Therapeutic platelets and methods

a technology of platelets and platelets, which is applied in the field of manipulation or modification of platelets, can solve the problems of blood transfusion centers under considerable pressure to produce platelets, rapid loss of platelet function, and limited shelf life under these conditions, so as to avoid platelet activation, effective platelet loading, and stable shelf life

Inactive Publication Date: 2006-06-22
RGT UNIV OF CALIFORNIA OFFICE OF TECH TRANSFER THE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0014] Methods of making and using inventive embodiments are also described. One such method is a process of preparing a dehydrated composition comprising providing a source of platelets, effectively loading the platelets with trehalose to preserve biological properties, cooling the trehalose loaded platelets to below their freezing point, and lyophilizing the cooled platelets. The trehalose loading includes incubating the platelets at a temperature from greater than about 25° C. to less than about 40° C. with a trehalose solution having up to about 50 mM trehalose therein. The process of using such a dehydrated composition further may comprise rehydrating the platelets. The rehydration preferably includes a prehydration step wherein the freeze-dried platelets are exposed to warm, moisture saturated air for a time sufficient to bring the water content of the freeze-dried p

Problems solved by technology

Blood transfusion centers are under considerable pressure to produce platelet concentrates for transfusion.
Today platelet rich plasma concentrates are stored in bloodbags at 22° C.; however, the shelf life under these conditions is limited to five days.
The rapid loss of platelet function during storage and risk of bacterial contamination complicates distribution and availability of platelet concentrates.
When activated they are substantial

Method used

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  • Therapeutic platelets and methods
  • Therapeutic platelets and methods
  • Therapeutic platelets and methods

Examples

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example 1

[0070] Washing of Platelets. Platelet concentrations were obtained from the Sacramento blood center or from volunteers in our laboratory. Platelet rich plasma was centrifuged for 8 minutes at 320×g to remove erythrocytes and leukocytes. The supernatant was pelleted and washed two times (480×g for 22 minutes, 480×g for 15 minutes) in buffer A (100 mM NaCl, 10 mM KCl, 10 mM EGTA, 10 mM imidazole, pH 6.8). Platelet counts were obtained on a Coulter counter T890 (Coulter, Inc., Miami, Fla.).

[0071] Loading of Lucifer Yellow CH into Platelets. A fluorescent dye, lucifer yellow CH (LYCH), was used as a marker for penetration of the membrane by a solute. Washed platelets in a concentration of 1-2×109 platelets / ml were incubated at various temperatures in the presence of 1-20 mg / ml LYCH. Incubation temperatures and incubation times were chosen as indicated. After incubation the platelets suspensions were spun down for 20× at 14,000 RPM (table centrifuge), resuspended in buffer A, spun down ...

example 2

[0079] Washing Platelets. Platelets were obtained from volunteers in our laboratory. Platelet rich plasma was centrifuged for 8 minutes at 320×g to remove erythrocytes and leukocytes. The supernatant was pelleted and washed two times (480×g for 22 minutes, 480×g for 15 minutes) in buffer A (100 mM NaCl, 10 mM KCl, 10 mM EGTA, 10 mM imidazole, 10 μg / ml PGE1, pH 6.8). Platelet counts were obtained on a Coulter counter T890 (Coulter, Inc., Miami, Fla.).

[0080] Loading Platelets with Trehalose. Platelets were loaded with trehalose as described in Example 1. Washed platelets in a concentration of 1-2×109 platelets / ml were incubated at 37° C. in buffer A with 35 mM trehalose added. Incubation times were typically 4 hours. The samples were gently stirred for 1 minute every hour. After incubation the platelet solutions were pelleted (25 sec in a microfuge) and resuspended in drying buffer (9.5 mM HEPES, 142.5 mM NaCl, 4.8 mM KCl, 1 mM MgCl2, 30 mM Trehalose, 1% Human Serum Albumin, 10 μg / ml...

example 3

[0092] We view trehalose as the main lyoprotectant in the drying buffer. However, other components in the drying buffer, such as albumin, can improve the recovery. In the absence of external trehalose in drying buffer, the numerical recovery is only 35%. With 30 mM trehalose in the drying buffer the recovery is around 65%. A combination of 30 mM trehalose and 1% albumin gave a numerical recovery of 85%.

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Abstract

A dehydrated composition is provided that includes freeze-dried platelets. The platelets are loaded with trehalose which preserves biological properties during freeze-drying and rehydration. The trehalose loading is conducted at a temperature of from greater than about 25° C. to less than about 40° C., most preferably at 37° C., with the loading solution having trehalose in an amount from about 10 mM to about 50 mM. These freeze-dried platelets are substantially shelf-stable and are rehydratable so as to have a normal response to an agonist, for example, thrombin, with virtually all of the platelets participating in clot formation within about three minutes at 37° C.

Description

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH AND DEVELOPMENT [0001] This invention was made with Government support under Grant No. HL67810-03, awarded by the National Institutes of Health. The Government has certain rights in this invention.FIELD OF THE INVENTION [0002] The present invention generally relates to the therapeutic uses of blood platelets, and more particularly to manipulations or modifications of platelets, such as in preparing freeze-dried compositions that can be rehydrated at the time of application and which when rehydrated have a normal response to thrombin and other agonists with respect to that of fresh platelets. The inventive compositions are useful in applications such as transfusion therapy, as hemostasis aids and for drug delivery. BACKGROUND OF THE INVENTION [0003] Blood transfusion centers are under considerable pressure to produce platelet concentrates for transfusion. The enormous quest for platelets necessitates storage of this blood component, sinc...

Claims

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Application Information

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IPC IPC(8): A61K35/14A01N1/02C12N5/08A61K35/19A61K47/48
CPCA01N1/0221A01N1/0284A61K35/19A61K45/06Y10T436/108331A61K47/48776A01N1/0231A61K2300/00A61K47/6901
Inventor WOLKERS, WILLEM F.CROWE, JOHN H.TABLIN, FERNOLIVER, ANN E.WALKER, NAOMI J.
Owner RGT UNIV OF CALIFORNIA OFFICE OF TECH TRANSFER THE
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