WO2004047807A1

WO2004047807A1 - Artificial blood on the basis of encapsulted haemoglobin

Info

Publication number: WO2004047807A1
Application number: PCT/HU2003/000008
Authority: WO
Inventors: István HORVÁTH
Original assignee: Horvath Istvan
Priority date: 2002-11-26
Filing date: 2003-01-31
Publication date: 2004-06-10
Also published as: HU0204065D0; EP1567129A1; HU226519B1; AU2003202717A1; HUP0204065A2

Abstract

The invention relates to a process for producing artificial blood, with haemoglobine encapsuled in a membrane as well as artificial blood produced this way, which is characterized by that, it involves haemoglobin encapsuled in pure cholesterol shell in the form of particules, which can be suspended in watery substance or can be made suitable for supplying blood for all vertebrata.

Description

ARTIFICIAL BLOOD ON THE BASIS OF ENCAPSULATED HAEMOGLOBIN

The invention relates to a process for producing artificial blood, with haemoglobine encapsuled in a membrane as well as artificial blood produced this way, which does not decompose in the organism, - but which is regarded as 'self by the immune system of the organism- from haemoglobine encapsuled in a membrane. During the process the artificial blood is produced with using haemoglobin encapsuled in a membrane, produced from a cholesterol of high purity degree, which can be integrated in the organism of all vertebrata, but which never decomposes in the organism. The artificial blood produced this way is suitable for supplying blood temporarily or for long term for any mammalia - human beings and animals - of any blood group or race drained of blood.

Demand for supplying blood had long arisen in medical science and in emergency medical attendance. The first results were achieved by restoring level of fluid content of blood in cases when considerable proportion of fluid content of blood was lost due to any physiological or pathological effects. In such cases intake of fluid ensured restoring the patient from severe cases and created favorable conditions for healing. In case of young children or elderly people it is very important to take this aspect into consideration, because loss of fluid could be fatal for the endangered organism. Often giving water can no longer help. In such cases patients should be supplied with water infusion injected directly to circulation. Prevention of drying out could be life saving in case of both young children and adults belonging to any age group suffering from infection caused by Salmonella sp., Vibrio cholera, certain viruses etc.

During loss of blood three essential basic materials leave the organism:

- Water, the fluid component of blood together with electrolytes solved in water.

- Proteins together with enzymes and antibodies.

- Cell elements, among them red blood cells accounting for supplying gases.

Possibility of blood transfusion has been made use of in almost every medical treatment as well as on battlefields since Landsteiner elaborated it in the knowledge of blood groups. Today, when pharmaceutical industry producing, and doctors using blood preparations must pay particular attention to the prevention of today still incurable virus-infections, applying of blood preparations or blood- supplementing materials offering provisional solutions are of particular significance.

In certain publications the following future tasks of producing artificial blood were designated:

- Haemoglobin should be encapsuled in a membrane, which is capable of resisting permanently enzymes in the blood.

- At the same time it should be a material acceptable for every organism, it should be regarded as 'self by the organism. In the state of art earlier processes are known, during which artificial membranes were produced trying to imitate chemical constituents of membranes in the organism. 19-22 % cholesterol could be found in the artificial membranes involving haemoglobin. Membranes built up mainly by using mixed lipoids, mainly phospholipids could not resist decomposing effect of enzymes. Combinations of lipoids promoted forming of artificial membranes, whereas membranes of pure cholesterol were difficult to form.

Artificial blood offers a possibility of solving the problem of one of the most significant functions of blood, the supply of carbon dioxide and oxygen gases and supply of fluid arising due to loss of blood. Artificial blood helps the patient suffered considerable loss of blood through life danger arising due to loss of blood. The preparation due to its nature and conditions of production contains beyond doubt no infectious organisms mentioned above. With avoiding possibility of infection the patient is definitely not exposed to an additional effect endangering life.

In the state of art the importance and significance of producing artificial blood have been recognized by many and several solutions were found to produce artificial blood with haemoglobin encapsuled in a liposome membrane (Djordjevich et al. PCT/US82/00899), and it can be clearly seen from works summerizing information in connection with artificial blood (Andreas M. Spiring et al: Drug of the future 1993. 18. (3) 249-253) that in forming the membrane of liposome several materials, first of all phospholipid are used.

The solutions available suffer from the limitation, that the membrane of the 'cells' was built of materials, which could be decomposed by enzymes and several of its lipoids might have an allergic effect.

When elaborating the solution according to the invention we aimed to work out a process as well as a blood supplementing suspension, in which haemoglobin is encapsuled in a membrane, which can not be decomposed by enzymes in the blood.

We realized, when we worked out the solution according to the invention, that a membrane made. from pure cholesterol meets these requirements fully, in case we use a single material for producing the membrane contrary to the processes in the state of art, and this material is cholesterol. So far cholesterol has not been applied for producing membranes in itself in pure state. In water solution, e.g. in a haemoglobin solution cholesterol crystallizes and is not able to produce a membrane which prevents it from being used for membrane production.

The invention is a process for producing artificial blood, which does not decompose in the organism, - but which is regarded as 'self by the immune system of the organism- from haemoglobine encapsuled in a membrane, which is suitable or can be made suitable for supplying blood for all vertebrata, which is characterized by that, during the process haemoglobin is encapsuled in a pure cholesterol shell and particles are created, which can be suspended in a watery substance in a way, that haemoglobin is put between two monomolecular lipoid films of properly arranged structure made from pure cholesterol and particles are formed by splitting the plan-parallel layers in a way that haemoglobin remain suitable for supplying oxygen and carbon dioxide.

The invention is furthermore a process for producing artificial blood, which does not decompose in the organism, - but which is regarded as 'self by the immune system of the organism- from haemoglobine encapsuled in a membrane, which is suitable or can be made suitable for supplying blood for all vertebrata, which is characterized by that, the haemoglobin is put on a monomolecular film and the film is split in a way, that the pieces of the film create a closed surface and encapsule the haemoglobin on their surface, so the haemoglobin does not decompose during circulation.

The invention further is artificial blood produced preferably by processes according to the invention, which is characterized by that, it involves haemoglobin encapsuled in pure cholesterol shell in the form of particles, which can be suspended in watery substance, which is suitable or can be made suitable for supplying blood for all vertebrata.

In one of the preferred realizations of the artificial blood according to the invention, the size of the particles remains below the size of the red-blood cells or red-blood corpuscles characteristic of the given species and said particles can permeate through the tubes of the capillaries owing to their size.

In another preferred realization of the artificial blood according to the invention, the haemoglobin used can derive directly from a haemoglobin solution produced from blood or can be haemoglobin dried by lyophilization.

In a further preferred realization of the artificial blood according to the invention, from the point of view of application of artificial blood the haemoglobin may be from the same species or deriving from other vertebrata.

In another preferred realization of the artificial blood according to the invention, during forming of the particles the material of the lipoid membrane encapsuling the haemoglobin is pure cholesterol.

In another preferred realization of the artificial blood according to the invention, forming of lipoid film from pure cholesterol takes place at an optimum temperature from an optimum solution. In another preferred realization of the artificial blood according to the invention, forming of cholesterol membrane can take place on solid, fluid surface or in g oa' seous substance.

In another preferred realization of the artificial blood according to the invention, during forming of particles splitting of the membrane into particle size takes place under the influence of variable physical energy.

The solution according to the invention is set forth by the following examples:

Example 1

Production of artificial blood

A solution of pure cholesterol is produced at a proper temperature, which is suitable for making a thin monomolecular layer on a smooth surface. The surface of the body, the temperature, the flowing speed of the solution should be optimum. The smooth surface can include dents of maximum 400 nm. The temperature can differ from the melting temperature by maximum 10% (obviously to the higher range). The flowing speed of cholesterol solution can be 1 mg/sec/50cm2 +-10% on a surface of a proper angle. The physical impacts consistent with the viscosity can range between electromagnetic waves (270 nm) of advantageously applicable frequency and energy through sound-waves of proper wavelength and energy to inaudible frequencies.

Determining the suitable frequency and wavelength takes place with choosing the values with adjustable equipment complying best with the temperature and angular offset. We bring haemoglobin molecules in connection with the cholesterol solution under ideal conditions for producing liposome. The physical force arising at the moment of encounter splits the cholesterol layer in the given size and it encapsules the haemoglobin. Liposomes produced this way are put into a liquid of around pH 7 value and are separated from haemoglobin molecules soluble or remained outside the lipoid shell. The haemoglobin solution is regained and the liposomes containing haemoglobin are collected for producing artificial blood.

Example 2

Fields of application of the artificial blood according to the invention: a./ It ensures oxygen supply of the organism during a severe operation, while genuine blood can be preserved for intake after the operation. b./ In case of an accident the ambulance man providing first aid can ensure oxygen supply of the organism drained of blood irrespective of blood group until the injured can get fully efficient medical treatment. z.l Under warring conditions it is suitable for supplying the injured soldier drained of blood with blood until he is taken to hospital. Life of the soldier can be saved by applying artificial blood. Canned artificial blood in lyophilized form may be the part of personal equipment. Doctors on the battlefield should only take along physiological solvent, mix it with the powdered blood and it can be immediately injectioned. d./ In case of special therapy it is suitable for replacing blood during a relatively short physical therapy, (chemotherapy, radiotherapy) e/ parasites of red blood cells (malaria, Babezia etc.) can not attack artificial blood produced according to the invention with haemoglobin encapsuled in a liposome

(HEL). f / gas supply can be increased in an organism demanding high oxygen consumption.

Example 3

Physical principle of production of artificial blood according to the invention:

Haemoglobin must be encapsuled into a semi-permeable lipoid film to retain its capability for supplying oxygen and carbon dioxide. During the process the haemoglobm is put between two monomolecular lipoid films of properly arranged structure made from pure cholesterol and liposomes are formed by splitting the plan-parallel layers The other possibility is to put the haemoglobin on a monomolecular film and the film is split in a way, that the pieces of the film roll up the haemoglobin on their surface, similarly as cigarette paper covers tobacco. The only condition so far to meet these requirements is the novelty of our invention, using membrane made from pure cholesterol. By packaging into the film it can be achieved, that the haemoglobin should not be dissolved during circulation, because dissolved haemoglobin is secreted in the kidneys.

The main characteristics of the artificial blood made by the process according to the invention are as follows:

- Haemoglobin of any animal species can be used

- Artificial blood can be applied in any animal species

- Pure cholesterol does not decompose in the organism, therefore circulation is ensured for a long term.

Cholesterol shell does not act as allergen

- Particles can be well lyophilized

- Blood parasites endangering red blood cells can not attack the artificial blood.

Immunologicai conditions of producing artificial blood :

Overriding immunologicai principle for the use of artificial blood is, that haemoglobin should be encapsuled in a liposome in such a way, that artificial blood should not have allergic influence. This requirement can be fulfilled so, that cholesterol existing in the organism is used in a form of high purity for forming the membrane substituting the cell membrane of the 'cells' of the artificial blood.

Difficulties entitled with the task: The complex containing haemoglobin may assume allergen characteristics in case the artificial 'cell membrane' acting as antigen is capable of stimulating anti-bodies damaging the organism in some way. This possibility must be taken into consideration even if cholesterol does not usually appear as allergen or appears as allergen to a small extent only. Nowadays it is not infrequent that materials earlier not considered allergen by allergologists in fact act as allergens. Anti cholesterol has been known so far as anti atherosclerotic, therefore its effect is accepted as favorable. Pure cholesterol was highlighted due to the fact that there is the least chance of harming the organism.

Possibility of overcoming side-effects

Our observations so far support our earlier working hypothesis, that a liposome can be produced with applying material with the least immunologicai power which can match the features of those of the natural cells or corpuscles produced in the organism. The raw material of the membrane liposome of the artificial blood produced by us is compiled exclusively of pure cholesterol that can be found in every genus of vertebrata. It has no extraneous element. No allergic side effect in animal tests could be noticed. On basis of our achievements so far it seems realistic to achieve the set aim, i.e. our preparation will prove to be suitable for filling the function of blood as supplier of oxygen and carbon dioxide in all organisms drained of blood irrespective of blood group.

As mentioned in the introduction, it is first of all suitable for meeting the demands of casualties and operations lasting for a relatively short time, but in a certain circle it ensures solution for medical treatment as well. On basis of the results of the animal tests carried out so far and keeping in mind the theoretical considerations already mentioned we find it suitable for use for human beings as well.

The advantage of the process according to the invention is, that the cholesterol membrane is not decomposed by enzymes of the organism. As it is regarded as 'self by the immune system of the organism, it is not toxic in the organism of vertebrata. The process according to the invention makes possible production of a Liposoma Encapsulated Haemoglobin (LEH) which can live long due to the fact, that there is no enzyme in the organism that could decompose it. The LEH particles of 200-500 nm size can easily get to every area of circulation. Cholesterol of high purity is suitable for encapsulating haemoglobin from any mammal organism. Due to this fact siim-le and inexpensive raw material can be ensured.

The membrane formed from pure cholesterol solved a task, which was the objective of the future only. Haemoglobin does not dissolve from the particles even in case of longer storing under proper conditions. The artificial blood produced this way can be stored in fluid form, in liophylized state, as powder and this powder suspended suitably provides artificial blood of full value. The pieces of the cholesterol film produced by one of the processes of the invention create a closed surface, encapsule the haemoglobin on their surface and cover it in a similar way to a cigarette paper covering tobacco. By packaging into the film it can be achieved, that the haemoglobin should not be dissolved during circulation, because dissolved haemoglobin is secreted in the kidneys.

LEH produced by a simple, inexpensive process can help an injured person through the danger of biological suffocation resulting from lack of blood. At the same time it restores level of fluid loss in the organism, recovering osmotic value of cells and enables haematogen organs to restore genuine composition of blood in the organism under conditions of physiological blood pressure and circulation as soon as possible. In case loss of blood is such an extent, that other constituents of blood (proteins, enzymes etc.) should be supplied without delay beside electrolytes, then there is the possibility of supplying genuine blood after artificial blood, which can be ensured under hospital conditions.

Solving the problem of artificial blood meets the requirements of an ancient demand, which seemed unreal many times, and which is one of the most significant achievements in sorting out problems arising due to loss of blood since the application of blood transfusion elaborated by Landsteiner armed with the knowledge of blood groups. The mutual possibility of application of haemoglobins of different species can be solved by applying artificial blood.

With the help of the process elaborated by us 'blood corpuscles" are given to the users, which creates possibility of making use of foreign species beside using it within the species. The possibility of using artificial blood mutually is given by the cholesterol, which can be found in every species with identical chemical constituents. As cholesterol acts in every mammal as 'self, it can not be decomposed in any species, haemoglobin encapsuled in cholesterol membrane can be used in every species providing artificial blood for the needy.

Further research can also benefit from this invention as well as users. Making use of corpuscles produced with using haemoglobm strange for species the needy can get access to artificial blood, which can be made in unlimited quantity. This can be done in a way, that presence and transfer of microorganisms endangering human organism (HIN, Ebola etc.) can be excluded with great safety.

Cholesterol did not show any variability during the phylogenesis. This phenomena has double benefits. The artificial blood produced is suitable not only for replacing the blood of only one species, namely that of human beings, but the same artificial blood can provide blood supply for almost all warm-blooded animals. The advantage of the theoretical and practical solution is, that any favorable effect recognized on an animal species can be transferred to a different species even without any modification. The human being, the Homo sapiens has obviously the greatest benefits from this, as the results of animal tests can be regarded identical value with observations on human beings.

Claims

Process for producing artificial blood, which does not decompose in the organism, - but which is regarded as 'self by the immune system of the organism- from haemoglobine encapsuled in a membrane, which is suitable or can be made suitable for supplying blood for all vertebrata, characterized by that, during the process haemoglobin is encapsuled in a pure cholesterol shell and particles are created, which can be suspended in a watery substance in a way, that haemoglobin is put between two monomolecular lipoid films of properly arranged structure made from pure cholesterol and particles are formed by splitting the plan-parallel layers in a way that haemoglobin remain suitable for supplying oxygen and carbon dioxide.

2. Process for producing artificial blood, which does not decompose in the organism, - but which is regarded as 'self by the immune system of the organism- from haemoglobine encapsuled in a membrane, which is suitable or can be made suitable for supplying blood for all vertebrata, characterized by that, the haemoglobin is put on a monomolecular film and the film is split in a way, that the pieces of the film create a closed surface and encapsule the haemoglobin on their surface, so the haemoglobin does not decompose during circulation.

3. Artificial blood produced preferably by processes according to claim 1 or 2, characterized by that, it involves haemoglobin encapsuled in pure cholesterol shell in the form of particles, which can be suspended in watery substance, which is suitable or can be made suitable for supplying blood for all vertebrata.

4. Artificial blood according to claim 3 characterized by that, the size of the particles remains below the size of the red-blood cells or red-blood corpuscles characteristic of the given species and said particles can permeate through the tubes of the capillaries owing to their size.

5. Artificial blood according to any of claims 1 to 4 characterized by that, the haemoglobin used can derive directly from a haemoglobin solution produced from blood or can be haemoglobin dried by lyophilization.

6. Artificial blood according to any of claims 1 to 5 characterized by that from the point of view of application of artificial blood the haemoglobin may be from the same species or deriving from other vertebrata.

7. A-rtificial blood according to any of claims 1 to 6 characterized by that during forming of the particles the material of the lipoid membrane encapsuling the haemoglobin is pure cholesterol.

8. Artificial blood according to claim 7 characterized by that, forming of lipoid film from pure cholesterol takes place at an optimum temperature from an optimum solution.

9. Artificial blood according to claim 6 characterized by that, forming of cholesterol membrane can take place on solid, fluid surface or in gaseous substance.

10. Artificial blood according to any of claims 3 to 9 characterized by that, during forming of particles splitting of the membrane into particle size takes place under the influence of variable physical energy.