Composition of platelet additive solution

Abstract

A composition for cold storage of blood platelets comprising at least one platelet additive solution; at least one fuel substrate; at least one pH balancer; and at least one buffer.

Description

[0001] COMPOSITION OF PLATELET ADDITIVE SOLUTION

[0002] This PCT international application claims priority to US Serial No. 63 / 532,451 , filed in the US Patent and Trademark Office on 14 August 2023, the entirety of which is incorporated herein by reference.

[0003] FIELD OF INVENTION

[0004] The present invention relates to a composition comprising a platelet additive solution for cold storage of blood platelets. In particular, the composition may comprise at least one platelet additive solution and at least one of a fuel substrate, a pH balancer, or a buffer.

[0005] BACKGROUND OF INVENTION

[0006] Platelet transfusions are lifesaving interventions for bleeding trauma patients. Increasing the shelf-life of platelets in cold storage increases the availability of platelets for transfusion, thereby increasing the potential for lives saved. This increase in shelf-life may also enable the transport of platelets for life-saving transfusions at greater distances from medical facilities and reduces waste of donated platelets due to degradation in storage.

[0007] Platelet Additive Solutions (PAS) are electrolyte solutions that contain ingredients for the preservation of platelet bioenergetic health during storage. Studies have demonstrated that long-term cold storage of platelets (e.g., 1-60C) in PAS for up to 13 days can support survival comparable or superior to those of platelets stored in plasma for no more than 7 days.

[0008] There remains a need for a composition comprising at least one PAS that has provides acceptable bioenergetic health for blood platelets and improved cold storage and shelflife properties. SUMMARY OF INVENTION

[0009] The invention provides in a first embodiment a composition for cold storage of blood platelets comprising at least one platelet additive solution; at least one fuel substrate; at least one pH balancer; and at least one buffer.

[0010] The invention provides in a second embodiment further to any of the previous embodiments a composition for cold storage of blood platelets in which the at least one platelet additive solution includes saline, citrate, phosphate, and acetate. The at least one platelet additive solution may include saline, acetate, magnesium, potassium, and gluconate, or may include saline, acetate, chloride, gluconate, magnesium, and potassium.

[0011] The invention provides in a third embodiment further to any of the previous embodiments a composition for cold storage of blood platelets including about 60 to about 80 vol. % of the at least one platelet additive solution.

[0012] The invention provides in a fourth embodiment further to any of the previous embodiments a composition for cold storage of blood platelets in which the at least one fuel substrate comprises at least one of a sugar, such as glucose, fructose, or sucrose, or sodium pyruvate.

[0013] The invention provides in a fifth embodiment further to any of the previous embodiments a composition for cold storage of blood platelets in which the at least one buffer comprises a phosphate buffer or acetate buffer.

[0014] The invention provides in a sixth embodiment further to any of the previous embodiments a composition for cold storage of blood platelets in which the composition has a pH of about 7 to about 8. The invention provides in a first method embodiment a method of increasing cold storage of stored blood platelets including adding the composition of any one of the previous embodiments to isolated blood platelets.

[0015] The invention provides in a second method embodiment further to any of the previous method embodiments a method of increasing cold storage of stored blood platelets in which the composition of any of the previous embodiments and blood platelets are stored at a temperature of about 1 to 6°C.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 A show compositions comprising unaltered Platelet Additive Solutions Isoplate, Intersol, Plasmalyte, Lactate Ringer’s solution (LR), and NaCI. FIG. 1 B shows these compositions with the addition of 100 mg / dL glucose fuel substrate.

[0018] FIG. 2A shows compositions comprising unaltered PAS Isoplate and Intersol and pH balanced Plasmalyte, LR, and NaCI. FIG. 2B shows these compositions with a phosphate buffer and pH balancer.

[0019] FIG. 3A shows a composition comprising unaltered PAS Isoplate and pH balanced Plasmalyte. FIG. 3B shows pH balanced Plasmalyte with differing amounts of glucose fuel substrate. FIG. 3C shows a comparison between the average luminescence of media at different glucose concentrations.

[0020] FIG. 4A shows unaltered PAS Isoplate and pH balanced Plasmalyte. FIG. 4B shows pH balanced Plasmalyte with a differing amounts of sodium pyruvate fuel substrate.

[0021] FIG. 4C shows a comparison between the average luminescence of media at different sodium pyruvate concentrations. DETAILED DESCRIPTION OF INVENTION

[0022] The present invention relates to a composition comprising a platelet additive solution for cold storage of blood platelets. In particular, the composition may comprise at least one platelet additive solution, and at least one of a fuel substrate, a pH balancer, or a buffer. In an embodiment, the composition comprises at least one platelet additive solution, at least one fuel substrate, at least one pH balancer, and at least one buffer.

[0023] In this detailed description, references to "one embodiment", "an embodiment", or “in embodiments” mean that the feature being referred to is included in at least one embodiment of the invention. Moreover, separate references to "one embodiment", "an embodiment", or “embodiments” do not necessarily refer to the same embodiment; however, neither are such embodiments mutually exclusive, unless so stated, and except as will be readily apparent to those skilled in the art. Thus, the invention can include any variety of combinations and / or integrations of the embodiments described herein.

[0024] As used herein “substantially”, “generally”, “about”, and other words of degree are relative modifiers intended to indicate permissible variation from the characteristic so modified (e.g., ±0.1 %, ±0.5%, ±1.0%, ±2%, ±5%, ±10%, ±20%). It is not intended to be limited to the absolute value or characteristic which it modifies but rather possessing more of the physical or functional characteristic than its opposite, and preferably, approaching or approximating such a physical or functional characteristic.

[0025] According to the present invention, the composition comprises at least one platelet additive solution. In embodiments, the at least one platelet additive solution may comprise saline and 1 ) at least one of citrate, phosphate, acetate, or any combination thereof (e.g., Intersol); 2) at least one of acetate, magnesium, potassium, gluconate, or any combination thereof (e.g., Isoplate, Plasmalyte), or 3) at least one of acetate, chloride, gluconate, magnesium, potassium or any combination thereof. The platelet additive solution may be Intersol, Isoplate, Plasmalyte, sodium chloride (NaCI), or Lactated Ringer’s solution (LR). In embodiments, a composition may comprise about 60 to about 80 vol.% PAS, for example, 65-70 vol.% along with other components, which may include plasma or a platelet concentrate.

[0026] According to an embodiment of the present invention, the composition comprises at least one fuel substrate. In embodiments, the fuel substrate may comprise at least one of a sugar, such as glucose, fructose, or sucrose; or sodium pyruvate. In specific embodiments, the amount of fuel substrate may be about 50 to about 500 mg / dL glucose or about 0.1 mM to about 5 mM sodium pyruvate.

[0027] In embodiments, the composition may comprise at least one pH balancer. In embodiments, the composition may have a pH of about 7.0 to about 8.0, for example about 7.4. In specific embodiments, the at least one pH balancer may be hydrochloric acid (HCI) or sodium hydroxide (NaOH).

[0028] In embodiments, the composition may comprise at least one buffer. In embodiments, the buffer may be a phosphate buffer or acetate buffer. In specific embodiments, the amount of buffer may be about 4 mM to about 10mM.

[0029] The following are non-limiting embodiments of the present invention. Four experiments were conducted to test the ability of different PAS additives to support platelet bioenergetic status. Bioenergetic health is sensitive to storage conditions.

[0030] Experimental Methods

[0031] Blood from healthy volunteer donors was collected via peripheral venipuncture into 3mL tubes containing sodium citrate anticoagulant. Tubes were then centrifuged at 200 x g for 10 minutes at room temperature to isolate a platelet-rich plasma (PRP), which was then divided into sample tubes for each PAS experimental treatment. PRP was then treated with 1 pg / mL prostaglandin I2 to prevent platelet activation, and the platelets were pelleted by centrifugation at 750 x g for 10 minutes at room temperature. The supernatant platelet-poor plasma (PPP) was aspirated to waste and platelet pellets were washed 3 times with PAS (specific to each treatment) and resuspended.

[0032] Samples were then diluted with PAS to 10x103platelets / mL. Aliquots of 100 mL of each sample were loaded in triplicate into wells of a 96-well plate, which was then covered with a breathable adhesive membrane and stored overnight at 4°C. After allowing the plates to warm to room temperature, a CellTiter-Glo® 2.0 luminescent cell viability assay was performed to assess ATP levels in platelets as an indicator of bioenergetic status. Luminescence values in arbitrary units were used to compare the relative metabolic health of the platelets following cold storage for each PAS treatment tested.

[0033] The first experiment (n=4 donors) tested 5 unaltered US food and Drug Administration (FDA)-approved solutions, which included Intersol, Isoplate, 0.9% sodium chloride (NaCI), lactated Ringer’s (LR), and Plasmalyte. All five solutions were tested with and without 100 mg / dL glucose.

[0034] The second experiment (n=5 donors) tested the same five solutions, with Isoplate and Intersol unaltered and with the remaining solutions pH balanced at physiological pH (pH 7.4), with and without phosphate buffering (4 mM). At this point, the PAS was down- selected to Isoplate and pH balanced and phosphate buffered Plasmalyte based on experimental results.

[0035] The third experiment (n=5 donors) tested the effects of different concentrations of glucose added to Isoplate, as well as to pH balanced and phosphate buffered Plasmalyte. The effects of treatment with 0 mg / dL, 50 mg / dL, 100 mg / dL and 500 mg / dL glucose were tested.

[0036] The fourth experiment (n=5 donors) tested the effects of different concentrations of sodium pyruvate added to Isoplate and to pH balanced and phosphate buffered Plasmalyte. Effects of treatments of 0 mM, 0.1 mM, 1 mM, and 5 mM sodium pyruvate were tested. Statistical analysis was performed using SAS / STAT 15.2. Generalized linear mixed modelling with PROC GLIMMIX was used to determine the effect of media type, additive amount, and their interaction, on luminescence, with a random effect for intercept and fixed effects for treatment, grouped by blood donor. Results were adjusted for multiple comparisons with a Tukey-Kramer adjustment. Statistical significance was accepted at p<0.05. Data is reported as mean ± standard deviation of luminescence in arbitrary units (x 10A6 AU).

[0037] Experimental Results

[0038] In the first experiment, as shown in FIG. 1A, luminescence values in Isoplate (3.82 ± 0.66) and Intersol (3.61 ± 0.68) were found to be higher than Plasmalyte (1.93 ± 0.23), NaCI (2.20 ± 0.39), and LR (2.86 ± 0.31 ; all adj. p<0.0001 ); Isoplate and Intersol were not significantly different from one another. As shown in FIG. 1 B, the addition of 100 mg / dL of glucose increased the average luminescence by 0.28, which was statistically significant (adj. p=0.005). There was no interaction between PAS media type and glucose concentration.

[0039] In the second experiment, as shown in FIG. 2A, luminescence in pH balanced Plasmalyte was greater than in pH balanced NaCI (3.45 ± 0.48 vs. 2.54 ± 0.31 , adj. p<0.0001 ) and in pH balanced LR (3.45 ± 0.48 vs. 2.08 ± 0.39, adj. p<0.0001 ).

[0040] Luminescence in pH balanced NaCI was also greater than that in pH balanced LR (2.54 ± 0.31 vs. 2.08 ± 0.39, adj. p=0.006). As shown in FIG. 2B, the increase in average luminescence after addition of phosphate was not statistically significant. There was no significant interaction between PAS media type and the addition of phosphate.

[0041] In the third experiment, as shown in FIGS. 3A-3C, luminescence in 500 mg / dL glucose (3.14 ± 0.68) was greater than that in the control group (2.86 ± 0.66, adj. p=0.007). Additionally, pH balanced and phosphate buffered Plasmalyte demonstrated a higher luminescence than Isoplate (3.09 ± 0.66 vs. 2.94 ± 0.62, adj. p=0.009). In the fourth experiment, as shown in FIGS. 4A-4C, luminescence in the group treated with 5 mM sodium pyruvate (2.81 ± 0.20) was greater than that in the groups treated with 0.1 mM sodium pyruvate (2.56 ±0.19, adj. p=0.0008) and the control group (2.51 ± 0.27, adj. p<0.0001 ). Similarly, the luminescence in the group treated with 1 mM sodium pyruvate (2.73 ± 0.25) was greater than the group treated with 0.1 mM pyruvate (2.56 ± 0.19, adj. p=0.03) and the control group (2.51 ± 0.27, adj. p=0.004).

[0042] TABLE - Summary of Experiments In the Table, (A) is a summary of Experiment 1 showing the effect of media and glucose; (B) is a summary of Experiment 2 showing the effect of phosphate buffering; (C) is a summary of Experiment 3 showing the effect of glucose on Isoplate versus pH balanced and buffered Plasmalyte; and (D) is a summary of Experiment 4 showing the effect of sodium pyruvate on Isoplate versus pH balanced and buffered Plasmalyte.

[0043] Thus, the inventors found that pH balancing and the addition of fuel substrates to PAS results in higher platelet ATP over one day in cold storage, with a particularly noticeable effect in Plasmalyte and Isoplate. These studies demonstrate that platelet bioenergetic status is sensitive to storage conditions, especially the pH of the storage media and the fuel substrates.

[0044] INDUSTRIAL APPLICABILITY

[0045] The present invention relates to a composition comprising a platelet additive solution. The composition may comprise a platelet additive solution and a fuel substrate, a pH balancer, and a buffer. The composition provides acceptable bioenergetic health for blood platelets and improved cold storage and shelf-life properties.

[0046] Although the present invention has been described in terms of particular exemplary and alternative embodiments, it is not limited to those embodiments. Alternative embodiments, examples, and modifications which would still be encompassed by the invention may be made by those skilled in the art, particularly in light of the foregoing teachings.

[0047] Those skilled in the art will appreciate that various adaptations and modifications of the exemplary and alternative embodiments described above can be configured without departing from the scope and spirit of the invention. Therefore, it is to be understood that, within the scope of the appended claims, the invention may be practiced other than as specifically described herein.

Claims

WHAT IS CLAIMED IS:1 . A composition for cold storage of blood platelets, comprising: at least one platelet additive solution; at least one fuel substrate; at least one pH balancer; and at least one buffer.

2. The composition of Claim 1 , wherein the at least one platelet additive solution comprises saline, citrate, phosphate, and acetate.

3. The composition of Claim 1 , wherein the at least one platelet additive solution comprises saline, acetate, magnesium, potassium, and gluconate.

4. The composition of Claim 1 , wherein the at least one platelet additive solution comprises saline, acetate, chloride, gluconate, magnesium, and potassium.

5. The composition of Claim 1 , comprising about 60 to about 80 vol.% of the at least one platelet additive solution.

6. The composition of Claim 1 , wherein the at least one fuel substrate comprises at least one of a sugar or sodium pyruvate.

7. The composition of Claim 1 , wherein the at least one fuel substrate comprises glucose, fructose, or sucrose.

8. The composition of Claim 1 , wherein the at least one fuel substrate comprises glucose.

9. The composition of Claim 1 , wherein the at least one fuel substrate comprises sodium pyruvate.

10. The composition of Claim 1 , wherein the at least one buffer comprises a phosphate buffer or acetate buffer.11 . The composition of any one of Claims 1 -6, comprising about 1 mM to about 5 mM sodium pyruvate.

12. The composition of any one of Claims 1-10, comprising about 50 mg / dL to about 500 mg / dL of the at least one fuel substrate.

13. The composition of any one of Claims 1 -10, wherein the pH is about 7 to about 8.

14. The composition of any one of Claims 1-10, wherein the composition contains no plasma.

15. A method of increasing cold storage of stored blood platelets, comprising adding the composition of any one Claims 1 -10 to isolated blood platelets.

16. A method according to Claim 15, wherein the composition and blood platelets are stored at a temperature of about 1 to 6°C.

Images