Recombinant apirase protein for use in the treatment of ischemic events at doses of 40-240 mg.

Abstract

This disclosure relates to dosage regimens for the administration of recombinant apirase proteins in the treatment of ischemic events in patients, such as ST-elevation myocardial infarction and acute ischemic stroke, and their medical use.

Description

Technical Field

[0001] The present disclosure relates to dosing regimens for the administration of recombinant apyrase proteins and their medical use in the treatment of ischemic events in patients such as ST-elevation myocardial infarction and acute ischemic stroke. The recombinant apyrase protein can be administered in combination with dual antiplatelet therapy including P2Y 12 inhibitors and aspirin.

Background Art

[0002] Myocardial infarction (MI) is a major cause of hospitalization and death worldwide (Asaria et al. 2017). Left untreated, MI results in irreversible damage to the myocardium due to lack of blood flow (ischemia) and thus lack of oxygen. Therefore, the main goal of therapy for MI is to promote the restoration of normal coronary blood flow for the purpose of reducing myocardial damage through reperfusion therapy. Reperfusion therapy typically involves the use of therapeutic agents to increase blood flow and reduce thrombosis, combined with surgical techniques such as percutaneous coronary intervention (PCI). Early reperfusion and PCI are preferred and are associated with improved outcomes, and guidelines suggest that PCI should be performed within 12 hours of the onset of MI symptoms (Ibanez et al. 2018).

[0003] One commonly used small molecule used to reduce thrombosis is aspirin. Another type of small molecule therapeutic agent is a P2Y 12 receptor inhibitor such as clopidogrel, ticagrelor, prasugrel, and cangrelol, which are known for their ability to inhibit platelets and prevent blood clotting. Aspirin and P2Y 12The combination therapy of receptor inhibitors, known as dual antiplatelet therapy (DAPT), has been shown to be clinically effective in preventing thrombotic events. For example, the National Institute for Health and Care Excellence (NICE) recommends ticagrelor in combination with low-dose aspirin for up to 12 months as a therapy for adults with acute coronary syndrome (ACS). However, these P2Y 12 Receptor inhibitors carry an increased risk of bleeding; therefore, care must be taken to ensure that patients receive this treatment in accordance with prescribing information and current guidelines.

[0004] P2Y such as Chikagrelor 12 Despite optimal antiplatelet and antithrombotic therapy with receptor inhibitors, approximately 10% of patients still experience a new myocardial infarction event annually, and increasing the intensity of these treatments increases the risk of bleeding without improving efficacy (Wallentin et al., 2009; Jernberg et al., 2015). Therefore, there is still a need for effective and safe treatment methods that can be used to treat ischemic events such as myocardial infarction.

[0005] Recent research has investigated the use of recombinant apirases as protein-based therapeutic agents. Apirases (ecto-ATP diphosphohydrolases) constitute a group of enzymes that catalyze the metabolism of ATP to ADP and ADP to AMP. In the body, AMP, produced by apirase-induced hydrolysis of ATP and ADP, is converted to adenosine by ubiquitously expressed extracellular CD73 / ecto-5'-nucleotidase. CD39, the first known human apirase, was initially identified as a cell surface protein on activated lymphocytes and endothelial cells, and various in vitro and in vivo studies have shown that apirases can maintain vascular integrity and physiologically inhibit inflammation and thrombus formation (Robson et al. 2005).

[0006] Moeckel et al. (2014) reported on 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 reported that this recombinant protein exhibited four times higher adenosine diphosphatase activity and a 50-fold longer plasma half-life than natural apirase, and that treatment with APT102 in animal models reduced infarct size without increasing bleeding time. International Publication No. 2022 / 038191 describes a method of treating ischemic events in patients, such as ST-elevation myocardial infarction and acute ischemic stroke, by administering AZD3366 in combination with a P2Y12 inhibitor.

[0007] To date, data on the behavior, safety, and efficacy of recombinant apirases containing AZD3366 in human patients, including dosage for administration, are not available. [Overview of the Initiative]

[0008] AZD3366 is a recombinant human apirase engineered to enhance the hydrolysis of extracellular adenosine tri / diphosphate (ATP / ADP) to adenosine monophosphate, followed by hydrolysis to adenosine. In preclinical studies conducted in canine, rodent, and porcine models (Moeckel et al. 2014 and unpublished results), AZD3366 demonstrated antithrombotic, anti-inflammatory, and tissue-protective properties. The inventors sought to determine for the first time how AZD3366 behaves when administered to humans at various doses.

[0009] The inventors have established through human clinical trials that AZD3366, used alone or in combination with aspirin and ticagrelor, is generally safe and well-tolerated when administered to humans. Furthermore, in vivo pharmacokinetic and safety data obtained from human trials were combined with preclinical efficacy data obtained from animal models to predict the dose of AZD3366 expected to achieve effective cardioprotective effects in humans without compromising patient safety. Moreover, it was established that these doses achieved complete platelet inhibition without a significant increase in bleeding-related adverse events when used in humans, demonstrating further clinical benefits attributable to the administration of AZD3366 in the treatment of ischemic events.

[0010] Therefore, administering AZD3366 at the doses described herein is expected to achieve clinical benefits when administered to humans for the treatment of acute coronary syndromes, including ST-elevation myocardial infarction, and for the treatment of ischemic events, including acute ischemic stroke.

[0011] Accordingly, one aspect of the present disclosure provides a method for treating an ischemic event in a patient, comprising administering a therapeutically effective dose of recombinant apirase protein to the patient, wherein the recombinant apirase protein is administered to the patient in a dose of 40 mg to 240 mg.

[0012] In another aspect, the disclosure provides a recombinant apirase protein for use in a method for treating an ischemic event in a patient, the method comprising administering the recombinant apirase protein to the patient in doses of 40 mg to 240 mg.

[0013] In another aspect, the disclosure provides the use of recombinant apirase protein in the manufacture of a pharmaceutical product for the treatment of ischemic events in a patient, wherein the treatment comprises administering the recombinant apirase protein to the patient in doses of 40 mg to 240 mg.

[0014] Recombinant apirase proteins may contain the amino acid sequence shown as SEQ ID NO: 2 (AZD3366).

[0015] Recombinant apirase protein (e.g., AZD3366) can be administered to patients in doses of 40 mg to 170 mg, 40 mg to 150 mg, 40 mg to 140 mg, or 40 mg to 100 mg.

[0016] Recombinant apirase protein (e.g., AZD3366) can be administered to patients in doses of 100 mg to 240 mg, 100 mg to 220 mg, or 100 mg to 200 mg.

[0017] Recombinant apirase protein (e.g., AZD3366) can be administered to a patient in doses of 100 mg to 140 mg, for example, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, or 140 mg. In one embodiment, recombinant apirase protein (e.g., AZD3366) is administered to the patient in a dose of 115 mg.

[0018] Recombinant apirase protein can be administered to patients by intravenous injection.

[0019] In some embodiments, the ischemic event to be treated is acute coronary syndrome. Acute coronary syndrome includes ST-elevation myocardial infarction (STEMI), non-ST-elevation myocardial infarction (NSTEMI), and unstable angina. In some embodiments, the acute coronary syndrome to be treated is ST-elevation myocardial infarction (STEMI) in the patient. In other embodiments, the ischemic event to be treated is acute ischemic stroke.

[0020] As demonstrated herein, AZD3366 can be safely administered to humans when used alone or in combination with aspirin and ticagrelor. Therefore, in some embodiments, the recombinant apirase protein is P2Y 12It is administered in combination with an inhibitor and / or aspirin. In some embodiments, the recombinant apyrase protein is administered in combination with dual antiplatelet therapy (DAPT), where DAPT 12 includes administration with both an inhibitor and aspirin. A skilled physician or other skilled healthcare provider can determine the most appropriate manner of administering each therapeutic agent to a patient using the exemplary dosages and treatment regimens further described herein.

[0021] P2Y 12 The inhibitor can be selected from the group consisting of ticagrelor, clopidogrel, ticlopidine, prasugrel, and cangrelol. In some embodiments, P2Y 12 The inhibitor is selected from the group consisting of ticagrelor, clopidrogrel, and prasugrel. In some embodiments, P2Y 12 The inhibitor is ticagrelor or clopidogrel. In some embodiments, P2Y 12 The inhibitor is ticagrelor.

[0022] When the recombinant apyrase protein is administered in combination with a P2Y 12 inhibitor and / or aspirin, the different agents are typically administered as separate formulations in sequential administrations. When sequential administration is used, the recombinant apyrase protein can be administered within 12 hours, 6 hours, or 2 hours of the administration of a loading dose of a P2Y 12 inhibitor and / or aspirin.

[0023] After administration of the recombinant apyrase protein and a loading dose of a P2Y 12 inhibitor and / or aspirin, a further maintenance dose of a P2Y 12Inhibitors and aspirin (e.g., for at least 2 days, at least 1 week, at least 6 weeks, or at least 6 months). As demonstrated herein, administration of a certain dose of AZD3366 with a loading dose of ticagrelor and aspirin resulted in an increase in capillary bleeding time (CBT) in humans, but this increase leveled off and became minimal as the maintenance doses of ticagrelor and aspirin increased.

[0024] This disclosure includes combinations of the described embodiments and preferred features, except where such combinations are clearly unacceptable or expressly avoided. [Brief explanation of the drawing]

[0025] Next, embodiments and experiments illustrating the principles of this disclosure will be described with reference to the attached drawings. [Figure 1] This shows the percentage of platelet aggregation inhibition from baseline over time (mean with 1 SEM error bar) from humans administered different doses of AZD3366 (10 mg, 30 mg, 90 mg, 180 mg, 360 mg, and 640 mg). Platelet aggregation was quantified in platelet-rich plasma (PRP) using the light-transmitted platelet aggregation assay (LTA) assay further described herein. Complete platelet inhibition was observed with dose-dependent duration at doses of 10 mg and above. [Figure 2] The following shows capillary bleeding time (CBT) data obtained from individuals administered AZD3366 at different doses (2 mg, 10 mg, 30 mg, 90 mg, 180 mg, 360 mg, and 640 mg). No significant increase in CBT was observed up to the 90 mg dose level. [Figure 3]The following shows capillary bleeding time (CBT) data obtained from humans administered AZD3366 in combination with aspirin (ASA) and ticagrelor (tica). Loading and maintenance doses of aspirin and ticagrelor were administered according to standard clinical practice, as further described in the examples. Baseline, A, B, and C represent the time of CBT measurement. The baseline panel provides CBT measurements taken before administration of either aspirin, AZD3366, or ticagrelor (0002:30PRE, or 2 hours and 30 minutes before AZD3366 administration). Panel A provides CBT measurements taken after administration of AZD3366 or placebo, and a loading dose of 324 mg aspirin (0000:10POST, meaning CBT measurements taken 10 minutes after AZD3366 or placebo administration). Panel B provides CBT measurements after administration of AZD3366 or placebo, and loading doses of 324 mg aspirin and 180 mg ticagrelor (0003:40 POST, meaning CBT measurements taken 3 hours and 40 minutes after administration of AZD3366 or placebo). Panel C provides CBT measurements after administration of AZD3366 or placebo, loading doses of 324 mg aspirin and 180 mg ticagrelor, and maintenance doses of 81 mg aspirin and 90 mg ticagrelor (0051:40 POST, meaning CBT measurements taken 51 hours and 40 minutes after administration of AZD3366). When loading doses of ASA and ticagrelor were given, there was a significant increase in CBT in the AZD3366 group compared to placebo, but this increase leveled off when healthy volunteers continued with the maintenance dose of DAPT. [Modes for carrying out the invention]

[0026] Next, aspects and embodiments of this disclosure will be described with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All references used herein are incorporated herein by reference.

[0027] Recombinant apirase protein Apirase (EC3.6.1.5) catalyzes the hydrolysis of phosphate anhydride bonded to adenosine triphosphate (ATP) to adenosine monophosphate (AMP) and to adenosine diphosphate (ADP) to AMP. The CD39 family members (also known as ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family members) represent some of the best-characterized apirases. Human CD39 family members include the native proteins shown in the table below.

[0028] [Table 1]

[0029] Human CD39L3 is a 529-amino acid protein shown in SEQ ID NO: 1. A specific exemplary soluble CD39L3 apirase protein has the amino acid sequence shown at positions 49-485 of SEQ ID NO: 1.

[0030] The design, production, and use of soluble recombinant apirase proteins, including CD39L3 (e.g., enhanced apirase), are described in U.S. Patent No. 7,247,300 (B1), European Patent No. 2,133,430 (B1), and European Patent No. 2,523,971 (B1), all of which are incorporated herein by reference in their entirety. The recombinant apirase proteins described herein may be any of the apirases described in those publications.

[0031] European Patent No. 2133430(B1) describes ADPase-enhanced apirases. These ADPase-enhanced apirases include modified forms of reference apirases, the modifications resulting in the same ADPase activity as the reference apirase, combined with increased ADPase activity compared to the reference apirase, or decreased ATPase activity compared to the reference apirase. Exemplary ADPase-enhanced apirases include those containing substitutions at positions 67 and 69 of CD39L3, with positional numbering following Sequence ID No. 1. Specifically, the ADPase-enhanced apirases include protein 8742 containing R67G and T69R substitutions, and protein 8906 containing R67A and T69R substitutions.

[0032] Exemplary ATPase and ADPase assays used to determine this activity are disclosed in European Patent No. 2133430(B1). For example, the ATPase and ADPase enzyme activity of purified soluble ADPase-enhanced apirase can be measured at 37°C in a 1 mL solution at pH 7.5 containing 8 mM CaCl2, 200 μM substrate (ATP for ATPase or ADP for ADPase), 50 mM imidazole, and 50 mM Tris (Picher et al., Biochem. Pharmacol. (1938) 51:1453). The reaction can be stopped, and the released inorganic phosphate can be measured by adding 0.25 mL of malachite green reagent (Baykov, et al., Anal. Biochem. (1988) 171:266). Based on spectrophotometric analysis at 630 nm, one unit of ATPase (or ADPase) corresponds to the release of 1 μmol of inorganic phosphate / min at 37°C. Key rate constants of enzymes such as Km and kcat can 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 and HPLC assays (J. Clin Invest. (1998) 101:1851-1859), or Marcus, A. Jet et al. (J. Clin Invest. (1991) 88:1690-1696).

[0033] Accordingly, the recombinant apirase proteins described herein may include one or more modifications (e.g., amino acid substitutions) compared to a reference apirase (i.e., a soluble version of CD39L3) having the amino acid sequence shown as positions 49-485 of SEQ ID NO: 1. These modifications may result in the same ADPase activity as the reference apirase, combined with increased ADPase activity compared to the reference apirase, or decreased ATPase activity compared to the reference apirase. In some embodiments, one or more modifications may include, or consist of, substitutions at positions 67 and 69, where these positions are numbered according to SEQ ID NO: 1. The substitution at position 67 may be a substitution to glycine, and the substitution at position 69 may be a substitution to arginine, or the substitution at position 67 may be a substitution to alanine, and the substitution at position 69 may be a substitution to arginine. In some embodiments, the substitution at position 67 is a substitution to glycine, and the substitution at position 69 is a substitution to arginine.

[0034] European Patent No. 2523971(B1) describes apirase having a uniform N-terminus and a method for producing such apirase, such that, for example, more than 80% of the apirase molecules have the same N-terminus containing an extravalent pleopropyl alcohol (EVLP). These proteins having a uniform N-terminus are described to have an average isoelectric point in the range of 3.0–4.5 and / or an enhanced half-life in rabbits and pigs.

[0035] Therefore, the recombinant apirase proteins described herein may contain homogeneous N-terminuses such that more than 80% of the apirase molecules have the same N-terminus and that the N-terminus is an extravalent long segment (EVLP), as described in European Patent No. 2523971(B1).

[0036] In certain embodiments, the recombinant apirase protein may further include one or more function-conserving substitutions (e.g., in addition to the substitutions in the ADPase-enhanced apirase described above). A function-conserving substitution is one that does not affect (or substantially affects) one or more functional properties (e.g., enzyme activity) compared to an equivalent non-substituted protein. In some embodiments, the recombinant apirase protein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 function-conserving substitutions.

[0037] In some embodiments, the recombinant apirase protein contains 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, wherein the amino acid residue at position 67 is glycine and the amino acid residue at position 69 is arginine, and the positions are numbered according to SEQ ID NO: 1. For example, recombinant apirase protein may contain the amino acid sequence of SEQ ID NO: 2 having 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) function-conserving substitutions, optionally with glycine at position 67 and arginine at position 69, the positions numbered according to SEQ ID NO: 1.

[0038] In an exemplary embodiment, the recombinant apirase protein contains the amino acid sequence of SEQ ID NO: 2 (AZD3366).

[0039] Treatment of ischemic events 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 specific part of the patient's body, such as the patient's heart or brain, and the restriction may be caused by a blood clot (thrombus). Examples of ischemic events in the heart include acute coronary syndrome. Examples of ischemic events in the brain include acute ischemic stroke (AIS). Acute coronary syndrome includes myocardial infarction classified as ST-elevation myocardial infarction (STEMI) or non-ST-elevation myocardial infarction (NSTEMI), and unstable angina. In some embodiments, the treatment is the treatment of ST-elevation myocardial infarction (STEMI) in a patient.

[0040] Myocardial infarction is generally classified clinically into STEMI and NSTEMI. These are based on changes in the electrocardiogram (ECG) and can be diagnosed by a physician or other skilled healthcare professional. The type of myocardial infarction may be defined according to or derived from the universal definition of myocardial infarction described by Thygesen et al. 2018.

[0041] In some embodiments, recombinant apirase protein is administered to the patient within 18 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or even 30 minutes after the onset of an ischemic event (e.g., acute coronary syndrome such as STEMI). The onset of an ischemic event as referred to herein may be at the time of the onset of one or more symptoms of the ischemic event (e.g., chest pain in the case of STEMI) or at the time of diagnosis (e.g., via electrocardiogram in the case of acute coronary syndrome), which may occur before or immediately after the patient arrives at a hospital (or equivalent) for treatment.

[0042] In some embodiments, patients suffering from acute coronary syndrome are administered recombinant apirase protein before undergoing surgical reperfusion therapy (e.g., percutaneous coronary intervention (PCI)).

[0043] PCI may include, but is not limited to, balloon angioplasty, stent implantation, rotational or laser atheroma resection, and / or close-range radiotherapy. If a stent is implanted, the stent may be, but is not limited to, a bare-metal stent, a drug-eluting stent, an absorbable stent, etc., as known in the art.

[0044] P2Y 12 The cardioprotective effects provided by the inhibitor may be useful in preventing and / or mitigating any damage to cardiac tissue or function resulting from the restoration of circulation after reperfusion therapy. Therefore, in some embodiments, the method further includes performing surgical reperfusion therapy (e.g., PCI) on the patient less than 48 hours, less than 24 hours, less than 12 hours, or less than 6 hours after administration of recombinant apirase protein.

[0045] The treatment of acute coronary syndrome can be demonstrated by a reduction in infarct size and / or restoration of blood flow to the affected area in the patient. Furthermore, treatment with recombinant apirase protein can result in inhibition of platelet aggregation in the patient. As demonstrated herein, complete platelet inhibition was achieved by AZD3366 at all doses tested, and dose-dependent sustained effects were reported. Methods for monitoring the success of acute coronary syndrome treatment include, for example, known P2Y 12 This will be understood by those skilled in the art, based on their knowledge of the treatment of acute coronary syndrome using inhibitors.

[0046] In other embodiments, the ischemic event to be treated is acute ischemic stroke. Each year, 100 new cases of ischemic stroke occur in a population of 70,000. Without treatment, 55 patients will die or require care within one year. Most of these 100 patients have a mild or transient stroke and are administered only antiplatelet agents to reduce recurrence. Approximately 25–35 patients receive reperfusion therapy, which saves 5–6 patients from death or requiring care and increases the number of patients without disability. Therefore, reducing the morbidity and mortality associated with acute ischemic stroke (AIS) is an unmet clinical need.

[0047] Preclinical and clinical trials are being conducted to treat and / or prevent stroke following an ischemic stroke event. 12 The use of inhibitors and aspirin is being evaluated. Furthermore, preclinical data have demonstrated that recombinant apirase protein AZD3366 can enhance reperfusion, reduce reocclusion, and decrease intracerebral hemorrhage in animal models of ischemic stroke (Sun et al., 2011; J. Tan et al., 2014). Therefore, administration of AZD3366 (optional, P2Y 12 Beneficial results obtained in the preclinical animal models of myocardial infarction described herein, related to inhibitors and / or in combination with aspirin, have been found to be potentially beneficial in the treatment of ischemic stroke. For example, the treatment reduced infarct size and / or brain damage in patients suffering from ischemic stroke, and P2Y 12 This effect can be achieved without significantly increasing the risk of bleeding compared to the inhibitor alone.

[0048] The term “treatment,” as used herein in the context of treating a condition, generally refers to the treatment and therapy of a human being, where several desired therapeutic effects (e.g., inhibition of the progression of a condition) are achieved, and include a reduction in the rate of progression, cessation of the rate of progression, regression of the condition, improvement of the condition, and cure of the condition. Treatment as a preventive measure (i.e., prophylaxis, prevention) is also included.

[0049] Dosage and treatment regimen The recombinant apirase proteins described herein may be administered, for example, by bolus injection, intravenous, intramuscular, subcutaneous, inhalation, continuous infusion, continuous release, or other pharmaceutically acceptable techniques. The recombinant apirase proteins described herein may be administered to a patient by intravenous injection, as performed in the AZD3366 Phase I trial reported in the Examples.

[0050] Recombinant apirase protein can be administered to patients in doses ranging from 20 mg to 640 mg. As described herein, doses of AZD3366 within this range have been safe and well-tolerated when administered to humans.

[0051] In some cases, recombinant apirase protein may be administered to patients in doses of 40mg-240mg, 50mg-240mg, 60mg-240mg, 70mg-240mg, 80mg-240mg, 90mg-240mg, 100mg-240mg, 110mg-240mg, 120mg-240mg, 130mg-240mg, 140mg-240mg, 150mg-240mg, 160mg-240mg, 170mg-240mg, 180mg-240mg, 190mg-240mg, or 200mg-240mg. In some cases, recombinant apirase protein may be administered in doses of 50mg-200mg, 60mg-200mg, 70mg-200mg, 80mg-200mg, 90mg-200mg, 100mg-200mg, 110mg-200mg, 120mg-200mg, 130mg-200mg, 140mg-200mg, 150mg-200mg, 160mg-200mg, 170mg-200mg, or 180mg-200mg. In some cases, recombinant apirase protein may be administered in doses of 50-180 mg, 60-180 mg, 70-180 mg, 80-180 mg, 90-180 mg, 100-180 mg, 110-180 mg, 120-180 mg, 130-180 mg, 140-180 mg, 150-180 mg, or 160-180 mg. No significant adverse events were observed at these doses, and greater therapeutic effects (e.g., cardioprotective effects) were expected at higher doses.

[0052] In some cases, recombinant apirase protein may be administered to patients in doses of less than 180 mg. As demonstrated herein, doses of AZD3366 of 180 mg or more did not induce any significant adverse events, but did result in increased capillary bleeding time (CBT) as measured in healthy volunteers. While CBT is not a definitive marker of bleeding in clinical practice, it may indicate increased bleeding. For example, recombinant apirase protein may be administered to patients in doses of 40 mg–170 mg, 50 mg–170 mg, 60 mg–170 mg, 70 mg–170 mg, 80 mg–170 mg, 90 mg–170 mg, 100 mg–170 mg, 110 mg–170 mg, 120 mg–170 mg, 130 mg–170 mg, 140 mg–170 mg, 150 mg–170 mg, or 160 mg–170 mg. In another example, recombinant apirase protein may be administered to a patient in doses of 40 mg to 160 mg, 50 mg to 160 mg, 60 mg to 160 mg, 70 mg to 160 mg, 80 mg to 160 mg, 90 mg to 160 mg, 100 mg to 160 mg, 110 mg to 160 mg, 120 mg to 160 mg, 130 mg to 160 mg, 140 mg to 160 mg, or 150 mg to 160 mg. As a further example, recombinant apirase protein can be administered to patients in doses of 40 mg to 150 mg, 50 mg to 150 mg, 60 mg to 150 mg, 70 mg to 150 mg, 80 mg to 150 mg, 90 mg to 150 mg, 100 mg to 150 mg, 110 mg to 150 mg, 120 mg to 150 mg, or 130 mg to 150 mg. As yet another example, recombinant apirase protein can be administered to patients in doses of 40 mg to 140 mg, 50 mg to 140 mg, 60 mg to 140 mg, 70 mg to 140 mg, 80 mg to 140 mg, 90 mg to 140 mg, 100 mg to 140 mg, 110 mg to 140 mg, 120 mg to 140 mg, or 130 mg to 140 mg.

[0053] In some cases, recombinant apirase protein may be administered in doses exceeding 90 mg. As demonstrated herein, there is a dose-dependent duration associated with platelet aggregation inhibition attributable to AZD3366, with doses exceeding 90 mg resulting in a significantly longer-lasting platelet aggregation inhibitory effect. For example, recombinant apirase protein may be administered to patients in doses of 100 mg–240 mg, 100 mg–230 mg, 100 mg–220 mg, 100 mg–210 mg, 100 mg–200 mg, 100 mg–190 mg, or 100 mg–180 mg. It will also be understood that doses exceeding 90 mg can be combined with the doses below 180 mg mentioned above. Therefore, in some cases, recombinant apirase protein may be administered to patients in doses of 100 mg–170 mg, 100 mg–160 mg, or 100 mg–150 mg.

[0054] Furthermore, while not bound by theory, combining in vivo pharmacokinetic and safety data obtained from human trials with preclinical efficacy data from animal models suggests that a dose range of 100 mg to 140 mg of AZD3366 likely represents the optimal range for achieving effective therapeutic effects (e.g., cardioprotection and / or antithrombotic effects) without compromising patient safety. In particular, this dose range is based on achieving in humans the same plasma exposure levels in porcine animal studies that were observed when significant improvements in infarct size were seen.

[0055] Therefore, depending on the circumstances, recombinant apirase may be administered to patients in doses of 100mg-140mg, 100mg-135mg, 100mg-130mg, 105mg-140mg, 105mg-135mg, 105mg-130mg, 110mg-140mg, 110mg-135mg, or 110mg-130mg. In some cases, recombinant apirase is administered to patients in doses of 105 mg to 125 mg, i.e., one of the following doses: 105 mg, 106 mg, 107 mg, 108 mg, 109 mg, 110 mg, 111 mg, 112 mg, 113 mg, 114 mg, 115 mg, 116 mg, 117 mg, 118 mg, 119 mg, 120 mg, 121 mg, 122 mg, 123 mg, 124 mg, and 125 mg. In some cases, recombinant apirase is administered to patients in doses of 110 mg to 120 mg, i.e., one of the following doses: 110 mg, 111 mg, 112 mg, 113 mg, 114 mg, 115 mg, 116 mg, 117 mg, 118 mg, 119 mg, and 120 mg. In one embodiment, recombinant apirase is administered to the patient at a dose of 110 mg. In another embodiment, recombinant apirase is administered to the patient at a dose of 115 mg. In a further embodiment, recombinant apirase is administered to the patient at a dose of 120 mg.

[0056] The precise dosage requirements for recombinant apirase protein may vary depending on age, race, weight, height, sex, duration of treatment, method of administration, biological activity of the recombinant apirase protein, and the severity of the condition or other clinical variables. While exemplary doses are provided above, other effective doses within the scope disclosed herein may be determined by a skilled physician or other skilled healthcare professional.

[0057] As described above, recombinant apirase is typically administered as a fixed dose, i.e., a dose not based on the individual patient's body weight. Alternatively, recombinant apirase may be administered in a dose calculated based on the patient's body weight in kilograms (kg). For example, a fixed dose of 120 mg corresponds to 2 mg / kg for a subject weighing 60 kg.

[0058] In some embodiments, the patient being treated has a body weight of 50-100 kg. In some embodiments, the patient being treated has a body weight of 18-30 kg / m². 2 They have a body mass index (BMI).

[0059] In some embodiments, the patients being treated are either Chinese or Japanese. Chinese subjects are Chinese men or women whose parents and all grandparents are Chinese and who have not lived outside of China for more than 10 years. Japanese subjects are Japanese men or women whose parents and all grandparents are Japanese and who have not lived outside of China for more than 10 years.

[0060] Recombinant apirase proteins may be administered as a pharmaceutical composition comprising the recombinant apirase protein and a pharmaceutically acceptable carrier or diluent.

[0061] As used herein, the term “pharmaceutically acceptable” refers to compounds, components, materials, compositions, dosage forms, etc., that are suitable for use in contact with the tissue of the subject of concern (e.g., human) without causing excessive toxicity, irritation, allergic response, or other problems or complications, within the bounds of reasonable medical judgment, and that have a reasonable benefit-risk ratio. Each carrier, diluent, excipient, etc., must also be “acceptable” in the sense that it is compatible with the other components of the formulation. Such diluents and excipients may consist of neutral buffered saline, antioxidants (e.g., ascorbic acid), low molecular weight polypeptides (e.g., polypeptides <10 amino acids), amino acids, carbohydrates (e.g., glucose, dextrose, sucrose, or dextran), chelating agents such as EDTA, stabilizers (e.g., glutathione), and co-substrates of recombinant apirase proteins, e.g., calcium (Ca 2+ ) may be administered at the time of administration for maximum enzyme activity. Such carriers and diluents are selected to be non-toxic to the patient at the recommended dosage and concentration.

[0062] Combination therapy In some of the therapies described herein, recombinant apirase protein is P2Y 12 It is administered concomitantly with the inhibitor and / or aspirin. The use of the term "concomitantly" in this context means that after administration (e.g., within 30 minutes, or within 1 hour, or within 2 hours, or within 3 hours), P2Y 12 It is intended that both the inhibitor and / or aspirin (and / or its metabolites) and the recombinant apirase protein are bioavailable in the patient's bloodstream (i.e., have an active effect). In animal models, administration of a specific dose of recombinant protein AZD3366 activates it within 5 minutes and does not return to baseline for 3-4 weeks, but P2Y 12 Inhibitors typically take longer to become active after administration. For example, P2Y 12 The maximum activity of the inhibitor ticagrelor is usually not observed until about 2 hours after administration, and this activity is maintained for more than 8 hours.

[0063] P2Y disclosed herein 12 The inhibitor may be selected from a list consisting of ticagrelor, clopidogrel, ticlopidine, prasugrel, and cangrelor. P2Y in this specification 12 References to inhibitors include any of these compounds as well as any metabolites, e.g., active metabolites. In some embodiments, P2Y 12 The inhibitor may be selected from a list consisting of ticagrelor, clopidogrel, ticlopidine, and prasugrel, for example, from a list consisting of ticagrelor, clopidogrel, and prasugrel. In some embodiments, P2Y 12 The inhibitor may be ticagrelor or clopidogrel. In some embodiments, P2Y 12 The inhibitor is ticagrelor.

[0064] Ticagrelor [(1S,2S,3R,5S)-3-[7-[[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-123-triazolo[4,5-d]pyrimidine-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol] is a reversibly binding oral P2Y(12) receptor antagonist under development for the prevention of thrombotic events in patients with acute coronary syndrome. Ticagrelor has the following chemical structure.

[0065] [ka]

[0066] Ticagrelor is the active ingredient in a drug product known as BRILINTA® (or BRILIQUE in Europe), which is approved for use in multiple jurisdictions, including the United States and Europe. Ticagrelor is currently marketed in the form of 60 mg and 90 mg immediate-release tablets. International Publication 2008 / 024045 discloses certain pharmaceutical formulations containing ticagrelor for oral administration. International Publication 2017 / 182589 discloses a rapidly disintegrating oral dosage form of ticagrelor.

[0067] 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, along with its metabolite (AR-C124910XX) of nearly equal potency, including the major circulating components in plasma. Plasma concentrations of ticagrelor and its active metabolite increase in a dose-dependent manner. Peak concentrations were achieved within approximately 1.5 and 2.5 hours, respectively. Maximum inhibition of platelet aggregation was observed approximately 2 hours after administration, and this is maintained for more than 8 hours post-administration. The mean elimination half-lives of ticagrelor and its active metabolite are listed on the drug label as 7 and 9 hours, respectively. After discontinuation, platelet activity returns to baseline after 5 days.

[0068] In some embodiments, ticagrelor is administered orally as a loading dose of 180 mg in combination with recombinant apirase protein. Ticagrelor may be administered in the form of orally disintegrating tablets (ODTs), as described, for example, in International Publication No. 2017 / 182589. One or more subsequent maintenance doses may be administered after the loading dose, for example, without recombinant apirase protein. As described below, following the initial loading dose of 180 mg, the prescribing information for ticagrelor states that a maintenance dose of 90 mg twice daily should be administered for the first year after an ACS event, and 60 mg twice daily thereafter. One or more subsequent maintenance doses may include 90 mg of ticagrelor twice daily or 60 mg of ticagrelor twice daily. The prescribing information further states that ticagrelor should be administered with a daily maintenance dose of 75–100 mg of aspirin.

[0069] Therefore, subsequent maintenance doses may further include administering a daily dose of 75-100 mg of aspirin.

[0070] P2Y 12 The inhibitor may be clopidogrel. Clopidogrel is typically administered via the oral route. In some embodiments, clopidogrel is administered in combination with recombinant apirase protein as a loading dose of 300 mg or 600 mg. One or more subsequent maintenance doses may contain about 75 mg of clopidogrel and may be administered after the loading dose, for example, without recombinant apirase protein. Similar to ticagrelor described above, the maintenance dose of clopidogrel may be administered as a daily dose of 75–100 mg of aspirin.

[0071] Clopidogrel is a prodrug and requires metabolic activation for its activity. Peak plasma concentrations of the active metabolite occur approximately 30–60 minutes after oral administration, and dose-dependent inhibition of platelet aggregation is observed approximately 2 hours after administration. After a single oral dose, dose-dependent inhibition of platelet aggregation can be observed 2 hours later. Clopidogrel has an elimination half-life of approximately 6 hours after a single 75 mg dose, but its active metabolite has an elimination half-life of approximately 30 minutes. After discontinuation, platelet aggregation and bleeding time gradually return to baseline in approximately 5 days.

[0072] P2Y 12 The inhibitor may be prasugrel. Prasugrel is typically administered via the oral route. In some embodiments, prasugrel is administered as a loading dose of 60 mg in combination with recombinant apirase protein. One or more subsequent maintenance doses may consist of about 5 mg or 10 mg of prasugrel and may be administered after the loading dose, for example, without recombinant apirase protein. Similar to ticagrelor described above, the maintenance dose of prasugrel may be administered with a daily dose of 75–100 mg of aspirin.

[0073] Prasugrel is a prodrug and is rapidly metabolized to pharmacologically active metabolites. Peak plasma concentrations of the active metabolite occur approximately 30 minutes after administration. It has a elimination half-life of approximately 7.4 hours.

[0074] P2Y 12 The inhibitor may be ticlopidine. Ticlopidine is typically administered via the oral route. In some embodiments, ticlopidine is typically administered via the oral route. In some embodiments, ticlopidine is administered as a loading dose of 500 mg in combination with recombinant apirase protein. One or more subsequent maintenance doses may contain about 250 mg of ticlopidine and may be administered after the loading dose, for example, without recombinant apirase protein. Similar to ticagrelor described above, the maintenance dose of ticlopidine may be administered as a daily dose of 75–100 mg of aspirin.

[0075] Peak plasma levels of ticlopidine are typically observed approximately 2 hours after oral administration. The half-life after a single dose ranges from 7 to 13 hours. The half-life after repeated doses is approximately 4 to 5 days.

[0076] P2Y 12 - The receptor inhibitor may be cangrerol. Cangrerol may be administered intravenously as a bolus, as a continuous infusion, or as a bolus followed by a continuous infusion. In some embodiments, cangrerol is administered as an intravenous bolus of 30 μg / kg, immediately followed by an intravenous infusion of 4 μg / kg per minute.

[0077] Cangrelol rapidly reaches steady-state plasma levels and inhibits platelet aggregation within 30 minutes of infusion initiation, and has a short plasma half-life of less than approximately 9 minutes. Maximum platelet inhibition is achieved within 15 minutes. The efflux half-life of cangrelol is approximately 3–6 minutes, and the platelet response typically returns to baseline within 15 minutes of interruption.

[0078] P2Y 12 The inhibitor is P2Y 12 It may be administered as a pharmaceutical composition comprising an inhibitor and a pharmaceutically acceptable carrier or diluent.

[0079] Recombinant apirase protein P2Y 12 Because it is administered in combination with the inhibitor, recombinant apirase protein and P2Y 12 It is not necessarily required that both inhibitors be administered physically at the same time. Rather, recombinant apirase protein and P2Y should be administered after administration. 12 Provided that both inhibitors are bioavailable in the patient's bloodstream, recombinant apirase protein and P2Y 12 One of the inhibitors may be administered first, followed by the other drug later (for example, more than one hour later).

[0080] Furthermore, some patients experiencing ischemic events may receive P2Y as part of their maintenance dose after a previous ischemic event, for example. 12Some patients may already be receiving regular inhibitors. Such patients are referred to as "P2Y" in this specification. 12 This is referred to as "currently receiving treatment with an inhibitor." For example, a 90 mg dose of ticagrelor is typically administered twice daily as part of a maintenance dose. In such patients administered recombinant apirase protein by the method described herein, the inhibitor (and / or its metabolites) is still considered to be bioactive in the patient's bloodstream, and therefore another dose of P2Y is administered. 12 Administering inhibitors is not always necessary. Alternatively, the bioactive P2Y2 in the bloodstream may be a factor. 12 To replenish the levels of the inhibitor, P2Y 12 A dose lower 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, in contrast to a 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 healthcare professional. In some embodiments, if the patient is currently P2Y 12 Even if you are receiving treatment with inhibitors, this method is P2Y 12 This still includes administering an inhibitor (e.g., a loading dose) to the patient.

[0081] P2Y 12 The inhibitor's final dose was P2Y 12 Mean efflux half-life of an inhibitor, P2Y 12 More than twice the mean efflux half-life of the inhibitor, or P2Y 12 More than three times the mean efflux half-life of the inhibitor, or P2Y 12 If an inhibitor is administered within a period corresponding to more than five times its mean elimination half-life, it may still be considered bioactive in the patient's bloodstream. For example, in the case of ticagrelor, the mean elimination half-life is 7 hours for ticagrelor itself and 9 hours for its active metabolite. Therefore, ticagrelor may be considered bioactive in the patient's bloodstream if the last dose was within the last 9 hours, 18 hours, 27 hours, 36 hours, or 45 hours. Alternatively, P2Y 12Inhibitors may be considered bioactive in the bloodstream until their activity returns to baseline after discontinuation of administration, which in the case of ticagrelor is 5 days later.

[0082] Other patients exhibiting ischemic events previously underwent P2Y 12 They may not have been given an inhibitor, or they may have previously undergone P2Y 12 It is possible that the inhibitor administration was interrupted, for example, P2Y 12 The inhibitor is no longer considered bioactive in the patient's bloodstream. These patients may be referred to as "naive" patients. In naive patients, recombinant apirase protein is P2Y 12 To administer in combination with an inhibitor, this method is P2Y 12 The process must include administering the inhibitor to the patient.

[0083] P2Y as described herein 12 Inhibitor administration is used for specific P2Y 12 The treatment depends on the inhibitor. For example, ticagrelor, clopidogrel, ticlopidine, and prasugrel are typically administered to patients in pharmaceutically acceptable oral forms, while cangrelor is typically administered to patients via intravenous injection.

[0084] Recombinant apirase protein and P2Y 12 The inhibitors may be administered as a combination formulation, for example, by intravenous injection. Alternatively, recombinant apirase protein and P2Y 12 The inhibitors may be administered to patients simultaneously or sequentially. As used herein, simultaneous administration refers to recombinant apirase protein and P2Y 12 This refers to administering both inhibitors to a patient essentially simultaneously (e.g., within 10 minutes, 5 minutes, or 1 minute) via different routes of administration of their choice. For example, P2Y administration provided by oral administration. 12 Intravenous injection of recombinant apirase protein within one minute of administering the inhibitor is considered a concurrent administration.

[0085] When sequential administration is used, recombinant apirase protein and P2Y 12 The inhibitors may be administered to each other within 18, 12, 6, or 2 hours. In some embodiments, recombinant apirase protein is administered first, followed by P2Y 12 The inhibitors are administered sequentially. Recombinant apirase proteins may be administered to the patient as illustrated herein, and P2Y 12 The inhibitor (e.g., ticagrelor) may be administered within 2 hours (e.g., 1 hour and 40 minutes). In other embodiments, P2Y 12 The inhibitor is administered first, followed by the recombinant apirase protein.

[0086] In some embodiments, recombinant apirase protein and P2Y 12 Both inhibitors are administered to the patient before surgical reperfusion therapy (e.g., PCI). In other embodiments, recombinant apirase protein is administered to the patient before surface reperfusion therapy (e.g., PCI), and P2Y 12 The inhibitor is administered immediately after surgical reperfusion therapy, for example, within 6 hours, 4 hours, 2 hours, or 1 hour. In some embodiments, recombinant apirase protein and P2Y 12 The inhibitors remain bioavailable in the patient's bloodstream during surgical reperfusion therapy (e.g., PCI).

[0087] This disclosure covers i) recombinant apirase polypeptide and P2Y 12 Embodiments include a combination of any of the above timings with respect to simultaneous or sequential administration of inhibitors, ii) timing of administration related to the onset of an ischemic event, and iii) timing of administration related to surgical reperfusion therapy (e.g., PCI). For example, the method may include administering recombinant apirase protein within 6 hours of the onset of an ischemic event, and the method further includes performing surgical reperfusion therapy (e.g., PCI) on the patient within 12 hours or 6 hours, and P2Y 12The inhibitor is administered to the patient within 6 hours of administration of recombinant apirase protein, and optionally, recombinant apirase protein and P2Y 12 Both inhibitors are administered to the patient before surgical reperfusion therapy (e.g., PCI) is performed.

[0088] Any of the methods described herein may further include administering aspirin to a patient. Aspirin is typically P2Y 12 The inhibitor and recombinant apirase protein are administered as separate formulations, and P2Y 12 The inhibitor and / or recombinant apirase protein are administered simultaneously or sequentially. In some embodiments, aspirin is P2Y 12 The inhibitor is administered within 24 hours, 18 hours, 12 hours, 6 hours, 2 hours, 1 hour, or 30 minutes after administration of the recombinant apirase protein. In some embodiments, aspirin is administered within 24 hours, 18 hours, 12 hours, 6 hours, 2 hours, 1 hour, or 30 minutes after administration of the recombinant apirase protein. As illustrated herein, aspirin is administered to the patient, followed by recombinant apirase protein 2 hours later, and then P2Y 12 An inhibitor (e.g., ticagrelor) may be administered within 2 hours (e.g., 1 hour and 40 minutes) of recombinant apirase protein administration.

[0089] In some embodiments, aspirin is P2Y 12 The inhibitor or recombinant apirase protein is administered simultaneously. In some embodiments, aspirin is administered within 18 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or even 30 minutes after the onset of an ischemic event (e.g., acute coronary syndrome such as STEMI), to induce P2Y 12 It is administered to patients in combination with an inhibitor.

[0090] Aspirin may be administered to a patient in doses of 50 mg to 325 mg, 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, 324 mg, 325 mg, or 350 mg. In some embodiments, aspirin is administered to a patient in doses of 50 mg to 200 mg, or 100 mg to 200 mg, for example, 162 mg. In some embodiments, aspirin is administered to a patient in a loading dose of 200 mg to 350 mg, or 250 to 325 mg, for example, 300 mg, 324 mg, or 325 mg. In some embodiments, aspirin is administered to a patient in a loading dose of 324 mg. In some embodiments, aspirin is administered to the patient in a maintenance dose of 75 mg to 150 mg, or 75 mg to 100 mg, for example, 81 mg.

[0091] This method uses recombinant apirase protein as a single effective dose and applies a suitable dose (e.g., loading dose) of P2Y 12 This may include administering inhibitors, and optionally aspirin, if present. While only a single effective dose of recombinant apirase protein is typically used, this method may be used as part of long-term treatment or maintenance therapy for P2Y 12 This may further include administering one or more oral doses of the inhibitor regularly, after a loading dose. For example, P2Y 12 The inhibitor may be administered once or twice daily for several weeks, months, or years after the initial loading dose, for example, at the maintenance dose described above. P2Y 12 The maintenance dose of the inhibitor may be administered with aspirin, as is known in the art (referred to as dual antiplatelet therapy or DAPT). P2Y after ischemic event 12 Long-term or maintenance therapy with inhibitors is well known in the art, and the appropriate dose and timing can be determined by a skilled physician or other skilled healthcare professional.

[0092] Features disclosed in the foregoing description, the following claims, or the accompanying drawings, expressed in a particular form or with respect to means for performing the disclosed functions, or methods or processes for obtaining the disclosed results, may be used, as appropriate, separately, or in any combination of such features, to implement the present disclosure in a variety of forms.

[0093] While this disclosure is described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will become apparent to those skilled in the art when given this disclosure. Therefore, the exemplary embodiments of this disclosure described above are illustrative and not limiting. Various modifications to the embodiments described can be made without departing from the spirit and scope of this disclosure.

[0094] To avoid any doubt, any theoretical explanations provided herein are provided for the purpose of improving the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0095] Any section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein.

[0096] Throughout this Spec., including the claims, unless the context requires otherwise, the terms “comprise” and “include,” as well as variations such as “comprises,” “comprising,” and “including,” will be understood to mean the inclusion of the integer or process or group of integers or processes stated, but not the exclusion of any other integer or process or group of integers or processes.

[0097] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless the context clearly indicates otherwise. Ranges may be expressed herein as “about” one particular value and / or “about” another particular value. Where such ranges are expressed, alternative embodiments include one particular value and / or other particular values. Similarly, where a value is expressed in an approximate form by the preceding “about,” it will be understood that the particular value forms an alternative embodiment. The term “about” with respect to numbers is optional and means, for example, + / - 10%. [Examples]

[0098] Example 1 - Phase 1 randomized, single-blind, placebo-controlled study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of AZD3366 in healthy men and women who are not capable of becoming pregnant. Research Overview This human first-dose (FiH) study was conducted to provide safety, tolerability, pharmacokinetic (PK), and pharmacodynamic (PD) data for AZD3366 in healthy men and women. This study has the ClinicalTrial.gov identifier: NCT04588727.

[0099] This study included participants weighing at least 50 kg and no more than 100 kg, with a body mass index (BMI) of 18-30 kg / m². 2 The study was conducted on healthy men and women aged 18 to 55 years, who were not likely to become pregnant.

[0100] Three groups were enrolled in this study: healthy subjects, healthy Japanese subjects, and healthy Chinese subjects. The healthy subjects group included healthy men and women (not of pregnancy potential) who did not originate from any indigenous group of the Far East, Southeast Asia, or the Indian subcontinent. The healthy Japanese subjects included healthy Japanese men and women (not of pregnancy potential) whose parents and all grandparents were Japanese and who had not lived outside of Japan for more than 10 years. The healthy Chinese subjects included healthy Chinese men and women (not of pregnancy potential) whose parents and all grandparents were Chinese and who had not lived outside of China for more than 10 years.

[0101] Part A of this study was a randomized, single-blind, placebo-controlled design to evaluate the safety, tolerability, pharmacokinetics (PK), and disease progression (inhibition of capillary bleeding time [CBT] and platelet aggregation) of a single escalating dose (SAD) of AZD3366 administered intravenously (IV) in healthy subjects, healthy Japanese subjects, and healthy Chinese subjects.

[0102] Part B of this study was a randomized, single-blind, parallel-group, placebo-controlled design to investigate the safety, tolerability, and PD (inhibition of CBT and platelet aggregation) of a single IV dose of AZD3366 in combination with ticagrelor and acetylsalicylic acid (ASA) in healthy subjects.

[0103] The co-administration of ASA and ticagrelor was selected based on the standard antiplatelet therapy regimen in patients with myocardial infarction.

[0104] Main purpose The main objectives of this study were as follows: Part A: ● To investigate the safety and tolerability of intravenous (IV) administration of AZD3366 for SAD in healthy subjects, healthy Japanese subjects, and healthy Chinese subjects. Part B: ● To investigate the safety and tolerability of a single IV dose of AZD3366 at a single dose level (160 mg) in healthy subjects with concomitant loading doses of ticagrelor and ASA, as well as with repeated administration.

[0105] secondary purpose The secondary objectives of this study were as follows: Part A: ● To characterize the pharmacokinetics (PK) of AZD3366 after a single intravenous administration in healthy subjects, healthy Japanese subjects, and healthy Chinese subjects. ● To characterize the progression of platelet aggregation (PD) of AZD3366 after a single intravenous administration of AZD3366 in relation to platelet aggregation (LTA) and inhibition of CBT in healthy subjects, healthy Japanese subjects, and healthy Chinese subjects. ● Investigate the immunogenicity of AZD3366 after intravenous administration. Part B: ● To study plasma exposure after IV administration of AZD3366 at a single dose level in healthy subjects with concurrent loading doses and repeated doses of ticagrelor and ASA, and to characterize the progression of platelet aggregation (PD) of AZD3366 with respect to inhibition of LTA and CBT. ● To study the effect of AZD3366 on the PK of Chicagrelor. ● Investigate the immunogenicity of AZD3366 after intravenous administration.

[0106] Dosage and treatment regimen Part A: Seven cohorts of eight participants each received a single short-duration intravenous infusion of AZD3366 (2-640 mg [n=6]) or placebo (n=2). Three additional cohorts of five healthy Japanese participants and one cohort of eight healthy Chinese participants were included within this dose range. In total, 103 participants were randomized. The mean age was 37.2 (±9) years, and 100 were male.

[0107] The first cohort received either 2 mg of AZD3366 or a placebo. Cohorts 2-7 received 10 mg, 30 mg, 90 mg, 180 mg, 360 mg, and 640 mg, respectively.

[0108] Part B: In Part B, another group of 24 subjects received a single IV dose of 160 mg of AZD3366 or placebo, either in combination with aspirin and ticagrelor (n=12) or with aspirin and ticagrelor alone (n=12).

[0109] Healthy volunteers were administered a loading dose of 324 mg of ASA, followed by a single IV dose of AZD3366 or placebo 2 hours later (t=0). One hour and forty minutes after administration of AZD3366 or placebo, healthy volunteers were administered a loading dose of 180 mg of ticagrelor. 90 mg of ticagrelor was administered every 12 hours after the loading dose (t=25 hours 40 minutes, t=37 hours 40 minutes, t=49 hours 40 minutes). ASA was administered daily at t=25 hours 40 minutes and t=49 hours 40 minutes. Capillary bleeding time was monitored at t=0 hours 10 minutes, t=3 hours 40 minutes, and t=51 hours 40 minutes.

[0110] Measurement and evaluation methods Light-transmitted platelet aggregation (LTA) assay to measure ADP-induced platelet response after administration of AZD3366 to healthy volunteers. ADP-induced platelet aggregation in platelet-rich plasma (PRP) was quantified using an LTA assay on a CHRONO-LOG® 490 4+ agglutination system equipped with an AGGGRO / LINK® interface. PRP was prepared by centrifugation of lithium heparin-treated blood samples at 100–170 g for 15 minutes, followed by transfer of the supernatant to a new polypropylene tube. To establish a 100% baseline in the LTA assay, platelet-poor plasma (PPP) was prepared by centrifugation of blood samples at 1500–2400 g for 20 minutes, followed by transfer of the supernatant to a new polypropylene tube. To stimulate platelet aggregation, 5 or 20 μmol / L of ADP was added to the PRP and PPP preparations in separate cuvettes containing a magnetic stirrer, and aggregation was tracked at 37°C for at least 5 minutes with constant agitation. Data were recorded as amplitude % (aggregation response in PRP samples expressed as % of PPP response) and area under the curve (AUC). For each dose and time point of AZD3366, inhibition of platelet aggregation was calculated compared to the pre-administration level of platelet aggregation.

[0111] Evaluation of capillary bleeding time (CBT) after administration of AZD3366 in healthy volunteers. CBT was evaluated by making a horizontal incision in the forearm and monitoring the time it took for bleeding to stop. A blood pressure cuff was placed on the upper arm and inflated to 40 mmHg. A standardized incision was then made using a Surgicutt® device, and bleeding was monitored at 30-second intervals by placing filter paper (Surgicutt® Bleeding Time Blotting paper) over the bleeding, not more than 0.5 mm from the incision site to avoid interfering with platelet plug formation. Bleeding was followed for 90 or 180 minutes, and it was determined to have stopped when the blood no longer stained the filter paper. If bleeding did not stop by the 90 or 180-minute interruption, it was determined that bleeding was still ongoing at that point. After the completion of the CBT analysis, the cuff was removed and the incision site was cleaned with an antiseptic swab.

[0112] Part A Results AZD3366 treatment alone did not yield any clinically relevant safety or tolerability findings.

[0113] Similar adverse event (AE) rates were observed in the AZD3366 and placebo groups, with no significant increase in bleeding events. A summary of AE outcomes for healthy volunteers treated with AZD3366 (across the entire dose) or placebo is provided in the table below.

[0114] [Table 2]

[0115] Peak AZD3366 plasma concentrations were reached within 0.5 hours post-administration, followed by a monophasic or biphasic decline, with a terminal PK half-life of approximately 140 hours. Complete ADP-stimulated platelet aggregation inhibition was achieved within 10 minutes post-administration. The duration of effect ranged from 4 hours at 2 mg to approximately 35 days at 640 mg (Figure 1).

[0116] With AZD3366 monotherapy, no increase in capillary bleeding time (CBT) was observed up to a 90 mg dose level, but a significant increase was observed at doses of 180 mg and above (Figure 2).

[0117] Part B Results Treatment with AZD3366 in combination with aspirin and ticagrelor (AZD3366+DAPT) was safe and well-tolerated. Similar to Part A, the adverse event rates were comparable in the AZD3366 and placebo groups, with no significant increase in bleeding events. A summary of AE results for the two groups is provided in the table below.

[0118] [Table 3]

[0119] When ASA and ticagrelor loading doses were administered, there was an increase in CBT in the AZD3366 group compared to placebo, but this increase leveled off when healthy volunteers continued with maintenance doses of DAPT (Figure 3).

[0120] conclusion AZD3366, either alone or in combination with aspirin and ticagrelor, was generally safe, well-tolerated, and achieved complete platelet inhibition with dose-dependent duration.

[0121] References To better explain and disclose the present invention and the latest art relating thereto, several publications are cited above. A complete citation of these references is provided below. Each of these references in its entirety is incorporated herein.

[0122] 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 Jernberg,T.,Hasvold,P.,Henriksson,M.,Hjelm,H.,Thursson,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。 Moeckel D,Jeong SS,Sun X,Broekman MJ,Nguyen A,Drosopoulos JH,Marcus AJ,Robson SC,Chen R,Abendschein D.(2014)Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk.Sci Transl Med.,6(248):248ra105.doi:10.1126 / scitranslmed.3009246. 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 for 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 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., Investigators of the PLATO trial,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,see Sambrook,J.,Russel,D.W.Molecular Cloning,A Laboratory Manual.3 ed.2001,Cold Spring Harbor,New York:Cold Spring Harbor Laboratory Press

[0123]

Table 4

Claims

1. A method for treating an ischemic event in a patient, comprising administering a therapeutically effective dose of recombinant apirase protein to the patient, The recombinant apirase protein comprises the amino acid sequence described in SEQ ID NO: 2, The method comprises administering the recombinant apirase protein to the patient in a dose of 40 mg to 240 mg.

2. Recombinant apirase protein for use in methods for treating ischemic events in patients, The recombinant apirase protein comprises the amino acid sequence described in SEQ ID NO: 2, The method comprises administering the recombinant apirase protein to the patient in a dose of 40 mg to 240 mg.

3. The method according to claim 1, wherein the method comprises administering the recombinant apirase protein to the patient in a dose of 40 mg to 170 mg, or the recombinant apirase protein for use according to claim 2.

4. The method according to claim 1, wherein the method comprises administering the recombinant apirase protein to the patient in a dose of 100 mg to 240 mg, or the recombinant apirase protein for use according to claim 2.

5. The method according to any one of claims 1 to 4, wherein the method comprises administering the recombinant apirase protein to the patient in a dose of 100 mg to 170 mg, or a recombinant apirase protein for use.

6. The method according to any one of claims 1 to 5, or recombinant apirase protein for use, wherein the method comprises administering the recombinant apirase protein to the patient in a dose of 100 mg to 140 mg, and optionally administering the recombinant apirase to the patient in a dose of 110 mg, 115 mg, or 120 mg.

7. The method according to any one of claims 1 to 6, or a recombinant apirase protein for use, wherein the ischemic event is acute coronary syndrome.

8. The method according to claim 7, or a recombinant apirase protein for use, wherein the ischemic event is ST-elevation myocardial infarction (STEM).

9. The method according to any one of claims 1 to 6, or a recombinant apirase protein for use, wherein the ischemic event is acute ischemic stroke.

10. The recombinant apirase protein, P2Y １２ A recombinant apirase protein for use in combination with an inhibitor, according to any one of claims 1 to 9.

11. The aforementioned P2Y １２ The method according to claim 10, or a recombinant apirase protein for use, wherein the inhibitor is selected from a list consisting of ticagrelor, clopidogrel, ticlopidine, prasugrel, and cangrelol.

12. The aforementioned P2Y １２ The method according to claim 11, or a recombinant apirase protein for use, wherein the inhibitor is ticagrelor, and optionally, the ticagrelor is administered in a loading dose of 60 to 200 mg.

13. The aforementioned P2Y １２ The method according to any one of claims 10 to 12, or a recombinant apirase protein for use, wherein the inhibitor is administered within two hours after the recombinant apirase protein is administered to the patient.

14. The method according to any one of claims 10 to 13, or recombinant apirase protein for use, further comprising administering aspirin to the patient, wherein optionally, the aspirin is administered in a loading dose of 250 to 325 mg.

15. The method according to claim 14, or a recombinant apirase protein for use, wherein the aspirin is administered within two hours after the recombinant apirase protein is administered to the patient.

16. For at least one week after administration of the recombinant apirase protein to the patient, a maintenance dose of P2Y was administered. １２ A method according to any one of claims 10 to 15, further comprising administering an inhibitor and aspirin, or a recombinant apirase protein for use.

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