CH638401A5 - Process for the preparation of haemoglobin preparations having an increased oxygen delivery - Google Patents

Description

The invention relates to a method for producing hemoglobin preparations suitable for intravenous injection with increased oxygen release compared to erythrocytes and hemoglobin solutions.

The oxygen semi-saturation pressure of hemoglobin (Hb), called the P50 value, is known to be 36 mbar in intact erythrocytes. In addition to the intraerythrocytic pH value of 7.25, the level of 2,3-diphosphoglycerate (DPG) is responsible for the level of willingness to release oxygen. For all infusion preparations based on freely dissolved hemoglobin, or derivatives thereof, the pH value of the blood plasma of about 7.4 is decisive, since they must inevitably transport the oxygen under these conditions. This fact alone leads to a left shift of the oxygen connection curve corresponding to a reduction in the P50 value to about 32 mbar in the best case. However, once such a hemoglobin solution has been infused, unbound 2,3-diphosphoglycerate is immediately excreted by the kidneys in the dissociation equilibrium due to its small molecular size. The DPG is bound to the Hb in a salt-like manner and rapidly dissociates accordingly, so that in a short time everything released in the bloodstream is exposed to DPG. As a result, the P50 value drops to 20-22 mbar. A higher P50 value indicates an increased willingness to give oxygen of a preparation.

So far, no hemoglobin preparation has been found that easily releases oxygen for an oxygen supply to the tissue. It is known that pyridoxal-5-phosphate (FIG. 1) is preferably also bound at the same point in the Hb molecule as DPG, but surprisingly it is bound to the hemoglobin considerably more strongly than diphosphoglycerate. The R.E. Benesch, R. Benesch, A. 40 Benk, R. Renthal and B. A. Bray in Proc. I. Interamer. Symp. Haemoglobins, Caracas (1969), "Genetical, functional and physical studies of hemoglobins", pgs. Model preparation 134-142 (Karger, Basel 1971) described from hemoglobin and pyridoxal phosphate cannot be used as an infusion solution because it triggers pyrogenic reactions.

It has now surprisingly been found that it is possible to produce a hemoglobin preparation suitable for intravenous injection with increased oxygen release compared to erythrocytes, which consists of a condensation product of hemoglobin and pyridoxal phosphate and does not cause a pyrogenic reaction. Such a preparation can be used therapeutically intravenously because of its favorable oxygen affinity. In this condensation product there is a hemoglobin tetramer with a statistical distribution of pyridoxal phosphate on the hemoglobin. The preparation has a PSo value of 42 mbar even at a pH of 7.4. The duration of such a preparation in the bloodstream is about 2 hours.

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- Such pyrogen-free hemoglobin preparations, which are effective in the blood circulation as excellent oxygen donors and can be used as infusion solutions (Hb / PP), can be obtained by using the hemoglobin recovery method from R.E. Benesch and R. Benesch [Proc. of the National Academy of Science USA 59, 526-532 (1968)] deviates and thereby proceeds according to the invention as defined in claim 1.

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The method defined in claim 2 provides preparations with a residence time in the blood of 4 to 9 hours.

300 ml or 500 ml infusion units can be produced directly in sterile filtration.

By adding IN sodium hydroxide solution and glucosemonohydrate, chemical processes are brought about in the processes according to the invention, by means of which the oxygen-binding properties of hemoglobin are changed and reductive enzymes are rendered ineffective.

The column chromatogram of such preparations (“Se-phadex” G-150.90 x 1.5 cm, 0.3 ml order volume) is shown in FIG. 2 of the drawings.

The P5 "values of the preparation produced according to the invention at a pH value of 7.45 are between 40 and 53.3 mbar and thus considerably exceed the p50 value of the hemoglobin in the erythrocytes.

It was further found that by further modification of those molecules which are capable of improving the oxygen release to the tissue by binding pyridoxal phosphate to hemoglobin and which have a residence time in the blood of about 2 hours, they are achieved in at least twice as long while maintaining their therapeutic function remain fully effective in the bloodstream. For this purpose, the procedure is expediently carried out as follows:

Human erythrocytes are treated with weakly alkaline wash solution at least 4 times, then hemolyzed and treated with a cation exchanger in H + form until the pH has dropped to 5.0-5.5, then freed from the resin and precipitated stromal mass by cooling centrifugation a desired hemoglobin concentration of 5-9% diluted with water, a pH value of 7-9 adjusted with 1N sodium hydroxide solution, then under a vacuum of 26.6 mbar for 10 minutes, then with introduction of nitrogen, stirred until the oxygen in The vessel is displaced by pure nitrogen, 0.25-1.25 g of pyridoxal-5-phosphate per liter of 5-9% hemoglobin solution is added and stirring is continued under nitrogen for 15 minutes to 2 hours at room temperature and then with 5 to 10 times the molar Amount of sodium borohydride compared to the pyridoxal phosphate stirred for a further 20 minutes at room temperature, then evacuated again, a dialdehyde of the formula OCH- (CH2) n-CHO, where n is 1-6, i n 9 times the molar amount added to the hemoglobin, stirred for 45 minutes under nitrogen, uncrosslinked hemoglobin then separated by salting out or ultrafiltration, the enriched crosslinked preparation adjusted to a pH of 7.6 at 20 ° C. with IN sodium hydroxide solution, using appropriate salt additives Blood plasma analog concentrations supplemented, 22 g glucose monohydrate added per liter and sterile filtered.

For the depletion of uncrosslinked hemoglobin there are e.g. two possibilities:

1. The 0.8 to 1.0 volume of the hemoglobin preparation of 4 molar ammonium sulfate solution is gradually stirred in to salt out the crosslinked hemoglobin. The centrifuged precipitate is dissolved in water and dialyzed against a 0.5% saline solution to remove the ammonium sulfate and all excess auxiliary chemicals. The last traces are removed by a mixed bed exchanger.

2. The hemoglobin preparation is passed through a hollow fiber cartridge Hl P 100 (manufacturer: Amicon Corporation, 21 Hostwell Avenue. Lexington, Massachusetts, USA). Ultrafiltration preferentially squeezes out uncrosslinked hemoglobin and depletes it in the unfiltrate.

In this process, pyridoxal phosphate is first allowed to act on hemoglobin and then the nascent hydrogen from sodium borohydride reduces the azomethine (Schiff base) formed to secondary amine bonds.

NaBH

(Hb) -N = CH- (Pyr)% (Hb) -NH-CH2- (Pyr)

A dialdehyde of the formula is then left

O = CH- (CH2) n-CH = O, n = 1-6

act on the molecules. As a result, the modified hemoglobin molecules are coupled together to form larger structures.

Suitable for this treatment are dialdehydes with in particular straight carbon chains of 3-8 carbon atoms, such as malondialdehyde, succindialdehyde, glutardialdehyde, adipindialdehyde, suberaldehyde, which correspond to the formula OCH- (CH2) n-CHO, in which n has a value of 1-6 means.

This process product (Hb / PP) m according to Fig. 3 of the drawing consists of

A = highly cross-linked pyridoxalized hemoglobin (m> 3) B = oligomer cross-linked pyridoxalized hemoglobin (m = 2-3)

C = uncrosslinked pyridoxalized hemoglobin (m = 1)

Expired blood bank erythrocytes can also be used as the starting material for both process modifications.

In addition to a flawless pyrogen test, the infusion preparation made of Hb / PP according to the invention was tested for acute toxicity in rats and mice. The rats were tolerated intravenously with 50 ml of an 8% solution per kg body weight without abnormal behavior and without conspicuous symptoms. After mice still survived an intravenous dose of 100 ml per kg body weight, it was determined according to Thaer: "Fundamentals of experimental drug research" (1965) that the

LD50 is more than 50 ml / kg body weight,

and the substance is classified as “relatively harmless”.

In contrast to the product from RE and R. Benesch, the preparation produced according to the invention was able to demonstrate, on the basis of a constant PS0 value, that the Schiff base binding of the pyridoxal phosphate to the amino group of the N-terminal valine of the β chain of the Hemoglobin is resistant to dialysis treatment in vitro. An exhaustive dialysis against 0.9% saline at 10 ° C was carried out.

Result

Preparation P50 (mbar)

before dialysis after dialysis

Hb / PP 731017 42.6 44

Hb / PP 731013 52 49.3

Compare literature quotation R.E. Benesch et al. 1971

The preparation Hb / PP also shows the superior effectiveness under the physiological conditions of the blood circulation in animal experiments. Even under a normal venous oxygen partial pressure of 53.3 mbar, the preparation already releases amounts of oxygen into the tissue, the released normal hemoglobin only under pathological conditions.

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sen, i.e. when the venous P02 has dropped to about 26.6 mbar (K. Messmer et al. in preparation).

The process according to the invention is further illustrated by the examples below. In the drawings, FIG. 1 shows the structural formula of pyridoxal-5-phosphate, and FIGS. 2-6 are chromatograms of preparations produced according to the invention.

example 1

The erythrocyte sediment of a 4-week stored blood (about 270 ml) is stirred for 10 minutes with 1/3 of its volume of 1.6% saline with 1.2% β-propiolactone content. The erythrocytes are centrifuged off at 0 C and, after aspirating off the supernatant and the platelet skin formed, are washed 4 more times with the 1 to 2-fold volume of the following solutions on the cooling centrifuge:

1. 3.8% sodium bicarbonate / water

2. 3.8% sodium bicarbonate / water

3. 1: 1 mixture 1.6% saline / 3.8% bicarbonate solution

4. 1.6% saline

For hemolysis, the washed erythrocytes are stirred into the amount of aqua pro injectione, which corresponds to the initial erythrocyte volume, and mixed with cation exchange suspension in the H + form - for example «DOWEX» 50 WX 8 - until the pH drops to 5. This is followed by dulling to a pH of 5.5-5.7 with 1N sodium hydroxide solution and separation of stroma and resin by cooling centrifugation and pouring off. The supernatant is diluted to 8.6% hemoglobin with aqua pro injectione, adjusted to pH 7.5 with IN sodium hydroxide solution and with 3.6 g of sodium chloride, 22 g of glucose monohydrate, 0.29 g of potassium chloride, 0.23 per liter of solution g of calcium chloride dihydrate and 0.25 g of magnesium sulfate heptahydrate were added.

After cooling again by centrifugation, the mixture is decanted and stirred in a closed vessel for 10 minutes under a vacuum of 26.6 mbar.

Passing through pure nitrogen gas expels the last oxygen, so that the hemoglobin is then predominantly in the deoxy form. This is reacted with 1.1 g of pyridoxal-5-phosphate monohydrate per liter of solution (corresponds to approximately 3.5 mol of pyridoxal phosphate per 1 mol of Hb-tetramer) by stirring for 1 hour at 20 ° C. under nitrogen, according to the previous one Add 2.12 g sodium bicarbonate per liter.

This is followed by a final adjustment with IN sodium hydroxide solution to a pH of 7.6 (at 20 ° C) and sterile filtration. Yield: About 400 ml of 8% Hb solution.

Pso value at pH 7.45: 42.6 mbar

Example 2

The combined erythrocyte sediments of six fresh blood (about 1.51) are treated according to Example 1, but for 5 minutes with 2.0% β-propiolactone / 1.6% sodium chloride solution. Hemolysis is carried out with twice the erythrocyte volume of about 3 liters of aqua pro injectione.

The pH adjustment with IN sodium hydroxide solution before the addition of - adjusted to the dilution - 0.82 g of pyridoxal-phosphate monohydrate per liter of solution takes place in deviation from Example 1 to 9.0, and the stirring time under nitrogen is 2 hours.

After again processing analogously result

4 liters of an Hb / PP solution, 6% Hb

Ps0 value: 42.6 mbar

Example 3

The erythrocyte sediment of a 10-day-old blood preserve is subjected to the 4 washing operations analogous to Example 1 without β-propiolactone pretreatment. However, the washed erythrocytes are hemolyzed in 450 ml aqua pro injectione (greater dilution). For reaction with 1.1 g pyridoxal-5-phosphate monohydrate per liter of solution (corresponding to 5 mol pyridoxal phosphate per 1 mol Hb-tetramer), the pH is adjusted to 7.3 and then under for 2 hours Nitrogen stirred at room temperature.

After the usual final electrolyte adjustment, about 700 ml of Hb / PP solution with a content of 5.5% Hb is obtained.

Pso value: 52 mbar

Example 4

Combined erythrocyte sediments from 25 expired blood (approximately 61) are stirred according to Example 1, but only for 5 minutes with the specified β-propiolactone / common salt solution. In contrast to Example 1, the pH is also adjusted to a value of 7.0 before the pyridoxal phosphate is added.

At the end of the further processing carried out analogously to Example 1, 10.5 liters of an Hb / PP preparation with an Hb content of 8%, which has a P50 value of 45.3 mbar, are obtained.

Example 5

100 ml of 6% hemoglobin solution were converted into the deoxy form by evacuating to 26.6 mbar and then flushing with nitrogen. 47 mg of pyridoxal phosphate was added with stirring and stirring continued for 15 minutes. Then 56 mg of sodium borohydride was added and stirring was continued for a further 20 minutes (room temperature). After the evacuation again, 0.8 ml of 10% glutaraldehyde was added and the mixture was stirred under nitrogen for 45 minutes. In order to separate the uncrosslinked hemoglobin, 90 ml of 4 molar ammonium sulfate solution were added within 7 minutes and stirring was continued for 10 minutes. The resulting precipitate was centrifuged off and the supernatant was discarded. The precipitate was dissolved in 20 ml of water. This was followed by a 3-hour dialysis with «Visking» dialysis tube type 10/32 against 50 times the volume of a 0.5% saline solution, the external solution being replaced 3 times. The dialyzed hemoglobin solution was sent through a mixed bed exchanger to remove the ammonium sulfate completely, consisting of a strongly acidic cation exchanger, for example “Dowex” WX 8 cation exchanger in the Na + form or “Dowex” 21 K anion exchanger in the Cl ~ form (supplier: DOW Chemical Company, Midland, Michigan, USA).

After adjustment to pH 7.5 with n-NaOH and to an isotonic electrolyte or glucose concentration (addition of 2 g glucose and 0.21 g sodium bicarbonate), the preparation was sterile filtered and stored in the refrigerator (5 ° C).

The preparation corresponded to a P50 value of 21.3 mbar and showed the column elution diagram over “Sepharo-se” 6 B (product from Pharmacia Fine Chemicals AB) according to FIG. 4.

Example 6

The procedure was as in Example 5, but with 3 times the batch size until the glutardialdehyde was added. After stirring, the preparation was passed through a hollow fiber ultrafiltration cartridge HIP 100 pre-filled with 0.9% NaCl solution at an inlet pressure of 666 mbar.

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pumps. The volume of Example 7 pressed by the ultrafiltration was continuously replaced by sterile water. The change was as in Example 5, but with the addition of 71 mg pumping time was about 5 hours. During this time pyridoxal phosphate was found. This means that full demineralization takes place simultaneously. The final product had a P50 value of 24 mbar and, as set in Example 5, had a P50 value of 25.3. 5 showed the gel chromatogram of FIG. 6.

mbar and showed the gel chromatogram of FIG. 5.

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Claims (3)

638401 2nd PATENT CLAIMS 1. Process for the preparation of hemoglobin preparations suitable for intravenous injection with increased oxygen release compared to erythrocytes and / or hemoglobin solution, characterized in that human erythrocytes are treated at least 4 times with a weakly alkaline washing solution, then hemolyzed and treated with a cation exchanger in H + form, until the pH value has dropped to 5.0-5.5, then the resin and precipitated stromal mass are removed by cooling centrifugation, diluted with water to a desired hemoglobin concentration of 5-9%, and a pH value of 7-9 is established with IN sodium hydroxide solution , the electrolytes are supplemented with analogous concentrations of salt additives on the blood plasma, 22 g of glucose monohydrate / 1 are added, then stirred under a vacuum of 26.6 mbar for 10 min, nitrogen gas is passed over it until the oxygen in the vessel is replaced by pure nitrogen, which Hemoglobin can react with 0.25 to 1.25 g pyridoxal-5-phosphate per liter 5-9% hemoglobin solution and stirring under nitrogen for 15 min to 2 h at room temperature, and finally adjusted to a pH of 7.6 at 20 ° C. with IN sodium hydroxide solution and sterile filtered. 2. The method according to claim I, characterized in that the erythrocytes are first treated at 10-15 ° C for 5-15 min with dilute solution of ß-propioIactone in a ratio of 4-12 g of ß-propioIactone per liter of erythrocyte sediment and then washing the treatment agent and its reaction products with weakly alkaline solutions. 3. Process for the preparation of hemoglobin preparations with a residence time of 4 to 9 h in the blood, characterized in that human erythrocytes are treated at least 4 times with a weakly alkaline washing solution, then hemolyzed and treated with a cation exchanger in H + form until the pH Value has dropped to 5.0-5.5, then freed of resin and precipitated stroma s by cooling centrifugation, diluted with water to a desired hemoglobin concentration of 5-9%, adjusted to pH 7-9 with IN sodium hydroxide solution, then stirring under a vacuum of 26.6 mbar for 10 minutes, nitrogen gas passed over it until the oxygen in the vessel is replaced by pure nitrogen, 0.25-1.25 g of pyridoxal-5-phosphate per liter of 5-9% hemoglobin solution is added and stirring under nitrogen for 1 h at room temperature, and then stirring with 5 to 10 times the molar amount of sodium borohydride compared to the pyridoxal-15 phosphate for a further 20 min at room temperature, then again evacuated, adding a dialdehyde of the formula OCH- (CH2) n-CHO, in which n is 1-6, in 9-fold molar amount to the hemoglobin, stirring for 45 minutes under nitrogen, then removing uncrosslinked hemoglobin by salting out 2o or ultrafiltration Set in sodium hydroxide solution to a pH value of 7.5 at 20 CC, electrolytes supplemented by analogous concentrations with salt additives on the blood plasma, 22 g of glucose monohydrate per liter added and sterile filtered. 25 4. The method according to claim 3, characterized in that the erythrocytes are first at 10-15 ° C for 5-15 minutes with dilute solution of β-propiolactone in a ratio of 4-12 g of β-propiolactone per liter of erythrocyte sediment treated and then the treatment agent and its reaction products washed out with weakly alkaline solutions.