MXPA99000402A - New agents of transfer of oxygen, conjugates of hemoglobin-hydroxyethylamidon that contain them, processes for its preparation and its use as substitutes sanguin - Google Patents

Abstract

The present invention relates to novel oxygen transfer agents comprising hemoglobin-hydroxyethylstarch conjugates and processes for their preparation. The invention further relates to the use of oxygen transfer agents as blood substitutes, plasma expanders, perfusion agents, hemodilution agents and / or cardioplegic solution. The invention relates in particular to oxygen transfer agents comprising a hemoglobin-hydroxyethylstarch conjugate in which the hemoglobin and the hydroxyethyl starch are selectively linked together via amide linkages between the free amino groups of the hemoglobin and the terminal reducing groups of hydroxyethyl starch, which is present in the oxidized form

Description

NEW AGENTS OF TRANSFER OF OXYGEN, CONJUGATES OF HEMOGLOBIN-HYDROXIETI STARCH THAT CONTAINS THEM, PROCESSES FOR PREPARATION AND USE AS BLOOD SUBSTITUTES FIELD OF THE INVENTION The present invention relates to the new oxygen transfer agents comprising hemoglobin-hydroxyethylalvidon conjugates and the processes for their preparation. The invention further relates to the use of oxygen transfer agents as blood substitutes, plasma expanders, perfusion agents, hemodilution agents and / or cardioplegic solution.

BACKGROUND OF THE INVENTION The development of stroma-free hemoglobin solutions, called "hemoglobin-based oxygen transporters" (HBOCs), which can be used as blood substitutes for a long time have been a pressing objective in pharmaceutical research and development. For example, blood loss as a result of an accident or an operation, in most cases is treated by allogeneic blood donation. Associated problems such as the uncontrolled transfer of pathogenic organisms, in particular of pathogenic viruses such as HIV or hepatitis and the need to typify P1003 / 98MX blood group before transfusion have been known to experts for a long time and have been described in the literature extensively. An HBOC product that can be used as a standard blood substitute would not only solve these problems, but could also be used as a plasma expander, perfusion agent, hemodilution agent and / or cardioplegic solution. Although the need for such a product was already recognized before (see Rabiner, J. Exp. Med. 126, (1967) 1127), none of the HBOC products known hitherto has achieved the status of being an approved medicament. The natural oxygen transfer agent is the blood pigment hemoglobin, a chromoprotein with molecular weight (MW) of 64 kilodalton (kDa). The protein consists of two chains of peptides a and ß, each of which has a bound heme as a prosthetic group. Hemoglobin is a porphyrin ring with a central iron atom. Isolated hemoglobin molecules are highly unstable and dissociate rapidly in the more stable α, β dimers (32 kDa MW). The biological half-life of the hemoglobin isolated in the blood circulation is approximately 1 hour, since the dimers are rapidly excreted via the kidneys. The dimers here cause nephrotoxic side effects (cf. Bunn &Jandl, J. Exp. Med.

P1003 / 98MX 129, (1967) 925-934). The HBOC products developed at the beginning also had a nephrotoxic potential, which was attributed to the contamination of these with constituents of the cell (see Relihan, Ann. Surg. 116, (1972) 700). In addition, an isolated hemoglobin composition lacks 2, 3-diphosphoglycerate (2,3-DPG), which is the allosteric activator of oxygen binding, which occurs naturally. This results in an increase in the affinity for the oxygen bonding of the isolated hemoglobin in these compositions and, together with this, a decrease in the oxygen release capacity. The development work on molecules derived from hemoglobin, therefore, was first directed to improve the properties of oxygen transfer from them and to avoid nephrotoxic symptoms. In this work, hemoglobin was cross-linked intramolecularly, intermolecularly linked to polymeric HBOC forms and / or coupled to polymers to provide conjugated HBOC forms. In this work, mixed forms of intra- and intermolecularly cross-linked hemoglobin derivatives were also prepared and investigated for planned use. The cross-linking of hemoglobin by means of bi- or polyfunctional crosslinking agents can P1003 / 98 X carried out selectively or non-selectively. In the form of selective cross-linking, two hemoglobin protein chains bind to each other intramolecularly, as a result of which it is stabilized in the natural tetrameric form of the isolated hemoglobin molecule. By selection of a suitable crosslinking agent, the affinity of hemoglobin for oxygen can also be adjd in such a way that the crosslinked hemoglobin can bind to oxygen reversibly under physiological conditions. Examples of these crosslinking agents are pyridoxal phosphate, diaspirin and derivatives thereof. Processes for hemoglobin cross-linking are described, for example, in Benesch (Meth. Enzymol., Vol 231 (1994), 267-274), Keipert et al. (Transfusion, vol 29 (1989), 767-773), Snyder et al. (Proc. Nati, Acad. Sci. USA, 8_4, (1987), 7280-7284 and Rogers et al. (Biochim et Biophys. Acta, 1248 (1995), 135-142) In a non-selective cross-linking, Intermolecularly cross-linked polymeric HBOC products are formed Suitable cross-linking agents and processes for their use are described for example in DE-26 07 706, EP-0 646 130 and Hai et al, (Art. Cells, Blood Subs. Immob. Biotech, 22 (3) (1994), 923- 931. An overview of various hemoglobin derivatives and the problems associated with their clinical use is given in Gould et al.

P1003 / 98MX al., Transfus. Sci. 16, (1995) 5-17, and Chang et al., Bio at., Art. Cells & Immob. Biotech., 2_0, (1992) 159-179. The known hemoglobin conjugates are described extensively in Xue and Wong (Meth. In Enzymol., 231 (1994), p.308-322) and for example, in DE 26 16 086 or DE 26 46 854. The latter exposes the processes by means of which hemoglobin binds to hydroxyethyl starch, by reaction of hydroxyethylstarch with sodium periodate. In this reaction, dialdehydes are formed and hemoglobin binds them. In contrast, DE 26 16 086 describes the coupling of hemoglobin to hydroxyethyl starch by a process in which first a crosslinking agent (eg, cyanogen bromide) binds to hydroxyethyl starch and then hemoglobin binds to the intermediate product. The affinity of hemoglobin for bonding with oxygen also depends on the ligand of the heme group during crosslinking and / or polymerization, as well as on the choice of suitable crosslinking and / or polymerization agents. Oxyhemoglobin is rapidly oxidized to methaemoglobin (Fe-III), which has an affinity for oxygen binding too high to be suitable as an oxygen transfer agent. Therefore the process for the preparation of HBOC derivatives mentioned has also been carried out with partial hemoglobin or P1003 / 98MX completely deoxygenated (cf. Benesch, R. E. loe. Cit). However, the processes that exist to date for the preparation of crosslinked and / or conjugated HBOC products have not made possible the selective binding of hemoglobin with a particular polymer. In all the processes a mixture of copolymers was formed, whose constituents had different biological activities. So far this has been possible for the products of the reaction or the composition of the mixture to which they were characterized only in approximate form. Both the high molecular weight (MW> 500 kDa) and the residual tetrameric forms caused toxic side effects. The elimination of certain portions of low and / or high molecular weight of the HBOC products, for example, with additional filtration steps, causes a considerable loss in performance, as a result of which the profitability of the preparation process is impaired in a considerable. Furthermore, the HBOC products tested to date have vascular side effects, which according to the latest clinical studies are attributed to HBOC forms of low molecular weight, for example, essentially the tetrameric HBOC forms (see Gould et al., Loc. Cit. ., and Alayash &Cashon, Molecular Medicine Today, 1, (1995) 122-127). These HBOC forms of low molecular weight are able to pass from P1003 / 98MX circulatory torrent to the endothelial cell layers of blood vessels. The high affinity of hemoglobin for the bond with nitric oxide (NO, also known as a relaxation factor that is derived from the endothelium, EDRF) causes the amount of NO that is available in free form in this tissue to be drastically reduced after the administration of HBOC derivatives. As a consequence of the local decrease in NO concentration, systemic vasoconstriction develops, leading to hypertension. Jia et al. (Nature, 380, (1996) 221-226) even attribute to hemoglobin a central role in the regulation of NO circulation. According to them, hemoglobin is oxygenated and S-nitrosylated cooperatively in the lungs. During the arteriovenous transfer, the NO group is transferred to other proteins, which as a result acquire a vasodilatory activity such as NO. However, as a rule HBOC products no longer have cooperative properties. Another toxic activity of the HBOC products tested to date was described, among others, by Alayash and Casho (see Molec. Med. Today, (1995) loe. Cit.). According to them, hemoglobin molecules outside the erythrocytes participate in redox reactions, in the P1003 / 98MX 'from which highly reactive hemoglobin and oxygen species are formed, which are responsible, in others, for the peroxidation of lipids. To suppress the toxic side effects of the HBOC products tested hitherto, administration forms were developed in which hemoglobin is packed into liposomes, resulting in synthetic transfer organelles analogous to erythrocytes (see Rudolph et al., Crit. Care Med 22, (1994) 142-150). However, the introduction of large amounts of phospholipids into the bloodstream is associated with additional risks for patients.

OBJECTS AND ADVANTAGES OF THE INVENTION In summary, it can be said that the HBOC products tested so far have not obtained their approval as medicines, since their clinical use as oxygen transfer agents has been prevented so far by an inadequate tolerability. The object of the present invention was therefore to provide an oxygen transfer agent that can be used clinically as a blood substitute. Another object of this invention is to provide a suitable preparation process for the oxygen transfer agent according to the invention.

P1003 / 98MX This object is achieved with an oxygen transfer agent comprising a hemoglobin-hydroxyethyl starch conjugate in which the hemoglobin and the hydroxyethyl starch are selectively bonded together via amide bonds between the free amino groups of the hemoglobin and the terminal reducing group of the hydroxyethyl starch, which is present in the oxidized form. Surprisingly, it has been found that the hemoglobin-hydroxyethyl starch conjugates according to the invention are outstandingly suitable as oxygen transfer agents, since in particular they are well tolerated. The agent has an affinity for bonding with oxygen which allows reversible bonds with this under physiological conditions (P50 from 20 to 80 mm Hg, preferably 20 to 50 mm Hg; P50 determination is carried out to enrichment maximum with pure oxygen - at least 150 mm Hg). The hemoglobin-hydroxyethylstarch conjugate is too large to penetrate the endothelial cell layers of blood vessels and therefore does not cause side effects of hypertension. The oxygen transfer agent contains neither antigenic nor pyrogenic constituents and does not cause nephrotoxic side effects either. According to the invention, it has surprisingly been found that the advantages in rheological properties P1003 / 98MX that have made hydroxyethylstarch a preferred agent for hemodilution and for volume replacement (see Weidler et al., Arzneim. -Forschung / Drug Res., 4JL, (1991) 494-498) are retained in the conjugate. The good tolerability of the oxygen transfer agent is therefore also based on a surprising combination of the advantages in the oxygen transfer properties of the hemoglobin and the hemodilution properties of the hydroxyethyl starch. The oxygen transfer agent has a long vascular persistence and the surface of the hemoglobin is protected with the substituents. Surprisingly, it has been found that in the hemoglobin-hydroxyethyl starch conjugate according to the invention, this protective effect prevents hemoglobin from participating in the toxic redox reactions. Another advantage of the oxygen transfer agent according to the invention is that the hydroxyethyl starch and the hemoglobin in conjugate form can be administered simultaneously at high concentrations, without the colloidal osmotic pressure being increased as a result. The oxygen transfer agent comprises the hemoglobin-hydroxyethyl starch conjugate in a concentration between 2 and 40 g / dl, preferably between 5 and 2 Q g / dl, and with particular preference in a concentration P1Q03 / 98MX between 8 and 20 g / dl. The oxygen transfer agent can also contain vehicles, physiologically tolerated known diluents or excipients. In the context of the present invention, hemoglobin free from stroma, purified and pasteurized, which can be obtained by the processes broadly described in the prior art, is preferably used for the preparation of the hemoglobin-hydroxyethyl starch conjugate. The hemoglobin can be crosslinked and / or polymerized. It can be of human, animal, plant or recombinant origin. In the context of the present invention, surprisingly, it has been found that the protective effects of hydroxyethyl starch prevent the immunological complications that could be expected if hemoglobin of animal origin is used. A preferred embodiment of the invention therefore relates to an oxygen transfer agent comprising a hemoglobin-hydroxyethylstarch conjugate in which the hemoglobin is of animal origin. The hemoglobin can be, for example, of bovine, porcine or equine origin. According to a particularly preferred embodiment of the invention, for the preparation of the hemoglobin-hydroxyethyl starch conjugate, bovine hemoglobin is used, which in its isolated form has the affinity for the bond with the preferred oxygen, even without crosslinking.

P1003 / 98MX If human hemoglobin is used, it must be stabilized in the tetrameric form by crosslinking and / or polymerization. With crosslinking and / or polymerization, human hemoglobin simultaneously becomes capable of reversibly binding to oxygen under physiological conditions. A large number of processes for crosslinking or polymerization are known to the experts. According to the invention, any desired process can be employed, provided that the hemoglobin is stabilized and the affinity for the oxygen that is desired results (P50 from 20 to 80 mm Hg, preferably P50 from 20 to 50 mm). , Hg). Preferred crosslinking processes include intramolecular cross-linking with bis-pyridoxal tetraphosphate (see Keipert et al., Transfusion, vol 29 (1989), 767-773) or diaspirin (see Snyder et al., Proc. Nati. Acad. Sci. USA, 84 (1987), 7280-7284) or cross-linking and polymerization with oxidized raffinose (cf. EP-0 646 130). According to a particularly preferred embodiment of the invention, before the coupling of the hydroxyethylstarch the hemoglobin is present in the deoxygenated or partially deoxygenated form. In the case of the partially deoxygenated form, compositions containing deoxyhemoglobin in a proportion of 20 to 80% and hemoglobin in other derivation states in one embodiment are preferred.

P1003 / 98MX ratio of 80 to 20%, in particular compositions containing deoxyhemoglobin in a proportion of 50 to 80% and hemoglobin in other derivation states in a proportion of 50 to 20% are preferred. Other derivative states of hemoglobin are, in particular, CO-, oxy- and / or methaemoglobin derivatives. To prepare the conjugate, hydroxyethylstarch having an average molecular weight of 1 to 40 kDa, preferably hydroxyethyl starch having an average molecular weight of 2 to 20 kDa, with particular preference hydroxyethyl starch having an average molecular weight of 5 to 20 kDa. The hydroxyethyl starch which is preferred is further characterized by a degree of molar substitution of 0.1 to 0.8 and a C2: C6 substitution ratio in the range of 2 to 20. The hydroxyethyl starch which is preferred according to the invention can be obtained by acid hydrolysis, for example with HCl, from a comparatively higher molecular weight hydroxyethylstarch that can be commercially available (Sigma). The hydroxyethylstarch is then subjected to one. precipitation reaction, in which, for example, acetone can be used. The molecular weight of the hemoglobin-hydroxyethyl starch conjugate according to the invention depends on the molecular weight or the molecular weight distribution of the P1003 / 98MX which is used, the molecular weight distribution of the hydroxyethylstarch used and the choice of reaction conditions. According to the invention, hemoglobin-hydroxyethyl starch conjugates with molecular weight between 100 and 700 kDa are preferred, particularly a molecular weight between 200 and 300 kDa is preferred. In the context of the present invention, it has been found that the known stabilizing effects of saccharides on hemoglobin (see Rudolph, Cryobiology, 2_5, (1988) 1-8) also occur in the conjugate if chain hydroxyethylstarch is used short. The oxygen transfer agents according to the invention therefore have better storage stability at 4 ° C and at room temperature, compared to unmodified HBOC products. The oxygen transfer agents therefore have surprisingly become themselves carriers of the advantageous stabilizing properties of the saccharides. The present invention also relates to processes for the preparation of oxygen transfer agents which comprise a hemoglobin-hydroxyethyl starch conjugate. These processes for the first time allow the selective binding of hemoglobin with hydroxyethyl starch, as a result of which an oxygen transfer agent is formed. The conjugate is prepared P1003 / 98 X in a multi-step process, in which the terminal reducing groups of the hydroxyethylstarch are first oxidized and then the hemoglobin is coupled to the terminal oxidized groups of the hydroxyethyl starch via amino groups via amide bonds. The raw material used for the process is preferably hydroxyethyl starch having an average molecular weight between 1 and 40 kDa, preferably hydroxyethyl starch having an average molecular weight between 2 and 20 kDa, with particular preference hydroxyethyl starch having an average molecular weight between 5 and 20 kDa. The hydroxyethylstarch that is preferred is further characterized by a degree of molar substitution between 0.1 and 0.8 and a C2: C6 substitution ratio in the range of 2 to 20. In the context of the invention, for the preparation of the oxygen transfer agent preferably, stromal-free hemoglobin, purified and pasteurized, cross-linked and / or polymerized is used. Here the hemoglobin can be of human, animal, plant or recombinant origin. In the context of the present invention, bovine hemoglobin is preferred, since the isolated form has an affinity for oxygen binding that allows reversible binding to oxygen under physiological conditions.

P1003 / 98MX According to a preferred process of the invention, the terminal reducing groups of the hydroxyethylstarch are first oxidized by mixing the hydroxyethylstarch with an iodine-containing solution and then adding potassium hydroxide solution. According to another preferred process of the invention, the hemoglobin is linked to the terminal oxidized groups of the hydroxyethyl starch in a second step. The reaction can be carried out, for example, by mixing the individual components at 40 ° C. Here a nucleophilic substitution reaction takes place between a free amino group of the hemoglobin and the lactone of the hydroxyethyl starch, to form an amide bond by means of which the hemoglobin is attached to the terminal oxidized reducing group of the hydroxyethyl starch.

DESCRIPTION OF THE PREFERRED MODALITIES According to the invention, it has therefore been found, surprisingly, that by the process of Hashimoto et al. (Kunststoffe, Kautschuk, Farsen, 9, (1992) 1271-1279) for the preparation of block polymers from polysaccharides and polyamides, purified hemoglobin which can be crosslinked intra- and / or intermolecularly, can be linked to the oxidized hydroxyethylstarch in such a way as to form an oxidizing agent.

P1003 / 98MX oxygen transfer particularly tolerated. By applying the principles according to the invention, for the first time the synthesis of a hemoglobin conjugate can be controlled to such an extent that the tetrameric forms of hemoglobin bind to the hydroxyethyl starch without a remarkable content of undesirable forms of high molecular weight hemoglobin. According to a preferred embodiment of the invention, the conditions of the reactions are selected in such a way that a hemoglobin-hydroxyethyl starch conjugate is formed having a molecular weight between 80 and 800 kDa, a molecular weight between 100 and 500 kDa is preferred and in particular between 150 and 400 kDa. An approximately quantitative reaction of the hemoglobin with the hydroxyethyl starch takes place by the preparation process according to the invention. Therefore, almost no form of low molecular weight hemoglobin remains in the reaction batch, a content of less than 5% of unconjugated hemoglobin forms is preferred. Accordingly, in another preferred embodiment of the invention, a non-expensive purification process is necessary to isolate the desired reaction product after the coupling of hemoglobin and hydroxyethyl starch. According to another preferred embodiment of the invention, before coupling to the hydroxyethyl starch hemoglobin P1003 / 98MX is present in either totally or partially deoxygenated form. In the case of the partially deoxygenated form, compositions comprising deoxyhemoglobin in a proportion between 20 and 80% and hemoglobin in other derivation states in a proportion between 80 and 20% are preferred., particularly compositions between 20 and 80% deoxyhemoglobin and between 80 and 20% hemoglobin in other derivation states are preferred. The deoxygenation of hemoglobin can be carried out by any chemical or physical process desired. In these, hemoglobin is treated either with chemical reducing agents, such as sodium ascorbate, glutathione, N-acetyl cysteine or N-acetyl methionine or is circulated countercurrently with an inert gas, such as N2, He or Ar, by a membrane permeable to gases. In a particularly preferred process, cysteine or acetyl cysteine is used as a reducing agent. The reduction is carried out until the oxyhemoglobin content is less than 5%, a content of less than 1% is preferred. The content of methemoglobin should be less than 5%, a content of less than 3 or 1% and in particular less than 0.5% is preferred. According to another particularly preferred embodiment of the invention, a hemoglobin solution is used in which the hemoglobin contains deoxyhemoglobin in a proportion P1003 / 98MX between 20 and 80% and hemoglobin in another derivation state in a ratio between 80 and 20% for the preparation of the hemoglobin-hydroxyethylstarch conjugate. To prepare that hemoglobin solution, oxyhemoglobin may be partially deoxygenated or deoxyhemoglobin may be partially oxygenated. Depending on the form derived from the initial hemoglobin solution and the desired preferred composition of the hemoglobin derivative according to the invention, a solution of hemoglobin can further be converted to the stable form CO with carbon monoxide and / or oxygenated with oxygen or gases which they contain 02 and / or deoxygenate with nitrogen or another inert gas. Here the exchange of gases can be carried out by any of the desired processes described in the prior art. Preferred processes include the gassing of a hemoglobin solution with oxygen or with an oxygen-containing gas or the partial chemical reduction of oxyhemoglobin with a reducing agent, such as, for example, sodium dithionate, sodium ascorbate or sodium bisulfite. . When the reaction is over, the reducing agent can be removed, for example by ultrafiltration. In a preferred embodiment of the invention, the ultrafiltration is carried out by means of a membrane that preserves the desired product in the material P1003 / 98MX retained. According to a particular preferred process of the invention, the hemoglobin is lyophilized with gassing with N2. In another particularly preferred embodiment of the invention, the hydroxyethyl starch is selectively oxidized in the terminal reducing groups first by the addition of 0.1-iodine solution to an aqueous solution of fractionated hydroxyethyl starch (MW <10 kDa). A solution of 0.1 N KOH is then added at room temperature (RT), until the color originating from the iodine disappears. This step is repeated once or several times and then the mixture is stirred for an additional 30 minutes. Then, the solution is subjected to dialysis, the dialysis membrane has an exclusion volume that preserves the desired product (in this case the oxidized hydroxyethyl starch) in the retentate. After purification by chromatography through a cation exchange column, the solution is lyophilized, it is also possible to perform the chromatography before the dialysis. According to another particularly preferred embodiment of the invention, the binding of the hemoglobin with the selectively oxidized hydroxyethylstarch is carried out first by placing the hemoglobin in DMSO or another suitable non-aqueous solvent and transferring the mixture to a three-necked round flask. For this, a solution in P1003 / 98MX DMSO is slowly added at 40 ° C to the hydroxyethyl starch oxidized by the above process. However, these steps can be performed in any desired order, ie the hemoglobin can also be added to the hydroxyethyl starch solution. After stirring at 40 ° C for 25 hours, the residue is purified by gel permeation chromatography (GPC) or dialysis and / or ultrafiltration and is thus freed from the solvent. The increase in the molecular weight of the hemoglobin preparations can be determined directly by means of SDS-PAGE and non-denaturing gel electrophoresis or ultracentrifugation (density gradient or sedimentation equilibrium by centrifugation). The usual chromatographic methods, such as SEC (size exclusion chromatography) or thin layer chromatography (TLC) are also suitable for determining molecular size. It is possible to use methods of affinity chromatography (HIC, RPC) and IEC (ion exchange chromatography), as well as IEF (isoelectric focus) to determine changes related to modification in the physicochemical properties of molecules. The degree of substitution can be quantified by H-NMR, C-NMR, mass spectrometry or capillary electrophoresis (CE). The colorimetric method for the determination of free amino groups in proteins by means of TNBS (Habeeb et al., Anal.

P1003 / 98MX Biochem., 14, 328 [1966]) in combination with a protein test (Bradford, Lowry, Biuret) or the determination of nitrogen by Kjeldahl is also suitable for this purpose. The invention also relates to oxygen transfer agents comprising conjugates of hydroxyethylstarch-hemoglobin and albumin. Here the albumin can be of human, animal or recombinant origin and is preferably used as an aqueous solution. The oxygen transfer agent preferably contains albumin in a concentration between 2 and 20 g / dl, concentrations between 5 and 15 g / dl are preferred. The weight ratio between hemoglobin-hydroxyethylstarch conjugate and albumin in the oxygen transfer agents that are preferred according to the present invention can be between 1:10 and 4: 1. Since albumin is considerably less expensive than the conjugate and which can be used to achieve the osmotic pressure that is desired in the oxygen transfer agent, in particular oxygen transfer agents which are comparatively high in oxygen content are preferred. albumin and low content of hemoglobin-hydroxyethylstarch conjugates. The invention further relates to oxygen transfer agents containing conjugates of P1003 / 98MX hemoglobin-hydroxyethylstarch and albumin and that have a particularly good vascular tolerability. According to a particular preferred embodiment of the invention, the conjugates described are mixed with albumin, preferably human serum albumin, which has been previously saturated with nitrogen monoxide. Hemoglobin and albumin have the property of complexing with NO in the N-nitroso form (see Keaney et al., J. Clin.Invest., 91, (1993) 1582-1589). As a rule, cross-linked HBOC products no longer have cooperative properties. Therefore, they lack the capacity for cooperative NO liaison. In the context of the present invention, it has surprisingly been found that this deficiency of the hemoglobin-hydroxyethyl starch conjugates can be compensated for by employing an oxygen transfer agent, which in addition to the conjugate contains an albumin solution that has formed complex with the NO. The saturation of albumin with NO is carried out by gassing a solution of albumin with NO excluding oxygen. The vascular tolerability of the product is further improved as a consequence. The present invention relates in particular to the use of the oxygen transfer molecules according to the invention and of the compositions of the hemoglobin-hydroxyethylstarch and albumin conjugates P1003 / 98MX as blood substitutes, plasma expanders, perfusion agents, hemodilution agents and / or as a cardioplegic solution.

EXAMPLE: Preparation of a hemoglobin-hydroxyethylamido conjugate.

A.1 Oxidation of terminal hydroxyethyl amide reducing groups: In a preferred process of the invention, the terminal reducing groups of the hydroxyethyl starch are selectively oxidized. First, 2 ml of a 0.1 N iodine solution was added dropwise to a solution of fractionated hydroxyethyl starch (MW <4 kDa, heavy amount approximately 0.56 mmol) which was made with less than 3 ml of deionized water. Then approximately 3.3 ml of a 0.1 N KOH solution are added at room temperature until the color originating from the iodine disappears. By repeating the previous step, 14 ml of iodine solution and 23 ml of the KOH solution were added to the reaction batch and the mixture was stirred for a further 30 minutes. The solution was purified by chromatography on a cation exchange column (Amberlite IR 120, form H). After diafiltration on a membrane of P1003 / 98MX regenerated cellulose (Millipore PLAC 076 10) with an exclusion limit of 1,000 Da, the partially concentrated solution was lyophilized. However, cation exchange chromatography can also be carried out after diafiltration. The yield was of the order of 80-90%.

A.2 Alternative process for the oxidation of hydroxyethylstarch terminal reducing groups: First, 2 ml of a 0.1 N iodine solution was added dropwise to a hydroxyethyl starch solution (MW <10 kDa, approx. less than 3 ml of deionized water. Then 0.1 N KOH solution was added at room temperature (RT) until the coloration originating from the iodine disappeared. By repeating the previous step, 14 ml of iodine solution and 23 ml of KOH solution were added to the reaction batch and the mixture was stirred for a further 30 minutes. Then, the solution was subjected to dialysis with an exclusion volume of the dialysis membrane of approximately 9 kDa. After purification by chromatography on a cation exchange column (Amberlite IR-120), the solution was lyophilized. The yield was of the order of 85%.

P1003 / 98MX B.1 Deoxygenation of hemoglobin by gassing: Bovine hemoglobin in a concentration of 6 g / dl in 0.5 M NaCl, 0.1 M Na2HP04 and 0.05 M NaHCO3 was deoxygenated by gassing. Hemoglobin was initially present in the CO form in a ratio between 94 and 96%. The deoxygenation was carried out in a closed vessel, in which the hemoglobin solution was circulated on a heat exchanger, while the membrane was first gassed first with 02 for partial oxygenation and then with N2 at 10 psi pressure. psi. The deoxygenation was finished until reaching a content of 70% deoxyhemoglobin. The deoxyhemoglobin was then lyophilized by gassing with N2.

B.2 Deoxygenation of hemoglobin by reducing chemical agents. Bovine hemoglobin at a concentration of 6 g / dl in 0.5 M NaCl, 0.1 M Na2HP04 and 0.05 M NaHC03, was chemically reduced. For this, 100 mM of sodium disulfite was added to the hemoglobin solution. After one hour, the resulting solution contained deoxyhemoglobin in a proportion of 75%. Sodium disulfite was removed by ultrafiltration to a 50 kDa membrane exclusion limit. The hemoglobin was lyophilized with gassing with N2.

P1003 / 98MX C Coupling of hemoglobin to the terminal oxidized groups of the hydroxyethyl starch: In each case about 1 to 1.5 g of the hemoglobin prepared according to steps Bl and B.2 were dissolved in 15 ml of DMSO and the mixture was transferred to a 100 ml three-necked round flask. A solution of 0.5 ml of DMSO and hydroxyethylstarch oxidized according to A was added slowly at 40 ° C. After stirring at 40 ° C for one to two days, the residue was freed from the solvent by dialysis and partially concentrated with the aid of diafiltration. The purity of the product, in particular the elimination of the starting substances, can be further improved by incorporating standard methods of chromatography and ultrafiltration. The success of the coupling reactions was confirmed with the help of gel permeation chromatography.

P1003 / 98MX

Claims (27)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. Oxygen transfer agent comprising a conjugate hemoglobin-hydroxyethyl starch, which is characterized wherein the hemoglobin and the hydroxyethyl starch in the conjugate are selectively linked together via amide linkages between the free amino groups of the hemoglobin and the terminal reducing groups of the hydroxyethyl starch, which is present in the oxidized form. Oxygen transfer agent according to claim 1, characterized in that the hemoglobin-hydroxyethyl starch conjugate is present in the oxygen transfer agent in a concentration between 2 and 20 g / dl. 3. Oxygen transfer agent according to claim 2, characterized in that the hemoglobin-hydroxyethylstarch conjugate is present in the oxygen transfer agent in a concentration between 5 and 20 g / dl. Oxygen transfer agent according to one of claims 1 to 3, characterized in that the P1003 / 98MX hemoglobin is of human, animal, plant or recombinant origin. Oxygen transfer agent according to one of claims 1 to 4, characterized in that the hemoglobin is of bovine origin. Oxygen transfer agent according to one of claims 1 to 5, characterized in that before coupling to hydroxyethylstarch, hemoglobin is present as deoxyhemoglobin and hemoglobin in other derivation states, such as CO-, 02- or methemoglobin. Oxygen transfer agent according to one of claims 1 to 6, characterized in that before coupling to hydroxyethyl starch, hemoglobin is present as a mixture of deoxyhemoglobin and oxyhemoglobin, the deoxyhemoglobin content is between 20 to 80% and the Hemoglobin content in other derivation states is between 80 and 20%. Oxygen transfer agent according to one of claims 1 to 7, characterized in that the hemoglobin is cross-linked and / or polymerized hemoglobin. Oxygen transfer agent according to one of claims 1 to 8, characterized in that the hydroxyethyl starch has an average molecular weight between 1 and 40 kDa. P1003 / 98MX 10. Oxygen transfer agent according to claim 9, characterized in that the hydroxyethyl starch has an average molecular weight between 2 and 20 kDa. Oxygen transfer agent according to one of claims 1 to 10, characterized in that the hydroxyethyl starch has a degree of molar substitution between 0.1 and 0.8 and a substitution ratio C2: C6 in the range between 2 and 20, in each case based on the hydroxyethyl groups. 12. Oxygen transfer agent according to one of claims 1 to 11, characterized in that the agent also contains albumin. 13. Oxygen transfer agent according to claim 12, characterized in that the albumin is serum albumin of human, animal, plant or recombinant origin. 14. Oxygen transfer agent according to one of claims 12 or 13, characterized in that the albumin is present in a concentration between 2 and 20 g / dl. Oxygen transfer agent according to one of claims 12 to 14, characterized in that the weight ratio between the conjugate hemoglobin-hydroxyethylstarch and the albumin is in the range between P1003 / 98MX 1:10 and 4: 1. Oxygen transfer agent according to one of claims 12 to 15, characterized in that before addition to the conjugate, the albumin is presented in aqueous solution saturated with nitrogen monoxide (NO). 17. Oxygen transfer agent according to one of claims 12 to 16, characterized in that the agent is presented as an aqueous solution or as a lyophilisate. 18. Process for the preparation of an oxygen transfer agent comprising a hemoglobin-hydroxyethylstarch conjugate, which is characterized in that the terminal reducing groups of the hydroxyethyl starch are first oxidized and then the hemoglobin is coupled to the terminal oxidized groups of the hydroxyethyl starch via free amino groups via amide bonds. 19. Process according to claim 18, characterized in that the oxidation of the terminal reducing groups of the hydroxyethylstarch is carried out first by mixing the hydroxyethylstarch with a solution containing iodine and then adding potassium hydroxide solution. Process according to one of claims 18 or 19, characterized in that the binding of the free amino groups of the hemoglobin with the reducing groups P1003 / 98MX Hydroxyethylstarch terminals which is presented in its oxidized form is carried out by mixing the individual components 40 ° C. 21. Process according to one of claims 18 to 20, characterized in that the hemoglobin is of human, plant animal or recombinant origin. 22. Process according to one of claims 18 to 21, characterized in that the hemoglobin is of bovine origin. 23. Process according to one of claims 22 to 22, characterized in that before coupling to hydroxyethyl starch, hemoglobin is presented as deoxyhemoglobin or as a mixture of deoxyhemoglobin and hemoglobin in other derivation states, such as CO-, 02- or methemoglobin. . Process according to one of claims 18 to 23, characterized in that before coupling to the hydroxyethyl starch, the hemoglobin is presented as a mixture of deoxyhemoglobin and hemoglobin in other derivation states, the deoxyhemoglobin content is between 20 and 80% and the content of hemoglobin in other derivation states is between 80 and 20%. 25. Process according to one of claims 18 to 24, characterized in that the hemoglobin is hemoglobin. reticulated and / or polymerized. P1003 / 98MX 26. Process according to one of claims 18 to 25, characterized in that the hydroxyethyl starch has an average molecular weight between 5 and 40 kDa, a degree of molar substitution between 0.1 and 0.8 and a substitution ratio C2: C6 in the range between 2 and 20, in each case based on the hydroxyethyl groups. 27. Use of an oxygen transfer agent according to claims 1 to 17 or prepared according to claims 18 to 25 as a blood substitute, plasma expander, perfusion agent, hemodilution agent and / or cardioplegic solution. P1003 / 98MX