-head

## Research Significance

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

## 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

***D’Agnillo, Am J Physiol. 287, 2004.***

## Vascular Endothelial Responses

- ● 	Vasculitis
- ● 	Vascular leakage

- to Anthrax Toxin

## Anthrax Lethal Toxin Induces Endothelial Barrier Dysfunction

**Time (h)**

**TEER**
**(% relative to control)**

**0**

**20**

**40**

**60**

**80**

**100**

**120**

**0**

**20**

**40**

**60**

**80**

**Medium**

**LT**

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

## Anthrax Lethal Toxin Alters Endothelial Adherens Junctions

**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