JPH01500524A - Hemoglobin polymer complex, its preparation method and its application fields - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Hemoglobin polymer complex, its Preparation method and its field of use The present invention provides a new polymer composite, its preparation method, and its use as an oxygen conductor. It concerns its field of use within the framework of blood transfusion work.

In the form of an intravenous injection, an aqueous hemoglobin solution without stroma and isotonic with blood is administered. We know that it is possible to inject.

However, one of the drawbacks of hemoglobin is its small size. The point is that it does not stay within the blood circulation path but diffuses outside the circulatory system. .

To correct this shortcoming, i.e., blood ``substitutes'' or oxygen conductors, Increasing the intravascular persistence of 16-hemoglobin within the framework of its use as a solute Therefore, several methods have been used.

For example, hemoglobin is added to a water-soluble polymer that is non-toxic, non-antigenic, and blood-compatible. It was done to fix it.

Thus, the number of hemoglobins modified by chemical fixation of water-soluble polymers Numerous examples show that it is possible to significantly increase the residence time of hemoglobin within an organism. It is known that

Of the various polymers used, the most common are polysaccharides, especially dex Tolan (French Patent No. 2328.478), hydroxyethyl starch (French Patent No. 2328.478), Lance Patent No. 2328.478), Inulin (European Patent Application No. 4) 3.675) and polyalkyl-1/-glycols, especially polyethylene glycols. It is a rule.

However, in any case, direct fixation of these polymers does not result in denaturation. Hemoglobin has oxygen-retaining properties that make it unsuitable for use in blood transfusions.

In fact, blood substitutes require that the compounds they form with oxygen are reversible, i.e. has the ability to fix (in this case hemoglobin is in its oxygenated form) Sometimes it can be easily desorbed from oxygen (in this case hemoglobin is in the deoxygenated form). only insofar as there is a role equivalent to that played by natural intraerythrocytic hemoglobin. I want to remind people that they can play a role.

This property for oxygen is present in the atmosphere to which hemoglobin is exposed. The curve corresponding to the change in the amount of oxygen retained by the mass unit of the conductor depending on the partial pressure of oxygen. It is characterized by a line (called a Barcroft curve).

For an aqueous solution of native hemoglobin (concentration 15 lmol per liter), this The changes are indicated by the reference line on Figure 1 at 25°C and pH 7.2. Ru.

One of the parameters tied to this curve is that the hemoglobin aqueous solution combines It is attached in such a way that it absorbs a mass of oxygen corresponding to 50% of the maximum amount that can be This is the half-saturated pressure (Pso), which is the partial pressure of oxygen that is essential. However, as mentioned above For modified hemoglobin (polymer-conjugated hemoglobin) such as The curve in Figure 1 showing the proportion of oxygen complexed to hemoglobin under oxygen partial pressure ( dotted line) to the left in relation to the curve corresponding to native hemoglobin (solid line). undergoes parallel movement.

This means that the pressure of denatured hemoglobin is Ps.

This means that the value of hemoglobin is lower than that of natural hemoglobin. In other words, degenerated hemo Globin has such a strong affinity for oxygen that oxygen is washed out. This results in the disadvantage of not being able to rapidly reintegrate into the organization Ru.

This modification of the oxygen retention properties of modified hemoglobin is inexplicable for the following reasons, among others. Natural hemoglobin is a deoxyhemoglobin inside red blood cells. The affinity of hemoglobin for oxygen by binding to amines in the allosteric site of Diphospho-2,3-glycerate (2 , 3DPG).

Improvements to correct this drawback include diphospho-2゜3-glycerin salt tolerance or hexyl The effects of transient effectors (or ligands) such as inositol salinate bond between the polymer and hemoglobin in the absence of oxygen It was presented by (French Patent No. 83145.45) The phosphated substance significantly duplicates the 70 steric sites of deoxyhemoglobin. It stabilizes deoxyhemoglobin, which acts as an oxygen transporter. protects the amino sites important for Seth.

However, these effectors are involved in the purification of the hemoglobin polymer complex. or purification may cause removal of the effector due to plasma dissociation conditions. If it is not purified, it is removed from the plasma.

To compensate for this drawback, it is necessary to resort to permanent effectors, e.g. Fixing Dogsal-5'-phosphate (Japanese Published Patent No. 59 10432) No. 3) was proposed.

It also significantly reduces its affinity for oxygen after immobilization on hemoglobin. It has also been proposed to use ligands with multiple phosphate groups.

However, one of the drawbacks of these compounds is that they require hemolysis to obtain them. Multiple operations must be used for the globin, in any case more than one operation. This may induce the formation of degradation products, especially methemoglobin. It's on point.

Furthermore, in European Patent Application Specification Nos. 142 and 125, on the other hand, on the other hand and from hemoglobin on the other hand. , the bond of the adduct compound in the hemoglobin Gantt polymer is an ionic bond. , ligand-polymer/hemoglobin ion complexes have also been proposed.

These complexes can reduce hemoglobin's affinity for oxygen .

However, purely ionic reactions between hemoglobin and ligand-polymer adducts This bond is only strong when hemoglobin is in its deoxygenated form. Ru. That is, when hemoglobin is re-diamined, a ligand-polymer addition compound The substance is released from the phosphate binding site of hemoglobin, and the adduct and hemoglobin are There will no longer be strong ionic interactions between the bottles.

When such an ionic complex is injected into an organism (living organism), it becomes highly specific to hemoglobin. Ligand-polymer adducts that are not bound to When the hemoglobin is not present, it exits the organism rapidly and the hemoglobin exits the vascular circulation. You can expect that.

The purpose of the present invention is to provide a system in which the extravascular diffusion rate is limited (and even eliminated). size, protected against plasma dissociative conditions, and its oxygen retention properties unchanged Improved over that of free hemoglobin, its preparation is a reaction to hemoglobin We propose a macromolecular complex of hemoglobin with a limited number of steps. The aim is to correct these shortcomings by

These objectives are achieved by the new polymeric conjugates according to the invention.

One of the objects of the present invention is to provide a physiologically compatible and reversible fixation of oxygen. The objective is to provide a new polymer complex that can be used with.

Another object of the present invention is to restore oxygen more easily than with 5M hemoglobin. The objective is to provide a new polymer complex that can be used with.

The present invention further proposes a new polymer complex that can be easily synthesized on an industrial scale. That is its purpose.

It is also an object of the invention that hemoglobin is present in at most two stages, generally is a new high-level hemoglobin obtained using a method that requires only one step. The purpose is to provide child complexes.

Another object of the invention is to provide blood substitutes, in particular blood transfusions or continuous vascular infusion of organs. A new hemoglobin polymer complex that can be used in surgeries requiring The objective is to propose a new aqueous solution that includes coalescence.

The present invention further provides that for M# hemoglobin, increasing Pw within the tester increases the Hemoglobin, whose oxygen-retaining properties remain stable in vivo Its purpose is to provide a solution.

Another object of the invention is to have a mild affinity for oxygen and a high hydrodynamic volume. At the same time, it was shown that hemoglobin remained in blood vessels due to suppression of hemoglobinuria during blood transfusion experiments. To propose a new hemoglobin polymer compound that causes an increase in the amount of hemoglobin. be.

The new novel hemoglobin according to the invention exhibits a lower affinity for oxygen than that of free hemoglobin. The hemoglobin polymer complex is a polymer with the following characteristics. - The polymer complex is composed of the following; - Hemoglobin that can be reversibly transferred from a deoxygenated form to an oxygenated form.

Φ Contains a polar group, preferably a hydroxyl group, a carboxyl group or an amine group, about 10 00 to approximately 500,000, preferably approximately 100,000 to approximately 100,000, non-toxic, and non-toxic. If so, the polymer P is non-antigenic, blood compatible and water soluble.

- the polymer complex is made of sulfates and/or phosphates and/or carboxylates; on the polymer P containing one or more negative charges possessed by the anionic groups consisting of Having a part Z that is fixed.

- the polymer P is bound to hemoglobin via: ・On the other hand, at least one of the sites Z of the polymer and hemoglobin through at least one ionic bond formed between ・On the other hand, a polymer and hemoglobin or one site (ZO) where a polymer exists and a hemoglobin - through at least one covalent bond formed between the moglobins - to the polymer. The number of covalent complexes between moglobins is about 70,000 to about 1 million, Preferably, it should have an average molecular weight of about 70,000 to about 500,000.

- the relationship between the number of negative charge 1 sites and the number of monomers is as follows: ・If each site contains only one anionic group consisting of sulfate or phosphate, the polymer There is at least one site for every 10 monomers.

・ At least one of the moieties consists of sulfate and/or phosphate. If both contain two anionic groups, at least one site on the polymer be.

・If each site contains a negative charge from the carboxylate, the same site Z 2 has at least two carboxyl (carboxylate) groups, and every fifth monomer has at least two carboxyl (carboxylate) groups There must be one or more parts Z.

- at least one of the sites contains three or more negative charges from the carboxylate; If so, there is at least one site for the polymer.

The polymer complex according to the invention is water-soluble, non-toxic and preferably non-antigenic. The conditions necessary for blood compatibility are included in the composition of the above-mentioned macromolecular complex. The potential polymer P is water-soluble, non-toxic, preferably non-antigenic, and blood-soluble. It turned out that they were compatible.

Furthermore, the part Z, which is the carrier of the anionic group that plays the role of a permanent effector, is Due to its presence on the chain, 50% of hemoglobin in solution is oxygenated. pressure will be increased, possibly due to the presence of free ligand. It was also confirmed that this cannot be said to be the case.

These permanent effectors are similar to the conformation of the hemoglobin molecule in its deoxygenated form. With strong stabilization, it converts hemoglobin from the deoxygenated form to the oxygenated form. allows for reverse migration, thus reducing hemoglobin's affinity for oxygen. It is something that causes a reduction.

In other words, these effectors allow hemoglobin to reversibly transport oxygen. , oxygen can be easily salted out, especially within tissues that are being washed out.

The covalent bond provides the macromolecular complex according to the invention with the ability to perform an oxygen retention function. There is no or very little life in the plasma medium for a critical period of time, i.e. 2-3 days. provides stability that prevents hemoglobin from being broken down and thus It is like eliminating extravascular spread.

In the macromolecular complex according to the invention, hemoglobin has at least one covalent Due to binding, however, there is an intermolecular reticulation phenomenon that particularly impairs the physical properties of hemoglobin. If the critical number of covalent bonds between the polymer and hemoglobin is exceeded, It's attached.

This critical number of covalent bonds is determined when the average molecular weight of the polymer complex according to the present invention is approximately 1 million. consistent with the fact that

The distribution of negative charge density is caused by the allosteric sites of hemoglobin, i.e. natural red blood cells. Internal effector, ionic bond with amine of diphospho-2,3-glycerate site It must be sufficient to form a

Statistically speaking, one sulfate or phosphate base per 10 monomers is required and sufficient. Although it is known that the ratio is 1/10 or more, the ratio may be greater than or equal to 1/l0.

Also, at least one of the sites consists of phosphate and/or T& salt ions. at a single site for all polymer chains if it contains at least two anionic groups. sufficient, but that does not exclude the possibility of having multiple sites on the chain. It's not like that.

Regarding the negative charge density in the case of carboxylates, at least two There must be 2 carboxylate ions for every 5 monomers. One site containing the ion is required.

If there are at least three carboxylate ions, the degree of charge at that site is One body part is sufficient for the body's frequency, but that does not mean that The possibility of having multiple sites on the chain is not excluded.

The polymers included in the composition of the macromolecular complex according to the invention that degrade in the plasma medium are about It has an average molecular weight of 1000 to about 500,000.

The polymers that enter into the macromolecular complexes according to the invention and do not degrade in organisms are about 1 It must have a molecular weight of less than 0,000, which means that when it comes to this belief, This is because polymers have difficulty crossing the kidney barrier and therefore accumulate within the organism. .

Polymers that do not biodegrade must have an average molecular weight of about 1000 or less. alkylene glycol, polyvinylpyrrolidine, polymethyl acrylate or This is the case for certain polysaccharides.

The polymer that enters into the construction of the macromolecular complex according to the invention is preferably non-antigenic. It must be used within the desired molecular weight range.

Therefore, in the case of dextran, its molecular weight must be less than about 70,000.

According to an advantageous embodiment of the invention, the internal Co〇- group and the NH,+ group of hemoglobin are The salt bridge between remains intact when hemoglobin is in its deoxygenated form .

Salt bridges are when hemoglobin is in its deoxygenated form. It refers to the type of intermolecular bond formed between the anion COO- and the anion COO-.

Salt bridges in the macromolecular complex according to the invention should preferably be intact. and This is because the ions that participate in the formation of these salt bridges are incorporated into other bonds. The transition of hemoglobin from the oxygenated to the deoxygenated form is This is because it occurs in a very difficult and imperfect way.

An "intact salt bridge" means that at least 50% of the salt bridge is unchanged and 80% of the salt bridge is ~90% and even lo.

It means that it is advantageous that the % does not change.

The criteria for determining whether a salt bridge is intact are, inter alia: : a) Affinity curve for oxygen; The affinity curve for oxygen (Parcroft curve) is similar to that of MM hemoglobin. If moved to the right in the relationship, this means that the salt bridge has not been modified. It implies that.

b) Hill coefficient (n); This parameter represents the sigmoid velocity of the Parkcroft curve, and can be used with some envy. That reflects the cooperative oxygen fixation properties. The value of n is the hemoglobin allos It makes it possible to assess the persistence of telic (molecular transformation) behavior. natural hemoglos For bins, this factor is between 2.7 and 3.0.

C) Bohr effect; This involves determining the oxygen retention behavior of hemoglobin at different pH levels and It consists of providing control over operations and being able to evaluate confusion.

According to another embodiment, the macromolecular conjugate according to the invention comprises a polysaccharide, in particular Inulin, a hydroxyalkyl starch in which the alkyl group has 2 to 4 carbon atoms , dextran and its derivatives, especially aminodextran, polyvinyl alcohol polymethacrylate and its derivatives, polypeptide, 2 to 5 alkene groups polyalkene glycols containing carbon atoms, especially polyethylene glycol and Selected from polypropylene glycol.

According to another embodiment of the invention, 2 originates from 0503H.

According to another embodiment of the invention, Z is pyridoxal sulfate, epinephrine Epinephrine 2'F&acid, epinephrine trisulfate, norepinephrine disulfate, norepinephrine Derived from nephrin trisulfate and phenolphthalein disulfate.

According to another embodiment of the invention, Z originates from OPO, H2.

According to another embodiment of the invention, Z is pyridoxal phosphate, adenosylated 3-phosphate, phosphotylosin, phosphoserine, inositol hexaphosphate From salts and derivatives thereof, nonositol 3-, 4-, 5-phosphate and derivatives thereof It is occurring.

According to another embodiment of the invention, Z is 0-CH(COOH)2, Co o)(.

-CH CH2 COOH arises from Note that n is a value from 1 to 4.

According to another embodiment of the invention, Z has about 2 to 10 carbon atoms in the main chain. polycarboxylic acids containing benzene carboxylic acids containing at least two carboxyl functional groups; Bonic acid, diphospho-2,3-glycerate, citric acid, propane 1,2,3) Derived from carboxylates or butane, tricarboxylate salts.

According to another embodiment of the invention, Z is a hydroxy-2-phot of the following formula: Lumyl-5-phosphoserine benzamide: CH20P03 H2 or the formula formyl-4-phosphoserine benzamide: H.O. CH20PO3H2 arises from.

According to another embodiment of the invention, site 2 includes at least three of the following: One of the functional groups is capable of immobilizing the Z group on the polymer. It is a functional group that makes it possible to

- One of the functional groups forms an ionic bond with the amine in the allosteric site of hemoglobin. A sulfate, phosphate or carboxylate functional group that can form.

- one of the functional groups is capable of forming a covalent bond with the NH2 group of hemoglobin Aldehyde, carboxyl or OH functionality.

The nature of the bond linking Z to the polymer will depend on the reaction elements present and reaction conditions. different.

The bond that integrates Z into the polymer is an ether, ester, amide or amine bond. It is.

For example, ester bonds are (after fixation H2SO4 gives one 0SO3H site, H3P0a gives one 0P03H site (gives 2): Ether bonds are obtained by chloride co /\ Obtained by the action of phosphoric acid or group grafting of acrylic acid (radical addition reaction) , the amide bond is between the NH2 group and the carboxyl (carboxylate) group of the Z group in the polymer. The amine bond is obtained by the reaction between the aldehyde of the NH2 group and the Z group of the polymer. Obtained by reaction between the groups followed by reduction. This last reaction is the same as the first reaction. It can also be done simultaneously (i.e. by reductive amination) . ).

The covalent bond between hemoglobin in the polymer is the carboxylic bond in the polymer or in the It is formed between the sil, aldehyde or hydroxyl group and the NH2 group of hemoglobin.

The bonds between polymeric hemoglobins are amide, imine, amine or carbamine. It is a combination of salts.

According to one embodiment of the invention, the covalent bond is directly attached to the aldehyde group of the polymer, the carboxyl group. It is formed between the xyl or hydroxyl group and the NH2 group of hemoglobin.

According to another embodiment of the invention, the covalent bond is a Z group fixed on the polymer. A form between the aldehyde, carboxyl or hydroxyl group of , and the NH2 group of hemoglobin will be accomplished.

According to a particularly advantageous embodiment of the invention, the covalent bond is with the NH2 group of hemoglobin. Formed between carboxyl groups.

The hemoglobin macromolecular complex according to the present invention is capable of producing 10 moles of glucobylanose. It is desirable that it contains about 5.4 moles of OSO,H groups, preferably about 40,000 moles. The polymer is composed of dextran with an average molecular weight, and the relationship between hemoglobin and the polymer is The covalent bonds between preferably about 18 aldehydes per 100 units of glucoside. Aldehyde groups preformed on dextran in proportion to NH of hemoglobin It is a macromolecular complex that is the result of a bond between two groups.

The hemoglobin macromolecular complex according to the present invention is capable of producing 10 units of glucopyranose. contains about 9.8 moles of 0303H groups and preferably has an average molecular weight of about 10,000. The polymer is composed of dextran with The bond is preferably in a ratio of about 14 aldehyde groups to 100 glucoside units. between the aldehyde groups preformed on dextran and the NH2 groups of hemoglobin. is a macromolecular complex that is the result of the combination of

Another hemoglobin polymer complex according to the invention is characterized in that the polymer preferably , contains about 1.7 sulfate ions per 10 units on glucopyrano and has an average molecular weight of Consisting of approximately 6000 dextrans, the covalent bonds between the polymer and hemoglobin are Preferably, the dextrin is present at a ratio of about 18 aldehyde groups per 100 glucoside units. Bond between the preformed aldehyde group on tolan and the NH2 group of hemoglobin It is a macromolecular complex that is the result of .

Another hemoglobin polymer complex according to the present invention is a polymeric hemoglobin complex containing 10 moles of polymer. Contains about 12 moles of OPOs H2 groups for lucopyranose, preferably about 4 It is composed of dextran with an average molecular weight of 0,000, and is composed of polymer and hemolyte. Preferably, the covalent bonds between the globins are about 18 per 100 glucoside units. Pre-formed aldehyde groups on dextran and hemoglobin in the proportion of aldehyde groups It is a macromolecular complex that is the result of bonds between the NH2 groups of the bottle.

Another hemoglobin-polymer complex according to the invention is characterized in that the polymer is preferably flat. It has an average molecular weight of approximately 40,000, is composed of aminodextran, and has ionic bonds. The carboxyl of benzenehexacarboxylic acid is immobilized on the polymer. The covalent bond is realized through the hexachloride (acid acid) group, and the covalent bond is not involved in the above ionic bond. via the carboxyl (carboxylate) group of carboxylic acid and the NH2 group of hemoglobin. It is a polymer composite, such as the one realized in

Another hemoglobin macromolecular complex according to the present invention is characterized in that the polymer consists of one monomer. contains an average of about 1 dibasic carboxylic acid per dibasic acid, preferably about 40,000 dibasic carboxylic acids. It is composed of polycarboxyl dextran with a uniform molecular weight, and the covalent bonds are Carboxyl group and NH of hemoglobin that are not involved in ionic bonding with hemoglobin It is a polymer complex realized through two groups.

To prepare the hemoglobin macromolecular complex according to the present invention, - In the first step, fixing the Z group on the polymer in the following way: ☆ If necessary, change the polymer to polyol and then fix the Z group by a known method. How to do it. Or ☆ Y is aldehyde, carboxyl, amine, hydroxyl, halo As active or activatable functional groups, such as hydrogen (e.g. chlorine), suitable Methods of using compound Z-Y using reagents. Or ☆ release of Z group to polymer P Methods of performing injectable grafting.

- Then, in a second step, in an aqueous medium of about 5 to 9 p)I, hemoglobin is Polymer P containing a Z group is reacted with hemoglobin under conditions that do not cause denaturation. to simultaneously form ionic and covalent bonds between the polymer and hemoglobin. .

Preparation of macromolecular composites according to the invention, consisting of fixing Z groups on polymers The first step is sulfuric acid esterification and phosphoric acid esterification of polymer P in the form of polyol. Alternatively, known methods such as carboxymethylation may be used. Polymer P is polyol If it is not a polyol, it must be converted into a polyol beforehand.

The first step in the preparation of the macromolecular complex according to the invention also involves replacing Y with an aldehyde, a carboxylic acid, Z-Y type conversion as xyl, amine, hydroxyl or halogen functional groups It can also be carried out using a compound. These Z-Y compounds can be prepared using conventional chemical techniques. For example, 5- or 6-carboxylic acid, diphosphoro-2,3-carboxylic acid, Pre-polyamined polymerization of lyceric acid, pyridoxal-5'-phosphate Can be fixed on the body.

In this case, remove all traces of the Z-Y compound by desalting with a gel filtration column, etc. It is essential to remove the In fact, even at low concentrations, the polymer complexes of the present invention reactions that, if encountered, can derail conclusions about the properties of this complex. The presence of Z-Y compounds that did not occur must be avoided.

Similarly, polyanionic chains can be immobilized onto polymers as in radiochemical grafting. Any other method can also be used.

The second stage mentioned above is the reaction stage between the polymer and hemoglobin, and if necessary In some cases, the activity of the polymer may be reduced before it is subjected to reaction with hemoglobin. The oxidation step may be performed first. However, in reality, this activation is It can be simultaneous with the reaction of moglobin.

During the second stage, the polymer reacts with hemoglobin, while site 2 of the polymer and hemoglobin react with hemoglobin. Ionic bonds between globin and covalent bonds between polymers and hemoglobin on the other hand form will be accomplished.

In this second stage, ☆ The carboxyl functional group, aldehyde functional group, or wood acid functional group of site Z is involved. Carboxylic or ☆ Carboxyl functional group, aldehyde functional group or wood acid functional group of polymer groups are involved.

If it is the carboxyl group that interacts with N H22 & of hemoglobin, then the carboxyl group To activate the xyl groups, water-soluble carbodiimides, especially N'-ethyl Ru-N-(3-dimethyl-aminopropyl)carbodiimide hydrochloric acid 1 (EDC I), N-←Droxysuccinimide or ethoxy-2-quinoline-1- Reagents traditionally used for peptide synthesis, such as ethyl carboxylate (EEDQ) Medicines are available.

The bond obtained at this time is an amide bond.

If it is an aldehyde group that interacts with the NH2 group of hemoglobin, for example, reduction Amination can be used.

Reductive amination of aldehydes forms imines through the action of amines, and N a B Ha, N a CN B H3, dimethylaminoporan or HCOOH It consists of simultaneously reducing an imine to an amine using a reducing agent such as.

The bond obtained at this time is an amine bond.

When it is an aldehyde group that interacts with the NH2 group of hemoglobin, the resulting bond is an imine bond, which in turn is activated by a mild reducing agent such as one of those mentioned above. working under conditions such that the amine must be stabilized by reduction with You can also do this.

Neither the Z group nor the polymer P contains an aldehyde or carboxyl group and is on an adjacent carbon. For example, if the polymer P contains some hydroxyl groups, the periodic acid resistance of the polymer P Formation of aldehyde groups on this polymer by can do.

At this time, the bonds obtained between the amines of hemoglobin and these aldehyde groups are , an imine bond, which can be reduced to an amine using, for example, NaBH, It must be stabilized.

If neither site Z nor polymer P contains an aldehyde group or a carboxyl group, an OH group containing polymers using a suitable reagent such as cyanogen bromide or carbonyldiimidazole. can act on the NH2 group of hemoglobin. In this case, you get The bond is a carbamate bond -〇-C-NH-.

Hemoglobin polymer complex where the covalent bond between the polymer and hemoglobin is an imine bond is not stable within the organism. So, for example, NaBH4.

The imine functional group was removed by reduction with NaCNBH3 or dimethylaminoporane. It is desirable to stabilize it with min.

Hemoglobin macromolecular complexes, whose covalent bonds are imine bonds, are particularly susceptible to reagents in vitro. This is a new intermediate compound that can be used as

If it is necessary to stabilize the covalent bonds, hemoglobin is in any case In other cases, the polymer will not undergo any of the above reactions, regardless of whether or not the polymer is activated. Number of reaction steps for hemoglobin, where moglobin undergoes only one reaction step In addition to reaction efficiency, limiting the hemoglobin (up to 2 steps) This is extremely advantageous as it is one of the important conditions for .

During the second stage, in fact, during the formation of the macromolecular complex according to the invention, hemoglobin In order to at least partially retain the oxygen retention properties of It is important to ensure that the relative mobility of the units does not undergo significant decreases or significant degeneration. It is.

The second reaction step is carried out at a temperature and for a sufficient period of time to adequately preserve the hemoglobin. If the amount exceeds this level, methemoglobin will be formed significantly (approximately 5% or more). Activators such as those described above are optionally activated in the absence of oxygen for a period of time shorter than aqueous, buffered or unbuffered, at a pH of about 5 to 9, in the presence of It is carried out within the medium.

Aqueous media can be used with conventional buffers to stabilize the pH at the desired value. buffered by

The reaction time is about 30 minutes to 20 hours, at a temperature of about 3°C to 30°C, about 1 hour. It is desirable that the time be between 8 hours and 8 hours.

The reaction time depends, inter alia, on the temperature at which the work is carried out.

The hemoglobin used is in deoxygenated form and has a 9% hemoglobin solubility. It is advantageous to use liquids.

The molar concentration ratio between the polymer and hemoglobin is further determined by the fact that the majority of hemoglobin molecules must be such that it covalently bonds with the polyanionic polymer.

As an example, in dissolved form in an aqueous medium with a pH of about 6.5, A dextran polymer with an average molecular weight of about 10,000 to 40,000 and hemo A mixture of globins (note that the moiety between dextran and hemoglobin in this mixture is (concentration ratio is approximately 0.5 to 2). The chromatography test revealed no free hemoglobin. It can be seen that a polymer composite according to the present invention is obtained.

The polymer complex according to the invention has no or almost no methemoglobin formation. It is easily synthesized (less than about 5%) and has only a few reactions to hemoglobin. It has the advantage that degeneration can be avoided.

Furthermore, the polymer complex according to the present invention has been shown to increase PSo in in vitro experiments. can be easily removed by extrarenal or extravascular diffusion during in vivo tests, and its efficacy is Incorporating unbound small molecular weight effectors whose effects are quickly canceled out. Not included.

The polymer composite of the present invention has only a moderate affinity for oxygen and a high wave mechanical Due to its large volume, hemoglobin remains in blood vessels due to suppression of hemoglobinuria during transfusion work. It has the advantage of increasing the availability.

Hemoglobin irreversibly attached to polyanionic polymers prepared according to the present invention The bottle complex, when dissolved in an aqueous solution of suitable composition, is particularly effective for blood transfusions or organ maintenance. It can serve as a blood substitute in surgeries that require continuous infusions.

The invention therefore also provides aqueous solutions containing the above-mentioned complexes, in particular e.g. Lord's solution (its composition is NaCQ8g/l:KCQo, 2g/l:CaC2) 0.2 g/sentence, MgC sentence 20-Lg/sentence: N aH2POa 0, 05 g/ Sentence; NaHCO31g/U; D, Curcose 1g/u) dialysis for hours and ultrafiltration until reaching a concentration of about 7% for hemoglobin. The purpose is also the isotonic solution of blood due to aIlii caused by .

The preparation of hemoglobin macromolecular complex obtained according to the present invention has a potential oxygen conductivity examined as a body. And that they can actually fix oxygen irreversibly , in particular, in Examples 1, 3, and 5 below as shown on Figure 5 and explained below. As the oxygen affinity curves of the listed products illustrate, *Release hemoglobin It has been demonstrated that oxygen can be restored more easily than In fact, these The characteristics of the formulation are that under the same conditions (NaCJI 0.05M, pH 7, 25℃) , the half-saturation (Pso) pressure is about 480 Pa for % native hemoglobin. It is extremely high (900-5000 Pa).

Thus, these compounds deliver large amounts of oxygen to tissues with local anemia. It can be used to supply Similarly, these compounds can be used in the presence of excipients (excipients). used in blood transfusions and administered to patients in the form of an isotonic aqueous solution of blood, whether or not You can also. Such compounds may also be used with or without added cryoprotectants. In this state, apply vacuum freeze-drying or make it into fine particles.

It can also be thawed before use.

The invention can be improved by using the examples given below without a limiting meaning. It will be well understood.

"Gender": Hemoglobin and WL acid dextran (dextran molecular weight: 40,000) Synthesis of covalent conjugates of: C,R,RrCKETTS, Biochemistry Journal, 1952 Year.

As shown in 51.129, the average portion ppsa Ia, Dextran sulfate sodium containing 8% T&X (10 moles of gluco Approximately 5.4 moles of sulfate ion per pyranose are prepared.

This activation of dextran sulfate is described in E, DEI, [, ACHERIE et al. , Biochemistry, Biophysics Act δ, 1983.

749.106, by periodate oxidation. use The amount of N a I O4 is.

Dextran/dialdehyde riM has glucobylanose of 100f1 However, it contains about 18 aldehyde groups.

This compound is dialyzed against water for a long time, first freeze-dried, and then kept at low temperature under vacuum. do.

Add 10g of this dextran dialdehyde sulfur 1111i to a 20% Na0 solution. 100-, and with this solution, sensitize it in the glove box and place it under vacuum. The first step is nitrogen! Perform 14 qi and repeat this process three times to remove oxygen in the same way. Add 1204 9% sensitized hemoglobin solution to the sensitized 0, IN solution. The p)l of the mixture obtained is adjusted to 7.5 by addition of thorium.

The solution is maintained at 20°C without stirring and the reaction is continued for 6 hours.

Then, prepare in situ with 200 mg of NaBH4 in io'N NaOH5-. Add 3.5 d of combined solution. Continue this reaction for 1 hour and remove excess reducing agent. Lower the pH to 7 for decomposition.

TS K, SW3000 by gel filtration method using Beckwan type column Examining the obtained chromatogram, (exclusion zone 300,000 daltons), It can be seen that there is very little unbound hemoglobin. corresponds to the elution volume. Reference may be made to FIG. 2, where the variation in optical density is shown. according to this , the amplitude of the peak corresponding to unbound hemoglobin is extremely small.

The affinity curve for oxygen of this conjugate according to the invention is shown in FIG. The affinity curve of M# hemoglobin for oxygen is represented using a dotted line.

Pso is 850 Pa (25°C, NaC, l 0.05M, pi (7).

The proportion of methemoglobin is 5%. This ratio is EVELYN K, A and HALLOY, Il, T c7) J, Biol.

Ches., 1938, 126,655. Ta.

Medical engineering: Synthesis of covalent complexes of hemoglobin and dextran sulfate, (Molecular weight of dextran = lOOOO) y4V of the above conjugate according to the present invention : 14 aldehyde groups and 12% sulfur ft (i.e. glucoside units) Contains 9.8 sulfate ions per lO unit of lucopyranose and has a molecular weight of 1000. dextran prepared according to the method described in Example 1 from 0 dextran Carry out the reaction of sodium aldehyde sulfate and deoxyhemoglobin.

Dextran dialdehyde sulfate tJil for 7 hours at 25°C in the absence of oxygen. og and a 9% deoxyhemoglobin solution (for the reaction described in Example 1) (same condition as the condition) is reversed by 2. All hemoglobin is bound to polymers and Coalescence (7) Pso is 1060 Pa (25°C, NaCMO, 05M, pH 7).

2) A mixture of dextran sulfate and hemoglobin prepared in 1) Comparison of covalent conjugates according to the invention of dextran ft salt and hemoglobin.

By steric exclusion chromatography, dextran sulfate (very good hemoglobin) is a macromolecular effector of Bin and therefore binds very strongly to deoxyhemoglobin ) and oxygenated hemoglobin at the output of the chromatography column. In the mouth, 2 corresponding to free hemoglobin and MM dextran sulfate, respectively. It was confirmed that this led to two separate peaks.

Figure 6 shows the results of injection of a mixture of dextran sulfate and hemoglobin. , the change in refractive index (ordinate) corresponding to the elution volume (abscissa) is represented. profit The resulting curve is represented by a solid line, which includes the first one (approximately 60.t elution volume) and a second one corresponding to free dextran sulfate. There are two peaks (approximately 90- elution volume).

Figure S6 also shows, in dotted lines, the device according to the invention, the synthesis of which is shown in l) above. The curve corresponding to the injection of a covalent conjugate of chytran ft-ate and hemoglobin is shown. It is.

The curves include those of M-free hemoglobin and free dextran sulfate, respectively. contains only a single eluting peak corresponding to a molecular weight larger than that of The corresponding abscissa is the excluded capacity).

The separation that makes it possible to obtain these two tree curves is performed under the following conditions at T55cm. Ultrogel Ac A 54IBF column (exclusion zone 90000) Performed in: Tris buffer 0.05M: NaC buffer 0.15M.

p) 17, 2, flow rate 30-7 hours).

In this way, the polymer-ligand is placed on hemoglobin to obtain the following situation: The conclusion is that it is necessary to fix irreversibly: a) The effector groups of the polymer act as soon as hemoglobin liberates its oxygen. In order to fulfill its role as a modulator for hemoglobin, It always remains near hemoglobin.

b) The polymer and hemoglobin remain bonded to each other and form a conduit. can be held within the circuit.

In addition, the affinity of hemoglobin for 02, which is fixed in the form of a covalent bond, is It was also observed that there was no increase compared to that of M-separated hemoglobin; In some cases - in particular, polycarbonate of dextran as effector polymer Acid salts (dextran benzene hexacarboxylate, polyacrylate dextra - The Pso of hemoglobin after binding with the polymer is is greater than the Pso of free hemoglobin in the presence of the same polymer. I found out that it is possible.

Therefore, it follows from this that the polymer binds hemoglobin in a covalent form, i.e. irreversibly. The complex according to the invention that is immobilized on a molecule is such that the hemoglobin after immobilization is It can be said that it exhibits superior functional activity. this is, During chemical reactions, hemoglobin is in a deoxygenated form and in this form , the chemical functional groups of the species are hidden and thus unaffected by bonding. This is because it becomes like this.

Medicine]: Synthesis of covalent conjugate of dextran sulfate and hemoglobin (dextran sulfate and hemoglobin) molecular weight = 6000) 18 aldehyde groups and 3% sulfur per 100 glucoside units. Molecules containing yellow (i.e. 1.7 sulfate ions per lO unit of glucopyranose) Dextra prepared according to the method described in Example 1 from an amount of 6000 dextran React sodium dialdehyde sulfate with deoxyhemoglobin. 6 hours , with 10 g of dextran φ dialdehyde sulfate in the absence of oxygen at 25°C. 9% deoxyhemoglobin solution 120. React J (with the reaction described in Example 1) same conditions). All hemoglobin is bound to the polymer and the complex PsO is 602 Pa (25°C, NaCl 0.05M, pH 7).

1: Synthesis of hemoglobin covalent conjugate and dextran phosphate.

(Molecular weight of dextran = 40,000) Industry by G, DAUL et al. al and Engineering Chemis-try (Industry Engineering Chemistry, 1954, 46.104 As shown in Figure 2, 12% phosphorus was extracted from dextran with a molecular weight of 40,000. containing dextran sodium phosphate (i.e. 10 moles of glucopyranose) (containing about 12 moles of phosphate ion).

As noted in Example 1, approximately 18 aldehyde groups are formed per 100 glucoside units. Activation of this dextran phosphate by periodate oxidation, as shown in Realize.

In exactly the same proportions and conditions as described in Example 1, this dextran dialde Reacts hydrophosphorus salt tolerance with deoxyhemoglobin. 6 hours of reaction, and After reduction with NaBH, as described in Example 1, all hemoglobin Bins are combined. The affinity curve for oxygen of this conjugate is shown in Figure 5. There is.

Complex (7) Pso is 1240 Pa (25°C, NaCjl.

0, 05M, pl(7), the proportion of methemoglobin is about 4% .

[: Synthesis of covalent conjugate of dextran polycarponate and hemoglobin (dextran polycarponate and hemoglobin) Molecular weight of strand = 40000).

Double work] 1 work P, ) ItlBERT et al., Proc, Natl^cad, Sci, U , S.A.

1978, 75.3143, activated by epichlorohydrin. Due to the action of ammonia on the strands, 5XlO' moles per tg of dry product (28 moles of NH per 100 moles on glucobylanol) Prepare chytran.

1 g of this aminodextran was dissolved in 20 - of water and P H is 6,5 with Ag1 clause 0 Next add 1.7g of benzenehexacarboxylic acid , followed by 1 g of N'-ethyl-N-(3-dimethyl-7minoprobyl)carbodi Add imide (EDCI), return pH to 6.5, and continue one reaction at 20°C for 3 days. After dialysis against a 0.5 M sodium acetate solution, the mixture Tolan is treated with anhydrous vinegar to fix the undetected amine functionality.

At this time, the solution was prepared using phospholeprosy buffer of pH 7, 20, and 2M. 1 molecular weight by desalting using an ACA 202 (IBF-7 Lance) column. It is purified by removing contaminants.

After prolonged dialysis against water, the solution containing the polyanionic polymer is lyophilized.

Compounds are stored at low temperature under vacuum.

Add 0.5 g of this dextran polycarboxylate to a 0.05 M NaC aqueous solution. Dissolve in 25.

Bring the pH to 6.5 with 0.1N sodium as described in Example 1. Deoxygenate the solution. Then 10% hemoglobin was deoxygenated under the same conditions. Add solution 15-. For mixtures held in a glove box under nitrogen scavenging. Then add 6 (1+g of EDCI) and continue the reaction at 20°C for 10 hours. Chroma obtained on ltragel ACA 34 (IBF-France) column No more togram! Make sure there is no hemoglobin present.

Reference may now be made to Figure 3, which shows the change in optical power as a function of elution volume. Wear. According to this diagram.

The amplitude of the peak corresponding to unbound hemoglobin should be small. I understand. The Pw of the complex is 1360 Pa (25°C, NaCJlO, 05M, p H7).

Kai J: Synthesis of covalent conjugate of hemoglobin and dextran polycarboxylate (Molecular weight of dextran = 40000) milk: Along the polygon, the oligomerization of the acrylic resin begins in an aqueous medium. of dextran containing polyacrylic oligomer chains by creating Prepare a polycarboxylic acid salt. This dextran sodium polycarboxylate is , purified by prolonged dialysis against water, and first freeze-dried.

This dextrin contains on average one dibasic carboxyl ring per glucoside unit. Ran's Sodium Polycarboxylate Ig and under exactly the same conditions as described in Example 5. React with deoxyhemoglobin under conditions.

The reaction was stopped after 4 hours and the mixture was.

Chromatograph on old trogel ACA 34 (IBF-7 Lance) column In this way, confirm that the 1i hemoglobin is gone. be able to. Here, please refer to Figure 4, which shows the change in optical density corresponding to the elution volume. can be seen. According to this diagram, the equivalent of unbound hemoglobin is The amplitude of the peak is small.

The affinity curve for oxygen of this complex is shown in FIG. P- is 550 0Pa (25℃.

NaCJl 0.05M, pH 7).

to quarrel The different polyanionic polymers mentioned in the examples were tested under the following conditions: The double conjugate injected into WISS-bred mice produced sperm at concentrations of 2.5 g/fL to 5 g/l. Dissolved in manufactured water, PH is 7.4 XJ! 4 sections 0 to each solution 0.5 d into 5 mice under 1M. After that, the behavior of these five mice was Observe for 7 days. These tests showed no signs of acute toxicity. won.

D, 0° 0.0° FIG.3 D, 0° FIG.4 HbO.

FIG.6 international search report 1+'mvm*mlAmljmm, PCT/FR87100262Als'! 9 Mi=XτOT:Mi:EIrE:Iko〉GoAτICNALSE2ARC:MiRE? CRTONFR;A-255166015103/85 None

Claims (21)

[Claims] 1. It exhibits a lower affinity for oxygen than that of free hemoglobin; biodegradation during the time it has to perform its oxygen retention (equilibrium) function in the plasma. A water-soluble polymer complex of hemoglobin that does not cause any or almost no reaction, The polymer complex is composed of the following: -Hemoglobin that can reversibly transition from a deoxygenated form to an oxygenated form ・Contains a polar group, preferably a hydroxyl group, a carboxyl group or an amine group, and contains about 1 000 to about 500,000, preferably about 1000 to about 100,000, non-toxic, a preferably non-antigenic, blood-compatible, water-soluble polymer P; - the polymer complex contains sulfate and/or phosphate and/or carboxylate; A site that contains one or more negative charges and is fixed on the polymer P having Z; - the polymer P is linked to hemoglobin via: - On the one hand, between at least one of the sites Z of the polymer and hemoglobin through at least one ionic bond formed in ・On the other hand, the polymer and hemoglobin or at least part of the site Z that the polymer has through at least one covalent bond formed between both one and hemoglobin, - The number of covalent bonds between the polymer and hemoglobin is approximately 70,000 to having an average molecular weight of about 1 million, especially about 70,000 to about 500,000. There is something - the relationship between negative charge, number of sites and number of monomers is as follows: and; ・If each site contains only one anionic group consisting of sulfate or phosphate, the polymer there is at least one site for every 10 monomers, - At least one of the moieties consists of sulfate and/or phosphate If it contains two anionic groups, there is at least one site on the polymer , ・If each site contains a negative charge from the carboxylate, on the same site Z at least two carboxyl (carboxylate) groups, also one for every five monomers There must be the above part Z, -At least one of the sites contains three or more negative charges from the carboxylate , there is at least one site for the polymer. Hemoglobin polymer complex. 2. The salt bridge between the internal COO- group of hemoglobin and NH3+ is remains intact when in deoxygenated form. 2. The polymer complex according to item 1. 3. The polymer P is a polysaccharide, especially a hydrogen whose alkyl group contains 2 to 4 carbon atoms. Droxyalkyl starch, inulin, dextran and its derivatives, especially amino acids Nodextran, polyvinyl alcohol, polyvinylpyrrolidone, polymethacrylate salts and their derivatives, polypeptides, alkene groups containing 2 to 5 carbon atoms. Containing polyalkene glycols, especially polyethylene glycol and polypropylene as claimed in claim 1, characterized in that it is selected from pyrene glycols. Polymer composites with 4. If the average molecular weight of the polymer P is composed of dextran and its derivatives, 70,000 or less, and it is polyalkene glycol, polyvinylpyrrolidone or If it is selected from polymethyl acrylates, it must be less than 10,000. The polymer composite according to claim 1, characterized in that: 5. The moiety Z is polymerized via an ester, ether, amide or amine functionality. 2. The polymer complex according to claim 1, which is bound to a body. 6. The site Z is OSO3H, OPO3H2, where n is a variable from 1 to 4. ， O-CH(COOH)2, -O-CH(COOH)-CH2-COOH, ▲There are mathematical formulas, chemical formulas, tables, etc. ▼ or pyridoxal sulfate, pyridoxal phosphate, adenosine triphosphate , phosphotyrosine, phosphoserine, inositol hexaphosphate and its derivatives, mainly Polycarboxylic acids containing 2 to 10 carbon atoms on the chain, with 3 carboxylic functional groups Benzenecarboxylic acid containing 2,3 diphosphoglycerates The polymer composite according to claim 1, characterized in that: 7. The hemoglobin contains on the one hand an aldehyde group, a carboxyl group or an OH group, and on the other hand, through the bonds formed between the NH2 groups of hemoglobin. Claim 1, characterized in that the polymer is bound to the polymer in the form of a polymer. polymer complex. 8. The covalent bond between the polymer and hemoglobin is an OH group possessed by the polymer, Formed directly between aldehyde or carboxyl groups and NH2 groups of hemoglobin or an aldehyde group, carboxylic acid group, which comes from the moiety Z fixed on the polymer. It is characterized by being formed from a sil group or a hydroxyl group and an NH2 group of hemoglobin. The polymer composite according to claim 1. 9. The moiety Z is characterized by containing three or more of the following functional groups: The polymer composite according to claim 1: - one of the functional groups is a functional group that allows moiety Z to be immobilized on the polymer - one of the functional groups is an amine in the allosteric site of hemoglobin A sulfate, phosphate or carboxylate salt that can form an ionic bond with is a functional group; - one of the functional groups forms a covalent bond with the NH2 group of hemoglobin; aldehyde, carboxyl or OH functional group. 10. The polymer preferably contains about 5.4 moles per 10 moles of glycopyranose. dextracontaining OSO3H groups, preferably having an average molecular weight of about 40,000. The covalent bond between hemoglobin and the polymer is preferably Preformed on dextran at a ratio of approximately 18 aldehyde groups to 100 side units obtained as a result of the bond between the aldehyde group and the NH2 group of hemoglobin. The polymer composite according to claim 1, characterized in that: 11. The polymer contains approximately 9.8 mol of OSO3 per 10 units of glucopyranose. composed of dextran containing H groups and preferably having an average molecular weight of about 10,000 The covalent bond between the polymer and hemoglobin is preferably a glucoside 10 Aldehyde groups preformed on the dextran in a ratio of about 14 aldehyde groups to 0 units. specifically obtained as a result of the bond between the aldehyde group and the NH2 group of hemoglobin. The polymer composite according to claim 1, characterized in that: 12. The polymer contains about 1.7 OSO3H per 10 units of glucopyranose. It is composed of dextran containing basic sulfate ions and preferably having an average molecular weight of about 6000. The covalent bond between the polymer and hemoglobin is preferably 100 units. Pre-formed alkalinity on dextran with a ratio of about 18 aldehyde groups per position Claims characterized in that the product is obtained as a result of a bond between dehyde groups. The polymer composite according to item 1 above. 13. The polymer contains approximately 12 moles of OPO3H per 10 moles of glucopyranose. 2 groups, preferably composed of dextran with an average molecular weight of about 40,000 The covalent bond between the polymer and hemoglobin is preferably a glucoside Preformed on dextran at a ratio of about 18 aldehyde groups to 100 units. obtained as a result of the bond between the aldehyde group and the NH2 group of hemoglobin. The polymer composite according to claim 1, characterized in that: 14. The polymer preferably consists of aminodextran having an average molecular weight of about 40,000. The ionic bond is formed by benzene hexacarbohydrate fixed on the polymer. The covalent bond is realized through the carboxyl (carboxylate) group of the acid, and the covalent bond is Other carboxyl groups of hexacarboxylic acid and hemoglyceride that are not involved in on complexation Claim 1, characterized in that the method is realized through Robin's NH2 group. Polymer complex as described. 15. The polymer contains about one dibasic carboxylic acid chain per monomer, Preferably a polycarboxyl dextran having an average molecular weight of about 40,000. Covalent bonds are molecules that are not involved in ionic bonding with hemoglobin. It is characterized by being realized through the ruboxyl group and the NH2 group of hemoglobin. The polymer composite according to claim 1. 16. A method for preparing a polymer composite according to claim 1, which is characterized by the following: Law: - in a first step, immobilizing the moieties Z on the polymer by: ・If necessary, change the polymer P to a polyol and then, for example, sulfate esterification, phosphoric acid A method of fixing site Z by esterification or carboxymethylation, or ・Y is an aldehyde, carboxyl, amine, hydroxyl or halogen. A method using a Z-Y compound as an active functional group or an activatable functional group, or - A method of radiochemically grafting the site Z onto the polymer P; - Next, in the second step, the polymer obtained as described above is added to hemoglobin. deoxygenated in an aqueous medium at a pH of about 5 to 9 under conditions such that the The polymer reacts with hemoglobin, forming ionic and covalent bonds between the polymer and hemoglobin. to form a joint at the same time; - In some cases, if the above reactions lead to imine functions, these The functional group of is, for example, NaBH4NaCNBH3, dimethylaminoborane or H Reduction with COOH stabilizes with amine functionality. 17. At the end of the first stage, the polymer having the moiety Z has reacted with hemoglobin. The method according to claim 16, characterized in that the method is activated before being applied. Law. 18. Said activation converts the OH function into an aldehyde group, for example by periodic acid oxidation. 18. A method according to claim 17, characterized in that it consists of changing. 19. The activation of the polymer having the site Z and its reaction with hemoglobin is actually 17. A method according to claim 16, characterized in that the method is performed at the same time. 20. The polymer having the site Z is -If neither polymer P nor site Z has aldehyde groups but contains hydroxyl functional groups, then bromide activated with cyanide or carbonyldiimidazole; or - If the polymer P or Z group has a carboxyl group, it is used in peptide synthesis being activated using a reagent; 20. A method according to claim 19, characterized in that: 21. above, for a time shorter than the time to induce up to about 5% methemoglobin. temperature that allows proper preservation of hemoglobin for up to 10 hours, e.g. that the reaction between the polymer and hemoglobin takes place at temperatures ranging from °C to 30 °C. The preparation method according to claim 20, characterized in that: