Tamaulipas 10-20071 Hemoglobin-Hyperpolymers A new type of an artificial oxygen carrier

Tamaulipas 10-20071

Hemoglobin-Hyperpolymers

A new type of an artificial oxygen carrier

Tamaulipas 10-20072

Acute Loss of BloodAcute Loss of Blood Deficient local CellularOxygen Supply

Deficient local CellularOxygen Supply

+ +Hyp-Oncotic

Oxygen Transporter

Blood Additive

Hyp-Oncotic

Oxygen Transporter

Blood Additive

Is-Oncotic Oxygen

Transporting Plasma

Substitute / Expander

Is-Oncotic Oxygen

Transporting Plasma

Substitute / Expander

Blood

LossDegeneration

Edema

Arteriosclerosis

Anaemia

Two very different artificial oxygen carriers forTwo very different reasons of hypoxia

Tamaulipas 10-20073

Blood Blood

Hemoglobin-Hyperpolymers

BloodAdditive

Application of the Blood Additive

Urin

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The indication field of the blood additiv is much bigger.

With blood loss Without blood loss• Accidents (war, traffic)• Invasive surgery

• 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 Oxygen Expander Therapy with Blood Additive

The indication fields for both artificial oxygen carriers

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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

The concept of the additive is unique in the world.

Tamaulipas 10-20076

PEGylated MultimeresPEGylated Multimeres

Polyme-risation

Surface Modification

Separation

Haemoglobin Multimeres

Haemoglobin Multimeres

MonomeresOligomeresMonomeresOligomeres

Haemo-globin

Haemo-globin

Hyper-polymeres

Hyper-polymeres

Production of the hemoglobin-hyerpolymers from pig blood

One-vessel- reaction

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In- vitro-Results:

Molecular structure of the artificial hemoglobin-hyperpolymer

Electron microscope foto

of the hyperpolymer

The loose structure and big volume of the molecule avoids extravasation, which is a general issue of artificial oxygen carriers.

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In-vitro-Results:

Molecular weight distribution of the artificial hyperpolymer

Weight average 900 000 Dalton

10% Percentile 180 000 Dalton

90% Percentile 3 600 000 Dalton

The molecular weight distribution

is sufficiently narrow.

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Hemoglobin System / DerivativeHalf 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)

In-vitro-Results:

Characteristics of oxygen binding in comparison

The oxygen binding of the blood additive is optimised

for oxygen delivery to the tissues.

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In-vitro-Results: Storage stability

The long-term storage stability of the additive

is two years at 4 degree Celsius.

It is stabilised as Carbonyl-Derivative.

The storage stability of the blood additive is sufficient.


Ex-vivo-Results:

Compatibility with fresh human blood plasma

The blood additive is compatible (no precipitation) with fresh human blood plasma between pH 6,6 to 8,2.


therapeutic concentration

In-vitro-Results: Oncotic pressure of the additive

in blood plasma

in electrolyte

At therapeutic concentration the oncotic pressure of the additive exerts only 5 mbar,which helps to keep „dry“ the tissues.


In blood plasma

In electrolyte solution

Therapeutic concentration

In-vitro-Results: Viscosity of the additive

At the therapeutic concentration the viscosity of the additive is lower than the viscosity of blood.


0

1

2

3

0 10 20 30 40 50

Verweilzeit (h)

Ko

nze

ntr

atio

n d

es

Trä

ger

s im

Blu

tpla

sma

(g/l)

Werte 5Werte 6

½½

Concentration in blood plasma (g/dL)

Time (h)

In-vivo-Results (rat): Lifetime of the additive

• 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.


The carrier keeps up the oxygen supply of the rat in small amount.It is more effective than blood.

by red blood cells

by the carrier

Overall

by solved oxygen

20

10

Hämatocrit in %

Spec. Oxygen uptake(mlSTPD · min-1 · kg-1)

In-vivo-Results (rat): Functinality of the carrier

DilutionExchange


The additive saved the life of the rats in lung shock.

+ Oleic acid + Oleic acid + Additive

0

2

4

6

8

All 5 rats without additive died after about 3 hours.All 5 rats with additive must be sacrified after about 8 hours.

1 2 3 4 5 6 7 8 9 10

SurvivalTime (h)

In-vivo-Results (rat): Efficacy of the additive in lung shock

Lung shock was induced by oleic acid before application of the additive.


without carrier

with carrier

arterial venous

L(ung)

L

L L

L

Cells

The mediator effect increases the geometric factor (F/d).

The diffusion area (Fef) increases and the diffusion distance (def) decreases.

1. Fick‘s Equation

mO2 = DO2,ef • O2,ef (PO2,ef,K – PO2,ef,G)

Fef

def

•

K

Why is the additive so effective?

Mediator-EffectBridging-Effect


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

Residence time (s)

Hb content 100 g/l

Red blood cells

Carrier

Mixture 1:1

The 1:1 mixture has the same oxygen releasing than pure carrier,behaving like the pure carrier.


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.

In sensitised mice the hyperpolymer has a small

immunogenic effect.


In-vivo-Results (mice): Induction of immuntolerance

Sensitised mice got completely tolerant

against hemoglobin and hyperpolymer

by oral application of hemoglobin or

by feeding pork meat.


Proband: ♂ , 70 kg Body WeightProband: ♂ , 70 kg Body Weight Proband: ♀ , 65 kg Body WeightProband: ♀ , 65 kg Body Weight

0

50

100

150

0 20 40 60 80 100 120 140Versuchszeit (min)

BD

(T

orr

) / H

F (

min

-1)

0

50

100

150


BD

(T

orr

) / H

F (

min

-1)

0

50

100

150


BD

(T

orr

) /

HF

(m

in-1

)0

50

100

150


BD

(T

orr

) /

HF

(m

in-1

)

syst. Blood Pressure

diast. Blood Pressure

Heart Rate

syst. BP

diast. BP

HR

Time (min)

BP(Torr)

HR(min-1)

BP(Torr)

HR(min-1)

Time (min)

Admin. 1,16 g Admin. 1,16 g

In-vivo-Results (humans): Blood pressure effect?

In rats hemoglobin causes an increase of systolic blood pressure by extravasation.

In humans the additive does not cause a blood pressure effect.

Low dose application of the additive on humans:


The additive does not change the transaminse levels significantly.

Proband W.B. Proband D.K.

0

4

8

12

161: 0,58 g 2: 2,32 g

1 2 1 2 1 2 3 1 2 3

GlutamatPyruvatTransaminase(norm < 22 U/l)

GlutamatOxalatTransaminase(norm < 18 U/l)

before

1, 2, 3: 1,16 g

after 24 h before after 24 h

In-vivo-Results (humans): Liver damage by the additive?

Transaminaseblood plasmalevel (U/l)

Measuring transaminase levels:

Administration


In-vivo-Results (humans): Immunogenicity

The additive was administered intravenously

in two persons three times with 14 days intervals

at dosages between 0,6 and 2,4g hyperpolymer.

No signs of adverse effects were seen

and especially no sign of immunogenicity.


Advantages of the additive

• 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


Short chronic of the project

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)


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Tamaulipas 10-20071 Hemoglobin-Hyperpolymers A new type of an artificial oxygen carrier