Overview of Hemoglobin-based Overview of Hemoglobin-based Oxygen Carriers (HBOCs)Oxygen Carriers (HBOCs)

Abdu I. Alayash, Ph.D.

Laboratory of Biochemistry and Vascular Biology, DH, OBRR, CBER, FDA

BPAC Meeting, December 14, 2006

Oxygen Therapeutics, “Blood Substitutes”

2,3 DPGPFC

PFC

PFC

RBC PFC Emulsion

Tetramer Conjugated Tetramer

Polymer Encapsulated Hb

HBOC Associated Pre-Clinical and Clinical HBOC Associated Pre-Clinical and Clinical Side EffectsSide 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)

HBOCs: Challenges Outside RBCsHBOCs: Challenges Outside RBCs

(A) Nature of chemical modification(A) Nature of chemical modificationnon-site specificity conformational & heme instability

(B) Vascular/oxidative effects of free Hb(B) Vascular/oxidative effects of free Hb a) consumes NO vasoconstriction/hypertension

b) generates/reacts with ROS vascular “injury”

ROS = Reactive Oxygen Species

OxyglobinOxyglobin®® (HBOC-301): Biochemical and (HBOC-301): Biochemical and Physiological Properties of an FDA Physiological Properties of an FDA

Approved HBOC for Veterinary MedicineApproved HBOC for Veterinary Medicine

Buehler et al., Anal Chem 77:3466, 2005

Molecular Size Distribution of HBOC-301 and its FractionsMolecular Size Distribution of HBOC-301 and its Fractions

0.0

0.2

0.4

0.6

0.8

1.0

1.0x104 1.0x105 1.0x106 1.0x107C

um

ula

tive

Wei

ght

Fra

ctio

n W

Molar Mass (g/mol)

HbBv 4 3 2 1

4

Ab

sorb

ance

0.000.100.200.300.400.500.600.700.800.901.001.101.201.30

0.00 10.00 20.00 30.00 40.00 50.00 60.00

1 23

HBOC-301 HbBv

(min)

Log (pO2) (mmHg)

0.5 1.0 1.5 2.0

n O2

0.0

0.2

0.4

0.6

0.8

1.0

pO2 (mmHg)0 10 20 30 40 50

n O2

0.40

0.45

0.50

0.55

0.60

Oxygen Equilibrium Curves (OECs) for HBOC-301 and its Fractions

Buehler et al., Anal Chem 77:3466, 2005

HBOCs: Challenges Outside RBCsHBOCs: Challenges Outside RBCs

(A) Nature of chemical modification(A) Nature of chemical modificationnon-site specificity conformational & heme instability

(B) Vascular/oxidative effects of free Hb(B) Vascular/oxidative effects of free Hb a) consumes NO vasoconstriction/hypertension

b) generates/reacts with ROS vascular “injury”

ROS = Reactive Oxygen Species

RBC Free Zone Plasma

RB

CNO

Ves

sel

Wal

l

Unstirred Layer

Blo

od

Flo

w

RBC Free Zone PlasmaR

BC

Unstirred Layer

NOHOOH

ONOO-

Hb

EC EC

Cell-free Hb and The Vasculature

(Alayash, A., Nature Rev. Drug Disc. 3:152-159, 2004)

HBOC-301 Associated Hypertension after 50% Exchange HBOC-301 Associated Hypertension after 50% Exchange TransfusionTransfusion

Time (Minutes)

-20 0 20 40 60 80 100 120 140

Me

an

Art

eri

al P

res

su

re (

mm

Hg

)

80

100

120

140

160

180

Baseline ET

Rat - MAP

Time (Minutes)

-20 0 20 40 60 80 100 120 140

Mea

n A

rter

ial

Pre

ssu

re (

mm

Hg

)

50

60

70

80

90

100

Baseline ET

Guinea pig - MAP

Baldwin et al. , AJP 283:H1292, 2002; Baldwin et al, AJP 284:H81, 2003; and Baldwin et al., JAP 96:893, 2004

CN

metH

b

HBOC Associated Oxidative Vascular InjuryHBOC Associated Oxidative Vascular Injury

Number of Mesenteric Leaks per Micron Venule

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

Area of Mesenteric Leaks per Micron Venule

Are

a of

Lea

ks (

0.00

0.02

0.04

0.06

0.08

0.10

0.12

HB

OC

-30

1

O-R

-Po

lyH

bA

0

-DB

BF

-Hb

CN

me

tHb

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 O2.