RED BLOOD CELL STORAGE SOLUTIONS, SOLUTION ADDITIVES, AND METHODS FOR IMPROVING THE STORAGE OF RED BLOOD CELLS

Abstract

Methods, additive kits and storage compositions for decreasing the deleterious effects of storage on pRBCs, for improving the aging process of stored pRBCs, and for preventing or ameliorating patient comorbidities following the transfusion or infusion of stored blood based on inhibiting acid sphingomyelinase during storage are provided.

Claims

1. A packed red blood cell (pRBC) storage solution comprising one or more acid sphingomyelinase (Asm) inhibitors.

2. The pRBC storage solution according to claim 1, wherein the one or more Asm inhibitors is selected from Table 1.

3. The pRBC storage solution according to claim 1, wherein the one or more Asm inhibitors comprises at least one functional Asm inhibitor.

4. The pRBC storage solution according to claim 1, wherein the Asm inhibitor comprises Amitriptyline.

5. The pRBC storage solution according to claim 1, wherein the Asm inhibitor comprises fluoxetine.

6. The pRBC storage solution according to claim 1, wherein the solution comprises an AS-3 solution.

7. An additive kit for formulating a pRBC storage solution comprising at least one Asm inhibitor.

8. The additive kit according to claim 7, wherein the at least one Asm inhibitor comprises Amitriptyline.

9. The additive kit according to claim 7, wherein the at least one Asm inhibitor comprises Fluoxetine.

10. A method for preserving red blood cells, the method comprising packing the red blood cells in a storage solution comprising at least one Asm inhibitor.

11. The method according to claim 10, wherein the Asm inhibitor comprises at least one Asm inhibitor set forth in Table 1.

12. The method according to claim 10, wherein the Asm inhibitor comprises at least one functional inhibitor of Asm.

13. The method according to claim 10, wherein the Asm inhibitor is Amitriptyline.

14. The method according to claim 10, wherein the Asm inhibitor is Fluoxetine.

15. A method for mitigating a complication associated with a transfusion or infusion of red blood cells, the method comprising storing the red blood cells in a storage solution comprising at least one Asm inhibitor.

16. The method according to claim 15, wherein the complication comprises lung inflammation.

17. The method according to claim 15, wherein the at least one Asm inhibitor is selected from the Asm inhibitors set forth in Table 1.

18. The method according to claim 15, wherein at least one Asm inhibitor comprises a functional Asm inhibitor.

19. The method according to claim 15, wherein the Asm inhibitor comprises Amitriptyline.

20. The method according to claim 15, wherein the Asm inhibitor comprises Fluoxetine.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1) Bar graph showing microparticle counts in human pRBCs at days 14, 28, and 42 of storage, as determined by Nanoparticle Tracking Analysis.

[0015] FIG. 2) Bar graph showing microparticle counts in mouse pRBCs stored for 14 days compared to fresh units, as determined by Nanoparticle Tracking Analysis.

[0016] FIG. 3) Bar graph showing that Amitriptyline reduces microparticle formation in stored human pRBCs, as determined by Nanoparticle Tracking Analysis.

[0017] FIG. 4) Bar graph showing that Amitriptyline reduces microparticle formation in stored mouse pRBCs, as determined by Nanoparticle Tracking Analysis.

[0018] FIG. 5) Comparison bars demonstrating that pRBCs from acid sphingomyelinase-deficient mice produce less microparticles than those from wild type mice, as determined by flow cytometry.

[0019] FIG. 6) Image of serum from mouse pRBCs at days 0, 3, 6, 9, 12, and 14, displaying increased cell-free hemoglobin over time.

[0020] FIG. 7) Cell-free Hgb as a function of Amitriptyline (AT) concentration showing reduction of hemolysis in stored human pRBCs.

[0021] FIG. 8) Cell-free Hgb as a function of AT concentration showing reduction of hemolysis in stored mouse pRBCs.

[0022] FIG. 9) Cell-free Hgb stored in AT versus PBS as a function of storage time showing reduction in hemolysis with AT additive over time in mouse pRBCs.

[0023] FIG. 10) Bar graph showing that maintenance of stored mouse pRBC size increases as a function of AT concentration.

[0024] FIG. 11) Bar graph showing that maintenance of stored mouse erythrocyte complexity increases as a function of AT concentration.

[0025] FIG. 12) Bar graph showing that AT dose-dependently prevents phosphatidylserine externalization in aged mouse erythrocytes.

[0026] FIG. 13) Bar graph showing that AT maintains band 3 protein integrity in stored mouse erythrocytes.

[0027] FIG. 14) Micrographs of lung cells showing that transfusion of pRBCs treated with amitriptyline causes significantly less lung edema and inflammatory cell recruitment in a murine model.

[0028] FIG. 15) Micrographs showing that transfusion of aged pRBCs results in tight junction disruption in a mouse model, which is prevented by amitriptyline treatment.

[0029] FIG. 16) Micrographs showing that transfusion of pRBCs from acid sphingomyelinase-deficient mice causes significantly less lung edema, inflammatory cell recruitment, and tight junction disruption when compared to pRBCs from wild type mice.

[0030] FIG. 17) Transfusion of aged pRBCs results in increased capillary leak, as measured by Bar graph showing Evans blue extravasation in the lungs is substantially prevented by treating pRBCs with amitriptyline.

[0031] FIG. 18) Bar graph showing that Asm inhibition by Fluoxetine treatment also reduced hemolysis in murine pRBCs during storage.

DETAILED DESCRIPTION

[0032] Generally, when whole blood is collected, it is separated into components and the red blood cell (RBC) fraction is stored as packed RBC's (pRBCs). Typically, RBCs are packed by centrifugation, and plasma is removed so that RBCs make up about 80 of the volume. The storage solution is added to the pRBC. As erythrocytes age in storage, they demonstrate several age-related changes including MP formation, hemolysis, decreased cell size and complexity, externalization of phosphatidylserine, and loss of band 3 protein.

[0033] Sphingolipids are a class of lipids containing a backbone of sphingoid bases. They play diverse roles in cellular processes and are significant components of cell membranes. Sphingomyelin (SM), which comprises 10% of the mammalian plasma membrane, is degraded by the hydrolyzing enzyme sphingomyelinase (SMase). In recent studies, membrane curvature and associated increases in mechanical bending stresses in RBCs, activated SMase, thereby reducing SM and increasing ceramide. Production of ceramide has a direct effect on a wide range of cellular processes and alters production of other immediately descendent metabolites such as sphingosine and sphingosine 1-phosphate (S1P), which are key regulators of inflammation. Furthermore, SMase has been implicated in lipid microdomain formation, membrane fragility, vesiculation, and MP formation.

[0034] Amitriptyline is a tricyclic antidepressant (TCA) that was initially introduced by Merck in 1961 for the treatment of major depressive disorder and is widely used in the clinic to treat a number of other disorders as well as neuropathic pain. In the 1970s the ability of tricyclic antidepressants to inhibit acid sphingomyelinase (Asm) was discovered. The enzyme Asm catalyzes the hydrolysis of sphingomyelin to ceramide. Asm and ceramide were shown to play a crucial role in a wide range of diseases, including cancer, cystic fibrosis, diabetes, Alzheimer's disease, and major depression, as well as viral (e.g., measles virus) and bacterial (e.g., Staphylococcus aureus, Pseudomonas aeruginosa) infections. Ceramide molecules may act in these diseases by the alteration of membrane biophysics, the self-association of ceramide molecules within the cell membrane and the ultimate formation of larger ceramide-enriched membrane domains/platforms. These domains were shown to serve the clustering of certain receptors such as CD95 and may also act in the above named diseases. Amitriptyline is known as a functional inhibitor of acid sphingomyelinase. The enzyme resides in the lysosome and is usually attached to the inner membrane leaflet by electrostatic forces. Membrane-bound ASM is active and degrades sphingomyelin, yielding ceramide.

[0035] Weak bases such as Amitriptyline passively diffuse across membranes in their neutral state. In acidic intracellular compartments like the lysosome, they become protonated. In this state, they can no longer cross the membrane and thus are trapped inside of the compartment. The accumulation of antidepressants in acidic compartments has been demonstrated experimentally. The accumulation of antidepressants like Amitriptyline or Desipramine in a lysosome interferes with the binding of ASM to the membrane, resulting in detachment of ASM and subsequent inactivation by proteolytic degradation.

[0036] Weak basicity and high lipophilicity are physicochemical properties Amitriptyline shares with other functional inhibitors of acid sphingomyelinase. These characteristics, rather than specific structural motives, appear to be the prerequisites for Asm inhibition by these drugs. Cell culture-based experiments have identified the compounds set forth in Table 1 as functional inhibitors of Asm(s). The relevant experiments used the human cell line H4. The Asm activity was measured using a radiolabel assay. (See, Kornhuber et al. “Identification of novel functional inhibitors of acid spingomyelinase” PLoS ONE (2011) 6(8), the entire disclosure of which is incorporated herein by this reference.) According to the “Kornhuber chemo-informatic prediction model”, in the absence of experimental data, identification of functional inhibitors of Asm based on certain molecular properties is nonetheless possible with a high degree of reliability.

[0037] As illustrated in the examples, many of the deleterious storage-related changes in RBCs are reduced by treatment of pRBCs with a functional Asm inhibitor. The solutions, methods and kits of the instant invention are exemplified by Amitriptyline (AT) and Fluoxetine (FT), however any functional inhibitor of Asm may be suitable with a reasonable degree of success, based on the Kornhuber chemo-informatic model (see also, e.g. Kornhuber, J. et al. (2008) “Identification of new functional inhibitors of acid sphingomyelinase using a structure-property-activity relation model” J Med Chem. 51: 219-237, the entire disclosure of which is incorporated herein by this reference). As shown below in the examples, AT treatment during storage reduced MP-rich serum in aged pRBCs. Ceramide accumulation is known to affect erythrocyte degradation, and by inhibiting this sphingolipid pathway, many of the age-related changes that occur during storage of pRBCs are prevented. As a result, much of the lung inflammation that is associated with transfusion of aged pRBCs is reduced. Similar results were produced when studying blood from Asm mice.

[0038] One embodiment of the invention provides a packed red blood cell (pRBC) storage solution comprising one or more acid sphingomyelinase (Asm) inhibitors. Suitable specific Asm inhibitors include functional Asm inhibitors, exemplified by Table 1 without limitation. Suitable specific inhibitors may also be identified by the Kornhuber chemo-informatics model and employed in the disclosed compositions, methods and kits with a reasonable expectation of success. According to very specific embodiments, at least one Asm inhibitor is selected from Amitriptyline, Fluoxetine, and combinations thereof. The solution may comprise an AS-3 solution.

[0039] Method embodiments for preserving red blood cells are also provided. The methods comprise packing the red blood cells in a storage solution comprising at least one Asm inhibitor. In some embodiments, the Asm inhibitor may be removed by filtration or other suitable means prior to transfusion/infusion into a patient. In specific embodiments, the Asm inhibitor comprises at least one Asm inhibitor set forth in Table 1. In more specific embodiments, the Asm inhibitor comprises at least one functional inhibitor of Asm, and in very specific embodiments the Asm inhibitor Amitriptyline, Fluoxetine, or both.

[0040] Other embodiments are directed to methods for mitigating a complication associated with a transfusion or infusion of red blood cells, the method comprising storing the red blood cells in a storage solution comprising at least one Asm inhibitor. According to specific embodiments, the complication comprises lung inflammation. Suitable Asm inhibitors are listed in Table 1, however as noted above, all functional Asm inhibitors may be predicted to provide a benefit to reducing the deleterious effects of storage on pRBCs. In very specific embodiments, the Asm inhibitor comprises Amitriptyline or Fluoxetine or both.

[0041] Kit embodiments are also contemplated. Kits comprise at least one Asm inhibitor as an additive for formulating a pRBC storage solution. Suitable specific Asm inhibitors are exemplified by Table 1, without limitation, or may identified by reference to the Kornhuber chemo-predictive model. In specific embodiments the functional Asm inhibitor comprises Amitriptyline or Fluoxetine or both.

[0042] Broadly, methods for improving the aging process of stored pRBCs and preventing or ameliorating patient comorbidities following the transfusion of stored blood are provided. Further embodiments are directed to treating subjects in need of a blood transfusion or infusion with red blood cells stored in accordance with the novel storage compositions disclosed herein, or treated with one or more Asm inhibitors. In some embodiments, the Asm inhibitor may be removed from the blood product prior to transfusion or infusion into the subject, for example by filtration or capture means.

[0043] The following Examples illustrate particular aspects and features of the invention disclosed herein and should not be taken as limiting the full scope of the invention as defined by the appended claims.

EXAMPLES

[0044] The following materials and methods apply to the Examples set forth herein.

Animal Model

[0045] Male C57BL/6 mice aged 8-10 weeks were purchased from Jackson Laboratories, fed standard laboratory diet and water ad libitum, and acclimated for 1 week in climate-controlled conditions with 12 hours light-dark cycles prior to use. Asm-deficient mice (Asm.sup.−/−) on a C57BL/6 background were used and compared to syngeneic C57Bl/6 wild type mice as controls. All experiments were approved by the Institutional Animal Care and Use Committee at the University of Cincinnati.

Blood Banking and Treatment

[0046] Human pRBCs were obtained from Hoxworth Blood Bank (Cincinnati, Ohio) and stored at 4° C. for 42 days according to standard blood banking practice. Murine pRBCs were obtained as previously described. Briefly, donor mice were anesthetized with intraperitoneal pentobarbital (0.1 mg/g body weight) and blood was collected via cardiac puncture into CP2D anticoagulant-coated syringes. CP2D was then added to the whole blood in a 1:7 ratio and gently mixed. Whole blood was centrifuged at 1,000 g for 10 minutes at 4° C. and platelet-rich plasma was discarded. Storage solution (AS-3) was added to erythrocytes in a 2:9 ratio using the original whole blood volume. Units were stored at 4° C. for 14 days.

[0047] Amitriptyline (Sigma-Aldrich, St Louis, Mo., USA) was dissolved in 0.9% sodium chloride and added to mouse and human pRBCs in a 1:10 dilution prior to storage. Except for dose-response experiments, AT treatment refers to a 125 μM concentration of AT in pRBCs.

Blood Component Isolation

[0048] Erythrocytes were pelleted from pRBC samples at 2,000 g for 10 minutes at 4° C. The supernatant from this spin was then centrifuged at 10,000 g for 10 minutes at 4° C. to pellet any remaining cells or platelets. The MP-rich supernatant from this second spin was used to characterize MP production and hemolysis. MPs were pelleted using a 20,000 g spin for 30 minutes at 4° C.

Erythrocyte Characterization

[0049] Hemoglobin in the MP-rich supernatant was quantified using a Coulter AcT diff Analyzer (Beckman Coulter Corp., Brea, Calif.). Cell size (forward scatter), complexity (side scatter), phosphatidylserine exposure (annexin V positivity), and band 3 protein integrity (eosin-5-maleimide.sup.68) were analyzed using an Attune flow cytometer (Life Technologies, Carlsbad, Calif.). Antibodies for ter119, CD235a, CD41, CD45, and Annexin V were acquired from BD Biosciences (San Diego, Calif.), and eosin-5-maleimide was obtained from Life Technologies (Carlsbad, Calif.).

Measurement of Lung Inflammation

[0050] To study the effect of AT treatment on lung inflammation following blood transfusion, mice received 200 μl IV injections of pRBCs or MPs. Lungs were taken 6 hours after transfusion for either histologic or Evans blue evaluation. Histologic lung sections were examined for evidence of lung edema with hematoxylin and inflammatory cells with anti-Gr1 antibodies (AbD Serotec, Raleigh, N.C.). Tight junction integrity was assessed by staining lung slides with Cy3-conjugated anti-ZO1 (Jackson ImmunoResearch, West Grove, Pa.). Endothelial cell permeability was assessed using the Evans blue method (source). In brief, mice were weighed and injected with 30 mg/kg Evans blue. After 10 minute of circulation time, mice were sacrificed and the pulmonary vasculature was flushed with 10 ml PBS. Lungs were dried at 55° C. for 24 hours and then incubated in 500 μl formamide for 48 hours at 55° C. Evans blue extraction calculated by measuring the optical density at 620 nm and comparing to a standard curve, and values were normalized to dry lung weight.

Example 1

Microparticle Formation

[0051] Mouse and human pRBCs generate MPs during storage (FIG. 1 and FIG. 2). Treatment of pRBCs with increasing doses of AT resulted in a dose-dependent reduction of MP formation (FIG. 3 and FIG. 4). Also, analysis of pRBCs acquired from Asm.sup.−/− mice revealed a significant decrease in MP production (FIG. 5). In addition, treatment of pRBCs with Fluoxetine, another known functional inhibitor of Asm (FIG. 18), demonstrated a similar reduction in microparticle formation, thereby confirming that the results generalize to functional Asm inhibitors.

Example 2

Erythrocyte Stability

[0052] During storage, erythrocytes experience age-related hemolysis, as measured by increased cell-free hemoglobin in pRBCs (FIG. 6). AT treatment resulted in a dose-dependent reduction in hemolysis in human and mouse pRBCs (FIG. 7, FIG. 8, and FIG. 9). Moreover, while aged erythrocytes demonstrate decreased cell size and complexity, these changes were mitigated with AT (FIG. 10 and FIG. 11).

[0053] As erythrocytes age in storage they are unable to maintain their membrane asymmetry and subsequently expose phosphatidylserine, usually on the inner plasma membrane, on their extracellular surface. AT treatment reduced this age-related phosphatidylserine expression in a dose dependent manner (FIG. 12). Also, while aged erythrocytes experience a degradation of band 3, a key structural protein, AT reduced this degradation by close to 50% (FIG. 13).

Example 3

Effect on Lung Inflammation

[0054] Research from this lab and others has shown that transfusion of aged pRBCs as well as MPs isolated from aged pRBCs results in lung inflammation in a mouse model. Mice that receive transfusions of aged pRBCs demonstrate increased lung edema as well as inflammatory cell recruitment to the lungs. These effects are greatly reduced when mice receive pRBCs that have been treated with AT (FIG. 14). Transfusion of aged pRBCs also resulted in marked disruption of tight junctions in the lung, as indicated by histologic evaluation of ZO1. This effect was mitigated by transfusing blood that had been treated with AT (FIG. 15). Transfusion of pRBCs from Asm.sup.−/− mice resulted in a similar reduction in lung edema, inflammatory cell recruitment, and tight junction disruption when compared to pRBCs from wild type mice (FIG. 16). As a result, mice receiving aged pRBCs also demonstrate increased Evans blue capillary leak, an effect not seen in mice receiving pRBCs treated with AT (FIG. 17).

TABLE-US-00001 TABLE 1 Exemplary functional Asm inhibitors Alverine Amiodarone Amitriptyline Amlodipine Aprindine Astemizole AY-9944 Benzatropine Bepridil Biperiden Camylofin Carvedilol Cepharanthine Chlorpromazine Chlorprothixene Cinnarizine Clemastine Clofazimine Clomiphene Clomipramine Cloperastine Conessine Cyclobenzaprine Cyproheptadine Desipramine Desloratadine Dicycloverine Dilazep Dimebon Doxepin Drofenine Emetine Fendiline Flunarizine Fluoxetine Flupentixol Fluphenazine Fluvoxamine Hydroxyzine Imipramine Lofepramine Loperamid Loratadin Maprotiline Mebeverine Mebhydrolin Mepacrine Mibefradil Norfluoxetine Nortriptyline Paroxetine Penfluridol Perhexiline Perphenazine Pimethixene Pimozide Profenamine Promazine Promethazine Protriptyline Sertindole Sertraline Solasodine Suloctidil Tamoxifen Terfenadine Thioridazine Tomatidine Trifluoperazin Triflupromazine Trimipramine Zolantidine

[0055] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims. It will be appreciated that the invention is in no way dependent upon particular results achieved in any specific example or with any specific embodiment. Articles such as “a”, “an” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims or from the description above is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more elements, limitations, clauses, or descriptive terms, found in any other claim that is dependent on the same base claim.

[0056] Where elements are presented as lists, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. For purposes of conciseness only some of these embodiments have been specifically recited herein, but the invention includes all such embodiments. It should also be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc.

[0057] Any particular embodiment, aspect, element, feature, etc., of the present invention, or any combination thereof, may be explicitly excluded from any one or more claims whether or not such exclusion is expressly recited herein. Applicants reserve the right to proviso out of the claims any specific solution component, ingredient, ingredient category, or combination thereof, whether or not such component, ingredient or ingredient category is recited herein.