Combination therapy

Abstract

To provide effective and safe treatment methods that may be used in treating ischemic events such as acute ischemic stroke (AIS) and myocardial infarction, and safe treatment methods for achieving prevention of relapsing ischemic events.SOLUTION: Ischemic events in a patient, such as ST-segment elevation myocardial infarction and acute ischemic stroke, are treated by administering a recombinant apyrase protein in conjunction with a P2Y12 inhibitor. The P2Y12 inhibitor is selected from a list consisting of ticagrelor, clopidogrel, ticlopidine, prasugrel and cangrelor.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a recombinant apyrase protein 12 The present invention relates to a method for treating an ischemic event in a patient, particularly an ST-segment elevation myocardial infarction and acute ischemic stroke, by administering the same in combination with an inhibitor. [Background technology]

[0002] Myocardial infarction (MI) is a leading cause of hospitalization and death worldwide (Non-Patent Document 1). If left untreated, MI leads to irreversible damage to the myocardium due to lack of blood flow (ischemia) and therefore lack of oxygen. Thus, the primary goal of treatment of MI is to promote the restoration of normal coronary blood flow with the aim of reducing myocardial damage through reperfusion therapy. Reperfusion therapy typically involves the use of therapies such as thrombolytic therapy to increase blood flow and reduce thrombosis, and is combined with surgical and non-surgical procedures such as percutaneous coronary intervention (PCI). Early reperfusion and PCI are preferred and have been associated with improved outcomes, and guidelines suggest that PCI should be performed within 12 hours of the onset of MI symptoms (Non-Patent Document 2).

[0003] Cerebrovascular disease refers to conditions that affect the circulation of blood to the brain and includes acute ischemic stroke (AIS) and transient ischemic attack (TIA). Cerebrovascular disease is the leading cause of death and severe long-term disability worldwide (Non-Patent Document 3 and Non-Patent Document 4). As with MI, the primary goal is to promote the restoration of normal cerebral blood flow with the goal of reducing brain tissue damage through reperfusion therapy. Reperfusion therapy involves the use of treatments such as thrombolytic therapy to increase blood flow and mitigate thrombosis, combined with interventional techniques such as thrombectomy. Early reperfusion is favorable and has been linked to improved outcomes, and guidelines recommend that thrombolytic reperfusion be performed within 4 to 5 hours of symptom onset in eligible patients, and thrombectomy be performed within 6 hours (Non-Patent Document 5).

[0004] To combat thrombus formation and prevent secondary thrombotic events, several therapeutic strategies are available and fall into two classes: protein-based therapeutics and small molecule therapeutics. Examples of small molecule therapeutics include P2Y inhibitors such as clopidogrel, ticagrelor, prasugrel, and cangrelor. 12 These include receptor inhibitors, which are known for their ability to inhibit platelets and prevent thrombosis. The efficacy of both clopidogrel and ticagrelor in reducing cardiovascular morbidity and mortality in secondary prevention has been demonstrated in clinical trials, and both drugs are approved for use in the prevention of thrombotic events. For example, the UK National Institute for Health and Care Excellence (NICE) recommends the combination of ticagrelor and low-dose aspirin for up to 12 months as treatment for adults with acute coronary syndrome (ACS). However, these P2Y 12 Receptor inhibitors are associated with bleeding risks, so care must be taken to ensure that patients receive this treatment in accordance with the prescribing information and current guidelines. The antiplatelet agent with the highest level of evidence in current treatment guidelines to reduce the risk of subsequent stroke after non-myoembolic AIS or TIA is aspirin, but clopidogrel is also used. Recently, guidelines in the United States (6) and China (7) have recommended the combination of aspirin and clopidogrel within 24 hours of AIS and TIA, and ticagrelor and aspirin have also shown benefits over aspirin alone (8).

[0005] Recent studies have explored the use of recombinant apyrase as a protein-based therapeutic. Apyrase (ecto-ATP diphosphohydrolase) constitutes a group of enzymes that catalyze the metabolism of ATP to ADP and ADP to AMP. In vivo, AMP, generated by apyrase-induced hydrolysis of ATP and ADP, is converted to adenosine by the ubiquitously expressed extracellular CD73 / ecto-5'-nucleotidase. The first known human apyrase, CD39, was originally identified as a cell surface protein on activated lymphocytes and endothelial cells, and various in vitro and in vivo studies have shown that apyrase can maintain vascular integrity and physiologically suppress inflammation and thrombosis ((Non-Patent Document 9)).

[0006] Apyrase inhibits the function of ATP and ADP by reducing the levels of ATP and ADP and all three platelet P2 receptors (P2X1, P2Y1, and P2Y 12 ), thus acting to inhibit platelet activation and mobilization. 12 In contrast to small molecule inhibitors of receptors, the use of apyrase to deplete ADP and ATP levels does not result in increased bleeding levels in preclinical models. In addition to its antiplatelet effect, apyrase also has cardioprotective and brain tissue protective effects, which are understood to be mediated by i) depletion of proinflammatory ATP and ADP molecules; and ii) increased levels of anti-inflammatory adenosine produced through downstream apyrase action. Further information on the mechanism of action of endogenous apyrase is provided in Figure 1 of (Non-Patent Document 10).

[0007] (Non-Patent Document 10) reports the design and production of a recombinant optimized form of soluble CD39L3, a member of the human CD39 family. The resulting recombinant protein is referred to as "APT102" or "AZD3366". The authors report that this recombinant protein exhibits 4-fold higher adenosine diphosphatase activity and 50-fold longer plasma half-life than native apyrase, and that treatment with APT102 in an animal model reduced infarct size without increasing bleeding time. The corresponding treatment with clopidogrel alone was not cardioprotective in this model, while combined APT102 and clopidogrel treatment did not alter the protective efficacy seen with APT102 alone (Figure 6 in (Non-Patent Document 10)). (Non-Patent Document 11) reports that combined tissue plasminogen activator (tPA) and APT102 reduced mortality and infarct volume compared to tPA or APT102 alone. The data also suggest that APT102 may safely extend the therapeutic window of tPA-mediated reperfusion without increasing hemorrhagic transformation.

[0008] P2Y such as ticagrelor 12 Despite optimal antiplatelet and antithrombotic therapy with receptor inhibitors, the risk of a new myocardial infarction event remains about 10% per year, and increasing the intensity of these treatments increases the bleeding risk without improving efficacy (Non-Patent Document 12; Non-Patent Document 13). Recent studies in AIS / TIA patients exploring the benefits of dual antiplatelet therapy with clopidogrel or ticagrelor and aspirin suggest a residual risk of new ischemic stroke events of 5-8% in the acute phase (1-3 months after the event) (Non-Patent Document 14; Non-Patent Document 15; Non-Patent Document 8). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 7,247,300B1 [Patent Document 2] European Patent No. 2133430B1 [Patent Document 3] European Patent No. 2523971B1 [Patent Document 4] International Publication No. 2008 / 024045 Brochure [Patent Document 5] International Publication No. 2017 / 182589 Brochure [Non-patent literature]

[0010] [Non-Patent Document 1] Asaria et al. (2017) [Non-Patent Document 2] Ibanez et al. (2018) [Non-Patent Document 3] Roth et al. (2018) [Non-Patent Document 4] Kyu et al. (2018) [Non-Patent Document 5] Powers et al. (2018) [Non-Patent Document 6] Power et al. (2018) [Non-Patent Document 7] Wang et al. (2017) [Non-Patent Document 8] Johnston et al. (2020) [Non-Patent Document 9] Robson et al. (2005) [Non-Patent Document 10] Moeckel et al. (2014) [Non-Patent Document 11] Tan et al. (2014) [Non-Patent Document 12] Wallentin et al. (2009) [Non-Patent Document 13] Jernberg et al. (2015) [Non-Patent Document 14] Wang et al. (2013)

Non-Patent Document 15

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Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Non-Patent Document 21

Non-Patent Document 22

Non-Patent Document 23

Non-Patent Document 24

Non-Patent Document 25

Non-Patent Document 26

Summary of the Invention

Problems to be Solved by the Invention

[0011] Thus, there remains a need for effective and safe therapies that can be used to treat ischemic events such as myocardial infarction and acute ischemic stroke (AIS), as well as safe therapies that can prevent recurrent ischemic events. The present invention has been devised in light of the above considerations. [Means for solving the problem]

[0012] The present inventors have 12 Administration of recombinant apyrase protein in combination with an inhibitor of P2Y 12 It has been determined that the combination therapy provides improved cardioprotective benefits compared to the individual use of the inhibitor alone.This suggests that the combination therapy can be used as an effective treatment for ischemic events in the heart, such as myocardial infarction (e.g., ST-segment elevation myocardial infarction), and ischemic events in the brain, such as acute ischemic stroke, for example, to prevent or ameliorate the damage caused by ischemic events (e.g., heart damage resulting from myocardial infarction, or brain damage resulting from acute ischemic stroke), and to prevent recurrent ischemic events.

[0013] In particular, the present inventors have 12 We have demonstrated that administration of recombinant apyrase protein (e.g., AZD3366) in combination with a P2Y inhibitor (e.g., ticagrelor) is surprisingly effective in reducing infarct size and improving cardiac function in animal models of myocardial infarction (MI). 12 This is contrary to the results reported in Figure 6 of (Non-Patent Document 10), which reports that the use of the inhibitor does not affect the cardioprotective effect observed with AZD3366 alone.

[0014] Furthermore, without wishing to be bound by theory, the improved cardioprotective effects provided by the combined treatment may be due to the P2Y receptor agonist, which leads to increased extracellular adenosine levels. 12The cardioprotective effect of AZD3366 is reported to also involve an increase in the levels of extracellular adenosine, suggesting that the two drugs act on the same pathway and modulate the levels of the same end product (P2Y 12 It was unexpected that the combination of a pyridoxalib and apyrase inhibitor (a pyridoxalib and a recombinant apyrase protein) induced a cardioprotective effect greater than that observed with the use of either agent alone.

[0015] It was also determined that administration of AZD3366 in combination with ticagrelor did not significantly increase bleeding levels in animal models compared to ticagrelor alone. This suggests that, for example, P2Y 12 P2Y to treat conditions for which inhibitors are already approved 12 This is important because it suggests that there is no significant additional risk in terms of increased bleeding with the use of combination therapy compared with the use of either inhibitor alone.

[0016] These results are in contrast to observations from previous clinical trials, where increasing antiplatelet activity or increasing antithrombotic activity on the same or complementary pathways was consistently observed to increase the risk of bleeding in combination with any beneficial efficacy effects.

[0017] Thus, the proposed combination represents the first unexpected example where net clinical benefit can be improved by targeting and enhancing effects arising from the same biological pathway in a setting such as MI.

[0018] Thus, one aspect of the present disclosure is to incorporate a recombinant apyrase protein into a P2Y 12 The present invention provides a method for treating an ischemic event in a patient comprising administering in combination with an inhibitor.

[0019] In another aspect, the disclosure provides a recombinant apyrase protein for use in a method of treating an ischemic event in a patient comprising administering to the patient a recombinant apyrase protein, the recombinant apyrase protein inhibiting P2Y 12It is administered in combination with an inhibitor.

[0020] In a further aspect, the present disclosure provides a recombinant apyrase protein for use in the manufacture of a P2Y 12 US20130232633A1 - Recombinant apyrase protein and a P2Y inhibitor for use in a method of treating an ischemic event in a patient comprising administering the protein to the patient in combination with a - 12 An inhibitor is provided.

[0021] In yet another aspect, the present disclosure provides a P2Y 12 - Patent application title: P2Y inhibitors for use in a method of treating an ischemic event in a patient comprising administering to the patient 12 Provides inhibitors of P2Y 12 The inhibitor is administered in combination with a recombinant apyrase protein.

[0022] In another aspect, the present disclosure provides a method for treating an ischemic event in a patient, comprising administering to a patient a recombinant apyrase protein and / or a P2Y 12 The present invention provides a method for treating amyloidosis by administering a recombinant apyrase protein to a mammalian animal, comprising administering to the mammal amyloidosis patient a therapeutically effective amount of the recombinant apyrase protein. 12 This includes administering to a patient in combination with an inhibitor.

[0023] P2Y 12 The inhibitor may be selected from the group consisting of ticagrelor, clopidogrel, ticlopidine, prasugrel, and cangrelor. 12 The inhibitor is selected from the group consisting of ticagrelor, clopidogrel, and prasugrel. 12 The inhibitor is ticagrelor or clopidogrel. Even more preferably, the P2Y 12 The inhibitor is ticagrelor.

[0024] The recombinant apyrase protein may be recombinant human CD39L3 apyrase, optionally a soluble recombinant human CD39L3 apyrase. The apyrase may be a soluble CD39L3 or an ADPase-enhanced apyrase as described in any one of U.S. Patent No. 5,399,233, U.S. Patent No. 5,399,233, and U.S. Patent No. 5,399,233. An exemplary recombinant human CD39L3 apyrase is provided herein as SEQ ID NO:2.

[0025] In some embodiments, the recombinant apyrase protein is a soluble CD39L3 protein comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to positions 49-485 of SEQ ID NO: 1, and the recombinant apyrase protein retains ADPase activity and ATPase activity. In some embodiments, the recombinant apyrase protein comprises one or more modifications (e.g., amino acid substitutions) compared to a reference apyrase, which may be a wild-type apyrase (e.g., an apyrase having the amino acid sequence set forth in SEQ ID NO: 1) or a soluble apyrase (e.g., a soluble apyrase having the amino acid sequence set forth in SEQ ID NO: 1).

[0026] These modifications may result in increased ADPase activity compared to the reference apyrase, or may result in the same ADPase activity as the reference apyrase combined with decreased ATPase activity compared to the reference apyrase. In some embodiments, one or more modifications may include or consist of substitutions at positions 67 and 69, positions numbered according to SEQ ID NO: 1. The substitution at position 67 may be glycine and the substitution at position 69 may be arginine, or the substitution at position 67 may be alanine and the substitution at position 69 may be arginine. Preferably, the substitution at position 67 is glycine and the substitution at position 69 is arginine.

[0027] In some embodiments, the recombinant apyrase protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:2 (AZD3366). In particularly exemplary embodiments, the recombinant apyrase protein comprises the amino acid sequence set forth in SEQ ID NO:2.

[0028] In some cases, when an ischemic event occurs, patients are already suffering from P2Y 12 Patients may be receiving treatment with a P2Y inhibitor, such as, for example, ticagrelor twice daily, when the ischemic event occurs. For example, the prescribing information for ticagrelor in the treatment of acute coronary syndrome (ACS) states that treatment is initiated with a "loading" dose of 180 mg, followed by a "maintenance" dose of 90 mg twice daily for the first year after the ACS event, and then 60 mg twice daily after one year. Thus, a patient may have experienced a previous ischemic event in the past two years, and may be receiving treatment with a P2Y inhibitor, such as, for example, ticagrelor twice daily, when the ischemic event occurs, as is treated herein. 12 Patients may currently be undergoing long-term treatment with inhibitors. As noted above, despite optimal antiplatelet and antithrombotic therapy, there remains a risk of developing a new ischemic event.

[0029] For example, the patient may receive P2Y 72 hours, or 36 hours, or 48 hours, or 24 hours, or 20 hours, or 16 hours, or 12 hours, or 8 hours, or 6 hours prior to carrying out the method. 12 The patient may be receiving (administered) an inhibitor of P2Y 12 In some embodiments, such as when the inhibitor is ticagrelor, the patient is administered P2Y 12 Are receiving an inhibitor (i.e., as part of a maintenance dose given twice daily).

[0030] In other cases, patients are currently 12 Patients may not have been treated with P2Y inhibitors. Such patients are described as “naive” patients.12 Inhibitor-naïve situations and when patients have previously received P2Y 12 This includes situations where the inhibitor has been administered, but not for at least 24 hours, 48 ​​hours, 72 hours, or one week prior to carrying out the method.

[0031] In a preferred embodiment, the method further comprises administering to the patient (e.g., a P2Y 12 Patients receiving treatment with P2Y inhibitors or other therapies, or naïve patients 12 Typically, the method involves administering a P2Y inhibitor to a patient in separate formulations that are administered separately, e.g., simultaneously or sequentially. 12 The method includes administering an inhibitor and a recombinant apyrase protein to a patient. A skilled physician or other skilled medical personnel can determine the optimal mode of administration of each therapeutic agent to a patient.

[0032] When sequential administration is used, recombinant apyrase protein and P2Y 12 The inhibitors are preferably administered within 24 hours, 12 hours, 1 hour, or more preferably within 30 minutes of each other. In some embodiments, the recombinant apyrase protein is administered first, and the P2Y 12 This is followed by sequential administration of the inhibitor. 12 The inhibitor is administered first, followed by the sequential administration of recombinant apyrase protein.

[0033] In some embodiments, the ischemic event being treated is an acute coronary syndrome, including ST-segment elevation myocardial infarction (STEMI), non-ST-segment elevation myocardial infarction (NSTEMI), and unstable angina. In a preferred embodiment, the ischemic event being treated is an ST-segment elevation myocardial infarction (STEMI) in a patient.

[0034] As demonstrated herein, recombinant apyrase protein (e.g., AZD3366) and P2Y 12Coadministration of inhibitors (e.g., ticagrelor) provided cardioprotection in preclinical animal models of myocardial infarction, which was superior to placebo and P2Y 12 Compared with treatment with P2Y inhibitors alone, P2Y inhibitors reduced infarct size and improved cardiac function. Thus, early reperfusion following myocardial infarction is favorable and is associated with improved outcome. Thus, the role of P2Y inhibitors in patients with acute coronary syndromes (e.g., STEMI) may be of benefit. 12 Administration of the apyrase protein in combination with an inhibitor should preferably be performed as soon as possible following the onset of an acute coronary syndrome, preferably as close as possible to the time the patient undergoes percutaneous coronary intervention (PCI), such as before the patient undergoes percutaneous coronary intervention (PCI).

[0035] In other embodiments, the ischemic event being treated is an acute ischemic stroke. Every year, 100 new patients per 70,000 population will develop an ischemic stroke. Without treatment, 55 patients will die or require care within one year. Most of the 100 patients will have a mild or transient stroke and only antiplatelet drugs will be administered to reduce recurrence. Approximately 25-35 patients will undergo reperfusion therapy, thereby saving 5-6 patients from death or needing care and increasing the number of patients without disability. Thus, reducing the morbidity and mortality associated with acute ischemic stroke (AIS) is an unmet clinical need.

[0036] Preclinical and clinical studies have demonstrated the potential of P2Y agonists to treat and / or prevent stroke following an ischemic stroke event. 12 The use of P2Y inhibitors and aspirin has been evaluated. Furthermore, preclinical data demonstrate that recombinant apyrase protein AZD3366 can be used to promote reperfusion, reduce reocclusion, and reduce intracerebral hemorrhage in animal models of ischemic stroke (Non-Patent Document 16; Non-Patent Document 11). Thus, the use of AZD3366 and P2Y 12It has been recognized that the beneficial results in the preclinical myocardial infarction animal model described herein associated with the combined administration of inhibitors may also be beneficial in the treatment of ischemic stroke. For example, combined therapy may reduce infarct size and / or reduce brain damage in patients who have suffered an ischemic stroke, an effect that may be in addition to preventing recurrent ischemic events, by inhibiting the activation of P2Y 12 This may be achieved without significantly increasing the risk of bleeding compared with the inhibitor alone.

[0037] As described in further detail below, oral P2Y 12 The onset of action of inhibitors is typically several hours, which is consistent with the increased upregulation of P2Y after an ischemic event (e.g., acute coronary syndrome). 12 Even if the inhibitor is administered very early, this means that there is a period of several hours during which the patient is not protected from associated damage (e.g., cardiac damage resulting from myocardial infarction) or the risk of recurrent ischemic events. In contrast, the recombinant apyrase protein AZD3366 has been shown in preclinical models to exhibit high levels of activity within minutes of intravenous administration and to maintain this activity for over 24 hours, depending on the dose (Non-Patent Document 17). Thus, early administration of recombinant apyrase protein can provide sufficient levels of P2Y 12 It has been recognized that the antithrombotic and tissue protective effects are provided in the early stages of myocardial infarction or AIS before the inhibitor becomes bioavailable in the patient's bloodstream to primarily exert its antithrombotic effects.

[0038] Thus, in some embodiments, the recombinant apyrase protein is capable of activating P2Y β-amyloid ... 12 In some embodiments, the method comprises administering to the patient a P2Y 12The method further comprises administering to the patient a surgical or non-surgical reperfusion therapy (e.g., PCI, thrombectomy, or thrombolytic therapy) less than 48 hours, less than 24 hours, less than 12 hours, less than 6 hours, less than 3 hours, less than 2 hours, or less than 1 hour following administration of the recombinant apyrase protein in combination with the inhibitor. In certain embodiments, the ischemic event is a myocardial infarction (e.g., STEMI) and the reperfusion therapy is PCI. In certain embodiments, the ischemic event is an AIS and the reperfusion therapy is thrombectomy. In certain embodiments, the ischemic event is a myocardial infarction (e.g., STEMI) or AIS and the reperfusion therapy is thrombolytic therapy (e.g., tissue plasminogen activator). In certain embodiments, the recombinant apyrase protein and P2Y 12 The inhibitor remains bioavailable in the patient's bloodstream during surgical or non-surgical reperfusion therapy (eg, PCI, thrombectomy, or thrombolytic therapy).

[0039] In some embodiments, the recombinant apyrase protein is administered in a dose of 10-1000 mg.

[0040] In some embodiments, the P2Y 12 The inhibitor is ticagrelor, which is administered at a dose of 60 to 200 mg, such as 60 mg, 90 mg, 120 mg, or 180 mg. In some embodiments, ticagrelor is administered at its recommended loading dose of 180 mg.

[0041] In some embodiments, the P2Y 12 The inhibitor is clopidogrel and is administered at a dose of 75-600 mg, e.g., 75 mg, 150 mg, 225 mg, 300 mg, 375 mg, 450 mg, 525 mg, or 600 mg, hi some embodiments, clopidogrel is administered at its recommended loading dose of 300 mg or 600 mg.

[0042] In some embodiments, the P2Y 12The inhibitor is ticlopidine and is administered at a dose of 250-500 mg, for example, 250 mg or 500 mg, hi some embodiments, ticlopidine is administered at its recommended loading dose of 500 mg.

[0043] In some embodiments, the P2Y 12 The inhibitor is prasugrel and is administered at a dose of 5 to 60 mg, e.g., 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, or 60 mg, hi some embodiments, prasugrel is administered at its recommended loading dose of 60 mg.

[0044] In some embodiments, the P2Y 12 The inhibitor is cangrelor, administered at a dose of 30 μg / kg intravenous bolus followed by 4 μg / kg intravenous infusion every minute.

[0045] In some embodiments, the method further comprises, for example, determining whether the recombinant apyrase protein is a P2Y 12 In these embodiments, the method further comprises administering to the patient aspirin, such that the inhibitor and aspirin are administered in combination. In these embodiments, the method typically comprises administering to the patient a recombinant apyrase protein, a P2Y inhibitor, and a P2Y agonist, as separate formulations that are administered separately, e.g., simultaneously or sequentially. 12 The method includes administering an inhibitor and aspirin, which may be administered in a dose of 50 to 325 mg.

[0046] In some embodiments, the method further comprises, for example, determining whether the recombinant apyrase protein is a P2Y 12 The method further comprises administering to the patient a thrombolytic therapy (e.g., tPA) to be administered in combination with the inhibitor and the thrombolytic therapy. In these embodiments, the method typically comprises administering to the patient a recombinant apyrase protein, a P2Y inhibitor, or ... 12 Inhibitors, and thrombolytic therapy.

[0047] Another aspect of the present invention is a method for producing a recombinant apyrase protein as described herein, comprising the steps of: 12 and an inhibitor, optionally the kit further comprises aspirin.

[0048] The present invention includes combinations of the described aspects and preferred features, except where such combinations are clearly contraindicated or explicitly avoided.

[0049] Reference will now be made to the accompanying drawings, in which embodiments and experiments illustrating the principles of the present invention will be discussed. [Brief description of the drawings]

[0050] [Figure 1] Illustrates the experimental protocol used to determine whether administration of recombinant apyrase protein AZD3366 provided additional benefits compared to administration of ticagrelor alone in terms of reducing infarct size and improving cardiac function in a porcine animal model of myocardial infarction. BSL = baseline; MI = myocardial infarction; Sac = sacrifice. [Diagram 2] Results from a mouse tail bleeding experiment showing the amount of bleeding measured following administration of ticagrelor (T), AZD3366 (AZD1) at 1 mg / kg, AZD3366 (AZD3) at 3 mg / kg, and combined administration of AZD3366 and ticagrelor (T + AZD1). The results demonstrate that combined administration of AZD3366 and ticagrelor did not increase the amount of bleeding beyond that seen with ticagrelor alone (T). Mean ± SEM is shown. ns; not significant, ***; P < 0.001 (one-way ANOVA + Dunnett's multiple comparison test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0052] Recombinant apyrase protein Apyrase (EC 3.6.1.5) catalyzes the hydrolysis of phosphate anhydrous bonds from adenosine triphosphate (ATP) to adenosine monophosphate (AMP) and from adenosine diphosphate (ADP) to AMP. CD39 family members (also referred to as ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family members) represent some of the best characterized apyrases. Human CD39 family members include naturally occurring proteins as listed in the table below.

[0053] [Table 1]

[0054] In some embodiments, the recombinant apyrase protein is a recombinant human apyrase protein or an engineered version thereof. In some embodiments, the recombinant apyrase protein is a fragment of a recombinant human apyrase protein or an engineered version thereof. In general, the term "fragment" as used herein with respect to the recombinant human apyrase protein of the present invention or an engineered version thereof refers to an N-terminally and / or C-terminally truncated protein that retains the enzymatic activity (such as ADPase and ATPase activity) of the full-length protein. In some embodiments, the recombinant apyrase protein is a member of the CD39 family, such as any of the native proteins or engineered versions thereof listed in the table above.

[0055] The recombinant apyrase protein may be a soluble recombinant human apyrase protein, such as an apyrase protein lacking some or all of the amino acid residues constituting the transmembrane domain of an apyrase protein that may be a member of the CD39 family. For example, human CD39L3 is a 529 amino acid residue protein set forth in SEQ ID NO: 1, and a particular exemplary soluble CD39L3 apyrase protein has the amino acid sequence set forth at positions 49 to 485 of SEQ ID NO: 1.

[0056] The term "engineered" or "engineered version thereof" in the context of recombinant apyrase protein means that the engineered protein has an amino acid sequence that is similar but not identical to the reference apyrase protein. Sequence identity is generally defined with reference to the algorithm GAP (Wisconsin GCG Package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximizing the number of matches and minimizing the number of gaps. Although the use of GAP may be preferred, other algorithms may also be used, such as, for example, BLAST (using the method of (Non-Patent Document 18)), FASTA (using the method of (Non-Patent Document 19)), or the Smith-Waterman algorithm ((Non-Patent Document 20)), generally using default parameters, or the TBLASTN program of (Non-Patent Document 18) supra. In particular, the psi-Blast algorithm may be used.

[0057] The reference apyrase protein may be a wild-type apyrase protein (e.g., a full-length CD39L3 apyrase protein) or a soluble apyrase protein (e.g., a protein having an amino acid sequence set forth at positions 49 to 485 of SEQ ID NO: 1). The engineered recombinant apyrase protein may contain one or more amino acid substitutions, such as, for example, substitutions that alter the enzymatic activity (e.g., ADPase activity) compared to the reference apyrase, and / or functionally conservative substitutions that do not substantially change the enzymatic activity of the reference apyrase. The engineered recombinant apyrase protein may contain, for example, one or more amino acid deletions or additions at the N- or C-terminus of the protein that do not substantially change the enzymatic activity (e.g., ADPase and ATPase activities) of the reference apyrase. An engineered recombinant apyrase protein may have an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference apyrase protein. For example, the recombinant apyrase protein set forth in SEQ ID NO:2 (AZD3366) is an engineered version of the soluble CD39L3 apyrase protein having the amino acid sequence set forth at positions 49 to 485 of SEQ ID NO:1.

[0058] In a preferred embodiment, the recombinant apyrase protein is a CD39L3 protein or an engineered version thereof. The recombinant apyrase protein may have an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1. In an even more preferred embodiment, the recombinant apyrase protein is a soluble CD39L3 protein or an engineered version thereof. The recombinant apyrase protein may have an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth at positions 49 to 485 of SEQ ID NO:1.

[0059] The design, production, and use of soluble recombinant apyrase proteins, including engineered versions of CD39L3 (e.g., enhanced apyrases), are described in U.S. Patent No. 5,393,636, U.S. Patent No. 5,393,636, and U.S. Patent No. 5,393,636, which are incorporated by reference herein in their entireties. The recombinant apyrase proteins described herein may be any of the apyrases described in these publications.

[0060] (Patent Document 2) describes ADPase-enhanced apyrases. These ADase-enhanced apyrases include modified forms of reference apyrases, where the modifications result in increased ADPase activity compared to the reference apyrase, or the same ADPase activity as the reference apyrase combined with decreased ATPase activity compared to the reference apyrase. Exemplary ADPase-enhanced apyrases include those that contain substitutions at positions 67 and 69 of CD39L3, where the position numbering is according to SEQ ID NO: 1. Specifically, ADPase-enhanced apyrases include protein 8742, which contains an R67G substitution and a T69R substitution; and protein 8906, which contains an R67A substitution and a T69R substitution.

[0061] Exemplary ATPase and ADPase assays used to determine this activity are disclosed in (Patent Document 2). For example, the ATPase and ADPase enzyme activity of purified soluble ADPase-enhanced apyrase can be measured at 37°C in 1 ml of solution containing 8 mM CaCl2, 200 μM substrate (ATP for ATPase or ADP for ADPase), 50 mM imidazole, and 50 mM Tris, pH 7.5 ((Non-Patent Document 21)). The reaction can be stopped and the released inorganic phosphate can be measured by the addition of 0.25 ml of Malachite Green Reagent ((Non-Patent Document 22)). Based on spectrophotometric analysis at 630 nm, one unit of ATPase (or ADPase) corresponds to the release of 1 μmole of inorganic phosphate per minute at 37°C. Important kinetic constants of the enzyme, such as Km and kcat, may be obtained, for example, by fitting the data to the Michaelis-Menten equation. Other assays useful for monitoring biochemical function include, but are not limited to, radiometric assays, both described by (Non-Patent Document 23), HPLC assays; or radio-TLC assays, both described by (Non-Patent Document 24).

[0062] Thus, the recombinant apyrase protein described herein may include one or more modifications (e.g., amino acid substitutions) compared to a reference apyrase having an amino acid sequence set forth as positions 49-485 of SEQ ID NO:1 (i.e., a soluble version of CD39L3). These modifications may result in increased ADPase activity compared to the reference apyrase, or may result in the same ADPase activity as the reference apyrase combined with decreased ATPase activity compared to the reference apyrase. In some embodiments, the one or more modifications may include or consist of substitutions at positions 67 and 69, the positions being numbered according to SEQ ID NO:1. The substitution at position 67 may be glycine and the substitution at position 69 may be arginine, or the substitution at position 67 may be alanine and the substitution at position 69 may be arginine. Preferably, the substitution at position 67 is glycine and the substitution at position 69 is arginine.

[0063] Patent Document 3, for example, describes an apyrase containing a homogenous N-terminus such that more than 80% of the apyrase molecules have the same EVLP-constituting N-terminus, and a method for producing the apyrase. These proteins with homogenous N-terminus are described to have an average isoelectric point in the range of 3.0 to 4.5 and / or an improved half-life in rabbits and pigs.

[0064] Thus, the recombinant apyrase protein described herein may comprise a homogenous N-terminus such that more than 80% of the apyrase molecules have the same N-terminus, as described in (Patent Document 3), and this N-terminus is an EVLP.

[0065] In certain embodiments, the recombinant apyrase protein may further comprise one or more functionally conservative substitutions (e.g., in addition to the substitutions in the ADPase-enhanced apyrase described above). A functionally conservative substitution is a substitution that does not affect (or does not substantially affect) one or more functional properties (e.g., enzymatic activity) compared to the equivalent unsubstituted protein. In some embodiments, the recombinant apyrase protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 functionally conservative substitutions.

[0066] In a preferred embodiment, the recombinant apyrase protein comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2, optionally wherein the amino acid residue at position 67 is a glycine and the amino acid residue at position 69 is an arginine, and wherein the positions are numbered according to SEQ ID NO:1. For example, the recombinant apyrase protein may comprise the amino acid sequence of SEQ ID NO:2, with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) functionally conservative substitutions, optionally wherein the amino acid residue at position 67 is a glycine and the amino acid residue at position 69 is an arginine, with the positions numbered according to SEQ ID NO:1. In an exemplary embodiment, the recombinant apyrase protein comprises the amino acid sequence of SEQ ID NO:2.

[0067] P2Y 12 Inhibitors P2Y 12 The inhibitor may be selected from the list consisting of ticagrelor, clopidogrel, ticlopidine, prasugrel, and cangrelor. 12 Reference to an inhibitor includes any of these compounds, as well as any metabolic products, such as active metabolites. 12 The inhibitor may be selected from the list consisting of ticagrelor, clopidogrel, ticlopidine, and prasugrel, for example selected from the list consisting of ticagrelor, clopidogrel, and prasugrel. In a preferred embodiment, the P2Y 12 The inhibitor may be ticagrelor or clopidogrel. In a particularly preferred embodiment, the P2Y 12 The inhibitor is ticagrelor.

[0068] Ticagrelor [(1S,2S,3R,5S)-3-[7-[[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol] is an oral reversibly binding P2Y(12) receptor antagonist. Ticagrelor has also been documented to increase local endogenous adenosine levels and enhance adenosine-induced responses by inhibiting equilibrative nucleoside transporter-1 (ENT-1). Ticagrelor is approved for the prevention of thrombotic events in patients with acute coronary syndrome and previous myocardial infarction, with regulatory applications pending for AIS and transient ischemic attacks. It has the following chemical structure: [ka]

[0069] Ticagrelor is the active ingredient of a pharmaceutical known as BRILINTA (registered trademark) (or BRILIQUE in Europe), which is approved for use in several jurisdictions, including the United States and Europe. Ticagrelor is currently available commercially in the form of 60 mg and 90 mg immediate release tablets. Patent document 4 discloses certain pharmaceutical formulations containing ticagrelor for oral administration. Patent document 5 discloses a rapidly disintegrating oral dosage form of ticagrelor.

[0070] Ticagrelor is typically rapidly absorbed after oral administration. Unlike clopidogrel and prasugrel, ticagrelor is not a prodrug and does not require metabolic activation for activity. Nevertheless, ticagrelor is extensively metabolized, and ticagrelor and its activity are approximately P2Y 12Equipotent metabolites (AR-C124910XX) constitute the major circulating components in plasma. Plasma concentrations of ticagrelor and its active metabolites increase in a dose-dependent manner; peak concentrations are achieved within approximately 1.5 and 2.5 hours, respectively. Maximum inhibition of platelet aggregation is observed approximately 2 hours after administration, and is maintained for more than 8 hours after administration. The mean elimination half-lives of ticagrelor and its active metabolites are listed on the drug label as 7 and 9 hours, respectively. Following discontinuation, platelet activity returns to baseline after 5 days.

[0071] In a preferred embodiment, ticagrelor is orally administered at a loading dose of 180 mg in combination with recombinant apyrase protein. Ticagrelor may be administered in the form of an orodispersible tablet (ODT), for example, as described in (Patent Document 5). One or more subsequent maintenance doses may be administered after the loading dose, for example, without recombinant apyrase protein. As described above, following an initial loading dose of 180 mg, the prescribing information for ticagrelor describes a maintenance dose of 90 mg twice daily for the first year after the ACS event, and 60 mg twice daily after one year. The one or more subsequent maintenance doses may include a dose of 90 mg ticagrelor twice daily, or a dose of 60 mg ticagrelor twice daily. The prescribing information further describes that ticagrelor is administered together with a maintenance dose of aspirin of 75 to 100 mg daily. Thus, the subsequent maintenance dose or doses may further comprise administering a daily dose of aspirin of 75-100 mg.

[0072] P2Y 12The receptor inhibitor may be clopidogrel. Clopidogrel is usually administered by oral route. In some embodiments, clopidogrel is administered in combination with recombinant apyrase protein at a loading dose of 300 mg or 600 mg. One or more subsequent maintenance doses may include about 75 mg of clopidogrel, and may be administered after the loading dose, for example, without recombinant apyrase protein. As with ticagrelor above, a maintenance dose of clopidogrel may be administered with a daily dose of aspirin of 75 to 100 mg.

[0073] Clopidogrel is a prodrug and requires metabolic activation for its activity. Peak plasma concentrations of the active metabolite occur approximately 30-60 minutes following an oral dose, with dose-dependent platelet aggregation inhibition observed at approximately 2 hours following administration, and peak platelet inhibition observed approximately 8 hours later. Following a single dose, dose-dependent platelet aggregation inhibition is observed at 2 hours. While clopidogrel has an elimination half-life of approximately 6 hours following a single 75 mg dose, its active metabolite has an elimination half-life of approximately 30 minutes. After cessation, platelet aggregation and bleeding times gradually return to baseline over approximately 5 days.

[0074] P2Y 12 The receptor inhibitor may be prasugrel. Prasugrel is typically administered by oral route. In some embodiments, prasugrel is administered in combination with recombinant apyrase protein at a loading dose of 60 mg. One or more subsequent maintenance doses may include about 5 mg or 10 mg of prasugrel, and may be administered after the loading dose, for example, without recombinant apyrase protein. As with ticagrelor above, a maintenance dose of prasugrel may be administered with a daily dose of aspirin of 75-100 mg.

[0075] Prasugrel is a prodrug and is rapidly metabolized to pharmacologically active metabolites. Peak plasma concentrations of the active metabolites occur approximately 30 minutes after administration, with peak platelet inhibition reached at approximately 2 hours. It has an elimination half-life of approximately 7.4 hours.

[0076] P2Y 12 The receptor inhibitor may be ticlopidine. Ticlopidine is typically administered by oral route. In some embodiments, ticlopidine is typically administered by oral route. In some embodiments, ticlopidine is administered in combination with recombinant apyrase protein at a loading dose of 500 mg. One or more subsequent maintenance doses may include about 250 mg of ticlopidine, and may be administered after the loading dose, for example, without recombinant apyrase protein. As with ticagrelor above, a maintenance dose of ticlopidine may be administered with a daily dose of aspirin of 75-100 mg.

[0077] Peak plasma levels of ticlopidine are typically observed approximately 2 hours after oral administration. The half-life following a single dose ranges from 7 to 13 hours. The half-life following repeated dosing is approximately 4 to 5 days.

[0078] P2Y 12 The receptor inhibitor may be cangrelor. Cangrelor may be administered intravenously as a bolus, as a continuous infusion, or as a bolus followed by a continuous infusion. In some embodiments, cangrelor is administered as an intravenous bolus of 30 μg / kg, immediately followed by an intravenous infusion of 4 μg / kg per minute.

[0079] Cangrelor rapidly reaches steady-state plasma levels and inhibition of platelet aggregation within 30 minutes of initiating the infusion, and has a short plasma half-life of less than approximately 9 minutes. Maximum platelet inhibition is achieved within 15 minutes. Cangrelor's elimination half-life is approximately 3-6 minutes, and platelet responses typically return to baseline within 15 minutes of cessation.

[0080] Treatment and Administration The methods and products for use described herein are for treating ischemic events in a patient (e.g., a human patient), including diseases and disorders in which blood supply is restricted to a particular part of the patient's body, such as, for example, the patient's heart or brain, and the restriction may be due to a blood clot (thrombus). Ischemic events in the heart include acute coronary syndromes. Ischemic events in the brain are referred to as acute cerebrovascular events and include acute ischemic stroke (AIS) and transient ischemic attack (TIA). Acute coronary syndromes include myocardial infarction, classified as ST-segment elevation myocardial infarction (STEMI) or non-ST-segment elevation myocardial infarction (NSTEMI), and unstable angina. In a preferred embodiment, the treatment is treatment of ST-segment elevation myocardial infarction (STEMI) in a patient.

[0081] Myocardial infarction is generally classified clinically as STEMI and NSTEMI, which are based on electrocardiogram (ECG) changes and may be diagnosed by a physician or other trained medical personnel. The types of myocardial infarction may be defined according to or derived from the universal definition of myocardial infarction described in (Non-Patent Document 25).

[0082] In the therapeutic methods described herein, the recombinant apyrase protein is 12 The use of the term "concomitant" in this context refers to administration of a P2Y inhibitor that is administered concurrently with the P2Y inhibitor following administration (e.g., within 30 minutes, within 1 hour, within 2 hours, or within 3 hours). 12 It is intended to mean that both the inhibitor (and / or its metabolites) and the recombinant apyrase protein are bioavailable (i.e., have an active effect) in the bloodstream of a patient. In animal models, administration of the recombinant apyrase protein AZD3366 resulted in significant enzyme activity, including antiplatelet activity, within 5 minutes (depending on dose) that did not return to baseline for 3-4 weeks (depending on dose), while suppressing P2Y 12Inhibitors typically take longer to become active following administration. For example, the maximum activity of ticagrelor is usually not observed until 2 hours after administration and is maintained for more than 8 hours.

[0083] Therefore, recombinant apyrase protein and P2Y 12 The physical coadministration of the recombinant apyrase protein and the P2Y inhibitor 12 It is not necessary for the recombinant apyrase protein and the P2Y inhibitor to be administered in combination. 12 Recombinant apyrase protein and P2Y inhibitors, provided that both are bioavailable in the patient's bloodstream. 12 Either one of the inhibitors may be administered first, with the other agent administered later (eg, one or more hours later).

[0084] In addition, some patients who present with an ischemic event may already be receiving regular P2Y therapy, for example as part of a maintenance dose after a previous ischemic event. 12 Such patients are referred to herein as "currently receiving a P2Y 12 For example, 90 mg of ticagrelor is typically administered twice daily as part of a maintenance dose. In such patients to whom a recombinant apyrase protein is administered according to the methods of the present invention, the inhibitor (and / or its metabolites) is considered to be still bioactive in the patient's bloodstream, and therefore, the P2Y 12 It may not be necessary to administer another dose of the inhibitor. Alternatively, bioactive P2Y 12 To replenish the levels of inhibitors, P2Y 12 A dose less than the usual loading dose of the inhibitor may be administered. For example, in the case of ticagrelor, a dose of 60 mg, 90 mg, or 150 mg may be administered, as opposed to the typical loading dose of 180 mg, if ticagrelor is still bioactive in the patient's bloodstream. The effective dose may be determined by a skilled physician or other skilled medical personnel. However, preferably, the patient is currently receiving a P2Y 12The method further comprises administering to the patient a P2Y 12 Still including administering an inhibitor (eg, a loading dose).

[0085] P2Y 12 The inhibitor should be administered until the last dose of the inhibitor is administered to the P2Y 12 Mean elimination half-life of inhibitors, P2Y 12 More than twice the mean elimination half-life of an inhibitor or P2Y 12 More than three times the mean elimination half-life of an inhibitor or P2Y 12 If the inhibitor is administered within a period of at least 5 times the mean elimination half-life of the inhibitor, or within a period equivalent to about 10 days, it may still be considered to be bioactive in the patient's bloodstream. For example, in the case of ticagrelor, the mean elimination half-life is 7 hours for ticagrelor and 9 hours for its active metabolite. Thus, ticagrelor may be considered to be bioactive in the patient's bloodstream if the last dose was administered within the last 9 hours, within the last 18 hours, within the last 27 hours, within the last 36 hours, or within the last 45 hours. Alternatively, P2Y 12 Inhibitors may be considered bioactive in the bloodstream until activity returns to baseline following cessation of administration, which in the case of ticagrelor is after 5 days.

[0086] Other patients presenting with ischemic events had previously 12 The patient may not have been administered an inhibitor, or may have been administered, for example, a P2Y 12 The former P2Y inhibitors are no longer considered bioactive in the patient's bloodstream. 12 Inhibitor administration may be discontinued. These patients may be referred to as "naive" patients. In naive patients, recombinant apyrase protein is administered to the P2Y 12 For administration in combination with a P2Y inhibitor, the method includes 12 It is necessary to include the step of administering the inhibitor to the patient.

[0087] Recombinant apyrase protein and P2Y 12The inhibitors may be administered as a combined formulation, for example, via intravenous injection. Alternatively, recombinant apyrase protein and P2Y 12 The administration of the inhibitors to the patient may be simultaneous or sequential. As used herein, simultaneous administration refers to administration of a recombinant apyrase protein and a P2Y 12 This refers to administering both P2Y inhibitors to a patient substantially simultaneously (e.g., within 10 minutes, 5 minutes, or 1 minute of each other), optionally via different routes of administration. 12 Intravenous injection of recombinant apyrase protein within 1 minute of the inhibitor would be considered co-administration.

[0088] When sequential administration is used, recombinant apyrase protein and P2Y 12 The inhibitors are preferably administered within 24 hours, 18 hours, 12 hours, 6 hours, 2 hours, 1 hour, or more preferably within 30 minutes of each other. In some embodiments, the recombinant apyrase protein is administered first, and the P2Y 12 This is followed by sequential administration of the inhibitor. 12 The inhibitor is administered first, followed by the sequential administration of recombinant apyrase protein.

[0089] In some embodiments, the recombinant apyrase protein is administered to the patient within 24 hours, or within 18 hours, or within 12 hours, or within 6 hours, or within 4 hours, or within 2 hours, or within 1 hour, or within 30 minutes after the onset of an ischemic event (e.g., an acute coronary syndrome such as STEMI). 12 The ischemic event is administered to the patient in combination with an inhibitor. The onset of an ischemic event referred to herein may be the onset of one or more symptoms of the ischemic event (e.g., chest pain in the case of STEMI), or may be the time of diagnosis (e.g., via an electrocardiogram in the case of acute coronary syndrome), which may be performed before or immediately after the patient arrives at the hospital for treatment.

[0090] Preferably, a recombinant apyrase protein is administered to a patient suffering from acute coronary syndrome or AIS before the patient is subjected to a surgical or non-surgical reperfusion therapy (e.g., percutaneous coronary intervention (PCI), thrombectomy or thrombolytic therapy). 12 It is administered in combination with an inhibitor.

[0091] PCI may include, without limitation, balloon angioplasty, stenting, rotational or laser atherectomy, and / or brachytherapy. In cases where a stent is placed, the stent may be, without limitation, a bare metal stent, a drug-eluting stent, an absorbable stent, etc., as known in the art.

[0092] Recombinant apyrase protein and P2Y 12 The protective effect on cardiac and brain tissues provided by the combination of inhibitors may be useful in preventing and / or mitigating any damage to tissues or function resulting from restoration of circulation following reperfusion therapy. Thus, in some embodiments, the method further comprises administering a surgical or non-surgical reperfusion therapy (e.g., PCI, thrombectomy, or thrombolytic therapy) to the patient less than 48 hours, less than 24 hours, less than 12 hours, or less than 6 hours following administration of the recombinant apyrase protein. In some embodiments, the recombinant apyrase protein and P2Y 12 Either inhibitor is administered to the patient prior to performing a surgical or non-surgical reperfusion therapy (e.g., PCI, thrombectomy, or thrombolytic therapy). In other embodiments, a recombinant apyrase protein is administered to the patient prior to performing a surgical or non-surgical reperfusion therapy (e.g., PCI), and a P2Y 12 The inhibitor is administered shortly after surgical reperfusion therapy has been performed, for example, within 6 hours, within 4 hours, within 2 hours, or within 1 hour. 12 The inhibitor remains bioavailable in the patient's bloodstream during surgical or non-surgical reperfusion therapy (eg, PCI, thrombectomy, or thrombolytic therapy).

[0093] The present disclosure relates to a method for the preparation of a recombinant apyrase protein and a P2Y 12 and iii) timing of administration relative to surgical or non-surgical reperfusion therapy (e.g., PCI, thrombectomy, or thrombolytic therapy). For example, the method may include administering a recombinant apyrase protein within 6 hours of onset of the ischemic event, the method further including administering to the patient a surgical or non-surgical reperfusion therapy (e.g., PCI, thrombectomy, or thrombolytic therapy) within less than 12 hours, or within less than 6 hours, and 12 The inhibitor is administered to the patient within 6 hours of administering the recombinant apyrase protein, and optionally, the recombinant apyrase protein and the P2Y 12 Both inhibitors are administered to the patient prior to undergoing surgical or non-surgical reperfusion therapy (eg, PCI, thrombectomy or thrombolytic therapy).

[0094] Any of the methods described herein may further comprise administering aspirin to the patient. Aspirin typically inhibits the P2Y 12 The inhibitor and the recombinant apyrase protein are administered as separate formulations, and 12 The inhibitor and the recombinant apyrase protein are administered simultaneously or sequentially with each other or both of them. In some embodiments, aspirin is administered in combination with the P2Y 12 The aspirin is administered within 24 hours, 18 hours, 12 hours, 6 hours, 2 hours, 1 hour, or more preferably within 30 minutes of administering the inhibitor. In some embodiments, the aspirin is administered within 24 hours, 18 hours, 12 hours, 6 hours, 2 hours, 1 hour, or more preferably within 30 minutes of administering the recombinant apyrase protein. In some embodiments, the aspirin is administered within 24 hours, 18 hours, 12 hours, 6 hours, 2 hours, 1 hour, or more preferably within 30 minutes of administering the recombinant apyrase protein. 12In some embodiments, aspirin is administered to inhibit P2Y activating the vasopressin receptor agonist (PSA) or vasopressin receptor agonist (PSA) within 24 hours, or within 18 hours, or within 12 hours, or within 6 hours, or within 4 hours, or within 2 hours, or within 1 hour, or within 30 minutes after the onset of an ischemic event (e.g., acute coronary syndrome (such as STEMI), AIS, or TIA). 12 It is administered to a patient in combination with an inhibitor.

[0095] Aspirin may be administered to a patient in a dose of 50 mg to 325 mg, such as, for example, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 162 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, or 350 mg. In some embodiments, aspirin is administered to a patient in a dose of 50 mg to 200 mg, or 100 mg to 200 mg, such as, for example, 162 mg. In some embodiments, aspirin is administered to a patient in a dose of 200 mg to 350 mg, or 250 to 325 mg, such as, for example, 300 mg or 325 mg. In some embodiments, aspirin is administered to a patient in a dose of 75 mg to 150 mg, or 75 mg to 100 mg.

[0096] The term "treatment" as used herein in the context of treating a condition generally relates to the care and treatment of a human in which some desired therapeutic effect is achieved, such as, for example, inhibiting the progression of a condition, including slowing the rate of progression, halting the rate of progression, regressing a condition, ameliorating a condition, and curing a condition. Treatment as a preventative measure (i.e., prophylaxis, prevention) is also included.

[0097] Administration of the recombinant apyrase protein described herein may be provided, for example, by bolus injection, intravenous, intramuscular, subcutaneous, inhalation, continuous infusion, sustained release, or other pharma- ceutically acceptable techniques. Ideally, the recombinant apyrase protein will be administered to the patient in a pharma- ceutically acceptable form containing a physiologically acceptable carrier, excipient, or diluent. Such diluents and excipients may include neutral buffered saline, antioxidants (e.g., ascorbic acid), low molecular weight polypeptides (e.g., polypeptides of less than 10 amino acids), amino acids, carbohydrates (e.g., glucose, dextrose, sucrose, or dextran), chelating agents such as EDTA, stabilizers (e.g., glutathione), etc. In addition, upon administration, for maximum activity of the enzyme, it is advisable to add, for example, calcium (Ca 2+ Co-substrates for the recombinant apyrase protein, such as , may also be administered. Such carriers and diluents are selected to be non-toxic to patients at the recommended dosages and concentrations.

[0098] The P2Y 12 Administration of the inhibitor depends on the specific P2Y 12 It will depend on the inhibitor. For example, ticagrelor, clopidogrel, ticlopidine, and prasugrel are typically administered to patients in a pharma- ceutically acceptable oral dosage form, while cangrelor is typically administered to patients via intravenous injection.

[0099] The dosage requirements of the recombinant apyrase protein may vary significantly depending on age, race, weight, height, sex, duration of treatment, method of administration, biological activity of the recombinant apyrase protein, and severity of the disease state or other clinical variables. Effective doses may be determined by a skilled physician or other skilled medical personnel.

[0100] The method comprises administering a single effective amount of a recombinant apyrase protein to a patient suffering from a P2Y 12This may include administering in combination with an appropriate dose (e.g., a loading dose) of a P2Y inhibitor, optionally in combination with aspirin, if present. While only a single effective dose of recombinant apyrase protein is typically used, the method may also include administering a loading dose followed by a P2Y inhibitor as part of a chronic or maintenance treatment. 12 The method may further include periodically administering one or more oral doses of an inhibitor of, for example, P2Y 12 The inhibitor may be administered once or twice daily, for example at the maintenance doses described above, for a period of weeks, months, or years, following an initial loading dose. 12 A maintenance dose of the inhibitor may be administered with aspirin, as is known in the art. 12 Long-term or maintenance treatment with inhibitors is known in the art, and appropriate dosages and timing can be determined by a skilled physician or other skilled medical personnel.

[0101] The recombinant apyrase protein may be administered as a pharmaceutical composition comprising the recombinant apyrase protein and a pharma- ceutically acceptable carrier or diluent. 12 Inhibitors include P2Y 12 It may be administered as a pharmaceutical composition comprising the inhibitor and a pharma- ceutically acceptable carrier or diluent.

[0102] The term "pharmacologically acceptable," as used herein, refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0103] The features disclosed in the above description, or disclosed in the following claims, or disclosed in the accompanying drawings, whether expressed in their specific form, or expressed in terms of means for performing a disclosed function, or expressed in terms of methods or steps for achieving a disclosed result, may be utilized separately or in any combination of such features to realize various forms of the invention.

[0104] While the present invention has been described in conjunction with the above exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the above exemplary embodiments of the present invention are considered to be illustrative and not limiting. Various changes may be made to the described embodiments without departing from the spirit and scope of the present invention.

[0105] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purpose of enhancing the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0106] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0107] Throughout this specification, including the claims which follow, unless the context requires otherwise, the words "comprise" and "include", as well as variations such as "comprises", "comprising", "including", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0108] It should 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 dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. The term "about" with respect to numerical values ​​is optional and may mean, for example, ±10%. EXAMPLES

[0109] Example 1 - Materials and methods of experiments performed in pigs Experimental design Landrace pigs (n=20; body weight approximately 40 kg) fed a normal chow diet were randomly (Excel random function) allocated into 4 groups and received: I) Oral placebo control (2 hours prior to induction of myocardial infarction (MI), n=5) II) Ticagrelor loading dose (180 mg; n = 5; orally 2 hours before MI induction) III) Ticagrelor loading dose (180 mg; n=5; orally 2 hours before MI induction and 1 mg / kg AZD3366 intravenously 10 minutes before reperfusion) IV) Ticagrelor loading dose (180 mg; n=5; orally 2 hours before MI induction and AZD3366 IV 3 mg / kg 10 minutes before reperfusion) The pigs then underwent a temporary (90 min) balloon occlusion of the left middle anterior descending coronary artery (experimental MI) followed by reperfusion essentially as described in (Non-Patent Document 26). Over the next 42 days, the pigs received daily oral maintenance doses of placebo (group I) or ticagrelor (90 mg / bid, groups II-IV), and serial cardiac magnetic resonance (CMR) assessments were performed on days 3 (early tissue repair phase) and 42 (late tissue repair phase) after MI.

[0110] This experimental design is illustrated diagrammatically in FIG.

[0111] Experimental induction of MI On the day of MI induction, animals were anesthetized by administering buprenorphine (0.03 mg / kg) and cefazolin (25 mg / kg) as prophylaxis for pain and wound infection, respectively, followed by an intramuscular injection of ketamine (30 mg / kg), xylazine (2.2 mg / kg), and atropine (0.05 mg / kg). Once sedated, animals were intubated endotracheally and anesthesia was maintained by inhalation of isoflurane (2%). Prior to the start of the MI induction procedure, an infusion (250 mL / h) of amiodarone (300 mg) and lidocaine (150 mg) in 1.000 mL saline was started through a line placed in the marginal ear vein as prophylaxis for malignant ventricular arrhythmias, and a bolus of heparin (100 U / kg) was administered to prevent clot formation in the catheter. MI was experimentally induced by minimally invasive closed thoracic myocardial balloon occlusion of the mid-portion of the left anterior descending coronary artery as previously described in (Non-Patent Document 26). Complete coronary ischemia (verified by angiography) was maintained for 90 min. At the end of the ischemic period, the balloon was fully deflated and the animals were allowed to recover. Electrocardiogram (ECG) and hemodynamic parameters were continuously recorded throughout the procedure, and early deterioration of left ventricular ejection fraction (LVEF, M-mode analysis using parasternal long axis view; (Non-Patent Document 26)) was recorded before and after MI induction using an echocardiography system (Phillips iE33) equipped with an S5-1 sector array transducer.

[0112] 3T-CMR acquisition and analysis: global and regional structural and functional analysis CMR studies were performed consecutively in all animals on days 3 (early tissue repair phase) and 42 (late tissue repair phase) after MI. The studies were performed on a 3.0T-CMR system (AchievaVR, Philips, Amsterdam, The Netherlands) and CMR image acquisition was performed by a CMR technician blinded to the treatment. For CMR studies, animals were anesthetized with an intramuscular injection of a cocktail consisting of ketamine, xylazine, and atropine and maintained by a continuous intravenous infusion of propofol to ensure mechanical ventilation. Animals were placed in a head-on supine position and a flexible phased array surface coil was placed over the chest, at which point static images of the heart were acquired using ECG gating. The following dedicated CMR sequences were acquired in all cases: "cine" balanced steady-state free precession (bSSFP) imaging sequence to assess wall motion (WM) and cardiac function; T2-weighted short tau inversion recovery (T2w-STIR) sequence to assess myocardial edema; early gadolinium enhancement (without reflow phenomenon) to test for microvascular obstruction; late gadolinium enhancement (LGE) to assess the amount and extent of myocardial necrosis. All CMR examinations followed an identical scheme. First, a scout image [T1-turbo field echo (TFE) sequence] was acquired to locate the true axis of the heart and define a field of view that included the entire heart. Then, bSSFP cine imaging was performed in both horizontal and vertical long axes (four-chamber and two-chamber views) and in multiple consecutive short-axis images covering the entire left ventricle (LV). In the short-axis cine sequence, we acquired 24 cardiac phases of all slices to ensure correct assessment of WM and cardiac function. At the time of acquisition of the cine sequence, a T2w-STIR sequence was obtained to evaluate myocardial edema. Gadolinium-based contrast agent (Gd-GTPA, MagnevistVR, Berlex Laboratories Inc., Wayne, NJ, USA) was then injected intravenously at a dose of 0.1 mmol / kg. An initial gadolinium-enhanced sequence was acquired 1 min after contrast administration. An LGE sequence was obtained 10 min after contrast administration.Details of the technical parameters for the CMR sequences and the analysis protocols for global and regional functional / anatomical parameters have been published previously (26).

[0113] Example 2 - Effects of AZD3366 and ticagrelor on cardiac injury, function, and bleeding in a porcine model of myocardial infarction Experiments were carried out to determine whether administration of AZD3366 (also called APT102), a recombinant soluble form of ADPase, would provide additional benefit to administration of ticagrelor alone in terms of reducing infarct size and improving cardiac function. Methods and analyses were carried out as described in Example 1.

[0114] Cardiac Damage Assessment The results of the cardiac injury evaluation experiments in a porcine animal model are shown in the table below.

[0115] [Table 2]

[0116] [Table 3]

[0117] The further reduction in necrotic area with ticagrelor + 3 mg / kg AZD3366 compared to ticagrelor alone, and the numerical reduction in necrotic area with ticagrelor + 1 mg / kg AZD3366 demonstrate the dose-dependent enhanced cardioprotective effect of the combination. Since this model is the best preclinical model of human disease, this is the strongest evidence suggesting a therapeutic effect in humans.

[0118] Cardiac function evaluation The results of the cardiac function evaluation experiments in the porcine animal model are shown in the table below.

[0119] [Table 4]

[0120] [Table 5]

[0121] The further improvement in wall motion score of ticagrelor + 3 mg / kg AZD3366 compared to ticagrelor alone demonstrates enhanced healing and recovery of cardiac function following myocardial infarction. The simultaneous occurrence of reduced infarct size and improved function links the acute cardioprotective action mode to improved cardiac functional recovery. Since this model is the best preclinical model of human disease, this is the strongest evidence suggesting a therapeutic effect in humans.

[0122] Example 3 - Effect of AZD3366 and ticagrelor on mouse tail bleeding The objective of this study was to evaluate the bleeding risk and platelet aggregation inhibition of AZD3366 alone or in combination with ticagrelor in vivo in mice.

[0123] Experimental protocol C57Bl6 mice were administered AZD3366 or vehicle as an intravenous bolus. In separate groups, mice were administered AZD3366 and ticagrelor, or ticagrelor alone as a bolus and ticagrelor continuous infusion. The administration groups, doses, and administration volumes were as described in the table below.

[0124] [Table 6]

[0125] At t = 20 min, a tail cut was performed and the bleeding volume and BT were recorded for 60 min. At the end of the experiment at t = 80 min, blood samples were taken from the abdominal aorta for platelet aggregation assessment and subsequent plasma concentration analysis of AZD3366 and ticagrelor.

[0126] Bleeding model Twenty minutes after administration, bleeding was initiated by cutting 5 mm of the tip of the tail. The tail was washed with water and placed in a hemoglobin-sensitive device. Light transmittance through the blood-water mixture was recorded and converted to absorbance using PharmLab software (V6.0, AstraZeneca R&D, Gothenburg, Sweden). Bleeding was defined as a transmittance lower than 95%. Total blood loss (area under the bleeding curve, t = 20 min to t = 80 min: cumulative blood loss vs. time, absorbance*s, abs*s) and bleeding time (BT) were measured over a 60-min period from the start of tail cut.

[0127] Blood and Plasma Analysis Blood samples from the abdominal aorta were collected into Omnifix®-F 1 mL syringes (Braun Medical AG, Emmenbrucke, Germany) fitted with Terumo® Neolus 27G, 0.4 x 20 mm needles (Terumo Europe NV, Leuven Belgium). 10 μL of lepirudin (Refludan® Bayer HealthCare Pharmaceuticals Inc), giving a final blood concentration of 7 μmol / L, 5 mg / mL in a maximum of 1 mL of blood, was used as anticoagulant. Whole blood was used for platelet aggregation using a Multiplate® impedance aggregometer. Plasma was prepared from the remaining blood by centrifugation at 10000*g for 5 min (Ole Dich, Hvidovre, Denmark). Plasma was transferred to new tubes and stored at -20°C for later analysis of total plasma concentrations of ticagrelor and AZD3366.

[0128] Immediately after blood collection, 175 μL of whole blood was added to a Multiplate® (Roche Diagnostics GmbH, Mannheim, Germany) mini test cell and mixed with 175 μL of saline (9 mg / mL, Fresenius Kabi AG, Bad Homburg, Germany) pre-warmed to 37° C. After 3 min, 12 μL of adenosine diphosphate (ADP, Roche Diagnostics, Mannheim, Germany) was added to a final assay concentration of 6.5 μmol / L. Responses were measured for 6 min and data are expressed as the mean area under the curve (AUC) of agglutination units (AU) recorded over time (AU*min).

[0129] Plasma concentrations of ticagrelor were measured using liquid chromatography-mass spectrometry (LC-MS / MS).

[0130] AZD3366 concentrations in plasma were measured by ELISA, involving capture of AZD3366 with a monoclonal mouse anti-AZD3366 antibody (mAb2567, Covance, Princeton, NJ, USA) coated onto 96-well plates and detection of bound AZD3366 with a horseradish peroxidase-conjugated polyclonal rabbit anti-AZD3366 antibody (pAb3939-HRP, Covance) using TMB as a substrate.

[0131] result The blood loss and BT results are presented in the table below and illustrated in FIG.

[0132] [Table 7]

[0133] Animals administered ticagrelor showed a 7.2-fold (p<0.001) and 5.2-fold (p<0.001) increase in bleeding volume and BT, respectively, compared to animals administered vehicle. In contrast, treatment with 1 or 3 mg / kg AZD3366 was not associated with a statistically significant increase in bleeding volume or BT compared to vehicle. Furthermore, there was no difference in bleeding volume and BT in the group administered ticagrelor in combination with AZD3366 compared to the group administered ticagrelor alone.

[0134] The platelet aggregation (PA) results are set forth in the table below.

[0135] [Table 8]

[0136] Thus, in contrast to ticagrelor (T), complete platelet aggregation (PA) inhibition by AZD3366 (1 and 3 mg / kg) was not associated with increased bleeding (Figure 2). Furthermore, coadministration of AZD3366 and ticagrelor did not increase bleeding more than that seen with ticagrelor alone.

[0137] The results of this study demonstrate that the combination of ticagrelor and AZD3366 does not increase bleeding beyond that seen with ticagrelor alone.

[0138] References Several publications are cited above in order to more fully describe and disclose the present invention and the state of the art to which it pertains. Full citations for these references are provided below. Each of these references is incorporated herein in its entirety. Asaria,P.,Elliott,P.,Douglass,M.,Obermeyer,Z.,Soljak,M.,Majeed,A.,& Ezzati,M.(2017).Acute myocardial infarction hospital admissions and deaths in England: a national follow-back and follow-forward record-linkage study.The Lancet.Public health,2(4),e191-e201.https: / / doi.org / 10.1016 / S2468-2667(17)30032-4 Ibanez,B.,James,S.,Agewall,S.,Antunes,M.J.,Bucciarelli-Ducci,C.,Bueno,H.,Caforio,A.,Crea,F.,Goudevenos,J.A.,Halvorsen,S.,Hindricks,G.,Kastrati,A.,Lenzen,M.J.,Prescott,E.,Roffi,M.,Valgimigli,M.,Varenhorst,C.,Vranckx,P.,Widimsky,P.,& ESC Scientific Document Group(2018).2017 ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation:The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology(ESC).European heart journal,39(2),119-177.https: / / doi.org / 10.1093 / eurheartj / ehx393 Roth G.A.et al,(2018)Global,regional,and national age-sex-specific mortality for 282 causes of death in 195 countries and territories,1980-2017: a systematic analysis for the Global Burden of Disease Study(2017)The Lancet,392,1736-1788,https: / / doi.org / 10.1016 / S0140-6736(18)32203-7. Kyu H.H.et al,(2018)Global,regional,and national disability-adjusted life-years(DALYs)for 359 diseases and injuries and healthy life expectancy(HALE)for 195 countries and territories,1990-2017: a systematic analysis for the Global Burden of Disease Study 2017.The Lancet,392,1859-2922 https: / / doi.org / 10.1016 / S0140-6736(18)32335-3. Powers W.J.et al.(2018)Guidelines for the Early Management of Patients With Acute Ischemic Stroke:2019 Update to the 2018 Guidelines for the Early Management of Acute Ischemic Stroke:A Guideline for Healthcare Professionals From the American Heart Association / American Stroke Association.Stroke,50,e344-e418.https: / / doi.org / 10.1161 / STR.0000000000000211 Wang Y.,Liu M.,Pu C.(2017)2014 Chinese guidelines for secondary prevention of ischemic stroke and transient ischemic attack:Compiled by the Chinese Society of Neurology,Cerebrovascular Disease Group.International Journal of Stroke,12(3),302-320.https: / / doi.org / 10.1177 / 1747493017694391. Johnston S.C.et al(2020)Ticagrelor and Aspirin or Aspirin Alone in Acute Ischemic Stroke or TIA.New England Journal of Medicine 383,207-17.https: / / doi.org / 10.1056 / NEJMoa1916870. Jernberg,T.,Hasvold,P.,Henriksson,M.,Hjelm,H.,Thuresson,M.,& Janzon,M.(2015).Cardiovascular risk in post-myocardial infarction patients:nationwide real world data demonstrate the importance of a long-term perspective.European heart journal,36(19),1163-1170.https: / / doi.org / 10.1093 / eurheartj / ehu505 Wang Y.et al(2013)Clopidogrel with aspirin in acute minor stroke or transient ischemic attack.New England Journal of Medicine,369(1),11-9.https: / / doi.org / 10.1056 / NEJMoa1215340 Johnston S.C.et al.(2018);Clopidogrel and Aspirin in Acute Ischemic Stroke and High-Risk TIA.New England Journal of Medicine,379,215-225.https: / / doi.org / 10.1056 / NEJMoa1800410. Robson,S.C.,Wu,Y.,Sun,X.,Knosalla,C.,Dwyer,K.,& Enjyoji,K.(2005).Ectonucleotidases of CD39 family modulate vascular inflammation and thrombosis in transplantation.Seminars in thrombosis and hemostasis,31(2),217-233.https: / / doi.org / 10.1055 / s-2005-869527 Sun,Guanghua & Zhao,Xiurong & Grotta,James & Savitz,Sean & Chen,Ridong & Aronowski,Jaroslaw.(2011).Apyrase,APT102,Improves the Beneficial Effect of rt-PA In Experimental Thromboembolic Stroke.E302-E302. Tan,Z.,Li,X.,Turner,R.C.,Logsdon,A.F.,Lucke-Wold,B.,DiPasquale,K.,Jeong,S.S.,Chen,R.,Huber,J.D.,& Rosen,C.L.(2014).Combination treatment of r-tPA and an optimized human apyrase reduces mortality rate and hemorrhagic transformation 6h after ischemic stroke in aged female rats.European journal of pharmacology,738,368-373.https: / / doi.org / 10.1016 / j.ejphar.2014.05.052 Thygesen,K.,Alpert,J.S.,Jaffe,A.S.,Chaitman,B.R.,Bax,J.J.,Morrow,D.A.,White,H.D.,& Executive Group on behalf of the Joint European Society of Cardiology(ESC) / American College of Cardiology(ACC) / American Heart Association(AHA) / World Heart Federation(WHF)Task Force for the Universal Definition of Myocardial Infarction(2018).Fourth Universal Definition of Myocardial Infarction(2018).Circulation,138(20),e618-e651.https: / / doi.org / 10.1161 / CIR.0000000000000617 Vilahur G, Gutierrez M, Casani L, Varela L, Capdevila A, Pons-Llado G, Carreras F, Carlsson L, Hidalgo A, Badimon L. Protective effects of ticagrelor on myocardial injury after infarction. Circulation 2016;134:1708-1719. Wallentin, L., Becker, R.C., Budaj, A., Cannon, C.P., Emanuelsson, H., Held, C., Horrow, J., Husted, S., James, S., Katus, H., Mahaffey, K.W., Scirica, B.M., Skene, A., Steg, P.G., Storey, R.F., Harrington, R.A., PLATO Investigators, Freij, A., & Thorsen, M. (2009). Ticagrelor versus clopidogrel in patients with acute coronary syndromes. The New England journal of medicine, 361(11), 1045-1057. https: / / doi.org / 10.1056 / NEJMoa0904327 For standard molecular biology techniques, refer to Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

[0139] Sequence

[0140] [Table 9] The present application includes the following aspects. [Aspect 1] Recombinant apyrase protein was expressed as P2Y 12 46. ​​A method of treating an ischemic event in a patient comprising administering in combination with an inhibitor. [Aspect 2] The recombinant apyrase protein is 12 20. A recombinant apyrase protein for use in a method for treating an ischemic event in a patient comprising administering to said patient in combination with an inhibitor. [Aspect 3] The recombinant apyrase protein is 12 A recombinant apyrase protein and a P2Y inhibitor for use in a method of treating an ischemic event in a patient comprising administering to said patient a recombinant apyrase protein and a P2Y inhibitor. 12 Inhibitors. [Aspect 4] The P2Y 12 administering to the patient a P2Y inhibitor 12 Patent application title: P2Y INHIBITOR FOR USE IN A METHOD FOR TREATING AN ISCHEMIC EVENT IN A PATIENT, WHEREIN: THE INHIBITOR IS ADMINISTERED IN CONJUNCTION WITH A RECOMBINANT APYRASE PROTEIN 12 Inhibitors. [Aspect 5] The recombinant apyrase protein and the P2Y 12 or co-administering to said patient said recombinant apyrase protein and said P2Y inhibitor. 12 The method of embodiment 1 or the recombinant apyrase protein and / or P2Y inhibitor for use according to any one of embodiments 2 to 4, comprising the step of sequentially administering, optionally on the same day, to said patient. 12 Inhibitors. [Aspect 6] a. administering the recombinant apyrase protein to the patient, followed by administering the P2Y 12 administering the inhibitors sequentially; or b. The P2Y 12 administering an inhibitor to said patient followed by sequential administration of said recombinant apyrase protein. The method according to embodiment 5 or the recombinant apyrase protein and / or P2Y for use according to embodiment 5, comprising 12 Inhibitors. [Aspect 7] The recombinant apyrase protein is administered to the patient. 12 The method according to any one of aspects 1, 5 or 6, or the recombinant apyrase protein and / or P2Y for use according to any one of aspects 2 to 6, wherein the recombinant apyrase protein and / or P2Y 12 Inhibitors. [Aspect 8] The method further comprises: 12 Starting at least 24 hours after administering the recombinant apyrase protein in combination with the P2Y inhibitor, 12 The method of any one of aspects 1 and aspects 5 to 7, or the recombinant apyrase protein and / or P2Y inhibitor for use according to any one of aspects 2 to 7, further comprising the step of administering the inhibitor to said patient at least daily, optionally wherein said daily administration continues for at least 1 month, at least 2 months, or at least 6 months. 12 Inhibitors. [Aspect 9] The P2Y 12 The method according to any one of the first and second to third aspects, or the recombinant apyrase protein and / or P2Y inhibitor for use according to any one of the first to third aspects, wherein the inhibitor is selected from the list consisting of ticagrelor, clopidogrel, ticlopidine, prasugrel and cangrelor. 12 Inhibitors. [Aspect 10] The P2Y 12 The method according to embodiment 9 or the recombinant apyrase protein and / or P2Y inhibitor for use according to embodiment 9, wherein the inhibitor is ticagrelor. 12 Inhibitors. [Aspect 11] The method according to any one of Aspects 1 and 5 to 10, or the recombinant apyrase protein and / or P2Y for use according to any one of Aspects 2 to 10, wherein the recombinant apyrase protein is a soluble CD39L3 protein comprising an amino acid sequence that is at least 80% identical to positions 49 to 485 of SEQ ID NO: 1. 12 Inhibitors. [Aspect 12] 12. The method of claim 11, or the recombinant apyrase protein and / or P2Y for use according to claim 11, wherein the recombinant apyrase protein comprises one or more modifications compared to a reference CD39L3 protein having an amino acid sequence set forth at positions 49 to 485 of SEQ ID NO: 1, which modifications result in an increased ADPase activity compared to the reference apyrase, or which modifications result in the same ADPase activity as the reference apyrase combined with a decreased ATPase activity compared to the reference apyrase. 12 Inhibitors. [Aspect 13] 13. The method according to embodiment 12, or the recombinant apyrase protein and / or P2Y for use according to embodiment 12, wherein said recombinant apyrase protein comprises substitutions at positions 67 and 69 compared to said reference CD39L3 protein, said positions being numbered according to SEQ ID NO: 1. 12 Inhibitors. [Aspect 14] 14. The method according to embodiment 13, or the recombinant apyrase protein and / or P2Y for use according to embodiment 13, wherein the substitution at position 67 is to glycine and the substitution at position 69 is to arginine. 12 Inhibitors. [Aspect 15] The method according to any one of aspects 1 and aspects 5 to 14, or the recombinant apyrase protein and / or P2Y for use according to any one of aspects 2 to 14, wherein the recombinant apyrase protein comprises the amino acid sequence set forth in SEQ ID NO:2. 12 Inhibitors. [Aspect 16] The method according to any one of aspects 1 and aspects 5 to 15, or the recombinant apyrase protein and / or P2Y for use according to any one of aspects 2 to 15, wherein the recombinant apyrase protein is administered in a dose of 10 mg to 1000 mg. 12 Inhibitors. [Aspect 17] a. The P2Y 12 the inhibitor is ticagrelor administered at a dose of 60-200 mg, optionally 180 mg; b. The P2Y 12 the inhibitor is clopidogrel administered at a dose of 75 to 600 mg, optionally 300 mg or 600 mg; c. Said P2Y 12 the inhibitor is ticlopidine administered at a dose of 250-500 mg, optionally 500 mg; d. The P2Y 12 the inhibitor is prasugrel and is administered in a dose of 5 to 60 mg, optionally 60 mg; or e. The P2Y 12 The method according to any one of aspects 1 and aspects 5 to 16, or the recombinant apyrase protein and / or P2Y inhibitor for use according to any one of aspects 2 to 16, wherein the inhibitor is cangrelor and is administered at a dose of 30 μg / kg intravenous bolus followed by an intravenous infusion of 4 μg / kg per minute. 12 Inhibitors. [Aspect 18] The method according to any one of aspects 1 and aspects 5 to 17, or the recombinant apyrase protein and / or P2Y for use according to any one of aspects 2 to 17, wherein the method further comprises the step of administering aspirin to the patient, optionally wherein the aspirin is administered in an amount of 50 to 325 mg. 12 Inhibitors. [Aspect 19] The method according to any one of aspects 1 and aspects 5 to 18, or the recombinant apyrase protein and / or P2Y for use according to any one of aspects 2 to 18, wherein the ischemic event is acute ischemic stroke or transient ischemic attack. 12 Inhibitors. [Aspect 20] The method according to any one of aspects 1 and aspects 5 to 18, or the recombinant apyrase protein and / or P2Y for use according to any one of aspects 2 to 18, wherein the ischemic event is an acute coronary syndrome. 12 Inhibitors. [Aspect 21] 21. The method according to embodiment 20 or the recombinant apyrase protein and / or P2Y for use according to embodiment 20, wherein said ischemic event is ST-segment elevation myocardial infarction (STEMI). 12 Inhibitors. [Aspect 22] The recombinant apyrase protein is 12 22. The method according to embodiment 20 or embodiment 21, or the recombinant apyrase protein and / or P2Y inhibitor for use according to embodiment 20 or embodiment 21, wherein the recombinant apyrase protein and / or P2Y inhibitor is administered to said patient within 24 hours, or within 12 hours, or within 6 hours after the onset of said ischemic event. 12 Inhibitors. [Aspect 23] The method further comprises: 12 23. The method according to any one of aspects 20 to 22, or the recombinant apyrase protein and / or P2Y inhibitor for use according to any one of aspects 20 to 22, further comprising administering to said patient a surgical or non-surgical reperfusion therapy less than 48 hours, less than 24 hours, less than 12 hours, less than 6 hours after administration of said recombinant apyrase protein and / or P2Y inhibitor in combination with said patient, and optionally said surgical or non-surgical reperfusion therapy is percutaneous coronary intervention (PCI), thrombectomy or thrombolytic therapy. 12 Inhibitors.

Claims

1. 1. A pharmaceutical composition for treating an ischemic event in a patient comprising a recombinant apyrase protein, The recombinant apyrase protein is administered to the patient in combination with ticagrelor; and The recombinant apyrase protein is a soluble CD39L3 protein comprising an amino acid sequence at least 90% identical to positions 49 to 485 of SEQ ID NO:

1. The pharmaceutical composition.

2. A pharmaceutical composition for treating an ischemic event in a patient comprising ticagrelor, The ticagrelor is administered to the patient in combination with a recombinant apyrase protein; and The recombinant apyrase protein is a soluble CD39L3 protein comprising an amino acid sequence at least 90% identical to positions 49 to 485 of SEQ ID NO:

1. The pharmaceutical composition.

3. A combination drug for treating an ischemic event in a patient, comprising a recombinant apyrase protein and ticagrelor, The recombinant apyrase protein is administered to the patient in combination with the ticagrelor; and The recombinant apyrase protein is a soluble CD39L3 protein comprising an amino acid sequence at least 90% identical to positions 49 to 485 of SEQ ID NO:

1. The combination drug.

4. The pharmaceutical composition of claim 1 or 2, or the combination pharmaceutical of claim 3, wherein the recombinant apyrase protein and the ticagrelor are administered to the patient simultaneously, or the recombinant apyrase protein and the ticagrelor are administered to the patient sequentially.

5. a. The recombinant apyrase protein is administered to the patient, followed by sequential administration of the ticagrelor; or b. The ticagrelor is administered to the patient, followed by sequential administration of the recombinant apyrase protein; A pharmaceutical composition or combination drug according to claim 4.

6. (i) the recombinant apyrase protein is administered to the patient within 24 hours or within 12 hours of administration of ticagrelor to the patient; and / or (ii) at least daily administration of the ticagrelor to the patient is initiated at least 24 hours after the recombinant apyrase protein is administered in combination with the ticagrelor; A pharmaceutical composition according to claim 1 or 2, or a pharmaceutical combination according to claim 3, or a pharmaceutical composition or pharmaceutical combination according to claim 4 or 5.

7. The pharmaceutical composition according to claim 1 or 2, or the combination pharmaceutical according to claim 3, or the pharmaceutical composition or combination pharmaceutical according to any one of claims 4 to 6, wherein the recombinant apyrase protein is a soluble CD39L3 protein comprising an amino acid sequence that is at least 95% identical to positions 49 to 485 of SEQ ID NO:

1.

8. The pharmaceutical composition or combination pharmaceutical of claim 7, wherein the recombinant apyrase protein contains one or more modifications compared to a reference CD39L3 protein having an amino acid sequence set forth at positions 49 to 485 of SEQ ID NO:1, and the modifications result in increased ADPase activity compared to the reference apyrase, or the modifications result in the same ADPase activity as the reference apyrase combined with decreased ATPase activity compared to the reference apyrase.

9. The pharmaceutical composition or combination pharmaceutical of claim 8, wherein the recombinant apyrase protein comprises substitutions at positions 67 and 69 compared to the reference CD39L3 protein, the positions being numbered according to SEQ ID NO:

1.

10. 10. The pharmaceutical composition or combination pharmaceutical of claim 9, wherein the substitution at position 67 is to glycine and the substitution at position 69 is to arginine.

11. The pharmaceutical composition according to claim 1 or 2, or the combination pharmaceutical according to claim 3, or the pharmaceutical composition or combination pharmaceutical according to any one of claims 4 to 10, wherein the recombinant apyrase protein comprises the amino acid sequence set forth in SEQ ID NO:

2.

12. (i) the recombinant apyrase protein is administered at a dose of 10 mg to 1000 mg; and / or (ii) the ticagrelor is administered in a dose of 60 to 200 mg; and / or (iii) aspirin is further administered to the patient. A pharmaceutical composition according to claim 1 or 2, or a combination pharmaceutical according to claim 3, or a pharmaceutical composition or combination pharmaceutical according to any one of claims 4 to 11.

13. The pharmaceutical composition according to claim 1 or 2, or the pharmaceutical combination according to claim 3, or the pharmaceutical composition or pharmaceutical combination according to any one of claims 4 to 12, wherein the ischemic event is acute ischemic stroke.

14. The pharmaceutical composition according to claim 1 or 2, or the combination pharmaceutical according to claim 3, or the pharmaceutical composition or combination pharmaceutical according to any one of claims 4 to 12, wherein the ischemic event is an acute coronary syndrome.

15. (i) the recombinant apyrase protein is administered to the patient in combination with the ticagrelor within 24 hours, or within 12 hours, or within 6 hours after the onset of the ischemic event; and / or (ii) the patient is administered a surgical reperfusion therapy less than 48 hours, or less than 24 hours, or less than 12 hours, or less than 6 hours after administration of the recombinant apyrase protein in combination with the ticagrelor; A pharmaceutical composition or pharmaceutical combination according to claim 14.

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