Milligan Blood Substitutes

7/28/2019 Milligan Blood Substitutes

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Why do we need blood substitutes?

Blood shortages

Public opinion on virus transmission / life insuranceissues

Aging population

Advancements in surgical procedures

HIV

New blood-borne diseases, eg nvCJD

Cost of safe transfusion in developing world


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Problems associated with blood transfusion

Blood shortages & donor recruitment

Compatibility need for cross-matching

Cost of blood processing

Shelf-life & storage

Human error

Unnecessary transfusions

Risk of disease transmission

Cultural & religious objection


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Risks of disease transmission

Risk factor Estimated frequency

per blood unit

transfused

Deaths per mil lion

units of blood

Hepatitis B 1 in 250,000

1 in 1,000,000


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Blood Substitutes

Blood substitutes are fluids which when

injected into the human blood streamcontribute significantly to the transport of

oxygen around the body

Cell-free oxygen carriers

Oxygen therapeutics

Red cell substitutes


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How would a blood substitute be used?

Coupling with autologous blood

Supporting transfusion service in developing

countries

Battlefield or natural disasters

Alternative to blood transfusion for patients with

religious objections


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Properties of an ideal blood substitute

Adequate oxygen uptake in the lungs

Adequate oxygen delivery to the tissues

Long circulation time

Non-toxic

Rapidly excreted without causing harm

Stable at room temperature

Easily sterilized

Cheap to manufacture

Long shelf-life & easy to store

Widely applicable w/o cross-matching

Free of side-effects


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Types of Blood Substitute

Biometric mimics natures way of

delivering oxygen to the bodys tissues,e.g. Hb based substitute

Abiotic use of totally synthetic chemicals

to deliver oxygen to the tissues, e.g. PFC

based substitiute


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Types of Blood Substitutes


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Hemoglobin Based Oxygen

Carriers (HBOCs)

Bodys natural O2 transporter Complex protein consisting of 4

subunit chains: 2 alpha and 2 beta

Each subunit contains an iron atom, which

binds oxygen reversibly

Inside the RBC Hb exists in a stableenvironment containing the enzymes it

requires to control O2 binding & other

characteristics


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O2 Delivery by hemoglobin


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Sources of Hemoglobin

Hemoglobin Explanation Advantages Disadvantages

Human Blood Involves extractionof Hb from donor

blood

Cheap; uses

discarded blood

Supply of waste

blood diminishing.

Unacceptable to

JWs

Cow Blood Uses Hb from cowblood

Cheap, plentiful.

Less chemical

modifications.

Acceptable to JWs

Unknown long-term

effects.

BSE cows & CJD

Micro-organisms

Genetically-modifiedbacteria & some

plants can be made

to produce Hb

Infinite supply.Avoids human

blood. Pure, virus-

free Hb

High productioncosts

TransgenicGenes for human

Hb inserted into a

developing animal

Potential infinite

supply of large

quantities of Hb

Ethical objections to

Hb factories. Hb

extraction difficulties


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Cell-free hemoglobin


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Characteristics of HBOCs

Size

Microvascular effects Vasoactivity

O2Affinity (P50) Oxidation

Absence of pro-inflammatory properties


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Size

64 kDa Hb tetramer dissociates into and

dimers

Filtered through renal glomerulus & disappearfrom circulation w/i few hours

Nephrotoxic Prolong T1/2 (12 36h) & minimize nephrotoxicity

by:

Stabilization of the tetramer

Polymerization of tetramers to oligomers

Surface conjugation to MW and diameter

Elimination then by RES


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Microvascular effects

Hb solutions: low viscosity, high oncoticpressure

Low viscosity shear on endothelial cells

vasodilators (endothelin & prostacyclin) localvasoconstriction & regional blood flow

Hamster skin fold model Hemopure(polymerized bovine HBOC) local tissue PO2compared with NS or Dextran


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Vasoactivity

Many HBOCs have systemic pressor effect

Pulmonary hypertension (in animals)

Mechanisms not fully understood

Free Hb closer to endothelium, binds nitric oxide &produces vasoconstiction

Greater vasoconstriction with lower MW products

Stimulate catecholamine release from adrenal medulla &potentiate response to norepinepherine

endothelin-1 levels

vasoconstriction cardiac output Sheep model of intra-op hemorrhage - HBOC produced

better volume expansion than RL, more rapid MAP,

CO in recovery phase, no DO2


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Oxygen affinity (P50)

Hb outside of RBC loses its 2,3-DPG

Affinity for O2 (P50 ) left-shift ofoxyhemoglobin dissociation curve

Reversal of left-shift attempted by pyridoxylationor Cl-

May be desirable property: HBOCs with low O2affinities which unload O2 at higher PO2 , may

trigger autoregulatory local vasoconstriction &impaired O2 delivery


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Absence of Pro-inflammatory Properties

Plasma from banked RBCs stored > 14 days

accumulates pro-inflammatory substances whichcan produce SIRS

HBOCs lack ability to activate WBCs in vitro

Trauma patients resuscitated with PolyHemeshowed no evidence of neutrophil priming in vivo


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Modified - hemoglobins


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Cross-linked HBOCs

HemAssist (Baxter) stabilized hemoglobin tetramer bydiaspirin linkage

Stored frozen

Phase III trials halted due to safety concerns (worseoutcome in stroke & trauma patients)

Similar outcome in CPB compared with RBCs

Animal TBI + hemorrhage CO, MAP, cerebral O2saturation

Somatogen (Optro, Baxter) recombinant human

hemoglobin cross-linked by single polypeptide consistingof 2 subunits, joined by shorter linker peptide to 2conventional -chains

Development discontinued during Phase I and II trials

(hypertension)

Polymerized HBOCs


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Polymerized HBOCs PolyHeme (Northfield) glutaraldehyde polymerized human hemoglobin;

pyridoxylated & extensively purified

Trauma patients who received PolyHeme required fewer transfusions of bankedblood

Case report: MVA-victim JW 5U PolyHeme for severe hemorrhage (Hb3.2g/dL) sustained for several days until hemorrhage controlled & erythropoesisstimulated by EPO compensated for blood loss

Hemopure (Biopure) glutaraldehyde polymerized bovine Hb.

Used as peri-op bridge

Slight pressor effect & CI

AA repair 27% receiving Hemopure avoided PRCs (cf none of controls)Licensed for clinical use in South Africa

Vetinary use FDA approved (Oxyglobin)

US Phase II on hold

Hemolink (Hemosol) polymerised human hemoglobin using oxidisedtrisaccharide, O raffinose followed by reduction step

Mild pressor effect

Phase II trials in dialysis and ANH

Cardiac surgery fewer transfusions at 5 days cf pentastarchPhase III trials completed in Canada, US & Europe.

Development discontinued due to MI

C j t d HBOC


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Conjugated HBOCs

PEG-Hemoglobin (Enzon) polyethylene glycol conjugated tobovine Hb tetramer

Much larger molecular radius

Longer T1/2 than most HBOCs (48h)

HyperoncoticUsed as sensitized for radiation treatment of solid tumours

PHP (Apex Bioscience) pyridoxilation of human Hb followed by

conjugation with polyoxyethyleneHypertensive effects

Phase III trials in septic & hemorrhagic shock

Hemospan (Sangart) human Hb tetramer conjugated topolyethylene glycol

Larger molecular diameter, high viscosity & high O2 affinity tominimize autoregulatory & vasoconstrictive effects

Phase III trials


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Perfluorocarbon based substitutes

P fl b


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Perfluorocarbons

Perfluorocarbons (PFCs) are organic compoundssimilar to hydrocarbons - fluorine, rather than

hydrogen atoms.

Clear, odourless fluids, chemically very

unreactive; linear, cyclic or polycyclic.

The stability of PFCs stem from the strength ofcarbon-fluorine bonds. Also responsible for the

inert nature of PFCs in the bloodstream.

2 most commonly uses PFCs are:

Perfluorodecalin (Flusol and Perftoran)

Perflubron (Oxygent)


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Gas carriage by PFC emulsions


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Microvascular effects of PFCs

Emulsion particles 0.2m diameter perfusesmallest capillaries (4 - 5m diameter) where noRBCs flow

Augment local O2 delivery much more thanwould be expected from in O2 content ofarterial blood

O2 in dissolved state higher PO2 inmicrocirculation driving pressure fordiffusion of O2 into tissues

O2 transported by PFCs is preferentially

metabolised due to its excellent unloadingcharacteristics


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Problems with perfluorocarbons

Immiscible with plasma, need to be prepared asemulsions (egg yolk phosphatide)

Require high FiO2 to dissolve adequatequantities of oxygen; limits applications to places

where supplementary O2 can be provided

Flu-like symptoms observed in human clinical

trials, delayed febrile reactions (due tophagocytosis by RES)

Thrombocytopenia at higher doses(no effect on coagulation or bleeding time)

Perfluorocarbon products


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Perfluorocarbon products

Fluosol-DA (Green Cross Corporation, Japan)Acute hemorrhage in patients who refuse blood transfusionfor religious reasons; performance disappointing

Approved for use following PTCA but cumbersome & lowefficacy

Oxygent (Alliance Pharmaceutical Corporation, San Diego)

ANH in dogs CO, mixed-venous PO2 & SatNear-fatal hemorrhage in pigs mortality (43% 13%)

Dogs undergoing CPB increased survival

Reduced transfusion requirements in orthpedic & urologicsurgery

Development on hold due to safety concerns (stroke)

Oxyfluor (HemaGen/PFC, Waltham, MA)Discontinued due to safety concerns

Current status of Blood Substitutes


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Current status of Blood Substitutes

Product class Product Company Technology Status

Perfluorocarbons Oxygent

Oxycyte

Oxyfluor

Alliance

Synthetic Blood

HemaGen

PFC Emulsion

PFC Emulsion

PFC Emulsion

On hold; safety (stroke)

Phase II

Discontinued; safety

Cross-linked Hb HemeAssist

rHb1.1

rHb2.0

Baxter

Somatogen

Baxter

Cross-linked Hb

Recombinant Hb

Recombinant Hb

Discontinued; safety (increased

mortality)


(hypertension)


Polymerized Hb PolyHeme

HBOC-201

(Hemopure)

Hemolink

Northfield Labs

Biopure

Hemosol

Glutaraldehyde,

pyridoxal Hb

Glutaraldehyde

bovine Hb

Polymerized Hb

Phase III (enrolling)

US phase II on clinical hold

Discontinued; safety (MI)

Conjugated Hb PHP

PEG-

Haemoglobin

Hemospan

Apex

Bioscience

Enzon

Sangart

PEG-human Hb

PEG-bovine Hb

PEG-human Hb

Phase III septic shock

Discontinued

Entering Phase III

Research Developments


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Research Developments

Hemoglobin from worms: Lumbricus

terrestris, Arenicola marina

Hb polymers, 50x larger than human

No modification required to remain

stable in bloodstream long enough

to oxygenate tissues

No breakdown and kidney damage

Pre-clinical testing in mice: normal

O2 carrying capacity & no allergic

reactions

?ease of extraction & purification in

sufficient quantities

?hypertension

Synthetic Red Blood Cells


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Synthetic Red Blood Cells

Encapsulation of hemoglobin in biodegradablepolymer membranes

Stabilizes Hb - prevents breakdown & kidneyproblems

Trap natural RBC enzymes with Hb, creating a

microenvironment for the Hb similar to normalblood

Ensure correct O2 and nitric oxide binding &

release Avoid hypertension

Micro-organisms, such as bacteria & fungi, willbe used to produce novel heme proteins

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