WO1997016967A1

WO1997016967A1 - Compositions and methods for promoting blood component survival

Info

Publication number: WO1997016967A1
Application number: PCT/US1996/017952
Authority: WO
Inventors: S. M. Hossein Sadrzadeh; Walter H. Dzik
Original assignee: Beth Israel Deaconess Medical Center, Inc.
Priority date: 1995-11-09
Filing date: 1996-11-08
Publication date: 1997-05-15

Abstract

Compositions and methods for promoting blood component survival are described. Typically, the compositions include a population of blood components, e.g., RBCs, and an antioxidant, e.g., a lazaroid. The methods of the invention include the steps of contacting, in vitro, a population of oxidatively stressed blood components with an antioxidant in an amount and over a period of time effective to protect against oxidative stress-induced blood component damage or in an amount and over a period of time effective to increase the viability of the blood components, e.g., for a period of time greater than 28 days. Methods for inhibiting oxidative stress-induced leukocyte-mediated damage to a population of blood components are also described. These methods include contacting a population of blood components which includes leukocytes and at least one additional type of blood component with an antioxidant in an amount and over a period of time effective to protect against oxidative stress-induced leukocyte-mediated blood component damage.

Description

COMPOSITIONS AND METHODS FOR PROMOTING BLOOD COMPONENT SURVIVAL

Background of the Invention Because blood carries oxygen, blood cells and proteins are at constant risk for oxidative damage. Blood and blood cells contain natural antioxidant enzymes to protect against damage from reactive oxygen species (ROS). When blood is stored ex vivo, however, the protective antioxidant effects diminish and blood cells and proteins are at increased risk for oxidative damage. In addition, when blood is treated ex vivo with chemicals or by processes designed for specific purposes, additional oxidative damage may occur. Examples of such treatments include treatments designed to prevent transmission of microbial pathogens and treatments designed to prevent transfusion-associated graft- versus- host-disease (TA-GVHD).

TA-GVHD is a devastating complication of blood transfusion. Brubaker, D.B. Pathogenesis and Diagnosis of Post-Transfusion Graft-vs.-Host Disease in: Baldwin, M.L. and Jeffries, L.C. eds. Irradiation of Blood Components (American Association of Blood Banks, Bethesda, 1992) pp. 1-30. Nearly always fatal, TA-GVHD results from engraftment and clonal expansion of allogeneic donor leukocytes. Brubaker, supra. Although no treatment has proved successful, TA-GVHD can be prevented by gamma irradiation of blood. Anderson, K.C. Clinical Indicators of Blood Component Irradiation in: Baldwin, M.L. and Jeffries, L.C. eds. Irradiation of Blood Components (American Association of Blood Banks, Bethesda, 1992) pp. 31-50. Gamma irradiation, however, is known to damage blood components such as red blood cells (RBC). Davey, R.J. The Effect of Irradiation on Blood Components in: Baldwin, M.L. and Jeffries, L.C. eds. Irradiation of Blood Components (American Association of Blood Banks, Bethesda, 1992) pp. 51-62. Evidence for this damage appears during storage of irradiated blood and includes the leakage of intracellular K⁺ and hemoglobin. Button, L.N. et al. (1981) Transfusion 21 :419- 426; Ramirez, A.M. et al. (1987) Transfusion 27:444-445; Rivet, C. et al. (1989) Transfusion 29:185; Brugnara, C. et al. (1990) Proc. AABT/ISBT (Los Angeles) p. 62; Peter, E.K. et al. (1991) Ann. Clin. Lab. Sci. 21 :420-425. In vivo, RBC survival studies have documented that irradiated blood which is stored for greater than 28 days has impaired post-transfusion survival compared with non-irradiated blood. Davey, R.J. The Effect of Irradiation on Blood Components in: Baldwin, M.L. and Jeffries, L.C. eds. Irradiation of Blood Components (American Association of Blood Banks, Bethesda, 1992) pp. 51-62; Davey, R. J. et al. (1992) Transfusion 32:525-528. As a consequence, the United States Food and Drug Administration (FDA) (FDA Memorandum, July 22, 1993) has recommended that the shelf-life of irradiated blood be limited to 28 days from the day of irradiation. Once limited to highly immunosuppressed recipients of transfusion, the indications for gamma irradiation have rapidly broadened in recent years. For example, all cellular components donated by blood relatives ofthe recipient must be irradiated. Reports of TA- GVHD in cancer patients with diagnosis other than leukemia/lymphoma have prompted some cancer centers to irradiate all cellular components. Research studies that have documented subclinical clonal expansion of allogeneic donor cells in immunologically normal recipients (Lee, T.H. et al. (1993) Transfusion 33:5 IS) have suggested that universal gamma irradiation of cellular components should be adopted. Moreover, in recent years, the dose of gamma irradiation recommended for treatment of blood has gradually increased from 10 grays (Gy: 1 Gy = 100 rad) to the current 25 Gy. The growing use of gamma irradiation and the increasing recommended doses of irradiation will result in an ever larger proportion ofthe blood supply having a reduced shelf-life as a result of irradiation-induced blood component damage.

Summary of the Invention

The present invention features compositions and methods for minimizing damage caused to blood components, such as RBCs, platelets, and granulocytes, by oxidative stress, e.g., the production of reactive oxygen species (ROS) and subsequent peroxidation ofthe membrane lipids and oxidation of membrane-bound proteins during or after gamma irradiation or initiated by a chemical or process described herein. The invention is based, at least in part, on the discovery that antioxidants can significantly reduce oxidative stress- induced blood component damage.

Accordingly, the present invention pertains to a composition which includes a population, e.g., an in vitro population, of blood components and an antioxidant. Populations of blood components which can be included in the compositions ofthe invention include cellular components, e.g., RBCs, granulocytes, lymphocytes, and platelets, and noncellular components, e.g., plasma proteins. Prior to, during, and/or after subjection to oxidative stress, e.g., induced by, for example, irradiation, e.g., gamma irradiation, treatment with a chemical or process for the purpose of inactivating microbial agents, e.g., a DNA intercalator, e.g., a photoactive agent which can be used as blood sterilizing agent, e.g., a psoralen, methylene blue, and gentian violet, a bleaching agent, an antimicrobial agent, or treatment with a chemical or process which minimizes or inhibits storage-related deterioration, damage and/or death of blood components, these blood components can be mixed or contacted with an antioxidant. Antioxidants which can be included in these compositions include enzyme antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase. Other examples of antioxidants which can be used in the compositions ofthe present invention include water soluble antioxidants such as vitamin E analogs and vitamin C, lipid soluble antioxidants such as vitamin E (both natural and synthetic forms, dα-tocopherol, dl-α tocopherol, tocopheryl acetate, and succinate) and caretenoids, e.g., beta carotene. Other antioxidants which can be used in the compositions and methods ofthe invention include trace elements such as selenium (which is a cofactor for glutathione peroxidase), thiol compounds such as cysteine, e.g., N-acetyl cysteine, and reducing substances such as butylated hydroxytoluene (BHT). Preferred antioxidants for use in the present invention include jazaroids, e.g., tirilazad mesylate. Typically, the compositions ofthe invention include a pharmaceutically acceptable carrier or diluent. In one embodiment, the composition is included within a container, e.g., a blood bag, for storage prior to use. In a preferred embodiment, the composition includes a population of RBCs, e.g., irradiated RBCs, and a lazaroid, e.g., tirilazad mesylate. Mixtures of antioxidants can also be included in the compositions ofthe invention.

The present invention also pertains to methods for promoting or increasing survival of a population of blood components, e.g., RBCs, which has been subjected to oxidative stress, e.g., irradiation, treatment with a chemical or process for the purpose of inactivating microbial agents, or treatment with a chemical or process which minimizes or inhibits storage-related deterioration, damage, and/or death of blood components. For example, as described above, the FDA recommended shelf-life of irradiated blood is 28 days from the day of irradiation. The shelf-life of irradiated blood which is treated according to the methods of this invention is increased to greater than 28 days, e.g., at least about 30 days, preferably at least about 35 days, still preferably at least about 40 days, and yet more preferably at least about 45 days, and most preferably at least about 50 days from the day of irradiation. These methods typically include contacting, in vitro, a population of oxidatively stressed blood components with an antioxidant in an amount and over a period of time effective to protect against oxidative stress-induced blood component damage or in an amount and over a period of time effective to increase the viability ofthe blood components, e.g., for a period of time greater than 28 days. These methods can be used to produce blood supplies with increased shelf-life and eliminate waste associated with having to discard blood supplies which are no longer viable, i.e., have not been used within the recommended time period. These methods have the further advantage of allowing a large build-up of stored blood. Antioxidants which can be used in these methods are described herein. A preferred antioxidant is a lazaroid, e.g., tirilazad mesylate. In another embodiment, the invention provides rneffiδds for increasing shelf-life of irradiated RBCs. These methods typically include contacting, in vitro, the irradiated RBCs with an antioxidant, e.g., a lazaroid, e.g., tirilazad mesylate, in an amount and over a period of time effective to protect against radiation-induced RBC damage. The present invention further pertains to methods for increasing survival of blood components in blood component transfusion recipient subjects. In these methods, a population of blood components is subjected to oxidative stress, and then contacted with an antioxidant to form a transfusion mixture. Alternatively, the population of blood components can be contacted with an antioxidant prior to and/or during subjection to oxidative stress to form a transfusion mixture. The transfusion mixture is then administered to a recipient subject. In one embodiment, the antioxidant is removed from the transfusion mixture prior to administration to the recipient subject. Preferably, the antioxidant is included in the transfusion mixture upon administration to the transfusion recipient. Other aspects ofthe invention are methods for inhibiting oxidative stress-induced leukocyte-mediated damage to a population of blood components. Blood leukocytes, and in particular, polymorphonuclear neutrophils (PMNs) are capable of generating ROS, e.g., superoxide anions, hydrogen peroxide, when activated, e.g., by exposure ofthe blood to oxidative stress induced by, for example, radiation, treatment with a chemical or process for the purpose of inactivating microbial agents, or treatment with a chemical or process which minimizes or inhibits storage-related deterioration, damage, and/or death of blood components. Such ROS damage other blood cells, e.g., RBCs, in the population of blood components. These methods include contacting a population of blood components, e.g., a blood sample, which includes leukocytes and at least one additional type of blood component, e.g., RBCs and/or platelets, with an antioxidant in an amount and over a period of time effective to protect against oxidative stress-induced leukocyte-mediated blood component damage or in an amount and over a period of time effective to increase the viability ofthe additional type of blood component, e.g., RBCs, e.g., for a period of time greater than 28 days. The antioxidant can be added to the population of blood components prior to, during and/or after the population of blood components is subject to oxidative stress. These methods can also be used to produce blood supplies with increased shelf-life and eliminate waste associated with having to discard blood supplies which are not longer viable. As removal of leukocytes from blood by filtration is costly and time consuming, the further advantage of these methods is that leukocyte removal is not necessary. The results described herein demonstrate that antioxidant treatment of blood components subjected to oxidative stress is at least as effective as leukocyte removal from blood in protecting blood components, e.g., RBCs, against oxidative stress-mediated damage.

Brief Description of the Drawings Figures 1A-1B are bar graphs showing the effects of increasing concentrations of t- butyl hydroperoxide (t-BHP) on the formation of thiobarbituric acid reactive substances (TBARS) (Figure 1 A) and on the hemoglobin oxidation (measured by formation of methemoglobin) (Figure IB) in RBC samples.

Figures 2A-2B are bar graphs showing the effects of gamma-irradiation on methemoglobin formation (Figure 2 A) and on the TBARS formation (Figure 2B) in RBC samples.

Figures 3A-3B are bar graphs showing the effects ofthe combined treatment of gamma-irradiation and oxidant treatment on methemoglobin formation (Figure 3A) and on TBARS formation (Figure 3B) in RBC samples. Figure 4 is a bar graph showing the level of TBARS formation in RBC samples treated with t-BHP alone, BHT, a lipid peroxidation inhibitor, and t-BHP, dithiothreitol (DTT), another lipid peroxidation inhibitor, and t-BHP as well as TBARS formation in an untreated RBC sample. Figures 5A-5B are bar graphs showing the level of methemoglobin formation

(Figure 5 A) and the level of TBARS formation (Figure 5B) in RBC samples over a 4 week storage period.

Figures 6A-6B are bar graphs showing the levels of methemoglobin foimation (Figure 6A) and TBARS formation (Figure 6B) in the RBC sample over the 4 week RBC storage period for RBC samples which were either exposed to gamma-irradiation and then stored in CPDA-1 at 4°C for 0-4 weeks or stored in CPDA-1 at 4°C for 0-4 weeks and then exposed to gamma irradiation.

Figure 7 is a bar graph depicting the level of methemoglobin formation, over a six hour period, in RBCs treated with an inhibitor of SOD and then irradiated. Figure 8 is a bar graph depicting the level of methemoglobin formation, over a six hour period, in RBCs treated with an inhibitor of glutathione peroxidase and then irradiated.

Figure 9 is a bar graph depicting the level of methemoglobin formation, over a six hour period, in RBCs treated with an inhibitor of catalase and then irradiated.

Figure 10 is a bar graph depicting the level of methemoglobin formation in RBCs irradiated in the presence and absence of PMNs.

Figures 11A-11B are bar graphs depicting the protective effect of vitamin E administration on radiation-induced methemoglobin formation (Figure 1 IA) and TBARS formation (Figure 1 IB) in a blood sample.

Figure 72 is a bar graph depicting the protective effect of tirilazad mesylate administration on radiation induced hemolysis of RBCs.

Figure 13 is a bar graph depicting the protective effect of tirilazad mesylate administration on radiation induced lipid peroxidation in RBCs.

Detailed Description of the Invention The present invention pertains to compositions which include a population of blood components, e.g., a population of blood components in vitro, and an antioxidant or a mixture of two or more antioxidants. The term "population" as used herein refers to a group of two or more blood components, e.g., two or more blood cells, e.g., two or more RBCs. The blood components can be obtained from whole blood from a living organism, e.g., a mammal, e.g., a human, a rodent, a pig. The blood components ofthe compositions and methods ofthe invention can be cellular, e.g., RBCs (also referred to herein as erythrocytes), granulocytes, lymphocytes, and platelets and/or noncellular components, e.g., blood proteins. Preferably, the blood components are RBCs. Antioxidants which can be used in the compositions and methods ofthe invention include compounds which can prevent, inhibit, minimize and/or at least partially ameliorate oxidative stress-induced blood component damage. Typically, the antioxidants prevent, inhibit, minimize and/or at least partially ameliorate detrimental effects of ROS on blood components, e.g., blood cells, e.g., RBCs. For example, inhibitors of lipid peroxidation are useful as antioxidants. Antioxidants which can be included in these compositions include enzyme antioxidants such as SOD, polyethylene glycol-SOD, catalase, and glutathione, e.g., glutathione peroxidase. Other examples of antioxidants which can be used in the compositions ofthe present invention include water soluble antioxidants such as vitamin E analogs and vitamin C, lipid soluble antioxidants such as vitamin E (both natural and synthetic forms, dα-tocopherol, dl-α tocopherol, tocopheryl acetate, and succinate) and caretenoids, e.g., beta carotene. Other antioxidants which can be used in the compositions and methods ofthe invention include trace elements such as selenium (which is a cofactor for glutathione peroxidase), thiol compounds such as cysteine, e.g., N-acetyl cysteine, and reducing substances such as butylated hydroxytoluene (BHT). Preferred antioxidants for use in the present invention include lazaroids, e.g., tirilazad mesylate. Lazaroids are a class of 21 -aminosteroids which inhibit membrane lipid peroxidation and also act as free radical scavengers. Preferred lazaroids include tirilazad and its mesylate salt, tirilazad mesylate (which is commercially available from Upjohn), 5α-tirilazad, 5β-tirilazad, 6α- hydroxytirilazad, 6β- hydroxytirilazad, and the salts thereof, e.g., the pharmaceutically acceptable salts thereof. For further description of these lazaroids, see e.g., U.S. Patent No. 5,547,949 and PCT publications WO 87/01706 and WO 96/06618, the contents of which are hereby incoφorated by reference. Examples of dosages or amounts of antioxidants which are included in the compositions ofthe invention are as follows: 1) vitamin E is included in the compositions ofthe invention in concentrations which range from about 10 mg to about 500 mg, preferably about 250 mg/unit of whole blood (i.e., about 250 mg/400ml of whole blood or packed red blood cells); 2) vitamin C is included in the compositions ofthe invention in concentrations which range from about 5 mg to about 50 mg per 100 ml of whole blood or packed red blood cells; 3) beta-carotene is included in the compositions ofthe invention in concentrations which range from about 5 mg to about 50 mg/400 ml of whole blood or packed red blood cells; and 4) tirilazad mesylate is included in the compositions ofthe invention in concentrations which range from about 0.01 mg/ml to about 0.2 mg/ml of whole blood or packed red blood cells.

In one embodiment ofthe invention, the compositions also include a pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable carriers and diluents include sterile saline and aqueous buffer solutions. The use of such carriers and diluents is well known in the art. The solutions are sterile and stable under the conditions of manufacture and storage. Pharmaceutically acceptable carriers or diluents suitable for use in the compositions ofthe present invention include standard media used to store and administer blood to human recipients. These media are known in the art and include citrate, phosphate, dextrose, adenine-formula 1 (CPDA-1) and bicarbonate buffers. For other examples of such media see Jeter, E.K et al. (1991) -4w?. Clin. Lab. Sci. 21(3):177-186. The compositions ofthe invention can be included in a container, e.g., a container suitable for storing the compositions ofthe invention, e.g., a blood bag.

Typically, the blood components ofthe compositions ofthe invention are oxidatively stressed. As used herein, the language "oxidatively stressed" refers to blood components which have been subjected to a treatment which results in oxidative stress of the blood components. Oxidative stress includes detrimental effects on the blood components which are caused by ROS and subsequent peroxidation ofthe membrane lipids and oxidation of membrane-bound proteins. Various factors can initiate the production of ROS. For example, exposure of a population of blood components to selected agents which confer a desired property on the blood components, e.g., irradiation, e.g., gamma irradiation which renders lymphocyte blood components nonreproductive, can initiate the production of ROS. Thus, when a population of blood components is exposed to irradiation, ROS form and the blood components are damaged, thereby reducing their viability and longevity. In addition, transmission of microbial pathogens is a major concern in blood transfusion science. Prion, viral, bacterial, fungal, and parasitic pathogens may be transmitted by blood component transfusion. Thus, in addition to screening out donors suspected of harboring transfusion-transmitted pathogens, additional benefit is gained by treating blood components with processes or chemicals which inactivate pathogens or render the pathogens non-infectious. Examples of such processes include, for example, blood component filtration and blood component purification. Examples ofthe chemicals which can be used to treat the blood components include photoactive dyes or agents used as blood sterilizing agents, e.g., psoralens (see U.S. Patent Nos. 5,399,719, 5,556,993, and

PCT publications WO 96/14737 and WO 95/19705, the contents of all of which are hereby incoφorated by reference), methylene blue, gentian violet, DNA intercalators, DNA inhibitors, bleaching agents, antimicrobial agents, protein transcription inhibitors, and enzyme inhibitors. The blood components can also be treated with chemicals or processes which minimize or inhibit storage-related deterioration (or decrease in viability, and/or damage) of blood components. Treatment ofthe blood components with such chemicals and processes can result in production of ROS and/or other agents which cause damage to the blood components and decrease their viability.

The present invention also pertains to methods for promoting or increasing the survival, e.g., survival in vitro, of a population of blood components, e.g., a population of RBCs, which has been subjected to oxidative stress. The shelf-life of oxidatively stressed blood components which are treated according to the methods of this invention is increased to at least about 30 days, preferably at least about 35 days, still preferably at least about 40 days, and yet more preferably at least about 45 days to about 48 days, and most preferably at least about 50 days or more from the day of initiation of oxidative stress. These methods include contacting, in vitro, a population of oxidatively stressed blood components with an antioxidant in an amount and over a period of time effective to protect against, e.g., inhibit or minimize, oxidative stress-induced blood component damage or in an amount and over a period of time effective to increase the viability ofthe blood components, e.g., for a period of time greater than 28 days, the FDA recommended shelf-life of oxidatively stressed, e.g., irradiated, blood. In one embodiment, the blood components are contacted with the antioxidant prior to subjection to the oxidative stress, e.g., radiation, e.g., treatment with a chemical or process for the purpose of inactivating microbial agents, treatment with a chemical or process which minimizes or inhibits storage-related deterioration and damage of blood components. Typically, the blood components, e.g., RBCs, are contacted with the antioxidant for a relatively short period of time. For example, the detrimental effects of oxidative stress on blood components can be decreased, inhibited, or minimized if the blood components are contacted or incubated with an antioxidant for as little as a few minutes, e.g., at least about 30 minutes, and at dosages and in amounts described herein. In other embodiments ofthe invention, the blood components are contacted with the antioxidant, or a combination of antioxidants, for a period of time greater than a few minutes, e.g., 30 minutes or more. Typically, blood components can be incubated with antioxidants for extended periods of time, e.g., hours and days, without causing detrimental effects on the blood cells or other side effects. In addition, the antioxidants which can be used as described herein can be administered to a human recipient together with the blood components. Examples of antioxidants which can be used in the methods ofthe invention are described herein.

The present invention further pertains to methods for increasing survival of blood components, e.g., RBCs, in blood component transfusion recipient subjects. These methods include contacting a population of blood components with at least one antioxidant to form a transfusion mixture, subjecting the transfusion mixture to oxidative stress and administering the oxidatively stressed transfusion mixture to a recipient subject. In a preferred embodiment, the population of blood components is subjected to oxidative stress prior to the addition ofthe antioxidant. In another embodiment, the antioxidant is removed from the transfusion mixture prior to the administering step. The blood components, e.g., RBCs, are contacted with the antioxidant or a combination of antioxidants in an amount and over a period of time effective to protect against oxidative stress or in an amount and over a period of time effective to increase the viability of blood components e.g., RBCs, e.g., for a period of time greater than 28 days. These amounts and time periods are described herein. As used herein, the phrase "transfusion mixture" refers to a population of blood components which includes an antioxidant or which has been contacted with an antioxidant.

In a preferred embodiment, the blood components are RBCs which are exposed to oxidative stress in the form of irradiation, e.g., gamma irradiation. The RBCs are typically exposed to about 15-50 grays (Gy: 1 Gy = 100 rad), preferably about 25 Gy, of gamma- irradiation from, for example, a cesium source. Presently, ofthe irradiated blood cells, e.g., RBCs, not treated according to the present invention that are transfused into a recipient subject, there is a blood cell, e.g., RBC, viability in the recipient subject after 24 hours of about 70%. Blood cells, e.g., RBCs, treated according to the present invention and then irradiated and transfused into a recipient subject exhibit a viability of greater than 70% after 24 hours. Preferably, the viability ofthe blood cells, e.g., RBCs, treated according to the methods ofthe present invention, 24 hours after transfusion is about 75%, more preferably about 80%, and most preferably about 90% or greater. The transfusion mixture is typically administered in a formulation which is compatible with the route of administration. An example of a suitable route of administration is intravenous injection (either as a single infusion or multiple infusions). The terms "subject" or "recipient subject" are used interchangeably herein and refer to mammals, particularly humans, who are to receive the compositions ofthe present invention. Alternatively, the antioxidant can be administered in vivo to a subject and the blood components removed from the subject and oxidatively stressed, e.g., irradiated. For example, blood components for use in the compositions and methods ofthe invention can be obtained from the blood of a human patient, e.g., an autologous donor, who has previously been administered an antioxidant for at least about one to two weeks or more. Still other aspects ofthe invention are methods for inhibiting white blood cell

(leukocyte) mediated oxidative stress-induced damage to a population of blood components. Blood leukocytes, and in particular, polymoφhonuclear neutrophils (PMNs) are capable of generating ROS, e.g., superoxide anions, hydrogen peroxide, when activated, e.g., by exposure ofthe blood to radiation or other chemicals or processes described herein. Such ROS damage other blood components, e.g., blood cells, e.g., RBCs, in the population of blood components. These methods include contacting a population of blood components which includes leukocytes and at least one additional type of blood component with an antioxidant in an amount and over a period of time effective to protect against oxidative stress-induced leukocyte-mediated blood component damage or in an amount and over a period of time effective to increase the viability of blood components, e.g., RBCs, e.g., for a period of time greater than 28 days. The antioxidant can be added to the population of blood components prior to, during, and/or after the population of blood components is subject to oxidative stress. Preferably, the antioxidant or a combination of antioxidants is added to the population of blood components prior to subjection to oxidative stress. The present invention is further illustrated by the following examples which in no way should be construed as being further limiting. The contents of all cited references (including literature references, issued patents, published patent applications, and co¬ pending patent applications) cited throughout this application are hereby expressly incoφorated by reference. EXAMPLES

The following materials and methods were used in the Examples:

Measuring Lipid Peroxidation in Red Blood Cells

Lipid peroxidation can damage RBC membrane structure with formation of membrane pores through which intracellular components, including hemoglobin and potassium ion, can leak. This increased pore formation results in leakage of potassium ion and altered water permeability and, eventually, in cell lysis. Lipid peroxidation also causes polymerization of membrane components and decreases cell deformability. Moreover, peroxidant injury, initiated in phospholipid and other lipid components ofthe membrane can be transmitted to neighboring substances such as membrane proteins. Spectrin, a major protein component ofthe RBC membrane skeleton, can be cross-linked in this manner, resulting in decreased RBC deformability.

Malonyldialdehyde (MDA), a secondary product of lipid peroxidation, is capable of cross-linking membrane components containing amino groups. MDA can increase membrane rigidity and decrease RBC deformability. The detection of MDA using its reaction with thiobarbituric acid is an indicator of lipid peroxidation. The product of this reaction, thiobarbituric acid reactive substances (TBARS), is a chromophore with a maximum absoφtion at 532 nm. Details ofthe TBARS assay for measuring oxidative damage to lipid membranes used herein can be found in Slater, T.F. et al. (1984) Meth. Enzymol. 105:283-293, the contents of which are hereby incoφorated by reference.

Measuring Hemoglobin Oxidation in Red Blood Cells

Hemoglobin can react with ROS to form methemoglobin which may form other oxidized products of hemoglobin. These products, which can be unstable and which may ultimately bind to the RBC membrane, include single chain hemoglobin, hemin, and hemichrome. Hemin has been shown to extensively bind to spectrin, to foster oxidative damage, and to mediate a direct detergent-like effect on the RBC membrane, potentially leading to disruption and lysis. Hemichromes induce topographical changes in the membrane surface and generate free hemin which affects the membrane further.

A common method for measuring hemoglobin oxidation in RBCs is by measuring the formation of methemoglobin. Methods for measuring methemoglobin formation are described in detail in Rice-Evans, C. and Baysal, E. (1987) Biochem. J. 244:191-196 and Harley, J. O. and Mauer, S.M. (1960) Blood 16:1722-1735, the contents of which are hereby incoφorated by reference. EXAMPLE I: EFFECTS OF OXIDANTS ON RED BLOOD CELLS

To determine the role of ROS in radiation-induced damage of RBCs, the effects of a known oxidant, t-butyl hydroperoxide (t-BHP) on human RBCs were studied. Blood was collected from normal blood donors under routine Blood Bank conditions and stored in citrate, phosphate, dextrose, adenine-formula 1 (CPDA-1) bags. Increasing amounts of t- BHP (0, 250, 500, 750, and 1000 μM) were added to the blood sample and the extent of oxidative damage was assessed by 1) measuring the formation of TBARS (as described above), which are indicators of lipid peroxidation in RBCs; and 2) measuring formation of methemoglobin (as described above), which is an indicator of hemoglobin oxidation in RBCs. The results of these experiments are shown in Figures IA and IB.

Figure 1 A is a bar graph showing the effects of increasing concentrations of t-BHP on the formation of TBARS in RBCs. As demonstrated by this Figure, lipid peroxidation in RBCs increases with increasing doses ofthe oxidant t-BHP. Figure IB is a bar graph showing the effects of increasing concentrations of t-BHP on methemoglobin formation. As shown in this Figure, methemoglobin formation and thus, hemoglobin oxidation, increases with increasing doses of t-BHP.

EXAMPLE II: EFFECTS OF GAMMA IRRADIATION ON RED BLOOD CELLS

In the first experiment, packed RBC samples (RBCs without plasma) were exposed to increasing doses of gamma-irradiation (15 to 75 Gy) and the extent of oxidative damage was assessed by methemoglobin formation. The results of this experiment are shown in

Figure 2A. Figure 2A is a bar graph showing the effects of increasing doses of gamma- irradiation on the methemoglobin formation. Figure 2 A demonstrates that the methemoglobin formation increases with increasing doses of gamma-irradiation. Methemoglobin formation is translated into percent hemoglobin oxidation according to the method described in Winterbourn, CC. "Reaction of Superoxide with Hemoglobin" in CRC Handbook of Methods for Oxygen Radical Research. Greenwald, R.A. ed. (CRC Press, Boca Raton, 1985) pp. 137-141, the contents of which are hereby incoφorated by reference.

In the second experiment, an RBC sample was exposed to gamma-irradiation (50 Gy) and the formation of TBARS was measured and compared to the formation of TBARS in an RBC sample which was not exposed to gamma-irradiation. The results of this experiment are shown in Figure 2B. Figure 2B is a bar graph showing the effect of gamma- irradiation on the level of TBARS formation in an RBC sample which was not irradiated and an RBC sample which was irradiated. As demonstrated by this Figure, the level of TBARS formation in the irradiated RBC sample was significantly greater than that in the non-irradiated RBC sample. EXAMPLE III: EFFECTS OF COMBINED TREATMENT WITH GAMMA-

IRRADIATION AND OXIDANT ON RED BLOOD CELLS

In the first experiment, two groups of RBC samples were used. The first group, which was not irradiated, was treated with increasing concentrations of t-BHP (0, 500, 750, and 1000 μM) and the second group was first exposed to 50 Gy of gamma-irradiation. Immediately after irradiation, d e second group was treated with increasing concentrations of t-BHP. The extent of oxidative damage in each group was then assessed by measuring methemoglobin formation in the RBCs. The results of this experiment are shown in Figure 3 A. Figure 3 A is a bar graph showing the effect of gamma-irradiation and t-BHP treatment on the level of methemoglobin formation in an RBC sample over various doses of t-BHP. Figure 3 A demonstrates that radiation treatment combined with t-BHP treatment resulted in increased methemoglobin formation as compared to treatment with t-BHP alone. This increase was observed over a range of increasing t-BHP concentrations. In the second experiment, two groups of RBC samples were also used. The first group, which was not irradiated, was treated with 1500 μM t-BHP and the second group was first treated with t-BHP and then exposed to 50 Gy of gamma-irradiation. The extent of oxidative damage in each group was then assessed by measuring TBARS formation in RBCs. The results of this experiment are shown in Figure 3B. Figure 3B is a bar graph showing the effect of gamma-irradiation and t-BHP treatment on the level of TBARS formation in an RBC sample. Figure 3B demonstrates that radiation treatment combined with t-BHP treatment resulted in increased TBARS formation compared to the sample treated with t-BHP alone.

EXAMPLE IV: EFFECTS OF ADDITION OF THE ANTIOXIDANTS BHT

AND DTT ON OXID ANT-INDUCED OXIDATIVE DAMAGE OF RED BLOOD CELLS

In this experiment, four RBC samples were treated as follows: one sample was treated with t-BHP (1 mM); another sample was treated with butylated hydroxytoluene

(BHT) (100 μM), a lipid peroxidation inhibitor, and then incubated with t-BHP (1 mM) for one half hour at 37°C; another sample was treated with dithiothreitol (DTT) (1 mM), which is a reducing agent, and then incubated with t-BHP for one half hour at 37°C; and the control sample was left untreated. The results of this experiment are shown in Figure 4. Figure 4 is a bar graph showing the level of TBARS formation in these four RBC samples. As demonstrated in Figure 4, addition of either BHT or DTT inhibited t-BHP-induced TBARS formation in the RBC samples. EXAMPLE V: EFFECTS OF STORAGE ON NON-IRRADIATED RED

BLOOD CELLS

In the first experiment, an RBC sample was stored in CPDA-1 at 4°C for 0-4 weeks. Each week, an aliquot ofthe RBC sample was taken and the formation of methemoglobin in the aliquot was measured. The results of this experiment are shown in Figure 5 A. Figure 5 A depicts a bar graph showing the levels of methemoglobin formation in the RBC sample over the 4 week RBC storage period. As shown in this Figure, methemoglobin formation increased as the storage period ofthe RBC samples increased. In the second experiment, an RBC sample was stored in CPDA-1 at 4°C for 0-4 weeks. Each week, an aliquot ofthe RBC sample was taken and the formation of TBARS in the aliquot was measured. The results of this experiment are shown in Figure 5B. Figure 5B depicts a bar graph showing the levels of TBARS formation in the RBC sample over the 4 week RBC storage period. As shown in this Figure, TBARS formation increased as the storage period ofthe RBC samples increased.

EXAMPLE VI: EFFECTS OF RADIATION AND STORAGE ON RED

BLOOD CELLS

In the first experiment, two identical RBC samples (collected in CPDA-1 ) were used. One ofthe RBC samples was exposed to 50 Gy gamma-irradiation and then stored at 4°C for 0-4 weeks. The second RBC sample was stored at 4°C for 0-4 weeks and then exposed to 50 Gy gamma-irradiation. Each week, the formation of methemoglobin in each sample was measured. The results of this experiment are shown in Figure 6A. Figure 6 A depicts a bar graph showing the levels of methemoglobin formation in the RBC sample over the 4 week RBC storage period for RBC samples which were either irradiated and then stored at 4°C for 0-4 weeks or stored at 4°C for 0-4 weeks and then irradiated. As shown in this Figure, methemoglobin formation increased as the storage period ofthe RBC samples increased in both the samples which were irradiated first and the samples which were first stored and then irradiated.

In the second experiment, two identical RBC samples (collected in CPDA-1) were used. One ofthe RBC samples was exposed to 50 Gy gamma-irradiation and then stored at 4°C for 0-4 weeks. The second RBC sample was stored at 4°C for 0-4 weeks and then exposed to 50 Gy gamma-irradiation. Each week, the formation of TBARS in each sample was measured. The results of this experiment are shown in Figure 6B. Figure 6B depicts a bar graph showing the levels of TBARS formation in the RBC sample over the 4 week RBC storage period for RBC samples which were either irradiated and then stored at 4°C for 0-4 weeks or stored at 4°C for 0-4 weeks and then irradiated. As shown in this Figure, TBARS formation increased in both the samples which were irradiated first and the samples which were first stored and then irradiated.

EXAMPLE VH: EFFECTS OF INHIBITION OF RED BLOOD CELL ANTIOXIDANT ENZYMES ON RED BLOOD CELLS

To determine the nature ofthe ROS involved in radiation-induced damage in RBCs, RBC antioxidant enzymes, such as SOD, catalase, and glutathione peroxidase (GSH-PX), in an RBC sample were specifically inhibited as follows. In one RBC sample, RBC catalase activity was specifically inhibited by preincubation of RBCs at 10% hematocrit, at 37°C for 14 hours with 50 mM 3-amino-l, 2, 4,-triazole in Hank's balanced salt solution (HBSS). Agar, N.S. et al. (1986) J Clin. Invest. 77:319-321. In another sample, RBC SOD activity was inhibited by incubation of RBCs at 10% hematocrit for 2 hours at 37°C with 50 mM diethyldithiocarbamate in HBSS. Heikkila, R.E. et al. (1976) J. Biol. Chem. 251 :2182-2185. In a third sample, RBC GSH was depleted by incubation ofthe cells in 10% hematocrit for 60 minutes with 2 mM 1 -chloro-2,4-dinitrobenzene in HBSS. Awathi, Y.C. et al. (1981) Blood 58:733-738. In all cases, the cells were washed twice following exposure to these inhibitors. After these washes, the cells were exposed to 50 Gy of gamma irradiation. The formation of methemoglobin was measured 2, 4, and 6 hours after irradiation.

The results of these inhibition experiments are shown in Figures 7, 8, and 9. Figure 7 is a bar graph depicting the level of methemoglobin formation, over a six hour period, in RBCs treated with an inhibitor of SOD and then irradiated. As demonstrated by this Figure, inhibition of SOD resulted in significant hemoglobin oxidation. Figure 8 is a bar graph depicting the level of methemoglobin formation, over a six hour period, in RBCs treated with an inhibitor of GSH. As demonstrated in this Figure, inhibition of GSH also resulted in significant hemoglobin oxidation. Figure 9 is a bar graph depicting the level of methemoglobin formation, over a six hour period, in RBCs treated with an inhibitor of catalase. As demonstrated by this Figure, inhibition of catalase resulted in hemoglobin oxidation but to a lesser extent than that showed with inhibitors of SOD and GSH. These results demonstrate that gamma irradiation induces oxidative damage in RBCs.

EXAMPLE VIII: EFFECTS OF RADIATION ON RED BLOOD CELLS IN THE

PRESENCE OR ABSENCE OF WHITE BLOOD CELLS

To investigate the source of ROS in RBC samples exposed to gamma-irradiation, the effect of gamma-irradiation on RBC in the presence or absence of white blood cells (leukocytes) was determined. Leukocytes, and in particular, polymoφhonuclear neutrophils (PMNs) are capable of generating ROS. PMNs were purified from whole blood. In the first sample, ten million of these purified PMNs were added to 1 ml of packed RBCs from the same donor. In the second sample, no PMNs were added to the packed RBCs. These packed RBC samples were then exposed to 50 Gy gamma-irradiation. The results of this experiment are shown in Figure 10. Figure 10 is a bar graph depicting the level of methemoglobin formation in irradiated RBCs in the presence and absence of PMNs. As demonstrated in this Figure, the extent of radiation-induced hemoglobin oxidation was much greater in the presence of leukocytes than in the absence of leukocytes. These data indicate that PMNs are responsible for the enhancement of radiation-induced damage of RBCs. Further experiments have demonstrated that irradiation of PMNs results in formation of superoxide anion, which is a ROS.

EXAMPLE IX: EFFECTS OF VITAMIN E ON RADIATION-INDUCED

DAMAGE OF RED BLOOD CELLS

To investigate the role of antioxidants against radiation-mediated RBC damage, the effects of vitamin E (dl-alpha tocopherol), a powerful antioxidant and lipid peroxidation inhibitor, were determined. In the first experiment, 150 ml of blood was taken from four individuals. The blood was pooled and exposed to 50 Gy gamma-iπadiation and then stored at 4°C. At weekly intervals up to 4 weeks of storage, aliquots ofthe blood from each sample were tested for formation of TBARS and methemoglobin.

In the second experiment, 150 ml of blood was taken from four individuals who had received 1000 IU of vitamin E per day for 4 weeks prior to removal ofthe blood. The blood was pooled and exposed to 50 Gy gamma-irradiation and then stored at 4°C. At weekly intervals up to 4 weeks of storage, aliquots ofthe blood from each sample were tested for formation of TBARS and methemoglobin. The results of these experiments are shown in Figures 11A-1 IB. Figure 1 1 A is a bar graph showing the effect of vitamin E administration on radiation-induced methemoglobin formation in the blood sample. As demonstrated in this Figure, vitamin E treatment reduced radiation-induced hemoglobin oxidation by about 50%. Figure 1 IB is a bar graph showing the effect of vitamin E administration on radiation-induced TBARS formation in the blood sample. As demonstrated in this Figure, vitamin E treatment significantly reduced radiation-induced TBARS formation (lipid peroxidation) in the blood sample.

EXAMPLE X: EFFECTS OF LAZAROIDS ON RADIATION-INDUCED DAMAGE OF RED BLOOD CELLS

To investigate the role of antioxidants against radiation-mediated RBC damage, the effects ofthe lazaroid tirilazad mesylate, a powerful antioxidant and lipid peroxidation inhibitor, were determined. In the first experiment, the ability of tirilazad mesylate to protect stored intact human red blood cells against radiation-induced damage was studied. Tirilazad mesylate (U-74006 F) was obtained from the Upjohn Company, as a pure white solid substance. A solution of 5 mg/ml (in 10 mM HCl) was prepared for use in the study. Approximately 450 ml fresh whole blood was collected from healthy adult individuals under the routine blood bank protocol, in a bag containing 63 ml CPDA-1 (Citrate, Phosphate, Dextrose and Adenine) as anticoagulant. The blood bag was centrifuged at 4°C for 8 minutes at 4000 RPM. Then the plasma was transferred to the attached satellite bag and discarded. The packed RBC was washed with PBS in three steps. In each step, the RBC suspension was centrifuged (at 3000, 4000, 5000 RPM) for 3 minutes at 4°C and each time the supernatant was discarded. The washed RBC was then divided into four equal aliquots. Tirilazad mesylate was added to two ofthe aliquots, #2 and #4, (Table 1) with a final concentration of 0.05 mg/ml RBC. All four aliquots were incubated at 37°C for 20 minutes. Following the incubation the aliquots #3 and #4 were irradiated with 50 Gy gamma-irradiation (Table 1).

TABLE 1 SUMMARY OF EXPERIMENT

Aliquot # Content Drug concentration Radiation dose

1 (control) RBC none none

2 RBC + Drug 0.05 mg/ml RBC none

3 RBC + Radiation none 50 Gy

4 RBC + Radiation 0.05 mg/ml RBC 50 Gy + Drug

All aliquots were stored at 4°C for 28 days. The oxidative damage to RBCs was assessed by measuring thiobarbituric acid reactive substances (TBARS), as an indicator of lipid peroxidation, and by determination of osmotic fragility of RBC expressed by % hemolysis. Storage of untreated intact RBC increased the osmotic fragility gradually. Irradiation caused progressive increase in osmotic fragility, which is very significant at day 28. The results of this experiment are shown in Table 2 and Figure 12. Figure 12 is a bar graph depicting the protective effect of tirilazad mesylate administration on radiation induced hemolysis of RBCs. Table 2 and Figure 12 demonstrate that the addition of tirilazad mesylate improved radiation induced hemolysis. Moreover, tirilazad mesylate not only improved radiation damage, but also improved the overall viability ofthe cells following storage. - 17 -

TABLE 2

OSMOTIC FRAGILITY OF RBCS IN 65% NACL FOLLOWING

STORAGE AND IRRADIATION

Sample % Hemolysis (mean ± SEM, n=3)

Day #l Day #7 Day #14 Day #21 Day #28

RBC 0 10.13 ± 3.04 30.53 ± 5.09 36.40 ± 14.06 39.33 ± 16.62

RBC + Drug 0 2.15 ± 0.55* 14.70 ± 5.70* 19.25 ± 2.55* 26.97 ± 5.09

RBC + 0 21.63 ± 3.31 41.05 ± 7.95* 46.33 ± 11.42 65.80 ± 2.20 Radiation

RBC + 0 10.20 ± 2.13 28.37 ± 2.09 24.73 ± 1 1.44 35.80 ± 8.53 Radiation + Drug

*n=2

The lipid peroxidation in intact human RBC was increased following storage and irradiation increased lipid peroxidation on both days one and 28 (See Table 3 and Figure 13). Figure 13 is a bar graph depicting the protective effect of tirilazad mesylate administration on radiation induced lipid peroxidation in RBCs. The results depicted in Table 3 and Figure 13 demonstrate that the addition of tirilazad mesylate protected the intact RBC against lipid peroxidation and irradiation. The effect of tirilazad mesylate is more pronounced in stored irradiated and non-irradiated cells.

TABLE 3 LIPID PEROXIDATION OF RBCS FOLLOWING STORAGE AND

IRRADIATION

Sample TBARS (concentration (mean ± SEM, n=3)

Day #l Day #28

RBC 2.57 ± 0.09 3.41 ± 0.30

RBC + Drug 2.95 ± 0.20* 2.99 ± 0.45

RBC + Radiation 3.00 ± 0.19 3.86 ± 0.32

RBC + Radiation + Drug 2.79 ± 0.11 2.91 ± 0.20

*n n-==2

The results from the experiments in this Example demonstrate that irradiation of red blood cells results in membrane lipid peroxidation and enhanced susceptibility to hemolysis. Addition of an antioxidant, such as tirilazad mesylate, to blood bags prior to irradiation significantly protects RBC against deleterious effects of radiation. Such treatment enhances RBC storage and RBC survival following transfusion.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents ofthe specific embodiments ofthe invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A composition comprising a population of blood components and an antioxidant.

2. The composition of claim 1 , wherein the population of blood components is oxidatively stressed.

3. The composition of claim 1 , further comprising a pharmaceutically acceptable carrier or diluent.

4. The composition of claim 1 , which is included in a container.

5. The composition of claim 4, wherein the container is a blood bag.

6. The composition of claim 1 , wherein the blood components are selected from the group consisting of red blood cells, platelets, and granulocytes.

7. The composition of claim 2, wherein the population of blood components is irradiated.

8. The composition of claim 2, wherein the population of blood components is treated with a chemical or process which inactivates microbial agents.

9. The composition of claim 2, wherein the population of blood components is treated with a chemical or process which inhibits storage-related blood component deterioration.

10. The composition of claim 1, wherein the antioxidant is a lazaroid.

11. The composition of claim 10, wherein the lazaroid is tirilazad mesylate.

12. The composition of claim 1, wherein the antioxidant is an enzyme antioxidant.

13. The composition of claim 12, wherein the enzyme antioxidant is selected from the group consisting of superoxide dismutase, catalase, and glutathione peroxidase.

14. The composition of claim 1 , wherein the antioxidant is a water soluble antioxidant.

15. The composition of claim 14, wherein the water soluble antioxidant is a vitamin E analog or vitamin C.

16. The composition of claim 1 , wherein the antioxidant is a lipid soluble antioxidant.

17. The composition of claim 16, wherein the lipid soluble antioxidant is selected from the group consisting of natural and synthetic forms of vitamin E, dα- tocopherol, dl-α tocopherol, tocopheryl acetate, succinate, and caretenoids.

18. The composition of claim 1 , wherein the antioxidant is selected from the group consisting of trace elements, thiol compounds, and reducing substances.

19. A composition comprising a population of red blood cells and an antioxidant.

20. The composition of claim 19, wherein the population of red blood cells is irradiated.

21. The composition of claim 19, further comprising a pharmaceutically acceptable carrier or diluent.

22. The composition of claim 19, which is included in a container.

23. The composition of claim 22, wherein the container is a blood bag.

24. The composition of claim 19, wherein the antioxidant is a lazaroid.

25. The composition of claim 24, wherein the lazaroid is tirilazad mesylate.

26. A method for promoting or increasing survival of a population of blood components which has been subjected to oxidative stress, comprising; contacting, in vitro, a population of oxidatively stressed blood components with an antioxidant in an amount and over a period of time effective to protect against oxidative stress-induced blood component damage.

27. The method of claim 26, wherein the population of blood components comprises a population of red blood cells.

28. The method of claim 26, wherein survival ofthe population of blood components is increased to greater than 28 days.

29. The method of claim 26, wherein the population of blood components is irradiated.

30. The method of claim 26, wherein the population of blood components is treated with a chemical or process which inactivates microbial agents.

31. The method of claim 26, wherein the population of blood components is treated with a chemical or process which inhibits storage-related blood component deterioration.

32. The method of claim 26, wherein the antioxidant is a lazaroid.

33. The method of claim 27, wherein the lazaroid is tirilazad mesylate.

34. The method of claim 26, wherein the antioxidant is an enzyme antioxidant.

35. The method of claim 26, wherein the antioxidant is a water soluble antioxidant.

36. The method of claim 26, wherein the antioxidant is a lipid soluble antioxidant.

37. A method for increasing shelf-life of irradiated red blood cells, comprising; contacting, in vitro, the irradiated red blood cells with an antioxidant in an amount and over a period of time effective to protect against radiation-induced red blood cell damage.

38. The method of claim 37, wherein the antioxidant is a lazaroid.

39. The method of claim 38, wherein the lazaroid is tirilazad mesylate.

40. A method for increasing survival of blood components in a blood component transfusion recipient subject, comprising; irradiating a population of blood components; contacting the irradiated population of blood components with an antioxidant to form an irradiated transfusion mixture; and administering the irradiated transfusion mixture to a recipient subject.

41. The method of claim 40, further comprising, prior to the administering step, the step of removing the antioxidant from the irradiated transfusion mixture.

42. The method of claim 40, wherein the iπadiating step is performed after the contacting step.

43. A method for inhibiting oxidative stress-induced leukocyte-mediated damage to a population of blood components, comprising; contacting a population of blood components which includes leukocytes and at least one additional type of blood component with an antioxidant in an amount and over a period of time effective to protect against oxidative stress-induced leukocyte-mediated blood component damage.