WO2003094953A1

WO2003094953A1 - Oxygen carrier, selected from haemoglobin, myoglobin and their derivatives, for treating organ dysfunction/tissue damage caused by an acute supply deficiency

Info

Publication number: WO2003094953A1
Application number: PCT/EP2003/003911
Authority: WO
Inventors: Wolfgang Barnikol; Harald Pötzschke
Original assignee: Sanguibiotech Gmbh
Priority date: 2002-05-11
Filing date: 2003-04-15
Publication date: 2003-11-20
Also published as: AU2003226812A1; DE10220992A1

Abstract

The invention relates to the use of one or more natural or modified oxygen carriers or their derivatives, for producing an agent for treating organ dysfunction caused by an acute supply deficiency and for treating/preventing tissue damage as a result of a dysfunction of this type. The inventive agent allows conditions caused by an acute oxygen and/or nutrient deficiency, such as tinnitus, cardiac infarction, stroke, sudden deafness, vertigo, placental insufficiency, renal shock or pulmonary shock to be treated. The oxygen carrier(s) can be of human or animal origin and can be used in the form of aqueous solutions containing, for example, the electrolyte concentrations that occur naturally or additional salts/additives. The oxygen carrier is used in solution in a concentration of between 2 and 200 g/litre, preferably between 10 and 80 g/litre over a period of between 1 day (e.g. single dose) and 6 weeks with multiple doses, according to requirements and prescription.

Description

OXYGEN CARRIER, SELECTED FROM HEMOGLOBIN, MYOGLOBIN AND DERIVATIVES THEREOF, FOR TREATING AN ORGAN FUNCTIONAL FAILURE / DAMAGE FROM ACUTE DEFAULT

description

Subject of the invention

The present invention relates to the use of one or more natural or modified oxygen carriers or derivatives thereof, for the preparation of an agent, for the treatment of an organ dysfunction due to an acute lack of supply and for the treatment / prevention of tissue damage as a result of such a disorder. In particular, acute oxygen and / or nutrient deficiency states such as tinnitus, heart attack, stroke, sudden hearing loss, dizziness, placenta insufficiency, kidney shock or lung shock can be treated according to the invention. The oxygen carrier (s) can be of human or animal origin and are used as aqueous solutions which, for example, have the naturally present electrolyte concentration or also further salts / additives. The oxygen carrier is used in a concentration of 2 to 200 g / liter solution, preferably 10 to 80 g / liter over a period of 1 day (e.g. single dose) up to 6 weeks with multiple doses depending on the need and indication.

Background of the Invention

Oxygen carriers and artificial oxygen carriers produced by modification of natural oxygen carriers such as hemoglobin or myoglobin for supplying oxygen to a living organism have been known for a long time, cf. DE 197 01 37, EP 97 100790, DE 44 18 937, DE 38 41 105, DE 37 14 351, DE 35 76 651. The hemoglobins or myoglobins are obtained in a known manner and can are crosslinked, resulting in crosslinked (intramolecular), polymeric and in particular hyperpolymeric products. In addition, the natural oxygen carriers, which can optionally also be crosslinked beforehand, can also be covalently linked to polyalkylene oxides, cf. Harris JM, Poly (Ethylene Glycol) Chemistry: Biotechnical and Biorrredfeal Applications, Plenum, New York, 1992).

PCT / US97 / 05088 (WO 97/35883) describes a process for producing an oxygen carrier substitute which is reacted and polymerized with pyridoxal phosphate. It is used as a substitute in particular for surgical interventions, i.e. blood loss.

DE-A1 100 31 740 (WO 02/00230), DE-A1 100 31 744 and DE-A1 100 31 742.1 describe modified oxygen carriers or special processes for their preparation which are crosslinked, polymerized and with polyalkylene oxides are implemented. The carriers thus produced are considered suitable, particularly for intravascular or biomedical use, e.g. as a replacement for the blood, described as an addition to this, since such oxygen carriers have, among other things, particularly good plasma tolerance. Furthermore, an application in the case of a chronic oxygen deficiency condition is also described here in general, but without specifying the type / location and amount or duration of such an application.

However, it is known that hemoglobin and, in this respect, artificial oxygen carriers are sensitive to oxidation reactions, resulting in the ineffective methemoglobin, which no longer allows oxygen transport, t. For example, EP-A 0 857 733 describes that artificial oxygen carriers are used to supply living systems particularly when the oxygen binding sites have been previously provided with a protective ligand such as carbon monoxide. This ligand is not removed before use, but while it is working. This is to ensure that the function of the oxygen carrier is released gradually, i.e. gradually, depending on the respective requirements, and that an undesired temporary oversupply of oxygen is avoided when used on a patient (see column 5, paragraph 2 in EP-A 857 733). The use of the protective ligament Kohteπrrrσπσxrd has the advantage that oxidation of the carrier can be prevented, but, as mentioned, a rapid or possibly desired temporal oversupply with oxygen is not possible, since carbon monoxide is very firmly ligated to the oxygen binding sites and is released slowly.

Acute shortages of an organ and consequent malfunctions with the result of a possible tissue damage can have different causes. These include e.g. B. nutrient deficiency situations, or acute lack of oxygen e.g. due to stress, acute vasoconstriction or also due to chronic deficiency such as chronic vasoconstriction. It is known that nutrient therapy can be used for tinnitus diseases or Meniere's syndrome. Here, hyperlipoproteinemia is specifically used to improve the ear diseases mentioned.

Reactive hypoglycemia can also be the cause of acute dysfunction such as that of the ear (tinnitus). Magnesium deficiency also applies e.g. as a factor in tinnitus development. In addition, electrolyte disorders are considered to be the cause of tinnitus.

Therefore, such shortages of supply have so far been e.g. by administration of solutions containing the above Nutrients, but especially insulin, are treated in appropriate doses.

Another method of treating the above-mentioned functional disorders is the direct respiratory administration of pure oxygen (gas), namely as hyperbaric oxygen therapy (HBO), or also by using electrical stimuli.

These methods are intended to quickly remedy such shortcomings in the supply, but damage to the tissue that is undersupplied is often unavoidable. In addition, when using pure oxygen, care must be taken to ensure that - although initially a large amount of this, i.e. a temporal oversupply may be necessary - oxidative toxification via over-concentrated (over-strained) oxygen must be avoided. Object of the invention

It is therefore an object of the present invention to find a means by which an organ dysfunction due to an acute lack of supply, in particular an oxygen supply crisis, can be treated in such a way that on the one hand the problem is effectively remedied and on the other hand permanent damage to the tissue as a result of the disorder and toxification can be treated or avoided.

Explanation of the Invention This object is achieved according to the invention by providing and using an agent for the organism which is dysfunctional due to an acute lack of supply and which has one or more oxygen carriers which are used in a concentration of 2 to 200 g / liter of medium, i.e. 0.2 to 20 wt.% Are included. The oxygen carrier or carriers are selected from hemoglobin, myoglobin or derivatives thereof. Accordingly, they are natural or preferably crosslinked, polymerized and / or pegylated, i.e. covalently linked with a polyalkylene oxide. Particularly preferred oxygen carriers are those which are both crosslinked, polymerized (intermolecularly crosslinked) and in particular also pegylated. The oxygen carriers can be of human or animal origin.

In the latter case, a reactive and / or non-reactive effector can in particular have been used in the production as described below. If appropriate, the oxygen carrier, in particular if it is both crosslinked, polymerized and pegylated, it being possible for effectors during production or chemical effectors to be linked, may also be carbonylated.

According to the invention, it was found that by means of the oxygen carriers mentioned, immediate treatment of the organ in a deficient state with sufficient amounts of de-energized oxygen, namely bioavailable oxygen, is possible, whereby toxification and also tissue damage can be avoided or treated. This is ensured in particular by the reversible binding to the carrier. The oxygen carrier is in particular as an infusion over a required period of z. B. 1 day to several days one or more times, possibly up to several weeks, until the acute deficiency has been remedied insofar as the organ concerned is again working properly. Administration may also continue thereafter, depending on the condition and conditions of the situation, to achieve a final cure. The oxygen carrier (s) can be administered in the amounts indicated as an infusion solution, preferably together with the additives described below. In particular, according to the invention, an acute lack of supply, above all due to a lack of oxygen (oxygen supply crisis), is treated by acute or chronic vasoconstriction, stress, spasm or arteriosclerosis. A lack of nutrients or combinations thereof with an oxygen deficiency can also be treated. The acute crises mentioned as a result of the oxygen deficiency states mentioned are dealt with in particular. A lack of nutrients can result from an undersupply of physiologically essential electrolytes and / or glucose or combinations thereof, the hormone insulin also playing a decisive role. Such a mode of action was not to be expected, since the state of the art indicated that artificial oxygen carriers in particular can be used in general in the case of chronic oxygen deficiency conditions, although other conditions exist than in the case of an acute supply deficiency state, such as, for example, with regard to vegetative regulation and the present concentration profiles of essential substances. It was therefore surprising that the amounts of oxygen carriers as described in accordance with the invention actually result in a controlled remedy of the deficiency situation, since on the one hand such deficiency states were assigned completely different mechanisms of remedial action and on the other hand a quick functional organ revitalization despite the initially high amount of oxygen present is achieved, especially since the determined range of the oxygen partial pressure then present is much smaller than when pure oxygen is introduced. This means that a physiological supply of bioavailable oxygen (at a low partial pressure) is possible without the slightest damaging effect. The production of such oxygen carriers of human or animal origin is known. Cell lysis takes place without freezing, so that the product is free of cell walls and plasma and free of stroma. After known cleaning, which is also explained in the abovementioned documents, the oxygen carrier can be used directly, for example in physiological sodium chloride solution or in other aqueous solutions as described below. The oxygen carrier is preferably crosslinked with a crosslinker, polymerized.

The oxygen carrier, which can be hemoglobin or myoglobin or mixtures thereof, preferably hemoglobin or also hemoglobin-myoglobin mixtures, can also be covalently linked to a polyalkylene oxide which is selected from polyethylene oxide, polypropylene oxide, or a copolymer of ethylene oxide and propylene oxide or a Esters, ethers or ester amides thereof. Oxygen carriers which are linked to a polyethylene oxide or a suitable derivative thereof are particularly preferably used.

It is further preferred if the covalently attached polyalkylene oxide has a molar mass of 200 to 5000 g / mol.

The carrier or carriers are very particularly preferably crosslinked, polymerized and covalently linked (pegylated) with a polyalkylene oxide, as described in DE-A1 100 31 740 (WO 02/00230), DE-A1 100 31 744 and DE-A1 100 31 742.1.

The oxygen carriers, especially the preferred ones, can optionally also be carbonylated. Particularly suitable oxygen carriers are hemoglobins or hemoglobins modified as described above with a molecular weight of 65,000 to, in particular from 70,000 to 15,000,000 g / mol, in particular 90,000 to 15,000,000, those with a molecular weight of 300,000 to 15,000 000, especially 300,000 to 10,000,000, in particular from 700,000 to 10,000,000, preferably 700,000 to 5,000,000 g / mol, are particularly preferred, or also myoglobins or modified derivatives thereof with a molecular weight of 15,000 g / Mol to 5,000,000 g / mol, preferably 100,000 to 3,000,000 or also 200,000 to 3,000,000 or mixtures of hemoglobin and myoglobin oxygen carriers as indicated. In the case of mixtures, the ratio of hemoglobin to myoglobin oxygen carrier or derivatives thereof can be from 20: 1 to 1:20, in particular 10: 1 to 1:10.

Likewise, the ratio of natural to modified oxygen carrier can be 20.1 to 1:20, especially 10.1 to 1.10.

The oxygen carrier (s) used according to the invention are effective when a concentration of 0.2 to 20% by weight, in particular 1 to 18% by weight, especially 3 to 15, preferably 3 to 12% by weight, in particular 5 to 10% by weight is present in the medium intended for the treatment. Accordingly, 2 to 200 g / liter or 10 to 180 g or 30 to 150 or 120 g or 50 to 100 g / liter of oxygen carriers are present in the medium. The medium is especially water.

If necessary, the oxygen carrier (s) can be saturated externally with oxygen shortly before use in a manner known per se via suitable exchangers and supplied to the cells. Various oxygen carriers of the type mentioned can also be added as a mixture, as described above. For example, those with an average molecular weight of 70,000 to, particularly 90,000 to 10,000,000, particularly 1,000,000, g / mol of porcine hemoglobin with an oxygen carrier made from myoglobin and having a molecular weight of 15,000 to 5,000,000, especially 100,000 up to 1,000,000 g / mol can be used together.

The oxygen carriers, in particular the artificial derivatives, of the type described can be produced as described in the abovementioned prior art, which is incorporated here. Particularly preferred are those which are produced as described in DE-A 100 31 740 (WO 02/00230), DE-A 100 31 742 and DE-A 100 31 744. For this purpose, moderately high molecular weight polyalkylene oxides are covalently bound to the hemoglobin or myoglobin molecules crosslinked with a crosslinker. Details of the process for the production of such artificial oxygen carriers are described above (e.g. in DE-A100 31 740), incorporated therein as indicated and can be summarized as follows:

Suitable as hemoglobin (or myoglobin) starting material for the production of the oxygen carriers used according to the invention is monomeric, native or with certain effectors, e.g. B. the oxygen affinity of hemoglobin (myoglobin) such as pyridoxal-5'-phosphate or 2-nor-2-formyl-pyridoxal-5'-phosphate, chemically converted and modified myoglobin or hemoglobin from humans, pigs or cattle. Human and in particular swine hemoglobin is preferred. The hemoglobin or myoglobin may optionally be deoxygenated by carbonylation before use.

The crosslinking of monomeric, native or effector-linked hemoglobin or myoglobin with a number of crosslinkers is known and has been described many times in the literature, cf. the above German applications. Examples include: divinyl sulfone, epichlorohydrin, butadiene diepoxide, hexamethylene diisocyanate, the dialdehydes glyoxal and glutardialdehyde and the diimido esters dimethyl suberimidate, dimethylmalonimidate and dimethyl adipimidate. Furthermore, reactions with dialdehydes, for example malondialdehyde, succindialdehyde, glutardialdehyde, adipindialdehyde and suberdialdehyde, and glyoxal, but also with structurally more complex compounds, which occur through oxidative ring opening of the cyclic half-acetal and half-ketal structures of the sugar molecules in monosaccharides and oligosaccharides and oligosaccharides and oligosaccharides, are also known which are produced by reaction with the dialdehydes o-adenosine and o-ATP, formed by ring-opening oxidation of the ribose in adenosine and in adenosine triphosphate. Different molecular weights can be obtained, cf. EP 0 201 618. In each case, based on monomeric hemoglobin / myoglobin, molar ratios of the crosslinking agent used - in particular the bifunctional crosslinking agent - of 3 to 60 times, preferably 6 to 35 times, are used. With regard to glutaraldehyde, a 7- to 10-fold molar excess of the glutaraldehyde is preferably used. Chemically unstable linkages, especially the Schiff bases, which arise in the reaction of functional aldehyde groups with amino groups of the hemoglobins, are reductively known in a known manner by reaction with suitable reducing agents, such as. As sodium borohydride, in a sufficient molar excess, based in each case on monomeric hemoglobin, preferably 2 to 100 times, particularly preferably 5 to 20 times, stabilized under suitable known conditions. The methods mentioned are known and incorporated herein. Bifunctional crosslinking agents are preferably selected for crosslinking the hemoglobins / myoglobins, for example butanedie epoxide, divinyl sulfone, a diisocyanate, in particular hexamethylene diisocyanate, cyclohexyl diisocyanate and 2,5-bisisocyanatobenzenesulfonic acid, a di-N-hydroxysuccinimidyl ester, a dialido ester, or a diimido ester, or a diimido ester, or a dialido ester, or a dialido ester, or a dialido ester, or a diimido ester or a the analog reacting glycol aldehyde, or glutardialdehyde. Glutardialdehyde is particularly preferred.

The reaction with the polyalkylene oxide, which in itself, but in particular additionally, ie before or after or ^' during the crosslinking, is likewise described in the above-mentioned German applications and incorporated therein. It is essentially reacted with a polyalkylene oxide or a derivative thereof, such as polyethylene oxide, polypropylene oxide, or copolymers of ethylene oxide and propylene oxide. The polyalkylene oxide derivative is particularly preferably an ether, an ester, or an ester amide with a short-chain aliphatic organic radical.

The covalently attached polyalkylene oxide preferably has a molar mass between 200 and 5000 g / mol, preferably between 500 and 2000 g / mol. For the covalent attachment of the polyalkylene oxides, preference is given to using those derivatives of the polyalkylene oxides which already contain a linking A ns - nιiL_ejneι ^ functional group and which have a direct chemical reaction with amino, alcohol or sulfhydryl groups of the hemoglobins to form covalent attachments of the polyalkylene oxides - for example polyalkylene oxides with reactive N-hydroxysuccinimidyl ester, epoxy (glycidyl ether), aldehyde, isocyanate, vinyl sulfone, iodoacetamide, imidazolyl format, tresylate groups, and the like. a. Many such monofunctionally activated polyethylene glycols are commercially available.

It is further preferred if the number of attached polyalkylene oxides in the product used according to the invention is between 1 and 40, in particular 4 to 15, molecules of polyalkylene oxide per molecule of the hemoglobin monomer. As described, the polyalkylene oxide can first be covalently linked and only then can the crosslinking be as described. Finally, a covalent attachment of a polyalkylene oxide can also be done both before the Networking, as well as after the networking. With these alternatives, processing and further processing can also be carried out unchanged as described.

The conditions for the attachment of the polyalkylene oxide are set out in detail in the abovementioned German applications and are therefore incorporated here.

In the production of the oxygen-transporting agents used according to the invention, chemically non-reacting effectors of the oxygen affinity of the hemoglobin / myoglobin can also be added to the reaction solution thereof before the covalent crosslinking of the hemoglobin. 2,3-bisphosphoglycerate, inositol hexaphosphate, inositol hexasulfate or mellitic acid are particularly suitable as effectors of the oxygen affinity of hemoglobin / myoglobin, 2,3-bisphosphoglycerate being particularly suitable. As mentioned, the conditions for the use of such non-reactive effectors are described in DE-A 100 31 742.

Oxygen carriers which have been produced (cf. DE 100 31 744/0) are particularly preferably used according to the invention in that highly purified hemoglobin or also myoglobin i) is first deoxygenated; ii) is then covalently reacted with an oxygen binding effector; iii) the solution is then mixed with a non-chemically reactive effector; and then iv) the hemoglobin with glutardialdehyde is stably covalently crosslinked with one another in an inverse concentration gradient reaction, based on the concentration of the crosslinking agent and the hemoglobin to be crosslinked, then the solution is diluted with water, and then v) a polyethylene oxide covalently tied up vi) the product obtained is worked up in a known manner. Crosslinking with glutardialdehyde, such as, for example, is very particularly preferred. B. in Pötzschke H. and Barnikol W. (1992), Biomaterials, Artificial Cells, and Immobilization Biotechnology 20: 287-291, or as described in the examples below. Effectors that react chemically or not chemically are described above or also in the abovementioned publication DE-A 100 31 744 or DE-A 100 31 742.

The molecular weight of the oxygen carriers produced as described is in the aforementioned range.

In particular, the oxygen carriers thus produced can also be cleaned as described, for. B. purified by chromatography (e.g. by preparative volume exclusion chromatography) by centrifugation, filtration or ultrafiltration, separated into fractions of different molecular weights and subsequently processed, cf. e.g. DE-A 100 31 740 and WO 02/00230.

If necessary, the oxygen carrier (s) can be oxygenated in a known manner before the use according to the invention.

It is further preferred if the oxygen carrier or carriers used has a partial pressure p50 of less than 26 torr, in particular 13 to 22 and preferably 15 to 20 torr. The oxygen partial pressure p50 is the pressure at which the carrier has bound half of the oxygen. In addition, the carrier should also have a sufficiently high level of cooperativity, which can be quantified as the so-called Hillian index. The normal value of the blood is 2.6. It has been shown that the cooperativity of the carrier used according to the invention should not be less than 2.0.

It is preferred if the optionally derivatized oxygen carrier used according to the invention is derived from cattle, pork or human hemoglobin. Pig hemoglobin is particularly preferred because of its structural and functional similarity. In addition, human hemoglobin is also preferred. The oxygen carrier can also be myoglobin or the product thereof modified as described above or mixtures thereof with hemoglobin derivatives. Pig hemoglobin, in particular human hemoglobin, is preferred. In the manner according to the invention, an immediate improvement in the deficiency state is surprisingly achieved by a currently sufficient but non-toxic amount of oxygen. A further improvement in the deficiency state can be achieved by adding further suitable additives such as salts, glucose, insulin, one or more natural amino acids or mixtures suitable for the patient to be treated.

Particularly preferred oxygen carriers or mixtures thereof are those which, if appropriate, reacted / treated with a chemically reactive / non-reactive effector, on the one hand crosslinked with glutardialdehyde and pegylated with a polyethylene oxide or derivative thereof with a molecular weight of 1500 to 2000 g / mol and have a total molecular weight of 300,000 to 15,000,000, in particular 700,000 to 10,000,000, based on hemoglobin or 100,000 to 3,000,000 or also 200,000 to 1,000,000 g / mol based on myoglobin. The agents according to the invention are produced by mixing the oxygen carrier (s) in the medium, in particular aqueous medium. The aqueous-based media can contain suitable additives, in particular 0 to 20% by volume, preferably 0.01 to 20% by weight, in particular 0.1 to 20% by weight and especially 0.1 to 15% , These are preferably selected from glucose, natural amino acids for the respective application, that is to say the amino acids natural for humans or animals, insulin, each in a physiological concentration or multiples thereof for the application in question, and also suitable known antibiotics, tissue factors and natural and / or artificial buffer substances such as TRIS, bicarbonate, phosphate and physiologically tolerable salts such as sodium chloride, potassium chloride, calcium magnesium chloride, sodium citrate, sodium lactate, likewise in a suitable, in particular physiological concentration or multiples thereof, or mixtures thereof, for the respective application. Potassium-calcium-magnesium chloride, sodium hydrogen (bi) carbonate, sodium citrate, sodium lactate, that is, the known electrolytes can be used, for example, in physiological concentration or in multiples thereof, e.g. B up to 10 times, ie in amounts of 0.1 to 30 or preferably 0.5 to 10% by weight, preferably 0.5 to 5% by weight, in particular 0.8 to 1.5% by weight, with this being the case sodium chloride is particularly suitable. The electrolytes can also be present in a mixture. The buffer substances can be used in such a way that a pH value as indicated, but above all 7.4. Glucose can be, for example, in amounts of 0.1 to 5% by weight, insulin in physiological dosage or in amounts of up to 25 IU / ml, the natural amino acids known for the respective application, that is to say those known for humans or for the respective animals Amino acids e.g. B. 0 or 0.01 up to 5 wt.%, Or tissue factors such as interleukins are present in physiological amounts up to 10 times the amount thereof. Particularly preferred additives are physiological sodium chloride solution (0.8 to 1.5%), in particular 0.9%). Glucose (in amounts as stated above, preferably for example 1%) and insulin in physiological dosage, if appropriate up to 5 times thereof, and also mixtures thereof. Above all, potassium chloride, magnesium chloride, sodium bicarbonate or mixtures thereof can also be added or additionally added in the amounts indicated above.

In particular, it has been shown that the oxygen carrier or carriers used according to the invention are effective over a wide pH range, namely from 5.5 to 9, in particular 6.5 to 8. In particular, when remedying a shortage of organ supplies, a pH of 7.4 can be present in the medium administered, such as the infusion.

As mentioned, the oxygen carrier (s) added to the medium / infusion release the oxygen by diffusion, so that the vessels are adequately supplied with oxygen, preferably oxygen and electrolytes, in such a way that permanent damage to tissue due to the deficiency can be avoided or treated , This takes place in the specified concentration range. This is checked during the treatment and, if necessary, the oxygen carrier is added in order to remain in the range according to the invention. The treatment can also be continued after the acute crisis has been remedied, the amount of oxygen carrier added then possibly being reduced. This concentration range is particularly important since neither an overdosing nor an underdosing is allowed. In particular, tinnitus, myocardial infarction, stroke, sudden hearing loss, dizziness, placenta insufficiency, kidney shock or pulmonary shock are treated according to the invention. To remedy a dysfunction of the heart, kidney, lung, brain, 5 to 150 g / liter medium, in particular 7 to 90 g / liter medium L, on oxygen carriers. In the case of dizziness, disorders of the ear or the placenta, 10 to 180 g / liter or 15 to 120 g / liter can be used.

In the case of stroke and heart attack, infusions are used especially during the acute phase of the event.

The oxygen carriers are particularly preferably administered in an amount of 10 to 80 g / liter, in particular 12 to 50 g / liter (infusion) medium, so that an average oxygen partial pressure of approximately 30 mm Hg is present in the tissue during administration, which does not increase is high in order to cause an oxidative, harmful oversupply, but is sufficient to remedy the acute deficiency state. In particular, the oxygen carriers according to DE-A1 100 31 740 (WO 02/00230), DE-A1 100 31 744 and DE-A1 100 31 742.1 and among them especially those with an average molecular weight of 700,000 to 5,000,000 g / mol (hemoglobins) or 100,000 to 1,000,000 g / mol (myoglobins) administered.

Examples

The invention is illustrated by the following examples.

Production of agents according to the invention

example 1

Human natural hemoglobin was freed from plasma and cell wall components by centrifugation and ultrafiltration and purified.

Of these, 8% by weight were dissolved in 100 ml of water containing 0.9% by weight of sodium chloride and 5% by weight of glucose and 20 IU / ml of insulin. Example 2

Pig hemoglobin, in a concentration of 330 g / L dissolved in an aqueous electrolyte of the composition 50 mM NaHCO ₃ and 100 rriM NaCl, was deoxygenated at 4 ° C. by stirring the solution under constantly renewed, pure nitrogen over the solution. Then 4 mol sodium ascorbate (as a 1 molar solution in water) per mol (monomeric) hemoglobin was added and allowed to react for 6 hours. The solution was titrated to a pH of 7.1 with 0.5 molar lactic acid, 1.1 mol of pyridoxal-5'-phosphate per mol of hemoglobin were added and the mixture was allowed to react for 16 h. Now a pH of 7.8 was set with 0.5 molar sodium hydroxide solution, 1.1 mol of sodium borohydride (as a 1 molar solution in 0.01 molar sodium hydroxide solution) was added and the mixture was left to react for one hour. Now a pH of 7.3 was set with 0.5 molar lactic acid, initially 1.1 mol of 2,3-bisphosphoglycerate per mol of hemoglobin and after a reaction time of 15 minutes, 8 mol of glutardialdehyde per mol of hemoglobin, dissolved in 1.8 L of pure water per liter of hemoglobin solution to crosslink the hemoglobin within 5 minutes and allow to react for 2.5 h. After titration with 0.5 molar sodium hydroxide solution to a pH of 7.8, 15 mol of sodium borohydride (as a 1 molar solution in 0.01 molar sodium hydroxide solution) were added per mol of hemoglobin for 1 h. 2 liters of water were added per liter of original hemoglobin solution. The pH was then 9.3 and 4 mol of methoxy-succinimidylpropionate-polyethylene glycol with a molecular weight of 2000 g / mol were added directly for 2 h. The nitrogen atmosphere above the solution was replaced by pure oxygen.

After 1 h, insoluble constituents were removed by centrifugation (20,000 g for 15 min). The electrolyte was then changed by volume exclusion chromatography (Sephadex G-25 - Gel, Pharmacia, D) to an aqueous electrolyte solution with the composition 125 mM NaCl, 4.5 mM KCI and 20 mM NaHCO ₃ .

The yield was 77%; the yield for molecular weights greater than 700,000 g / mol is 28%. Measurements of the characteristic of oxygen binding under physiological conditions (a temperature of 37 ° C, a carbon dioxide partial pressure of 40 Torr and a pH of 7.4) gave the product a p50 of 22 Torr and an n50 of 1.95.

This oxygen carrier is particularly suitable for use according to the invention in aqueous solution as described in Example 1.

Example 3

The synthesis of the human hemoglobin crosslinked with glutardialdehyde was carried out as in Example 2, but using concentrated human hemoglobin and using a 16-fold molar excess of the crosslinking agent. Polymers were obtained by fractionating the solution of the crosslinking products with the aid of preparative volume exclusion chromatography (according to EP-A 95 10 72 80.0: “Process for the production of uniform molecular hyperpolymeric hemoglobins” with Sephacryl S-300 HR gel, Pharmacia Biotech, Freiburg, D ) (here as the first eluted 57 mass% of the cross-linked hemoglobin). The cross-linked hemoglobins were divided into two parts A and B. Hemoglobin A (see Figure 3) was found to be predominantly polymeric hemoglobin with a modal value of the molecular weight distribution of 950 kg / mol (cf. Example 1.) Covalent attachment of monofunctionally active mPEG-SPA-1000 was carried out analogously to the procedure described for cross-linked pig hemoglobin in Example 2. After the addition of sodium bicarbonate (up to 150 mM) to the solution of the polymers, one could React a 12-fold molar excess of mPEG-SPA-1000 with the hemoglobin monomers Following a reaction time of one hour, lysine was added in a 60-fold molar excess to 'trap' active molecules of the mPEG-SPA-1000. Both the crosslinked hemoglobin according to solution A and the crosslinked and pegylated product according to solution B are suitable for the use according to the invention.

Example 4

Cross-linked bovine hemoglobin was produced by cross-linking concentrated bovine hemoglobin with a 14-fold molar excess of glutardialdehyde according to Example 2, a molecular fractionation of the synthesis products, the binding of mPEG-SPA-1000 according to Examples 2 and 3. Figure 5 shows a molecular weight distribution of the unmodified hemoglobin polymer, namely an eluogram of a volume exclusion chromatography (on the gel “Sephacryl S-400 HR”, Pharmacia Biotech, Freiburg, D), the modal value of the molecular weight distribution here is 810 kg / mol ,

Example 5

Concentrated, deoxygenated porcine hemoglobin dissolved in an aqueous electrolyte with the composition 50 mmol / L NaHCO ₃ and 100 mmol / L NaCl was reacted at room temperature with a 14-fold molar excess of glutardialdehyde. Sodium cyanoborohydride, added in a 10-fold molar excess to the (monomeric) hemoglobin, reduced the Schiff bases formed during the crosslinking and stabilized the covalent crosslinking. The crosslinked hemoglobin solution obtained was divided into three parts (A, B and C) and processed differently. Part A remained unchanged, the determination of the molecular weight distribution (according to Pötzschke H. et al. (1996, Macromolecular Chemistry and Physics 197, 1419-1437, and Pötzschke H. et al. (1996, Macromolecular Chemistry and Physics 197, 3229-3250) using volume exclusion chromatography with the gel Sephacryl S-400 HR (Pharmacia Biotech, Freiburg, D) gave a modal value of the molecular weight distribution of 520 kg / mol for the crosslinked porcine hemoglobin.

The polymers of fraction B were covalently linked with monofunctionally active mPEG-SPA-1000 (Shearwater Polymers Europe, Enschede, NL): First, sodium bicarbonate was added as a solid substance to a final concentration of 150 mmol / L to dissolve the crosslinked hemoglobins, followed by Add mPEG-SPA-1000 in a 12-fold molar excess (based on the hemoglobin monomers) also as a solid substance. After a reaction time of one hour, lysine was added in a 60-fold molar excess (based on hemoglobin) and reacted with still active mPEG-SPA-1000 molecules. Part C: The solution of the crosslinked hemoglobins was carried out in exactly the same way as described for Part B, but using mPEG-SPA-2000 (Shearwater Polymers Europe, Enschede, NL).

This was followed by a solvent exchange in the three solutions A, B and C (using ultrafiltration, “Ultraminisette 10 kDa”, Pall Gelman Sciences, Roßdorf, D, or volume exclusion chromatography on gel “Sephadex G-15 M”, Pharmacia Biotech , Freiburg, D) to a solution in an aqueous electrolyte (StLg) of the composition: 125 mM NaCl, 4.5 mM KCI and 3 mM NaN ₃ . All products according to solution A, B or C are suitable for the purpose according to the invention.

Example 6

Intramolecularly cross-linked hemoglobin was produced as described in Example 2, but in a 0.1% concentration.

Example 7

Commercial natural human myoglobin (e.g. from Sigma, D) was purified by gel chromatography. According to the invention, this can be used as such or modified as described above.

Example 8

12% of an unmodified human hemoglobin as described in Example 1 and 6% by weight of a modified product as described in Example 2 were dissolved in 100 ml of purified water containing 0.9% by weight of sodium chloride, 0.2% by weight of sodium bicarbonate, 1% by weight % Glucose given. The solution is immediately ready for use.

Example 9

10% by weight of a human myoglobin modified with polyethylene glycol, prepared according to Example 3, solution A, was added to 100 ml of purified water containing 0.9% by weight of sodium chloride and 5% by weight of glucose, insulin 20 IU / ml. The solution is immediately ready for use and especially durable. II. Application examples

Example 10 A solution according to Example 2 was administered to a male patient, 67 years, 3 times over a period of 6 weeks at intervals of 14 days. There was no increase in the transaminase blood level in any case of administration. There are also no signs of an immune reaction.

Example 11

The same treatment as in Example 10 was carried out in a female patient aged 65 years. Again, there was no increase in the level of transaminase and no sign of an immune reaction.

Claims

claims

1. Use of one or more oxygen carriers selected from hemoglobin, myoglobin or derivatives thereof, for the preparation of an agent for the treatment of an organ dysfunction due to an acute lack of supply and for

Treatment / prevention of tissue damage due to such a disorder.

2. Use according to claim 1, characterized in that the acute lack of supply due to lack of oxygen, a lack of nutrients or

Combinations of these exist.

3. Use according to claim 2, characterized in that there is a lack of oxygen due to acute or chronic vasoconstriction, stress, vascular spasm or arteriosclerosis.

4. Use according to claim 2, characterized in that the lack of nutrients is due to the lack of electrolytes, glucose or insulin or combinations.

5. Use according to one of claims 1 to 4, characterized in that the acute lack of care is tinnitus, heart attack, stroke, sudden hearing loss, dizziness, placental insufficiency, kidney shock or pulmonary shock.

6. Use according to one of claims 1 to 5, characterized in that the oxygen carrier (s) of human or animal origin or modified derivatives thereof or mixtures thereof.

7. Use according to one of claims 1 to 6, characterized in that the oxygen carrier or carriers are selected from natural or modified human or pig hemoglobin or mixtures thereof.

8. Use according to one of claims 1 to 7, characterized in that modified or natural myoglobin or mixtures thereof is used.

9. Use according to one of claims 1 to 8, characterized in that hemoglobin and myoglobin or modified derivatives thereof are used in a mixing ratio of 1:20 to 20: 1.

10.Use according to one of claims 1 to 9, characterized in that the modification of the or the oxygen binder is an intra-, intermolecular

Cross-linking, a pegylation, a reaction with chemically reactive or chemically non-reactive effectors or a combination thereof.

11.Use according to claim 10, characterized in that the modification is an intermolecular crosslinking, a pegylation or a combination thereof.

12. Use according to claim 10, characterized in that as a modification there is additionally a reaction with a chemically non-reactive or a chemically reactive effector or a combination thereof.

13. Use according to one of claims 1 to 12, characterized in that the oxygen carrier (s) are selected in the form of an aqueous solution containing 0.2 to 20% by weight of the oxygen carrier (s) and 0.01 to 20% by weight of additives from physiologically acceptable salts,

Buffer substances and amino acids, glucose, insulin, tissue factors, or mixtures thereof, can be used.

H. Use according to claim 13, characterized in that 0.1 to 20% by weight of additives are present and the physiologically tolerable salts are selected from sodium chloride, sodium hydrogen sodium bicarbonate, Potassium chloride, calcium-magnesium chloride, sodium citrate, sodium lactate, or mixtures thereof.

15. Use according to one of claims 13 or 14, characterized in that the additives are selected from 0.9% sodium chloride and 1%

Glucose and insulin in physiological doses.