WO1986004240A1 - Plasma substitute - Google Patents

Abstract

A plasma substitute is comprised of a water-soluble polymer which is biocompatible with phosphate, sulphate and/or sulphonate groups. These products are obtained by coupling the polymer with compounds containing phosphate, sulphate and sulphonate groups, which compounds are capable of fixing the hemoglobine by adsorption. Preferably, dextrane is used as polymer constituent and inositolhexaphosphate is used as hemoglobine-related compound.

Description

 Blood substitute

description

The invention relates to a blood substitute based on biocompatible polymers and hemoglobin.

Oxygen-transporting preparations with and without plasma expander effects are already known as blood substitutes. If you leave the fluorocarbons because of their fully synthetic

Of character, there are still a number of semi-synthetic and natural products.

The simplest preparation to produce is the stroma-free hemoglobin solution. Since this contains neither membrane structures nor antigen determinants, the problems of typing and sensitization are eliminated. The main disadvantage of the stroma-free hemoglobin solution is the too short intravascular half-life, which should be six to twelve hours for a blood or plasma substitute. The average circulating half-life for batches of 6% stroma-free hemoglobin solutions is 100 to 140 minutes. 240 minutes after the exchange of 2 ml / kg of whole blood, only approx. 20% stroma-free hemoglobin solution can be detected in the bloodstream. Another disadvantage of the stroma-free hemoglobin solution is the oxygen binding curve that is clearly shifted to the left in vitro compared to the oxygen dissociation curve of normal blood. This means that the oxygen affinity of the hemoglobin is significantly increased and that the oxygen in the tissue is released too difficult again. The stroma-free hemoglobin solution can only be accepted as a blood substitute if, on the one hand, it is possible to increase the intravascular residence time and on the other hand the release of oxygen from to facilitate the oxygen-loaded hemoglobin tetramer.

Methods for solving the problem of the intraversal residence time of the stroma-free hemoglobin solutions being too short are known. You can e.g. B. either bind the hemoglobin tetramer one or more times via covalent chemical bonds to a polymeric soluble support or polymerize to shorter units with other hemoglobin tetramers to form larger units. This procedure gives units which mostly contain several hemoglobin tetramers and are so large that they can no longer be excreted by the kidney without prior chemical-physiological degradation. Apart from the different residues that are necessary for the covalent bond or act as a polymeric carrier, the hemoglobin tetramer is always changed by the covalent bond at at least one reactive site. However, since the accessible reactive groups in hemoglobin are also responsible for their typical biological properties such as oxygen and carbon dioxide transport, a covalent bond to these groups also necessarily leads to a different biological function of the hemoglobin. Although preparations could be made whose intravascular half-life is sufficient for twelve hours, there is usually an increase in oxygen affinity at the same time. On the other hand, a stroma-free hemoglobin solution can be prepared by coupling pyridoxal phosphate to the terminal amino groups with a permanently reduced oxygen affinity. To improve the intravascular half-life, there are products in which several hemoglobin molecules are chemically covalently linked to one another by binding units. Through this process of covalent linkages, these products are in principle comparable to the polymer-bound products.

The invention has for its object a hemoglobin to be developed with an increased intravascular residence time and reduced oxygen affinity.

This object is achieved in that the hemoglobin is absorptively bound with a water-soluble polymer via phosphate, sulfate and / or sulfonate groups. Subclaims 2 to 4 relate to advantageous developments of the blood substitute according to the invention.

The agent according to the invention is produced by activating a water-soluble polymer by introducing free OH, NH, SH and / or COOH groups and then with a compound which has the ability to bind hemoglobin absorptively and phosphate and sulfate - And / or contains sulfonate groups, is reacted and that the resulting product is then brought together with hemoglobin. Further developments of the method according to the invention are described in subclaims 6 to 10.

According to the invention, affinors which have a high affinity for hemoglobin and at the same time shift the oxygen dissociation curves to the right are covalently bound to various polymers which act as a carrier compound. It is thereby achieved that the polymer molecule is adsorbed firmly on hemoglobin via the affinity component and thus the elimination of the hemoglobin via the kidney is made more difficult by the higher molecular weight. At the same time, the oxygen affinity of the adsorptively bound hemoglobin is reduced compared to the stroma-free hemoglobin.

According to the invention, any known plasma substitute can be used as the polymer. However, polysaccharides, in particular dextran, are preferably used.

In particular, phosphoric acid or derivatives 8-hydroxy-1,3,6-pyrenetrisulfonate, pyridoxal phosphate, N- (2,4-diphosphobenzyl) -1-amino-5-naphthalenesulfonic acid, 2,3-disphosphoglycerate, adenosine triphosphate, inositol tetraphosphate, inositol pentaphosphate, inositol hexasulfate and preferably inositol hexaphosphate are suitable. On the one hand, inositol hexaphosphate in the series 2,3-diphosphoglycerate / adenosine triphosphate / inositol tetraphosphate / inositol pentaphosphate / inositol hexasulfate / inositol hexaphosphate causes the greatest decrease in the oxygen affinity of hemoglobin. On the other hand, the hemoglobin / inositol hexaphosphate complex has the lowest dissociation constant in the series: 2,3-diphosphoglycerate / inositol pentaphosphate / inositol hexasulfate / inositol hexaphosphate. Apart from these suitability factors, inositol hexaphosphate occurs naturally in the human organism. In principle, there are compounds that have an even greater affinity for hemoglobin and an even smaller one

Dissociation constants of the corresponding complex have as inositol hexaphosphate. These are above all analogue substances which have more than six phosphoric acid or sulfate residues.

The water-soluble polymer is activated to produce the blood substitute according to the invention. Using the example of dextran, this can be carried out synthetically most simply and with the best conversion rates by catalytic reaction with epichlorohydrin. As a catalyst e.g. Zinc tetrafluoroborate used. In this way, the non-reactive compound (I) is initially created, which does not already lead to crosslinking during production. In the alkaline medium, this compound (I) converts to the very reactive epoxy variant (II):

Die Kopplung des Hämoglobin-Affinors, z.B. Inositolhexaphosphats (IHP), an lösliches Dextran kann dann z.B. entsprechend folgender Reaktion erfolgen:

The hemoglobin affinor, for example inositol hexaphosphate (IHP), can then be coupled to soluble dextran, for example, in accordance with the following reaction:

where R represents an inositol ring with five remaining phosphoric acid groups.

Likewise, a product according to the invention can be prepared starting from 3-amino-2-hydroxypropyl-dextran (IV), which is obtained by reacting 3-chloro-2-hydroxypropyidextran (I) or the epoxy variant (II) in an aqueous medium with ammonia can be. In this case the unreactive amino group of 3-amino-2-hydroxypropyl-dextran (IV) has to be activated, e.g. by dicyclohexylcarbodiimide (DCC) or epichlorohydrin.

wobei R die obige Bedeutung besitzt.

where R has the meaning given above.

To achieve an increase in the number of phosphate groups in the affinor, several activated inositol hexaphosphate groups or simply phosphoric acid groups can be used

Polyamines, e.g. Triethylenetetramine or polyethyleneimine groups can be locally enriched on the modified dextran.

For this epoxy activated dextran e.g. reacted with triethylenetetramine. This has two terminal primary amino groups, one of which is reacted in the reaction with 2,3-epoxypropyldextran. The resulting product contains 3 secondary and one primary amino group. These amino groups serve as reaction centers in the reaction with the

Epichlorohydrin activated phosphate groups, the inositol hexaphosphate activated with epichlorohydrin and the alkali metal hydrogen phosphate also treated with epichlorohydrin.

The invention is illustrated by the following examples:

Example 1

Activation of dextran with zinc tetrafluoroborate / Epi chlorhvdrin

In a 100 ml three-necked flask with reflux condenser and dropping funnel, 5 g of dextran (M = 81600) are dissolved in a mixture of 5 ml of water and 7.5 ml of 25% zinc tetrafluoroborate solution while heating in an oil bath. When the temperature has reached 80 ° C, a total of 25 ml of epichlorohydrin is slowly added dropwise with vigorous stirring. The mixture is stirred at 80 ° C for three hours and then at room temperature for about 10 hours.

The polymer is precipitated by pouring it dropwise into 1 liter of acetone, filtered off, washed with acetone and dried in vacuo. For further purification, the product is repeatedly dissolved in a little water and alternately precipitated by dropping it in acetone or methanol. 4 g of 3-chloro-2-hydroxypropyl-dextran are obtained as a white powder which is soluble in water. Average chlorine content: c a. 3%.

Reaction of 3-chloro-2-hydroxypropyl-dextran with triethylenetetramine

10 g of 3-chloro-2-hydroxypropyl-dextran (M = 4oooo) are dissolved in 1 liter of water (pH 9). After stirring for a while, 10 ml of triethylenetetramine are added and stirring is continued at room temperature overnight. The mixture is then, as usual, ultrafiltered and the concentrate is precipitated by stirring in acetone. Elemental analysis nitrogen: 4.92%

Chlorine: 0.44%

Production of activated inositol hexaohoschat

54 g of sodium phytate pH 7 are dissolved in 100 ml of water, 10 ml of epichlorohydrin are added and the mixture is magnetically stirred at 30 ° C. for 36 hours. Then the solution is on Rotary evaporator he evaporated to dryness. A fine, white powder is obtained. Elemental analysis chlorine: 10.6% phosphorus: 15.1%

Reaction of triethylenetetramine-modified dextran with activated inositol hexaohosphate

3-Chloro-2-hydroxypropyl-dextran modified with triethylenetetramine is dissolved in water, mixed with an aqueous solution of the activated phosphate and stirred. The reaction solution is then concentrated in an ultrafiltration cell over a PM-10 membrane. The reaction product is precipitated by pouring it into methanol. After the precipitation has settled, the product is filtered off with suction, washed with methanol and dried. Average phosphorus content: approx. 6%

Example 2

Preparation of 3-amino-2-hvdroxvoropyl-dextran

4 g of 3-chloro-2-hydroxypropyl-dextran are prepared as described in Example 1 and dissolved in a 200 ml Erlenmeyer flask in a mixture of 60 ml of water and 20 ml of 25% aqueous ammonia solution and magnetically stirred at room temperature for 20 hours. The solution is then added dropwise to 1 liter of methanol. The precipitate formed is filtered off, washed with acetone and dried in a vacuum desiccator. For cleaning, the product is also repeatedly dissolved in a little water and precipitated by dropping in 1 1 of methanol. 3.5 g of white powder are obtained. Average nitrogen content: approx. 1%.

Reaction of 3-amino-2-hydroxyprooyl-dextran with sodium phytate 18 g of sodium phytate are gradually introduced into 200 ml of water with stirring and simultaneous cooling with ice water. After the addition has ended, the mixture is stirred until the solution is clear. Then it is adjusted to pH 7 with hydrochloric acid and 2 g of 3-amino-2-hydroxypropyl-dextran and 300 ml of hexamethylphosphoric triamide are added with stirring.

10 g of dicyclohexylcarbodiimide are mixed in a mixture of 40 ml of hexamethylphosphoric triamide and 20 ml

Dissolved water (possibly with heating) and then added dropwise to the above solution. After the addition has ended, the solution is stirred at room temperature for 48 h. The hexamethylphosphoric triamide is removed in vacuo and the aqueous solutions are ultrafiltered. The concentrates are freeze-dried. Average phosphorus content: approx. 8%.

Example 3

Implementation of Phosohatouffer oH 7 with epichlorohydrin

142 g (1 mol) of disodium hydrogen phosphate and 136 g (1 mol) of potassium hydrogen phosphate are dissolved in 2 l of water and the solution is adjusted to pH 7 with dilute sodium hydroxide solution. Then 111 g (1.2 mol) of epichlorohydrin are added and the mixture is stirred for 72 hours at room temperature. The volume of the solution is then concentrated to one liter on a rotary evaporator. For the analysis, 50 ml of this solution are evaporated to dryness.

Elemental analysis chlorine: 8.2% phosphorus: 14.4%

The activated phosphate is reacted with triethylenetetramine-modified dextran by the process described in Example 1. Average phosphorus content: approx. 5% Example 4

Reaction of phosphoric acid with epichlorohydrin

10 g of epichlorohydrin are added dropwise to 20 g of 84% phosphoric acid with stirring and ice cooling within 45 minutes. After the addition is complete, the mixture is stirred for a further 30 minutes. It is then diluted with water to 350 ml and the solution is then gradually added with a total of 75 g of barium hydroxide with stirring until an alkaline reaction.

The further reaction with activated dextran takes place according to the method described in Example 1 or 3. Average phosphorus content: approx. 5%

Example 5

The inositol hexaphosphate / dextran coupling products are examined by gel chromatography for the coupling and their suitability for adsorption on hemcglobin. Different mixtures of the coupling products with hemoglobin are separated by gel chromatography according to their molecular weight. In contrast to pure hemoglobin, in which only a single sharp peak always occurs, the chromatograms of the mixtures of hemoglobin with the coupling products always show two peaks which are more or less separate depending on the chromatography conditions (gels, eluents, etc.). This shows that, in addition to the pure hemoglobin, the fraction of a higher molecular weight, hemoglobin-containing product is present, which can be separated off by gel chromatography.

The coupling products cause the expected right shift of the oxygen dissociation curve of the hemoglobin. activated phosphoric acid or with activated 8-hydroxy-1,3,6-pyrene-trisulfonate, pyridoxal phosphate, N- (2,4-diphosphobenzyl) -1-amino-5-naphthalenesulfonic acid, 2,3-diphosphoglycerate, adenosine triphosphate, inositol tetraphosphate, inositol pentaphosphate and / or inositol hexasulfate, preferably inositol hexaphosphate is reacted.

10. The method according to any one of claims 5 to 9, characterized in that a plasma substitute, preferably dextran, is used as the polymer.

Claims

Biospatial patent applications 1. blood replacement based on biocompatible polymers and hemoglobin, characterized in that the hemoglobin with a water-soluble polymer Phosphate, sulfate, and / or sulfonate groups is bound by adsorption. 2. Blood substitute according to claim 1, characterized in that for the adsorptive binding of hemoglobin the polymer at several points with phosphoric acid groups or with 8-hydroxy-1,3,6-pyrentrisulfonate, pyridoxal phosphate, N- (2,4-diphosphobenzyl) -1 -amino-5-naphthalene-sulfonic acid, 2,3-diphosphoglycerate, adenosine triphosphate, inositol tetraphosphate, inositol pentaphosphate and inositol hexasulfate, preferably inositol hexaphosphate, is chemically bound. 3. Blood substitute according to claim 1 or 2, characterized in that 4 to 20 phosphate, sulfate and / or sulfonate groups are present at each binding site. 4. Blood substitute according to one of claims 1 to 3, characterized in that the polymer is a plasma substitute, preferably dextran. 5. A method for producing the blood substitute according to one of claims 1 to 4, characterized in that a water-soluble polymer is activated by introducing free OH, NH, SH and / or COOH groups and then with a compound which has the ability has to bind hemoglobin adsorptively and contains phosphate, sulfate and / or sulfonate groups, is reacted and that the resulting product is then brought together with hemoglobin. Process according to claim 5, characterized in that the compound which adsorbs the hemoglobin is activated before the reaction with the polymer, preferably with epichlorohydrin. 7. The method according to claim 5 or 6, characterized in that the polymer is activated by reaction with epichlorohydrin in the presence of a catalyst or with dicyclohexylcarbodiimide. 8. The method according to any one of claims 5 to 7, characterized in that the polymer activated with epichlorohydrin is reacted with a low molecular weight polyamine, preferably with triethylene tetramine or polyethyleneimine, for the further introduction of reactive groups. 9. The method according to any one of claims 5 to 8, characterized in that the activated polymer with