Active Ingredient of an Erythrocytes-containing Composition

Abstract

The present invention relates to an active ingredient of an erythrocytes-containing composition, an erythrocytes-containing composition comprising said active ingredient, and a method of treatment of a human or animal being suffering from and/or threatened by blood loss or a blood-formation disorder.

Claims

1. A method of treatment of a human or animal being suffering from or threatened by blood loss, comprising the administration to said being of an erythrocytes-containing composition comprising chelerythrine or a derivative thereof as an active ingredient.

2. The method according to claim 1, wherein said erythrocytes-containing composition is a blood conserve.

3. The method according to claim 1, wherein said composition is configured for a non-cryopreserved storage.

4. The method according to claim 1, wherein said chelerythrine or derivative thereof supports the viability or functional efficiency of said erythrocytes.

5. The method according to claim 1, wherein said chelerythrine or derivative thereof inhibits eryptosis or hemolysis.

6. The method according to claim 1, wherein said chelerythrine or derivative thereof increases the shelf-life of said composition.

7. The method according to claim 1, wherein said chelerythrine or derivative thereof is contained in the composition in a concentration of approx. ≥0.5 μM.

8. The method according to claim 1, wherein said chelerythrine or derivative thereof is contained in the composition in a concentration of approx. ≥10 μM.

9. The method according to claim 1, wherein said blood loss is acute blood loss.

10. The method of claim 1, wherein said human or animal being is suffering from or threatened by a blood-formation disorder.

11. An erythrocytes-containing composition comprising chelerythrine or a derivative thereof as an active ingredient.

12. The erythrocytes-containing composition of claim 11, which is a blood conserve.

13. The erythrocytes-containing composition of claim 8, wherein said composition is configured for non-cryopreserved storage.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1: Sketch illustrating problems arising from prolonged blood storage.

[0054] FIG. 2: A-B and D-E: Costunolide-induced eryptosis and cell shrinkage in mature human erythrocytes. C: Costunolide induced eryptosis is not associated with hemolysis. Number of independent experiments: n=3.

[0055] FIG. 3: Pre-incubation of human erythrocytes with chelerythrine results in inhibition of costunolide-induced eryptosis and cell shrinkage. Incubation time for chelerythrine (26 h) and costunolide (24 h). Number of independent experiments: n=3.

[0056] FIG. 4: Inhibition of glucose depletion-induced eryptosis and cell shrinkage by treatment of human erythrocytes with chelerythrine. Incubation time: 24 h. Number of independent experiments: n=2.

[0057] FIG. 5: Complete depletion of both GSH and GSSG after 24 h treatments of human erythrocytes with costunolide. Number of independent experiments: n=3.

[0058] FIG. 6: Complete inactivation of glucose-6-phosphate dehydrogenase (G6PDH) activity after 24 h treatment of human erythrocytes with costunolide. Number of independent experiments: n=3.

[0059] FIG. 7: Chelerythrine does not affect costunolide-induced GSH- and GSSG-depletion. In order to make the extremely low GSSG values visible, they were indicated as pmol instead of nmol. The incubation time for chelerythrine-treated erythrocytes lasted 26 h and 24 h for costunolide-treated erythrocytes. Number of independent tests: n=5.

[0060] FIG. 8: Chelerythrine as a potent and selective inhibitor of the protein kinase C alpha (PKC-α), inhibits costunolide-induced eryptosis.

EXAMPLES

1. Material and Methods

Study Design

[0061] Single treatment: Erythrocytes were treated with costunolide or chelerythrine for 24 hours (see FIGS. 2, 4, 5 and 6). Double treatments: Erythrocytes were first pre-treated with chelerythrine for two hours, followed by treatment with costunolide for further 24 hours (see FIGS. 3 and 7). Thus, the incubation time for chelerythrine-treated erythrocytes lasted 26 h and 24 h for costunolide-treated erythrocytes. In single (0.2% v/v DMSO) and double treatments (0.4% v/v DMSO), DMSO-treated cells served as negative controls.

Erythrocytes

[0062] Highly purified erythrocyte suspensions from healthy volunteers with white blood cell (WBC) or thrombocyte contaminations below 0.1% (Lang P.A. et al. 2005; J. Cell. Sci. 118: 1233-1243 “Stimulation of erythrocyte ceramide formation by platelet-activating factor”) were provided by the blood bank of the University of Tubingen. Aliquots of the individual erythrocyte concentrates were either used directly at 0.6% hematocrit (Hct) or stored at 4° C. for up to one week. The study was approved by the ethics committee of the University of Tubingen, the study was performed in agreement with the declaration of Helsinki, and volunteers gave written consent (184/2003 V).

Solutions

[0063] Experiments analyzing annexing V binding and cell volume (0.6% Hct) were carried out in Ringer solution. Staining of erythrocytes with annexin-V-FLUOS was performed in annexin binding buffer. Ringer solution was composed of (in mM): 125 NaCl, 5 KCl, 1.2 MgSO.sub.4, 32 N-2-hydroxyethyl-piperazine-N′-ethanesulfonic acid (HEPES)/NaOH (pH 7.4), 5 glucose, and 1 CaCl.sub.2. Annexin-binding buffer contained (in mM): 125 NaCl, 10 HEPES/NaOH (pH 7.4), and 5 CaCl.sub.2.

Chemicals

[0064] 1 mg chelerythrine (chloride) was dissolved in 520 μl and 5 mg costunolide in 538 μl DMSO to achieve 5 mM and 40 mM stock solutions, respectively. These stocks were subsequently aliquoted and stored at −20° C. for up to one month. Annexin-V-FLUOS was also aliquoted and stored at −20° C. for several months. Chelerythrine, costunolide, DMSO, annexin-V-FLUOS, N-Ethylmaleimide (NEM) and EGTA were purchased from Sigma (Taufkirchen, Germany).

Annexin-V-FLUOS Working Concentration

[0065] On the day of the measurements the required annexin-V-FLUOS was diluted in annexin binding buffer in a ratio of 1:33. From this batch 48 μl were taken to stain each sample with 3×10.sup.6 erythrocytes (for more details see flow cytometry).

Flow Cytometry

[0066] At the end of the incubation period, 0.1 ml erythrocytes (3×10.sup.6) were added in 500 μl annexin wash buffer, mixed thoroughly and pelleted by centrifuging. Erythrocytes pellets were gently vortexed to obtain a homogeneous cell suspension. To detect the exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane (a measure of the intensity of eryptosis), erythrocytes were stained with 48 μl of diluted annexin-V-FLUOS and carefully vortexed. After an incubation period of 20 minutes in the dark at room temperature, 200 μl annexin binding buffer was added to each sample, thoroughly vortexed to obtain single cell suspensions, and analyzed by flow cytometry on a FACS Calibur (Becton Dickinson, Heidelberg, Germany). In the FL1-channel, binding of annexin-V-FLUOS (eryptosis) was measured. Erythrocyte volume was determined by forward scatter (FSC). To this end, corresponding erythrocytes suspensions were immediately analysed by flow cytometry.

Hemolysis Measurement

[0067] After 24 hours treatment of erythrocytes (0.6% Hct) with costunolide and/or chelerythrine, hemolysis was determined. 600 μl cell suspension form each condition containing 3.6×106 erythrocytes centrifuged for 8 min at 420×g, 4° C., and the supernatants were harvested. As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatants was determined photometrically at 405 nm. The absorption of the supernatant of DMSO-treated erythrocytes lysed in 600 μl distilled water was defined as 100%. From this the standard curve was made by serial dilution.

Intracellular GSH and GSSG Analysis

[0068] For single (FIG. 5) or double treatments (FIG. 7), pure RBCs (0.6% Hct) were suspended in 30 ml Ringer solution and treated with varying concentrations of costunolide (1-80 μM) or different concentrations of chelerythrine (1-10 μM) and a fixed costunolide concentration (80 μM). DMSO-treated RBCs served as negative control. After incubation, 58 μl from a 310 mM NEM stock was given to each condition, gently mixed for 1 min and then centrifuged 10 min long at 10 C and 228×g. Supernatants were removed and the cell pellets were stored at −20° C. until analyses. GSH and GSSG were measured on the clear supernatant obtained by treatment of 0.1 ml RBCs with 0.12 ml 15% (w/v) trichloroacetic acid. For GSH analysis one aliquot (0.05 ml) of supernatant was loaded onto HPLC and the GS-NEM conjugate was revealed by a diode-array detector at 265 nm wavelength (Giustarini D. et al. 2011; Anal. Biochem. 415: 81-83, “Detection of glutathione in whole blood after stabilization with N-ethylmaleimide”). GSSG was measured at the spectrophotometer by the GSH recycling method with slight modifications (Giustarini D. et al. 2013; Nat. Protoc. 8: 1660-1669, “Analysis of GSH and GSSG after derivatization with N-ethylmaleimide”). One aliquot of RBCs (10 ml) was hemolyzed by a 1:200 dilution with H.sub.2O for haemoglobin determination (Di lorio E.E. 1981; Methods Enzymol. 76: 57-72 “Preparation of derivatives of ferrous and ferric hemoglobin”). The HPLC analyses were carried out by an Agilent series 1100 instrument. The spectrophotometric analyses were performed by a Jasco V-530 instrument.

Determination of G6PDH Activity

[0069] Pure RBCs (0.6% Hct) were incubated in 20 ml Ringer solution and treated with DMSO or different concentrations of costunolide. 24 hrs later, cell suspensions were centrifuged at 10° C., 228×g. Supernatants were removed and the cell pellets were stored at −80° C. until analyses. G6PDH activity was measured in lysates according to methods recommended by the International Committee for Standardization in Hematology (Di lorio E.E. 1981; l.c.). The activity was expressed in units per grams of hemoglobin (U/g Hb) (Ghashghaeinia M. et al. 2012; l.c.). RBC pellet was diluted 1:20 with distilled water. After 10 min (for GR 20 min) incubation at 37° C., the reactions were started by adding substrate or hemolysate and mixed properly. Then, 200 μL of each reaction mixture was transferred into a 96-well plate and changes in absorbance were recorded at 340 nm at 1 min intervals for 20 min at 37° C. (Infinite 200 Nanoquant, Tecan). All assays were run in triplicates and specific enzyme activity was calculated using the Lambert-Beer law. Activity =(ΔA×V/(ϵ×l×t))×f.

Compositions of the Assay Mixtures

6PGA Assay

[0070] 0.1 M Tris-HCl/0.5 mM EDTA, pH=8.0; 0.01 M MgCL2; 0.2 mM NADP.sup.+; 20 μL of 1:20 hemolysate; 10 min incubation at 37° C.; start by addition of 0.6 mM 6PGA; 20 min measurement, blank without 6PGA.

G6PDH Assay

[0071] 0.1 M Tris-HCl/0.5 mM EDTA, pH=8.0; 0.01 M MgCl2; 0.2 mM NADP.sup.+; 20 μL of 1:20 hemolysate; 10 min incubation at 37° C.; start by addition of 0.6 mM 6PGA+0.6 mM G6P

Statistical Analysis

[0072] Data are presented as the mean values±SEM of at least 3 independent experiments with different blood samples. A total of 19 different blood samples were used in this study. One-way ANOVA with Dunnet's post test was used for statistical comparisons of treated samples with controls. Differences of the means were considered to be statistically significant when the calculated p value was less than 0.05 (*P<0.05, **P<0.01,***P<0.001,****P<0.0001).

2. Results

[0073] Chelerythrine is a potent, selective and cell-permeable inhibitor of the protein kinase C alpha (PKC-α); Herbert J.M. et al., 1990, I.c., and Hu B. et al., 2017, I.c. Human erythrocytes possess four isoforms of PKCs: alpha, zeta, mu and iota, of which only the subtype PKC-α translocates to the plasma membrane in order to perform biological (physiological) activities there (Govekar R.B. and Zingde S.M. 2001; Ann. Hematol. 80: 531-534, “Protein kinase C isoforms in human erythrocytes”); i.e. induction of eryptosis.

[0074] Therefore, the addition of the pharmacological substance chelerythrine (a natural product of plant origin) to blood bags (erythrocyte concentrates) is an ideal way and an effective measure to curb the increasing intensity of eryptosis during the storage of blood preserves. Thus, the blood recipients receive better, higher quality and more functional erythrocytes, thus saving their lives and relieving their immune system.

[0075] The sketch depicted in FIG. 1 is intended to illustrate the problem of weeks of blood storage and the associated negative consequences as well as the advantages of adding chelerythrine to blood bags.

[0076] In order to prove the correctness of this assumption as well as the necessity and effectiveness of adding chelerythrine to blood bags (erythrocyte concentrates), human erythrocytes were submitted to a stress test.

[0077] For this purpose, human erythrocytes were treated with the most various physiological concentrations of the natural product costunolide (a sesquiterpene) and after 24 hours its influence on eryptosis, cell shrinkage and hemolysis were evaluated. Increasing concentrations of costunolide were associated with increased rates of eryptosis and cell shrinkage (FIG. 2).

[0078] Increasing concentrations of costunolide partially simulated the increasing concentrations of stress factors that occur during the weeks of storage of human erythrocytes in blood bags, which in turn trigger the eryptosis machinery.

[0079] The second step was to examine whether chelerythrine, a natural benzophenanthridine alkaloid, can inhibit costunolide-induced eryptosis and cell shrinkage. To achieve this aim, human erythrocytes were first treated for two hours with various concentrations of chelerythrine (1 to 10 μM) and then the highest physiological concentration of costunolide (80 μM) was added. In fact, chelerythrine was able to inhibit costunolide-induced eryptosis and cell shrinkage (FIG. 3). The incubation time for chelerythrine-treated erythrocytes lasted 26 h and 24 h for costunolide-treated erythrocytes. DMSO-treated erythrocytes served as a control group.

[0080] It is to be noted that the addition of chelerythrine even reduces the basal eryptosis rate: DMSO (1.69%); chelerythrine (1.1%) (FIG. 3A). As a result, chelerythrine will inhibit both basal and storage-related activation of the eryptosis machinery in blood bags (erythrocyte concentrates).

[0081] The most important aspect of this invention concerns its applicability in transfusion medicine. Chelerythrine is added to blood bags in a standardized procedure. This corresponds to the pre-incubation of human erythrocytes with chelerythrine in the experimental setup.

[0082] Obviously, chelerythrine (0.5 to 10 μM) is able to downregulate any kind of stressor-induced eryptosis and cell shrinkage (FIG. 4). This experiment served as proof of principle.

[0083] The next step was to investigate if costunolide-induced eryptosis is caused by impairment of the redox balance of human erythrocytes. For this, the influence of costunolide on glutathione (GSH) levels as well as on the activity of the pro-survival enzyme glucose-6-phosphate dehydrogenase (G6PDH) was studied. Costunolide was indeed able to deplete both the reduced (GSH) and the oxidized (GSSG) form of glutathione in a concentration-dependent manner (FIG. 5) and to inhibit G6PDH activity completely (FIG. 6).

[0084] Moreover, it was investigated if chelerythrine can affect the costunolide-induced GSH depletion. This question could be answered with a clear “no” (FIG. 7). This demonstrates that in the experimental setup chelerythrine acts downstream of the glutathione cascade and has no influence on GSH synthesis machinery. In other words: chelerythrine specifically inhibits the activity of the enzyme PKC-α in human erythrocytes.

[0085] The complete results of this work are shown in FIG. 8.

3. Conclusions

[0086] The inventor has surprisingly realized that chelerythrine, a natural product of plant origin, or derivatives thereof can be used in vitro as a beneficial active ingredient of an erythrocytes-containing composition, such as a blood conserve. The addition of the PKC inhibitor chelerythrine to an erythrocytes-containing composition can/will extend the storage durability of blood bags, support the vitality and functional efficiency of human erythrocytes and strongly inhibit the storage-related increase of eryptosis machinery and the intensification of hemolytic processes. As a result, the recipients of the erythrocyte concentrates receive better, higher quality and more functional erythrocytes, saving their lives and relieving their immune system.

[0087] The advantages of adding the chelerythrine to erythrocytes-containing or blood conserves (erythrocyte concentrates), which may be stored in a standardized erythrocyte-specific preservation solution (the so-called stabilizing additive solution), as discovered by the inventor, can be summarized as follows: [0088] a) Achieving better vitality and functionality of human erythrocytes; [0089] b) Significant inhibition of storage-related eryptosis (erythrocyte death); [0090] c) Significant inhibition of calcium-dependent activation of cation channels; [0091] d) Significant inhibition of the storage-related shrinkage of human erythrocytes; [0092] e) Increasing the durability of human erythrocytes; [0093] f) Significant reduction of storage-related hemolytic processes in human erythrocytes; [0094] g) Significant relief of the immune system of the blood recipient.