Methods and pharmaceutical compositions for the treatment of acute ischemic stroke

Abstract

This invention provides a method for treating acute ischemic stroke (AIS) in patients requiring treatment for AIS, and a pharmaceutical composition for such treatment. [Solution] A combination therapy in which a patient is administered a therapeutically effective combination of t-PA and DNAse, wherein the administration of this combination produces enhanced therapeutic efficacy compared to the administration of t-PA alone.

Description

Technical Field

[0001] The present invention relates to methods and pharmaceutical compositions for the treatment of acute ischemic stroke.

Background Art

[0002] Background of the Invention Acute ischemic stroke (AIS) is the sudden interruption of adequate blood flow to a region of the brain, usually caused by a thrombus or other embolus that has clogged or formed in one of the arteries supplying blood to the brain. If the blockage is not removed promptly, ischemia can lead to permanent neurological deficits or death. Intravenous (IV) administration of recombinant tissue plasminogen activator (t-PA; alteplase, Actilyse®, Boehringer Ingelheim) is the only drug therapy that has been confirmed to be effective in AIS. This treatment is inefficient in terms of early recanalization. Since 2015, this treatment has been associated with the realization of endovascular treatment (EVT) that enables an increase in the early recanalization rate and a significant improvement in the functional outcome at the third month in the case of proximal intracranial artery occlusion. The time frame for AIS treatment is within 4.5 hours for IV t-PA infusion and within 6 hours for EVT. Even within this time, there is strong evidence that the shorter the time between symptom onset and treatment, the better the result. EVT for AIS consists of mechanical removal of thrombus using a thrombus removal device such as a stent retriever or a direct aspiration catheter.

[0003] The primary objective of AIS management is to achieve the earliest possible recanalization of the occluded artery. This early recanalization is, in fact, the main predictor of neurological clinical outcomes. International guidelines for AIS treatment recommend IV thrombolysis with t-PA within 4 hours and 30 minutes of the onset of EVT-related symptoms in cases of proximal anterior circulation occlusion up to 6 hours. However, IV thrombolysis is not very effective in recanalizing proximal occlusions. In fact, recent reports indicate rates of 5% in internal carotid artery occlusions and 20% in proximal middle cerebral artery occlusions. The effectiveness of IV t-PA infusion is limited to 10% due to the dilution of the potent compound in whole blood, inhibition of t-PA by circulatory inhibitors such as PAI-1 during plasma transport, low initial levels of t-PA binding to thrombus, and an increased risk of delayed hemorrhagic changes. Furthermore, while the thrombolytic effect of t-PA is beneficial, the toxicity of t-PA in the high-dose range currently used is a concern. While EVT is associated with high recanalization rates, it is overly specialized, expensive, and therefore difficult to use in emergencies. Easily administered drug treatments to increase recanalization rates associated with IV thrombolysis would represent a significant advance in the management of AIS.

[0004] Neutrophil extracellular traps (NETs) have recently been identified as a major trigger and structural factor in various forms of thrombosis. NETs are extracellular networks primarily composed of neutrophil-derived DNA. Recently, a study identified extracellular DNA threads from NETs as a potential therapeutic target to enhance the effectiveness of t-PA-induced thrombolysis in acute coronary syndrome (Mangold, A., Alias, S., Scherz, T., Hofbauer, T., Jakowitsch, J., Panzenboeck, A. & Mascherbauer, J. (2015). Coronary Neutrophil Extracellular Trap Burden and Deoxyribonuclease Activity in ST-Elevation Acute Coronary Syndrome Are Predictors of ST-Segment Resolution and Infarct Size. Circulation research, 116(7), 1182-1192). In recent years, DNAse 1 alone has been shown to significantly reduce infarct volume compared to the vehicle in mouse models of cerebral ischemia-reperfusion (De Meyer, SF, Suidan, GL, Fuchs, TA, Monestier, M., & Wagner, DD (2012). Extracullular chromatin is an important mediator of ischemic stroke in mice. Arteriosclerosis, thrombosis, and vascular biology, 32(8), 1884-1891). Therefore, this study disclosed that DNAse 1 may be suitable for improving downstream effects induced by ischemia, but it did not disclose that this enzyme has a favorable effect on proximal arterial recanalization rates. [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention relates to methods and pharmaceutical compositions for the treatment of AIS. In particular, the present invention is defined by the claims. [Modes for carrying out the invention]

[0006] Detailed description of the invention Neutrophil extracellular traps (NETs) are DNA extracellular networks decorated with histone and granule proteins produced by activated neutrophils. NETs have been identified as major triggers and structural factors of thrombosis. Our objective was to evaluate the presence of NETs in thrombi recovered during endovascular therapy in patients with acute ischemic stroke (AIS) and their effect on t-PA-induced thrombolysis. Accordingly, we analyzed thrombi from 108 AIS patients treated with endovascular therapy. The thrombi were characterized by hematoxylin / eosin staining, immunohistochemistry, and ex vivo enzyme assays. In addition, we evaluated the effect of deoxyribonuclease 1 (DNAse 1) on thrombolysis of AIS thrombi ex vivo. Histological analysis revealed that NETs contributed to the composition of all AIS thrombi, particularly their composition in the outer layer. Quantitative measurement of thrombus NETs content was not associated with clinical outcomes or the pathogenesis of AIS, but it was significantly correlated with the length of endovascular procedures and the number of device passes. Ex vivo, recombinant DNAse 1 promoted t-PA-induced thrombolysis, while DNAse 1 alone was ineffective. This study suggests that thrombus NETs content, regardless of its cause, may contribute to resistance to reperfusion, including mechanical or pharmacological approaches with intravenous t-PA. These results also suggest that combination therapies involving t-PA and DNAse 1 may have a synergistic effect that enables the efficacy of t-PA.

[0007] A first object of the present invention relates to a method for treating a patient requiring treatment for acute ischemic stroke (AIS), the method comprising administering t-PA and DNAse to the patient. In one embodiment, the method of the present invention comprises administering to the patient a therapeutically effective combination of t-PA and DNAse.

[0008] As used herein, the terms “therapeutic dose,” “therapeutic combination,” or “pharmaceutical dose” refer to the amount or dose of t-PA, the amount or dose of DNAse, or both in the combination according to the present invention, which is intended to (1) delay or prevent the onset of AIS; (2) reduce the severity or incidence of AIS; (3) slow or halt the progression, worsening, or exacerbation of one or more symptoms of AIS; (4) bring about improvement of the symptoms of AIS; or (5) cure AIS, without causing serious negative or adverse side effects in the subject. The therapeutic dose or combination may be administered before the onset of AIS. Alternatively or additionally, the therapeutic dose or combination may be administered after the onset of AIS.

[0009] In one embodiment, the administration of t-PA and DNAse in combination results in enhanced therapeutic efficacy compared to the administration of t-PA alone.

[0010] A further object of the present invention is a method for enhancing the efficacy of t-PA administered as part of a treatment regimen to a patient suffering from AIS, the method comprising administering a pharmaceutically effective amount of t-PA to the patient in combination with DNAse.

[0011] A further object of the present invention relates to a method for enhancing the efficacy of t-PA administered as part of a treatment regimen to a patient suffering from AIS, the method comprising administering DNAse to the patient treated with t-PA. In one embodiment, a therapeutically effective dose of DNAse is administered to the patient. In one embodiment, the method of the present invention makes it possible to reduce the dose of t-PA that will be administered to the patient.

[0012] A further object of the present invention relates to a method for achieving recanalization of an occluded intracranial artery in a patient suffering from AIS, comprising administering t-PA and DNAse to the patient. In one embodiment, the method of the present invention comprises administering to the patient a therapeutically effective combination of t-PA and DNAse.

[0013] The present invention further relates to a combination of t-PA and DNAse, comprising or essentially comprising them, for use in the treatment of patients requiring treatment for AIS.

[0014] As used herein in relation to the combination, the term "essentially derived from" means that t-PA and DNase are the only therapeutic or biologically active compounds in the combination of the present invention.

[0015] Another object of the present invention is a pharmaceutically acceptable combination comprising, consisting of, or essentially comprising t-PA and DNAse and at least one pharmaceutically acceptable carrier, for use in the treatment of patients requiring treatment for AIS.

[0016] Another object of the present invention is a parts kit comprising a first part containing t-PA and a second part containing DNAse for use in the treatment of patients requiring treatment for AIS.

[0017] Another object of the present invention is a parts kit comprising a first part comprising a pharmaceutical composition comprising t-PA and at least one pharmaceutically acceptable carrier, for use in the treatment of a patient requiring treatment for AIS, and a second part comprising a pharmaceutical composition comprising DNAse and at least one pharmaceutically acceptable carrier.

[0018] Another object of the present invention is a medicament comprising the combination of t-PA and DNAse described above herein, the pharmaceutical combination described above herein, or the parts kit described above herein for use in its treatment in patients requiring treatment for AIS.

[0019] As used herein, the term "acute ischemic stroke" or "AIS" refers to a patient having or at risk of having a "definite acute ischemic cardiovascular syndrome (AICS)" as defined by the diagnostic criteria of Kidwell et al., "Acute Ischemic Cerebrovascular Syndrome: Diagnostic Criteria," Stroke, 2003, 34, pp. 2995-2998 (incorporated herein by reference). Thus, acute ischemic stroke refers to the acute onset of any severity of neurological dysfunction consistent with focal cerebral ischemia.

[0020] In one embodiment, the patient is diagnosed with AIS.

[0021] In another embodiment, the patient is at risk of developing AIS. Examples of risk factors for developing AIS include, but are not limited to, atrial fibrillation or other cardioembolic diseases, atherosclerotic plaques or stenosis occurring in the aortic arch, carotid artery or intracranial artery, spontaneous cervical dissection, genetic factors, family history of AIS, and others.

[0022] In one embodiment, the patient is human. In one embodiment, the patient is male. In another embodiment, the patient is female.

[0023] In one embodiment, the patient has been previously treated by EVT or is planned to receive treatment by EVT. Thus, according to one embodiment, the method of the present invention includes administering t-PA and DNAse to the patient and performing EVT on the patient.

[0024] As used herein, the terms “treatment” or “to treat” refer to both prophylactic and curative or disease-modifying treatments, including treatment of patients at risk of or suspected of being afflicted with the disease, and patients diagnosed as being afflicted or suffering from a disease or medical condition, including the suppression of clinical relapses. Treatments may be administered to subjects who have or may eventually acquire a medical disability in order to prevent, cure, delay, reduce the severity of, or improve one or more symptoms of the disability or relapse of the disability, or to extend the subject’s survival beyond the survival expected without such treatment. “Treatment regimen” means a pattern of treatment for a disease, for example, a pattern of medication used in treatment. A treatment regimen may include an induction regimen and a maintenance regimen. The terms “induction regimen” or “induction period” refer to a treatment regimen (or a portion of a treatment regimen) used for the initial treatment of the disease. The general goal of an induction regimen is to provide the patient with a high level of medication during the initial period of the treatment regimen. An induction regimen may employ (partially or entirely) a “loading regimen,” which may include administering a dose of the drug at a higher dose than the physician uses during the maintenance regimen, administering the drug at a higher frequency than the physician uses during the maintenance regimen, or both. The terms “maintenance regimen” or “maintenance period” refer to a treatment regimen (or portion of a treatment regimen) used to maintain a patient during treatment of a disease, for example, to keep the patient in remission for an extended period (months or years). A maintenance regimen may employ continuous therapy (i.e., administering the drug at regular intervals, such as weekly, monthly, or yearly) or intermittent therapy (i.e., intermittent treatment, intermittent treatment, relapse treatment, or treatment upon achieving a specific predetermined criterion [e.g., disease onset]).

[0025] As used herein, the term “t-PA” has its general meaning in the art and refers to a tissue-type plasminogen activator. This term includes native t-PA and recombinant t-PA, as well as modified forms of t-PA that retain the enzymatic or fibrinolytic activity of native t-PA. The enzymatic activity of t-PA can be measured by evaluating the molecule’s ability to convert plasminogen to plasmin. The fibrinolytic activity of t-PA may be determined by any in vitro clot-forming activity known in the art. Recombinant t-PA is widely described in the prior art and is known to those skilled in the art. t-PA is commercially available as alteplase (Activase® or Actilyse®). Modified forms of t-PA ("modified t-PA") are characterized and known to those skilled in the art. Modified t-PAs include, but are not limited to, variants with amino acid or domain deletions or substitutions, variants conjugated or fused with other molecules, and variants with chemical modifications such as modified glycosylation. Several modified t-PAs are described in PCT International Publication No. 93 / 24635;EP352,119;EP382174. In some embodiments, the modified form of t-PA is tenecteplase. As used herein, the term "tenecteplase," also known as the tissue-type plasminogen activator variant of the TNK-t-PA or TNKASE® brand, refers to the t-PA variants known as T103N, N117Q, K296A, H297A, R298A, and R299A t-PA, available from Genentech, Inc. (South San Francisco, Calif.), in which Thr103 of wild-type t-PA is changed to Asn (T103N), Asn117Gln of wild-type t-PA is changed to (N117Q), and Lys-His-Arg-Arg 296-299 of wild-type t-PA is changed to Ala-Ala-Ala-Ala (KHRR296-299AAAA).Tenecteplase is a genetically engineered variant of human t-PA cloned and expressed in Chinese hamster ovary cells (see generally Keyt et al., Proc. Natl. Acad. Sci USA, 91: 3670-3674 (1994) and Verstraete, Am. J. Med, 109: 52-58 (2000) for an overview of third-generation thrombolytic drugs). Tenecteplase has been engineered to have increased fibrin specificity and increased half-life compared to alteplase.

[0026] In one embodiment, the t-PA is a mutant t-PA as described in International Publication No. WO 2013 / 034710. In one embodiment, the mutant t-PA comprises the sequence of SEQ ID NO: 1 (corresponding to the human wt t-PA mature form) or SEQ ID NO: 2 (corresponding to the human wt t-PA first chain of tc-t-PA), preferably the association of SEQ ID NO: 1 or SEQ ID NO: 2 with SEQ ID NO: 3 (corresponding to the human wt t-PA second chain of tc-t-PA), or consists of a variant having at least 80%, 85%, 90%, 95% or more identity thereto, wherein the sequence comprises a mutation consisting of the substitution of any amino acid of the lysine binding site of SEQ ID NO: 1 or SEQ ID NO: 2 with a hydrophilic amino acid selected from arginine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, serine, threonine, tyrosine and histidine, preferably by arginine, and / or a mutation consisting of the substitution of arginine at position 275 of SEQ ID NO: 1 or SEQ ID NO: 2 with serine.

[0027] In one embodiment, the mutant t-PA includes the sequence of SEQ ID NO: 1 (corresponding to the mature form of human wt t-PA) or SEQ ID NO: 2 (corresponding to the first human wt t-PA of double-stranded t-PA), and preferably consists of an association of SEQ ID NO: 1 or SEQ ID NO: 2 and SEQ ID NO: 3 (corresponding to the second human wt t-PA of double-stranded t-PA), or a variant thereof having at least 80%, 85%, 90%, 95% or more identity, in which case the sequence includes a mutation consisting of a substitution of tryptophan at position 253 of SEQ ID NO: 1 or SEQ ID NO: 2 with a hydrophilic amino acid selected from arginine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, serine, threonine, tyrosine, and histidine, preferably arginine, and / or a mutation consisting of a substitution of arginine at position 275 of SEQ ID NO: 1 or SEQ ID NO: 2 with serine.

[0028] In one embodiment, the mutant t-PA further has the following mutations: - Arginine substitution of proline at position 125 of sequence number 1 or sequence number 2. - Deletion of the N-terminal finger domain and / or EGF-like domain in SEQ ID NO:1 or SEQ ID NO:2, and / or substitution of asparagine at position 117 of SEQ ID NO:1 or SEQ ID NO:2 with glutamine. - Substitution of threonine at position 103 of SEQ ID NO: 1 or SEQ ID NO: 2 with asparagine, and / or substitution of asparagine at position 117 of SEQ ID NO: 1 or SEQ ID NO: 2 with glutamine, and / or substitution of lysine-histidine-arginine-arginine (KHRR) at positions 296-299 of SEQ ID NO: 1 with alanine-alanine-alanine-alanine (AAAA), - Serine substitution of cysteine ​​at position 84 of sequence number 1 or sequence number 2. - Substitution of arginine at position 275 of SEQ ID NO:1 or SEQ ID NO:2 with glutamate or glycine, and / or deletion of the kringle 1 domain in SEQ ID NO:1 or SEQ ID NO:2 It includes at least one of the following.

[0029] As used herein, the terms “identity” or “identical” in reference to the relationship between two or more amino acid sequences refer to the degree of sequence relevance between amino acid sequences, determined by the number of matches between sequences of two or more amino acid residues. “Identity” measures the percentage of identical matches between the smaller of the two or more sequences by gapped alignment (if any) processed by a specific mathematical model or computer program (i.e., “algorithm”). The identity of related amino acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Arthur M. Lesk, Computational Molecular Biology: Sources and Methods for Sequence Analysis (New-York: Oxford University Press, 1988); Douglas W. Smith, Biocomputing: Informatics and Genome Projects (New-York: Academic Press, 1993); Hugh G. Griffin and Annette M. Griffin, Computer Analysis of Sequence Data, Part 1 (New Jersey: Humana Press, 1994); Gunnar von Heinje, Sequence Analysis in Molecular Biology: Treasure Trove or Trivial Pursuit (Academic Press, 1987); Michael Gribskov and John Devereux, Sequence Analysis Primer (New York: M. Stockton Press, 1991); and Carillo et al., 1988. SIAM J. Appl. Math. 48(5):1073-1082. The preferred method for determining identity is designed to give the greatest match between the test sequences. Methods for determining identity are described in publicly available computer programs.Preferred computer programming methods for determining the identity between two sequences include the GCG program package, which includes GAP (Devereux et al., 1984. Nucl. Acid. Res. 12(1 Pt 1):387-395; Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, WI), BLASTP, BLASTN, TBLASTN, and FASTA (Altschul et al., 1990. J. Mol. Biol. 215(3):403-410). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., 1990. J. Mol. Biol. 215(3):403-410). The well-known Smith-Waterman algorithm is also sometimes used to determine identity.

[0030] As used herein, the term “DNAse” has its general meaning in the art and refers to, including, all enzymes having phosphodiesterase activity and the ability to hydrolyze DNA. Any suitable DNAse may be used in the present invention. Most preferably, the DNAse is DNAse 1 (EC 3.1.21.1). However, in some embodiments, the DNAse may be DNAse II (EC 3.1.21.1). Any DNAse present in several species and capable of cleaving DNA may be used in the present invention. In one embodiment, the DNAse is recombinant DNAse. The DNAse may be of animal origin, such as from cattle or pigs. The DNAse may be of plant, fungal, or microbial origin. However, typically and most preferably, the DNAse is of human origin, preferably recombinant human DNAse. Commercially available DNAse preparations, such as Dornase® and Pulmozyme®, may be used in embodiments of the present invention.

[0031] As used herein, the term “combination” is intended to refer to all forms of administration in which a first drug is administered together with a further (second, third, etc.) drug.

[0032] The drugs may be administered simultaneously, separately, sequentially, and in any order. In one embodiment, t-PA is administered before DNAse. In another embodiment, DNAse is administered before t-PA. In yet another embodiment, t-PA and DNAse are administered simultaneously.

[0033] Drugs administered in combination have biological activity in the target to which they are delivered. Therefore, in the context of this invention, a combination comprises at least two different drugs, one of which is at least t-PA and the other is DNAse. As used herein, the expression "enhancing the potency of t-PA" refers to the ability of recombinant DNAse to increase t-PA-induced fibrinolysis. In particular, DNAse is suitable for increasing the recanalization rate provided by t-PA. Thus, the combination of the two drugs results in enhanced fibrinolysis of thrombi.

[0034] As used herein, the term “therapeutically effective combination” means an amount or dose of t-PA combined with an amount or dose of DNAse that is sufficient to treat acute ischemic stroke, in particular to achieve recanalization of an occluded artery. The amount of t-PA or DNAse in a given therapeutically effective combination may vary for different individuals and different diseases and will depend on one or more additional drugs or treatments included in the combination. The “therapeutic effective dose” is determined using procedures routinely employed by those skilled in the art to produce an “improved therapeutic outcome.” However, it will be understood that the total daily dose of the compounds and compositions of the present invention is to be determined by the attending physician within the bounds of sound medical judgment. A specific therapeutically effective dose level for any particular subject will depend on a wide variety of factors, including the disorder being treated and its severity; the activity of the particular compound being employed; the particular composition being employed, the subject’s age, weight, overall health, sex, and diet; the timing of administration, route of administration, and excretion rate of the particular compound being employed; the duration of treatment; drugs used in combination with or in conjunction with the particular polypeptide being employed; and similar factors well known in medicine. For example, it is well within the skill of those skilled in the art to start with a compound dose lower than the dose required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dose of the product may vary over a wide range of 0.01 mg to 1,000 mg / day per adult. Typically, compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient to adjust the dosage to the target symptoms to be treated. Pharmaceuticals typically contain about 0.01 mg to about 500 mg of the active ingredient, preferably 1 mg to about 100 mg. The effective amount of the drug is usually supplied at dosage levels of 0.0002 mg / kg to about 20 mg / kg body weight / day, particularly about 0.001 mg / kg to 7 mg / kg body weight / day.In one embodiment, a single dose of t-PA, DNAse, or a combination of the present invention is administered (or will be administered) to the subject, preferably during the acute phase of AIS. In one embodiment, multiple doses of t-PA, DNAse, or a combination of the present invention are administered (or will be administered) to the subject. Therefore, in one embodiment, the combination of the present invention is administered as divided doses. In one embodiment, the dose of t-PA administered (or will be administered) to the subject falls between approximately 0.01 mg / kg and approximately 5 mg / kg. In one embodiment, the dose of t-PA administered to the subject falls between approximately 0.1 mg / kg and approximately 1 mg / kg. In one embodiment, the dose of alteplase (t-PA of the present invention) administered to the subject is equal to approximately 0.9 mg / kg. In one embodiment, the dose of tenecteplase (t-PA of the present invention) administered to the subject falls between approximately 0.1 mg / kg and approximately 0.4 mg / kg. In one embodiment, the dose of the DNAse of the present invention administered to the subject is between about 10 μg / kg and about 500 μg / kg, preferably between about 50 μg / kg and about 250 μg / kg. In one embodiment, the dose of rhDNAse 1 (the DNAse of the present invention) administered to the subject is equal to about 125 μg / kg. Thus, in one embodiment, the method of the present invention includes administering a dose of t-PA to the subject in the range of about 0.01 mg / kg to about 5 mg / kg, and administering a dose of DNAse to the subject in the range of about 0.1 μg / kg to about 500 μg / kg. The term “about” as used herein prior to a number means ±10% of the value of the number.

[0035] Typically, the drug of the present invention (i.e., t-PA or DNAse or a combination thereof) is administered to a subject in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0036] As used herein, the term “pharmaceutically acceptable carrier” refers to an excipient that does not produce adverse reactions, allergic reactions, or other undesirable reactions when administered to mammals, preferably humans. This includes any and all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic and absorption retardants, and others. A pharmaceutically acceptable carrier refers to a non-toxic solid, semi-solid, or liquid extender, diluent, encapsulating material, or any type of formulation aid. For human administration, the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by regulatory authorities such as the FDA or EMA.

[0037] pharmaceutically acceptable carriers that may be used with these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, serum proteins such as lecithin and human serum albumin, buffers such as phosphates, glycine, sorbic acid and potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica and magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin. For use in administration to a subject, the compositions will be formulated for administration to the subject.

[0038] The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, orally, vaginally, or via an implanted reservoir.

[0039] In one embodiment, the t-PA and DNAse, combination thereof or pharmaceutically appropriate combination, pharmaceutical or parts kit according to the present invention would be formulated for administration to a subject. The t-PA and DNAse, combination thereof or pharmaceutically appropriate combination, or pharmaceutical according to the present invention may be administered orally, parenterally, topically, by inhalation spray, rectally, nasally, orally, vaginally, or via an implanted reservoir.

[0040] In one embodiment, t-PA and DNAse are administered to the patient via the same route of administration. In another embodiment, t-PA and DNAse are administered to the patient via different routes of administration.

[0041] In one embodiment, t-PA and DNAse according to the present invention, their combination or pharmaceutically appropriate combination, pharmaceutical or parts kit, are administered by injection. Those used herein include subcutaneous, intravenous, intramuscular, intra-articular, intra-sacral, intrasternal, subarachnoid, intrahepatic, intrafocal, and intracranial injection or infusion techniques. The sterile injection forms of the compositions or combinations of the present invention may be aqueous or oily suspensions. These suspensions may be formulated by techniques known in the art using appropriate dispersants or wetting and suspending agents. The sterile injection preparations may also be sterile injections or suspensions in a non-toxic, parenterally acceptable diluent or solvent, such as a 1,3-butanediol solution. Acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixatives are conventionally employed as solvents or suspensions. For this purpose, any non-irritating fixative containing synthetic monoglycerides or synthetic diglycerides may be employed. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectable preparations, as are pharmaceutically acceptable natural oils such as olive oil or castor oil, especially their polyoxyethylene derivatives. These oily solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants commonly used in the formulation of pharmaceutically acceptable drug forms, including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans, and other emulsifiers, or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solids, liquids, or other drug forms, may also be used for formulation purposes.

[0042] The compositions or combinations of the present invention may be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, or aqueous solutions. For tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. Diluents useful for oral administration in capsule form include, for example, lactose. If an aqueous suspension is required for oral use, the active ingredient is mixed with emulsifiers and suspending agents. Certain sweeteners, flavorings, or colorings may also be added, if desired.

[0043] Alternatively, the compositions or combinations of the present invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing a drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0044] The compositions or combinations of the present invention may also be administered topically, particularly when the target of treatment includes areas or organs that are easily accessible by topical application, such as diseases of the eyes, skin, or lower gastrointestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. For topical application, the compositions may be formulated as suitable ointments containing the active components suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the compositions may be formulated as suitable lotions or creams containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Topical application for the lower gastrointestinal tract can be achieved as a rectal suppository (see above) or as an appropriate enema. Patches may also be used.

[0045] The compositions of the present invention may also be administered by nasal aerosol or inhalation. Such compositions may be prepared by techniques well known in pharmaceutical formulation technology and may be prepared as a solution in saline solution using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, carbon fluoride, and / or other conventional solubilizers or dispersants.

[0046] The present invention is further illustrated by the following drawings and embodiments. However, these embodiments and drawings should not be construed in any way as limiting the scope of the present invention. [Brief explanation of the drawing]

[0047] [Figure 1]This figure shows the correlation between neutrophil extracellular trap (NETS) load, acute thrombolytic therapy, and stroke cause. To assess the correlation between NETs load and characteristics of acute ischemic stroke patients, thrombus NETs content was determined by quantifying neutrophil elastase release using endonuclease-treated thrombi. (A) Compared to patients who did not receive t-PA treatment, NETs load was slightly but significantly reduced in patients treated with IV t-PA. (B) According to the TOAST classification, there was no association between stroke cause and NETs load (LAA: large vessel atherosclerosis; CE: cardiac embolism; LAC: pit-type; OTH: other causes; UND: undetermined cause). (C) and (D) Interestingly, NETs load showed a positive correlation with the length of EVT intervention and the number of device passes. [Figure 2] This figure shows the ex vivo stroke clot lysis assay. The thrombolytic efficacy of tPA and DNAse, administered alone or in combination, was compared in vitro. For these experiments, thrombi recovered by thrombectomy were incubated in PBS supplemented with tPA (1 μg / mL) and / or DNAse (100 U / mL). The weight of the thrombus was assessed before the addition of tPA and DNAse, and 10, 30, and 60 minutes after addition. Results are expressed as a percentage of the initial thrombus weight. Data are presented as median (interquartile range). [Figure 3] This figure shows that neutrophil extracellular traps are constitutively present in thrombi of acute ischemic stroke. The presence of NETs in AIS thrombi was investigated by measuring DNA-associated neutrophil elastase activity. The dot plot shows the elastase activity measured in the supernatant of thrombi before and after endonuclease treatment (n=23; p<0.0001). [Figure 4]This figure shows that the extracellular DNA content of neutrophils in thrombi is not associated with the cause of AIS, but is associated with the characteristics of the endovascular procedure. A-B. Comparison of neutrophil-derived extracellular DNA content (n=72) between thrombi classified according to (A) the cause of stroke (LAA: atherosclerosis; CE: cardiac embolism; OTH: other causes; UND: undetermined) or (B) whether or not IV t-PA treatment was administered before endovascular therapy (p=0.03). C-D. Correlation between the neutrophil-derived extracellular DNA content of thrombi (n=72) and the length of the endovascular procedure (C); and the number of device passes achieved (D). [Figure 5] This figure shows that DNAse 1 ex vivo enhances t-PA-induced thrombolysis. Acute ischemic stroke thrombi recovered by endovascular therapy were incubated with t-PA and / or DNAse 1, and their lysis was tracked by measuring the change in wet weight of the thrombi over time. The mean baseline weight of the thrombi was 14.6 ± 8.4 mg. The thrombolytic effect of t-PA alone or in combination with DNAse 1 was compared. (n=13; mean [SD]; 10 min: t-PA=105.3% [7.02] vs. t-PA+DNase I=97.73% [5.62] (p=0.022), 30 min: t-PA=104.8% [13.45] vs. t-PA+DNase I=75.13% [17.39] (p=0.001), 60 min: t-PA=82.71% [20.08] vs. t-PA+DNase I=41.71% [26.43] (p=0.007). t-PA alone was associated with a slight but significant reduction in thrombus weight at 60 min compared to baseline (p=0.003). B. Comparison of thrombolytic effects of DNase I alone or in combination with t-PA (n=11, mean [SD]; 10 min: DNase I DNAse I = 104.6% [12.73] compared to DNAse I + t-PA = 92.91% [17.55] (30 min: DNAse I = 95.66% [21.29] compared to DNAse + t-PA = 69.39% [21.65] (60 min: DNAse I = 83.36% [33.89] compared to DNAse I + t-PA = 37.83% [17.65]). DNAse I alone did not affect thrombus weight at 60 min compared to baseline (p=0.06). [Example 1]

[0048] Neutrophil extracellular traps (NETs) have recently been identified as major triggers and structural factors in various forms of thrombosis. NETs are extracellular networks primarily composed of neutrophil-derived DNA. Recently, a study identified extracellular DNA strands from NETs as a potential therapeutic target to enhance the effectiveness of t-PA-induced thrombolysis in acute coronary syndrome. The aim of this study was to evaluate the impact of NET load in thrombi recovered during endovascular therapy in patients with acute ischemic stroke (AIS) on t-PA-induced thrombolysis, clinical outcomes, and the etiology of AIS.

[0049] The inventors analyzed thrombi from 150 AIS patients treated with endovascular therapy. Thrombuses were characterized by HE staining, immunohistochemistry, and enzyme assays. Histological analysis revealed that NETs (non-organic steroids) contribute to the scaffolding of most thrombi, particularly their superficial layers. In vitro endonuclease treatment of thrombi revealed significant release of free DNA (p<0.001) and neutrophil elastase (p<0.001) into the supernatant, reflecting quantitative NETs load on the thrombi. NETs load on thrombi did not correlate with clinical outcomes or the cause of AIS. Recombinant deoxyribonuclease 1 (DNAse 1) promoted t-PA-induced thrombolysis in an in vitro lysis assay (p=0.02), whereas t-PA or DNAse 1 alone were ineffective in inducing significant thrombolysis of these thrombi.

[0050] This study suggests that the NETs inclusion structure of AIS thrombi may be involved in t-PA-induced thrombolytic resistance, independently of their cause and localization. The effectiveness of a strategy involving the co-administration of DNAse 1 in addition to t-PA to promote AIS thrombofibrinolysis may be of great interest in the treatment of AIS. [Example 2]

[0051] method Sample Selection Patients who underwent endovascular therapy (EVT) and successfully had thrombus removal performed at the inventors' facility between December 2015 and December 2016 were enrolled in this study. The local ethics committee approved this study protocol.

[0052] Data collection Using structured questionnaires (ETIS registry, endovascular procedures in ischemic stroke), we prospectively collected patient demographics, vascular risk factors, imaging findings, pre-procedure vital signs, AIS severity, and clinical outcomes. The TOAST classification was used to categorize the causes of stroke.

[0053] Endovascular therapy procedures In the first example, the interventional physician had the discretion to choose an EVT procedure using either a stent retriever or the direct aspiration first path technique. Detailed technical procedures have been previously published. 1

[0054] Collection and treatment of thrombi from acute ischemic stroke Thrombosis from acute ischemic stroke (AIS) was collected at the end of EVT. They were immediately frozen at -20°C, then stored at -80°C and / or fixed in 3.7% paraformaldehyde, and then embedded in paraffin. 108 patients were included. Thrombosis from 74 patients was used for lysis assays (n=24) and / or for determining NETs content, assessed by measuring NE release after endonuclease treatment. NE release was quantified by measuring NE antigen (n=72) and / or activity (n=23). Thrombosis from 34 patients was fixed in paraformaldehyde and used for immunohistoanalysis.

[0055] Histology and immunohistochemistry After deparaffinization, tissue sections were permeabilized, washed, and incubated with a primary antibody against MPO (rabbit anti-human MPO antibody, A0398, Dako) and citrullinated histone H4 (rabbit anti-human histone H4 (citrulline 3) antibody, 07-596, Millipore), followed by incubation with a fluorescent secondary antibody and counterstaining with DAPI (Sigma-Aldrich). Each AIS thrombus was also stained with hematoxylin / eosin (H&E).

[0056] ExVivo's NETs rating A commercially available NETs assay kit (Cayman Chemical) was used. This assay kit measures neutrophil elastase activity in tissue supernatant before and after incubation with endonuclease. The released neutrophil elastase corresponds to extracellular DNA-associated neutrophil elastase. To quantify the NETs content of AIS thrombi, a human neutrophil elastase ELISA kit (HycultBiotech) was used to measure neutrophil elastase antigen in the supernatant of thrombi recovered after endonuclease treatment.

[0057] Ex vivo thrombolysis assay The following protocol was a modification of Mangold et al. (2). Briefly, frozen AIS thrombi were glycolyzed, divided into two equal parts, and incubated in PBS in the presence of recombinant t-PA (1 μg / mL, Actilyse, Boehringer Ingelheim, Ingelheim am Rhein, Germany) and / or recombinant DNAse 1 (100 IE / ml, Pulmozyme, Roche, Basel, Switzerland) on a thermomixer at 500 rpm and 37°C. The weight of the thrombi was measured using an ultraprecise balance before the start of the procedure and at 10, 30, and 60 minutes. Thrombolysis was expressed as a percentage of the baseline thrombus weight.

[0058] statistical analysis Data were analyzed using nonparametric ANOVA (Kruskal-Wallis), followed by the Wilcoxon rank-sum test for paired data comparisons or the Mann-Whitney U test for unpaired data comparisons. Results are presented as mean ± SD for continuous variables and as numerical values ​​(percentages) for qualitative variables. Statistical analysis was performed using PrismGraph 4.0 software (GraphPad Software, San Diego, CA). Values ​​of P < 0.05 were considered statistically significant.

[0059] result Patient characteristics The inventors included 108 patients in this study between December 2015 and December 2016. The patient characteristics are shown in Table I. These characteristics are similar to those of the recently published EVT clinical trial.

[0060] Neutrophil extracellular traps are constitutively present in thrombi of acute ischemic stroke. Morphometric analysis of AIS thrombi after H&E staining revealed the presence of numerous polymorphonuclear cells in all thrombi examined. Extracellular nucleic acid strands suggestive of NETs were found in all 34 thrombi histologically analyzed, particularly on their surface (data not shown).

[0061] Immunofluorescence detection confirmed that regions containing extracellular DNA co-localized with citrullinated histones and granular neutrophil proteins (MPO) corresponding to NETs (data not shown). To confirm the presence of NETs in AIS thrombi, the inventors developed an ex vivo NETs assay method in 23 thrombi. For this assay method, the presence of NETs was examined by measuring the NE activity released from the thrombi after endonuclease treatment. Since NE activity increased after endonuclease incubation, the presence of NETs was confirmed in all thrombi (Figure 3).

[0062] The neutrophil-derived extracellular DNA content correlates with the length of the EVT procedure and the number of device passes. Thrombotic neutrophil-derived extracellular DNA content was quantified by measuring the NE antigen released from thrombi treated with endonucleases (n=72). There was no significant correlation between NETs content and stroke cause, 3-month functional outcome, or final TICI score (Figure 4A). NETs content from patients previously treated with IV t-PA was significantly reduced compared to thrombi from patients who did not receive IV t-PA therapy (Figure 4B). Finally, NETs content showed a positive correlation with endovascular procedure length and device pass count (Figures 4C-D).

[0063] Targeting NETs using DNAse 1 promoted ex vivo t-PA-induced thrombolysis. To test whether targeting NETs with DNAse 1 can enhance thrombolysis, the inventors performed an ex vivo thrombolysis assay on 24 AIS thrombi. In the first experiment, the inventors compared t-PA alone with t-PA + DNAse 1 (n=13). Adding DNAse 1 to t-PA significantly promoted ex vivo thrombolysis. In the second experiment, the inventors compared DNAse 1 alone with t-PA + DNAse 1 (n=11). DNAse 1 alone was ineffective in inducing significant thrombolysis (Figures 5A-B).

[0064] Consideration Our research has shown that (1) all AIS thrombi contain NETs regardless of the cause of stroke, (2) NETs are mainly localized in the outer layer of AIS thrombi, (3) NET content is related to the length of the endovascular procedure and the number of device passes, and (4) simultaneous administration of t-PA and DNAse 1 promotes ex vivo thrombolysis compared to t-PA or DNAse 1 alone.

[0065] Our findings are consistent with recent reports indicating that NETs are an important component of AIS thrombi. 6The latter study found that the amount of NETs was significantly higher in cardiac-derived AIS thrombi compared to non-cardiac thrombi. In this study, we did not find a correlation between NETs and the cause of stroke, but perhaps more importantly, we showed that NETs contribute to the scaffolding of thrombi, regardless of the origin of the thrombus. This particular construction may be involved in t-PA resistance, as suggested by the dramatic increase in ex vivo t-PA-induced thrombolysis in the presence of DNAse 1. Our results support recent evidence that DNAse 1 can also help enhance t-PA-induced thrombolysis of human coronary thrombi and AIS thrombi. Notably, we show herein that treatment with DNAse 1 alone does not have an ex vivo thrombolytic effect, which indicates that both fibrin and neutrophil-derived extracellular DNA matrix must be targeted to induce successful thrombolysis.

[0066] Previous studies have shown that extracellular DNA and histones actually modify the structure of fibrin, making it more resistant to mechanical and enzymatic disruption. 7 This fact may explain the observed correlation between NETs content and the number of device passes required to achieve successful recanalization. In this perspective, NETs may be involved in interactions between the thrombus and the arterial wall or between the thrombus and the EVT device, thus increasing the difficulty for successful thrombectomy during EVT.

[0067] Interestingly, previous experimental studies have already evaluated the effects of DNAse1 injection in ischemia-reperfusion models. In a mouse model of cerebral ischemia-reperfusion, DNAse1 alone was found to significantly reduce infarct volume compared to the vehicle. 8 In a rat myocardial ischemia-reperfusion model, DNAse 1 alone was ineffective, but simultaneous administration of DNAse 1 and t-PA significantly reduced infarct size. 9These inconsistent results may be explained by the endogenous expression of t-PA in cerebral capillaries, which would favor the effect of DNAse 1 alone in the brain. 10 Therefore, the administration of DNAse 1 in addition to IV t-PA may have a favorable effect not only on proximal arterial recanalization rates but also on downstream microvascular thrombosis.

[0068] Further optimization of thrombolysis may be achieved by evaluating the endothelial contribution to t-PA resistance. For example, the protein C pathway plays a crucial role in coagulation and inflammation. Indeed, previous studies have shown that increased soluble endothelial protein C receptors are associated with thrombolytic resistance in AIS patients with large vessel occlusion. 11

[0069] Ultimately, these findings support a “pharmacological cocktail” of future AIS treatments, including therapies that target thrombi embedded in large vessels, activated endothelium, and downstream microangiogenic thrombosis. 12

[0070] conclusion NETs, ​​at least partially, form a scaffold responsible for thrombus t-PA resistance. Our data support the idea that DNAse 1 injection may have a synergistic effect in improving the effectiveness of IV t-PA-induced thrombolysis in AIS.

[0071] [Table 1]

[0072] Table I. Clinical characteristics of acute ischemic stroke patients (n=108) from whom thrombi were collected and used for this study. SD: Standard deviation; LAA: Large artery atherosclerosis; CE: Cardiac embolism; OTH: Other causes; UND: Undetermined; IV: Intravenous; MCA: Middle cerebral artery; ICA: Internal carotid artery; BA: Basilar artery; NIHSS: National Institutes of Health Stroke Scale; TICI: Thrombolysis in cerebral infarction.

[0073] References Throughout this application, various references describe the current state of the art to which the present invention pertains. The disclosures of these references are incorporated herein by reference. [Table 2]

Claims

1. A combination comprising t-PA and DNAse for use in the treatment of patients requiring treatment for AIS.

2. The combination according to claim 1, wherein t-PA is recombinant t-PA.

3. The combination according to claim 1 or claim 2, wherein t-PA is a modified form of natural t-PA that retains the enzymatic activity or fibrin-lytic activity of natural t-PA.

4. The combination according to any one of claims 1 to 3, wherein t-PA is a modified form of natural t-PA, in which Thr103 of wild-type tPA is changed to Asn (T103N), Asn117 of wild-type tPA is changed to Gln (N117Q), and Lys-His-Arg-Arg 296-299 of wild-type tPA are changed to Ala-Ala-Ala-Ala.

5. The combination according to any one of claims 1 to 4, wherein t-PA is alteplase or tenecteplase.

6. The combination according to any one of claims 1 to 5, wherein DNAse is DNAse 1.

7. The combination according to any one of claims 1 to 6, wherein the DNAse is recombinant.

8. The combination according to any one of claims 1 to 7, wherein the DNAse is of human origin.

9. The combination according to any one of claims 1 to 8, wherein DNAse is dorunase or pulmozyme.

10. A method for treating acute ischemic stroke (AIS) in a patient requiring treatment for AIS, comprising administering to the patient a therapeutically effective combination of t-PA and DNAse, wherein the administration of the combination produces enhanced therapeutic efficacy compared to the administration of t-PA alone.

11. A method for enhancing the efficacy of t-PA administered as part of a treatment regimen to a patient suffering from AIS, comprising administering to the patient a pharmaceutically effective amount of t-PA in combination with DNAse.

12. A method for achieving recanalization of an occluded intracranial artery in a patient with AIS, comprising administering to the patient a therapeutically effective combination of t-PA and recombinant DNAse.

13. The method according to any one of claims 10 to 12, wherein t-PA is recombinant t-PA.

14. The method according to any one of claims 10 to 13, wherein t-PA is a modified form of natural t-PA that retains the enzymatic activity or fibrin-solubilating activity of natural t-PA.

15. The method according to any one of claims 10 to 14, wherein t-PA is a modified form of natural t-PA, in which Thr103 of wild-type tPA is changed to Asn (T103N), Asn117 of wild-type tPA is changed to Gln (N117Q), and Lys-His-Arg-Arg 296-299 of wild-type tPA are changed to Ala-Ala-Ala-Ala.

16. The method according to any one of claims 10 to 15, wherein t-PA is alteplase or tenecteplase.

17. The method according to any one of claims 10 to 16, wherein DNAse is DNAse 1.

18. The method according to any one of claims 10 to 17, wherein the DNAse is recombinant.

19. The method according to any one of claims 10 to 18, wherein the DNAse is of human origin.

20. The method according to any one of claims 10 to 19, wherein the DNAse is a dolnase or a pulmozyme.

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