NUCLEOSIDE-CONTAINING COMPOSITIONS AND METHODS FOR TREATING RED BLOOD CELLS

Abstract

Blood storage and/or rejuvenating compositions that include D-ribose and a nucleoside other than inosine (e.g., guanosine) are disclosed herein. Such compositions can be useful in methods for treating (e.g., storing and/or rejuvenating) red blood cells.

Claims

1. A blood storage and/or rejuvenating composition comprising a nucleoside and D-ribose, with the proviso that the nucleoside is not inosine.

2. The composition of claim 1 wherein the nucleoside is selected from the group consisting of adenosine, deoxyadenosine, guanosine, deoxyguanosine, 5′-methyluridine, thymidine, uridine, deoxyuridine, cytidine, and deoxycytidine.

3. The composition of claim 1 wherein the nucleoside is guanosine.

4. The composition of claim 1 further comprising sodium pyruvate.

5. The composition of claim 1 further comprising an inorganic phosphate.

6. The composition of claim 1 wherein the composition is an aqueous solution.

7. A blood storage and/or rejuvenating composition comprising 75 to 1500 mM guanosine.

8. The composition of claim 7 further comprising D-ribose.

9. The composition of claim 8 wherein the concentration of the D-ribose is 75 to 1500 mM.

10. The composition of claim 7 further comprising sodium pyruvate.

11. The composition of claim 10 wherein the concentration of the sodium pyruvate is 75 to 1500 mM.

12. The composition of claim 7 further comprising an inorganic phosphate.

13. The composition of claim 12 wherein the concentration of the inorganic phosphate is 75 to 1500 mM.

14. The composition of claim 7 further comprising inosine.

15. The composition of claim 7 further comprising L-arginine.

16. The composition of claim 15 wherein the concentration of the L-arginine is 75 to 1500 mM.

17. A blood storage and/or rejuvenating composition comprising: 225 mM guanosine; 300 mM D-ribose; 300 mM sodium pyruvate; and 300 mM inorganic phosphate.

18. A method of storing blood, the method comprising contacting red blood cells with a blood storage and/or rejuvenating composition according to claim 1.

19. The method of claim 18 wherein the red blood cells are packed red blood cells or in whole blood.

20. A method of rejuvenating blood, the method comprising contacting red blood cells with a blood storage and/or rejuvenating composition according to claim 1.

21. The method of claim 20 wherein the red blood cells are packed red blood cells or in whole blood.

22. A method of rejuvenating blood, the method comprising: providing red blood cells having a 2,3-diphosphoglycerate value lower than the value for freshly drawn blood; and mixing the red blood cells with a blood storage and/or rejuvenating composition under conditions effective to increase the 2,3-diphosphoglycerate value, wherein the blood storage and/or rejuvenating composition comprises guanosine.

23. The method of claim 22 wherein conditions effective comprise incubating the cells in the blood storage and/or rejuvenating composition at a temperature of 4° C. to 37° C.

24. The method of claim 23 wherein the temperature is room temperature.

25. The method of claim 22 wherein conditions effective comprise incubating the cells in the blood storage and/or rejuvenating composition for a time of at least 10 minutes.

26. The method of claim 25 wherein the time is 10 minutes to 48 hours.

27. The method of claim 26 wherein the time is 10 minutes to 4 hours.

28. The method of claim 27 wherein the time is 30 minutes to 2 hours.

29. The method of claim 22 wherein the blood storage and/or rejuvenating composition further comprises D-ribose.

30. The method of claim 22 wherein the red blood cells are packed red blood cells or in whole blood.

31. A method of rejuvenating blood, the method comprising: providing red blood cells having an adenosine triphosphate value lower than the value for freshly drawn blood; and mixing the red blood cells with a blood storage and/or rejuvenating composition under conditions effective to increase the adenosine triphosphate value, wherein the blood storage and/or rejuvenating composition comprises guanosine.

Description

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0025] In 1915, blood transfusion was first attempted from a direct donor to a recipient. During the years following World War I, the practice improved with the use of a citrate glucose solution to collect the blood, the use of refrigeration, and blood typing. Then, in the 1960's and 1970's, improvements continued when glass bottle storage was replaced with durable plastic bags, better anticoagulants were developed, and the addition of mannitol and adenine allowed for storage of RBCs for 42 days. See, for example, Bartlett et al., J. Clin. Invest. 1960; 39:56; Bunn et al., J. Clin. Invest. 1969; 48:311; Akerblom et al., Scand. J. Clin. Lab. Invest. 1968; 21:245-248; and Delivoria-Papadopoulos et al., Science 1969; 165:601-602. Today, blood is still preserved with various anticoagulant solutions that include adenine and citrate. The blood is stored at 4° C., collected in plasticized blood bags, and discarded if not used within 42 days because over this six week storage period RBC viability is largely lost. As RBCs die, the lysed cells release the more durable hemoglobin molecule, which has a low P50, and which presents a barrier to oxygen diffusion.

[0026] Today some estimate that there are approximately 16 million units of RBCs transfused annually in the United States. This number constitutes an average of 34,000 units used every day to support patients undergoing surgery (especially heart, liver, and kidney), cancer treatment, complications of sickle cell anemia, trauma, sepsis, and various conditions requiring critical care. Although several complications have been associated with RBC transfusion, the overall transfusion rate among patients in intensive care is reported to be 44% (Corwin et al., Crit. Care Med., 2004; 32:39-52). Certain of the reported complications result from inherent properties of the blood products transfused, while others are a consequence of RBC storage.

[0027] RBCs undergo major biochemical and biomechanical changes during storage that affect their post transfusion performance and recent studies have drawn attention to possible adverse effects from older stored blood. See, for example, Walsh et al., Crit. Care Med. 2004; 32:364-371; Van de Watering et al., Transfusion 2006; 46:1712-1718; Vamvakas et al., Transfusion 2000; 40:101-109; and Hebert et al., Anesthesia & Analgesia. 2005; 100:1433-1438. The RBC storage lesion is evidenced by the loss of 2,3-DPG, the principal organic phosphate of the human erythrocyte. The 2,3-DPG content within the cell correlates with the position of the oxygen-hemoglobin dissociation curve, as reflected by the P50 (the partial pressure of oxygen at which hemoglobin is 50% saturated) in a variety of clinical conditions, including hypoxic states such as exposure to high altitude and cyanotic heart disease; a variety of anemia's; hyperthyroidism; septic shock; and the changes associated with blood storage (Oski et al., Blood 1971; 37:52-58). In blood stored under conventional blood bank conditions, the 2,3-DPG level drops sharply, and by 10 days of storage 2,3-DPG levels are only 20-25 percent of their original level. Within 21 days of storage they fall to 10 percent of their initial content (Van de Watering et al., Transfusion 2006; 46:1712-1718; and Vamvakas et al., Transfusion 2000; 40:101-109.

[0028] Storage lesions remain a significant concern and a major focus of research in transfusion medicine. Research evidence suggests that storage of RBCs for long periods of time results in reduced oxygen delivery, and transfusion of older blood (i.e., greater than 14-days of storage) has been identified as an independent risk factor for the development of multiple organ failure. See, for example, (Fitzgerald et al., Crit. Care Med. 1997; 25:726-732; Marik et al., JAMA, 1993; 269:3024-3029; Raat et al., Crit. Care Med., 2005; 33:39-45; and Zallen et al., Am. J. Surg., 1999; 178:570-572).

[0029] Based on the results of early studies (e.g., Van de Watering et al., Transfusion 2006; 46:1712-1718; and Vamvakas et al., Transfusion 2000; 40:101-109), it has been assumed that 2,3-DPG levels in RBCs are rejuvenated within 24-hours of transfusion. These studies were performed in normal volunteers with no circulatory problems and with normal blood volume. It is not known whether such recovery would occur in patients suffering from severe blood loss, circulatory issues, or problems associated with underlying medical conditions. Further, the inability of transfused RBCs to deliver oxygen to tissue during the critical time in the early hours following transfusion may have a significant impact on clinical outcome. Although certain studies indicate that the age of transfused RBCs has little or no effect on clinical outcomes in certain conditions (e.g., Hebert et al., Anesthesia & Analgesia. 2005; 100:1433-1438), others suggest the opposite, showing that the duration of storage of RBCs is associated with adverse outcome (Oski et al., Blood 1971; 37:52-58).

[0030] A predominance of the literature suggests the development of an RBC storage solution(s) that would limit or reverse storage lesions would be of considerable consequence to transfusion medicine and could help make RBC transfusion safer and more effective. See, for example, Fitzgerald et al., Crit. Care Med. 1997; 25:726-732; Marik et al., JAMA, 1993; 269:3024-3029; Raat et al., Crit. Care Med., 2005; 33:39-45; Zallen et al., Am. J. Surg., 1999; 178:570-572; Buetler et al., J. Lab. Clin. Med., 1969; 74:300; and Valerie et al., J. Lab. Clin. Med., 1969; 73:722-733. It is postulated that presently disclosed RBC storage and/or rejuvenating compositions will provide such a restorative benefit.

[0031] In one aspect, the present disclosure provides a blood storage and/or rejuvenating composition. In one embodiment, the composition includes a nucleoside and D-ribose, with the proviso that the nucleoside is not inosine. In certain embodiments, the nucleoside includes one or more of adenosine, deoxyadenosine, guanosine, deoxyguanosine, 5′-methyluridine, thymidine, uridine, deoxyuridine, cytidine, and deoxycytidine. In preferred embodiments, the nucleoside is guanosine. Optionally, the composition can further include sodium pyruvate and/or inorganic phosphate. In certain embodiments the composition is an aqueous solution. In preferred embodiments, the composition is an aqueous composition having a pH of 6 to 8.5.

[0032] In another embodiment, the blood storage and/or rejuvenating composition includes 75 to 1500 mM guanosine. Optionally, the composition can further include D-ribose at a concentration of, for example, 75 to 1500 mM. Optionally, the composition can further include sodium pyruvate at a concentration of, for example, 75 to 1500 mM. Optionally, the composition can further include an inorganic phosphate at a concentration of, for example, 75 to 1500 mM. Optionally, the composition can further include L-arginine at a concentration of, for example, 75 to 1500 mM. Optionally, the composition can further include inosine at a concentration of, for example, 75 to 1500 mM. When used to store and/or rejuvenate blood, the composition is typically diluted approximately 30-fold to provide a final concentration of 2.5 to 50 mM guanosine; and optionally 2.5 to 50 mM D-ribose, 2.5 to 50 mM sodium pyruvate, 2.5 to 50 mM inorganic phosphate, 2.5 to 50 mM L-arginine, and/or 2.5 to 50 mM inosine.

[0033] In certain preferred embodiments, the blood storage and/or rejuvenating composition includes 150 to 900 mM guanosine. Optionally, the composition can further include D-ribose at a concentration of, for example, 150 to 900 mM. Optionally, the composition can further include sodium pyruvate at a concentration of, for example, 150 to 900 mM. Optionally, the composition can further include an inorganic phosphate at a concentration of, for example, 150 to 900 mM. Optionally, the composition can further include L-arginine at a concentration of, for example, 150 to 900 mM. Optionally, the composition can further include inosine at a concentration of, for example, 150 to 900 mM. When used to store and/or rejuvenate blood, the composition is typically diluted approximately 30-fold to provide a final concentration of 5 to 30 mM guanosine; and optionally 5 to 30 mM D-ribose, 5 to 30 mM sodium pyruvate, 5 to 30 mM inorganic phosphate, 5 to 30 mM L-arginine, and/or 5 to 30 mM inosine.

[0034] In other preferred embodiments, the blood storage and/or rejuvenating composition includes 300 to 600 mM guanosine. Optionally, the composition can further include D-ribose at a concentration of, for example, 300 to 600 mM. Optionally, the composition can further include sodium pyruvate at a concentration of, for example, 300 to 600 mM. Optionally, the composition can further include an inorganic phosphate at a concentration of, for example, 300 to 600 mM. Optionally, the composition can further include L-arginine at a concentration of, for example, 300 to 600 mM. Optionally, the composition can further include inosine at a concentration of, for example, 300 to 600 mM. When used to store and/or rejuvenate blood, the composition is typically diluted approximately 30-fold to provide a final concentration of 10 to 20 mM guanosine; and optionally 10 to 20 mM D-ribose, 10 to 20 mM sodium pyruvate, 10 to 20 mM inorganic phosphate, 10 to 20 mM L-arginine, and/or 10 to 20 mM inosine.

[0035] In another embodiment, the blood storage and/or rejuvenating composition includes: 225 mM guanosine; 300 mM D-ribose; 300 mM sodium pyruvate; and 300 mM inorganic phosphate. When used to store and/or rejuvenate blood, the composition is typically diluted approximately 30-fold to provide a final concentration of 7.5 mM guanosine, 10 mM D-ribose, 10 mM sodium pyruvate, and 10 mM inorganic phosphate.

[0036] The compositions described herein can be used, for example, in a method of storing blood. In certain embodiments, the method includes contacting RBCs with a blood storage and/or rejuvenating composition as described herein.

[0037] Alternatively, or in addition to, the compositions described herein can be used, for example, in a method of rejuvenating blood. In certain embodiments, the method includes contacting RBCs with a blood storage and/or rejuvenating composition as described herein.

[0038] In certain preferred embodiments, the method of rejuvenating blood includes: providing RBCs (e.g., packed RBCs or in whole blood) having a 2,3-DPG value lower than the value for freshly drawn blood; and mixing the RBCs with a blood storage and/or rejuvenating composition under conditions effective to increase the 2,3-DPG value, wherein the blood storage and/or rejuvenating composition includes guanosine. In certain embodiments, conditions effective to increase the 2,3-DPG value include incubating the cells in the blood storage and/or rejuvenating composition at a temperature of 4° C. to 37° C., and in certain preferred embodiments at a temperature of room temperature. In certain embodiments, conditions effective to increase the 2,3-DPG value include incubating the cells in the blood storage and/or rejuvenating composition for a time of at least 10 minutes, in preferred embodiments for a time of 10 minutes to 48 hours, in certain preferred embodiments for a time of 10 minutes to 4 hours, and in other preferred embodiments for a time of 30 minutes to 2 hours. Exemplary conditions effective to increase the 2,3-DPG value include incubating the cells in the blood storage and/or rejuvenating composition at 37° C. for 10 minutes to four hours. Other exemplary conditions effective to increase the 2,3-DPG value include incubating the cells in the blood storage and/or rejuvenating composition at room temperature for 10 minutes to four hours. In preferred embodiments, the blood storage and/or rejuvenating composition includes one or more of the blood storage and/or rejuvenating compositions described herein.

[0039] In certain preferred embodiments, the method of rejuvenating blood includes: providing RBCs (e.g., packed RBCs or in whole blood) having an ATP value lower than the value for freshly drawn blood; and mixing the RBCs with a blood storage and/or rejuvenating composition under conditions effective to increase the ATP value, wherein the blood storage and/or rejuvenating composition includes guanosine. In certain embodiments, conditions effective to increase the ATP value include incubating the cells in the blood storage and/or rejuvenating composition at a temperature of 4° C. to 37° C., and in certain preferred embodiments at a temperature of room temperature. In certain embodiments, conditions effective to increase the ATP value include incubating the cells in the blood storage and/or rejuvenating composition for a time of at least 10 minutes, in preferred embodiments for a time of 10 minutes to 48 hours, in certain preferred embodiments for a time of 10 minutes to 4 hours, and in other preferred embodiments for a time of 30 minutes to 2 hours. Exemplary conditions effective to increase the ATP value include incubating the cells in the blood storage and/or rejuvenating composition at 37° C. for 10 minutes to four hours. Other exemplary conditions effective to increase the ATP value include incubating the cells in the blood storage and/or rejuvenating composition at room temperature for 10 minutes to four hours. In preferred embodiments, the blood storage and/or rejuvenating composition includes one or more of the blood storage and/or rejuvenating compositions described herein.

[0040] By increasing 2,3-DPG concentration in stressed RBCs, it is postulated RBC storage and/or rejuvenating compositions as disclosed herein will decrease oxygen affinity and increase oxygen delivery to affected tissue following transfusion. Further, by maintaining cellular energetics, it is hypothesized that the storage and/or rejuvenating compositions disclosed herein will decrease cell fragility and increase deformability, thereby improving flow through the capillaries. The net result will be a decrease in storage lesions and greater oxygen delivery to affected tissue following transfusion.

[0041] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLES

Example 1

[0042] A storage and/or rejuvenating composition that includes D-ribose, inosine, sodium pyruvate, and inorganic phosphate, all at a 300 mM concentration as a slurry was prepared. When used to store and/or rejuvenate RBCs, the slurry is diluted 30-fold to a final concentration of 10 mM to form a solution. The composition exhibits significant results in restoring 2,3-DPG levels in stored RBCs and increasing the ATP content from the baseline value. In a study, RBCs were collected and stored for an average of 21 days at 4° C. according to standard blood banking practice. Various RBC storage and/or rejuvenating compositions were added to the 21-day old stored RBCs and held at 37° C. for one to four hours before being tested for 2,3-DPG concentrations. 2,3-DPG levels were measured in all the examples using a diagnostic 2,3-diphosphoglycerate (DPG) kit available from Roche Diagnostics Corp. (Cat. #10148334001). A normal 2,3-DPG concentration of RBC immediately upon harvest is 4.0 μmol/ml. Following storage the average 2,3-DPG concentration fell to 0.17 μmol/ml (Table 1). The addition of RBC storage and/or rejuvenating composition restored the concentration to greater than 50% of normal post-harvest level. An increase to 1.0 μmol/ml is considered a significant improvement. This series of experiments demonstrated a RBC storage and/or rejuvenating composition increased ATP content and 2,3-DPG levels. The practicality of the solution for adoption in blood bank practice may be limited, however, due to the low solubility of inosine as well as the need for warming of the blood (e.g., for one hour) prior to transfusion.

TABLE-US-00001 TABLE 1 2,3-DPG Level of Stored RBCs with RBC Storage and/or Rejuvenating Composition Average Time Point 2,3-DPG Range % ATP (Hours) (μmol/ml) (μmol/ml) N = increase 0 0.17 0.07-0.35 5 1 2.4 1.8-3.3 10 24 2 1.8 1.2-2.7 10 22

[0043] Experimental protocols were designed to find methods to increase the solubility of inosine. It was found that L-arginine in an equimolecular solution enhanced the inosine solubility such that inosine remained in solution at concentrations above 50 mM at room temperature. A storage and/or rejuvenating composition including 300 mM each of inosine, L-arginine, sodium pyruvate, D-ribose, and inorganic phosphate was diluted 30-fold into 21-day old stored RBCs. One set of stored blood samples was incubated for 60 minutes at room temperature and another was incubated for 60 minutes at 37° C. As shown in Table 2, the L-arginine containing storage and/or rejuvenating compositions successfully restored 2,3-DPG and ATP levels regardless of mode of warming. This result demonstrates the rejuvenation of 2,3-DPG and ATP using room temperature incubation and a solution devoid of problems associated with slurries and washing of the red cells prior to transfusion.

TABLE-US-00002 TABLE 2 2,3-DPG and ATP of stored RBC in L-arginine containing solution Average 2,3-DPG Range % ATP Sample (μmol/ml) (μmol/ml) N = increase Control 0.29 N.A. 1 60 minutes 3.2 2.9-3.5 3 29 at 37° C. 60 minutes 1.3 1.0-1.5 3 27 at Room Temperature

Example 2

[0044] An additional experimental protocol was designed to determine if nucleosides other than inosine can successfully aid in restoring 2,3-DPG and ATP levels and, potentially, reduce formation of the breakdown products, hypoxanthine and uric acid. Guanosine, a purine nucleoside consisting of guanine linked by its N9 nitrogen to the C1 carbon of ribose, was chosen as the test nucleotide. Table 3 presents data obtained using a storage and/or rejuvenating composition including a 10 mM final concentration each of guanosine, sodium pyruvate, inorganic phosphate, and D-ribose.

TABLE-US-00003 TABLE 3 2,3-DPG and ATP of stored RBC in a guanosine containing solution Average Time Point 2,3-DPG Range % ATP (Hours) (μmol/ml) (μmol/ml) N = increase Control 0.25 N.A. 1 60 minutes 1.94 1.51-2.43 3 43 at 37° C. 60 minutes 0.51 0.28-0.83 3 32 at Room Temperature

[0045] The 10 mM guanosine solution was capable of restoring 2,3-DPG levels when heated to 37° C. for 60 minutes. The concentrated guanosine composition was not completely soluble and, upon dilution, did not restore 2,3-DPG levels following room temperature incubation, although ATP levels were elevated.

Example 3

[0046] Table 4 presents data obtained using a storage and/or rejuvenating composition including 10 mM each of inorganic phosphate and D-ribose, and guanosine at the indicated concentration. The solutions did not include sodium pyruvate. Rejuvenation was observed for 60 minutes incubation at 37° C.

TABLE-US-00004 TABLE 4 2,3-DPG (μmol/ml) Control 0.20 1 mM Guanosine 0.47 5 mM Guanosine 1.30 10 mM Guanosine  1.10

Example 4

[0047] Table 5 presents data obtained using a storage and/or rejuvenating composition including a final concentration of 10 mM each of L-arginine, inosine, D-ribose, sodium pyruvate, and inorganic phosphate. Rejuvenation was observed after 10 minutes and 60 minutes of incubation at 37° C.

TABLE-US-00005 TABLE 5 2,3-DPG (μmol/ml) Control −0.17 10 minutes at 37° C. 1.39 60 minutes at 37° C. 4.25

Example 5

[0048] Rejuvenation was not observed for 60 minutes incubation at 37° C. when inosine was replaced with varying concentrations of inosine monophosphate in a storage and/or rejuvenating composition including 10 mM each of D-ribose, sodium pyruvate, and inorganic phosphate.

Example 6

[0049] Rejuvenation was not observed for 60 minutes incubation at 37° C. when inosine was replaced with varying concentrations of ribose-5-phosphate in a storage and/or rejuvenating composition including 10 mM each of D-ribose, sodium pyruvate, and inorganic phosphate.

Example 7

[0050] Rejuvenation was not observed for 10 minutes incubation at 4° C. in a storage and/or rejuvenating composition including 10 mM each of inosine, D-ribose, sodium pyruvate, and inorganic phosphate.

[0051] The complete disclosure of all patents, patent applications, and publications, and electronically available material (e.g., GenBank amino acid and nucleotide sequence submissions; and protein data bank (pdb) submissions) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.