WO2013103567A2 - Methods of blocking platelet activation during extracorporeal circulation using cangrelor - Google Patents

Description

METHODS OF BLOCKING PLATELET ACTIVATION DURING

EXTRACORPOREAL CIRCULATION USING CANGRELOR

TECHNICAL FIELD

[0001] The invention relates to methods of blocking activation of platelets during medical procedures involving extracorporeal circulation (ECC) of blood, hypothermia, or both.

BACKGROUND OF INVENTION

[0002] Extracorporeal circulation (ECC) is employed in many cardiac surgical procedures to maintain stable circulatory parameters of the patient. In addition, hypothermia ranging between 28°C and 32°C is widely employed as adjunct to ECC to increase the patient's ischemia tolerance. However, platelet activation is a potentially hazardous side effect that can result when either or both of the techniques are used during cardiac procedures.

[0003] For example, contact of blood with artificial ECC surfaces leads to deposition of blood proteins including fibrinogen at the interface. Fibrinogen is the main ligand of the platelet receptor glycoprotein (GP) Ilb/IIIa (CD41/CD61) and mediates binding of platelets in aggregates. As a consequence of contact with blood proteins on ECC surfaces, platelets release their granule contents, form aggregates, adhere to ECC surfaces, and thereby become unavailable for adequate blood coagulation. This consequential dysfunction of platelets can play an important role in severe bleeding 1 and thromboembolic complications associated with ECC.2-^"5 [0004] In addition, shear stress within the ECC circuit induces substantial release of the platelet agonist adenosine diphosphate (ADP) from platelets⁹ and erythrocytes¹⁰ in amounts that are sufficient to induce platelet aggregation.¹¹ Released ADP recruits additional circulating platelets and amplifies the platelet activation cascade. This phenomenon is also called ADP augmentation pathway. 12 ' 13 ADP metabolism is also decreased under hypothermic conditions 13 which is often the manner in which ECC -based procedures are performed. Indeed, it has been recently shown that ADP plays a central role in hypothermia-induced platelet activation and that pharmacological blockade of the platelet ADP receptor P2Y12 inhibits hypothermia-induced platelet activation. 13 ADP-mediated platelet activation is therefore of particular importance during ECC. [0005] Pharmacological inhibition of the GPIIb/IIIa receptor has been proposed to protect platelet function temporarily during ECC.⁶' ⁷ However, this approach, which has been termed "platelet anaesthesia," has not become established in clinical routine because the half-lives of commercially available GPIIb/IIIa receptor blockers still range within 2 hours and may therefore promote bleeding complications. The optimal agent for platelet protection would therefore be a short-acting substance to achieve safe therapy control during hypothermic ECC.

[0006] Therefore, there is a need to establish a pharmacological strategy suitable for platelet protection during medical procedures that include ECC or hypothermia, or both.

BRIEF SUMMARY OF INVENTION

[0007] In a first embodiment, the present invention is directed to a method of inhibiting activation of platelets in the blood of a subject undergoing an extracorporeal circulation (ECC)- based medical procedure, a hypothermia-based medical procedure, or a hypothermic ECC-based medical procedure, wherein the method comprises administering an effective amount of a pharmaceutical composition comprising cangrelor to a subject undergoing such a procedure, thereby inhibiting activation of platelets in the blood of the subject.

[0008] In a second embodiment, the invention is directed to a method of inhibiting platelet granule release in the blood of a subject undergoing an ECC-based medical procedure, a hypothermia-based medical procedure, or a hypothermic ECC-based medical procedure, wherein the method comprises administering an effective amount of a pharmaceutical composition comprising cangrelor to a subject undergoing such a procedure, thereby inhibiting platelet granule release in the blood of the subject

[0009] In a third embodiment, the invention is directed to a method of inhibiting platelet- leukocyte aggregation in the blood of a subject undergoing an ECC-based medical procedure, a hypothermia-based medical procedure, or a hypothermic ECC-based medical procedure, wherein the method comprises administering an effective amount of a pharmaceutical composition comprising cangrelor to a subject undergoing such a procedure, thereby inhibiting platelet- leukocyte aggregation in the blood of the subject.

[0010] In a fourth embodiment, the invention is directed to a method of inhibiting platelet loss from the blood of a subject undergoing an ECC-based medical procedure, a hypothermia- based medical procedure, or a hypothermic ECC-based medical procedure, wherein the method comprises administering an effective amount of a pharmaceutical composition comprising cangrelor to a subject undergoing such a procedure, thereby inhibiting platelet loss from the blood of the subject.

[0011] In each of the embodiments of the present invention, the ECC-based medical procedure is a medical procedure selected from the group consisting of surgery, hemodialysis, hemofiltration, apheresis, extracorporeal membrane oxygenation, and ventricular assist devices. In a particular aspect, the ECC-based medical procedure is cardiopulmonary bypass surgery.

[0012] In particular aspects, the methods of the present invention further comprise administering an anticoagulant to the subject. Suitable anticoagulants include heparin and bivalirudin. The anticoagulant may be administered to the subject prior to, concurrent with, or after the pharmaceutical composition comprising cangrelor is administered to the subject. The anticoagulant may be administered to the subject orally, as an intravenous bolus, as a continuous intravenous infusion, or as an intravenous bolus followed by a continuous intravenous infusion. When the anticoagulant is administered as an intravenous bolus, the bolus may comprise between about 0.1 and 10 mg/kg bivalirudin, such as about 0.75 mg/kg bivalirudin, for example. When the anticoagulant is administered as a continuous intravenous infusion, the infusion may comprises between about 0.5 and 10 mg/kg/h bivalirudin, such as about 1.75 mg/kg/h bivalirudin, for example.

[0013] In particular aspects, the pharmaceutical composition further comprises a

pharmaceutically acceptable carrier or diluent.

[0014] In particular aspects, the pharmaceutical composition is administered to the subject in an oral dosage form, an intravenous dosage form, or both.

[0015] In particular aspects, the pharmaceutical composition is administered to the subject orally, as an intravenous bolus, as a continuous intravenous infusion, or as an intravenous bolus followed by a continuous intravenous infusion.

[0016] In particular aspects, the pharmaceutical composition is administered to the subject prior to beginning the medical procedure as an intravenous bolus and during the procedure as a continuous intravenous infusion.

[0017] In particular aspects, the pharmaceutical composition comprises between about 20 and 40 μg/kg. [0018] In particular aspects, the pharmaceutical composition comprises about 30 μg/kg cangrelor.

[0019] In particular aspects, when the pharmaceutical composition comprising cangrelor is administered as an intravenous bolus, the bolus comprises between about 20 and 40 μg/kg cangrelor.

[0020] In particular aspects, when the pharmaceutical composition comprising cangrelor is administered as an intravenous bolus, the bolus comprises about 30 μg/kg cangrelor.

[0021] In particular aspects, when the pharmaceutical composition comprising cangrelor is administered as a continuous intravenous infusion, the infusion comprises between about 1 and 10 μg/kg/min cangrelor.

[0022] In particular aspects, when the pharmaceutical composition comprising cangrelor is administered as a continuous intravenous infusion, the infusion comprises about 4 μg/kg/min cangrelor.

[0023] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described herein, which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that any conception and specific embodiment disclosed herein may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that any description, figure, example, etc. is provided for the purpose of illustration and description only and is by no means intended to define the limits the invention.

BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 : ECC- and hypothermia-induced platelet granule release, platelet- granulocyte binding and platelet loss inhibited by ADP-receptor blockade ex vivo. Human blood was left untreated ("baseline") or treated ex vivo with PBS ("control"), cangrelor ("cangrelor") or 2-MeSAMP ("2-MeS"). All treated samples were circulated in an ECC model for 30 minutes at 28°C and 37°C, respectively. The percentage of platelets with positive expression of the platelet activation marker P-selectin was evaluated in flow cytometry (A, n=8). β-TG plasma

concentrations (IE ml^"1) were measured using ELISA (B, n=4). To evaluate ADP plasma concentrations (μΜ), a bioluminometric assay was used (C, n=8). The formation of platelet- granulocyte aggregates was measured in flow cytometry (geo mean fluorescence intensity; D, n=5). Platelet counts (xlO 3 μΓ -1 ) were measured in all blood samples (E, n=8). Data are given as means and SEM; mean values of all baseline samples were transformed to 100 % and data measured post-ECC are given as percentages of adjusted baseline values of each temperature group; groups were compared using RM-ANOVA with Bonferroni's multiple comparison test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0025] Figure 2: Platelet interaction with the ECC surface during hypothermic ECC is not inhibited by ADP-receptor blockade ex vivo. Human blood was left untreated ("baseline") or treated ex vivo with either vehicle (PBS) as control ("control"), the P₂Yi₂ antagonist cangrelor ("cangrelor"), or the P₂Y₁₂ antagonist 2-MeSAMP ("2-MeS"). All treated samples were circulated ex vivo in the Chandler loop ECC model for 30 minutes at 28°C and 37°C, respectively. Platelet binding to ECC surface was measured in optical density units employing a specialized ELISA (A; n=5). Binding of an anti-CD42ba-PE mAb (geo mean fluorescence intensity; B; n=8) was measured using flow cytometry. Data are given as means and SEM; mean baseline values were transformed to 100% and data measured post-ECC are given as percentages of adjusted baseline values of each temperature group; groups were compared using RM- ANOVA with Bonferroni's multiple comparison test (*p<0.05).

[0026] Figure 3: Timeline of hypothermic cardiopulmonary bypass (CPB) in pigs. Tl-6 indicate blood sampling time points: Tl: skin incision before median sternotomy; T2: 10 minutes after initiation of cangrelor or placebo infusion and heparin application directly before start of cardiopulmonary bypass (CPB); T3: 45 minutes after start of CPB; T4: directly before end of CPB; T5: 10 minutes after end of CPB; T6: 60 minutes after end of CPB.

[0027] Figure 4: Haematocrit and haemoglobin values as well as platelet counts. Prior to, during and following hypothermic CPB in vehicle ("placebo") and cangrelor-treated (0.075 μg kg^"1 min^"1) pigs changes in haematocrit (A) and haemoglobin concentration (B) were measured. Data are given as means and SEM; in both groups, baseline values (Tl) were compared to T2-T6 of the respective group using RM-ANOVA with Bonferroni's multiple comparison test

(**p<0.01; ***p<0.001; ****p<0.0001; n=5). Platelet counts (C) were measured at all time points (Tl-6). Data are given as means and SEM. For platelet counts a haematocrit correction was performed for values measured after start of CPB (T2-6) to adjust for haemodilution caused by the priming volume of the heart lung machine. Mean baseline values were adjusted to 100% and data measured during and after CPB are given in relation to the adjusted baseline value in each treatment groups; groups were compared using RM-ANOVA with Bonferroni's multiple comparison test. Tl: skin incision before median sternotomy; T2: 10 minutes after initiation of cangrelor or placebo infusion and heparin application directly before start of CPB; T3: 45 minutes after start of CPB; T4: directly before end of CPB; T5: 10 minutes after end of CPB; T6: 60 minutes after end of CPB.

[0028] Figure 5: ADP-receptor blockade with cangrelor prevents an increase of platelet aggregation. Whole blood samples were taken at different time points prior to, during and after hypothermic CPB (Tl-6) from vehicle ("placebo") and cangrelor infused (0.075 μg kg^"1 min^"1) pigs. Platelet-rich plasma was prepared from all blood samples and ADP-induced (20 μΜ) platelet aggregation was analyzed. Data are given as means and SEM. In placebo and cangrelor treated groups baseline values (Tl) were adjusted to 100% and groups were compared using RM-ANOVA with Bonferroni's multiple comparison test (*p<0.05; **p<0.01; ***p<0.001; n=5). Tl: skin incision before median sternotomy; T2: 10 minutes after initiation of cangrelor or placebo infusion and heparin application directly before start of CPB; T3: 45 minutes after start of CPB; T4: directly before end of CPB; T5: 10 minutes after end of CPB; T6: 60 minutes after end of CPB.

[0029] Figure 6: ADP-receptor blockade with cangrelor prevents an increase in fibrinogen binding. Platelet fibrinogen binding was measured in flow cytometry in the vehicle ("placebo") and cangrelor-treated (0.075 μg kg^"1 min^"1) groups at different time points during hypothermic CPB in pigs. Data are given as means and SEM. In both groups baseline values (Tl) were adjusted to 100% and groups were compared using RM-ANOVA with Bonferroni's multiple comparison test (*p<0.05; n=5). Tl: skin incision before median sternotomy; T2: 10 minutes after initiation of cangrelor or placebo infusion and heparin application directly before start of CPB; T3: 45 minutes after start of CPB; T4: directly before end of CPB; T5: 10 minutes after end of CPB; T6: 60 minutes after end of CPB.

DETAILED DESCRIPTION OF THE INVENTION

[0030] Exposure of blood to artificial ECC surfaces during medical procedures can result in platelet dysfunction, which can be followed by deleterious bleeding complications,

thromboembolic events, myocardial infarction and increased mortality. 2 ' 3 ' 6 ' 20 ' 21 This effect is due, in part, to ECC-induced release of the platelet agonist ADP and it can be compounded under hypothermic conditions due, in part, to decreased ADP metabolism. 13 The present invention is directed to methods of protecting platelet function during medical procedures that include ECC or hypothermia, or both, and that address these problems.

[0031] In particular, the present invention is directed to methods that utilize the non- thienopyridine adenosine triphosphate analogue cangrelor, which reversibly binds to and inhibits the P₂Y₁₂ ADP receptor. Cangrelor is direct- acting, reversible, and selective, and it has a short half-life. It is metabolized through dephosphorylation pathways and has a plasma half-life of 3-5 minutes; platelet function returns to normal within 30-60 minutes of drug termination. 23 When given as a bolus plus infusion, it quickly and consistently inhibits platelets to a high degree with normalization of platelet function shortly after discontinuation. A phase 2 trial in patients undergoing PCI demonstrated dose-dependent platelet inhibition similar to that achieved with abciximab, less bleeding time prolongation, and more rapid return to platelet function. 24

[0032] As described herein, the rapid on- and off- set of this agent allows targeted and short- term platelet protection in the setting of ECC and/or hypothermia. Regarding potential bleeding complications, the data show that no influence of cangrelor on haemoglobin values as an indicator for blood loss. These findings indicate that short- acting P₂Yi₂ blockade may be safely performed during medical procedures that include ECC and/or hypothermia.

[0033] The invention is therefore directed to methods of protecting platelets during medical procedures that include ECC or hypothermia, or both. The methods comprise administering an effective amount of a pharmaceutical composition comprising cangrelor to a subject undergoing a medical procedure that includes ECC or hypothermia, or both. In embodiments of the invention, the invention is directed to methods of protecting platelets in the blood of a subject undergoing an ECC -based medical procedure, a hypothermia-based medical procedure or a hypothermic ECC-based medical procedure, where the method comprises administering an effective amount of a pharmaceutical composition comprising cangrelor to a subject undergoing such a procedure, thereby protecting platelets in the blood of the subject. The protection of platelets through the methods of the present invention includes, but is not limited to, inhibiting activation of platelets, inhibiting platelet granule release, inhibiting platelet-leukocyte aggregation (including platelet-granulocyte aggregation), inhibiting platelet aggregation and inhibiting platelet loss from the blood of the subject.

[0034] In one embodiment, the invention is directed to a method of inhibiting activation of platelets in the blood of a subject undergoing an ECC-based medical procedure, a hypothermia- based medical procedure, or a hypothermic ECC-based medical procedure, wherein the method comprises administering an effective amount of a pharmaceutical composition comprising cangrelor to a subject undergoing such a procedure, thereby inhibiting activation of platelets in the blood of the subject.

[0035] In a second embodiment, the present invention is directed to a method of inhibiting platelet granule release in the blood of a subject undergoing an ECC-based medical procedure, a hypothermia-based medical procedure, or a hypothermic ECC-based medical procedure, wherein the method comprises administering an effective amount of a pharmaceutical composition comprising cangrelor to a subject undergoing such a procedure, thereby inhibiting platelet granule release in the blood of the subject.

[0036] In a third embodiment, the present invention is directed to a method of inhibiting platelet-leukocyte aggregation in the blood of a subject undergoing an ECC-based medical procedure, a hypothermia-based medical procedure, or a hypothermic ECC-based medical procedure, wherein the method comprises administering an effective amount of a pharmaceutical composition comprising cangrelor to a subject undergoing such a procedure, thereby inhibiting platelet-leukocyte aggregation in the blood of the subject. In one aspect, the platelet-leukocyte aggregation is platelet-granulocyte aggregation.

[0037] In a fourth embodiment, the present invention is directed to a method of inhibiting platelet loss from the blood of a subject undergoing an ECC-based medical procedure, a hypothermia-based medical procedure, or a hypothermic ECC-based medical procedure, wherein the method comprises administering an effective amount of a pharmaceutical composition comprising cangrelor to a subject undergoing such a procedure, thereby inhibiting platelet loss from the blood of the subject.

[0038] In each of the embodiments of the present invention, the term "cangrelor"

encompasses the compound of Formula I, as well as tautomeric, enantiomeric and diastereomeric forms thereof, and racemix mixtures thereof, other chemically active forms thereof, and pharmaceutically acceptable salts of these compounds, including a tetrasodium salt.

Formula I

These alternative forms and salts, processes for their production, and pharmaceutical

compositions comprising them, are well known in the art and set forth, for example, in U.S. Patent No. 5,721,219. Additional disclosure relevant to the production and use of cangrelor may be found in U.S. Patent Nos. 5,955,447, 6,130,208 and 6,114,313, as well as in U.S. Appln. Publication No. 2006/0270607.

[0039] In each of the methods disclosed herein, the medical procedure may be any which involve extracorporeal circulation (ECC), hypothermia or hypothermic ECC. Medical procedures involving ECC include, but are not limited to, surgery, such as cardiopulmonary bypass surgery, and hemodialysis, such as kidney dialysis. The medical procedures also include hemofiltration, apheresis (including plasmapheresis, erythrocytapheresis, plateletpheresis and leukapheresis), extracorporeal membrane oxygenation (ECMO), and ventricular assist devices.

[0040] The skilled artisan will understand that in many medical procedures that include ECC and/or hypothermia, an anticoagulant is administered to the subject. Each of the methods disclosed herein may therefore include the additional step of administering an anticoagulant in addition to the pharmaceutical composition comprising cangrelor. Suitable anticoagulants include heparin and bivalirudin. The manner and timing of anticoagulant administration will vary and depend on the medical procedure being performed on the subject. Thus, anticoagulant administration may be prior to, concurrent with, or after administration of cangrelor.

[0041] The anticoagulant bivalirudin is a potent, reversible inhibitor of the serine protease thrombin. Thrombin is critical in the thrombotic process, cleaving fibrinogen into fibrin monomers and converting Factor XIII to Factor Xllla, thereby allowing fibrin to develop into a covalently cross-linked framework which leads to clot formation. The chemical structure of bivalirudin is shown in Formula II.

Formula II

[0042] In each of the embodiments of the present invention, the term "bivalirudin" encompasses the compound of Formula II as well as pharmaceutically acceptable salts thereof. Salts of bivalirudin, processes for the production of bivalirudin, and pharmaceutical

compositions comprising bivalirudin, are well known in the art and set forth, for example, in U.S. Patent No. 5,196,404.

[0043] In each of the methods of the present invention where the medical procedure includes hypothermia or hypothermic-ECC, the blood of the subject is subjected to a hypothermic temperature, or the subject is subjected to a hypothermic temperature, or both. As used herein, a hypothermic temperature means a temperature lower than the average core body temperature of the subject. For example, a hypothermic temperature includes, but is not limited to, a temperature of less than about 37°C, 36°C, 35°C, 34°C, 33°C₅ 32°C₅ 31°C₅ 30°C₅ 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21°C, 20°C, 19°C, 18°C, 17°C, 16°C, or 15°C. The hypothermic temperature may also be defined as a temperature of about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C.

[0044] It will be clear to the skilled artisan that medical procedures that include ECC can be conducted under conditions of normothermia, that is, where cooling is not applied to the subject or to the blood of the subject. In certain embodiments, ECC can be conducted under conditions of hyperthermia where the subject or to the blood of the subject is warmed to either maintain the core temperature of the subject or to elevate the core temperature of the subject.

[0045] In each aspect and embodiment of the present invention, cangrelor is formulated and administered to a subject in the form of a pharmaceutical composition comprising the active agent and, optionally, a pharmaceutically acceptable carrier, diluent and/or excipient. Thus, the present invention encompasses a pharmaceutical composition comprising cangrelor, and optionally a pharmaceutically acceptable carrier, diluent and/or excipient. Similarly, an anticoagulant, such as heparin or bivalirudin, may be formulated and administered to a subject in the form of a pharmaceutical composition comprising the active agent and, optionally, a pharmaceutically acceptable carrier, diluent and/or excipient. Thus, the present invention also encompasses a pharmaceutical composition comprising an anticoagulant, such as heparin or bivalirudin, and optionally a pharmaceutically acceptable carrier, diluent and/or excipient. The invention also encompasses pharmaceutical compositions comprising both cangrelor and an anticoagulant, such as heparin or bivalirudin, in the same composition. As used herein, the term "medicament" is synonymous with "pharmaceutical composition."

[0046] Suitable carriers and diluents are well known to those skilled in the art and include saline, such as 0.9% NaCl, buffered saline, dextrose (e.g., 5% dextrose in water), water, Water- for-Injection (WFI), glycerol, ethanol, propylene glycol, polysorbate 80 (Tween-80™), 0.002% polysorbate 80 (Tween-80™), poly(ethylene)glycol 300 and 400 (PEG 300 and 400), PEGylated castor oil (e.g. Cremophor EL), poloxamer 407 and 188, a cyclodextrin or a cyclodextrin derivative (including HPCD ((2-hydroxypropyl)-cyclodextrin) and (2-hydroxyethyl)- cyclodextrin, hydrophilic and hydrophobic carriers, and combinations thereof. Hydrophobic carriers include, for example, fat emulsions, lipids, PEGylated phospholipids, polymer matrices, biocompatible polymers, lipospheres, vesicles, particles, and liposomes. The terms specifically exclude cell culture medium. [0047] Excipients included in the pharmaceutical compositions have different purposes depending, for example on the nature of the drugs, and the mode of administration. Examples of generally used excipients include, without limitation: stabilizing agents, solubilizing agents and surfactants, buffers, antioxidants and preservatives, tonicity agents, bulking agents, lubricating agents, emulsifiers, suspending or viscosity agents, inert diluents, fillers, disintegrating agents, binding agents, wetting agents, lubricating agents, antibacterials, chelating agents, sweeteners, perfuming agents, flavouring agents, coloring agents, administration aids, and combinations thereof.

[0048] The pharmaceutical compositions may contain common carriers and excipients, such as cornstarch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, alginic acid, croscarmellose sodium, and sodium starch glycolate.

[0049] The particular carrier, diluent or excipient used will depend upon the means and purpose for which the active ingredient is being applied.

[0050] The pharmaceutical compositions of the present invention may be formulated, for example, for oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, topical or parenteral administration. Parenteral modes of administration include without limitation, intradermal, subcutaneous (s.c, s.q., sub-Q, Hypo), intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.), intra-arterial, intramedulary, intracardiac, intra- articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids). Any known device useful for parenteral injection or infusion of drug formulations can be used to effect such administration. In noted aspects and embodiments of the present invention, administration of the pharmaceutical compositions is via parenteral administration, preferably intravenous administration, or oral administration.

[0051] In intravenous (IV) administration, a sterile formulation of the pharmaceutical compositions of the present invention and optionally one or more additives, including solubilizers or surfactants, can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion. Intravenous fluids include, without limitation, physiological saline, 0.9% NaCl, phosphate buffered saline, 5% dextrose in water, 0.002% polysorbate 80 (Tween-80™) in water or Ringer's™ solution.

[0052] In intramuscular preparations, a sterile formulation of the pharmaceutical compositions of the present invention can be dissolved and administered in a pharmaceutical diluent such as Water-for-Injection (WFI), physiological saline, 0.9% NaCl or 5% dextrose in water.

[0053] Pharmaceutical compositions comprising cangrelor include pharmaceutical compositions comprising from about 0.1 to about 50 mg/ml of cangrelor. Particular examples of pharmaceutical compositions comprising cangrelor include the following: (i) cangrelor at a concentration of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/mL in 0.9% NaCl, and (ii) cangrelor at a concentration of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/mL in 5% dextrose. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier or diluent.

[0054] Pharmaceutical compositions comprising bivalirudin include pharmaceutical compositions comprising from about 0.1 to about 50 mg/ml of bivalirudin. Particular examples of pharmaceutical compositions comprising bivalirudin include the following: (i) bivalirudin at a concentration of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/mL in 0.9% NaCl, and (ii) bivalirudin at a concentration of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/mL in 5% dextrose. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier or diluent.

[0055] In preferred aspects, the pH of the pharmaceutical compositions of the present invention ranges from about 5 to about 8. In specific examples, the pH is about 5, 5.5, 6, 6.5, 7, 7.5, or 8.

[0056] As used herein, the terms "dose", "dosage", "unit dose", "unit dosage", "effective dose", "effective amount", "protective amount" and related terms refer to physically discrete units that contain a predetermined quantity of cangrelor and/or an anticoagulant calculated to produce a desired protective or therapeutic effect. These terms are synonymous with

therapeutically-effective amounts and amounts sufficient to achieve the stated goals of the methods disclosed herein.

[0057] Particular doses of the pharmaceutical compositions comprising cangrelor and/or anticoagulant of the present invention will vary depending upon the stated goals of the methods, the physical characteristics of the subject, existence of related or unrelated medical conditions, the composition of the formulation, the means used to administer the drug to the subject and the location of administration, whether directly to the subject or to equipment used during the medical procedure. The specific dose for a given subject will generally be set by the judgment of the attending physician.

[0058] When administered as an intravenous (IV) formulation, a pharmaceutical composition comprising cangrelor may be administered as a bolus, as a continuous infusion, or as a bolus followed by a continuous infusion. For example, a bolus may be administered shortly before the start of the medical procedure, followed by continuous infusion during the procedure. When administered as a bolus, a dose of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μg/kg cangrelor, or more, is administered to the subject. In preferred embodiments, between about 20 and 40 μg/kg cangrelor is administered, more preferably about 30 μg/kg. When administered as a continuous infusion, cangrelor may be administered at about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 μg/kg/min, or more, to the subject. In preferred embodiments, between about 1 and 10 μg/kg/min cangrelor is administered, more preferably about 4 μg/kg/min. The skilled artisan will understand that different dosages may be

administered during different time points of the medical procedure being performed on the subject.

[0059] Similarly, when administered as an intravenous (IV) formulation, a pharmaceutical composition comprising bivalirudin may be administered as a bolus, as a continuous infusion, or as a bolus followed by a continuous infusion. When administered as a bolus, a dose of about 0.05, 0.1, 0.25, 0.5, 0.75, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mg/kg bivalirudin, or more, is administered to the subject. In preferred embodiments, between about 0.1 and 10 mg/kg bivalirudin is administered, more preferably about 0.75 mg/kg. When administered as a continuous infusion, bivalirudin may be administered at about 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 mg/kg/h, or more, to the subject. In preferred embodiments, between about 0.5 and 10 mg/kg/h bivalirudin is administered, more preferably about 1.75 mg/kg/h. The skilled artisan will understand that different dosages may be administered during different points of the medical procedure being performed on the subject.

[0060] IV formulations comprising both cangrelor and an anticoagulant, such as bivalirudin, may be prepared using the same guidelines above for IV formulations comprising either cangrelor or an anticoagulant alone.

[0061] In each of the embodiments where the pharmaceutical composition is administered as continuous intravenous infusion, the infusion may continue for the duration of the medical procedure, or may continue for at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340 or 360 minutes, or more. The skilled artisan will understand that the period of time over which the pharmaceutical composition is administered may be shorter or longer than the indicated times due to the particular characteristics of a subject or medical procedure.

[0062] Where the pharmaceutical composition is administered during a medical procedure, the bolus may be administered within about 360, 300, 240, 180, 120, 90, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute prior to the beginning of the medical procedure, such as before the beginning of ECC and/or hypothermia when the medical procedure includes either or both. The bolus may also be administered after the start of the medical procedure, or after the start of ECC or hypothermia. The course of treatment associated with the methods of the present invention will depend on the particular medical procedure being practiced. However, the course of treatment will generally begin prior to the start of the medical procedure and continue for the duration of the procedure. When the medical procedure is stopped, administration of the pharmaceutical composition comprising cangrelor will also generally cease. However, under some circumstances administration of the pharmaceutical composition comprising cangrelor and/or an anticoagulant to the subject may continue for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more hours, for about 1, 2, 3, 4, 5, 6, 7 or more days, for about 1, 2, 3, 4 or more weeks, or for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months.

[0063] As used herein, a "subject" upon which the methods of the present invention may be practiced refers to an animal, such as a mammalian or an avian species, including a human, a non-human primate, a horse, a cow, a sheep, a goat, a dog, and a cat, or other animal of agricultural importance.

Examples

Materials and methods

Ex vivo Chandler loop model

[0064] Blood sampling procedures were approved by the ethics committee of the University of Tubingen, Germany. Blood from non-medicated male subjects, who gave signed informed consent, was collected by venipuncture. Blood was anticoagulated with heparin (final

concentration (fc): 3 I.U. ml^"1). In each experiment, 20 ml of heparinized blood served for measurement of baseline values before circulation in the ECC model. The effect of ECC on platelets was analyzed ex vivo using a well-established closed-loop ECC model (Chandler loop).¹⁴ PVC tubings without additional coating (Jostra, Hirrlingen, Germany) were filled with 20 ml of heparinized whole blood, which was treated with PBS (control), the short-acting P₂Y₁₂ antagonist cangrelor (fc: 1 μΜ; The Medicines Company, Parsippany, NJ) or the irreversible P₂Y₁₂ inhibitor 2-MeSAMP (fc: 100 μΜ; Sigma- Aldrich). Each tubing (tubing length: 50 cm, diameter: 3/8 x 3/32 inch) was closed into a circuit and circulated for 30 minutes in a water bath (at 30 rpm) at 28°C and 37°C, respectively.

Animals and anesthesia

[0065] The study was approved by the animal ethics committee of the University of

Tubingen, Germany. Ten pigs ("German Landrace") weighing 68.1 kg (range 62.1 to 79.9 kg; n=5; placebo group) and 67.5 kg (range 64.5 to 73.6 kg; n=5; cangrelor group) were purchased from a local specific pathogen-free breeding facility. Animals were pre-medicated with a combination of atropine (0.05 mg kg^"1 body weight (b.w.) intramuscular (im); Dr. Franz Kohler Chemie GmbH, Bensheim, Germany) and azaperone (2.0 mg kg^"1 b.w. im; Janssen-Cilag GmbH, Neuss, Germany), followed by midazolam (0.05 mg kg^"1 b.w. im; Ratiopharm GmbH, Ulm, Germany) and xylazine (30-50 μg kg^"1 b.w. im; Eurovet Animal Health BV, Bladel,

Netherlands). An ear vein was cannulated and anesthesia was induced with an intravenous (iv) bolus injection of propofol (2 mg kg^"1; Fresenius Kabi, Bad Homburg, Germany). Animals were connected to a respirator ("Kion", Siemens, Solna, Sweden) to maintain ventilation. Anesthesia was maintained using a combination of the following drugs: propofol (4 mg kg^"1 h^"1 iv; Fresenius Kabi), fentanyl (0.02-0.15 mg kg^"1 h^"1 iv; Ratiopharm GmbH) and midazolam (0.2 mg kg^"1 h^"1 iv; Ratiopharm GmbH).

[0066] At the end of each experiment the animals were euthanized by a lethal dose of potassium chloride (1 mmol kg^"1 iv; B. Braun Melsungen AG, Melsungen, Germany).

Cardiopulmonary bypass

[0067] An arterial catheter ("Leader-Cath", 4,5 Fr, Vygon GmbH&Co.KG, Aachen,

Germany) and a central venous catheter (7 Fr, Arrow International Inc., PA, USA) were inserted in the left carotid artery and the left internal jugular vein, respectively. To prepare connection of the heart-lung machine (HLM) median sternotomy was performed. An aortic cannula (16 Fr, Jostra) was inserted into the ascending aorta and a venous cannula ("MECC-Set", 32/40 Fr, Jostra) was inserted into the right atrium according to standard procedures. Heparin (Ratiopharm GmbH) was administered in a dose of 500 IE kg^"1 to prevent blood clotting inside the HLM and to achieve a desired activated clotting time (ACT) in the range of > 400 seconds. ACT measurements were performed using the Hemochrom Jr. II system (Life Systems, Hamburg, Germany) and were repeated at least each 30 minutes. If necessary, additional heparin boli were administered. Cardiopulmonary bypass (CPB) was established using a heart-lung machine ("S3 System Slimline"; Stockert, Munich, Germany) containing the following elements: 3/8 inch tubing ("Bioline coating", Jostra), bag reservoir (Medistad, Medemblik, Netherlands), and oxygenator (Quadrox, "Bioline coating", Jostra). Blood flow within the CPB circuit was maintained by a roller pump (Stockert). A suction device served to return any blood, which occurred during the procedure inside the pericardial cavity to the animal's circulation. The oxygenator was connected to a heat exchanger to establish desired blood temperatures in the range of 37-28 °C. The priming volume (1 liter) consisted to one third of isotonic saline

(Fresenius Kabi) and to two thirds of 6% hydroxyethyl starch (Fresenius Kabi) and was heparinized (heparin concentration: 5 I.E ml^"1).

[0068] CPB was performed with an average flow of 3 to 3.5 1 min^"1 to establish a mean arterial blood pressure of 60 mmHg. Aortic clamping was not performed and the heart was left beating throughout the whole procedure. After connection of the HLM, the animal was cooled down to 28°C using the CPB circuit. After rewarming to 37°C, weaning from CPB was performed under administration of noradrenaline infusion (maximum value of 0.1 μg kg^"1 min^"1) to establish a minimal mean arterial blood pressure of 60 mmHg. After weaning from CPB protamine was administered in a dosage of 400 IE kg^"1.

Administration of study medication and blood sampling time points

[0069] Cangrelor (The Medicines Company) was dissolved in aqua ad injectionem to achieve a final concentration of 0.05 mg ml^"1. Continuous infusion of weight-adjusted rates of cangrelor (0.075 μg kg^"1 min^"1; "cangrelor-treated group") or placebo (0.0015 ml kg^"1 min^"1 solvent, which equals 0.075 μg kg^"1 min^"1 of the cangrelor solution; "placebo group") was started 10 minutes before initiation of CPB and was terminated when weaning from CPB was performed.

[0070] Blood was sampled at skin incision before sternotomy (Tl), 10 minutes after initiation of cangrelor or placebo infusion and heparin application directly before start of CPB (T2), 45 minutes after start of CPB (T3), directly before end of CPB (T4), 10 minutes after the end of CPB (T5), and 60 minutes after end of CPB (T6).

Flow cytometry

[0071] Human platelets were analyzed directly after blood sampling ("baseline") and after 30 minutes of ex vivo ECC in baseline and circulated samples. Expression of P-selectin, CD42ba as well as activated GPIIb/IIIa on human platelets was analyzed according to previously described methods. 13 ' 15 For detection of platelet-granulocyte binding, 45 μΐ of whole blood was incubated for 20 minutes with 10 μΐ of an anti-CD41-FITC mAb (Beckman Coulter, Marseille, France) and 10 μΐ of an anti-CD15-PE mAb (Beckman Coulter). Afterwards, samples were treated with FACS Lysing Solution (BD Biosciences, Heidelberg, Germany), centrifuged at 200 g for 5 minutes and washed with PBS (Invitrogen GmbH, Karlsruhe, Germany). Samples were fixed with CellFix® (BD Biosciences).

[0072] Fibrinogen binding on porcine platelets was analyzed prior to, during and after hypothermic CPB in both groups. Whole blood was diluted (1:50 in modified Tyrode's buffer) and 50 μΐ were incubated with 10 μΐ of a FITC-labeled chicken anti-fibrinogen antibody

(Immunsystem AB, Uppsala, Sweden) according to previously described methods.¹⁶

Samples were incubated for 20 minutes at 37°C and then fixed with CellFix® (BD Biosciences). Flow cytometry was performed within 6h using a FACScan® cytometer (BD Biosciences) according to standard procedures. 13 ' 15 ' 17

Analysis of platelet adhesion to the ECC surface

[0073] Binding of the platelet- specific protein CD41 is a measure for the deposition of platelets to the ECC surface. CD41-ECC binding was detected as previously described using a specially designed ELISA method.¹⁴ Briefly, ECC tubings were collected, washed and blocked after 30 min of circulation in the ECC-model. Surface-bound CD41 was detected using a primary anti-CD41 antibody (Sigma, Deisenhofen, Germany) and an alkaline phosphatase conjugated secondary antibody (Immunotech/Coulter, Marseille, France). The chromogenic reaction was stopped by addition of NaOH and light absorbance determined with an ELISA reader MR 5000 (Dynatech, Denkendorf, Germany) at 405 nm.

Bioluminometric determination of ADP levels

[0074] ADP plasma levels were determined according to a previously described standard method (ATP bioluminescence assay kit CLS II; Roche, Mannheim, Germany). 13 ' 18

Measurement of plasma β-thromboglobulin (β-TG) and thrombin-antithrombin (TAT) complex concentrations

[0075] Analyses of β-TG as well as TAT complex concentrations were performed as previously described using ELISA kits from Diagnostica Stago (Asnieres, France) for β-TG levels and Enzygnost TAT micro (Siemens, Marburg, Germany), respectively.¹⁴

Platelet aggregometry

[0076] Ex vivo platelet aggregation was measured as previously described 17 in porcine platelet-rich plasma at the indicated sampling time points. In short, platelet-rich plasma was prepared and aggregation was induced by ADP (fc: 20 μΜ) and measured using a 4-channel aggregometer (PAP-4, Biodata Corp, Horsham, PA, USA).

Whole blood count analysis

[0077] Human or porcine whole blood was anti-coagulated using EDTA (EDTA- Monovette®, Sarstedt, Niimbrecht, Germany) and blood count analysis was performed using an ABX Micros 60 blood analyzer (Axon Lab AG, Baden-Dattwil, Switzerland).

Statistical analysis

[0078] Data are depicted as means with standard errors of the mean (SEM). For experiments in the ex vivo ECC model, mean baseline values were transformed to 100% to compare ECC- related effects between the two temperature groups. Data measured after ECC are given in relation to the adjusted baseline values. [0079] To adjust for haemodilution caused by the ECC priming volume a "haematocrit correction" was performed for porcine platelet counts, which were sampled during and after porcine CPB. The "haematocrit correction factor" was calculated by dividing the baseline haematocrit (Tl) by haematocrit values measured during and after CPB (T2-T6). Values measured at T2-T6 were multiplied by the respective "haematocrit correction factor".

Mean baseline (Tl) values of both treatment groups were adjusted to 100%, if indicated. In each group data measured at times points T2 to T6 are given in relation to these adjusted baseline values.

[0080] To analyze differences between data sets repeated measures ANOVA with

Bonferroni's multiple comparison test was performed. A p-value of <0.05 was defined as statistically significant.

Results

[0081] To analyze the effect of P₂Y₁₂ blockade during ECC and hypothermia on platelets, a Chandler loop model was employed to mimic ECC at 37°C and 28°C.

P₂Y₁₂ receptor blockade inhibits platelet granule release, platelet-granulocyte binding, and platelet loss during ex vivo hypothermic ECC

[0082] The release of platelet alpha granules during normothermic and hypothermic ex vivo ECC was determined by evaluating surface expression of P-selectin as well as plasma β-TG concentrations. Investigation of these markers is a reliable method for the detection of platelet degranulation and therefore platelet activation.

[0083] Hypothermic ECC increased levels of the platelet activation markers P-selectin 5.9- fold and β-TG 43.9-fold (p<0.0001; Figure 1A and B). Normothermic ECC caused a 16.2-fold increase of β-TG plasma levels (p<0.01), whereas no significant increase in P-selectin expression was observed. In the hypothermic group, treatment with the P₂Y₁₂ antagonists cangrelor or 2- MeSAMP significantly decreased levels of the platelet activation markers P-selectin and β-TG, respectively (p<0.0001; Figure 1A and B). P₂Y₁₂ blockade decreased β-TG release during normothermic ECC, however without reaching statistical significance.

[0084] To investigate platelet dense granule release during normothermic and hypothermic ECC, plasma ADP concentrations were determined after 30 minutes of ex vivo ECC at 37°C and 28°C. Only hypothermic ECC resulted in a significant 1.5-fold increase (p<0.05) of ADP levels. Cangrelor as well as 2-MeSAMP significantly decreased (p<0.01) ADP concentrations close to values observed at baseline (Figure 1C). The finding that ADP plasma concentrations decreased in the presence of P₂Y₁₂ blockers can be explained by the fact that P₂Y₁₂ blockade inhibits platelet dense granule release, which consequently results in lower ADP plasma concentrations.

As previously shown, ADP is continuously metabolized in plasma and whole blood. 13 ' 22 This phenomenon contributes to explain the observed tendency for lower ADP levels in P₂Y₁₂ blocker- treated samples compared to baseline values.

[0085] Platelet-granulocyte aggregate formation was significantly increased during hypothermic ECC (p<0.01, Figure ID). Treatment with the P₂Y₁₂ antagonists cangrelor significantly reduced this effect (p<0.05).

[0086] Moreover, ECC resulted in a significant loss of circulating platelets (Figure IE; p<0.0001) in vehicle (PBS) treated blood (control), which was further increased by hypothermia (p<0.001). This effect was significantly reduced by both cangrelor (p<0.001) and 2-MeSAMP (p<0.0001). The reason why platelet loss was decreased, but not completely abolished, may be explained by the fact that P₂Y₁₂ blockade inhibits platelet-granulocyte binding and thereby loss of single platelets in cell aggregates, but does not prevent adhesion of platelets to the ECC surface.

Effects of ex vivo ECC and P₂Yi₂ blockade on platelet-ECC binding, CD42ba expression and GPIIb IIIa activation.

[0087] Hypothermic ECC resulted in a significant increase in platelet adhesion to the artificial ECC surface (p<0.05; Figure 2A). P₂Y₁₂ blockers (cangrelor or 2-MeSAMP) had no effect on platelet-ECC interaction. Neither ECC at 37°C or 28°C nor P₂Yi₂ blockade had a significant effect on expression of the platelet von Willebrand factor receptor CD42ba (Figure 2B).¹⁹ Hypothermic ECC resulted in activation of the platelet fibrinogen receptor GPIIb/IIIa (p<0.001). This effect was decreased by P₂Y₁₂ blockade, however without reaching statistical significance (data not shown). P₂Y₁₂ blockade has no effect on thrombin-antithrombin (TAT) complex formation during

ECC

[0088] As an index for thrombin generation indicating activation of the plasma coagulation cascade, TAT complex formation was measured before and after ECC in all groups.

Normo thermic as well as hypothermic ECC induced mild increases in TAT levels. Treatment with P₂Y₁₂ blockers had no effect on this phenomenon (n=4; data not shown).

Effects of short-acting P₂Yi₂ blockade during hypothermic CPB in vivo

Components and duration of CPB

[0089] Whether the inhibitory effects of P₂Y₁₂ blockade on ex vivo ECC- and hypothermia- induced platelet activation would also be valid for the in vivo setting was investigated. The effect of cangrelor during hypothermic CPB in pigs was therefore evaluated. A heart-lung machine consisting of heparin-coated components ("Bioline") was employed, as is used in adult cardiac surgery. CPB was performed with a median duration of 85 minutes (interquartile range (IQR): 22.5 minutes) in the placebo group and 75 minutes (IQR: 15 minutes) in the cangrelor- treated group (p=0.1443 derived from paired t-test). The cangrelor concentration used during

hypothermic CPB was determined in preliminary experiments employing cangrelor in concentrations ranging from 0.025 to 2 μg kg^"1 min^"1 (data not shown). A concentration of 0.075 μg kg^"1 per minute was found to sufficiently inhibit platelet function and allowing rapid reversibility of P₂Y₁₂ blockade at the same time.

[0090] A timeline indicating CPB procedures as well as blood sampling time points is shown in Figure 3.

Effects of CPB on haematocrit values, haemoglobin concentrations and platelet counts

[0091] The CPB priming volume causes haemodilution and hence decreased haematocrit values (Figure 4A). Therefore, 45 minutes after start of CPB haematocrit values were

significantly lower in both treatment groups in comparison to pre-CPB values. Notably, at each time point no differences in haematocrit (Figure 4A) and haemoglobin values (Figure 4B) between both treatment groups were observed. Furthermore, no significant differences in platelet counts (Figure 4C) were measured between control- and cangrelor-treated animals during and after hypothermic CPB. Effects on platelet aggregation

[0092] A major concern in the setting of pharmacological platelet inhibition during CPB is the potential induction of platelet dysfunction and bleeding. Therefore, the duration of platelet inhibition during CPB was evaluated using the short-acting platelet P2Y12 inhibitor cangrelor. ADP-induced platelet aggregation was investigated at different time points prior to, during and after CPB (Figure 5). Cangrelor infusion significantly inhibited ADP-induced platelet aggregation directly after start of infusion prior CPB (T2, p<0.01), after 45 minutes of CPB (T3, p<0.001), and before the end of CPB (T4, p<0.05). Notably, 10 minutes after cangrelor infusion was terminated, platelet function fully returned to values observed in placebo-treated controls.

Effects of P?Y_j blockade on fibrinogen binding to porcine platelets

[0093] Platelet activation is associated with a conformational switch of the GPIIb/IIIa receptor allowing fibrinogen binding, which can be detected in flow cytometry. 17 In order to evaluate potential activating effects of CPB in vivo, binding of fibrinogen to platelets was determined (Figure 6). In placebo-treated animals, an increase in platelet fibrinogen binding was observed during hypothermic CPB reaching statistical significance (p<0.05) 60 minutes after

CPB. This phenomenon was inhibited by cangrelor treatment (p<0.05).

* * * *

[0094] While the invention has been described with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention. The scope of the appended claims is not to be limited to the specific embodiments described.

[0095] All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from

consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims. REFERENCES

1. Rajagopalan S, Mascha E, Na J, Sessler DI. The effects of mild perioperative

hypothermia on blood loss and transfusion requirement. Anesthesiology 2008; 108: 71-7.

2. Weerasinghe A, Taylor KM. The platelet in cardiopulmonary bypass. Ann Thome Surg 1998; 66: 2145-52.

3. Paparella D, Brister SJ, Buchanan MR. Coagulation disorders of cardiopulmonary bypass: a review. Intens Care Med 2004; 30: 1873-81.

4. Xavier RG, White AE, Fox SC, Wilcox RG, Heptinstall S. Enhanced platelet aggregation and activation under conditions of hypothermia. Thromb Haemost 2007; 98: 1266-75.

5. Augoustides JG. Fatal intraoperative thrombosis in contemporary adult thoracic aortic surgery requiring deep hypothermic circulatory arrest: observations from the literature, 1993- 2006. / Thorac Cardiovasc Surg 2007; 134: 1069-70.

6. Hiramatsu Y, Gikakis N, Anderson HL, 3rd, et al. Tirofiban provides "platelet anesthesia" during cardiopulmonary bypass in baboons. / Thorac Cardiovasc Surg 1997; 113: 182-93.

7. Straub A, Schiebold D, Wendel HP, et al. Platelet anaesthesia during extracorporeal circulation: differential effects of GP Ilb/IIIa blockers on platelet activation marker P-selectin expression at hypothermia. Thromb Res 2008; 122: 383-9.

8. Harder S, Klinkhardt U, Alvarez JM. Avoidance of bleeding during surgery in patients receiving anticoagulant and/or antiplatelet therapy: pharmacokinetic and pharmacodynamic considerations. Clin Pharmacokinet 2004; 43: 963-81.

9. Brown CH, 3rd, Leverett LB, Lewis CW, Alfrey CP, Jr., Heliums JD. Morphological, biochemical, and functional changes in human platelets subjected to shear stress. J Lab Clin Med 1975; 86: 462-71.

10. Reimers RC, Sutera SP, Joist JH. Potentiation by red blood cells of shear-induced platelet aggregation: relative importance of chemical and physical mechanisms. Blood 1984; 64: 1200-6.

11. Hall MW, Goodman PD, Solen KA, Mohammad SF. Formation of occlusive platelet aggregates in whole blood caused by low concentrations of ADP. Asaio J 2000; 46: 693-5.

12. Offermanns S. Activation of platelet function through G protein-coupled receptors. Circ Res 2006; 99: 1293-304. 13. Straub A, Krajewski S, Hohmann JD, et al. Evidence of platelet activation at medically used hypothermia and mechanistic data indicating ADP as a key mediator and therapeutic target. Arterioscler Thromb Vase Biol 2011; 31: 1607-16.

14. Straub A, Wendel HP, Dietz K, et al. Selective inhibition of the platelet phosphoinositide 3-kinase pi lObeta as promising new strategy for platelet protection during extracorporeal circulation. Thromb Haemost 2008; 99: 609-15.

15. Straub A, Breuer M, Wendel HP, et al. Critical temperature ranges of hypothermia- induced platelet activation: Possible implications for cooling patients in cardiac surgery. Thromb Haemost 2007; 97: 608-16.

16. Krajewski S, Kurz J, Wendel HP, Straub A. Flow cytometry analysis of porcine platelets: Optimized methods for best results. Platelets 2011: Oct. 13. ePub.

17. Peter K, Kohler B, Straub A, et al. Flow cytometric monitoring of glycoprotein Ilb/IIIa blockade and platelet function in patients with acute myocardial infarction receiving reteplase, abciximab, and ticlopidine: continuous platelet inhibition by the combination of abciximab and ticlopidine. Circulation 2000; 102: 1490-6.

18. Gorman MW, Marble DR, Ogimoto K, Feigl EO. Measurement of adenine nucleotides in plasma. Luminescence 2003; 18: 173-81.

19. Bergmeier W, Chauhan AK, Wagner DD. Glycoprotein Ibalpha and von Willebrand factor in primary platelet adhesion and thrombus formation: lessons from mutant mice. Thromb Haemost 2008; 99: 264-70.

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subpopulation. Am J Hematol 2002; 69: 45-55.

21. Gawaz M. Role of platelets in coronary thrombosis and reperfusion of ischemic myocardium. Cardiovasc Res 2004; 61: 498-511.

22. Coade SB, Pearson JD. Metabolism of adenine nucleotides in human blood. Circ Res 1989; 65: 531-7.

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24. Greenbaum A.B. et al., Am Heart J 151:689.el-10 (2006).

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting activation of platelets in the blood of a subject undergoing an extracorporeal circulation (ECC)-based medical procedure, a hypothermia-based medical procedure, or a hypothermic ECC-based medical procedure, said method comprising

administering an effective amount of a pharmaceutical composition comprising cangrelor to a subject undergoing such a procedure, thereby inhibiting activation of platelets in the blood of the subject.

2. A method of inhibiting platelet granule release in the blood of a subject undergoing an ECC-based medical procedure, a hypothermia-based medical procedure, or a hypothermic ECC-based medical procedure, said method comprising administering an effective amount of a pharmaceutical composition comprising cangrelor to a subject undergoing such a procedure, thereby inhibiting platelet granule release in the blood of the subject

3. A method of inhibiting platelet-leukocyte aggregation in the blood of a subject undergoing an ECC-based medical procedure, a hypothermia-based medical procedure, or a hypothermic ECC-based medical procedure, said method comprising administering an effective amount of a pharmaceutical composition comprising cangrelor to a subject undergoing such a procedure, thereby inhibiting platelet-leukocyte aggregation in the blood of the subject.

4. A method of inhibiting platelet loss from the blood of a subject undergoing an ECC-based medical procedure, a hypothermia-based medical procedure, or a hypothermic ECC- based medical procedure, said method comprising administering an effective amount of a pharmaceutical composition comprising cangrelor to a subject undergoing such a procedure, thereby inhibiting platelet loss from the blood of the subject.

5. The method of any one of claims 1-4, wherein the ECC-based medical procedure is a medical procedure selected from the group consisting of surgery, hemodialysis,

hemofiltration, apheresis, extracorporeal membrane oxygenation, and ventricular assist devices.

6. The method of claim 5, wherein the ECC-based medical procedure is

cardiopulmonary bypass surgery.

7. The method of any one of claims 1-6, further comprising administering an anticoagulant to the subject.

8. The method of claim 7, wherein the anticoagulant is heparin or bivalirudin.

9. The method of claim 7, wherein the anticoagulant is administered to the subject prior to, concurrent with, or after the pharmaceutical composition comprising cangrelor is administered to the subject.

10. The method of any one of claims 7-9, wherein the anticoagulant is administered to the subject orally, as an intravenous bolus, as a continuous intravenous infusion, or as an intravenous bolus followed by a continuous intravenous infusion.

11. The method of any one of claims 1-6, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or diluent.

12. The method of any one of claims 1-6, wherein the pharmaceutical composition is administered to the subject in an oral dosage form, an intravenous dosage form, or both.

13. The method of any one of claims 1-6, wherein the pharmaceutical composition is administered to the subject orally, as an intravenous bolus, as a continuous intravenous infusion, or as an intravenous bolus followed by a continuous intravenous infusion.

14. The method of any one of claims 1-6, wherein the pharmaceutical composition is administered to the subject prior to beginning the medical procedure as an intravenous bolus and during the procedure as a continuous intravenous infusion.

15. The method of any one of claims 1-6, wherein the pharmaceutical composition comprises between about 20 and 40 μg/kg cangrelor.

16. The method of any one of claims 1-6, wherein the pharmaceutical composition comprises about 30 μg/kg cangrelor.

17. The method of claim 13 or 14, wherein when the pharmaceutical composition comprising cangrelor is administered as an intravenous bolus, the bolus comprises between about 20 and 40 μg/kg cangrelor.

18. The method of claim 13 or 14, wherein when the pharmaceutical composition comprising cangrelor is administered as an intravenous bolus, the bolus comprises about 30 μg/kg cangrelor.

19. The method of claim 13 or 14, wherein when the pharmaceutical composition comprising cangrelor is administered as a continuous intravenous infusion, the infusion comprises between about 1 and 10 μg/kg/min cangrelor.

20. The method of claim 13 or 14, wherein when the pharmaceutical composition comprising cangrelor is administered as a continuous intravenous infusion, the infusion comprises about 4 μg/kg/min cangrelor.

21. The method of claim 10, wherein when the anticoagulant is administered as an intravenous bolus, the bolus comprises between about 0.1 and 10 mg/kg bivalirudin.

22. The method of claim 10, wherein when the anticoagulant is administered as an intravenous bolus, the bolus comprises about 0.75 mg/kg bivalirudin.

23. The method of claim 10, wherein when the anticoagulant is administered as a continuous intravenous infusion, the infusion comprises between about 0.5 and 10 mg/kg/h bivalirudin.

24. The method of claim 10, wherein when the anticoagulant is administered as a continuous intravenous infusion, the infusion comprises about 1.75 mg/kg/h bivalirudin.