Recombinant adenosine triphosphate bisphosphatase proteins at doses of 40 MG to 240 MG for use in treatment of ischemic events

Abstract

The present disclosure relates to dosage regimens for administering recombinant adenosine triphosphate bisphosphatase proteins and medical uses thereof in the treatment of ischemic events such as ST segment elevation myocardial infarction and acute ischemic stroke in a patient.

Description

Technical Field

[0001] The present disclosure relates to dosing regimens for administering recombinant apyrase proteins and their medical use in treating ischemic events in patients such as ST-segment elevation myocardial infarction and acute ischemic stroke. The recombinant apyrase proteins can be co-administered with dual antiplatelet therapy comprising a P2Y 12 inhibitor and aspirin. Background Art

[0002] Myocardial infarction (MI) is a leading cause of hospitalization and death worldwide (Asaria et al., 2017). Without treatment, MI causes irreversible damage to the myocardium due to lack of blood flow (ischemia) and thus lack of oxygen. Therefore, the main aim of MI treatment is to accelerate the restoration of normal coronary blood flow, with the aim of reducing myocardial damage through reperfusion therapy. Reperfusion therapy typically involves the combined use of therapeutic agents with surgical techniques such as percutaneous coronary intervention (PCI) to increase blood flow and reduce thrombosis. Early reperfusion and PCI are preferred and are associated with improved outcomes, and guidelines recommend that PCI should be performed within 12 hours of the onset of MI symptoms (Ibanez et al. 2018).

[0003] One commonly used small molecule for reducing thrombosis is aspirin. Another type of small molecule therapeutic agent is a P2Y 12 receptor inhibitor such as clopidogrel, ticagrelor, prasugrel, and cangrelor, which are known to be able to inhibit platelets and prevent blood clots. The co-administration of aspirin and a P2Y 12 receptor inhibitor is called dual antiplatelet therapy (DAPT), which 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 the treatment of adults with acute coronary syndrome (ACS) for up to 12 months. However, these P2Y 12 receptor inhibitors do carry an increased risk of bleeding, and thus care is needed to ensure that patients receive this treatment in accordance with the prescription information and current guidelines.

[0004] Despite optimal antiplatelet and antithrombotic treatment involving P2Y 12 receptor inhibitors such as ticagrelor, there is still a risk of approximately 10% of new myocardial infarction events per year, 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 studies have investigated the use of recombinant apyrases as protein-based therapeutics. Apyrases (ecto-ATP diphosphohydrolases) constitute a group of enzymes that catalyze the metabolism of ATP to ADP and the metabolism of ADP to AMP. In vivo, the AMP generated by the hydrolysis of ATP and ADP induced by apyrases is converted to adenosine by the widely expressed extracellular CD73 / ecto-5'-nucleotidase. The first known human apyrase, CD39, was initially identified as a cell surface protein that activates lymphocytes and endothelial cells, and various in vitro and in vivo studies have shown that apyrases can maintain vascular integrity and physiologically inhibit inflammation and thrombosis (Robson et al. 2005).

[0006] Moeckel et al. (2014) reported the design and production of a recombinant optimized form of soluble CD39L3 (a human CD39 family member). The resulting recombinant protein is referred to as "APT102" or "AZD3366". The authors reported that this recombinant protein has four-fold higher ADPase activity and a 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. WO 2022 / 038191 describes a method for treating ischemic events (such as ST-segment elevation myocardial infarction and acute ischemic stroke) in patients by co-administering AZD3366 with a P2Y12 inhibitor.

[0007] Data on the behavior, safety, and efficacy of recombinant apyrases (including AZD3366), including dosing, in human patients have not been obtained to date. SUMMARY OF THE INVENTION

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

[0009] The inventors have confirmed through human clinical trials that when administered to humans, AZD3366, used alone or in combination with aspirin and ticagrelor, is generally safe and well-tolerated. In addition, the in vivo pharmacokinetic and safety data obtained from human trials were combined with the preclinical efficacy data obtained from animal models to predict the doses of AZD3366 that are expected to achieve effective cardioprotection in humans without compromising patient safety. Additionally, it has been confirmed that these doses achieve complete platelet inhibition in humans without a significant increase in bleeding-related adverse events, indicating further clinical benefits resulting from the administration of AZD3366 in the treatment of ischemic events.

[0010] Accordingly, when administered to humans for the treatment of ischemic events, including the treatment of acute coronary syndromes such as ST-segment elevation myocardial infarction and the treatment of acute ischemic stroke, it is expected that the administration of the described doses of AZD3366 will provide clinical beneficial effects.

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

[0012] In another aspect, the present disclosure provides a recombinant apyrase protein for use in a method of treating an ischemic event in a patient, wherein the method comprises administering the recombinant apyrase protein to the patient at a dose of 40 mg to 240 mg.

[0013] In another aspect, the present disclosure provides the use of recombinant apyrase protein in the manufacture of a medicament for treating an ischemic event in a patient, the treatment comprising administering the recombinant apyrase protein to the patient at a dose of 40 mg to 240 mg.

[0014] The recombinant apyrase protein may comprise the amino acid sequence as shown in SEQ ID NO:2 (AZD3366).

[0015] The recombinant apyrase protein (e.g., AZD3366) may be administered to a patient at a dose of 40 mg to 170 mg, 40 mg to 150 mg, 40 mg to 140 mg, or 40 mg to 100 mg.

[0016] The recombinant apyrase protein (e.g., AZD3366) may be administered to a patient at a dose of 100 mg to 240 mg, 100 mg to 220 mg, or 100 mg to 200 mg.

[0017] Recombinant apyrase protein (such as AZD3366) can be administered to a patient at a dose of 100 mg to 140 mg, such as at a dose of 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, or 140 mg. In one embodiment, the recombinant apyrase protein (such as AZD3366) is administered to a patient at a dose of 115 mg.

[0018] The recombinant apyrase protein can be administered to a patient by intravenous injection.

[0019] In some embodiments, the ischemic event being treated is acute coronary syndrome. Acute coronary syndrome includes ST-segment elevation myocardial infarction (STEMI), non-ST-segment elevation myocardial infarction (NSTEMI), and unstable angina. In some embodiments, the acute coronary syndrome being treated is ST-segment elevation myocardial infarction (STEMI) in the patient. In other embodiments, the ischemic event being 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. Thus, in some embodiments, the recombinant apyrase protein is administered in combination with a P2Y 12 inhibitor and / or aspirin. In some embodiments, the recombinant apyrase protein is administered in combination with dual antiplatelet therapy (DAPT), where DAPT includes administration with both a P2Y 12 inhibitor and aspirin. A skilled physician or other skilled medical personnel can determine the most suitable manner of administering each therapeutic agent to the patient, and exemplary doses and treatment regimens are further described herein.

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

[0022] In the case where the recombinant apyrase protein is administered in combination with a P2Y 12 inhibitor and / or aspirin, the different agents are generally administered as separate formulations administered sequentially. In the case of sequential administration, the recombinant apyrase protein can be administered after administration of a loading dose of P2Y 12Administration within 12 hours, 6 hours, or 2 hours of the inhibitor and / or aspirin.

[0023] After administration of recombinant apyrase protein and administration of a loading dose of P2Y 12 After administration of the inhibitor and / or aspirin, a maintenance dose of P2Y 12 Inhibitor and aspirin (e.g., for at least 2 days, at least one week, at least 6 weeks, or at least 6 months). As demonstrated herein, although an increase in capillary bleeding time (CBT) was observed in humans upon administration of a certain dose of AZD3366 with a loading dose of ticagrelor and aspirin, this increase tended to plateau and became insignificant with continued administration of the maintenance dose of ticagrelor and aspirin.

[0024] The present disclosure includes combinations of the described aspects and preferred features, unless such combinations are clearly impermissible or expressly avoided. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying drawings, wherein:

[0026] Figure 1 Illustrates the percentage of platelet aggregation inhibition relative to baseline over time in humans administered different doses (10 mg, 30 mg, 90 mg, 180 mg, 360 mg, and 640 mg) of AZD3366 (mean ± 1 SEM error bars). Platelet aggregation in platelet-rich plasma (PRP) was quantified using the light transmission aggregometry (LTA) assay further described herein. Complete platelet inhibition was observed at 10 mg and above doses, with a dose-dependent duration.

[0027] Figure 2 Illustrates capillary bleeding time (CBT) data obtained from humans administered different doses (2 mg, 10 mg, 30 mg, 90 mg, 180 mg, 360 mg, and 640 mg) of AZD3366. No significant increase in CBT was observed at dose levels up to 90 mg.

[0028] Figure 3Illustrated are 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 routine clinical practice, as further described in the Examples. Baseline, A, B, and C represent time points at which CBT was measured. The baseline plot provides a CBT measurement prior to administration of any of aspirin, AZD3366, or ticagrelor (0002:30 PRE, or 2 hours 30 minutes prior to administration of AZD3366). Plot A provides a CBT measurement taken after administration of AZD3366 or placebo and a loading dose of 324 mg aspirin (0000:10 POST, meaning a CBT measurement taken 10 minutes after administration of AZD3366 or placebo). Plot B provides a CBT measurement taken after administration of AZD3366 or placebo and a loading dose of 324 mg aspirin and 180 mg ticagrelor (0003:40 POST, meaning a CBT measurement taken 3 hours 40 minutes after administration of AZD3366 or placebo). Plot C provides a CBT measurement taken after administration of AZD3366 or placebo, a loading dose of 324 mg aspirin and 180 mg ticagrelor, and maintenance doses of 81 mg aspirin and 90 mg ticagrelor (0051:40 POST, meaning a CBT measurement taken 51 hours 40 minutes after administration of AZD3366). When loading doses of ASA and ticagrelor were administered, CBT in the AZD3366 group increased significantly compared to placebo, but this increase plateaued when healthy volunteers continued to receive maintenance doses of DAPT. Detailed Description

[0029] Aspects and embodiments of the present disclosure will now be discussed with reference to the accompanying figures. Additional aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

[0030] Recombinant Apyrase Protein

[0031] Adenosine triphosphate diphosphatase (EC 3.6.1.5) catalyzes the hydrolysis of the phosphoanhydride bond of adenosine triphosphate (ATP) to adenosine monophosphate (AMP) and catalyzes the hydrolysis of the phosphoanhydride bond of adenosine diphosphate (ADP) to AMP. CD39 family members (also known as extracellular nucleotide triphosphate diphosphohydrolase (E-NTPDase) family members) represent some of the most well-characterized adenosine triphosphate diphosphatases. Human CD39 family members include the native proteins listed in the following table:

[0032] Name of the native protein Alternative names NCBI accession number CD39 ATPDase, ecto-apyrase, NTPDase 1 U87967.1 CD39L1 NTPDase 2, ecto-ATPase AF144748.1 CD39L2 NTPDase 6 AY327581.1 CD39L3 NTPDase 3, CD39L3, HB6 AF034840.2 CD39L4 NTPDase 5, ER-UDPase, PCPH AF039918.1 LALP70 UDPase, NTPDase 4 AF016032.1 LALP1 NTPDase 7 AF269255.1 Hepatocytic canalicular ecto-ATPase NTPDase 8, hATPDase AY430414.1

[0033] Human CD39L3 is a protein of 529 amino acid residues as set forth in SEQ ID NO:1. A specific exemplary soluble CD39L3 adenosine triphosphate diphosphatase protein has the amino acid sequence as set forth in positions 49 - 485 of SEQ ID NO:1.

[0034] The design, production and use of soluble recombinant adenosine triphosphate diphosphatase proteins (including engineered forms of CD39L3) such as enhanced adenosine triphosphate diphosphatase are described in US7247300B1, EP2133430B1 and EP2523971B1, all of which patents are incorporated herein by reference in their entireties. The recombinant adenosine triphosphate diphosphatase proteins described herein can be any of the adenosine triphosphate diphosphatases described in those publications.

[0035] EP2133430B1 describes ADPase - enhanced adenosine triphosphate diphosphatases. These ADPase - enhanced adenosine triphosphate diphosphatases include modified forms of a reference adenosine triphosphate diphosphatase, wherein the modification results in increased ADPase activity compared to the reference adenosine triphosphate diphosphatase, or the same ADPase activity as the reference adenosine triphosphate diphosphatase, and decreased ATPase activity compared to the reference adenosine triphosphate diphosphatase. Exemplary ADPase - enhanced adenosine triphosphate diphosphatases include those that contain substitutions at positions 67 and 69 of CD39L3, wherein the position numbering is consistent with SEQ ID NO:1. Specifically, ADPase - enhanced adenosine triphosphate diphosphatases include Protein 8742 that contains R67G and T69R substitutions; and Protein 8906 that contains R67A and T69R substitutions.

[0036] Exemplary ATPase and ADPase assays for determining such activity are disclosed in EP2133430B1. For example, the ATPase and ADPase activities of purified soluble ADPase-enhanced apyrase can be assayed at 37 °C in 1 ml of a 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) (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 micromole of inorganic phosphate per minute at 37 °C. Key kinetic constants of the enzyme such as Km and kcat can be obtained by fitting the data to, for example, the Michaelis-Menten equation. Other assays that can be used to monitor biochemical function include, but are not limited to, radiometric assays, HPLC assays (J. Clin Invest. (1998) 101:1851-1859) or radio-TLC assays (J. Clin Invest. (1991) 88:1690-1696), all described by Gayle Ill et al. or by Marcus, A.J. et al., respectively.

[0037] Thus, compared to a reference apyrase having the amino acid sequence shown in positions 49-485 of SEQ ID NO:1 (i.e., the soluble form of CD39L3), the recombinant apyrase proteins described herein can contain one or more modifications (e.g., amino acid substitutions). These modifications can result in increased ADPase activity compared to the reference apyrase, or the same ADPase activity as the reference apyrase, and decreased ATPase activity compared to the reference apyrase. In some embodiments, one or more modifications can comprise 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 can be glycine, the substitution at position 69 can be arginine, or the substitution at position 67 can be alanine and the substitution at position 69 can be arginine. In some embodiments, the substitution at position 67 is a glycine substitution and the substitution at position 69 is an arginine substitution.

[0038] EP2523971B1 describes adenosine triphosphate diphosphatase and methods for producing adenosine triphosphate diphosphatase comprising a homogeneous N-terminus, such that more than 80% of the adenosine triphosphate diphosphatase molecules have the same N-terminus comprising EVLP. These proteins with a homogeneous N-terminus are described as having an average isoelectric point in the range of 3.0 to 4.5 and / or an extended half-life in rabbits and pigs.

[0039] Thus, as described in EP2523971B1, the recombinant adenosine triphosphate diphosphatase proteins described herein may comprise a homogeneous N-terminus such that more than 80% of the adenosine triphosphate diphosphatase molecules have the same N-terminus, which is EVLP.

[0040] In certain embodiments, the recombinant adenosine triphosphate diphosphatase protein may further comprise one or more functionally conservative substitutions (e.g., in addition to the substitutions in the ADPase-enhanced adenosine triphosphate diphosphatase described above). Functionally conservative substitutions are substitutions that do not affect (or substantially do not affect) one or more functional properties (such as enzyme activity) compared to the equivalent unsubstituted protein. In some embodiments, the recombinant adenosine triphosphate diphosphatase 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.

[0041] In some embodiments, the recombinant adenosine triphosphate diphosphatase 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 glycine and the amino acid residue at position 69 is arginine, where these positions are numbered according to SEQ ID NO:1. For example, the recombinant adenosine triphosphate diphosphatase protein may comprise the amino acid sequence of SEQ ID NO:2 having 1 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 glycine and the amino acid residue at position 69 is arginine, where these positions are numbered according to SEQ ID NO:1.

[0042] In an exemplary embodiment, the recombinant adenosine triphosphate diphosphatase protein comprises the amino acid sequence of SEQ ID NO:2 (AZD3366).

[0043] Treatment of ischemic events

[0044] Methods and products for use in the uses described herein for treating an ischemic event in a patient (such as a human patient), the ischemic event including diseases and disorders in which blood supply is restricted to a particular part of the patient's body (such as the patient's heart or brain), where the restriction can be caused by a blood clot (thrombus). Ischemic events of the heart include acute coronary syndromes. Ischemic events in the brain include acute ischemic stroke (AIS). Acute coronary syndromes include myocardial infarctions classified as ST-segment elevation myocardial infarction (STEMI) or non-ST-segment elevation myocardial infarction (NSTEMI) and unstable angina. In some embodiments, the treatment is for a patient's ST-segment elevation myocardial infarction (STEMI).

[0045] Myocardial infarctions are typically clinically classified as STEMI and NSTEMI. These are based on changes in an electrocardiogram (ECG) and can be diagnosed by a physician or other skilled medical personnel. The type of myocardial infarction can be defined or derived from the universal definition of myocardial infarction proposed by Thygesen et al. in 2018.

[0046] In some embodiments, recombinant apyrase protein is administered to the patient within 18 hours or less, or 12 hours or less, or 6 hours or less, or 4 hours or less, or 2 hours or less, or 1 hour or less, or even 30 minutes or less after the onset of an ischemic event (such as an acute coronary syndrome, such as STEMI). As mentioned herein, the onset of an ischemic event can be at 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 an acute coronary syndrome), and the diagnosis can be made before or shortly after the patient arrives at a hospital (or its equivalent) for treatment.

[0047] In some embodiments, recombinant apyrase protein is administered to a patient before surgical reperfusion treatment (such as percutaneous coronary intervention (PCI)) of a patient with an acute coronary syndrome.

[0048] PCI can include, but is not limited to, balloon angioplasty, stent implantation, rotational atherectomy, or laser atherectomy and / or brachytherapy. In the case of implanting a stent, the stent can be, but is not limited to, a bare metal stent, a drug-eluting stent, an absorbable stent, etc., as known in the art.

[0049] By P2Y 12The cardioprotective effect provided by the inhibitor can be used to prevent and / or mitigate damage to cardiac tissue or function caused by the restoration of circulation after reperfusion therapy. Thus, in some embodiments, the method further comprises subjecting the patient to surgical reperfusion therapy (e.g., PCI) within less than 48 hours, less than 24 hours, less than 12 hours, or less than 6 hours after administration of the recombinant apyrase protein.

[0050] Treatment of acute coronary syndrome can be demonstrated by reduction of the infarct size in the patient and / or by restoration of blood flow to the affected area. Additionally, treatment with the recombinant apyrase protein can result in inhibition of platelet aggregation in the patient. As demonstrated herein, AZD3366 achieved complete platelet inhibition at all tested doses, with a dose-dependent sustained effect reported. Skilled practitioners will understand methods of monitoring the success of treatment of acute coronary syndrome, such as based on their knowledge of using known P2Y 12 inhibitors to treat acute coronary syndrome.

[0051] In other embodiments, the ischemic event being treated is acute ischemic stroke. In a population of 70,000, there are 100 new patients with ischemic stroke each year. Without treatment, 55 patients die or become dependent within one year. The majority of the 100 patients have a mild or transient stroke and only receive antiplatelet drugs to reduce recurrence. Approximately 25 to 35 patients receive reperfusion therapy, which spares 5 to 6 patients from death or dependence and increases the number of patients without disability. Thus, reducing the morbidity and mortality associated with acute ischemic stroke (AIS) is an unmet clinical need.

[0052] Preclinical and clinical trials have evaluated the use of P2Y 12 inhibitors and aspirin for the treatment and / or prevention of stroke after ischemic stroke events. Additionally, preclinical data have demonstrated that the recombinant apyrase protein AZD3366 can be used to enhance reperfusion, reduce re-occlusion, and reduce intracerebral hemorrhage in animal models of ischemic stroke (Sun et al., 2011; Tan et al., 2014). Thus, it is recognized that the beneficial outcomes associated with administration of AZD3366 (optionally in combination with a P2Y 12 inhibitor and / or aspirin) in the preclinical myocardial infarction animal model described herein may also be beneficial for the treatment of ischemic stroke. For example, compared to a P2Y 12 inhibitor alone, the treatment can reduce the infarct size and / or reduce brain injury in patients with ischemic stroke and achieve this effect without significantly increasing the risk of bleeding.

[0053] As used herein, the term "treatment" in the context of treating a medical condition generally refers to the treatment and therapy of a human, in which some desired therapeutic effect is achieved, such as inhibiting the progression of the medical condition, and includes a reduction in the rate of progression, a halt in the rate of progression, regression of the medical condition, improvement of the medical condition, and cure of the medical condition. Treatment as a preventive measure (i.e., prophylaxis, prevention) is also included.

[0054] Dose and treatment regimen

[0055] Administration of the recombinant apyrase protein described herein can be carried out by, for example, bolus injection, intravenous, intramuscular, subcutaneous, inhalation, continuous infusion, sustained release, or other pharmaceutically acceptable techniques. As carried out in the AZD3366 Phase I study reported in the examples, the recombinant apyrase protein described herein can be administered to patients by intravenous injection.

[0056] The recombinant apyrase protein can be administered to patients at a dose of 20 mg to 640 mg. As described herein, doses of AZD3366 within this range are safe and well tolerated when administered to humans.

[0057] In some cases, the recombinant apyrase protein can be administered to a patient at a dose of 40 mg to 240 mg, 50 mg to 240 mg, 60 mg to 240 mg, 70 mg to 240 mg, 80 mg to 240 mg, 90 mg to 240 mg, 100 mg to 240 mg, 110 mg to 240 mg, 120 mg to 240 mg, 130 mg to 240 mg, 140 mg to 240 mg, 150 mg to 240 mg, 160 mg to 240 mg, 170 mg to 240 mg, 180 mg to 240 mg, 190 mg to 240 mg, or 200 to 240 mg. In some cases, the recombinant apyrase protein can be administered at a dose of 50 mg to 200 mg, 60 mg to 200 mg, 70 mg to 200 mg, 80 mg to 200 mg, 90 mg to 200 mg, 100 mg to 200 mg, 110 mg to 200 mg, 120 mg to 200 mg, 130 mg to 200 mg, 140 mg to 200 mg, 150 mg to 200 mg, 160 mg to 200 mg, 170 mg to 200 mg, or 180 mg to 200 mg. In some cases, the recombinant apyrase protein can be administered at a dose of 50 mg to 180 mg, 60 mg to 180 mg, 70 mg to 180 mg, 80 mg to 180 mg, 90 mg to 180 mg, 100 mg to 180 mg, 110 mg to 180 mg, 120 mg to 180 mg, 130 mg to 180 mg, 140 mg to 180 mg, 150 to 180 mg, or 160 to 180 mg. No significant adverse events were observed at these doses, and a greater therapeutic (e.g., cardioprotective) effect is expected at higher doses.

[0058] In some cases, the recombinant apyrase protein can be administered to a patient at a dose of less than 180 mg. As demonstrated herein, although doses of AZD3366 of 180 mg or greater did not elicit any significant adverse events, an increase in capillary bleeding time (CBT) was measured in healthy volunteers. Although CBT is not a definitive marker of bleeding in a clinical setting, it may indicate increased bleeding. For example, the recombinant apyrase protein can be administered to a patient at a dose of 40 mg to 170 mg, 50 mg to 170 mg, 60 mg to 170 mg, 70 mg to 170 mg, 80 mg to 170 mg, 90 mg to 170 mg, 100 mg to 170 mg, 110 mg to 170 mg, 120 mg to 170 mg, 130 mg to 170 mg, 140 mg to 170 mg, 150 mg to 170 mg, or 160 mg to 170 mg. As another example, the recombinant apyrase protein can be administered to a patient at a dose 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, the recombinant apyrase protein can be administered to a patient at a dose 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 a further example, the recombinant apyrase protein can be administered to a patient at a dose 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.

[0059] In some cases, the recombinant apyrase protein can be administered at a dose greater than 90 mg. As demonstrated herein, there is a dose-dependent duration associated with the inhibition of platelet aggregation caused by AZD3366, and a dose greater than 90 mg results in a significantly more persistent inhibition of platelet aggregation. For example, the recombinant apyrase protein can be administered to a patient at a dose of 100 mg to 240 mg, 100 mg to 230 mg, 100 mg to 220 mg, 100 mg to 210 mg, 100 mg to 200 mg, 100 mg to 190 mg, or 100 mg to 180 mg. It should also be understood that a dose greater than 90 mg can be combined with the above doses less than 180 mg. Thus, in some cases, the recombinant apyrase protein is administered to a patient at a dose of 100 mg to 170 mg, 100 mg to 160 mg, or 100 mg to 150 mg.

[0060] Furthermore, without wishing to be bound by theory, by combining in vivo pharmacokinetic and safety data obtained from human trials with preclinical efficacy data using animal models, it is believed that a dose of AZD3366 between 100 mg and 140 mg may represent the optimal range between achieving an effective therapeutic effect (e.g., cardioprotection and / or antithrombosis) without compromising patient safety. In particular, this dose range is based on achieving a similar plasma exposure in humans as in porcine animal studies when a significant improvement in infarct area is observed.

[0061] Thus, in some cases, recombinant apyrase is administered to a patient at a dose of 100 mg to 140 mg, 100 mg to 135 mg, 100 mg to 130 mg, 105 mg to 140 mg, 105 mg to 135 mg, 105 mg to 130 mg, 110 mg to 140 mg, 110 mg to 135 mg, or 110 mg to 130 mg. In some cases, recombinant apyrase is administered to a patient at a dose of 105 mg to 125 mg, i.e., at any one of 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 apyrase is administered to a patient at a dose of 110 mg to 120 mg, i.e., at any one of 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 example, recombinant apyrase is administered to a patient at a dose of 110 mg. In another example, recombinant apyrase is administered to a patient at a dose of 115 mg. In additional examples, recombinant apyrase is administered to a patient at a dose of 120 mg.

[0062] The exact dosage requirements for the recombinant apyrase protein can vary according to age, race, weight, height, gender, duration of treatment, method of administration, the biological activity of the recombinant apyrase protein, and the severity of the medical condition or other clinical variables. While exemplary dosages are provided above, other effective dosages within the scope disclosed herein can be determined by a skilled physician or other skilled medical personnel.

[0063] As described above, recombinant apyrase is typically administered at a flat dose, i.e., a dose not based on the individual body weight of the patient. Alternatively, recombinant apyrase can be administered at a dose calculated based on the body weight of the patient in kilograms (kg). For example, in a subject with a body weight of 60 kg, a flat dose of 120 mg corresponds to 2 mg / kg.

[0064] In some embodiments, the patient being treated has a body weight between 50 kg and 100 kg. In some embodiments, the patient being treated has a body mass index (BMI) between 18 kg / m 2 and 30 kg / m 2 .

[0065] In some embodiments, the patient being treated is a Chinese or Japanese subject. A Chinese subject refers to a male or female Chinese whose parents and grandparents are both Chinese and who has lived outside of China for no more than 10 years. A Japanese subject refers to a male or female Japanese whose parents and grandparents are both Japanese and who has lived outside of China for no more than 10 years.

[0066] The recombinant apyrase protein can be administered as a pharmaceutical composition comprising the recombinant apyrase protein and a pharmaceutically acceptable carrier or diluent.

[0067] As used herein, the term "pharmaceutically acceptable" pertains to compounds, ingredients, materials, compositions, dosage forms, etc. that are suitable, within the scope of reasonable medical judgment, for contact with the tissues of the subject being discussed (e.g., a human) without undue toxicity, irritation, allergic response, or other problems or complications, and that are 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. Such diluents and excipients can consist of a neutral buffered salt solution, an antioxidant (e.g., ascorbic acid), a low molecular weight polypeptide (e.g., a polypeptide of < 10 amino acids), amino acids, carbohydrates (e.g., glucose, dextrose, sucrose, or dextran), a chelating agent (such as EDTA), a stabilizer (such as glutathione). Additionally, a cosubstrate of the recombinant apyrase protein, such as calcium (Ca 2+ ), can be administered at a dose that achieves maximal enzyme activity. Such carriers and diluents are selected to be non-toxic to the patient at the recommended doses and concentrations.

[0068] Combination therapy

[0069] In some of the treatment methods described herein, the recombinant apyrase protein is administered in combination with a P2Y 12 inhibitor and / or aspirin. As used herein, the term "in combination" means that after administration (e.g., within 30 minutes, or within one hour, or within 2 hours, or within 3 hours), the P2Y 12 inhibitor and / or aspirin (and / or its metabolites) and the recombinant apyrase protein are bioavailable (i.e., have an active effect) in the bloodstream of the patient. In an animal model, administration of certain doses of the recombinant protein AZD3366 renders it active within 5 minutes and it does not return to baseline for 3 to 4 weeks, while P2Y 12 inhibitors generally take longer to become active after administration. For example, the maximal activity of the P2Y 12 inhibitor ticagrelor is generally observed about 2 hours after dosing and this is maintained for more than 8 hours.

[0070] The P2Y disclosed herein12 The inhibitor can be selected from the list consisting of ticagrelor, clopidogrel, ticlopidine, prasugrel, and cangrelor. The P2Y 12 inhibitor mentioned herein includes any one of these compounds and any metabolites (such as active metabolites). In some embodiments, the P2Y 12 inhibitor can 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 some embodiments, the P2Y 12 inhibitor can be ticagrelor or clopidogrel. In some embodiments, the P2Y 12 inhibitor is ticagrelor.

[0071] 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 a reversible-binding oral P2Y(12) receptor antagonist developed for the prevention of thrombotic events in patients with acute coronary syndrome. It has the following chemical structure:

[0072]

[0073] Ticagrelor is the active ingredient in a drug product known as (or BRILIQUE in Europe), which has been approved for use in multiple jurisdictions, including the United States and Europe. Ticagrelor is currently sold in the form of 60 mg and 90 mg immediate-release tablets. WO 2008 / 024045 discloses certain pharmaceutical formulations containing ticagrelor for oral administration. WO 2017 / 182589 discloses a fast-disintegrating oral dosage form of ticagrelor.

[0074] Ticagrelor is usually rapidly absorbed after oral administration. Different from clopidogrel and prasugrel, ticagrelor is not a prodrug and does not require metabolic activation to exert its activity. Ticagrelor is still widely metabolized, and ticagrelor and its active and approximately equivalent metabolites (AR-C124910XX) constitute the main circulating components in plasma. The plasma concentrations of ticagrelor and its active metabolite increase in a dose-dependent manner; peak concentrations are reached within about 1.5 hours and 2.5 hours, respectively. Maximum inhibition of platelet aggregation is observed approximately 2 hours after dosing, and this is maintained for more than 8 hours after dosing. The average elimination half-lives of ticagrelor and its active metabolite are described as 7 hours and 9 hours, respectively, in the drug label. After discontinuation of the drug, platelet activity returns to baseline after 5 days.

[0075] In some embodiments, ticagrelor is orally administered in a loading dose of 180 mg in combination with recombinant apyrase protein. Ticagrelor can be administered in the form of an orally disintegrating tablet (ODT), such as described in WO 2017 / 182589. One or more subsequent maintenance doses can be administered after the loading dose, for example, without administering recombinant apyrase protein. As described below, after an initial loading dose of 180 mg, the prescribing information for ticagrelor describes a maintenance dose of 90 mg administered twice daily during the first year after an ACS event, and 60 mg administered twice daily after one year. One or more subsequent maintenance doses can include two daily doses of 90 mg of ticagrelor, or two daily doses of 60 mg of ticagrelor. The prescribing information further describes ticagrelor being administered with aspirin having a daily maintenance dose of 75 mg to 100 mg.

[0076] Accordingly, one or more subsequent maintenance doses can also include administering aspirin having a daily dose of 75 mg to 100 mg.

[0077] P2Y 12 The P2Y inhibitor can be clopidogrel. Clopidogrel is typically administered via the oral route. In some embodiments, clopidogrel is administered in a loading dose of 300 mg or 600 mg in combination with recombinant apyrase protein. One or more subsequent maintenance doses can comprise approximately 75 mg of clopidogrel and can be administered after the loading dose, for example, without administering recombinant apyrase protein. Similar to ticagrelor described above, the maintenance dose of clopidogrel can be administered with aspirin having a daily dose of 75 mg to 100 mg.

[0078] Clopidogrel is a prodrug and its activity requires metabolic activation. The peak plasma concentration of the active metabolite occurs approximately 30 minutes to 60 minutes after oral administration, and dose-dependent platelet aggregation inhibition is observed approximately 2 hours after administration. Dose-dependent platelet aggregation inhibition can be observed within 2 hours after a single oral dose. Clopidogrel has an elimination half-life of approximately 6 hours after a single dose of 75 mg, while its active metabolite has an elimination half-life of approximately 30 minutes. After discontinuation, platelet aggregation and bleeding time gradually return to baseline within about 5 days.

[0079] P2Y 12The inhibitor can be prasugrel. Prasugrel is typically administered via the oral route. In some embodiments, prasugrel is administered in a loading dose of 60 mg in combination with recombinant apyrase protein. One or more subsequent maintenance doses may comprise about 5 mg or 10 mg of prasugrel and may be administered after the loading dose, for example, without administering recombinant apyrase protein. Similar to ticagrelor described above, the maintenance dose of prasugrel can be administered with aspirin at a daily dose of 75 mg to 100 mg.

[0080] Prasugrel is a prodrug and is rapidly metabolized to a pharmacologically active metabolite. The peak plasma concentration of the active metabolite occurs approximately 30 minutes after dosing. It has an elimination half-life of about 7.4 hours.

[0081] P2Y 12 The inhibitor can 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 in a loading dose of 500 mg in combination with recombinant apyrase protein. One or more subsequent maintenance doses may comprise about 250 mg of ticlopidine and may be administered after the loading dose, for example, without administering recombinant apyrase protein. Similar to ticagrelor described above, the maintenance dose of ticlopidine can be administered with aspirin at a daily dose of 75 mg to 100 mg.

[0082] The peak plasma level of ticlopidine is typically observed about 2 hours after oral administration. The half-life after a single dose ranges from 7 hours to 13 hours. The half-life after repeated dosing is about 4 days to 5 days.

[0083] P2Y 12 The P2Y-receptor inhibitor can be cangrelor. Cangrelor can be administered intravenously by bolus injection, continuous infusion, or a combination of bolus injection followed by continuous infusion. In some embodiments, cangrelor is administered as a 30 μg / kg intravenous bolus injection, followed immediately by a 4 μg / kg / min intravenous infusion.

[0084] Cangrelor rapidly reaches steady-state plasma levels and platelet aggregation inhibition within 30 minutes after the start of infusion, and has a short plasma half-life, approximately less than 9 minutes. Maximum platelet inhibition is achieved within 15 minutes. The elimination half-life of cangrelor is about 3 minutes to 6 minutes, and platelet responsiveness typically returns to baseline within 15 minutes after discontinuation of the drug.

[0085] P2Y 12 The inhibitor can be administered as a pharmaceutical composition comprising a P2Y 12 inhibitor and a pharmaceutically acceptable carrier or diluent.

[0086] For the combined administration of recombinant apyrase protein and a P2Y 12 inhibitor, the recombinant apyrase protein and the P2Y 12 inhibitor do not always have to be physically administered simultaneously. Instead, one of the recombinant apyrase protein and the P2Y 12 inhibitor can be administered first, and then the other agent can be administered later (e.g., one hour or later), provided that both the recombinant apyrase protein and the P2Y 12 inhibitor are bioavailable in the patient's bloodstream after administration.

[0087] In addition, some patients presenting with ischemic events may already be regularly administered a P2Y 12 inhibitor, for example as part of a maintenance dose after a previous ischemic event. Such patients are referred to herein as "currently being treated with a P2Y 12 inhibitor". For example, a 90 mg dose of ticagrelor is typically administered twice daily as part of a maintenance dose. In such patients being administered recombinant apyrase protein according to the methods herein, it may not always be necessary to administer another dose of the P2Y 12 inhibitor, as the inhibitor (and / or its metabolite) is still considered to be biologically active in the patient's bloodstream. Alternatively, a reduced dose of the P2Y 12 inhibitor compared to the normal loading dose can be administered to increase the level of the biologically active P2Y 12 inhibitor in the bloodstream. For example, in the case of ticagrelor, if ticagrelor is still biologically active in the patient's bloodstream, a dose of 60 mg or 90 mg or 150 mg can be administered instead of the typical loading dose of 180 mg. The effective dose can be determined by a skilled physician or other skilled medical personnel. In some embodiments, even if the patient is currently undergoing treatment with a P2Y 12 inhibitor, the method still includes administering a P2Y 12 inhibitor (e.g., a loading dose) to the patient.

[0088] If the last dose of the inhibitor is administered within a period corresponding to more than twice the average elimination half-life of the P2Y 12 inhibitor, more than three times the average elimination half-life of the P2Y 12 inhibitor, or more than five times the average elimination half-life of the P2Y 12 inhibitor, or more than five times the average elimination half-life of the P2Y 12 inhibitor, then the P2Y 12The inhibitor is biologically active in the patient's bloodstream. For example, in the case of ticagrelor, the mean elimination half-life of ticagrelor is 7 hours, and the mean elimination half-life of its active metabolite is 9 hours. Thus, ticagrelor can be considered to be biologically active in the patient's bloodstream if the last dose was 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, the P2Y 12 inhibitor can be considered to be biologically active in the bloodstream until the activity returns to baseline after dosing is stopped, which in the case of ticagrelor, occurs after 5 days.

[0089] Other patients who exhibit ischemic events may not have previously received a P2Y 12 inhibitor, or may have had a previous P2Y 12 inhibitor administration interrupted, such that the P2Y 12 inhibitor is no longer considered to be biologically active in the patient's bloodstream. These patients can be referred to as "untreated" patients. In untreated patients, in order to co-administer recombinant apyrase protein with a P2Y 12 inhibitor, the method must include the step of administering a P2Y 12 inhibitor to the patient.

[0090] The administration of the P2Y 12 inhibitor described herein will depend on the specific P2Y 12 inhibitor used. For example, ticagrelor, clopidogrel, ticlopidine, and prasugrel are typically administered to patients in pharmaceutically acceptable oral dosage forms, while cangrelor is typically administered to patients via intravenous injection.

[0091] Recombinant apyrase protein and a P2Y 12 inhibitor can be administered as a combination formulation, such as via intravenous injection. Alternatively, the administration of recombinant apyrase protein and a P2Y 12 inhibitor to the patient can be simultaneous or sequential. As used herein, simultaneous administration refers to administering both recombinant apyrase protein and a P2Y 12 inhibitor to the patient at substantially the same time (e.g., within 10 minutes of each other, within 5 minutes of each other, or within 1 minute of each other), optionally via different routes of administration. For example, intravenous injection of recombinant apyrase protein within 1 minute of oral administration of a P2Y 12 inhibitor would be considered simultaneous administration.

[0092] When sequential administration is used, recombinant apyrase protein and a P2Y 12The inhibitors can be administered within 18 hours, 12 hours, 6 hours, or 2 hours of each other. In some embodiments, the recombinant apyrase protein is administered first, followed sequentially by the P2Y 12 inhibitor. As exemplified herein, the recombinant apyrase protein can be administered to a patient, and the P2Y 12 inhibitor (e.g., ticagrelor) can be administered within 2 hours (e.g., 1 hour and 40 minutes) later. In other embodiments, the P2Y 12 inhibitor is administered first, followed sequentially by the recombinant apyrase protein.

[0093] In some embodiments, both the recombinant apyrase protein and the P2Y 12 inhibitor are administered to the patient before a surgical reperfusion treatment (e.g., PCI). In other embodiments, the recombinant apyrase protein is administered to the patient before a surgical reperfusion treatment (e.g., PCI), and the P2Y 12 inhibitor is administered within 6 hours, 4 hours, 2 hours, or 1 hour, e.g., shortly after the surgical reperfusion treatment. In some embodiments, both the recombinant apyrase protein and the P2Y 12 inhibitor remain bioavailable in the patient's bloodstream during a surgical reperfusion treatment (e.g., PCI).

[0094] The present disclosure includes embodiments that combine any timing of i) simultaneously or sequentially administering a recombinant apyrase polypeptide and a P2Y 12 inhibitor; ii) a timing of administration related to the onset of an ischemic event; and iii) a timing of administration related to a surgical reperfusion treatment (e.g., PCI). For example, the method can include administering the recombinant apyrase protein within 6 hours of the onset of an ischemic event, wherein the method further includes performing a surgical reperfusion treatment (e.g., PCI) on the patient within less than 12 hours or less than 6 hours, and wherein the P2Y 12 inhibitor is administered to the patient within 6 hours of administering the recombinant apyrase protein, optionally wherein both the recombinant apyrase protein and the P2Y 12 inhibitor are administered to the patient before the surgical reperfusion treatment (e.g., PCI).

[0095] Any method described herein can further include administering aspirin to the patient. Aspirin is typically administered as a formulation separate from the P2Y 12 inhibitor and the recombinant apyrase protein, and is administered simultaneously or sequentially with one or both of the P2Y 12 inhibitor and the recombinant apyrase protein. In some embodiments, the aspirin is administered after administering the P2Y 12Aspirin is administered within 24 hours, 18 hours, 12 hours, 6 hours, 2 hours, 1 hour, or 30 minutes of the inhibitor. In some embodiments, aspirin is administered within 24 hours, 18 hours, 12 hours, 6 hours, 2 hours, 1 hour, or 30 minutes of the administration of recombinant apyrase protein. As exemplified herein, aspirin can be administered to a patient, followed by recombinant apyrase protein 2 hours later, and then P2Y 12 inhibitor (e.g., ticagrelor) within 2 hours (e.g., 1 hour and 40 minutes) of the administration of recombinant apyrase protein.

[0096] In some embodiments, aspirin is co-administered with P2Y 12 inhibitor or recombinant apyrase protein. In some embodiments, aspirin and P2Y 12 inhibitor are co-administered to a patient after the onset of an ischemic event (e.g., acute coronary syndrome such as STEMI) within 18 hours or less, or 12 hours or less, or 6 hours or less, or 4 hours or less, or 2 hours or less, or 1 hour or less, or even 30 minutes or less.

[0097] Aspirin can be administered to a patient at a dose between 50 mg and 325 mg, such as 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 at a dose between 50 mg and 200 mg, or between 100 mg and 200 mg, such as 162 mg. In some embodiments, aspirin is administered to a patient at a loading dose between 200 mg and 350 mg, or between 250 mg and 325 mg, such as 300 mg, 324 mg, or 325 mg. In some embodiments, aspirin is administered to a patient at a loading dose of 324 mg. In some embodiments, aspirin is administered to a patient at a maintenance dose between 75 mg and 150 mg, or between 75 mg and 100 mg, such as 81 mg.

[0098] The method can include co-administering a single effective dose of recombinant apyrase protein with a suitable dose (e.g., a loading dose) of P2Y 12 inhibitor and optionally aspirin (if present). Although typically only a single effective dose of recombinant apyrase protein is used, the method can also include administering one or more oral doses of P2Y periodically and after the loading dose.12 An inhibitor as part of long-term or maintenance therapy. For example, a P2Y 12 inhibitor may be administered once or twice daily, for example at the maintenance dose described above, after an initial loading dose, for several weeks, months or even years. The maintenance dose of P2Y 12 inhibitor may be administered together with aspirin, as is known in the art (referred to as dual antiplatelet therapy, or DAPT). Long-term or maintenance therapy with a P2Y 12 inhibitor after an ischemic event is known in the art, and appropriate dosages and timing can be determined by a skilled physician or other skilled medical personnel.

[0099] ***

[0100] Features disclosed in the foregoing description, or in the appended claims, or in the drawings, expressed in their specific form or according to the means for performing the disclosed function or the method or process for obtaining the disclosed result, may be used appropriately alone or in any combination of such features in their various forms to implement the present disclosure.

[0101] Although the present disclosure has been described in connection with the above exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art when the present disclosure is given. Accordingly, the exemplary embodiments shown above are considered illustrative rather than restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the present disclosure.

[0102] To avoid any doubt, any theoretical explanations provided herein are for enhancing the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0103] Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0104] Throughout the specification, including the following claims, unless the context requires otherwise, the words "comprise", "include" and variations such as "comprises", "comprising" and "including" will be understood to imply the inclusion of the 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.

[0105] It should be noted that, as used in the specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents. 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 one particular value and / or to another particular value. Similarly, when values are expressed as approximations by use of the antecedent "about", it should be understood that the particular value forms another embodiment. The term "about" associated with a numerical value is optional and means, for example, + / - 10%.

[0106] Examples

[0107] Example 1 – A Phase 1, randomized, single-blind, placebo-controlled study evaluating the safety, tolerability, pharmacokinetics and pharmacodynamics of AZD3366 in infertile healthy men and women

[0108] Study overview

[0109] This first-in-human (FiH) study was conducted to provide data on the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of AZD3366 in healthy male and female subjects. The ClinicalTrial.gov identifier for this study is: NCT04588727.

[0110] This study was conducted in healthy male and female (non-fertile) subjects aged 18 to 55 years, weighing at least 50 kg and not more than 100 kg (including the end values), and with a body mass index (BMI) in the range of 18 kg / m 2 to 30 kg / m 2 (including the end values).

[0111] Three groups (healthy subjects, healthy Japanese subjects, and healthy Chinese subjects) were enrolled in this study. The healthy subject population consisted of healthy male and female (non-fertile) subjects who did not have any native ancestry from the Far East, Southeast Asia, or the Indian subcontinent. The healthy Japanese population group consisted of healthy male and female (non-fertile) Japanese subjects whose parents and grandparents were Japanese and who had lived outside Japan for no more than 10 years. The healthy Chinese population group consisted of healthy male and female (non-fertile) Chinese subjects whose parents and grandparents were Chinese and who had lived outside China for no more than 10 years.

[0112] Part A of this study was a randomized, single-blind, placebo-controlled design to evaluate the safety, tolerability, PK, and PD (capillary bleeding time [CBT] and platelet aggregation inhibition) of single ascending doses (SAD) of AZD3366 administered intravenously (IV) in healthy subjects, healthy Japanese subjects, and healthy Chinese subjects.

[0113] Part B of this study is a randomized, single-blind, parallel-group placebo-controlled design to investigate the safety, tolerability, and PD (CBT and platelet aggregation inhibition) of single IV administration of AZD3366 in combination with ticagrelor and acetylsalicylic acid (ASA) in healthy subjects.

[0114] The combination of ASA and ticagrelor was selected according to the standard of care for antiplatelet treatment regimens in patients with myocardial infarction.

[0115] Primary objectives

[0116] The primary objectives of the study were:

[0117] Part A:

[0118] · To investigate the safety and tolerability of the SAD of IV administration of AZD3366 in healthy subjects, healthy Japanese subjects, and healthy Chinese subjects.

[0119] Part B:

[0120] · To investigate the safety and tolerability of single IV administration of one dose level (160 mg) of AZD3366 in combination with loading dose and repeated dosing of ticagrelor and ASA in healthy subjects.

[0121] Secondary objectives

[0122] The secondary objectives of the study were:

[0123] Part A:

[0124] · To characterize the PK of AZD3366 after IV administration of a single dose of AZD3366 in healthy subjects, healthy Japanese subjects, and healthy Chinese subjects.

[0125] · To characterize the PD of AZD3366 relative to light transmission aggregometry

[0126] [LTA] and CBT after IV administration of a single dose of AZD3366 in healthy subjects, healthy Japanese subjects, and healthy Chinese subjects.

[0127] · To explore the immunogenicity after IV administration of AZD3366.

[0128] Part B:

[0129] · In healthy subjects, AZD3366 was administered intravenously (IV) at one dose level while a loading dose and repeated doses of ticagrelor and ASA were co-administered, and plasma exposure was studied and the pharmacodynamics (PD) of AZD3366 was characterized relative to platelet aggregation inhibition by light transmission aggregometry (LTA) and impedance aggregometry (CBT).

[0130] · The effect of AZD3366 on the pharmacokinetics (PK) of ticagrelor was studied.

[0131] · The immunogenicity after IV administration of AZD3366 was explored.

[0132] Dose and treatment regimen

[0133] Part A:

[0134] Seven groups of 8 subjects received a single short-term IV infusion of AZD3366 (2 mg to 640 mg [n = 6]) or placebo (n = 2). In this dose range, an additional 3 groups of 5 healthy Japanese / 1 group of 8 healthy Chinese subjects were included. A total of 103 subjects were randomly allocated; the mean age was 37.2 (±9) years and 100 were male.

[0135] The first group received 2 mg of AZD3366 or placebo. Groups 2 to 7 received 10 mg, 30 mg, 90 mg, 180 mg, 360 mg, and 640 mg, respectively.

[0136] Part B:

[0137] In Part B, a single group of 24 subjects received a single IV dose of 160 mg of AZD3366 or placebo in combination with aspirin and ticagrelor (n = 12) or aspirin and ticagrelor alone (n = 12).

[0138] A loading dose of 324 mg of ASA was administered to healthy volunteers, and a single IV dose of AZD3366 or placebo was administered 2 hours later (t = 0). One hour and 40 minutes after the administration of AZD3366 or placebo, a loading dose of 180 mg of ticagrelor was administered to healthy volunteers. 90 mg of ticagrelor was administered every 12 hours after the loading dose (t = 25 h 40 m, t = 37 h 40 m, t = 49 h 40 m). ASA was administered daily at t = 25 h 40 m and t = 49 h 40 m. Capillary bleeding time was monitored at t = 0 h 10 m, t = 3 h 40 m, and t = 51 h 40 m.

[0139] Measurement and evaluation methods

[0140] Light transmission aggregometry (LTA) for measuring ADP-induced platelet responses after administration of AZD3366 to healthy volunteers method (LTA)

[0141] In those with AGGGRO / CHRONO- of the interface Quantification of ADP-induced platelet aggregation in platelet-rich plasma (PRP) using LTA on a CHRONO-490 4+ aggregation system. PRP was prepared by centrifuging heparin lithium anticoagulated blood samples at 100 g to 170 g for 15 minutes, and then transferring the supernatant to a fresh polypropylene tube. To set 100% baseline in the LTA assay, platelet-poor plasma (PPP) was prepared by centrifuging blood samples at 1500 g to 2400 g for 20 minutes, and then transferring the supernatant to a fresh polypropylene tube. To stimulate platelet aggregation, 5 μmol / L or 20 μmol / L of ADP was added to the PRP and PPP preparations in separate cuvettes with a magnetic stirrer, and then aggregated for at least 5 minutes with continuous stirring at 37 °C. Data were recorded as percent amplitude (the aggregation reaction in the PRP sample expressed as a percentage of the PPP reaction) and area under the curve (AUC). For each AZD3366 dose and time point, platelet aggregation inhibition was calculated relative to the pre-dose platelet aggregation level.

[0142] Assessment of capillary bleeding time (CBT) after administration of AZD3366 to healthy volunteers

[0143] CBT was evaluated by making a horizontal incision on the forearm and monitoring the time to cessation of bleeding. A blood pressure cuff was placed on the upper arm and inflated to 40 mmHg. Then a standardized incision was made with the device, and bleeding was monitored at 30-second intervals by bringing filter paper ( bleeding time blotting paper) into contact with the bleeding site, but not less than 0.5 mm from the incision to avoid interfering with the formation of the platelet plug. Bleeding was continued for up to 90 minutes or 180 minutes, and bleeding was judged to have stopped when the filter paper was no longer contaminated with blood. If bleeding did not stop within the 90-minute or 180-minute cut-off time, bleeding was judged to be continuing at that time point. After completion of the CBT analysis, the cuff was removed and the incision site was cleaned with a sterile cotton swab.

[0144] Results of Part A

[0145] Treatment with AZD3366 alone did not result in any clinically relevant safety or tolerability outcomes.

[0146] Comparable adverse event (AE) rates were observed in the AZD3366 and placebo groups, and there was no significant increase in bleeding events. A summary of the AE results in healthy volunteers treated with AZD3366 (all doses) or placebo is provided in the table below:

[0147]

[0148] Peak plasma concentration of AZD3366 is reached within 0.5 hour after dosing, followed by a mono- or bi-phasic decline, with a final PK half-life of approximately 140 hours. Complete inhibition of ADP-stimulated platelet aggregation is achieved within 10 minutes after dosing; the duration ranges from 4 hours at 2 mg to approximately 35 days at 640 mg( Figure 1 ).

[0149] In monotherapy with AZD3366, no increase in capillary bleeding time (CBT) was observed at dose levels up to 90 mg, while a significant increase was observed at 180 mg and above( Figure 2 ).

[0150] Results of Part B

[0151] AZD3366 treatment in combination with aspirin and ticagrelor (AZD3366 + DAPT) is safe and well tolerated. As in Part A, there is a comparable rate of adverse events in the AZD3366 and placebo groups, and no significant increase in bleeding events. A summary of the AE results for the two groups is provided in the table below:

[0152]

[0153] When a loading dose of ASA and ticagrelor is administered, CBT in the AZD3366 group increases compared to placebo, but this increase levels off when healthy volunteers continue to receive the maintenance dose of DAPT( Figure 3 ).

[0154] Conclusion

[0155] AZD3366, alone or in combination with aspirin and ticagrelor, is generally safe, well tolerated, and achieves complete platelet inhibition with a dose-dependent duration.

[0156] References

[0157] Numerous publications are cited above in order to more fully describe and disclose the present invention and the state of the art to which the present invention pertains. The complete citations of these references are provided below. The entire contents of each of these references are incorporated herein.

[0158] 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

[0159] 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

[0160] 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

[0161] 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.

[0162] 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

[0163] 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。

[0164] 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

[0165] 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

[0166] 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., & Thorsén, 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

[0167] For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press

[0168] Sequences

[0169]

[0170]

Claims

1. A method for treating an ischemic event in a patient, the method comprising administering to the patient a therapeutically effective amount of a recombinant apyrase protein, wherein the recombinant apyrase protein comprises the amino acid sequence shown in SEQ ID NO:2, and wherein the method comprises administering the recombinant apyrase protein to the patient at a dose of 40 mg to 240 mg.

2. A recombinant apyrase protein for use in a method for treating an ischemic event in a patient, wherein the recombinant apyrase protein comprises the amino acid sequence shown in SEQ ID NO:2, and wherein the method comprises administering the recombinant apyrase protein to the patient at a dose of 40 mg to 240 mg.

3. The method according to claim 1, or the recombinant apyrase protein used according to claim 2, wherein the method comprises administering the recombinant apyrase protein to the patient at a dose of 40 mg to 170 mg.

4. The method according to claim 1, or the recombinant apyrase protein used according to claim 2, wherein the method comprises administering the recombinant apyrase protein to the patient at a dose of 100 mg to 240 mg.

5. The method or the recombinant apyrase protein used according to any one of claims 1 to 4, wherein the method comprises administering the recombinant apyrase protein to the patient at a dose of 100 mg to 170 mg.

6. The method or the recombinant apyrase protein used according to any one of claims 1 to 5, wherein the method comprises administering the recombinant apyrase protein to the patient at a dose of 100 mg to 140 mg, optionally wherein the recombinant apyrase protein is administered to the patient at a dose of 110 mg, 115 mg or 120 mg.

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

8. The method or the recombinant apyrase protein used according to claim 7, wherein the ischemic event is ST-segment elevation myocardial infarction (STEMI).

9. The method or the recombinant apyrase protein used according to any one of claims 1 to 6, wherein the ischemic event is acute ischemic stroke.

10. The method according to any one of claims 1 to 9 or the recombinant apyrase protein used, wherein the recombinant apyrase protein binds to a P2Y 12 inhibitor.

11. The method according to claim 10 or the recombinant apyrase protein used, wherein the P2Y 12 inhibitor is selected from the list consisting of ticagrelor, clopidogrel, ticlopidine, prasugrel, and cangrelor.

12. The method according to claim 11 or the recombinant apyrase protein used, wherein the P2Y 12 inhibitor is ticagrelor, optionally wherein the ticagrelor is administered at a loading dose between 60 mg and 200 mg.

13. The method according to any one of claims 10 to 12 or the recombinant apyrase protein used, wherein the P2Y 12 inhibitor is administered within 2 hours of administering the recombinant apyrase protein to the patient.

14. The method or the recombinant apyrase protein used according to any one of claims 10 to 13, wherein the method further comprises administering aspirin to the patient, optionally wherein the aspirin is administered at a loading dose between 250 mg and 325 mg.

15. The method according to claim 14 or the recombinant apyrase protein used, wherein the aspirin is administered within 2 hours of administering the recombinant apyrase protein to the patient.

16. The method or the recombinant apyrase protein used according to any one of claims 10 to 15, wherein the method further comprises administering a maintenance dose of a P2Y 12 inhibitor and aspirin for at least 1 week after administering the recombinant apyrase protein to the patient.

Images