DE102007005329A1 - Use of adenosine for erythrocytes protection, and for prevention of sepsis, hemolytic uremic syndrome, microcirculation disturbances, diabetic vascular disease or anemia, has antibiotic or cytostatic drug - Google Patents

Abstract

Use of adenosine for erythrocytes protection, is claimed. The concentration of the adenosine is 1-3000 mg, preferably 500 mg. Independent claims are included for: (1) a method for producing a medium for erythrocytes protection, which involves supplying adenosine, and formulation of the adenosine in a pharmaceutical acceptable carrier; and (2) a method for the therapeutic and/or preventive treatment of an organism, which is affected by a erythrocytes apoptosis, which involves supplying adenosine, and bringing the adenosine into the organism. ACTIVITY : Antibacterial; Immunosuppressive; Antianemic; Nephrotropic; Hemostatic. MECHANISM OF ACTION : Adenosine-A1-receptor antagonist; Nitrobenzylthioinosine inhibitor; Phosphatase inhibitor.

Description

The The present invention relates to an erythrocyte protection agent, a process for producing such an agent, a process for the therapeutic and / or prophylactic treatment of a living being, which is affected by erythrocyte apoptosis and a composition for the preservation of stored blood and erythrocyte-containing preparations.

medium for erythrocyte protection, process for their preparation, process for the treatment of patients suffering from erythrocyte apoptosis as well as compositions for the preservation of stored blood and erythrocyte-containing preparations are in the prior art well known.

Under Erythrocyte apoptosis or eryptosis becomes a strictly regulated physiological Process in the nature of a "cell suicide" of red blood cells or Erythrocytes also understood as the programmed erythrocyte cell death referred to as. In an organism, the erythrocyte apoptosis is under certain circumstances, especially in stressful situations, by exogenous as well as endogenous stimuli, to protect the whole organism triggered. For example. are erythrocytes during their Life cycle exposed to various stress factors. Average Once a minute, they pass the lungs, where they are in oxidative Experience stress. They go through this more than once an hour Renal medulla, where it suffers an osmotic shock. erythrocytes are pressed through capillaries, which have a diameter which is smaller than their own. The integrity The erythrocytes is therefore permanently exposed to challenges.

at Damage to the erythrocyte membrane becomes hemoglobin as well as other intracellular proteins in the extracellular space or the extracellular fluid released. This process is called hemolysis. The extracellular Fluid is filtered by kidney glomeruli, it being in the acidic lumen of the tubules for precipitation of leaked hemoglobin can come. That precipitated Hemoglobin in turn can damage the tubules and thereby lead to kidney failure. Furthermore, can the leakage of intracellular proteins inflammatory reactions in the organism.

Of the Erythrocyte apoptosis or eryptosis is u. a. attributed the function, the aforementioned complications resulting from hemolysis result, to prevent. The erythrocyte apoptosis occurs to a shrinkage of the cell and the formation of protuberances on the erythrocyte membrane. Furthermore, proteases are activated and phosphatidylserine, this almost exclusively in non-apoptotic erythrocytes located on the inside of the cell membrane, is located on the Outside of the cell membrane exposed. The exposed phosphatidylserine is recognized by macrophages, which changed them in such a way Erythrocytes phagocytose and degrade. In this physiological process It prevents it from releasing hemoglobin or of intracellular proteins from the cell interior the erythrocytes into the extracellular medium with the consequence an inflammatory reaction and the above further complications comes.

In addition to the physiologically desired function of preventing hemolysis, however, the erythrocyte apoptosis also plays an important pathophysiological role. There is evidence that sickle cell anemia is associated with increased erythrocyte apoptosis activity; see. Lang et al. (2002), Enhanced erythrocytes apoptosis in sickle cell anemia, thalassemia and glucose-6-phosphate dehydrogenase deficiency, Cell. Physiol. Biochem. 12, pages 365-372 , It has also been shown that iron deficient erythrocytes are more sensitive to erythrocyte apoptosis; see. Kempe et al. (2006), Enhanced programmed cell death of iron-deficient erythrocytes. FASER J. 20, pages 368-370 , The erythrocyte apoptosis also appears to ensure that the malaria plasmodium falciparum pathogen survives inside the erythrocytes; see. Duranton et al. (2003), Physiological properties of the plasmodium falciparum-induced cationic conductance of human erythrocytes. Cell. Physiol. Biochem. 13, pages 189-198 , Furthermore, after erythrocyte exposure to hemolysin Kanagawa from Vidrio Parahaemolyticus, a marine bacterium that can be ingested via contaminated seafood, it appears that eryptosis is induced; see. Lang et al. (2004), Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes. Cell. Microbiol. 6, pages 391-400 ,

Increased eryptosis appears to be associated with hemolytic uremic syndrome (HUS) [ Lang et al. (2006a), Suicidal death of erythrocytes in recurrent hemolytic uremic syndrome. J. Mol. Med. 84, pages 378-388 ], sepsis [ Kempe et al. (2006), Suicidal erythrocyte death in sepsis. J. Mol. Med. ], diabetes [ Nicolay et al. (2006), Stimulation of suicidal erythrocyte death by methylgoyoxal. Cell. Physiol. Biochem. 18, pages 223-232 ], or in the treatment with cytostatic agents such as paclitaxel [ Lang et al. (2006b), Stimulation of erythrocyte phosphatidylserine exposure by paclitaxel. Cell Physiol. Biochem. 18, pages 151-164 ].

Against this background, there are efforts to find agents with which targeted the Erythrocytenapoptose can be inhibited to treat the aforementioned diseases or pathological conditions or to prevent them. For example, in the international application WO 2005/087216 It has been proposed to use for the inhibition of erythrocyte apoptosis phosphate, iron, inhibitors of sphingomyelinase, inhibitors of phospholipase A2 and inhibitors of cyclooxygenase. However, phosphate and iron act only in the case of eryptosis due to phosphate or iron deficiency, sphingomyelinase inhibitors only in ceramide-induced eryptosis and cycloxygenase inhibitors only if the cause of eryptosis is the release of PGE ₂ . A universal treatment of eryptosis is not possible with these compounds.

In front In this context, it is the object of the present invention to provide an agent having erythrocyte-protective properties and with which the disadvantages of the prior art known compounds and agents for inhibiting erythrocyte apoptosis can be avoided. In particular, such should Provided means that are inexpensive to produce and as poor as possible to side effects.

These Task is accomplished by the use of adenosine to produce a Dissolved by means of erythrocyte protection. The task is further characterized by a process for the preparation of an erythrocyte protection agent solved, comprising the following steps: (1) provision of adenosine, and (2) formulation of adenosine into a pharmaceutical acceptable carrier.

Pharmaceutically acceptable carriers are fully described in the art. For example, reference is made to Bauer et al. (1999), Textbook of Pharmaceutical Technology, 6th Edition, Wissenschaftliche Verlagsgesellschaft mbH Stuttgart ; Rowe et al. (2006), Handbook of pharmaceutical excipients, 5th edition, Pharmaceutical Press and American Pharmacist Association , The content of the aforementioned publications is incorporated by reference into the application.

The Recognizing that adenosine is effective in erythrocytes from apoptosis could protect, was therefore surprising because of this Compound previously attributed completely different effects were.

Adenosine or 6-aminopurine-9-β-D-ribofuranoside, 9-D-ribofuranosyl-9-H-purine-6-amine, C ₁₀ H ₁₃ N ₅ O ₄ , M _R = 267.24, is a nucleoside which is composed of adenine and β-D-ribose. Adenosine is a component of ribonucleic acids.

For Adenosine has been described to dilate this peripheral blood vessel and vagotropic. Adenosine is used as a medicine, for example used for the treatment of AV nodal tachycardia.

For a variety of different nucleated cell types has been described that adenosine inhibits apoptosis, z. Of hepatocytes [ Blom et al. (1999), Prevention of cycloheximide-induced apoptosis in hepatocytes by adenosine and by caspase inhibitors. Biochem. Pharmacol. 58, pages 1891-1898 ], Brain cells [ Boucher et al. (2006), Reduction of apoptosis in the amygdala by A2A adenosine receptor agonist following myocardial infarction. Apoptosis 11, pages 1067-1074 ], transformed mast cells [ Gao et al. (2001), A3 adenosine receptor activation triggers phosphorylation of protein kinase B and protects rat basophilic leukemia 2H3 mast cells from apoptosis. Mol. Pharmacol. 59, pages 76-82 ], Cardiocytes [ Goldenberg et al. (2003), Adenosine protects against angiotensin II-induced apoptosis in rat cardiocyte cultures. Mol. Cell. Biochem. 252, pages 133-139 ], transformed cells from the adrenal medulla [ Huang NK. et al. (2001), Activation of Protein Kinase A and Atypical Protein Kinase C by A (2A) adenosine receptor antagonizes apoptosis due to serum deprivation in PC12 cells. J. Biol. Chem. 276, pages 13838-13846 ], Neutrophils [ Ottonello et al. (2002), synovial fluid from patients with rheumatoid arthritis inhibits neutrophil apoptosis: role of adenosine and proinflammatory cytokines. Rheumatology (Oxford), 41, pages 1249-1260 ] and cells from the lung tissue [ Rivo et al. (2004), Activation of A3 adenosine receptor protection against ischemia-reperfusion injury associated with reduction in apoptosis. At the. J. Transplant. 4, pages 1941-1948 ].

Nevertheless, it was not to be expected that adenosine also exhibits an erythrocyte inhibiting effect on erythrocytes. Thus, erythrocytes not only have no cell nuclei but also no mitochondria. Both organelles are central elements of the apoptotic machinery of nucleated cells. Thus, it has been thought that erythrocytes are eliminated by a mechanism different from that of apoptosis of nucleated cells; see. Lang et al. (2005a), Mechanism of suicidal erythrocyte death, Cell. Physiol. Biochem. 15, pages 195-202 , This seems to be evidenced by the fact that in eryptosis of erythrocytes after incubation with ionomycin or exposure to hyperosmotic shock the activation of caspases does not occur, but with corresponding induction of apoptosis of nucleated cells; see. Lang et al. (2004), Involvement of Ceramides in Hyperosmotic Shock-Induced Death of Erythrocytes. Cell. Death. Differ. 11, pages 231-243 ,

The The object underlying the invention is hereby completely solved.

at the agent is preferably a drug which additionally a pharmaceutically acceptable carrier having.

by virtue of the good availability and the small size of adenosine, which allows a straightforward formulation, is such a drug can be produced inexpensively. The low side effects make it possible for the first time purposefully to treat such pathophysiological conditions or to prevent such on an increased erythrocyte apoptosis decline.

there it is preferred if the drug is for treatment and / or Prevention of a disease or a pathological condition is provided, which is selected from the group from: sepsis, hemolytic uremic syndrome (HUS), diabetic vascular damage, microcirculation disorders and anemia.

These Embodiment has the advantage that such a drug provided will be used to treat the major diseases or can be prevented, which is based on an increased erythrocyte apoptosis decline.

Further It is preferred if the drug is part of a treatment provided with cytostatics.

So It is known that the administration of cytotoxic agents, for example Paclitaxel, in the treated patients to the undesirable Enhancement of erythrocyte apoptosis results; Long et al. (2006b, supra). The invention provides effective remedy by the erythrocyte apoptosis is purposefully inhibited without the Effect of the cytostatic is impaired.

According to the invention it is further preferred if the medicament for an application is formed, selected from the group consisting of: oral, rectal, parenteral, local, transdermal.

These Measure has the advantage that the drug depending on desired application form already in a suitable manner is designed. The attending physician thereby has a choice various forms of the drug are available, whereby the concrete form depends on the condition of the patient, the respective one Treatment program and other factors.

In front In this background, it is also preferable if the drug is formed in a form which is selected from the Group consisting of: powder, tablet, juice, drops, capsule, suppositories, Solution, solution for injection, aerosol, ointment, conditioner, Plaster, pellet, dragee.

With This measure is already in an advantageous manner provided a formulation variant that the immediate Use of adenosine for the treatment or prevention of allows such pathological conditions, the associated with increased erythrocyte apoptosis.

at the use according to the invention or the inventive It is further preferred method, when the drug is adenosine in a concentration selected from the group consisting of (in each case given in weight of the active substance Adenosine relative to the total weight of the drug): 1 μg / g, 10 μg / g, 100 μg / g, 1 mg / g, 10 mg / g, 100 mg / g, 1 g / g.

These Measure has the advantage that the drug is the active ingredient Adenosine already has a concentration that is needed for treatment or prevention of diseases or pathological conditions particularly suitable with increased erythrocyte apoptosis are associated.

Further it is preferred if the absolute amount of the active substance is adenosine in a dosage unit of the drug between about 1 and about 3000 mg, preferably between about 10 and about 1500 mg, more preferably between about 100 and 750 mg, and most preferably at about 500 mg.

With Adenosine is already in one of these measures Amount provided that in the patient readily the desired Adenosine levels can be achieved. With a dosage unit is according to the invention one of the above formulation variants denotes, for example, a tablet, a dragee, a capsule Etc.

Further it is in the use according to the invention or preferred method of the invention, if the drug additionally has another active substance.

These Measure has the advantage that by adding the other Active substance the effects of the invention Drugs can still be improved. That's the way it is possible by combining with another substance to cause an intervention in the respective clinical picture in one place at the adenosine shows no effect to the disease through synergistic To combat effects even more effectively.

at the further active substance is preferably an antibiotic.

This measure has the advantage that such a further substance is provided, which the The most common cause of sepsis or hemolytic uremic syndrome, namely a bacterial infection, independently fought. The medicament according to the invention is thus directed specifically against the amplification of the erythrocyte apoptosis and simultaneously shuts off the cause of the sepsis.

alternative it is preferred if the further active substance is a cytostatic is.

These Measure has the advantage that for the first time a combination preparation is provided with the one hand, an effective cystostat-based Treatment of a patient is possible without it at the same time to an undesired increased erythrocyte apoptosis comes.

According to the invention it is further preferred if the agent is a preservative for stored blood and erythrocyte-containing preparations is.

These Embodiment has the advantage that such a means the life of stored blood and erythrocyte-containing preparations can increase. It is known that stored blood and erythrocyte-containing Preparations have a limited useful life. This goes u. a. on the fact that the erythrocytes the eryptosis go through and thereby the number of erythrocytes in the preparations over the time is decreasing. Preserved blood and erythrocyte-containing preparations can therefore by the addition of adenosine in terms their shelf life can be significantly improved. It goes without saying that the preservative may contain further constituents, common in blood and erythrocyte preparations are, such as saline.

In front This background is another object of the present invention Invention a composition for the preservation of blood products and erythrocyte-containing preparations, the adenosine and a having pharmaceutically acceptable carrier.

there it is preferred if in the preservative adenosine in a Concentration is about 0.1 μM to about 1 mM, preferably at about 1 μM to about 500 μM, further preferably at about 10 μM to 250 μM, most preferably at about 100 uM.

The Inventors have found from cell culture experiments that the specified concentration ranges optimal erythrocyte protection guarantee.

The Composition preferably has adenosine in a concentration at about 0.1 μM to about 1 mM, preferably at about 1 μM to about 500 μM, more preferably at about 10 μM to 250 μM, most preferred at about 100 μM is.

As As stated above, the inventors could by means of Cell culture experiments find that the protective effect of Adenosine is particularly good in the concentration ranges mentioned.

In front This background concerns a further subject matter of the present invention Invention a method for therapeutic and / or prophylactic treatment of a living being affected by erythrocyte apoptosis or / and at risk of erythrocyte apoptosis, comprising the following steps: (1) providing adenosine, (2) introduction of adenosine into the animal, and (3) optionally repeating steps (1) and (2) several times.

The Adenosine can in this process in the form of the invention Provided drug and introduced into the living being. The pertaining to the features, features and advantages described above the medicament according to the invention apply insofar for this procedure accordingly.

It it is understood that the above and the following yet to be explained features not only in each case specified combination, but also in other combinations or can be used in isolation, without the scope of the present To leave invention.

The The invention will now be described in more detail with reference to exemplary embodiments explains what makes up more features, features and benefits. Reference is made to the attached Figures showing the following:

• A. Histogramme zur Annexin-V-Bindung an Erythrozyten in einem repräsentativen Experiment mit Erythrozyten aus gesunden Freiwilligen, die für 24 Stunden in Gegenwart (obere Graphen) oder in Abwesenheit (untere Graphen) von Glucose und in Abwesenheit (linke Graphen) oder Gegenwart (rechte Graphen) von 10 μM Adenosin inkubiert wurden.
• B. Arithmetische Mittel ± SEM (n = 4–7) der prozentualen Bindung von Annexin-V an Erythrozyten nach 24-stündiger Inkubation in Gegenwart (Ringer, unausgefüllter Balken) oder Abwesenheit (grauer oder schwarzer Balken) von Glucose und in Abwesenheit (grauer Balken) oder Gegenwart (schwarze Balken) von Adenosin [10 μM, 30 μM und 100 μM Adenosin (ADO)]. * Zeigt einen signifikanten Unterschied bei Gegenwart von Glucose an, # zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Adenosin an (ANOVA unter Verwendung des Dunnetts-Test als post hoc Test; P ≤ 0,05).
• C. Arithmetische Mittel ± SEM (n = 4–7) des prozentualen Anteils der Annexin-V-Bindung an Erythrozyten nach einer Inkubation für 48 Stunden in Gegenwart (Ringer, unausgefüllte Balken) oder Abwesenheit (grauer bzw. schwarzer Balken) von Glucose und in Abwesenheit (grauer Balken) oder Gegenwart (schwarze Balken) von Adenosin (10 μM, 30 μM und 100 μM ADO). * Zeigt einen signifikanten Unterschied bei Gegenwart von Glucose an, # zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Adenosin an (ANOVA unter Verwendung des Dunnetts-Test als post hoc Test; P ≤ 0,05).

1 Stimulation of phosphatidylserine exposure by glucose depletion in the presence and absence of adenosine.

• A. Histograms for annexin V binding to erythrocytes in a representative experiment with erythrocytes from healthy volunteers maintained for 24 hours in the presence (upper graphs) or in the absence (lower graphs) of glucose and in the absence (left graphs) or present ( right graphene) of 10 μM adenosine were incubated.
• B. Arithmetic mean ± SEM (n = 4-7) of the percent binding of annexin-V to erythrocytes after 24 hours incubation in the presence (Ringer, open bar) or absence (gray or black bar) of glucose and in the absence (gray Bars) or presence (black bars) of adenosine [10 μM, 30 μM and 100 μM adenosine (ADO)]. * Indicates a significant difference in the presence of glucose, # indicates a significant difference from the absence of adenosine (ANOVA using the Dunnetts test as a post hoc test, P ≤ 0.05).
• C. Arithmetic mean ± SEM (n = 4-7) of the percentage of annexin-V binding to erythrocytes after incubation for 48 hours in the presence (wrestlers, open bars) or absence (gray and black bars) of glucose and in the absence (gray bar) or presence (black bars) of adenosine (10 μM, 30 μM and 100 μM ADO). * Indicates a significant difference in the presence of glucose, # indicates a significant difference from the absence of adenosine (ANOVA using the Dunnetts test as a post hoc test, P ≤ 0.05).

• A. Histogramme von Erythrozyten-Vorwärtsscatter in einem repräsentativen Experiment mit Erythrozyten aus gesunden Freiwilligen, die für 24 Stunden in Gegenwart (obere Graphen) oder in Abwesenheit (untere Graphen) von Glucose und in Abwesenheit (linke Graphen) oder Gegenwart (rechte Graphen) von 10 μM Adenosin inkubiert wurden.
• B. Arithmetische Mittel ± SEM (n = 4–7) der Erythrozyten-Vorwärtsscatter nach 24-stündiger Inkubation in Gegenwart (Ringer, unausgefüllter Balken) oder Abwesenheit (grauer oder schwarzer Balken) von Glucose und in Abwesenheit (grauer Balken) oder Gegenwart (schwarze Balken) von Adenosin (10 μM, 30 μM und 100 μM ADO). * Zeigt einen signifikanten Unterschied bei Gegenwart von Glucose an, # zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Adenosin an (ANOVA unter Verwendung des Dunnetts-Test als post hoc Test; P ≤ 0,05).
• C. Arithmetische Mittel ± SEM (n = 4–7) der Erythrozyten-Vorwärtsscatter nach 48-stündiger Inkubation in Gegenwart (Ringer, unausgefüllter Balken) oder Abwesenheit (grauer bzw. schwarzer Balken) von Glucose in Abwesenheit (grauer Balken) oder Gegenwart (schwarze Balken) von Adenosin (10 μM, 30 μM und 100 μM ADO). * Zeigt einen signifikanten Unterschied bei Gegenwart von Glucose an, # zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Adenosin an (ANOVA unter Verwendung des Dunnetts-Test als post hoc Test; P ≤ 0,05).

2 Decrease of the forward scatter by glucose depletion in the presence and absence of adenosine

• A. Histograms of erythrocyte forward caterpillar in a representative experiment with erythrocytes from healthy volunteers exposed for 24 h in the presence (upper graphs) or in the absence (lower graphs) of glucose and in the absence (left graphs) or presence (right graphs) of 10 μM adenosine were incubated.
• B. Arithmetic mean ± SEM (n = 4-7) of the red cell forward scan after 24 hours incubation in the presence (Ringer, open bar) or absence (gray or black bar) of glucose and in the absence (gray bar) or presence ( black bars) of adenosine (10 μM, 30 μM and 100 μM ADO). * Indicates a significant difference in the presence of glucose, # indicates a significant difference from the absence of adenosine (ANOVA using the Dunnetts test as a post hoc test, P ≤ 0.05).
• C. Arithmetic mean ± SEM (n = 4-7) of the erythrocyte forward scatter after 48 hours incubation in the presence (Ringer, open bar) or absence (gray and black bars, respectively) of glucose in the absence (gray bar) or presence ( black bars) of adenosine (10 μM, 30 μM and 100 μM ADO). * Indicates a significant difference in the presence of glucose, # indicates a significant difference from the absence of adenosine (ANOVA using the Dunnetts test as a post hoc test, P ≤ 0.05).

• A. Histogramme der Annex-V-Bindung an Erythrozyten in einem repräsentativen Experiment mit Erythrozyten aus gesunden Freiwilligen, die für 24 Stunden in Gegenwart (obere Graphen) oder in Abwesenheit (untere Graphen) von Cl^– (ersetzt durch Gluconat) und in Abwesenheit (linke Graphen) oder Gegenwart (rechte Graphen) von 10 μM Adenosin inkubiert wurden.
• B. Arithmetische Mittel ± SEM (n = 4–11) des prozentualen Anteils von Hämolyse (schwarze Balken) und des prozentualen Anteils der Annexin-V-Bindung an Erythrozyten (unausgefüllte Balken) nach 24- stündiger Inkubation in Gegenwart (Ringer) oder Abwesenheit (0 Chlorid) von Cl^– und in Abwesenheit und Gegenwart von Adenosin (10 μM, 30 μM und 100 μM ADO). * Zeigt einen signifikanten Unterschied bei Gegenwart von Chlorid an, # zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Adenosin an (ANOVA unter Verwendung des Dunnetts-Test als post hoc Test; P ≤ 0,05).

3 Stimulation of exposure of phosphatidylserine and hemolysis by Cl ^- depletion in the presence and absence of adenosine

• A. Histograms of Annex V binding to erythrocytes in a representative experiment with erythrocytes from healthy volunteers exposed for 24 hours in the presence (upper graphs) or in the absence (lower graphs) of Cl ^- (replaced by gluconate) and in the absence ( left graphene) or presence (right graphene) of 10 μM adenosine were incubated.
• B. Arithmetic mean ± SEM (n = 4-11) of the percentage of hemolysis (black bars) and the percentage of annexin V-binding on erythrocytes (open bars) after 24 hours incubation in the presence (Ringer) or absence (0 chloride) of Cl ^- and in the absence and presence of adenosine (10 μM, 30 μM and 100 μM ADO). * Indicates a significant difference in the presence of chloride, # indicates a significant difference from the absence of adenosine (ANOVA using the Dunnetts test as a post hoc test, P ≤ 0.05).

• A. Histogramme von Erythrozyten Vorwärtsscatter in einem repräsentativen Experiment mit Erythrozyten aus gesunden Freiwilligen, die für 24 Stunden in Gegenwart (obere Graphen) oder Abwesenheit (untere Graphen) von Cl^– (ersetzt durch Gluconat) in Abwesenheit (linke Graphen) oder Gegenwart (rechte Graphen) von 10 μM Adenosin inkubiert wurden.
• B. Arithmetische Mittel ± SEM (n = 4–11) von Erythrozyten-Vorwärtsscatter nach 24-stündiger Inkubation in Gegenwart (Ringer, unausgefüllte Balken) oder Abwesenheit (grauer bzw. schwarzer Balken) von Cl^– und in Abwesenheit (grauer Balken) oder Gegenwart (schwarze Balken) von Adenosin (10 μM, 30 μM und 100 μM ADO). * Zeigt einen signifikanten Unterschied bei Gegenwart von Chlorid an, # zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Adenosin an (ANOVA unter Verwendung des Dunnetts-Test als post hoc Test; P ≤ 0,05).

4 Decrease of the forward scissors by Cl ^- depletion in the presence and absence of adenosine

• A. Histograms of erythrocyte forward scatter in a representative experiment with erythrocytes from healthy volunteers exposed for 24 h in the presence (upper graph) or absence (lower graph) of Cl ^- (replaced by gluconate) in the absence (left graph) or presence (right) Graphene) of 10 μM adenosine were incubated.
• B. Arithmetic mean ± SEM (n = 4-11) of red cell forward scan after 24 hours incubation in the presence (Ringer, open bars) or absence (gray and black bars, respectively) of Cl ^- and in the absence (gray bar) or Presence (black bars) of adenosine (10 μM, 30 μM and 100 μM ADO). * Indicates a significant difference in the presence of chloride, # indicates a significant difference from the absence of adenosine (ANOVA using the Dunnetts test as a post hoc test, P ≤ 0.05).

5 Effect of Cl ^- depletion on cytosolic Ca ^{2 +} activity in the presence and absence of adenosine

Arithmetic mean ± SEM (n = 4) of fluo3-dependent fluorescence in erythrocytes maintained for 180 minutes (A) or 360 minutes (B) in Ringer's solution in the presence (open bars) or absence (gray bars) of Cl ^- ( replaced by gluconate) and in the absence (light gray bar) or presence (dark gray bar) of 100 μM adenosine or with 1 μM ionomycin (Iono) as positive control. * Indicates a significant difference from a presence of Cl ^- (ANOVA using the Dunnetts test as a post hoc test; P ≤ 0.05).

• A. Histogramme der Annexin-V-Bindung an Erythrozyten in einem repräsentativen Experiment mit Erythrozyten aus gesunden Freiwilligen, die für 24 Stunden in Abwesenheit (obere Graphen) oder Gegenwart (untere Graphen) von Okadasäure (1 μM) und in Abwesenheit (linke Graphen) oder Gegenwart (rechte Graphen) von 10 μM Adenosin inkubiert wurden.
• B. Arithmetische Mittel ± SEM (n = 4) der prozentualen Annexin-V-Bindung an Erythrozyten nach 24-stündiger Inkubation in Abwesenheit (Ringer, unausgefüllte Balken) oder Gegenwart (graue oder schwarze Balken) von Okadasäure (1 μM) und in Abwesenheit (grauer Balken) oder Gegenwart (schwarze Balken) von Adenosin (10 μM, 30 μM und 100 μM ADO). * Zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Okadasäure an, # zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Adenosin an (ANOVA unter Verwendung des Dunnetts-Test als post hoc Test; P ≤ 0,05).

6 Stimulation of the exposure of phosphatidylserine by okadaic acid in the presence and absence of adenosine

• A. Histograms of annexin V binding to erythrocytes in a representative experiment with erythrocytes from healthy volunteers exposed for 24 hours in the absence (upper graphs) or presence (lower graphs) of okadaic acid (1 μM) and in the absence (left graphs) or presence (right graphene) of 10 μM adenosine were incubated.
• B. Arithmetic mean ± SEM (n = 4) of percent annexin-V binding to erythrocytes after 24 hours incubation in the absence (wrestlers, open bars) or presence (gray or black bars) of okadaic acid (1 μM) and in the absence (gray bar) or presence (black bars) of adenosine (10 μM, 30 μM and 100 μM ADO). * Indicates a significant difference from the absence of okadaic acid, # indicates a significant difference from the absence of adenosine (ANOVA using the Dunnetts test as a post hoc test, P ≤ 0.05).

• A. Histogramme von Erythrozyten-Vorwärtsscatter in einem repräsentativen Experiment mit Erythrozyten aus gesunden Freiwilligen, die für 24 Stunden in Abwesenheit (obere Graphen) oder Gegenwart (untere Graphen) von Okadasäure (1 μM) und in Abwesenheit (linke Graphen) oder Gegenwart (rechte Graphen) von 10 μM Adenosin inkubiert wurden.
• B. Arithmetische Mittel ± SEM (n = 4) von Erythrozyten-Vorwärtsscatter nach 24-stündiger Inkubation in Abwesenheit (Ringer, unausgefüllter Balken) oder Gegenwart (graue oder schwarze Balken) von Okadasäure (1 μM) und in Abwesenheit (grauer Balken) oder Gegenwart (schwarze Balken) von Adenosin (10 μM, 30 μM und 100 μM ADO). * Zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Okadasäure an, # zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Adenosin an (ANOVA unter Verwendung des Dunnetts-Test als post hoc Test; P ≤ 0,05).

7 Decrease of the forward scatter by okadaic acid in the presence and absence of adenosine

• A. Histograms of erythrocyte forward caterpillar in a representative experiment with erythrocytes from healthy volunteers exposed for 24 hours in the absence (upper graphs) or presence (lower graphs) of okadaic acid (1 μM) and in the absence (left graph) or presence (right) Graphene) of 10 μM adenosine were incubated.
• B. Arithmetic mean ± SEM (n = 4) of red cell forward scan after 24 hours incubation in the absence (Ringer, open bar) or presence (gray or black bars) of okadaic acid (1 μM) and in the absence (gray bar) or Presence (black bars) of adenosine (10 μM, 30 μM and 100 μM ADO). * Indicates a significant difference from the absence of okadaic acid, # indicates a significant difference from the absence of adenosine (ANOVA using the Dunnetts test as a post hoc test, P ≤ 0.05).

• A. Histogramme der Annexin-V-Bindung an Erythrozyten in einem repräsentativen Experiment mit Erythrozyten aus gesunden Freiwilligen, die für 24 Stunden in Abwesenheit (obere Graphen) oder Gegenwart (untere Graphen) von Phorbol-12-myristat-13-acetat (3 μM) und in Abwesenheit (linke Graphen) oder Gegenwart (rechte Graphen) von 10 μM Adenosin inkubiert wurden.
• B. Arithmetische Mittel ± SEM (n = 4) des prozentualen Anteils der Annexin-V-Bindung an Erythrozyten nach 24-stündiger Inkubation in Abwesenheit (Ringer, unausgefüllter Balken) oder Gegenwart (grauer oder schwarzer Balken) von Phorbol-12-myristat-13-acetat (3 μM) und in Abwesenheit (grauer Balken) oder Gegenwart (schwarze Balken) von Adenosin (10 μM, 30 μM und 100 μM ADO). * Zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Phorbol-12-myristat-13-acetat an, # zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Adenosin an (ANOVA unter Verwendung des Dunnetts-Test als post hoc Test; P ≤ 0,05).

8th Stimulation of exposure of phosphatidylserine by phorbol 12-myristate 13-acetate in the presence and absence of adenosine

• A. Histograms of annexin V binding to erythrocytes in a representative experiment with erythrocytes from healthy volunteers exposed for 24 hours in the absence (upper graphs) or presence (lower graphs) of phorbol 12-myristate 13-acetate (3 μM ) and in the absence (left graphs) or presence (right graphs) of 10 μM adenosine were incubated.
• B. Arithmetic mean ± SEM (n = 4) of the percentage of annexin-V binding to erythrocytes after 24 hours incubation in the absence (Ringer, open bar) or presence (gray or black bar) of phorbol-12-myristate- 13-acetate (3 μM) and in the absence (gray bar) or presence (black bars) of adenosine (10 μM, 30 μM and 100 μM ADO). * Indicates a significant difference from the absence of phorbol 12-myristate 13-acetate, # indicates a significant difference from the absence of adenosine (ANOVA using the Dunnetts test as a post hoc test; P ≤ 0.05) ,

• A. Histogramme von Erythrozyten-Vorwärtsscatter in einem repräsentativen Experiment mit Erythrozyten aus gesunden Freiwilligen, die für 24 Stunden in Abwesenheit (obere Graphen) oder Gegenwart (untere Graphen) von Phorbol-12-myristat-13-acetat (3 μM) und in Abwesenheit (linke Graphen) oder Gegenwart (rechte Graphen) von 10 μM Adenosin inkubiert wurden.
• B. Arithmetische Mittel ± SEM (n = 4) von Erythrozyten-Vorwärtsscatter nach 24-stündiger Inkubation in Abwesenheit (Ringer, unausgefüllter Balken) oder Gegenwart (grauer oder schwarzer Balken) von Phorbol-12-myristat-13-acetat (3 μM) und in Abwesenheit (grauer Balken) oder Gegenwart (schwarze Balken) von Adenosin (10 μM, 30 μM und 100 μM ADO). * Zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Phorbol-12-myristat-13-acetat an.

9 Decrease of the forward scatter by phorbol 12-myristate 13-acetate in the presence and absence of adenosine

• A. Histograms of red cell forward scan in a representative experiment with erythrocytes from healthy volunteers exposed for 24 hours in the absence (upper graphs) or presence (lower graphs) of phorbol 12-myristate 13-acetate (3 μM) and in the absence (left graphs) or presence (right graphs) of 10 μM adenosine were incubated.
• B. Arithmetic mean ± SEM (n = 4) of red cell forward scan after 24 hours incubation in the absence (Ringer, open bar) or presence (gray or black bar) of phorbol 12-myristate 13-acetate (3 μM). and in the absence (gray bar) or presence (black bars) of adenosine (10 μM, 30 μM and 100 μM ADO). * Indicates a significant difference from the absence of phorbol 12-myristate 13-acetate.

• A. Arithmetische Mittel ± SEM (n = 4–8) des prozentualen Anteils der Annexin-V-Bindung an Erythrozyten nach 24-stündiger Inkubation in Gegenwart (Ringer) oder Abwesenheit (0 Chlorid) von Cl^–, in Abwesenheit (– Adenosin) oder Gegenwart (+ Adenosin) von Adenosin (30 μM), in Abwesenheit (unausgefüllte Balken) oder Gegenwart von 10 μM NBMPR (graue Balken) oder 2,5 μM CGS-15943 (schwarze Balken). * Zeigt einen signifikanten Unterschied gegenüber der Gegenwart von Chlorid an, # zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Adenosin an (ANOVA unter Verwendung des Dunnetts-Test als post hoc Test; P ≤ 0,05).
• B. Arithmetische Mittel ± SEM (n = 4–8) des Erythrozyten-Vorwärtsscatters nach 24-stündiger Inkubation in Gegenwart (Ringer) oder Abwesenheit (0 Chlorid) von Cl^–, in Abwesenheit (– Adenosin) oder Gegenwart (+ Adenosin) von Adenosin (30 μM), in Abwesenheit (unausgefüllte Balken) oder in Gegenwart von NBMPR (graue Balken) oder CGS-15943 (schwarze Balken). * Zeigt einen signifikanten Unterschied gegenüber der Gegenwart von Chlorid an, # zeigt einen signifikanten Unterschied gegenüber der Abwesenheit von Adenosin an (ANOVA unter Verwendung des Dunnetts-Test als post hoc Test; P ≤ 0,05).

10 Stimulation of exposure of phosphatidylserine and decrease of forward scatter by Cl ^- depletion in the presence and absence of adenosine and in the presence and absence of a uptake inhibitor (NBMPR) and purinergic receptor antagonist (CGS-15943)

• A. Arithmetic mean ± SEM (n = 4-8) of the percentage of annexin V binding to erythrocytes after 24 hours incubation in the presence (Ringer) or absence (0 chloride) of Cl ^- , in the absence (- adenosine) or presence (+ adenosine) of adenosine (30 μM), in the absence (open bars) or presence of 10 μM NBMPR (gray bars) or 2.5 μM CGS-15943 (black bars). * Indicates a significant difference from the presence of chloride, # indicates a significant difference from the absence of adenosine (ANOVA using the Dunnetts test as a post hoc test, P ≤ 0.05).
• B. Arithmetic mean ± SEM (n = 4-8) of the erythrocyte forward scatter after 24 hours incubation in the presence (Ringer) or absence (0 chloride) of Cl ^- , in the absence (- adenosine) or presence (+ adenosine) of Adenosine (30 μM), in the absence (open bars) or in the presence of NBMPR (gray bars) or CGS-15943 (black bars). * Indicates a significant difference from the presence of chloride, # indicates a significant difference from the absence of adenosine (ANOVA using the Dunnetts test as a post hoc test, P ≤ 0.05).

• A. Arithmetische Mittel ± SEM (n = 6) des prozentualen Anteils der Annexin-V-Bindung an Erythrozyten nach 24-stündiger Inkubation in Abwesenheit von Cl^–, in Abwesenheit (0 Cl) oder Gegenwart (0 Cl + Adenosin) von Adenosin (100 μM), in Abwesenheit (unausgefüllte Balken) oder Gegenwart (ausgefüllte Balken) von 20 μM FSCPX. * Zeigt einen signifikanten Unterschied gegenüber den jeweiligen Kontrollen ohne Adenosin an (wiederholte Messungen ANOVA).
• B. Arithmetische Mittel ± SEM (n = 8) des prozentualen Anteils der Annexin-V-Bindung an Erythrozyten nach 24-stündiger Inkubation in Abwesenheit von Cl^– (0 Cl) oder Gegenwart (0 Cl + Adenosin) von Adenosin (100 μM), in Abwesenheit (unausgefüllte Balken) oder Gegenwart (geschlossene Balken) von 4 μM VUF5574. * Zeigt einen signifikanten Unterschied gegenüber den jeweiligen Kontrollen ohne Adenosin an (wiederholte Messungen ANOVA).

11 Phosphatidylserine exposure during Cl ^- depletion in the presence and absence of adenosine and in the presence and absence of the purinergic receptor antagonist FSCPX or VUF5574

• A. Arithmetic mean ± SEM (n = 6) of the percentage of annexin V binding to erythrocytes after 24 hours incubation in the absence of Cl ^- , in the absence (0 Cl) or presence (0 Cl + adenosine) of adenosine ( 100 μM), in the absence (open bars) or presence (solid bars) of 20 μM FSCPX. * Indicates a significant difference from the controls without adenosine (repeated measurements ANOVA).
• B. Arithmetic mean ± SEM (n = 8) of the percentage of annexin-V binding to erythrocytes after 24 hours incubation in the absence of Cl ^- (O Cl) or presence (0 Cl + adenosine) of adenosine (100 μM) , in absence (open bars) or presence (closed bars) of 4 μM VUF5574. * Indicates a significant difference from the controls without adenosine (repeated measurements ANOVA).

embodiments

1. Material and methods

1.1 erythrocytes, solutions and chemicals

The Experiments were performed at 37 ° C with isolated erythrocytes carried out from healthy volunteers. The volunteers declared after appropriate education her consent. The study was by the Ethics Committee University of Tübingen (184 / 2003V).

The Ringer's solution contained (in mM): 125 NaCl, 5 KCl, 1 MgSO ₄ , 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, 1 CaCl ₂ ; pH 7.4. If indicated, chloride was removed from the medium and replaced with gluconate or glucose. The removal of Cl ^- leads to the escape of KCl and osmotically caused by water and thereby cell shrinkage, which is a known trigger of eryptosis; see. Lang et al. (2005a, supra).

Adenosine was used at concentrations ranging from 10 to 100 μM, the inhibitor of nucleoside transport S- (4-nitrobenzyl) -6-thioinosine (NBMPR) at 10 μM, the purinergic receptor antagonist CGS-15943 at 2.5 μM, the adenosine -A1 receptor antagonist 8-Cylcopentyl-3- [3 -] [4- (fluorosulfonyl) benzoyloxypropyl] -1-1-propylxanthine (FSCPX) at concentrations of 20 μM, the adenosine A3 receptor antagonist N- (2-methoxyphenyl) -N '- (2- (3-pyridyl) quinazolin-4-yl) urea (VUF5574) at concentrations of 4.4 μM, the phosphatase inhibitor okadaic acid at a concentration of 1 μM, and the phorbol ester phorbol 12-myristate 13-acetate at a concentration of 3 μM. If indicated, the final concentration of dimethylsulfoxide (DMSO) solvent was 0.1%. Okada acid, phorbol 12-myristate 13-acetate, S- (4-nitrobenzyl) -6-thioinosine (NBMPR), CGS 15943 and adenosine were purchased from Sigma (Taufkirchen, Germany) and the Ca ^{2 +} -dye Fluo-3 / AM from Calbiochem (Bad Soden, Germany).

1.2 Hemolysis

The effect of removing Cl ^- on the integrity of the cell membrane of the erythrocytes was determined by measuring the hemoglobin concentration in the supernatant (at 405 nm, Tecan Eliza Reader) after centrifugation at 500 g for 5 minutes at 4 ° C. The respective values are expressed as the percentage of hemoglobin concentration after complete lysis of the cells by exposure to pure water (set as 100% hemolysis). At the same time, the absorptions of the corresponding concentrations of adenosine were determined and subtracted from the absorbances of the supernatant.

1.3 FACS analysis of annexin-V binding and forward scatter

The FACS analysis was performed as previously described; see. Andree et al. (1990), Einding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers. J. Biol. Chem. 265, pages 4923-4928 , After incubation under the respective experimental condition, the cells were washed in Annexin V binding buffer containing (in mM): 125 NaCl, 10 HEPES / NaOH (pH 7.4) and 5 CaCl ₂ . The erythrocytes were suspended in a solution containing Annexin V-Fluos (Roche Diagnostics, Mannheim, Germany) and Annexin V buffer (1:50 dilution). After 10 minutes of incubation, the samples were again diluted 1: 5 in Annexin V binding buffer and purified by flow cytometry analysis on a Becton FACS Calibur Dickinson (Heidelberg, Germany). Cells were analyzed by forward scatter and Annexin V fluorescence intensity was measured in FL-1 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

1.4 Measurement of intracellular Ca ⁺⁺

Intracellular Ca ⁺⁺ measurements were performed as previously described; see. Lang et al. (2005b) Inhibition of erythrocyte "apoptosis" by catecholamines. Naunyn Schmiedeberg's Arch. Pharmacol. 372, pages 228-235 , The erythrocytes were loaded with Fluo-3 / AM (Calbiochem) by adding 2 μl of a Fluo-3 / AM stock solution (2.0 mM in DMSO) to 1 ml of erythrocyte suspension (0.16% hematocrit in Ringer). The cells were incubated at 37 ° C for 15 minutes protected from light. Then, another 2 μl aliquot of Fluo-3 / AM stock solution was added and the cells were incubated for 25 minutes. Fluo-3 / AM-loaded erythrocytes were centrifuged at 1000 g for 5 minutes at 22 ° C and washed twice with Ringer's solution containing 0.5% bovine serum albumin (Sigma) and once with Ringer's solution. Fluo-3 / AM-loaded erythrocytes were resuspended in 0.5 ml of Ringer's solution (0.16% hematocrit) in the presence and absence of chloride containing various concentrations of adenosine and incubated at 37 ° C for various periods of time. Subsequently, the Ca ⁺⁺ -dependent fluorescence intensity was measured by FACS analysis in the fluorescence channel FL-1 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. As a positive control for increased Ca ^{+ +} activity, Fluo-3-labeled erythrocytes were incubated in Ringer's solution containing 1 μM ionomycin.

1.5 Statistics

The Data is expressed as arithmetic mean ± SEM and the statistical analysis was done by paired or unpaired t-test or ANOVA as required.

2 results

In intact erythrocytes is phosphatidylserine on the inside of the Limited cell membrane, while in erythrocytes, undergoing apoptosis, a restructuring of the cell membrane takes place, leading to the exposure of phosphatidylserine to the cell surface leads; see. Lang et al. (2005a, supra). phosphatidylserine binds specifically and with high affinity annexin-V, which was therefore added to suicidal, d. H. to identify eryptotic erythrocytes.

Of the percentage of annexin-V binding to erythrocytes from fresh withdrawn blood is low (0.51 ± 0.05%, n = 4), what the maintenance of phosphatidylserine asymmetry in the majority of circulating erythrocytes. After 24 hours Incubation in Ringer's solution showed a tendency to Increase in the percentage of annexin-binding erythrocytes (0.81 ± 0.16%, n = 4). In the presence of 10 μM adenosine, the annexin binding was erythrocytes tend to be lower (0.59 ± 0.05%, n = 4).

The eryptosis is triggered by energy depletion, which is accompanied by deprivation of glucose for 24 or 48 hours; see. Klarl et al. (2006), Protein kinase C mediates erythrocyte "programmed cell death" following glucose depletion. At the. J. Physiol. Cell. Physiol. 290, pages C244-C253 , Despite the inhibition of Na ⁺ / K ⁺ -ATPase, which eventually leads to cell swelling, glucose depletion initially leads to cell shrinkage, a result of Ca ⁺⁺ entry and subsequent activation of Ca ⁺⁺ sensitive K ⁺ channels; see. Kim et al. (1998), The effect of pyrimidines nucleosides on adenosine-induced apoptosis in HL-60 cells. J. Cancer. Res. Clin. Oncol. 124, pages 471-477 , Glucose depletion also results in exposure to phosphatidylserine, which identifies damaged erythrocytes and promotes their elimination from circulating blood; see. Kim KT et al. (1998, supra). Therefore, after energy depletion, eryptosis precedes erythrocyte swelling, thus favoring the elimination of energy-depleted cells prior to hemolysis; Lang et al. (2005a, supra).

As in 1 demonstrated withdrawal of glucose for 24 hours ( 1B ) or 48 hours ( 1C ) in fact significantly the percentage of annexin-binding erythrocytes. The annexin-V binding was attenuated in the presence of adenosine, this effect reaching statistical significance at ≥30 μM adenosine.

As in 2 illustrated, the glucose withdrawal decreased the forward scatter, which represents the cell shrinkage, this effect reaching statistical significance after 48 hours ( 2C ). Adenosine led to the reversal of the shrinkage effect by glucose depletion, with the effect reaching ≥ 30 μM adenosine statistical significance ( 2C ).

The eryptosis is further triggered by hyperosmotic or iso-osmotic cell shrinkage; see. Lang et al. (2005a, supra). This is achieved by removing extracellular Cl ^- , resulting in cellular loss of Cl ^- and K ⁺ associated with osmotically induced water; see. Lang et al. (2003), Cation channels trigger apoptotic death of erythrocytes. Cell Death Differ 10, pages 249-256 , Treatment is followed by activation of Ca ⁺⁺ -permeable erythrocyte cation channels, leading to the entry of Ca ⁺⁺ and, consequently, a Ca ⁺⁺ -sensitive Cell membrane reorganization results; see. Lang et al. (2003, supra).

As in 3 In fact, removal of Cl ^-, in fact, leads to an increase in the percentage of annexin V-binding erythrocytes, the effect being significantly attenuated by the presence of adenosine (≥10 μM). This treatment was associated with a slight but statistically significant hemolysis which was significantly attenuated again in the presence of 100 μM adenosine ( 3 ).

As expected, the removal of Cl ^- further leads to a significant decrease of the forward scatter ( 4 ), but this effect is slightly attenuated significantly in the presence of adenosine (≥30 μM).

Adenosine, on the other hand, does not significantly modify the effect of Cl ^- removal on fluo-3 fluorescence, which is an indicator of cytosolic Ca ⁺⁺ activity. Instead, the increase in fluo-3 fluorescence after Cl ^- removal in the presence and absence of adenosine was the same ( 5 ). As a positive control, the Ca ⁺⁺ ionophore ionomycin led to a significant increase in fluorescence fluorescence ( 5 ).

There the energy depletion leads to activation of protein kinase C, which is involved in the induction of eryptosis, which on glucose withdrawal follows, cf. Klarl et al. (2006, supra) carried out additional experiments to elucidate whether adenosine interferes with signaling of protein kinase C (PKC). Specifically, the eryptosis was after the exposure of the PKC stimulator Phorbol 12-myristate 13-acetate (PMA, 3 μM) and the phosphatase inhibitor Okadaic acid (1 μM) in the presence and absence examined by adenosine.

As in 6 In fact, the presence of okadaic acid (1 μM) indeed resulted in significant annexin binding, with the effect attenuated in the presence of adenosine. The effect of adenosine reached statistical significance at concentrations of adenosine at ≥30 μM. Annexin binding in the presence of okadaic acid was accompanied by a significant decrease in the forward scatter ( 7 ), the effect being significantly attenuated again in the presence of adenosine (≥10 μM).

The treatment of erythrocytes with PMA (3 μM) likewise led to a significant annexin binding ( 8th ), this effect being attenuated again in the presence of adenosine (≥10 μM).

The PMA-induced annexin binding was accompanied by a significant decrease in the forward scatter ( 9 ), which was not significantly attenuated in the presence of adenosine (≥ 10 μM).

Further Experiments were performed to investigate whether the effect of adenosine sensitive to the nucleoside transport inhibitor Nitro-benzylmercapto-purin-ribose (NBMPR) or the blocking of purinergic receptors by the nonspecific receptor antagonist CGS-15943, the adenosine A1 receptor antagonist 8-cyclopentyl-3- [3 -] [4- (fluorosulfonyl) benzoyloxypropyl] -1-1-propylxanthine (FSCPX) or the adenosine A3 receptor antagonist N- (2-methoxyphenyl) -N '- (2- (3-pyridyl) quinazolin-4-yl) urea (VUF5574) was.

As in the 10 and 11 As illustrated, neither NBMPR (10 μM), CGS-15943 (2.5 μM), FSCPX (20 μM) nor VU5574 (4.4 μM) significantly interferes with the inhibitory effect of adenosine on annexin-V binding to erythrocytes 24 hours incubation in Cl ^- -free medium.

3. Conclusion

The Inventors were able to demonstrate by means of in vitro experiments that Adenosine has erythrocyte-protective properties and therefore is suitable as an active ingredient for a drug with which diseases or treated or prevented pathological conditions which are associated with erythrocyte apoptosis. Examples such diseases are sepsis, hemolytic uremic syndrome (HUS), diabetes, microcirculatory disorders and anemia. Furthermore, it could be demonstrated that adenosine was involved is such a compound, as the active substance of an agent for Preservation of stored blood or erythrocyte-containing preparations suitable is.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• WO 2005/087216 [0008]

Cited non-patent literature

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Claims (21)

Use of adenosine for the production of a By means of erythrocyte protection. Use according to claim 1, characterized that the agent is a medicine that has an additional having pharmaceutically acceptable carrier. Use according to claim 2, characterized that the drug is for treatment and / or prevention of sepsis is provided. Use according to claim 2, characterized that the drug is for treatment and / or prevention hemolytic uremic syndrome (HUS) is. Use according to claim 2, characterized that the drug is for treatment and / or prevention provided by diabetic vascular damage is. Use according to claim 2, characterized that the drug is for treatment and / or prevention is provided by microcirculation disorders. Use according to claim 2, characterized that the drug is for treatment and / or prevention of anemia is provided. Use according to claim 2, characterized that the drug is part of a treatment with cytotoxic drugs is provided. Use according to one of claims 2 to 8, characterized in that the medicament is for a Application is formed, which is selected from the Group consisting of: oral, rectal, parenteral, local, transdermal. Use according to one of claims 2 to 9, characterized in that the drug is formed in a mold which is selected from the group consisting of: powder, Tablet, juice, drops, capsule, suppositories, solution, Solution for injection, aerosol, ointment, conditioner, patch, Pellet, dragee. Use according to one of claims 2 to 10, characterized in that the drug adenosine in a Concentration, which is selected from the group consisting of (in each case given in weight of the active substance adenosine based on the total weight of the drug): 1 μg / g, 10 μg / g, 100 μg / g, 1 mg / g, 10 mg / g, 100 mg / g, 1 g / g. Use according to one of claims 2 to 11, characterized in that the absolute amount of the active substance adenosine in a dosage unit of the drug between about 1 and about 3000 mg, preferably between about 10 and about 1500 mg, more preferably between about 100 and about 750 mg, and most preferably at about 500 mg. Use according to one of claims 2 to 12, characterized in that the medicament is another active substance having. Use according to claim 13, characterized that the further active substance is an antibiotic. Use according to claim 13, characterized that the further active substance is a cytostatic. Use according to claim 2, characterized that the agent is a preservative for blood and erythrocyte-containing preparations. Use according to claim 16, characterized that the preservative has adenosine in a concentration at about 0.1 μM to about 1 mM, preferably at about 1 μM to about 500 μM, more preferably about 10 μM up to 250 μM, most preferably at about 100 μM lies. Process for the preparation of an erythrocyte protection agent, which has the following steps: (1) Provision of Adenosine, and (2) Formulation of Adenosine in a Pharmaceutical acceptable carrier. Method for therapeutic and / or prophylactic Treatment of a living being, by an erythrocyte apoptosis affected and / or at risk of erythrocyte apoptosis consists of the following steps: (1) Provision from adenosine, (2) introduction of adenosine into the animal, and (3) if necessary, repeated repetition of steps (1) and (2). Composition for the preservation of preserved blood and erythrocyte-containing preparations containing adenosine as well a pharmaceutically acceptable carrier, preferably one physiological saline solution. A composition according to claim 20, characterized in that it has adenosine in a concentration ranging from about 0.1 μM to about 1 mM, preferably about 1 μM to about 500 μM, more preferably about 10 μM to 250 μM, highest before zugt at about 100 uM is.