Synergistic targeted composition for treating arterial and venous thrombosis

Abstract

The combination of apyrase and annexin, including the fusion protein, is a synergistic antithrombotic agent that does not induce unwanted bleeding.

Description

Technical Field

[0001] The present invention relates to the field of treating conditions characterized by undesirable thrombosis, such as heart attacks, strokes, and surgical complications where bleeding should be minimized. Specifically, the present invention aims to administer, together with apilase, a factor that competes with the prothrombinase complex and binds to phosphatidylserine on the surface of activated platelets for preventing or treating thrombosis.

Background Art

[0002] The related conditions treated by the method of the present invention are both arterial and venous thrombosis.

[0003] In the case of arterial thrombosis, high - concentration unfractionated heparin is commonly used to prevent intraoperative blood coagulation in patients undergoing coronary artery bypass grafting (CABG). Despite using protamine antagonism, heparin treatment increases postoperative bleeding, and approximately 30% of patients require blood transfusion after CABG. Other antithrombotic therapies also significantly increase the risk of postoperative bleeding. Clopidogrel (trade name: Plavix), prasugrel (trade name: Effient), ticagrelor (trade name: Brilinta), and ibuprofen are generally interrupted for several days before coronary artery surgery. Patients taking warfarin (trade name: Coumadin), apixaban (trade name: Eliquis), rivaroxaban (trade name: Xarelto), edoxaban (trade name: Savaysa), or dabigatran (trade name: Pradaxa) should discontinue their medication before surgery. Therefore, an antithrombotic treatment that does not cause intraoperative or postoperative bleeding is highly needed.

[0004] Current adjuvant antithrombotic therapy during percutaneous coronary intervention (PCI), formerly known as stent angioplasty, also increases the risk of massive bleeding. Coronary artery disease can be diagnosed by the presence of ST-elevation acute myocardial infarction (STEMI) on an electrocardiogram. Platelets play a central role in thrombotic complications (Non-Patent Literature 1). Treatment for this condition aims to restore normal coronary blood flow and maximize the salvage of functional myocardium.

[0005] Current US and European guidelines recommend primary PCI with adjuvant therapy including dual antiplatelet and anticoagulant regimens. However, currently available antiplatelet agents have a delayed onset of action in approximately 40-50% of STEMI patients (Non-Patent Literature 2), and all combinations have mechanisms of action that increase bleeding. Recent clinical trials have shown that 11-12% of patients experience massive bleeding (Non-Patent Literature 3). In addition, none of the current antithrombotic agents (i.e., combinations of antiplatelet and anticoagulant agents) protect against reperfusion injury, defined as myocardial infarction following the restoration of coronary blood flow after ischemia. Reperfusion injury accounts for up to 50% of the final size of myocardial infarction and leads to associated cardiac dysfunction. This can explain why, despite optimal coronary reperfusion, the mortality rate after AMI is nearly 10% and the incidence of heart failure is almost 25% (Non-Patent Literature 1, Non-Patent Literature 4, Non-Patent Literature 5).

[0006] Therefore, there has long been a pressing and unmet medical need for a fast-acting therapeutic agent to adjuvant PCI that promotes reperfusion without increasing bleeding, simultaneously reduces reperfusion injury, improves myocardial salvage and left ventricular function recovery, and thereby reduces the incidence of heart failure.

[0007] A left ventricular assist device (LVAD) is a pump used in patients who have reached end-stage heart failure. An LVAD is a battery-powered mechanical pump that is surgically implanted to assist the left ventricle (the heart's primary pumping chamber) in pumping blood to the rest of the body. The LVAD may be used until the heart becomes functional again, or the patient may receive long-term treatment with the LVAD to prolong their life and improve their quality of life. Despite significant improvements in survival, functional capacity, and quality of life, pump thrombosis, stroke, and bleeding remain common complications. Because all current antithrombotic drugs increase the risk of bleeding, finding the optimal antithrombotic therapy that balances thrombosis prevention with increased bleeding risk remains an ongoing challenge.

[0008] For patients undergoing surgery and receiving chronic antithrombotic therapy, current clinical recommendations suggest discontinuing antithrombotic therapy before surgery because these medications increase the risk of excessive bleeding. For example, discontinuing aspirin around the time of surgery in patients requiring CABG, discontinuing unfractionated heparin 4-6 hours before surgery, and administering the final dose of low molecular weight heparin 24 hours before surgery. However, discontinuing these antithrombotic treatments also increases the risk of intraoperative thrombosis, such as pulmonary embolism, which can be highly fatal. Currently, there are no antithrombotic treatments that can be safely used for intraoperative management.

[0009] Similar disadvantages are seen with anticoagulant therapy for venous thromboembolism, in that treatment is plagued by an increased risk of massive bleeding events and provides minimal protection against venous wall thickening or fibrosis. Venous thromboembolism, including deep vein thrombosis (DVT) and pulmonary embolism (PE), is a leading source of morbidity and mortality worldwide.

[0010] Current prevention and treatment of DVT involves the administration of parenteral anticoagulants, such as heparin, which is then followed by oral anticoagulants such as warfarin (Non-Patent Document 1, Non-Patent Document 6). Other drugs are also used; for example, rivaroxaban is approved for both short-term and long-term treatment (Non-Patent Document 7, Non-Patent Document 3), but all of these have mechanisms of action that cause bleeding, so the doses that can be administered are limited. In addition, current treatments do not effectively protect against post-thrombotic syndrome (PTS), which is characterized by blood reflux, overflow obstruction, and tissue hypoxia caused by impaired venous valves that follows venous wall thickening and fibrosis (Non-Patent Document 8, Non-Patent Document 9, Non-Patent Document 10).

[0011] The impact of deep vein thrombosis (DVT) is increasing with the aging population. In 2005, the U.S. Senate designated March as "Deep Vein Thrombosis Awareness Month." Clearly, the health and economic burden of DVT is significant, and DVT patients would greatly benefit from effective antithrombotic drugs that can reduce post-traumatic stress syndrome (PTS) without being limited by the increased bleeding risk.

[0012] As described below, the compositions and methods of the present invention also provide solutions to these unmet medical needs.

[0013] The use of apirase, specifically soluble CD39L3 and its modifications, for treating conditions associated with thrombosis is disclosed in Patent Documents 1, 2, and 3. The use of apirase as a therapy for bleeding conditions is disclosed in Patent Document 4, and improved forms of apirase based on soluble CD39L3 are disclosed in Patent Document 5. In addition, apirase therapy for fibroproliferative disorders, pulmonary hypertension, and heart failure is disclosed in Patent Document 6, which is currently pending.

[0014] Annexin V, a protein known to bind to phosphatidylserine on the surface of activated platelets in competition with the binding of the prothrombinase complex, is considered relevant to the prevention and treatment of thrombosis, given the allegations that antibodies reacting to annexin V affect arterial and / or venous thrombosis, for example, in patients with systemic lupus erythematosus (Non-Patent Literature 11). In practice, the primary current use of annexin V is as a marker for apoptosis, but it is understood that annexin V can inhibit prothrombin activation and prevent thrombus formation under normal venous and arterial blood flow conditions. Antibodies reacting to annexin V have been shown to cause thrombotic complications in patients with type 1 diabetes (Non-Patent Literature 12).

[0015] It is now known that annexin V, or other annexins that bind to phosphatidylserine on the surface of activated platelets, and apirase, when administered individually or in combination, exert a synergistic antithrombotic effect without promoting undesirable bleeding.

[0016] Therefore, this combination provides a solution to the problems currently associated with the treatment of both arterial and venous thrombosis. Specific embodiments of annexin V conjugated with apirase as a fusion protein have also been found to exhibit favorable properties with respect to recombinant production.

[0017] All documents cited herein are incorporated in their entirety by reference. [Prior art documents] [Patent Documents]

[0018] [Patent Document 1] U.S. Patent No. 7,247,300 [Patent Document 2] U.S. Patent No. 7,390,485 [Patent Document 3] U.S. Patent No. 8,021,866 [License 4] U.S. Patent No. 8,535,622 [Patent Document 5] U.S. Patent No. 8,771,683 [License 6] U.S. Charter of Issuance No. 14 / 666,121 [Non-licensed literature]

[0019] [Non-licensed Document 1] Heit,JA,J Thromb Haemost(2005)3:1611-1617 [Non-licensed Document 2] Riteau,N.,et al.,Am J Respir Crit Care Med(2010)182:774-783 [Non-licensed Document 3] Ufer,M.,Thromb Haemost(2010)103:572-585 [Non-licensed Document 4] Spyropoulos,AC,et al.,J Manag Care Pharm(2007)13:475-486 [Non-licensed Document 5] Marcus,AJ,et al.,Semin Thromb Hemost(2005)31:234-246 [Non-licensed Document 6] Kyrle,PA,et al.,Lancet(2005)365:1163-1174 [Non-licensed Document 7] Perzborn,E.,et al.,Nature Rev. Drug Discovery(2011)10:61-75 [Non-licensed Document 8] Popuri,RK,et al.,Arterioscler Thromb Vasc Biol(2011)31:479-484 [Non-licensed Document 9] Saarinen, J., et al., J Cardiovasc Surg (2000) 41:441-446 [Non-Patent Document 10] Deatrick, KB, et al., J Vasc Surg 2011 53:139-146 [Non-Patent Document 11] Esposito, G., et al., Autoimmunity Rev (2005)4:55-60 [Non-Patent Document 12] Bakar, F., et al., J Clin Endocrinol Metab(2014)99:932-937 [Overview of the Initiative] [Means for solving the problem]

[0020] In one embodiment, the present invention relates to a composition comprising apirase and annexin bound to phosphatidylserine (PS) on the surface of activated platelets, wherein the apirase and annexin together are administered in an amount effective to inhibit thrombosis without increasing bleeding.

[0021] The combination of annexin and apirase can also be administered in the form of a single molecule in which apirase and annexin are covalently bonded.

[0022] Regarding the ease of producing such covalent combinations, it is advantageous that apirase and annexin covalently bond via a linker peptide to form a fusion protein, and that the linker peptide is particularly resistant to hydrolysis in plasma and / or cell culture supernatant.

[0023] The present invention also aims to provide recombinant production in a covalent form of apirase and annexin, comprising materials related to such recombinant production, and a method for treating subjects experiencing or at risk of thrombosis using compositions of the present invention. [Brief explanation of the drawing]

[0024] [Figure 1] Figures 1A and 1B show the effects of as-is apirase APT102 and its fusion protein APT402 on platelet aggregation in an ex vivo reaction mixture containing human platelet-rich plasma (PRP) activated with 20 μM ADP. Equivalent inhibitory activity against ADP-induced human platelet aggregation is demonstrated by APT102 and APT402. The X-axis represents assay time (6 minutes), and the Y-axis represents light transmittance. (A) APT102 dose-dependently inhibits platelet aggregation induced by 5 mM ADP. (B) APT402 dose-dependently inhibits platelet aggregation induced by 5 mM ADP. Arrows indicate the addition of ADP and apirase. [Figure 2] Figure 2 shows the inhibitory activity of APT402 versus annexin V against factor X activation in PBMCs. Equivalent inhibitory activity of LPS-induced procoagulant activity by APT402 versus annexin V is demonstrated. Peripheral blood mononuclear cells were incubated with LPS (1 mg / ml) to induce tissue factor expression and FX activation, as measured in the presence of FVII, FX, and the chromogenic substrate S2765. [Figure 3] Figure 3 shows the synergistic effect of Annexin V and APT102 on the attenuation of thrombin generation. A thrombogram of nanomolar thrombin generated in human platelet-rich plasma activated with 20 mM ADP is shown. Treatment with APT402 or APT102 and Annexin V shows comparable inhibitory effects. These results are superior to those obtained with APT102 or Annexin V alone. [Figure 4] Figure 4 shows the PK analysis of APT402 in rabbits (n=2) after a 0.4 mg / kg IV bolus injection. [Figure 5] Figure 5 shows the PK analysis (ELISA) of APT402 in rabbits using a 0.2 mg / kg bolus injection followed by a 120-minute continuous IV infusion. [Figure 6]Figure 6 shows the inhibition of ADP-induced (50 μM) platelet aggregation in rabbit platelet-rich plasma using a 0.2 mg / kg bolus of APT402 followed by 24 μg / kg / min for 120 minutes. Aggregation was effectively inhibited at 60 and 110 minutes and returned to baseline levels at 180 minutes. [Figure 7] Figure 7 shows the attenuation of thrombin generation in rabbits using a 0.2 mg / kg bolus of APT402 followed by a 24 μg / kg / min infusion over 120 minutes. Thrombin generation was effectively attenuated at 60 and 110 minutes, but returned to baseline levels at 180 minutes. [Figure 8] Figures 8A-C show that APT402 preferably localizes to the thrombus site of an injured artery. A. APT402 conjugated with a fluorescent dye (LS288); B. Fluorescence intensity in injured arteries of rabbits treated with a 0.2 mg / kg bolus followed by 12 mg / kg / min of APT402 for 120 minutes. Each bar represents the average of three readings. C. APT402 preferably binds to thrombi. [Figure 9] Figure 9 shows the occlusion rates in the control and various treatment groups in the electrical injury model (EIM) in rabbits. Treatment with APT402 at 12 or 24 μg / kg / min completely prevented occlusion. [Figure 10] Figure 10 shows the arterial thrombus weight (mg) of the control and various treatment groups in rabbits undergoing EIM. Treatment with APT402 at 24 μg / kg / min was more effective than any other treatment, both alone and in combination. [Figure 11] Figure 11 shows that APT402, at a dose that completely prevents occlusion, did not prolong bleeding time (BT) (BT measurement limit: 5 minutes), while clopidogrel, LMWH, bivalirudin, and ticagrelor (alone or in combination with bivalirudin) prolonged bleeding time. [Figure 12] Figure 12 shows that the effective dose of APT402 did not affect prothrombin time in rabbits, while bivalirudine, both alone and in combination with ticagrelor, increased prothrombin time. [Figure 13] Figure 13 shows that the effective dose of APT402 did not affect partial thromboplastin time in rabbits, while LMWH, vivalirudine, and ticagrelor (alone or in combination with vivalirudine) increased partial thromboplastin time. [Figure 14] Figure 14 shows that APT402 does not affect mean arterial pressure (millimeters of mercury; mmHg). [Figure 15] Figure 15 shows that APT402 does not affect heart rate (pulse per minute; BPM). [Figure 16] Figures 16A-D show the effect of APT402 on the sequelae of EIM in deep vein thrombosis (DVT). [Figure 17] Figure 17 shows the effect of APT402 on collagen deposition in DVT reduced by EIM. [Modes for carrying out the invention]

[0025] This invention utilizes the combined action of apirase and annexin, antiplatelet and anticoagulant proteins, which have an unexpected synergistic effect in the prevention or treatment of both arterial and venous thrombosis. This combination solves the problems associated with commercially available antithrombotic drugs and, more specifically, has a more favorable profile in terms of avoiding increased bleeding compared to these commercially available antithrombotic drugs. In addition, this invention includes a unique fusion protein in which apirase and annexin are linked by an additional amino acid sequence. When produced as a fusion protein, this invention includes its advantageous form in terms of its expression level when recombinantly produced, and its resistance to proteolysis in both recombinant cultures and plasma. These features appear to depend on the linker provided between the annexin and apirase components.

[0026] Components In general, any apirase that inhibits platelet activation and aggregation can be used. Considerations regarding such apirases can be found in U.S. Patent No. 7,390,485. Certain variants of the soluble form of CD39L3 are particularly useful.

[0027] A particularly useful form of apirase is the soluble form of CD39L3, the sequence of which is disclosed in U.S. Patent No. 7,390,485. The full-length CD39L3 and its putative amino acid sequence are SEQ ID NOs. 55 and 56, respectively. The soluble form is also disclosed in this patent, as well as in the sequence of the coding nucleotide as SEQ ID NOs. 59 and the sequence of the putative amino acid sequence as SEQ ID NOs. 60.

[0028] Patent 485 also describes site-directed mutagenesis of both soluble CD39 and soluble CD39L3. Of particular use in the present invention are mutant forms of soluble CD39L3 in which the amino acids at positions 67 and 69 are mutated. Two of these mutants of soluble CD39L3 are R67A T69R and R67G T69R. R67G T69R of soluble CD39L3 is a particularly preferred double mutant. This “improved” form of the sequence is disclosed in U.S. Patent No. 8,771,683, and the relevant nucleotide and amino acid sequences are replicated herein as SEQ ID NOs. 1 and 2, respectively. This embodiment is designated APT102.

[0029] The annexin component useful in the present invention is annexin V, which binds to phosphatidylserine on activated platelets. Annexins are members of a large family of proteins that have similar structures but exhibit diverse physiological activities. Any annexin exhibiting the above properties of annexin V can be used in the present invention. The structures of these annexins, including the native or optimized nucleotide sequences encoding them, are known in the art. In some embodiments, the selected annexin will be of the same species as the subject being treated.

[0030] Production of components of the present invention Generally, it is convenient to produce the components useful in the present invention using recombinant technology. This is especially true in embodiments in which apirase and annexin are covalently linked. Recombination techniques for such proteins are well known in the art at present, and recombinant production can be carried out in various cells and environments, including animal, plant, and microbial cells, in situ in cultures and multicellular organisms. Accordingly, the present invention includes recombinant materials for producing these components, such as suitable regulatory sequences, vectors, and expression systems including host cells having them.

[0031] In vivo administration of the composition of the present invention The compositions of the present invention can be administered to warm-blooded animals, including humans and other mammals, as well as domestic avian species. These may include, for example, companion animals or livestock. When treating human diseases, a qualified physician will use established protocols to determine how the compositions of the present invention should be used in terms of dosage, schedule, and route of administration. In other species, a veterinarian is assumed to play a similar role. In such applications, dose increases may be used.

[0032] Preferably, the pharmaceutical composition of the present invention is administered parenterally, i.e., intra-arterial, intravenous, intraperitoneal, subcutaneous, or intramuscular. More preferably, the pharmaceutical composition is administered intravenously or intraperitoneally by bolus or infusional injection.

[0033] The pharmaceutical compositions of the present invention may contain water, buffer water, physiological saline, glycine, dextrose, isotonic sucrose solution, etc., and may also contain glycoproteins to improve stability, such as albumin, lipoprotein, globulin, etc. These compositions may be sterilized by conventional known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered and freeze-dried under sterile conditions, and the freeze-dried preparations may be mixed with a sterile aqueous solution before administration. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters, and buffers, isotonic agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

[0034] The concentration of the components of the present invention in the pharmaceutical formulation starts at less than about 0.05% by weight and can vary widely, usually from about 2-5% by weight or at least from about 2-5% by weight to about 10-30% by weight, and is selected according to a specific administration method chosen, mainly based on the volume of liquid, viscosity, etc. For example, the concentration may be increased to reduce the fluid load associated with the treatment.

[0035] Preferably, the pharmaceutical composition of the present invention is administered intravenously. The dosage of the delivery vehicle formulation varies depending on the ratio of drug to lipid, as well as the opinion of the administering physician based on the patient's age, weight, and condition. One preferred protocol involves a bolus IV administration followed by a prolonged IV infusion.

[0036] In addition to pharmaceutical compositions, suitable formulations for veterinary use can be prepared and administered in a manner appropriate to the target. Preferred veterinary targets include mammalian species such as non-human primates, dogs, cattle, horses, sheep, and poultry. Other potential targets include laboratory animals, such as rats, rabbits, mice, and guinea pigs.

[0037] Indications The compositions of the present invention are useful in patients who wish to treat or prevent thrombosis without causing excessive bleeding. These include, but are not limited to, surgical procedures such as thoracic surgery including CABG, and major surgeries with a high risk of thrombosis, or surgical procedures or medical research (e.g., electrophysiological studies, pacemaker implantation, percutaneous cardiac valve placement or repair) in which antithrombotic therapy is administered as a preventive measure against coagulation due to the placement of an indwelling catheter or device. Patients who wish to be treated with the compositions of the present invention include patients with myocardial infarction, including STEMI, who are undergoing vascular regeneration / reperfusion therapy using PCI or thrombolysis, and patients with stable or unstable coronary artery disease who are undergoing coronary artery regeneration using PCI. Suitable subjects include patients with peripheral vascular disease undergoing vascular regeneration procedures, such as peripheral vascular grafting or intraluminal balloon angioplasty, with or without stent placement, as well as patients receiving long-term antithrombotic therapy as a bridging strategy / intraoperative management during fasting and surgical procedures when oral antithrombotic therapy cannot be sustained, such as patients with atrial fibrillation who are under treatment / prevention for venous thromboembolism (VTE) and are therefore at high risk of thrombotic complications.

[0038] This composition is also useful for the prevention or treatment of VTE, including deep vein thrombosis and pulmonary embolism, including orthopedic surgery and fracture treatment, as well as prophylactic treatment associated with immobile subjects, acute or chronic diseases (including cancer), and subjects at high risk of thrombosis. In addition, the composition of the present invention is used to treat subjects with heparin-induced thrombocytopenia (HIT), including before, during, or after potential reperfusion therapy (but also when reperfusion therapy is not used), as well as during treatment or pulmonary angioplasty in subjects with chronic thromboembolic pulmonary disease / hypertension (CTEPH), and subjects with acute ischemic stroke requiring additional antithrombotic therapy in addition to standard treatment.

[0039] Stroke is defined as loss of neurological function resulting from cerebral ischemia or intracranial hemorrhage, including, but not limited to, acute ischemic stroke, major and / or small vessel occlusive ischemic stroke associated with reperfusion by thrombolysis or thrombectomy, prevention of tissue damage associated with embolic stroke, and for improved neurological function and outcomes, and for prevention of recurrent stroke in patients with acute transient ischemic attack who are at high risk of stroke.

[0040] kit The apirase and annexin used in the compositions of the present invention may be individually combined or covalently combined in the same composition. The individual compositions may be administered to a subject simultaneously or sequentially. A kit may be subsequently packaged in individual containers, with a first composition containing apirase in a first container and a second composition containing a suitable annexin in a second container.

[0041] The kit also includes instructions on how to administer the compositions to a subject, including a description of the ratio of at least the amount of each composition to be administered. In one embodiment, equimolar amounts are administered. However, the molar ratio of apirase to annexin varies depending on the selection of these components and can range from 10:1 to 1:10 in the case of apirase:annexin. Alternatively, or in addition, the kit is constructed such that the amount of composition in each container is pre-measured so that the amount in one container, combined with the amount in the other container, represents the correct ratio. Alternatively, or in addition, the containers may be marked with a scale so that the appropriate amount can be dispensed based on a visible scale. The containers may be ready for administration themselves; for example, the kit may contain appropriate amounts of each composition in individual syringes. Compositions containing pre-formulated therapeutic agents in the correct ratios may also be packaged in this manner, so that the composition can be administered directly from syringes pre-packaged within the kit.

[0042] Unless otherwise defined, all technical terms, notations, and other scientific or scientific terminology used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. In some cases, terms that have a commonly understood meaning are defined herein for clarity and / or for immediate reference, but the inclusion of such definitions herein should not necessarily be interpreted as representing a fundamental difference from the commonly understood meaning in the art. Many of the techniques and procedures described or referenced herein are well understood by those skilled in the art and are commonly used by conventional methods. Unless otherwise specified, procedures, including the use of commercially available kits and reagents, are commonly performed according to the manufacturer's defined protocols and / or parameters. All patents, applications, published applications, and other publications referenced herein are incorporated in their entirety by reference. If any definitions set forth in this section conflict with or contradict any definitions set forth in any patent, application, published application, or other publication incorporated herein by reference, the definitions set forth in this section shall prevail over the definitions incorporated herein by reference.

[0043] As used herein, “a” or “an” means “at least one” or “one or more.”

[0044] The following examples are illustrative of the present invention and do not limit it. Furthermore, the following scientific discussions on the pathogenesis gathered from the data do not mean to limit the present invention as described herein.

[0045] Abbreviations: BT: bleeding time; aPTT: activated partial thromboplastin time; TCT: thrombin coagulation time; ACT: activated coagulation time; PT: prothrombin time; LMWH: low molecular weight heparin; FX: factor X; DVT: deep vein thrombosis; EIM: electrical injury model; IVC: inferior vena cava. [Examples]

[0046] Example 1 Design of apirase-annexin fusion products and linkers The apirase drugs used in these examples are soluble apirase drugs prepared from a fabrication encoding soluble CD39L3 R67G T69R, and have a homogenized N-terminus as described in U.S. Patent No. 8,771,683, designated herein as APT102 (SEQ ID NO: 2).

[0047] To optimize expression levels and prevent degradation, nucleic acids encoding annexin V were fused to the C-terminus of apirase drugs with linkers of varying lengths, providing sequences encoding the mouse IgGκ sequences shown in SEQ ID NOs. 3 and 4 herein for various repeats, and inserted into the expression plasmid pSecTag2c.

[0048] HEK293 cells were stably transformed using a linearized expression plasmid that provides expression of the apirase annexin V fusion protein by modifying the linker sequence.

[0049] The transformants were adapted for serum-free suspension culture and continuously divided into larger flasks. A typical suspension culture was administered using 0.5 × 10⁶ cells. 6 When inoculated at a concentration of cells / mL, HEK293 cells typically reach 3.5 × 10⁶ cells within 5-6 days. 6 Cells grew to over 1 / mL. Cells were divided every 3-4 days, and the fusion protein was collected in the conditioned medium. Apirase annexin V fusion production using a 20AA linker was scaled up to a 3L spinner.

[0050] The proteins were purified to homogenization using IMAC, Q, and SP columns. The amino acid sequences of the various designed linkers are shown below. >m0 (original design: cleaved by protease) [ka] >m1 (cleaved by protease) [ka] >m2 (No disconnection detected) [ka] >m3 (No disconnection detected) [ka]

[0051] In the case of the initially designed linker, m0, approximately 50% of the purified protein was degraded in the linker region by protease activity present in the culture medium. Further studies showed that lower pH (e.g., 5.0) accelerated degradation compared to pH 7.4. The protease-resistant fusion protein was designed by introducing a G-to-T substitution, as shown in m1, which was still susceptible to protease degradation when the pH of the purified supernatant was reduced to 5 with sodium citrate. When this mutation was combined with a serine substitution with alanine or glutamine, as shown in m2 and m3, degradation was minimized.

[0052] Based on a comparison of expression levels, it was also shown that the 20-amino acid linker was the most advantageous.

[0053] Expression levels were compared to flexible linkers with amino acid residue lengths of 5, 10, 15, and 20, or to a rigid linker with 9 amino acid residues. [ka]

[0054] The proteins derived from the purified supernatant of the conditioned medium described above were separated by SDS-polyacrylamide gel electrophoresis. Analysis showed that the expression of the fusion protein was positively correlated with the length of the 20AA linker, resulting in the highest expression. The rigid linker variant also showed lower expression than the variant with the 20AA flexible linker. The amino acid and nucleotide sequences of this 20AA linker are shown in Sequence ID No. 3.

[0055] The final preparations used in Examples 2-10 had the nucleotide sequence of Sequence ID No. 7, where the signal sequence encoded the bovine α-lactalbumin signal peptide. The encoded apirase-linker-annexin V fusion is designated APT402.

[0056] Example 2 Expression and purification of APT402 from CHO cells The apirase drug in the preparation for producing the fusion protein APT402 is APT102. The apirase annexin V fusion with a 20AA linker, as described in Example 1, was produced in CHO cells using the signal sequence of the bovine α-lactalbumin signaling peptide derived from the preparation shown in Sequence ID No. 7. As described, the apirase-linker-annexin protein was designated as APT402. The producing cell line was created by transducing the CHO parent cell line four times with a retrovector and expression retrovector plasmid prepared from Catalent (Madison, USA). The pooled population of transduced cells was named CHO-S-APT402-R 4× pool. Samples of the pooled population cell line were cryopreserved.

[0057] To produce 10 L, the cell line CHO-S-APT402-R 4× pool was subcultured every 3-4 days during the exponential growth phase to scale up a 10 L Braun bioreactor in PFCHO LS (HyClone). Cells were seeded into two 10 L bioreactors at a cell density of approximately 300,000-400,000 cells / mL in PFCHO LS (HyClone) medium. The fed batch supplements used in this study were HyClone PS307 (12% (w / v) solution), AGT CD CHO 5X Feed Medium Complete (Invitrogen), 20% glucose solution, 200 mM L-glutamine, 50x solution of L-asparagine (15 g / L) / L-serine (10 g / L), and 50x solution of L-tyrosine (4 g / L) / L-cystine (2 g / L).

[0058] ANX chromatography. Harvesting of 10LBRX-3380-4×pool-001 from a 10L container was performed on day 12 of culture. The column was washed with 10mM Tris-Cl, pH 7.4 for equilibration. Triton-treated medium was diluted with an equal volume (6.60L) of WFI to prepare the ANX input. The input (13.2L) was applied to the column. Subsequently, the column was washed with 10mM Tris, 100mM NaCl, pH 7.4, and 3L of this washing solution was collected. The APT402 fraction was eluted with 10mM Tris, 270mM NaCl, pH 7.4 and collected (2.37L).

[0059] SP Sepharose chromatography. The column was sterilized with 1M NaOH for 1 hour, followed by washing with WFI. The column was equilibrated in 10 mM Tris, 50 mM NaCl, 20 mM CaCl2, pH 7.4. The input (ANX eluent added and stirred for 20 minutes + 9.4 L of 10 mM Tris, pH 7.4 + 34.5 g of CaCl2; 11.75 L) was applied to the column, passed through, washed, and recovered (approximately 12 L). The column was washed with 10 mM Tris, 50 mM NaCl, pH 7.4 to re-establish the baseline. The column was eluted with 10 mM Tris, 300 mM NaCl, pH 7.4, and the eluted peak was recovered as a clear, colorless solution (1.0 L).

[0060] Heparin Hyper D chromatography. The column was washed with 10 mM Tris, 1 M NaCl, pH 7.4. Subsequently, the column was equilibrated in 10 mM Tris, pH 7.4. The feed (buffer exchange SP pool, 0.88 L) was applied to the column, perfused, washed, and recovered (1.40 L). Residual protein was removed from the column with 10 mM Tris, 1 M NaCl, pH 7.4, and this was also recovered (about 100 mL). The chromatogram is shown below. The heparin perfusion volume (1.40 L) was buffer-exchanged to 10 mM Tris, 150 mM NaCl, pH 7.4 in discontinuous mode using a Masterflex pump and a Pellicon Polysulfone 10K membrane. The SP pool was concentrated approximately 7-fold to 200 mL, diluted approximately 10-fold to 2.0 L with 10 mM Tris, 150 mM NaCl, pH 7.4, concentrated again to 200 mL, and diluted again to 2.0 L, approximately 10-fold, with 10 mM Tris, 150 mM NaCl, pH 7.4. After concentrating to approximately 10 mL in the third concentration, the filter was flushed with 200 mL of 10 mM Tris, 150 mM NaCl, pH 7.4, and this was added to the retentate. The final volume of the concentrate was 300 mL, and approximately 4.9 L of permeate water was recovered (about 24.5 times the initial concentrate volume). The retentate aqueous solution was further concentrated using an Amicon stirred cell device with a 10 kD regenerated cellulose membrane.

[0061] The protein was purified until homogeneous. The final recovery rate was approximately 54% overall. The bioburden was 0 CFU, and the endotoxin in the formulated bulk was assayed at 1.0 < X < 2.0 EU / mL or 0.6 < X < 1.2 EU / mg.

[0062] Example 3 APT402 showed ex vivo antiplatelet activity equivalent to APT102 and ex vivo factor X activation inhibition equivalent to annexin V. APT402 was designed to maintain the enzymatic and biological activity of both APT102 and Annexin V. Using a malachite green assay, the inventors found that the enzymatic parameters of APT402 for ATP and ADP were equivalent to those of APT102. Similarly, APT402 inhibited ADP-induced human platelet aggregation with equivalent potency to APT102 (Figure 1). Inhibitory activity against FX activation was assayed using peripheral blood mononuclear cells (PBMCs) purified by standard methods from normal human donors and anesthetized rabbits. Similar dose-dependent reductions in LPS-induced FX activation were observed with both APT402 and Annexin V in rabbit and human PBMCs (Figure 2). As expected, APT102 had no effect on the procoagulant activity of PBMCs.

[0063] Example 4 APT402 demonstrated synergistic inhibition of thrombin generation from activated human plasma-rich platelets compared to APT102 and Annexin V alone. Thrombin formation in citrate-treated human platelet-rich plasma (PRP) was quantified by calibrated automated thrombography (CAT) using a Thrombinoscope system (Synapse) according to a method developed by Hemker et al. Aliquots of PRP were incubated in wells of a 96-well plate with a drug at room temperature for 15 minutes, followed by 5 minutes at 37°C. Thrombin formation was initiated by adding 0.5 pM tissue factor and CaCl2 to bring the concentration to 16.7 mM, and monitored using a microtiter plate fluorometer (Fluoroskan Ascent, Thermo Electron Corp., Vantaa, Finland). Thrombinoscope software was used to generate thrombograms (thrombin in nM units versus time), and important thrombin formation parameters were also generated: thrombin formation initiation delay time (min), peak thrombin concentration (nM), peak thrombin time (min), and endogenous thrombin production capacity (ETP; integrated region under the thrombogram curve).

[0064] The effects of equimolar concentrations of APT102, Annexin V, APT102 + Annexin V, and APT402 on thrombin generation from 20 μM ADP-activated human platelets were compared using a CAT assay (Figure 3). Annexin V significantly increased peak thrombin time but somewhat decreased peak thrombin concentration. APT102 had some inhibitory effect on both parameters. However, APT102 + Annexin V synergistically inhibited thrombin generation, significantly increasing peak thrombin time by a factor of 4 and simultaneously significantly decreasing peak thrombin by a factor of 3. These characteristics were mimicked by APT402.

[0065] Example 5 APT402 showed rapid onset of action after a single bolus administration in healthy rabbits. APT402 was administered intravenously as a single bolus (0.40 mg / kg) to two rabbits. Pharmacokinetic modeling showed the best fit to the biphasic exponential curve of ADPase activity (Figure 4). Maximum activity was detected in plasma 30 minutes post-administration. Distribution phase and efflux half-life (t) of APT402 were also observed. 1 / 2 The half-lives were 30 minutes and 6 hours, respectively, which is significantly longer compared to the 5-minute distribution phase half-life and 20-minute efflux half-life (18-fold) of Annexin V. Administration of APT402 inhibited 95% of 20 μM ADP-induced ex vivo platelet aggregation by 10 minutes after IV administration, and showed a rapid onset of action inhibiting 90% and 80% at 1 and 6 hours after administration, respectively. These data suggest that, due to its relatively short half-life, APT402 should be administered as a bolus and then as an IV infusion to ensure stable thrombosis reduction.

[0066] Example 6 Continuous intravenous infusion of APT402 resulted in rapid onset and cessation of its effects in healthy rabbits. Preliminary data indicate that APT402 has a short distribution phase half-life. Subsequently, rabbits were intravenously injected with APT402 as a single bolus (0.2 mg / kg), followed by intravenous infusions at 12 and 24 μg / kg / min for 120 minutes. ELISA data showed that APT402 was detectable in plasma up to 30 minutes after administration. APT402 concentrations, inhibition of ADP-induced platelet aggregation, and thrombin generation were maintained for up to 120 minutes during the infusion, and then significantly reduced 60 minutes after discontinuation of APT402 infusion (Figures 5, 6, 7). There were no differences between animals treated with APT402 and control animals in terms of bleeding time, ACT, PT, aPTT, or platelet count. The data indicate that optimal treatment with APT402 is achieved with 30 minutes of pretreatment to ensure attenuation of both local and systemic thrombosis.

[0067] Example 7 APT402 selectively localizes to the thrombus site of injured arteries in rabbits. Near-infrared (NIR) fluorescent dye (LS288,Ex / Em773 / 793) in methanol was conjugated with the functional amine of APT402 (approximately 1:1). The purified bioconjugate showed a single fluorescence band (Figure 8A). A custom-made fiber-based portable videorate system was used as described for in vivo imaging of thrombi by fluorescence molecular tomography (FMT) (CITE). Thrombosis was initiated 30 minutes after a bolus of APT402 (NIR-labeled and unlabeled APT402) and subsequent 12 μg / kg / min IV injection (maintaining patency of the injured carotid artery for 2 hours) by applying an anodic current to the carotid needle electrode. Mean fluorescence values ​​and confidence intervals for three rabbits are shown in Figure 8B. The injury site showed an approximately 6-7-fold increase in fluorescence after baseline due to the thrombus formed around the transvascular needle electrode. Importantly, APT402 was found to bind to thrombi (Figure 8C). In contrast, there was no increase in signaling in undamaged control carotid arteries (data not shown). Therefore, APT402 is specifically targeted to sites of arterial injury and thrombosis. The tissue distribution of NIR-labeled APT402 indicates that the majority of the label was taken up by the liver and spleen.

[0068] Example 8 APT402, both alone and in combination, was more effective in reducing arterial thrombosis in rabbits than other antiplatelet or anticoagulant agents, without increasing the risk of bleeding. Rabbits were randomized to one of the following 11 groups, initiating treatment 30 minutes before electrical injury.

[0069] [Table 1]

[0070] Electrical injury generated occlusion in 60% of rabbits treated with saline within 2 hours. The mean thrombotic weight was 7.8 mg. Treatment with 12 or 24 μg / kg / min of APT402, ticagrelor (alone or in combination with Angiomax) completely prevented occlusion (Figure 9). Thrombotic weight decreased in a dose-dependent manner with APT402 (Figure 10). Notably, treatment with 24 μg / kg / min of APT402 resulted in the lowest thrombotic weight compared to all other treatments, including the combination of ticagrelor and bivalirudine. On the other hand, APT402 did not affect bleeding time (Figure 11), prothrombin time (Figure 12), partial thromboplastin time (Figure 13), blood pressure (Figure 14), or heart rate (Figure 15).

[0071] Example 9 APT402 attenuated venous thrombosis, specifically deep vein thrombosis (DVT), in an acute mouse electrical injury model (EIM) without increasing bleeding or inducing decreased coagulation ability. An electrical injury model of venous thrombosis was used for acute testing (Diaz JA et al. Thromb Haemost. 104:366-375, 2010).

[0072] The mice were randomized into the following four groups (n=10 / group).

[0073] [Table 2]

[0074] EIM consistently induced IVC thrombosis in all mice, with an average thrombus weight of 22.5 mg. Thrombus weight, BT, APTT, and TCT were measured 48 hours after DVT induction.

[0075] Treatment with LMWH reduced thrombus weight by 57% compared to the control group and resulted in a significant lengthening of bleeding time: 3 times longer in aPTT, 2.5 times longer in TCT, and 2 times longer in TCT (Figure 16).

[0076] APT402 reduced thrombus weight by 44% and 65% at low and high doses, respectively, in a dose-dependent manner. Importantly, APT402 did not result in any detectable prolongation of BT, aPTT, or TCT (Figure 16). Similarly, APT402 administered as a daily ip bolus of 1.0 mg / kg for two days also reduced thrombus weight by 53% without increasing bleeding time.

[0077] These data suggest that APT402 is safe because it does not cause dose-limiting bleeding, and that it is more effective than enoxaparin in treating DVT.

[0078] Example 10 APT402 reduced fibrosis in a chronic mouse electrolytic injury (EIM) model of DVT without increasing bleeding. In the chronic fibrosis study, mice were randomized and blinded into the following groups (n=5-12 / group).

[0079] [Table 3]

[0080] Collagen deposition was stained using Masson's three-color staining method 14 days after DVT induction. EIM consistently induced IVC fibrosis in all mice and increased average collagen deposition by approximately six times compared to healthy mice (Figure 17).

[0081] Treatment with APT402 and APT102 significantly reduced collagen deposition by 33% and 11%, respectively. There was one death in the placebo group, but no deaths, increased bleeding, or serious side effects were observed in the APT402 and APT102 groups.

[0082] These data suggest that APT402 is safe and could be an effective treatment for post-thrombotic syndrome.

[0083] Sequence List Soluble CD39L3 R67G T69R(APT102) Sequence ID 1 - Nucleotide SEQ ID NO: 2 - Amino Acids [ka] APT402 has an optimized codon and a mouse Igκ signal peptide sequence. Sequence ID 3 - Nucleotide SEQ ID NO: 4 - Amino Acids [ka] [ka] [ka] 20AA Linker Sequence ID 5 - Nucleotide SEQ ID NO: 6 - Amino Acids [ka] APT402 with optimized codon and bovine α-lactalbumin signal peptide Sequence ID 7 - Nucleotide SEQ ID NO: 8 - Amino Acids [ka] [ka] [ka] APT402 with optimized codons Sequence ID 9 - Nucleotide SEQ ID NO: 10 - Amino Acids [ka] [ka] [ka]

Claims

1. A composition for preventing or treating thrombosis, comprising apirase and annexin V bound to phosphatidylserine (PS) on the surface of activated platelets, wherein the apirase and annexin V form a fusion protein.

2. The composition according to claim 1, wherein the apirase is soluble CD39L3 or a modified form thereof having enhanced ADPase activity.

3. The composition according to claim 2, wherein the apirase is a modified form of soluble CD39L3, and the modification consists of substitutions at amino acid positions 67 and 69.

4. The composition according to claim 3, wherein the substitutions are R67G and T69R.

5. The composition according to claim 4, wherein the apirase is APT102 (SEQ ID NO: 2).

6. The composition according to claim 1, wherein the apirase and annexin V are covalently bonded via a linker peptide to form a fusion protein.

7. The composition according to claim 6, wherein the linker peptide contains 3 to 30 amino acids.

8. The composition according to claim 7, wherein the linker peptide contains 15 to 25 amino acids.

9. The composition according to claim 6, wherein the linker peptide is resistant to hydrolysis in plasma and / or cell culture supernatant.

10. The composition according to claim 6, wherein the linker peptide has the amino acid sequence of SEQ ID NO:

6.

11. The composition according to claim 1, wherein the fusion protein is APT402 (SEQ ID NO: 10).

12. A nucleic acid encoding a fusion protein as defined in any one of claims 1 to 11.

13. The nucleic acid according to claim 12, further comprising a regulatory sequence for expression.

14. Recombinant host cells or vectors containing the nucleic acid described in claim 13.

15. A method for producing a fusion protein comprising apirase and annexin V covalently linked via a linker peptide, comprising culturing the host cells described in claim 14, and recovering the fusion protein from the culture.

16. A fusion protein containing apirase and annexin V covalently linked via a linker peptide.

17. A composition according to any one of claims 1 to 11 for treating subjects who have experienced thrombosis or who are undergoing surgery at risk of excessive bleeding.

18. The composition according to claim 17, which is administered intravenously (IV).

19. The composition according to claim 17, administered by bolus injection and subsequent IV infusion.

20. The use of a fusion protein of apirase and annexin V, which binds to phosphatidylserine (PS) on the surface of activated platelets, in the manufacture of a pharmaceutical product for the treatment of subjects experiencing thrombosis or undergoing surgery at risk of excessive bleeding.

21. The use according to claim 20, wherein the apirase is APT102 (SEQ ID NO: 2).

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