Milligan Blood Substitutes
Transcript of 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)
Discontinued; safety
(hypertension)
Discontinued; safety
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