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Hemoglobin-Hyperpolymers
A new type of an artificial oxygen carrier
Tamaulipas 10-2007

Two very different artificial oxygen carriers for
Two very different reasons of hypoxia
Acute Loss of Blood
Deficient local Cellular
Oxygen Supply
2
Blood
Loss
+
Is-Oncotic Oxygen
Transporting Plasma
Substitute / Expander
Degeneration
Edema
Arteriosclerosis
Anaemia
+
Hyp-Oncotic
Oxygen Transporter
Blood Additive
Tamaulipas 10-2007

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Application of the Blood Additive
Blood
Additive
Hemoglobin-
Hyperpolymers
Blood Blood
Urin
Tamaulipas 10-2007

The indication fields for both artificial oxygen carriers
With blood loss
•
Accidents (war, traffic)
•
Invasive surgery
Therapy with Oxygen Expander
Without blood loss
•
Lung shock
•
Heart infarction
•
Stroke
•
Arterial occlusion
•
Renal shock
•
Liver shock
•
Tinitus
•
Sudden deafness
•
Blindness
•
Vertigo
•
Plazenta-Insufficiency
•
Diabetes/complications
•
Tumor-Oxygenation
•
Anemias
Therapy with Blood Additive
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The indication field of the blood additiv is much bigger.
Tamaulipas 10-2007

Natural model for the additiv is the erythrocruorin of the earthworm.
Electron microscope foto of erythrocruorin
Erythrocruorin is a huge hyp-oncotic hemoglobin-hyperpolymer solved in blood and transporting the oxygen.
26 nm
Molecular weight: 3,3 Mio Dalton
High oxygen affinity (p50): 9Torr
High cooperativity (n50): 12
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The concept of the additive is unique in the world.
Tamaulipas 10-2007

Production of the hemoglobin-hyerpolymers from pig blood
Haemoglobin
Haemoglobin
Multimeres
PEGylated
Multimeres
Monomeres
Oligomeres
Polymerisation
Surface
Modification
Separation
One-vessel- reaction
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Hyperpolymeres
Tamaulipas 10-2007

In- vitro-Results:
Molecular structure of the artificial hemoglobin-hyperpolymer
Electron microscope foto of the hyperpolymer
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The loose structure and big volume of the molecule avoids extravasation, which is a general issue of artificial oxygen carriers.
Tamaulipas 10-2007

In-vitro-Results:
Molecular weight distribution of the artificial hyperpolymer
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Weight average 900 000 Dalton
10% Percentile 180 000 Dalton
90% Percentile 3 600 000 Dalton
The molecular weight distribution is sufficiently narrow.
Tamaulipas 10-2007

In-vitro-Results:
Characteristics of oxygen binding in comparison
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Hemoglobin System / Derivative
Half Saturation Pressure p50 / Torr
Cooperativity n50
Human Full Blood
Human Hemoglobin
Porcine Hemoglobin
Blood Additiv
25
15
15
16
2.5
2.5
2.5
2.1
Measured under Physiological Conditions:
(T = 37
°
C, pH = 7.4, pCO2 = 40 Torr, Absence of 2,3-DPG and ATP)
The oxygen binding of the blood additive is optimised for oxygen delivery to the tissues.
Tamaulipas 10-2007

The long-term storage stability of the additive is two years at 4 degree Celsius.
It is stabilised as Carbonyl-Derivative.
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The storage stability of the blood additive is sufficient.
Tamaulipas 10-2007

Ex-vivo-Results:
Compatibility with fresh human blood plasma
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The blood additive is compatible (no precipitation) with fresh human blood plasma between pH 6,6 to 8,2.
Tamaulipas 10-2007

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In-vitro-Results: Oncotic pressure of the additive in blood plasma therapeutic concentration in electrolyte
At therapeutic concentration the oncotic pressure of the additive exerts only 5 mbar, which helps to keep „dry“ the tissues.
Tamaulipas 10-2007

Therapeutic concentration
In blood plasma
In electrolyte solution
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At the therapeutic concentration the viscosity of the additive is lower than the viscosity of blood.
Tamaulipas 10-2007

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In-vivo-Results (rat): Lifetime of the additive
Concentration in blood plasma (g/dL)
3
Werte 5
Werte 6
2
1
½
½
0
0 10 20 30 40 50
•
In alert rats the lifetime increases with the dosages.
•
With therapeutic concentration the lifetime reaches 30 hours.
• Transfered to humans gives a lifetime of about 60 hours.
Tamaulipas 10-2007

In-vivo-Results (rat): Functinality of the carrier
Spec. Oxygen uptake
(ml
STPD
· min -1 · kg -1 )
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Overall
Exchange Dilution by red blood cells
10 by the carrier by solved oxygen
Hämatocrit in %
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The carrier keeps up the oxygen supply of the rat in small amount.
It is more effective than blood.
Tamaulipas 10-2007

In-vivo-Results (rat): Efficacy of the additive in lung shock
Lung shock was induced by oleic acid before application of the additive.
+ Oleic acid + Oleic acid + Additive
Survival
Time (h)
8
6
4
16
2
1 2 3 4 5 6 7 8 9 10
0
All 5 rats without additive died after about 3 hours.
All 5 rats with additive must be sacrified after about 8 hours.
The additive saved the life of the rats in lung shock.
Tamaulipas 10-2007

Why is the additive so effective?
L(ung)
L without carrier K
Cells with carrier arterial
L
L
L venous
Mediator-Effect
Bridging-Effect
1. Fick‘s Equation
• mO
2
= DO
2 ,ef
• a
O
2 ,ef
F ef d ef
(PO
2,ef,K
– PO
2,ef,G
)
The diffusion area ( F ef
) increases and the diffusion distance ( d ef
) decreases.
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The mediator effect increases the geometric factor ( F/d ).
Tamaulipas 10-2007

In-vitro-Results: Proof of the bridging effect
Oxygen release from an artificial capillary
Page et al. Microv. Research 55 (1998) 54-64
Oxygen
Saturation
Hb content 100 g/l
Red blood cells
Carrier
Mixture 1:1
Residence time (s)
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The 1:1 mixture has the same oxygen releasing than pure carrier, behaving like the pure carrier.
Tamaulipas 10-2007

In-vivo-Results (mice): Immunogenicity of the additive
• Mice were sensitised against Hemoglobin.
• Five sensitised mice received hemoglobin and hyperpolymer three times with 14 days intervall.
• The level of entire Ig, IgG1, and IgG2a in blood plasma was determined after each application.
• After the 3. application the hyperpolymer induced a slight increase of the Ig levels.
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In sensitised mice the hyperpolymer has a small immunogenic effect.
Tamaulipas 10-2007

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In-vivo-Results (mice): Induction of immuntolerance
Tamaulipas 10-2007

In-vivo-Results (humans): Blood pressure effect?
In rats hemoglobin causes an increase of systolic blood pressure by extravasation.
Low dose application of the additive on humans:
Proband:
♂
, 70 kg Body Weight Proband:
♀
, 65 kg Body Weight
Admin. 1,16 g
BP
(Torr)
150
HR
(min -1 ) syst. Blood Pressure
BP
(Torr)
150
HR
(min -1 )
Admin. 1,16 g syst. BP diast. Blood Pressure
100 100
50
Heart Rate
50 diast. BP
HR
0
0 20 40 60 80
Versuchszeit (min)
100 120 140
Time (min)
0
0 20 40 60 80
Versuchszeit (min)
100 120 140
Time (min)
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In humans the additive does not cause a blood pressure effect.
Tamaulipas 10-2007

In-vivo-Results (humans): Liver damage by the additive?
Measuring transaminase levels:
Proband W.B.
1: 0,58 g 2: 2,32 g
Transaminase blood plasma level (U/l)
16
12
Proband D.K.
1, 2, 3: 1,16 g
G lutamat
P yruvat
T ransaminase
(norm < 22 U/l)
8
4
G lutamat
O xalat
T ransaminase
(norm < 18 U/l)
0
1 2 before
1 2 after 24 h
1 2 3 before
1 2 3 after 24 h Administration
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The additive does not change the transaminse levels significantly.
Tamaulipas 10-2007

The additive was administered intravenously in two persons three times with 14 days intervals at dosages between 0,6 and 2,4g hyperpolymer.
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No signs of adverse effects were seen and especially no sign of immunogenicity.
Tamaulipas 10-2007

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• Simple production: without chromatography
• High yield: about 20%
• Valuable byproduct: Oligomers as oxygen expander
• Universal: Application independent from blood group
• Independent from blood stores
• No danger of infection (hepatitis, AIDS)
• No burden on the reticulo-endothelial system
Tamaulipas 10-2007

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1969 • First publication on hemoglobin associates
1988 • Formulation of the hyperpolymer concept
• First synthesis of hyperpolymers
1996 • First proof of functionality in animals
2000 • Prescription for production of the additive
2001 • First sterile production at lab scale
2002 • First application in humans
• First proof of efficacy in lung shock (rats)
Tamaulipas 10-2007

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Tamaulipas 10-2007