NZ738372B2 - Platelet storage methods and compositions for same - Google Patents

Abstract

Disclosed are compositions and methods for slowing, preventing, or reversing platelet damage, particularly as may occur during blood banking or during refrigeration of platelets. The composition may include one or more of a RAC inhibitor, a CDC42 inhibitor, a RHOA inhibitor (Rhosin or G04), or a combination thereof. The compositions may further include a pharmaceutically acceptable carrier. In certain embodiments, the RAC inhibitor is NSC23766, the CDC42 inhibitor is CASIN (Formula IIa), and the RHOA inhibitor is Rhosin (G04; Formula III). bination thereof. The compositions may further include a pharmaceutically acceptable carrier. In certain embodiments, the RAC inhibitor is NSC23766, the CDC42 inhibitor is CASIN (Formula IIa), and the RHOA inhibitor is Rhosin (G04; Formula III).

Description

ET STORAGE METHODS AND COMPOSITIONS FOR SAME CROSS REFERENCE TO D APPLICATIONS This application claims priority to and benefit of US Patent Application Serial No. 14/743,213, filed June 18, 2015, of same title, in its entirety for all purposes.

BACKGROUND Patients with low platelet counts often require platelet transfusion. This is particularly crucial in the treatment of patients with cancer or e trauma. The use of platelet transfusions has increased dramatically since 1980s, but a safe, long-term platelet storage method remains unavailable. The demonstration of sful, refrigerated storage of platelets for extended lengths of time, for example, 3’ days or longer, would dramatically change the current practice of platelet transfusion in the n World. Approximately 3,000,000 doses of platelets are used in the United States every year, and account for sales of ~$1.5 Billion annually. The current short shelf—life represents a major handicap to convert platelet ts into ive commodities.

Depending on the time of the year, month or even week, up to 20% of products can be wasted due to expiration. In the meantime, there are moments of platelets shortages due to unpredictable increased usage.

The extension of platelet product shelf-life would strengthen the national inventory of platelets for oncological and trauma patients. An estimated 10-fold se in the need of platelet and plasma products is expected by the US government in war casualties and massive trauma patients due to the 1 red cellzl plateletzl plasma product transfusion policy.

Current practice has platelets stored at 20 to 24°C after preparation, which has a d lifetime up to 5 days, primarily due to concerns about ial contamination. Bacterial contamination of platelet products for usion is a major safety problem in blood g.

The consequence of transfusion of contaminated products is increased morbi-mortality among a susceptible population of cancer patients (1).

Different technologies have been developed aiming to minimize the risk of bacterial ination including diversion pouches for collection, bacterial detection with automatic culture systems and pathogen reduction systems (2—6). While there has been a icant reduction in the number of cases of platelet transfusion associated sepsis, the risk of transfusion-associated sepsis ranges between 1 in ,000 to 86,000 platelet transfusions (7, 8). Storage of platelets in cold temperatures, as is done for red cells, would reduce the proliferation of most bacteria and allow a longer period of storage (9), minimizing the current shortages (10) that the short storage time (5- day) for platelets approved by the FDA (11). Conventional cold storage of platelets, however, has been ed by the discovery that the 24-hour recovery of chilled platelets was significantly reduced (14).

The development of a method to prevent platelet damage upon refrigeration is a much needed, and long sought after e in blood banking. Such development would revolutionize the current method of platelet storage. The instant disclosure solves one or more of these deficiencies in the art.

BRIEF SUMMARY Disclosed are compositions and methods for one or more of slowing, preventing, or ing platelet damage, particularly as may occur during blood banking or during refrigeration of ets. The composition may e one or more of a RAC inhibitor, a CDC42 inhibitor, a RHOA inhibitor, or a combination thereof. The compositions may further include a ceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS 1A— 1B depict a schematic model of “cold receptor” initiated ellular events involving Rho GTPases. 1A. Cold causes the actomyosin changes, Ca2+ mobilization, loss of spectrin anchorage, and Gplb clustering. 1B. “Cold receptor” may stimulate Cdc42, Rac, and/or Rho activation, which in turn control lipid raft assembly and actomyesin reorganization, resulting in Gplb clustering.

FIG 2 depicts Rho GTPases inhibitor chemical structures and s.

FIGS 3A—D depict a g model of Casin bound to Cdc42 surface groove and data demonstrating Casin activity. 3A. Docking model of Casin bound to a Cdc42 surface groove required for activation. 3B.

Collagen—induced Cdc42 activation is inhibited by Casin (10 pM) in a GST-PAK pulldown assay. 3C. Fibrinogen mediated platelet actin filopedia structure is blocked by Casin (10 HM). Rhodamine conjugated phalloidin—staining of platelets d to coated collagen surface. 3D. Collagen d aggregation is blocked by 10 “M Casin (upper panel) and is reversible (up to 30 uM Casin) upon a wash of the inhibitor treated platelets (lower panel).

FIGS 4A—4E depict a docking model of Rac inhibitor NSC23766 and data showing NSC23766 activity. 4A. X—ray ure of 66 bound to a Racl surface groove ed for GEF activation. 4B. Racl activity is inhibited by NSC23766 (50 uM) in a GST-PAK pulldown assay. 4C. Fibrinogen-mediated platelet actin lamellopodia structure is blocked by NSC23766 (50 uM). ine conjugated phalloidin- staining of platelets adhered to coated collagen surface. 4D. Collagen induced aggregation is blocked by NSC23766 in a dose-dependent fashion. 4E. Inhibition of collagen induced platelet aggregation by NSC23766 (SOuM) is reversible upon a wash of the inhibitor treated platelets.

FIGS 5A—5D depict a docking model of Rho inhibitor G04 and data showing GO4 activity. 5A. Docking model of G04 bound to a RhoA surface groove required for GEF tion. Upper panel: low resolution; Lower panel: high resolution binding domain. 5B.

Collagen-induced RhoA activity is inhibited by G04 (50 uM) in a GST-Rhotekin pulldown assay. 5C. U46629 (10 mM/Fibrinogen (3pM)—mediated platelet actin opodia structure is blocked by G04 (30 uM). ine conjugated phalloidin-staining of platelets adhered to coated collagen surface. 5D. Collagen induced aggregation is inhibited by G04 (upper panel). Similar to collagen, thrombin d aggregation (data not shown) is blocked by G04 in a dose- dependent fashion (upper panel) and is reversible upon a wash of the inhibitor treated platelets (lower panel).

FIGS 6A—6D depict the effects of Rho GTPase inhibitor treatment on platelet transfusion. 6A. Experimental s. 6B-6D. 24-hour recovery and al of platelets in different conditions. 6B. r recovery. 6C-6D. Platelet recovery at different time points. Donor platelets were stored at room temperature (squares) or pretreated with a mixed Rho GTPase inhibitors (50 uM NSC23766, 10 pM CASIN and/or 75 ”M G04) or no drug (control). Data are presented as mean SD. *p<0.01 (Anova test with Bonferroni correction, between refrigerated with no inhibitor and refrigerated with triple inhibitor combination).

FIG 7 depicts effects of Rho GTPase inhibitor combinations on platelet survival upon transfusion. Platelet recovery at different time points is shown. Donor platelets were stored at room room temp (squares) or pretreated with a mixed Rho GPase tors (50 uM NSC23766, 10 uM CASIN and/or 75 uM G04) or no drug (Ctrl). Data are presented as mean iSD. *p<0.01 (Anova test with Bonferroni correction, between refrigeratied with no inhibitor and refrigerated with triple inhibitor combination).

FIG 8 depicts total microparticle and G plb+ article counts from platelets stored in different conditions. Results are representative of three independent experiments with similar results of platelets stored for 6 days in 50 11M NSC23766, 10 pM CASIN and/or 75 ”M G04, or no drug (Vehicle control). The left panel shows articles/mcL of refrigerated stored ets for 6 days; the right panel shows Gp1b+ article counts of refrigerated stored platelets for 6 days.

FIG 9 depicts amelioration of lipid raft formation by Rho GTPase inhibitor combinations. Platelets were stored at RT or 4°C for 3 or 7 days in different combinations of inhibitors (50 ”M NSC23766, 10 pM CASIN and/or 75 pM G04) or no drug (vehicle control) at RT or 4°C.

Platelet lysates were analyzed in the presence (lipid rafts) or absence (whole cell lysate) of Triton-X-lOO mediated extraction. Results are representative of two independent experiments with similar results.

Lowe panels represent normalized quantification of lipid raft formation.

FIG lOA—lOC depicts ry (%) of human platelets transfused to pre-treated, thrombocytopenic NSG immunodevicient mice (FIG 10A and 10B); FIG C shows differential recovery between test and control in a randomized, crossover trial of treated refrigerated platelets in atologous transfusion of 7—day stored Rhesus monkey ets.

FIG 11A—11B depicts RhoA deletion (RhoAA/A) after administratoin of poly I:C compared with pre- poly I:C RhoA expresion levels in platelets. FIG 11B depicts Flotillin-l and actin expression in lipid rafts (upper panel) and in whole platelet lysates (lower panel).

FIG l2A-12D depict the effect of Rho family tors on murine wild-type and RhoA-deficient platelets (12A); the effect of Rho family inhibitors on human ets (12B); and the effect of Rho family inhibitors on galactosyl- (12C) and sialyl- (12D) transferase activities to the platelet membranes.

DETAILED PTION Unless defined otherwise, all technical and scientific terms used herein have the same g as commonly understood by one of ordinary skill in the art to which the embodiments belong. gh any methods and materials similar or equivalent to those described herein may also be used in the practice or g of the ments, the preferred methods and materials are now described. All publications mentioned herein are expressly incorporated by reference in their entireties.

DEFINITIONS It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly es otherwise. Thus, for example, nce to “a et” includes a plurality of such platelets and reference to “the carrier” includes reference to one or more carriers and equivalents thereof known to those skilled in the art, and so forth.

The term “about” or “approximately” means within an acceptable error range for the particular value as ined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

An “amide” is a chemical moiety with formula —(R)n—C(O)NHR' or —(R)n—NHC(O)R', where R and R' are independently selected from WO 04809 the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), and where n is 0 or 1.

The term “aromatic” refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes both carbocyclic aryl (e.g., phenyl) and heterocych aryl groups (e.g., pyridine). The term es monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups. The term “carbocyclic” refers to a compound which contains one or more covalently closed ring ures, and that the atoms forming the backbone of the ring are all carbon atoms. The term thus distinguishes carbocyclic from heterocyclic rings in which the ring backbone contains at least one atom which is different from carbon. The term “heteroaromatic” refers to an aromatic group which contains at least one cyclic ring.

As used herein, the term “alkyl” refers to an tic hydrocarbon group. The alkyl moiety may be a “saturated alky group, which means that it does not n any alkene or alkyne moieties. The alkyl moiety may also be an “unsaturated alkyl” moiety, which means that it contains at least one alkene or alkyne moiety. An “alkene” moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond, and an “alkyne” moiety refers to a group consisting of at least two carbon atoms and at least one carbon- carbon triple bond. The alkyl , whether saturated or rated, may be branched, ht chain, or cyclic.

The alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated). The alkyl group may also be a medium size alkyl having 1 to 10 carbon atoms. The alkyl group could also be a lower alkyl having 1 to 5 carbon atoms. The alkyl group of the compounds of the invention may be designated as “C1-C4 alkyl” or similar designations.

By way of example only, “C1-C4 alkyl” indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso—propyl, n-butyl, tyl, sec-butyl, and t-butyl.

The alkyl group may be substituted or unsubstituted. When substituted, the substituent group(s) ) one or more group(s) individually and ndently selected from cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, hio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N—carbamyl, O-thiocarbamyl, N—thiocarbamyl, C-amido, N—amido, S- sulfonamido, N—sulfonamido, C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, and amino, ing mono- and di-substituted amino groups, and the protected derivatives thereof. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, l, ropyl, utyl, cyclopentyl, cyclohexyl, and the like. Wherever a tuent is described as being “optionally substituted” that substitutent may be substituted with one of the above substituents.

The term “ester” refers to a chemical moiety with formula —(R)n— COOR', where R and R' are independently selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), and where nisOorl.

The substituent “R” appearing by itself and without a number designation refers to a substituent selected from the group consisting of en, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicycljc (bonded through a ring carbon).

An “O—carboxy” group refers to a RC(=O)O— group, where R is as defined herein.

A “C-carboxy” group refers to a —C(=O)OR groups where R is as d herein.

An “acety ” group refers to a —C(=O)CH3, group.

A “trihalomethanesulfony ” group refers to a X3CS(=O)2— group where X is a halogen.

A “cyano” group refers to a —CN group.

An “isocyanato” group refers to a —NCO group.

A “thiocyanato” group refers to a —CNS group.

An “isothiocyanato” group refers to a —NCS group.

A “sulfinyl” group refers to a —S(=O)—R group, with R as defined herein.

A “S-sulfonamido” group refers to a —S(=O)2NR, group, with R as d herein.

A fonamido” group refers to a RS(=O)2NH— group with R as defined herein.

A lomethanesulfonamido” group refers to a X3CS(=O)2NR— group with X and R as defined herein.

An “O-carbamyl” group refers to a —OC(=O)—N(R)2, group—with R as defined herein.

An “N—carbamyl” group refers to a ROC(=O)NH— group, with R as defined herein.

An “O-thiocarbamyl” group refers to a —OC(=S)—N(R)2, group with R as defined herein.

An “N—thiocarbamyl” group refers to an ROC(=S)NH— group, with R as defined herein.

A do” group refers to a —C(=O)—N(R)2 group with R as defined herein.

An “N—amido” group refers to a RC(=O)NH— group, with R as defined herein.

The term “perhaloalky ” refers to an alkyl group where all of the hydrogen atoms are replaced by halogen atoms.

The term “acylalky ” refers to a RC(=O)R'— group, with R as defined herein, and R' being a diradical alkylene group. Examples of acylalkyl, without limitation, may include CH3C(=O)CH2—, CH3C(=O)CH2CH2—, CH3CH2C(=O)CH2CH2—, CH3C(=O)CH2CH2CH2—, and the like.

Unless otherwise indicated, when a substituent is deemed to be “optionally substituted,” it is meant that the tutent is a group that may be substituted with one or more group(s) dually and independently selected from cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, to, alkylthio, arylthio, cyano, halo, yl, thiocarbonyl, O—carbamyl, amyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N—sulfonamido, C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, and amino, WO 04809 including mono— and di-substituted amino groups, and the protected tives thereof.

The terms “protecting group” and “protecting groups” as used herein refer to any atom or group of atoms that is added to a molecule in order to prevent existing groups in the molecule from undergoing unwanted chemical ons. The protecting group moiety may be chosen in such a way, that they are stable to the reaction conditions applied and readily removed at a convenient stage using methodology known from the art. A non-limiting list of protecting groups include ; substituted ; alkylcarbonyls (e.g., t—butoxycarbonyl (BOC)); arylalkylcarbonyls (e.g., benzyloxycarbonyl, benzoyl); tuted methyl ether (e.g. methoxymethyl ether); substituted ethyl ether; a substituted benzyl ether; tetrahydropyranyl ether; silyl ethers (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, t—butyldimethylsilyl, or t- butyldiphenylsilyl); esters (e.g. benzoate ester); carbonates (e.g. methoxymethylcarbonate); sulfonates (e.g. te, mesylate); acyclic ketal (e.g. dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane or 1,3- dioxolanes); acyclic acetal; cyclic acetal; acyclic hemiacetal; cyclic hemiacetal; and cyclic dithioketals (e.g., 1,3-dithiane or 1,3- dithiolane).

As used herein, the term “cycloalkyl” is intended to cover three-, four—, five-, six—, seven—, and eight— or more membered rings sing carbon atoms only. A cycloalkyl can optionally contain one or more rated bonds situated in such a way, however, that an ic pi— on system does not arise. Some examples of “cycloalkyl” are the carbocycles cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclopentadiene, cyclohexane, cyclohexene, 1,3—cyclohexadiene, 1,4- cyclohexadiene, cycloheptane, or cycloheptene.

As used herein, ocyclyl” means a cyclic ring system comprising at least one heteroatom in the ring system backbone. The heteroatoms are independently selected from oxygen, sulfur, and nitrogen.

Heterocyclyls may include multiple fused rings. Heterocyclyls may have any degree of saturation provided that at least one ring in the ring system is not aromatic. cyclyls may be substituted or unsubstituted, and are attached to other groups via any available valence, preferably any available carbon or en. Preferred monocyclic heterocycles are of 5 or 6 members. In six membered monocyclic heterocycles, the heteroatom(s) are selected from one up to three of oxygen, sulfur, and nitrogen, and wherein when the heterocycle is five membered, preferably it has one or two heteroatoms selected from oxygen, sulfur, and nitrogen.

A heterocyclyl can further n one or more carbonyl or thiocarbonyl functionalities, so as to make the definition include oxo— systems and thio—systems such as lactams, lactones, cyclic imides, cyclic ides, cyclic carbamates, and the like. Some examples of “heterocyclyls” include, but are not limited to, tetrahydrothiopyran, 4H-pyran, tetrahydropyran, dine, 1,3-dioxin, 1,3-dioxane, 1,4- dioxin, 1,4—dioxane, piperazine, 1,3-oxathiane, athiin, 1,4- oxathiane, tetrahydro—1,4-thiazine, 2H-1,2—oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, oin, dihydrouracil, morpholine, trioxane, hexahydro—1,3,5- triazine, tetrahydrothiophene, tetrahydrofuran, pyrroline, pyrrolidine, pyrrolidone, pyrrolidione, pyrazoline, pyrazolidine, imidazoline, imidazolidine, 1,3—dioxole, 1,3-dioxolane, 1,3-dithiole, 1,3-dithiolane, isoxazoline, isoxazolidine, oxazoline, idine, oxazolidinone, thiazoline, thiazolidine, and 1,3-oxathiolane. The attachment point of a heterocycle radical can be at the position of a nitrogen heteroatom or via a carbon atom of the heterocycle.

The term “ary ” means a carbocych aromatic ling or ring system.

Moreover, the term “aryl” includes fused ring systems wherein at least two aryl rings, or at least one aryl and at least one C3-8—cycloalkyl share at least one chemical bond. Some examples of “aryl” rings include ally substituted phenyl, naphthalenyl, phenanthrenyl, anthracenyl, tetralinyl, fluorenyl, indenyl, and indanyl. The term “ary ” s to aromatic, including, for example, benzenoid groups, ted via one of the ring-forming carbon atoms, and optionally carrying one or more substituents selected from heterocyclyl, heteroaryl, halo, hydroxy, amino, cyano, nitro, alkylamido, acyl, C1-6 alkoxy, C1-6 alkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, C1-6 alkylamino, alkylsulfenyl, alkylsulfinyl, alkylsulfonyl, sulfamoyl, or trifluoromethyl. The aryl group can be substituted at the para and/or meta positions. In other embodiments, the aryl group can be substituted at the ortho position. Representative examples of aryl groups include, but are not limited to, phenyl, 3-halophenyl, 4-halophenyl, 3- hydroxyphenyl, 4—hydroxyphenyl, 3-aminophenyl, 4-aminophenyl, 3- methylphenyl, 4—methylphenyl, 3-methoxyphenyl, 4—methoxyphenyl, 4—trifluoromethoxyphenyl 3-cyanophenyl, 4—cyanophenyl, dimethylphenyl, naphthyl, hydroxynaphthyl, hydroxymethylphenyl, romethylphenyl, alkoxyphenyl, 4-morpholin-4—y1—pheny1, 4- pyrrolidin-l-ylphenyl, 4-pyrazoly1pheny1, 4—triazoly1phenyl, and 4—(2— oxopyrrolidin— 1-yl)phenyl.

As used herein, the term “heteroaryl” means an ic radical having one or more heteroatom(s) (e.g., oxygen, sulfur, or nitrogen) in the ring backbone and may include a single ring (e.g., pyridine) or multiple condensed rings (e.g., quinoline). aryl groups can carry one or more substituents, each independently ed from halo, hydroxy, amino, cyano, nitro, cycloalkyl, kyl, aryl, heterocyclyl, mercapto, alkylamido, acyl, C1—6-alkoxy, C1alkyl, C1 hydroxyalkyl, C1aminoalkyl, lkylamino, alkylsulfenyl, ulfinyl, alkylsulfonyl, sulfamoyl, and trifluoromethyl.

Representative examples of heteroaryl groups e, but are not limited to, optionally substituted derivatives of furan, benzofuran, thiophene, benzothiophene, pyrrole, pyridine, indole, oxazole, azole, isoxazole, benzisoxazole, le, hiazole, isothiazole, imidazole, benzimidazole, pyrazole, indazole, tetrazole, quionoline, isoquinoline, pyridazine, pyrimidine, purine and pyrazine, furazan, 1,2,3-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, tn'azole, benzotn'azole, pteridine, phenoxazole, oxadiazole, benzopyrazole, quinolizine, cinnoline, phthalazine, quinazoline, and quinoxaline. In some embodiments, the substituents can be halo, hydroxy, cyano, O— C1alkyl, C1-6—alkyl, hydroxy—C1a1kyl, and C1a1kyl.

The term “platelet” is used here to refer to a blood platelet. A platelet can be described as a minisule protoplasmic disk occurring in vertebrate blood. Platelets play a role in blood clotting. The platelet may be d from any source including a human blood supply, or the patient's own blood.

As used herein, the term “effective amount” means the amount of one or more active components that is sufficient to show a desired effect.

This es both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether stered in combination, serially or simultaneously.

Refrigerated storage is ed to reduce platelet life-span due to sed temperature that cause glycoprotein-1b (GPIb) receptors to cluster on specific microdomains of the platelet membrane. Applicant has found that recognition of specific ed/syalylated residues on clustered glycoproteins by macrophage B2 ins and hepatocyte Ashwell—Morell receptors results in platelet phagocytosis by the host and removal from circulation. Thus, Applicant has identified prevention of rotein clustering as a useful target for chemical ention.

Platelet glycoproteins are intimately associated with intracellular cytoskeleton. Clustering of platelet glycoproteins depends on the formation of lipid raft in the platelet membrane, which in turn depends on the dynamics of the highly ted processes of actomyosin assembly/disassembly. Rho family GTPases, including RhoA, Racl and Cdc42, are a class of GTP—binding enzymes that are central tors of F—actin polymerization/depolymerization, and have been shown to control lipid raft formation and composition. Therefore, Applicant postulates that changes in Rho GTPase activities may influence platelet ne lipid raft assembly and glycoprotein composition. Reversible targeting of Rho family GTPases by small molecule inhibitors may prevent cytoskeleton—dependent refrigeration storage lesions in platelets and result in increased platelet survival.

The mechanisms of how cold temperatures affect platelet survival are not completely understood, though significant information has been collected in the past decade. The s of cold ature on platelets are believed to be complex and involve shape change, cytoskeletal reorganization, activation, cell surface protein clustering and changes in the carbohydrate structures of e glycoproteins (15-18). Refrigeration-induced changes ing filopodia or lamellipodia are accompanied by an increase in the fraction of total cellular actin in a polymeric state (F—actin) (12, 18, 19) and disappearance of a peripheral microtubule coil (20). Isolated prevention of microtubule polymerization using colchicine has not resulted in shape change prevention upon activation (21, 22).

Prevention of isolated actin cs using cytochalasin B s in reversion of discoid shape (18) but not in ed et survival in baboons (23), suggesting that irreversible blockade of actin polymerization does not prevent the refrigeration damage.

Presence of platelet cold receptors has been postulated as an explanation for both tatic and clinical effects when platelets are submitted to temperatures below 16°C. Cold temperature is believed to induce deglycosylation of glycoprotein Ib ectodomain ng N- acetyl—D—glucosamine residues (17), which sequesters GM1 gangliosides in lipid rafts. Raft-associated glycoprotein Iba forms clusters upon binding of 1432 adaptor proteins to its cytoplasmic tail, a process accompanied with mitochondrial damage and PS exposure (apoptosis—like)(24). The mechanisms of platelet clearance are believed to be associated with lipid—raft associated GPIb clustering and prevention of clustering prevents platelet nce (15, 25).

Intimately associated with intracellular cytoskeleton, GPIb clustering depends on the ion of omains (so—called “lipid rafts”) in the et ne which in turn depends on the dynamics of the highly regulated processes of acto—myosin assembly/disassembly at multiple levels. In summary, refrigeration results in multiple, complex platelet s that may be closely associated with cytoskeletal impairments at multiple levels (FIG 1A).

Actin cytoskeletal rearrangements responsible for lipid rafts and GPIb clustering in lipid rafts depends on the coordinated activities of Cdc42, Racl and RhoA GTPases, which control specific downstream effectors in regulating polymerization and depoymerization of F—actin, actomyosin contraction, tubulin polymerization, and spectrin anchorage. The Rho family GTPases are a class of GTP—binding enzymes that act as signaling switches in spatial/temporal transduction and amplification of signals from platelet receptors to the intracellular ing pathways that drive platelet function. Among the direct Rho GTPase effectors, WASPs, forrnins and PAKs that control n polymerization/depolymerization have been shown to be crucial in the l of lipid raft formation and composition and tubular polymerization of platelets (27) (FIG 1B). Therefore, changes in Rho GTPase activities may influence platelet ne microdomain assembly and glycoprotein composition. Earlier studies using dominant negative mutants of Cdc42 and Racl found no effect on prevention of cold-induced platelet damage (28), but the limitation of the tools used has prevented investigators from manipulating actin/actomyosin dynamics in a specific, reversible fashion.

Without intending to be limited by theory, it is believed that reversible inhibition of multiple Rho family of GTPases by al inhibitors can significantly improve platelet survival and transfusion on after refrigerated storage by erence with actomyosin dynamics and membrane microdomains to prevent GPIb clustering.

Compositions and methods useful for et survival and/or quality, usion, and associated issues are disclosed . In one aspect, a composition for platelet storage or treatment is described. The composition may comprise a RAC inhibitor, for example, those described in US. Patent No. 7,517,890 and US. Patent No. 7,612,080, a CDC42 inhibitor, for example, those described in US. Patent No. 8,383,124; a RHOA inhibitor, and combinations thereof.

In one aspect, the RAC inhibitor may comprise a nd having the structure of Formula I or a pharmaceutically acceptable salt thereof: _ (Formula I) wherein R1 to R2 are independently selected from the group ting of H, — X—Alk, —X—A1k-X', and —X—Y—X'; wherein X is —CR7Rg; X' is —CHR7R8; Alk is a C2-C18 substituted or unsubstituted hydrocarbon chain; Y is a C2-C3 substituted or unsubstituted alkylene chain; R6 is H or (C1-C4) alkyl; and R7 and R3 are independently ed from the group consisting of H and (C1-C4) alkyl; or a salt f; In one aspect, the RAC inhibitor may comprise the structure of Formula Ia or a pharmaceutically acceptable salt thereof.

(Formula Ia) wherein: R10 to R12 are independently selected from the group consisting of H, halo, (C1-C4) alkyl, branched (C3-C4) alkyl, halo (C1-C4) alkyl, (C1-C4) , N02, and NH2.

In one aspect, the RAC inhibitor may comprise Formula lb or a pharmaceutically acceptable salt thereof. la Ib) In one aspect, the CDC42 inhibitor may comprise Formula II or a pharmaceutically acceptable salt thereof.

(Formula H) wherein Y is selected from the group consisting of 0R7, NRgRg, and NNR8R9; R7 is selected from the group consisting of C1_(, alkyl, (CH2)uC3-7 cycloalkyl, C2_5 alkenyl, C1.6 alkoxy, hydroxy-C1-5 alkyl, phenyl, C1_6 alkyl substituted with up to 5 fluoro, and C16 alkoxy substituted with up to 5 fluoro, said C1-6 alkyl, (CH2)uC3_7 cycloalkyl, C2_(, alkenyl, C1_(, , hydroxy—C1_(, alkyl, phenyl are each optionally substituted with one or more substituents each independently selected from the group consisting of halo, —CN, —OH, C1_(, alkoxyl, aryl, R19, and ORzo; R3 and R9 may each be separately a hydrogen, or separately selected from the group consisting of C1.6 alkyl, C34 lkyl, and phenyl, said C1-6 alkyl, C3-7 cycloalkyl, and phenyl, each optionally substituted with one or more substituents each independently ed from the group consisting of halo, cyano, nitro, hydroxy, C1_(, alkyl, —(CH2)uC3_7cycloalkyl, C2_5 alkenyl, WO 04809 C1_6 alkoxy, hydroxy—C1-6 alkyl, R19, ORzo, C1-6 alkyl substituted with up to 5 fluoro, and C1_(, alkoxy substituted with up to 5 fluoro, each optionally substituted with one or more substituents each independently selected from the group consisting of halo, cyano, nitro, hydroxy, C1_6 alkyl, and C1_6 alkoxy; or R8 and R9 are optionally taken together with the nitrogen to which they are attached to form indolinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl, each optionally substituted with one or more substituents each ndently selected from the group consisting of halo, cyano, nitro, hydroxy, C1_(, alkyl, (CH2)uC3_7cycloalkyl, C24, alkenyl, C145 alkoxy, hydroxy—C1_(, alkyl, phenyl, CH; alkyl substituted with up to 5 fluoro, and C1-6 alkoxy substituted with up to 5 fluoro; or R8 and R2 come together to be C1_3 alkyl linking together as a ring; each u is independently 0, 1, 2, 3, or 4; R2 is a hydrogen, or selected from the group consisting of C1_(, alkyl, C3_7 lkyl, and phenyl, said C1_(, alkyl, C3_7 cycloalkyl, and phenyl, each optionally substituted with one or more substituents each ndently selected from the group ting of halo, cyano, nitro, hydroxy, C1_(, alkyl, —(CH2)uC3_7 cycloalkyl, CM alkenyl, Cm , hydroxy—C1_(, alkyl, phenyl, C1_6 alkyl substituted with up to 5 fluoro, CH, alkoxy substituted with up to 5 fluoro, and —O(CH2)uphenyl optionally substituted with one or more substituents each independently selected from the group consisting of halo, cyano, nitro, hydroxy, C1_(, alkyl, and C1_(, alkoxy; or R8 and R2 come er to be C1.3 alkyl linking together as a ring; R3, R4, R5 and R6 are each independently selected from the group consisting of: hydrogen, halo, cyano, nitro, hydroxy, C 1.6 alkyl, (CH2)uC3_7cycloa1kyl, —O(CH2)uC3_7cycloalkyl, C2_(, alkenyl, C1_(, alkoxy, y—C1.6 alkyl, phenyl, C1.5 alkyl substituted with up to 5 fluoro, and C1_(, alkoxy substituted with up to 5 fluoro, said C1- 6 alkyl, (CH2)uC3_7cycloalkyl, —O(CH2)uC3_7cycloalkyl, C24, alkenyl, C1_(, alkoxy, hydroxy—C1.6 alkyl, and phenyl, each ally substituted with one or more substituents each independently selected from the group consisting of halo, cyano, nitro, hydroxy, C1_(, alkyl, —(CH2)uC3_7cycloalkyl, C24, alkenyl, C14, alkoxy, hydroxy—CH; alkyl, phenyl, C1_(, alkyl substituted with up to 5 fluoro, and C1-5 alkoxy substituted with up to 5 fluoro, said phenyl optionally substituted with one or more tuents each ndently selected from the group consisting of halo, cyano, nitro, y, C145 alkyl, —(CH2)uC3_7cycloa1ky1, C2_5 alkenyl, C16 alkoxy, hydroxy—C1-6 alkyl, phenyl, CH; alkyl substituted with up to 5 fluoro, and C1_(, alkoxy substituted with up to 5 fluoro; R19 is aryl optionally substituted with one or more substituents each independently selected from the group consisting of halo, cyano, nitro, hydroxy, C1_(, alkyl optionally substituted with up to 5 fluoro, and C1.5 alkoxy optionally substituted with up to 5 fluoro; R20 is hydrogen or aryl optionally tuted with one or more substituents each independently selected from the group consisting of halo, cyano, nitro, hydroxy, C1_(, alkyl optionally substituted with up to 5 fluoro, and C1-6 alkoxy optionally tuted with up to 5 fluoro; and wherein when Y is NRgRg then R3 and R2 optionally come together to be C1_3 alkyl g together as a ring, with the proviso when R3 comes together with R2 to be C1_3 alkyl linking together as a ring then R4, is not substituted with yl.

In one aspect, the CDC42 inhibitor may comprise Formula H, or a pharmaceutically acceptable salt thereof (Formula H) wherein: Y is NRgRg, R8 is en; and R9 is C1_(, alkyl, said C1_(, alkyl, optionally substituted with one or more substituents each ndently selected from the group consisting of hydroxy, R19 or ORzo; R19 is phenyl optionally substituted with one or more substituents each independently selected from the group ting of halo, cyano, C1.5 alkyl optionally tuted with up to 5 fluoro, and C1.6 alkoxy optionally substituted with up to 5 fluoro; and R20 is hydrogen or phenyl optionally substituted with one or more substituents each independently selected from the group consisting of halo, cyano, nitro, hydroxy, C1.5 alkyl optionally substituted with up to fluoro, and C1.6 alkoxy optionally substituted with up to 5 fluoro.

In one aspect, the CDC42 inhibitor may comprise Formula Ha (CASIN), or a pharmaceutically acceptable salt thereof m (Formula IIa) In one aspect, the RHOA inhibitor may comprise (Formula III)(G04), or a pharmaceutically acceptable salt thereof.

HEN”? ““2 H \-".' 1 . , \ _ )1; X?”I “my. #1»wang NN gray“ ¢ ”‘ch y Ni}, s i. ‘ “:33? i} U j “x”JK‘Nf ’ la HI)(GO4) In one aspect, the composition may comprise the Formula 1b (NSC23766) or a pharmaceutically acceptable salt thereof, Formula IIa (CASIN) or a pharmaceutically acceptable salt f; Formula HI (G04) or a pharmaceutically acceptable salt thereof, and ations f.

Any amine, hydroxy, or carboxyl side chain on the compounds of the may be esterified or amidified. The procedures and specific groups to be used to achieve this end are known to those of skill in the art.

Synthesis of one or more of the above-referenced compounds may be describes in, for example, US. Patent No. 124 to Zheng, issued February 26, 2013; US. Patent No. 7,612,080 to Zheng et al., issued November 3, 2009; and US. Patent No.7,517,890 to Zheng et a1, issued April 14, 2009, the contents of which are incorporated by reference for all purposes.

The active agent can form salts, which are also within the scope of the red embodiments. Reference to a compound of the active agent herein is understood to include reference to salts thereof, unless otherwise indicated. The term “salt(s)”, as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases. In addition, when an active agent contains both a basic moiety, such as, but not limited to an amine or a pyridine or imidazole ring, and an acidic moiety, such as, but not limited to a ylic acid, zwitterions (“inner salts”) can be formed and are included within the term “salt(s)” as used herein. Pharrnaceutically acceptable (e.g., non- toxic, physiologically acceptable) salts are preferred, gh other salts are also , e.g., in isolation or purification steps, which can be employed during preparation. Salts of the nds of the active agent can be formed, for example, by reacting a compound of the active agent with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

The active agents which contain a basic moiety, such as, but not limited to an amine or a pyridine or imidazole ring, can form salts with a variety of organic and inorganic acids. ary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, ates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, tes, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides (formed with hydrochloric acid), hydrobromides (formed with en bromide), hydroiodides, 2- WO 04809 hydroxyethanesulfonates, es, maleates (formed with maleic acid), methanesulfonates (formed with methanesulfonic acid), 2- naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, ylpropionates, phosphates, picrates, pivalates, nates, salicylates, succinates, sulfates (such as those formed with sulfuric acid), sulfonates (such as those mentioned ), tartrates, thiocyanates, toluenesulfonates such as tosylates, undecanoates, and the like.

The active agents that contain an acidic , such as, but not limited to a carboxylic acid, may form salts with a variety of organic and inorganic bases. Exemplary basic salts include ammonium salts, alkali metal salts such as , lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines d with N,N-bis(dehydro— abietyl)ethylenediamine], N—methyl—D-glucamines, N—methyl—D- glucamides, t—butyl amines, and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl s (e.g., benzyl and phenethyl bromides), and others.

Active agent, and salts f, can exist in their tautomeric form (for example, as an amide or imino ether). All such tautomeric forms are contemplated herein as part of the preferred embodiments.

] All stereoisomers of the present compounds, such as those, for e, which can exist due to asymmetric carbons on any of the substituents, including omeric forms (which can exist even in the absence of asymmetric carbons) and diastereomeric forms, are contemplated and within the scope of the preferred embodiments.

Individual stereoisomers of the compounds of the preferred embodiments can, for example, be substantially free of other isomers, or can be admixed, for example, as racemates or with all other or other selected, isomers. The chiral centers of the preferred embodiments can have the S or R configuration as defined by the IUPAC 1974 Recommendations.

When the compounds are in the forms of salts, they may comprise pharmaceutically acceptable salts. Such salts may include pharmaceutically acceptable acid addition salts, ceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like.

Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, c, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, esulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, ic, stearic, palmitic, EDTA, glycolic, p—aminobenzoic, ic, benzenesulfonic, p— toluenesulfonic acids, sulphates, nitrates, phosphates, perchlorates, borates, acetates, benzoates, hydroxynaphthoates, glycerophosphates, ketoglutarates and the like. es of metal salts include lithium, sodium, potassium, magnesium salts and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts and the like. Examples of organic bases include , arginine, guanidine, diethanolamine, choline and the like.

The ceutically acceptable salts may be ed by s known to one of ordinary skill in the art. For example, by reacting the active agent with 1 to 4 lents of a base such as sodium hydroxide, sodium methoxide, sodium hydride, potassium t—butoxide, calcium hydroxide, magnesium hydroxide and the like, in solvents like ether, THF, methanol, t—butanol, dioxane, panol, ethanol, etc.

Mixture of ts can be used. Organic bases like lysine, arginine, diethanolamine, choline, guandine and their derivatives etc. can also be used. Alternatively, acid addition salts Wherever applicable are prepared by treatment with acids such as hloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, p- toluenesulphonic acid, esulfonic acid, fonic acid, acetic acid, citric acid, maleic acid salicylic acid, ynaphthoic acid, ic acid, palmitic acid, succinic acid, benzoic acid, esulfonic acid, tartaric acid and the like in solvents like ethyl acetate, ether, alcohols, acetone, THF, dioxane, etc. Mixture of solvents can also be used.

In one aspect, any of the above-described compositions may comprise a physiologically acceptable carrier. In one aspect, the physiologically acceptable carrier may comprise a buffer. In one aspect, the physiologically acceptable carrier may be selected from saline, phosphate buffered saline, Tris ed saline, Hank's buffered saline, water, or a combination thereof. In one aspect, the physiologically acceptable carrier may comprise an electrolyte solution.

In one aspect, the RAC inhibitor, the CDC42 inhibitor, and the RHOA inhibitor may be present in a carrier at a concentration, in ation or separately, of at least about 10 ”M, or at least about 25 “M, at least about 50 ”M, at least about IOOuM, at least about 200 ”M, at least about 500 “M, at least about 1 mM, at least about 10 mM, at least about 50 mM, at least about 100 mM, or at least about 1 M.

In one aspect, the RAC inhibitor may be t at a concentration of from about 10 “M to about 500 uM, or from about 25 uM to about 400 11M, or from about 50 pM to about 300 uM, or from about 75 11M to about 200 “M, or from about 100 ”M to about 150 “M, or about 50 ] In one aspect, the CDC42 inhibitor may be present at a concentration of from about 5 uM to about 500 uM, or from about 10 uM to about 400 ”M, or from about 25 tho about 300 pM, or from about 50 ”M to about 200 “M, or from about 75 uM to about 150 “M, or about 10 In one aspect the RHOA inhibitor may be present at a concentration of from about 10 pM to about 500 uM, or from about 25 pM to about 400 pM, or from about 50 “M to about 300 uM, or from about 75 ”M to about 200 “M, or from about 100 uM to about 150 “M, or about 75 ] In further aspects, the described compositions may comprise an additive selected from NaCl, KCl, CaC12, MgClz, MgSO4, Na3 citrate, citric acid, NaHC03, Na phosphate, Na acetate, Na gluconate, glucose, maltose, mannitol, and combinations thereof. The described compositions may comprise an additive selected from NaCl, KCl, WO 04809 CaClz, MgClz, MgSO4, Na3 citrate, citric acid, NaHCO3, Na phosphate, Na acetate, Na ate, glucose, maltose, mannitol, and combinations thereof, wherein said additive may be present in an amount of from about 0.5 mmol/L to about 150 mmol/L.

In further aspects, the described compositions may comprise one or more ingredients selected from D-ribose, D-glucose, Hanks solution, Hepes solution, bovine serum albumin, tic anticoagulant peptide and sterile water, or combinations thereof.

In one aspect, the composition may comprise an ary substance selected from pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and ations thereof.

In one aspect, the composition may have a pH of from about 5 to about 8, or from about 6 to about 7, or about 6.8 to about 7.4.

In one aspect, the composition may be isotonic.

In yet further aspects, the composition may comprise an additional therapeutic agent.

A method for storing platelets is also described. In this aspect, the method comprises the step of g ets in a composition as disclosed herein.

In one aspect, the method may comprise ting platelets with a solution comprising an RAC tor, an CDC42 inhibitor, and/or a RHOA inhibitor, n the one or more actives are present in a carrier at a concentration, in ation or separately, of at least about 10 uM, or at least about 25 uM, at least about 50 uM, at least about 100pM, at least about 200 ”M, at least about 500 ”M, at least about 1 mM, at least about 10 mM, at least about 50 mM, at least about 100 mM, or at least about 1 M.

In one aspect, the method may se contacting platelets with a solution comprising an RAC inhibitor present in a solution at a concentration of from about 10 uM to about 500 “M, or from about 25 “M to about 400 ”M, or from about 50 uM to about 300 “M, or from about 75 “M to about 200 “M, or from about 100 uM to about 150 pM, or about 50 uM.

In one aspect, the method may comprise contacting platelets with a solution comprising a CDC42 inhibitor present in a solution at a concentration of from about 5 “M to about 500 ”M, or from about 10 “M to about 400 uM, or from about 25 uM to about 300 “M, or from about 50 uM to about 200 “M, or from about 75 [AM to about 150 uM, or about 10 “M.

In one aspect, the method may comprise contacting platelets with a solution comprising an RHOA inhibitor present in a on at a concentration of from about 10 ”M to about 500 “M, or from about 25 pM to about 400 ”M, or from about 50 uM to about 300 ”M, or from about 75 ”M to about 200 “M, or from about 100 ”M to about 150 “M, or about 75 “M.

In one aspect, the storage step may be carried out at a temperature of from about 1°C to about 20°C, or at about 1°C, or about 2°C, or about 3°C, or about 4°C, or about 5°C, or about 6°C, or about 7°C, or about 8°C, or about 9°C, or about 10°C, or about 11°C, or about 12°C, or about 13°C, or about 14°C, or about 15°C, or about 16°C.

Ine aspect, the storage step may be carried out for a period of time of from about 7 to 20 days, or from about 10 to 15 days, or greater than 7 days, or greater than 8 days, or greater than 9 days, or greater than 10 days, or greater than 11 days, or r than 12 days, or greater than 13 days, or greater than two weeks.

In one , the composition may be used in an amount sufficient to inhibit a platelet damaging activity selected from polymerization of F— actin, depolymerization of n, actomyosin contraction, n polymerization, spectn'n anchorage, or combinations thereof.

In one aspect, applying the described s, the platelet survival may be greater than about 65%, or greater than about 70%, or greater than about 75%, or greater than about 80%, or greater than about 85%, or greater than about 90%, or greater than about 95%, or greater than about 99% after a storage period of 24 hours at 5C.

In one aspect, a method of reversing platelet activation is also disclosed, comprising contacting ted platelets with a composition as described herein. In one aspect, the ting step may be canied out at a ature of about 0°C. In a yet further aspect, the disclosed compositions may be used to reverse refrigeration storage lesions in platelets. In this aspect, platelets having refrigeration storage lesions may be contacted with a composition as disclosed herein, for a period of time sufficient to reverse refrigeration storage lesions.

Examples Applicant has tested the ability of three chemical inhibitors, CASIN, NSC23766 and G04, targeting Cdc42, Racl and RhoA, respectively (FIG 2), to t the activity of the GTPases and the consequent cytoskeleton-dependent functions of human platelets. A rationally developed inhibitor of Cdc42 with activity on activation by guanine nucleotide exchange factors is CASIN (29, Nature Biotech under revision). CASIN was ly discovered by Zheng group as a small le that recognizes a pocket domain that ically blocks the ability of GEF interaction with Cdc42 (FIG 3A). CASIN is found to suppress of hematopoietic stem cell aging through its specific Cdc42 inhibitory activity (29). In platelets, it inhibits Cdc42 activation (FIG 3B) and prevents collagen-induced platelet shape changes (FIG 3C).

These s depend on integrin signaling and involve F—actin rization and filopodia formation as demonstrated by phalloiding staining (FIG 3C, lower panels). Cdc42 inhibition by CASIN results in prevention of collagen-induced platelet ation (FIG 3D) that can be reversed by a washout of the inhibitor (FIG 3D, upper and lower panels).

Similarly, Applicant has identified an inhibitor of Rac, NSC23766 (30— 34), with an ability to impair Racl activation by le activating signals which may be l in platelet signaling associated with Gpr/GpX (35). NSC23766 was discovered in a structure-based virtual screening of compounds that fit into a surface groove of Racl known to be critical for guanine nucleotide exchange factor specification (34) (FIG 4A). NSC23766 can effectively block Racl activation (FIG 4B) and Rac-dependent cytoskeletal rearrangements (actin lamellopodia) of platelets stimulated by collagen (30, 32, 35), indicating its ability for ssing collagen-integrin dependent signaling (FIG 4C). Finally, 66 reversibly inhibits, in a dose- dependent fashion, platelet aggregation d by collagen (FIG 4D, upper and lower panels).

Finally, G04/Rhosin was also developed by us as a RhoA GTPase activation site inhibitor that transiently and specifically blocks RhoA activity and RhoA-mediated signaling functions (36, 37). G04 ns two aromatic rings tethered by a , and it binds to the surface area sandwiching Trp58 of RhoA (FIG 5A) with a submicromolar Kd and effectively ts GEF-catalyzed RhoA activation. In platelets, G04 2015/066252 specifically inhibits en-induced RhoA activity (FIG 5B) and RhoA-mediated ar functions including fibrinogen-dependent platelet spreading (FIG 5C) and collagen-dependent platelet ation (FIG 5D). Effect by collagen or thrombin is reversible (FIG 5D, lower).

In addition to the reversibility, each inhibitor transiently mimics the effects of Cdc42, Racl, or RhoA gene knockout in ets, respectively, and does not show any additive effects in the respective knockout cells (data not shown), indicating their specificity and a lack of toxicity.

A combination of CASIN (10 uM), G04 (75 uM) and/or NSC23766 were used in C57Bl/6 murine carboxyfluorescein—lateled platelets incubated at 0°C or 5°C for 3.5 hours. After re—warming, platelets were infused in 5 congenic mice (per group) and compared with control (room temperature stored) and refrigerated, ted platelet group (FIG 7). While the combination of NSC23766, G04 and CASIN result in close—to-complete reversal of the survival deficiency induced by refrigerated storage, NSC23766 alone or NSC23766 in combination with CASIN or G04 did not result in significant reversal of the e lesion of platelets (Figure 7). In a second experiment focused on the specific ations with more activity to reverse the survival impairment associated with the use of Rho GTPase inhibitors, we analyzed the 24-hour recovery (%) and survival (hours) of platelets that had been stored in presence of several combinations of drugs (Figure 6A).

Similarly, treatment of refrigerated platelets with CASIN or G04 alone did not improve platelet r recovery (FIG 6B) or survival (FIG 6C) in vivo. However, a triple combination of CASIN, G04 and NSC23766 resulted in a significant improved ry (FIG 6B) and survival (FIG 6C) of refrigerated platelets after infusion. Use of the same triple inhibitor combination during storage of refrigerated platelets at 5°C instead of 0°C resulted in complete reversal of the recovery and survival of refrigerated platelets in vivo (FIG 6D) as determined by hit regression analysis (COSTCO software) and ANOVA test with Bonferroni correction for statistical differences.

Applicant found that the r recovery of platelets refrigerated was significantly reduced (~20%) compared with the group control (stored at room temperature, p<0.01). stingly, the shortened recovery was completely reversed by incubation with the cocktail of three inhibitors (FIG 6D). Altogether, these data indicate that the inhibitory ation of Cdc42, Racl, and RhoA with CASIN, NSC23766 and G04 can specifically and reversibly inhibit platelet activation and may be useful in a reversal of refrigeration e lesion in platelets.

Finally, Rho family GTPase inhibitors can prevent human platelet storage lesion in vitro as assessed by article ion and content of Gpr (CD42b) in microparticles. Applicant found that the use of tors resulted in reversal of the refrigeration dependent microparticle generation (Figure 8A—B). Finally, Applicant analyzed the formation of lipid raft microdomains on the platelet membrane by differential analysis in lysates enriched in Triton—X—lOO resistant membrane fragments in human platelets stored refrigerated for 3 and 7 days e 9). We found a complete correlation between the results of survival of erated platelets in presence of inhibitor ations with the formation of lipid raft microdomains, suggesting that the inhibitor combination containing G04, NSC23766 and CASIN s in reversal of refrigeration-induced lipid raft formation. Interestingly, washing out GO4, NSC23766 and CASIN s in restoration of the same levels of lipid rafts as found in 3 or 7 day stored ets at room temperature (Figure 9), suggesting that the removal of these inhibitors results in platelet membrane rheological modifications similar to the ones observed in unprocessed, standard storage platelets for the same period of time (Figure 9). These data provide proof-of—concept that the long-term storage (6 days) of platelets in plasma containing Rho GTPase inhibitors is not rious of platelets but can further prevent their storage lesion-associated activation.

Thus, Applicant’s data strongly support that reversible inhibition of multiple Rho family of GTPases by chemical inhibitors can significantly t refrigerated storage damage and improve platelet survival and transfusion function after refrigeration by interference with lipid raft microdomain formation, actomyosin cs and GPIb clustering in membrane microdomains.

Applicant further analyzed and confirmed that the reversible inhibition of multiple Rho family of GTPases by chemical tors can significantly prevent refrigerated storage damage and improve platelet survival and transfusion function after refrigeration. The ism is believed to be through ting Rho GTPase—regulated lipid raft microdomain formation, actomyosin dynamics and GPIb clustering in ne omains.

To further validate our results from murine refrigerated platelet survival improvement in primates, Applicant first analyzed the survival of human pooled (n=10 donors per experiment) platelets transfused in inbred thrombocytopenic, irradiated, hage-depleted NSG mice.

These immunodeficient mice allow the survival of human platelets for a short period of time (<24 hours) but sufficiently long to allow comparison. In this model, Applicant found that, in two independent experiments (n=4 mice per group and ment), the incubation of platelets during storage with the inhibitors G04 (RhoA inhibitor), NSC23766 (NSC; Rac inhibitor) in absence of wash step or with a wash step at room temperature of the inhibitors used in vitro.

Interestingly, Casin (Cdc42 inhibitor) did not modify the poor survival of refrigerated platelets (FIG 10A-10B). Secondly, these results were further ted by applying a combination of G04, NSC and Casin in outbred Rhesus monkey platelets that were biotinylated and autologously transfused after 7—day refrigerated storage. As seen in FIG 10C, a randomized, crossover trial where the same monkeys were transfused with control (not treated with inhibitors, control) or inhibitors (test), demonstrated that the inhibitor combination resulted in improved (positive recoveries) in the test group.

To better define the effect of RhoA inhibition and to rule out any off- target effect of G04, we analyzed ets from wild-type (Wt) mice and from mice with interferon-induced (polylzC) genetic deficiency of RhoA (FIG 11A). G04, and to a lesser degree NSC, were able to reduce the level of Flot—l in the membrane lipid raft (microdomains) of refrigerated Wt platelets (FIG 11B). Interestingly, the levels of membrane-bound Flot-l in RhoA—deficient (RhoANA) platelets was r to those found in G04 treated Wt refrigerated platelets (FIG 10C), and NSC does not appear to add any significant further inhibition.

Next, Applicant analyzed r the phagocytosis of refrigerated murine platelets by activated monocytic THP—1 cells was modified by pre-treatment with the Rho family inhibitors. Pre—treatment of refrigerated platelets with G04 or the combination of G04, Casin and NSC reduce the y to be recognized by activated THP—1 cells (FIG 12A). Use of murine RhoA genetically deficient platelets (FIG 12A, inset) were ant to the phagocytosis induced by eration and showed no response to G04 or the ation of G04, Casin and NSC (FIG 12A). These results strongly indicate that G04 blocks the ability of refrigerated platelets to present ligands susceptible of recognition by hage cells and this effect is exclusively dependent on RhoA activity. These results were confirmed in human platelets.

Phagocytosis by THP—1 cells of refrigerated human platelets was completely prevented by storage with G04 or combination of G04, Casin and NSC even after wash out of the inhibitors at room temperature (FIG 12B).

Finally, Applicant examined whether the absence of translocation of lipid raft microdomains to the membrane (FIG 9) ates with changes in galactosyl-transferase and sialyl-transferase activities.

These activities should reduce by transfer to other substrates the galactosyl and sialyl residues of Gpr on the membrane, a crucial step in the s of refrigeration storage lesion of platelets. Applicant found that G04 or G04 in combination with Casin and NSC completely prevented the presence of osyl- (FIG 12C) or sialyl— (FIG 12D) transferase activities on the platelet membrane. This effect is completely reversible since washing of the inhibitors restores the transport of these enzymes to the membrane (FIG 12C-D).

Altogether, these new data strongly support the sion that Rho GTPase inhibitor G04 alone or G04 in combination with NSC23766 WO 04809 and/or Casin will prevent refrigerated e lesion of platelets. This effect is reproduced in mouse, human and non-human primate platelets. The mechanism involves the prevention of formation of galactosyl— and sialyl—transferase rich membrane lipid rafts on refrigerated platelet membranes through a modulation of actomyesin dynamics.

All percentages and ratios are calculated by weight unless otherwise indicated.

All percentages and ratios are calculated based on the total ition unless otherwise indicated.

It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical tion, as if such lower numerical limitations were expressly written herein. Every m numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written . Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower cal ranges were all expressly written herein.

The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such ion is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.” Every document cited herein, including any cross referenced or related patent or ation, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with t to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. r, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

While particular ments of the present invention have been illustrated and described, it would be s to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

What is claimed is: 1. A composition, comprising platelets, a RHOA inhibitor selected from: , or a salt f, , or a salt thereof, or combinations thereof; and an acceptable carrier. 2. The composition according to claim 1, wherein the carrier comprises a buffer. 3. The composition according to claim 1 or 2, wherein the carrier is selected from saline, phosphate-buffered saline, tris-buffered saline, Hank’s buffered saline, water, or a combination thereof. 4. The composition according to any one of claims 1-3, wherein the carrier comprises an electrolyte solution.

. The composition according to any one of claims 1-4, further comprising an additive selected from NaCl, KCl, CaCl2, MgCl2, MgSO4, sodium citrate, citric acid, NaHCO3, sodium phosphate, sodium e, sodium gluconate, glucose, maltose, mannitol, D-ribose, D-glucose, Hank’s solution, HEPES solution, bovine serum albumin, tick anticoagulant peptide and sterile water, a pH adjusting agent, a pH buffering agent, a ty adjusting agent, a stabilizer, a g agent, and combinations f. 6. The composition according to claim 5, wherein the additive comprises a sodium ion, potassium ion, acetate ion, magnesium ion, chloride ion, gluconate ion, glucose, ate ion, citrate ion, calcium ion, and combinations f. 1003747565 7. The composition according to claim 5 or 6, n said additive is t in an amount of from 0.5 mmol/L to 150 mmol/L. 8. The composition according to any one of claims 1-7, wherein the pH of the composition is from 5 to 8. 9. The composition according to any one of claims 1-8, wherein the composition is isotonic.

. The ition ing to any one of claims 1-9, further comprising a therapeutic agent. 11. The composition according to any one of claims 1-10, wherein the RHOA inhibitor is , or a salt thereof. 12. The composition according to any one of claims 1-10, wherein the RHOA inhibitor is , or salt thereof. 13. The composition according to any one of claims 1-12, wherein the RHOA inhibitor is present at a concentration of at least 10 μM. 14. The composition according to any one of claims 1-13, wherein the RHOA inhibitor is present at a concentration of from 10 μM to 500 μM.

. The composition according to any one of claims 1-14, wherein the RHOA inhibitor is present at a tration of from 25 μM to 400 μM. 16. The composition ing to any one of claims 1-15, wherein the RHOA inhibitor is present at a concentration of from 50 μM to 300 μM. 17. The composition according to any one of claims 1-16, wherein the RHOA inhibitor is present at a concentration of from 75 μM to 200 μM. 18. The composition according to any one of claims 1-17, wherein the RHOA inhibitor is present at a concentration of from 100 μM to 150 μM. 19. A method for storing platelets comprising forming the composition of any one of claims 1-18, and storing said composition. 1003747565 . A method of reversing platelet activation comprising forming the composition of any one of claims 1-18, wherein the platelets added to the composition are activated. 21. A method of reversing refrigeration storage lesion in ets comprising forming the composition of any one of claims 1-18, wherein the platelets in the composition have eration storage lesions prior to preparation of the composition. 22. A method for slowing platelet damage comprising forming the composition of any one of claims 1-18. 23. The method according to any one of claims 19-22, wherein the composition is stored in cold storage at 1 °C to 20 °C. 24. The method according to any one of claims 19-22, wherein the composition is stored in cold storage at 1 °C to 15 °C.

. The method according to any one of claims 19-22, wherein the composition is stored in cold storage at 1 °C. 26. The method according to any one of claims 19-25, wherein the composition is stored in cold e for a period of time from 7 to 20 days. 27. The method according to any one of claims 19-25, n the composition is stored in cold storage for a period of time from 10 to 15 days. 28. The method according to any one of claims 19-24, wherein platelet survival is greater than 65% after a storage period of 24 hours at 5 °C. 29. The method according to any one of claims 19-24, wherein platelet survival is greater than 95% after a storage period of 24 hours at 5 °C.

. The method according to any one of claims 19-29, wherein the composition ts platelet damaging activity wherein the platelet damaging activity is selected from: polymerization of F-actin, depolymerization of F-actin, actomyosin contraction, tubulin polymerization, and spectrin anchorage. 31. The method ing to any one of claims 19-30, n the RHOA inhibitor or salt thereof is the only et preserving ingredient. 32. Use of the composition of any one of claims 1-18 in the manufacture of a medicament for administering ets to a subject in need thereof. 1003747565 33. The use of claim 32, wherein the medicament is for infusion of the platelets in the subject in need f. 34. The use of claim 32, wherein the medicament is for transfusion of the platelets into the subject in need thereof, wherein the platelet survival upon transfusion is improved.

. The use according to any one of claims 32-34, n the RHOA inhibitor or a salt thereof is essentially the only platelet preserving ingredient. 36. Use of a RHOA inhibitor selected from: , or a salt thereof, , or a salt thereof, or combinations thereof and one or more platelets in the manufacture of a ment sing a composition according to any one of claims 1-18 for ing platelet survival upon transfusing platelets into a subject, wherein the platelet survival improvement is in comparison with the tage platelet survival for platelets that are not contacted with at least one of the aforementioned RHOA inhibitors. 37. The use of claim 36, wherein the RHOA inhibitor or a salt thereof is the only platelet preserving ingredient. 1003747565 1/11 FIG 1 A B GpIb “Cold receptor” cluster Cold Lipid raftfilopodia Lipid raftlamellipodia Cdc42 Stress fibers Uncapping actin assembly Actomyosin Increase [Ca2+ ] Tubulin depolymerized Loss of spectrin anchorage GpIb clustering FIG 2 Rho GTPase Inhibitors COMPOUNDS TARGET 66 RAC CASIN CDC42 G04 RHOA 1003747571 2/11 FIG 3.

A D B D 7571 3/11 FIG 4.

A D NSC23766 NSC23766 (M) Leu67 c Leu70Pro73Gln74 Arg68Ser71 Tyr64 Lys5 Asp59 Asp76 Thr58Val7Trp56 a Gly54 Asp57 r Rac1 f NSC23766 treated, B p then washed out No 66 s C 0 M NSC23766 50 M NSC23766 s 1003747571 4/11 FIG 5.

A C 7571 /11 FIG 6 37 C RT 0-5 C RT Treatment 0 C-Control 0 C-G04 0 C-Casin 0 C-Casin/ * G04/NSC23766 Platelet ry (%) 100 0 20 40 60 80 Time (hours post-infusion) 1003747571 6/11 FIG 7 8847 7/11 FIG 8 8847 8/11 FIG 9 7571 9/11 FIG 10 7571 /11 FIG 11 7571 11/11 FIG 12 7571

Claims (37)

What is claimed is:

1. A composition, comprising platelets, a RHOA inhibitor selected from: , or a salt f, , or a salt thereof, or combinations thereof; and an acceptable carrier.

2. The composition according to claim 1, wherein the carrier comprises a buffer.

3. The composition according to claim 1 or 2, wherein the carrier is selected from saline, phosphate-buffered saline, tris-buffered saline, Hank’s buffered saline, water, or a combination thereof.

4. The composition according to any one of claims 1-3, wherein the carrier comprises an electrolyte solution.

5. The composition according to any one of claims 1-4, further comprising an additive selected from NaCl, KCl, CaCl2, MgCl2, MgSO4, sodium citrate, citric acid, NaHCO3, sodium phosphate, sodium e, sodium gluconate, glucose, maltose, mannitol, D-ribose, D-glucose, Hank’s solution, HEPES solution, bovine serum albumin, tick anticoagulant peptide and sterile water, a pH adjusting agent, a pH buffering agent, a ty adjusting agent, a stabilizer, a g agent, and combinations f.

6. The composition according to claim 5, wherein the additive comprises a sodium ion, potassium ion, acetate ion, magnesium ion, chloride ion, gluconate ion, glucose, ate ion, citrate ion, calcium ion, and combinations f. 1003747565

7. The composition according to claim 5 or 6, n said additive is t in an amount of from 0.5 mmol/L to 150 mmol/L.

8. The composition according to any one of claims 1-7, wherein the pH of the composition is from 5 to 8.

9. The composition according to any one of claims 1-8, wherein the composition is isotonic.

10. The ition ing to any one of claims 1-9, further comprising a therapeutic agent.

11. The composition according to any one of claims 1-10, wherein the RHOA inhibitor is , or a salt thereof.

12. The composition according to any one of claims 1-10, wherein the RHOA inhibitor is , or salt thereof.

13. The composition according to any one of claims 1-12, wherein the RHOA inhibitor is present at a concentration of at least 10 μM.

14. The composition according to any one of claims 1-13, wherein the RHOA inhibitor is present at a concentration of from 10 μM to 500 μM.

15. The composition according to any one of claims 1-14, wherein the RHOA inhibitor is present at a tration of from 25 μM to 400 μM.

16. The composition ing to any one of claims 1-15, wherein the RHOA inhibitor is present at a concentration of from 50 μM to 300 μM.

17. The composition according to any one of claims 1-16, wherein the RHOA inhibitor is present at a concentration of from 75 μM to 200 μM.

18. The composition according to any one of claims 1-17, wherein the RHOA inhibitor is present at a concentration of from 100 μM to 150 μM.

19. A method for storing platelets comprising forming the composition of any one of claims 1-18, and storing said composition. 1003747565

20. A method of reversing platelet activation comprising forming the composition of any one of claims 1-18, wherein the platelets added to the composition are activated.

21. A method of reversing refrigeration storage lesion in ets comprising forming the composition of any one of claims 1-18, wherein the platelets in the composition have eration storage lesions prior to preparation of the composition.

22. A method for slowing platelet damage comprising forming the composition of any one of claims 1-18.

23. The method according to any one of claims 19-22, wherein the composition is stored in cold storage at 1 °C to 20 °C.

24. The method according to any one of claims 19-22, wherein the composition is stored in cold storage at 1 °C to 15 °C.

25. The method according to any one of claims 19-22, wherein the composition is stored in cold storage at 1 °C.

26. The method according to any one of claims 19-25, wherein the composition is stored in cold e for a period of time from 7 to 20 days.

27. The method according to any one of claims 19-25, n the composition is stored in cold storage for a period of time from 10 to 15 days.

28. The method according to any one of claims 19-24, wherein platelet survival is greater than 65% after a storage period of 24 hours at 5 °C.

29. The method according to any one of claims 19-24, wherein platelet survival is greater than 95% after a storage period of 24 hours at 5 °C.

30. The method according to any one of claims 19-29, wherein the composition ts platelet damaging activity wherein the platelet damaging activity is selected from: polymerization of F-actin, depolymerization of F-actin, actomyosin contraction, tubulin polymerization, and spectrin anchorage.

31. The method ing to any one of claims 19-30, n the RHOA inhibitor or salt thereof is the only et preserving ingredient.

32. Use of the composition of any one of claims 1-18 in the manufacture of a medicament for administering ets to a subject in need thereof. 1003747565

33. The use of claim 32, wherein the medicament is for infusion of the platelets in the subject in need f.

34. The use of claim 32, wherein the medicament is for transfusion of the platelets into the subject in need thereof, wherein the platelet survival upon transfusion is improved.

35. The use according to any one of claims 32-34, n the RHOA inhibitor or a salt thereof is essentially the only platelet preserving ingredient.

36. Use of a RHOA inhibitor selected from: , or a salt thereof, , or a salt thereof, or combinations thereof and one or more platelets in the manufacture of a ment sing a composition according to any one of claims 1-18 for ing platelet survival upon transfusing platelets into a subject, wherein the platelet survival improvement is in comparison with the tage platelet survival for platelets that are not contacted with at least one of the aforementioned RHOA inhibitors.

37. The use of claim 36, wherein the RHOA inhibitor or a salt thereof is the only platelet preserving ingredient.