DE102017218847A1 - Method for improving the stability of blood and blood products - Google Patents

Abstract

The present invention relates to a method for improving the stability of blood and / or blood products. Furthermore, the present invention relates to blood and / or blood products, which are improved by a special additive in their stability. Moreover, the present invention relates to the use of specific additives for improving the stability of blood and / or blood products.

Description

The present invention relates to a method for improving the stability of blood and / or blood products. Furthermore, the present invention relates to blood and / or blood products, which are improved by a special additive or a special combination of additives in their stability. Moreover, the present invention relates to the use of specific additives for improving the stability of blood and / or blood products.

Concern about national and global blood supply is growing steadily. Human blood products are expensive and have only limited shelf life. One reason for this is the relatively short storage stability of blood and blood products. In the context of the present invention, blood products (also referred to as hemoproducts) are understood as meaning the cellular or plasmatic blood components or whole blood obtained from human blood which have been prepared for transmission (transfusion) to a recipient.

Blood and blood products are usually obtained from blood donations.

During blood donation, the blood is passed into a closed bag system containing an anticoagulant and a nutrient solution for the cells. The bag system is designed so that the individual blood components can be transferred into separate bags that are already firmly connected without having to open the system. As a result, reactions with the air and contamination with germs or dust particles can be avoided.

The next step in the process of obtaining blood products usually takes place at a blood donation center. There, the separation of the blood donation into its components is achieved by centrifugation: The cells and blood components are separated by their different weight in layers. The erythrocytes (red blood cells) are found in the lowest layer, above the leukocytes (white blood cells), then the platelets (platelets) and at the top of the cell-free blood plasma. After centrifugation, the individual components are located in respective separate pouch areas and are then split by light sensor-controlled pressing into an erythrocyte concentrate, a fresh plasma preparation and the intermediate layer (buffy coat) containing the white blood cells and the platelets. The erythrocyte concentrate can be stored at +4 ° C for 42 days. Alternatively, the erythrocyte concentrate may also be incorporated with glycerol and frozen, for example, at minus 196 ° C, so that it is durable for years. Before a transfusion, the glycerin must then be washed out of the mixture again, so that this process is very expensive and expensive.

The erythrocyte concentrate thus obtained is colloquially referred to as blood conserve.

These conserved blood is, as we have seen, a limited-life biomedical product. A specific problem with erythrocyte-containing blood conserves is hemolysis. During hemolysis, the cell walls of the erythrocytes dissolve to release hemoglobin. The number of erythrocytes is about 5 million / μL, so that the reduction of the erythrocyte concentration by the hemolysis for the blood bank is actually unproblematic; however, the free hemoglobin produced by hemolysis has a toxic effect and thus represents a crucial problem in the storage of stored blood.

Hemoglobin is an iron-containing metal protein consisting of four globins, each of which contains a heme complex as a covalently bonded oxygen-transport functional group. By deoxyhemoglobin is meant hemoglobin without bound oxygen, and by oxyhemoglobin is meant hemoglobin which has bound oxygen.

Free hemoglobin can be bound by serum haptoglobin, a protein, in a molar ratio of 1: 1, thereby rendering it harmless. In vivo, the formed adduct is removed from the circulation with a half-life of 8 minutes. However, when this "buffer" of haptoglobin, which is expected to be between 0.3 to 2.0 g / L in humans, is consumed (for example, in a blood bank), toxic free hemoglobin will be produced in an uncontrolled manner. Therefore, a too low haptoglobin level can be indirectly understood as evidence of progressive hemolysis.

For example, in the Federal Republic of Germany, for legal reasons, the degree of hemolysis of a blood bank must be less than 0.8%, otherwise the use of the blood bank for transfusion is not permitted.

The degree of hemolysis [%], one of the most important parameters for assessing the "freshness" of a stored blood, can be calculated using the following formula: $$HämOlyse\left[\%\right]=100-HämOkrit-Wert\left[\%\right]\cdot \left(\frac{freiesHämOblObin}{Gesamt-HämOGlObin}\right)$$

The hemotocrit value is the volume fraction of cellular elements in the blood which can be determined by centrifugation and is expected to be about 40 to 52%.

The storage life of an erythrocyte-containing blood reservoir under ideal storage conditions and temperatures of ± 4 ° C is, as mentioned above, depending on the storage medium, only up to 42 days. The need for blood or blood products tends to increase, for example, in the Federal Republic of Germany is hardly covered by the donated amount. Since donor readiness is stagnating or declining slightly, this problem will increase in the future. Improved methods and compositions for their storage for prolonging the stability of blood and / or blood products are thus still required.

For this purpose, various methods and compositions have been developed in the past.

The WO 97/16967 A discloses the use of antioxidants to reduce oxidative stress-induced damage to blood components. However, the sole use of antioxidants is not sufficient to solve the task described below.

Another method for improving storage stability and resistance to hemolysis is described in U.S. Pat US 2001/0049089 A disclosed. According to this document, the blood product is stored as a suspension with a conventional preservative solution which additionally contains L-carnitine or an alkanoyl derivative. In this document, blood stabilization is addressed only indirectly by reducing harmful secondary reactions, such as ATP depression and, as a result, pH lowering.

The US 2010/0021879 A discloses a composition for preserving red blood cells which contains, inter alia, antioxidants, potassium-sparing drugs and potassium channel blockers. However, the sole use of antioxidants is not sufficient to solve the task described below.

The US 2011/0151428 A discloses a method of improving the stability of blood and blood products based on a specific composition for reducing hemolysis in a blood sample or blood product sample. Exact information on the mechanism of action is not disclosed.

The methods known from the prior art for improving the stability of blood and blood products are unsatisfactory for a variety of reasons.

Therefore, despite the various methods known in the art, it remains an object to provide further methods and compositions for improving the stability of blood and blood products to enable the storage of blood and blood products for periods longer than 42 days.

It is therefore an object of the present invention to provide a method and composition for improving the stability of blood and / or blood products such that blood or blood products are provided for longer than the possible time periods to date can.

The method and the compositions are intended to be as simple as possible and to dispense with agents that lead to undesirable side effects, especially in humans, who receive the blood and / or blood products.

By "improving the stability" of blood and / or blood products is meant in the context of the present invention in particular the avoidance of decomposition products during storage and the prolongation of the shelf life of the blood and / or blood product.

In the context of the present invention, "blood products" is understood as meaning the cellular or plasmatic blood components or whole blood obtained from human blood which have been prepared for transmission (transfusion) to a recipient. The term "blood and / or blood product" therefore also includes blood.

The term blood or blood products includes human, but also animal blood or blood products. In the context of the present invention, blood and / or blood products do not mean any blood still present in the human or animal body. For the purposes of the present invention, blood and / or blood products are present outside the human or animal body.

The solution to this problem underlying the present invention is to slow down the erythrocyte decomposition reaction and to trap toxic free hemoglobin.

In the context of the present invention, it has been recognized that the erythrocyte decomposition mechanism is based on homolytic cleavages of electron-pair bonds. This creates radicals that accelerate the process autocatalytically.

Based on this finding, the decomposition rate of the erythrocytes is effectively reduced according to the invention by adding butylhydroxytoluene (BHT) to the blood and / or blood product.

The present invention therefore relates to a method for improving the stability of blood and / or blood products, the method according to the invention being characterized in that butylhydroxytoluene (BHT) is added to the blood and / or blood product.

Butylhydroxytoluene (BHT) is a chemical compound from the group of toluene derivatives or phenols, which does not occur naturally, but is produced and used industrially in considerable quantities. It is a colorless and odorless, crystalline solid, practically insoluble in water and is used in many consumer products, e.g. As paints, waxes, cosmetics, drugs or packaging materials and also as an approved food additive under the name E 321 used. Butylhydroxytoluene (BHT) can be purchased commercially.

According to the invention, a concentration of 0.10 to 20.00 ppm of BHT is preferably added to the blood and / or blood product in order to achieve sufficient stability of the blood and / or blood product. It is further preferred if the concentration of BHT in the blood and / or blood product is 1.00 to 15.00 ppm. Even more preferably, a concentration of 5.00 to 12.00 ppm BHT in the blood and / or blood product is realized. Particularly preferred is a concentration of 8.00 to 10.00 ppm in the blood and / or blood product.

In vivo experiments have shown that at these concentrations of BHT no harmful effects of humans on transfusion is to be expected. Therefore, at these concentrations BHT in the context of the present invention is free of side effects.

Since BHT is a crystalline solid, it is preferably added to the blood and / or blood product together with a pharmaceutically acceptable carrier or diluent. Preferably, the pharmaceutically acceptable carrier or diluent is a lipophilic carrier or lipophilic diluent. BHT is preferably initially charged with 1% by weight of lecithin as an emulsifier as an aqueous system.

For the purposes of the present invention, the term "pharmaceutically acceptable" is understood to mean that the carrier or diluent for blood and / or blood products is inert and does not cause any undesirable side reactions and, in particular, is non-toxic.

Furthermore, it has been found according to the invention that it is preferred if haptoglobin is added to the blood and / or blood product in addition to the BHT. As stated previously, haptoglobin is used in vivo to bind and remove harmful free hemoglobin from the human body. However, this known mode of functioning of the haptoglobin is based on haptoglobin formed in vivo and must be distinguished from the mode of functioning of a haptoglobin additized to a blood and / or blood product,

A certain amount of haptoglobin is already inherent in the body or in the blood. It is produced by the liver and released into the blood as needed. The concentration of haptoglobin is a maximum of about 2.50 g / l in the human body. The reference range (normal range) in serum is age- and sex-dependent and is approximately 0.50 to 2.50 g / l. Haptoglobin can be obtained from human urine via liquid chromatography and is therefore available for adding blood or blood products in sufficient quantities.

According to the invention, the scavenging function of the toxic free hemoglobin is decisively improved by the proposed increase in the concentration of haptoglobin. Preferably, the concentration of haptoglobin in the blood and / or blood product after the addition is 0.40 to 8.00 g / l. Particularly preferably, the concentration of haptoglobin in the blood and / or blood product after the addition is 2.00 to 6.00 g / l. More preferably, the concentration of haptoglobin in the blood and / or blood product after the addition is 3.00 to 5.00 g / l.

Toxicity assessments with descriptor software as used for risk assessments show no harmful interactions for the intended concentration range of haptoglobin.

Both measures (ie the addition of BHT and haptoglobin) at least lead to a doubling of the shelf life of blood and / or blood products (for example of blood), whereby by optimization (enlargement) of the selected contents according to the basic biochemical knowledge further improvements appear possible because of Buffer area is expanded.

Thus, in a preferred embodiment, the present invention provides a method for improving the stability of blood and / or blood products, which comprises adding to the blood and / or blood product butylhydroxytoluene (BHT) and haptoglobin.

In the method of the present invention, when adding BHT and haptoglobin, the addition of butylhydroxytoluene and haptoglobin to the blood and / or blood product may take place simultaneously or successively, with either BHT and then haptoglobin or haptoglobin and then BHT added. The added amounts of BHT and haptoglobin are based on the above quantities.

Therefore, further, the method is further preferably characterized by butylhydroxytoluene (BHT) at a concentration of 0.10 to 20.00 ppm, preferably 1.00 to 15.00 ppm, more preferably 5.00 to 12.00 ppm preferably from 8.00 to 10.00 ppm, and the concentration of haptoglobin after the addition is 0.40 to 8.00 g / l, more preferably 2.00 to 6.00 g / l, more preferably 3.00 to 5.00 g / l.

• Ausführungsform 1:
  • Butylhydroxytoluol (BHT) in einer Konzentration von 0,10 bis 20,00 ppm und Haptoglobin in einer Konzentration von 0,40 bis 8,00 g/l;
• Ausführungsform 2:
  • Butylhydroxytoluol (BHT) in einer Konzentration von 1,00 bis 15,00 ppm und Haptoglobin in einer Konzentration von 2,00 bis 6,00 g/;
• Ausführungsform 3:
  • Butylhydroxytoluol (BHT) in einer Konzentration von 8,00 bis 10,00 ppm und Haptoglobin in einer Konzentration von 3,00 bis 5,00 g/;
• Ausführungsform 4:
  • Butylhydroxytoluol (BHT) in einer Konzentration von 0,10 bis 20,00 ppm und Haptoglobin in einer Konzentration von 3,00 bis 5,00 g/;
• Ausführungsform 5:
  • Butylhydroxytoluol (BHT) in einer Konzentration von 8,00 bis 10,00 ppm und Haptoglobin in einer Konzentration von 0,40 bis 8,00 g/l.

In particular, the following concentrations of BHT and haptoglobin are encompassed by the present invention:

• Embodiment 1:
  • Butylhydroxytoluene (BHT) in a concentration of 0.10 to 20.00 ppm and haptoglobin in a concentration of 0.40 to 8.00 g / l;
• Embodiment 2:
  • Butylhydroxytoluene (BHT) at a concentration of 1.00 to 15.00 ppm and haptoglobin at a concentration of 2.00 to 6.00 g /;
• Embodiment 3:
  • Butylhydroxytoluene (BHT) at a concentration of 8.00 to 10.00 ppm and haptoglobin at a concentration of 3.00 to 5.00 g / l;
• Embodiment 4:
  • Butylhydroxytoluene (BHT) at a concentration of 0.10 to 20.00 ppm and haptoglobin at a concentration of 3.00 to 5.00 g / l;
• Embodiment 5:
  • Butylhydroxytoluene (BHT) in a concentration of 8.00 to 10.00 ppm and haptoglobin in a concentration of 0.40 to 8.00 g / l.

The inventive method is particularly suitable for improving the stability of blood and / or blood products in a blood bank.

In addition, the usual additives of blood and / or blood products can be used in the method according to the invention, in particular additives selected from the group consisting of citrate, phosphate, dextrose, adenine, EDTA and combinations thereof. The addition of the additives BHT and haptoglobin according to the invention can be done separately in time or in parallel with the usual additives.

In a further aspect, the present invention provides blood and / or a blood product containing butylhydroxytoluene (BHT) in a concentration of 0.10 to 20.00 ppm, preferably 1.00 to 15.00 ppm, more preferably 5.00 to 12.00 ppm, more preferably from 8.00 to 10.00 ppm.

As described above, the blood and / or blood product preferably contains haptoglobin at a concentration of 0.4 to 8 g / l, more preferably 2 to 6 g / l, still more preferably 3 to 5 g / l.

The blood or blood product may further contain at least one additional additive selected from the group consisting of citrate, phosphate, dextrose, adenine, EDTA, and combinations thereof.

Moreover, the present invention relates to the use of butylhydroxytoluene (BHT) in blood or blood products to improve stability.

In the context of the use according to the invention, the BHT is preferably used in combination with haptoglobin.

The amounts of BHT and optionally haptoglobin are as already explained several times. The human toxicological safety and scavenging function in the proposed concentration for BHT can be derived from in vivo experiments and computer simulation based on the structure-activity relationships (descriptor software). This also applies to haptoglobin.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 97/16967 A [0015]
• US 2001/0049089 A [0016]
• US 2010/0021879 A [0017]
• US 2011/0151428 A [0018]

Claims (9)

Process for improving the stability of blood and / or blood products, characterized in that butylhydroxytoluene (BHT) is added to the blood and / or blood product. Method according to Claim 1 , characterized in that haptoglobin is added to the blood and / or blood product. Method according to Claim 2 , characterized in that the addition of butylhydroxytoluene (BHT) and haptoglobin to the blood and / or blood product takes place simultaneously or successively. Method according to Claim 1 to 3 , characterized in that butylhydroxytoluene (BHT) in a concentration of 0.10 to 20.00 ppm is added to the blood and / or blood product. Method according to one of Claims 1 to 4 , characterized in that the concentration of haptoglobin after the addition is 0.40 to 8.00 g / l in the blood and / or blood product. Method according to one of Claims 1 to 5 , characterized in that the blood and / or blood product is a blood conserve. Method according to Claim 1 to 6 characterized in that butylhydroxytoluene (BHT) or butylhydroxytoluene (BHT) and haptoglobin are added to the blood and / or blood product together with a pharmaceutically acceptable carrier or diluent. Blood and / or blood product, characterized by a content of butylhydroxytoluene (BHT) of 0.10 to 20.00 ppm and optionally a content of haptoglobin of 0.40 to 8.00 g / l. Use of butylhydroxytoluene (BHT) and optionally haptoglobin to improve the stability of blood and / or blood products.