Methods and pharmaceutical compositions for treatment of acute ischemic stroke

Abstract

To provide methods and pharmaceutical compositions for treatment of acute ischemic stroke (AIS).SOLUTION: The present invention relates to a method of treating an acute ischemic stroke (AIS), comprising administering a therapeutically effective combination of t-PA and DNAse.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] 2. Background of the Invention Acute ischemic stroke (AIS) is the sudden interruption of adequate blood flow to a compartment of the brain, usually caused by a thrombus or other embolus lodged or forming in one of the arteries supplying blood to the brain. If the blockage is not quickly removed, the ischemia may lead to permanent neurological damage or death. Intravenous (IV) administration of recombinant tissue-type plasminogen activator (t-PA; alteplase, Actilyse®, Boehringer Ingelheim) is the only pharmacological therapy with proven efficacy in AIS. This treatment is less efficient in terms of early recanalization. Since 2015, this treatment is associated with the realization of endovascular treatment (EVT), which allows an increase in early recanalization rates in cases of proximal intracranial arterial occlusion and a significant improvement in functional outcomes at 3 months. 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 the onset of symptoms and treatment, the better the outcome.EVT for AIS consists in the mechanical removal of the thrombus with a thrombus removal device such as a stent retriever or direct aspiration catheter.

[0003] The main goal of AIS management is to obtain as rapid and early recanalization of the occluded artery as possible. This early recanalization is, in fact, the main predictor of the neurological clinical outcome. International guidelines for AIS management 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 of up to 6 hours. However, IV thrombolysis is less effective in recanalization in the setting of proximal occlusion. In fact, rates of 5% of cases of internal carotid artery occlusion and 20% of cases of proximal middle cerebral artery occlusion have been recently reported. The efficacy of IV infusion of t-PA is limited to 10% due to dilution of the potent compound in whole blood, inhibition of t-PA by circulating inhibitors such as PAI-1 during plasma transport, low initial levels of t-PA binding to the thrombus, and an increased risk of delayed hemorrhagic transformation. Furthermore, although the thrombolytic effect of t-PA is beneficial, the toxicity of t-PA at the required high dose range currently used is problematic. EVT is associated with high recanalization rates but is overly specialized, expensive, and therefore difficult to access on an emergency basis. An easily administered pharmacological treatment to increase the recanalization rates associated with IV thrombolysis would be a major advance in the management of AIS.

[0004] Neutrophil extracellular traps (NETs) have been identified in recent years as the main trigger and structural factor of various forms of thrombosis. NETs are extracellular nets composed mainly of neutrophil-derived DNA. Recently, a study designated extracellular DNA threads from NETs as a potential therapeutic target to increase the efficacy of t-PA-induced thrombolysis in acute coronary syndromes (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). Recently, in a mouse model of cerebral ischemia-reperfusion, DNAse 1 alone was found to significantly reduce infarct volume compared to vehicle (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). Thus, this study demonstrated that DNAse 1 may be suitable for improving downstream effects induced by ischemia, but failed to disclose that this enzyme favorably impacts proximal arterial recanalization rates. Summary of the Invention [Problem to be solved by the invention]

[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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Detailed Description of the Invention Neutrophil extracellular traps (NETs) are DNA extracellular networks decorated with histones and granule proteins generated by activated neutrophils. NETs have been identified as the main trigger and structural factor of thrombosis. Our aim was to evaluate the presence of NETs in thrombi retrieved during endovascular therapy in patients with acute ischemic stroke (AIS) and their impact on t-PA-induced thrombolysis. Therefore, we analyzed thrombi from 108 AIS patients treated with endovascular therapy. Thrombi were characterized by hematoxylin / eosin staining, immunostaining, and ex vivo enzymatic assays. Additionally, we evaluated the impact 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, especially in their outer layer. Quantitative measurements of thrombus NETs content were not associated with clinical outcome or pathogenesis of AIS, but were significantly correlated with the length of the endovascular procedure and the number of passes of the device. Ex vivo, recombinant DNAse 1 enhanced t-PA-induced thrombolysis, whereas DNAse 1 alone was ineffective. This study suggests that the NETs content of thrombi, regardless of their cause, may contribute to resistance to reperfusion, including mechanical or pharmacological approaches with intravenous t-PA. These results also suggest that combination therapy involving t-PA and DNAse 1 may have a synergistic effect that allows for the efficacy of t-PA.

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

[0008] The term "therapeutically effective amount" or "therapeutically effective combination" or "pharmacologically effective amount" as used herein refers to an amount or dose of t-PA, an amount or dose of DNAse, or an amount or dose of both in a combination according to the invention, that aims to (1) delay or prevent the onset of AIS; (2) reduce the severity or incidence of AIS; (3) slow or stop the progression, worsening, or exacerbation of one or more symptoms of AIS; (4) bring about an improvement in the symptoms of AIS; or (5) cure AIS, without causing significant negative or adverse side effects in the subject. A therapeutically effective amount or combination may be administered prior to the onset of AIS. Alternatively or additionally, a therapeutically effective amount or combination may be administered after the onset of AIS.

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

[0010] 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, comprising administering to the patient a pharmacologic effective amount of t-PA in combination with DNAse.

[0011] 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, comprising administering DNAse to the patient treated with t-PA. In one embodiment, a therapeutically effective amount of DNAse is administered to the patient. In one embodiment, the method of the present invention allows for a reduction in the dose of t-PA to be administered to the patient.

[0012] A further object of the present invention is a method for achieving recanalization of an occluded intracranial artery in a patient suffering from AIS, comprising administering to the patient t-PA and DNAse, hi 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 comprising, consisting of or consisting essentially of t-PA and DNAse for use in the treatment of AIS in a patient in need thereof.

[0014] The term "consisting essentially of" as used herein with respect to a combination means that t-PA and DNase are the only therapeutic or biologically active compounds in the combination of the invention.

[0015] Another object of the present invention is a pharmaceutical combination comprising, consisting of or consisting essentially of t-PA and DNAse and at least one pharma- ceutically acceptable carrier for use in the treatment of AIS in a patient in need thereof.

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

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

[0018] Another object of the invention is a medicament comprising a combination of t-PA and DNAse as described herein above or a pharmaceutical combination as described herein above or a kit of parts as described herein above for use in the treatment of AIS in a patient in need thereof.

[0019] The term "acute ischemic stroke" or "AIS" as used herein refers to a patient having or at risk for "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, which is incorporated herein by reference. Acute ischemic stroke thus refers to the acute onset of neurological dysfunction of any severity consistent with focal cerebral ischemia.

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

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

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

[0023] In one embodiment, the patient has previously been treated with EVT or is scheduled to undergo treatment with EVT. Thus, according to one embodiment, the method of the present invention comprises administering t-PA and DNAse to a patient and performing EVT on said patient.

[0024] The term "treatment" or "treating" as used herein refers to both preventative or prophylactic treatment and curative or disease-modifying treatment, including treatment of patients at risk of or suspected of being afflicted, and patients diagnosed as being ill or suffering from a disease or medical condition, including suppression of clinical recurrence. Treatment may be administered to a subject who has a medical disorder or who may eventually acquire a disorder to prevent, cure, delay, reduce the severity of, or ameliorate the onset of one or more symptoms of the disorder or the recurrence of the disorder, or to extend the subject's survival beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant a pattern of treatment of a disease, such as a dosing pattern used during treatment. Therapeutic regimens may include induction regimens and maintenance regimens. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to the patient during the initial period of the treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen" which may include administering a dose of a drug that exceeds the dose employed by the physician during a maintenance regimen, administering a drug more frequently than the frequency with which the physician administers the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used for the maintenance of a patient during disease treatment, for example, to keep the patient in remission for an extended period of time (months or years). A maintenance regimen may employ continuous therapy (i.e., administering a drug at regular intervals, such as weekly, monthly, yearly, etc.) or intermittent therapy (i.e., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of a certain predetermined criterion (e.g., disease manifestation, etc.).

[0025] The term "t-PA" as used herein has its general meaning in the art and refers to tissue-type plasminogen activator. The term includes native 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 assessing the ability of this molecule to convert plasminogen to plasmin. The fibrinolytic activity of t-PA may be determined by any in vitro clot lysis activity known in the art. Recombinant t-PA has been widely described in the prior art and is known to the skilled artisan. t-PA is commercially available as alteplase (Activase® or Actilyse®). Modified forms of t-PA ("modified t-PA") have been characterized and are known to the skilled artisan. Modified t-PA includes, but is not limited to, variants in which amino acids or domains are deleted or substituted, variants conjugated or fused with other molecules, and variants with chemical modifications such as modified glycosylation. Some modified t-PAs are described in PCT Publication Nos. WO 93 / 24635; EP 352,119; EP 382174. In some embodiments, the modified form of t-PA is tenecteplase. As used herein, the term "tenecteplase," also known as TNK-t-PA or TNKASE™ brand tissue-type plasminogen activator variant, refers to a t-PA variant designated T103N, N117Q, K296A, H297A, R298A, R299A t-PA available from Genentech, Inc., South San Francisco Calif., in which Thr103 of wild-type t-PA has been changed to Asn (T103N), Asn117Gln of wild-type t-PA has been changed to N117Q, and Lys-His-Arg-Arg 296-299 of wild-type t-PA has been 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 (for a review of third generation thrombolytic drugs generally, see Keyt et al., Proc. Natl. Acad. Sci USA, 91: 3670-3674 (1994) and Verstraete, Am. J. Med, 109: 52-58 (2000)). Tenecteplase has been engineered to have increased fibrin specificity and an increased half-life compared to alteplase.

[0026] In one embodiment, the t-PA is a mutant t-PA as described in WO 2013 / 034710. In one embodiment, said mutant t-PA comprises the sequence of SEQ ID NO:1 (corresponding to human wt t-PA mature form) or SEQ ID NO:2 (corresponding to human wt t-PA first chain of tc-t-PA), preferably consisting of an association of SEQ ID NO:1 or SEQ ID NO:2 with SEQ ID NO:3 (corresponding to human wt t-PA second chain of tc-t-PA) or a variant thereof having at least 80%, 85%, 90%, 95% or more identity, wherein said sequence comprises a mutation consisting of the substitution of any amino acid in the lysine binding site of SEQ ID NO:1 or SEQ ID NO:2 by 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 by serine.

[0027] In one embodiment, said mutant t-PA comprises 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 chain of human wt t-PA of two-chain t-PA), preferably consisting of an association of SEQ ID NO:1 or SEQ ID NO:2 with SEQ ID NO:3 (corresponding to the second chain of human wt t-PA of two-chain t-PA) or a variant thereof having at least 80%, 85%, 90%, 95% or more identity, wherein said sequence comprises a mutation consisting of a substitution of tryptophan at position 253 of SEQ ID NO:1 or SEQ ID NO:2 by 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 a substitution of arginine at position 275 of SEQ ID NO:1 or SEQ ID NO:2 by serine.

[0028] In one embodiment, the mutant t-PA further comprises the following mutation: - substitution of proline at position 125 of SEQ ID NO:1 or SEQ ID NO:2 by arginine, - a deletion of the N-terminal finger domain and / or a deletion of the EGF-like domain in SEQ ID NO:1 or SEQ ID NO:2 and / or a substitution of the asparagine at position 117 of SEQ ID NO:1 or SEQ ID NO:2 by glutamine, - substitution of threonine at position 103 of SEQ ID NO:1 or SEQ ID NO:2 by asparagine and / or substitution of asparagine at position 117 of SEQ ID NO:1 or SEQ ID NO:2 by glutamine and / or substitution of lysine-histidine-arginine-arginine (KHRR) at positions 296 to 299 of SEQ ID NO:1 by alanine-alanine-alanine-alanine (AAAA), - substitution of the cysteine ​​at position 84 of SEQ ID NO:1 or SEQ ID NO:2 by serine, - a substitution of arginine at position 275 of SEQ ID NO:1 or SEQ ID NO:2 with glutamic acid or glycine, and / or a deletion of the kringle 1 domain in SEQ ID NO:1 or SEQ ID NO:2 Includes at least one of the following:

[0029] As used herein, the term "identity" or "identical" when applied to the relationship between sequences of two or more amino acid sequences refers to the degree of sequence relatedness between amino acid sequences, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences, with alignments, with gaps (if any), processed by a particular mathematical model or computer program (i.e., an "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. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs.Preferred computer program methods for determining 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 may also be used to determine identity.

[0030] The term "DNAse" as used herein has its general meaning in the art and refers to and includes all enzymes that have phosphodiesterase activity and the ability to hydrolyze DNA. Any suitable DNAse may be used in the present invention. The DNAse is most preferably DNAse 1 (EC 3.1.21.1). However, in some embodiments, the DNAse may be DNAse II (EC 3.1.21.1). DNAses are present in several species, and any DNAse capable of cleaving DNA may be used in the present invention. In one embodiment, the DNAse is a recombinant DNAse. The DNAse may be of animal origin, such as bovine or porcine origin. The DNAse may be of plant, fungal, or microbial origin. However, typically and most preferably, the DNAse is of human origin, and is preferably a recombinant human DNAse. Commercially available DNAse preparations, such as Dornase™ and Pulmozyme™, may be used in embodiments of the present invention.

[0031] The term "combination" as used herein is intended to refer to all forms of administration that provide a first drug together with an additional (second, third, ...) drug.

[0032] The drugs may be administered simultaneously, separately or sequentially, and in any order. In one embodiment, t-PA is administered prior to administration of DNAse. In another embodiment, DNAse is administered prior to administration of t-PA. In another embodiment, t-PA and DNAse are administered simultaneously.

[0033] The drugs administered in combination have biological activity in the subject to which they are delivered.Therefore, in the context of the present invention, the combination comprises at least two different drugs, where one drug is at least t-PA and the other drug is DNAse.As used herein, the expression "enhance the efficacy of t-PA" refers to the ability of recombinant DNAse to enhance the fibrinolysis induced by t-PA.In particular, DNAse is suitable for enhancing the recanalization rate provided by t-PA.Therefore, the combination of the two drugs results in the promotion of fibrinolysis of thrombus.

[0034] As used herein, the term "therapeutically effective combination" refers to an amount or dose of t-PA together with an amount or dose of DNAse sufficient to treat acute ischemic stroke, particularly 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 the one or more additional agents or treatments included in the combination. A "therapeutically effective amount" 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 usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a wide variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed, the age, weight, general health, sex and diet of the subject; the administration time, route of administration, and excretion rate of the specific compound employed; the duration of treatment; drugs used in combination or concordance with the specific polypeptide employed; and similar factors well known in medicine. For example, it is well within the skill of the art to start the dosage of the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the product may vary over a wide range of 0.01 mg to 1,000 mg per day per adult. Typically, the composition contains 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 symptoms of the subject to be treated. The medicament typically contains about 0.01 mg to about 500 mg of the active ingredient, preferably 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is usually supplied at a dosage level 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 the combination of the present invention is (or will be) administered to the subject, preferably during the acute phase of AIS. In one embodiment, multiple doses of t-PA, DNAse or the combination of the present invention are (or will be) administered to the subject. Thus, in one embodiment, the combination of the present invention is administered as a split dose. In one embodiment, the dose of t-PA administered (or will be) to the subject is comprised between about 0.01 mg / kg and about 5 mg / kg. In one embodiment, the dose of t-PA administered to the subject is comprised between about 0.1 mg / kg and about 1 mg / kg. In one embodiment, the dose of alteplase (t-PA of the present invention) administered to the subject is equal to about 0.9 mg / kg. In one embodiment, the dose of tenecteplase (t-PA of the present invention) administered to the subject is comprised between about 0.1 mg / kg and about 0.4 mg / kg. In one embodiment, the dose of DNAse of the present invention administered to the subject is comprised 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 (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 comprises administering to the subject a dose of t-PA ranging from about 0.01 mg / kg to about 5 mg / kg, as well as administering to the subject a dose of DNAse ranging from about 0.1 μg / kg to about 500 μg / kg. The term "about" as used herein preceding a number means ±10% of the value of said number.

[0035] Typically, the agents of the present invention (ie, t-PA or DNAse or a combination thereof) are administered to a subject in the form of a pharmaceutical composition that includes a pharma- ceutically acceptable carrier.

[0036] The term "pharmaceutically acceptable carrier" as used herein refers to an excipient that does not produce adverse, allergic or other untoward reactions when administered to a mammal, preferably a human. This includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Pharmaceutically acceptable carriers refer to non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or formulation auxiliary of any kind. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards required by regulatory authorities such as the FDA or EMA.

[0037] Pharmaceutically acceptable carriers that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, phosphates, buffer substances such as glycine, sorbic acid, 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, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat. For use in administration to a subject, the composition will be formulated for administration to the subject.

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

[0039] In one embodiment, t-PA and DNAse, combinations or pharmaceutical combinations thereof, medicaments or kits of parts according to the present invention will be formulated for administration to a subject. t-PA and DNAse, combinations or pharmaceutical combinations thereof, or medicaments according to the present invention may be administered orally, parenterally, topically, by inhalation spray, rectally, nasally, bucally, 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, their combination or pharmaceutical combination, medicament or kit of parts according to the present invention are injected. As used herein, includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Sterile injectable 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 suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as solvents or suspending media. For this purpose, any bland fixed oil may be employed, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful for the preparation of injectables, as are pharma- ceutically acceptable natural oils such as olive oil or castor oil, especially its polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants commonly used in the formulation of pharma- ceutically acceptable dosage 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 pharma-ceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes.

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

[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 the 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 glycols.

[0044] The compositions or combinations of the present invention may also be administered locally, especially when the target of treatment includes areas or organs that are easily accessible by topical application, including diseases of the eye, skin, or lower gastrointestinal tract. Suitable topical formulations are easily prepared for each of these areas or organs. For topical application, the compositions may be formulated as a suitable ointment 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 a suitable lotion or cream containing the active components suspended or dissolved in one or more pharma- ceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Topical application for the lower intestinal tract can be accomplished in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used.

[0045] Compositions of the invention may also be administered by nasal aerosol or inhalation.Such compositions are prepared by techniques well known in the pharmaceutical formulation art and may be prepared as a solution in saline employing benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0046] The present invention is further illustrated by the following figures and examples, which should not, however, be construed as limiting the scope of the present invention. [Brief description of the drawings]

[0047] [Figure 1]Correlation between neutrophil extracellular trap (NETS) burden, acute thrombolytic therapy, and cause of stroke. To evaluate the correlation between NETs burden and acute ischemic stroke patient characteristics, thrombus NETs content was determined by quantifying the release of neutrophil elastase by endonuclease-treated thrombi. (A) NETS burden was slightly but significantly reduced in patients treated with IV t-PA compared to patients who did not receive t-PA treatment. (B) There was no association between stroke cause and NETS burden according to TOAST classification (LAA: large vessel atherosclerosis; CE: cardioembolic; LAC: foveolar; OTH: other causes; UND: undetermined cause). (C) and (D) Interestingly, NETs burden was positively correlated with the length of EVT intervention and the number of device passes. [Diagram 2] Figure 1. 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 retrieved by thrombectomy were incubated in PBS supplemented with tPA (1 μg / mL) and / or DNAse (100 U / mL). Thrombi weight was assessed before and 10, 30 and 60 min after addition of tPA and DNAse. Results are expressed as a percentage of initial thrombi weight. Data are presented as median (interquartile range). [Diagram 3] Neutrophil extracellular traps are constitutively present in thrombi from acute ischemic stroke. The presence of NETs in AIS thrombi was examined by measuring DNA-associated neutrophil elastase activity. Dot plots show elastase activity measured in the supernatant of thrombi before and after endonuclease treatment (n=23; p<0,0001). [Figure 4]FIG. 1: Thrombus neutrophil-derived extracellular DNA content is not associated with the cause of AIS but is associated with endovascular procedure characteristics. A-B. Comparison of neutrophil-derived extracellular DNA content (n=72) between thrombi classified according to (A) cause of stroke (LAA: atherosclerosis; CE: cardioembolism; OTH: other causes; UND: undetermined) or (B) having or not received IV t-PA treatment prior to endovascular therapy (p=0.03). C-D. Correlation of thrombus neutrophil-derived extracellular DNA content (n=72) with the length of the endovascular procedure (C); and the number of device passes achieved (D). [Diagram 5] Figure 1: DNAse 1 enhances t-PA-induced thrombolysis ex vivo. Acute ischemic stroke thrombi retrieved by endovascular therapy were incubated with t-PA and / or DNAse I and their lysis was followed by measuring the wet weight change of the thrombi over time. The mean baseline weight of the thrombi was 14.6±8.4 mg. Comparison of the thrombolytic effect of t-PA alone or in combination with DNAse I. (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 small but significant reduction in thrombus weight at 60 min compared to baseline (p = 0.003). B. Comparison of the thrombolytic efficacy of DNAse I alone or in combination with t-PA (n = 11, mean [SD]; 10 min: DNAse (10 min: DNAse I = 104.6% [12.73] vs. DNAse I + t-PA = 92.91% [17.55], 30 min: DNAse I = 95.66% [21.29] vs. DNAse I + t-PA = 69.39% [21.65], 60 min: DNAse I = 83.36% [33.89] vs. DNAse I + t-PA = 37.83% [17.65]). DNAse I alone did not affect clot weight after 60 min compared to baseline (p = 0.06). Example 1

[0048] Neutrophil extracellular traps (NETs) have been identified in recent years as the main trigger and structural factor of various forms of thrombosis. NETs are extracellular nets composed mainly of neutrophil-derived DNA. Recently, a study designated extracellular DNA strands from NETs as a potential therapeutic target to increase the efficacy of t-PA-induced thrombolysis in acute coronary syndromes. The aim of this study was to evaluate the impact of NETs burden in thrombi retrieved during endovascular therapy in patients with acute ischemic stroke (AIS) on t-PA-induced thrombolysis, clinical outcomes, and the pathogenesis of AIS.

[0049] We analyzed thrombi from 150 AIS patients treated with endovascular therapy. Thrombi were characterized by HE staining, immunostaining, and enzyme assays. Histological analysis revealed that NETs contributed to the scaffolding of most thrombi, especially in their superficial layers. In vitro endonuclease treatment of thrombi revealed a significant release of free DNA (p<0.001) and neutrophil elastase (p<0.001) into the supernatant, reflecting the quantitative thrombus NETs burden. Thrombus NETs burden did not correlate with clinical outcome or cause of AIS. Recombinant deoxyribonuclease 1 (DNAse 1) enhanced t-PA-induced thrombolysis in an in vitro lysis assay (p=0.02), whereas both t-PA or DNAse 1 alone were ineffective in inducing significant thrombolysis of these thrombi.

[0050] This study suggests that the NETs-enclosed structure of AIS thrombi may also be involved in t-PA-induced thrombolysis resistance, independent of their cause and localization.The efficacy of a strategy involving the co-administration of DNAse 1 in addition to t-PA to promote AIS thrombus fibrinolysis may also be of great interest for the treatment of AIS. Example 2

[0051] method Sample Selection Patients who underwent successful thrombectomy and were treated with endovascular therapy (EVT) at our institution between December 2015 and December 2016 were enrolled in this study. The local ethical committee approved the study protocol.

[0052] Collecting data Patient demographics, vascular risk factors, imaging findings, preprocedural vital signs, severity of AIS, and clinical outcomes were prospectively collected using a structured questionnaire (ETIS Registry, Endovascular Procedures in Ischemic Stroke). Cause of stroke was classified using the TOAST classification.

[0053] Endovascular Therapy Procedures In the first instance, an EVT procedure was chosen using either a stent retriever or a direct aspiration first path technique at the discretion of the interventional physician. The detailed technical procedure has been published previously.1

[0054] Collection and processing of acute ischemic stroke thrombi Acute ischemic stroke (AIS) thrombi were 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. Thrombi from 74 patients were used for lysis assays (n=24) and / or for determination of NETs content assessed by measurement of NE release after endonuclease treatment. NE release was quantified by measurement of NE antigen (n=72) and / or activity (n=23). Thrombi from 34 patients were fixed in paraformaldehyde and used for immunohistochemical analysis.

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

[0056] Ex vivo NETs assessment A commercially available NETs assay kit (Cayman chemical) was used, which consists in measuring neutrophil elastase activity in tissue supernatants 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 supernatants of thrombi collected after endonuclease treatment.

[0057] Ex vivo thrombolysis assay The following protocol was adapted from Mangold et al. (2). Briefly, frozen AIS thrombi were lysed, 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. Thrombi were weighed using an ultraprecision balance before the start of the treatment and after 10, 30, and 60 min. Thrombi lysis was expressed as a percentage of baseline thrombi weight.

[0058] statistical analysis Data were analyzed using nonparametric analysis of variance (Kruskal-Wallis) followed by Wilcoxon rank sum test for comparison of paired data or Mann-Whitney U test for comparison of unpaired data. Results are presented as mean ± SD for continuous variables and numbers (percentages) for qualitative variables. For statistical analysis, PrismGraph 4.0 software (GraphPad Software, San Diego, CA) was used. A value of P<0.05 was considered statistically significant.

[0059] result Patient characteristics We included 108 patients in this study between December 2015 and December 2016. Patient characteristics are shown in Table I. These characteristics are similar to those of recently published EVT trials.

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

[0061] Immunofluorescence detection confirmed that the areas containing extracellular DNA were colocalized with citrullinated histones and granular neutrophil protein (MPO), which correspond to NETs (data not shown). To confirm the presence of NETs in AIS thrombi, we performed an ex vivo NETs assay in 23 thrombi. For this assay, the presence of NETs was examined by measuring the NE activity released from the thrombi after endonuclease treatment. The activity of NE increased after endonuclease incubation, confirming the presence of NETs in all thrombi (Figure 3).

[0062] Neutrophil-derived extracellular DNA content correlates with EVT procedure length and number of device passes Thrombus neutrophil-derived extracellular DNA content was quantified by measuring NE antigen released from endonuclease-treated thrombi (n=72). There was no significant correlation between NETs content and cause of stroke, 3-month functional outcome, or final TICI score (Figure 4A). Thrombus content of NETs 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 positively correlated with the length of the endovascular procedure and the number of device passes (Figure 4C-D).

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

[0064] Consideration Our studies show that (1) all AIS thrombi contain NETs, ​​regardless of stroke cause; (2) NETs are primarily localized in the outer layer of AIS thrombi; (3) NETs content is associated with the length of the endovascular procedure and the number of device passes; and (4) coadministration of t-PA and DNAse 1 enhances ex vivo thrombolysis compared with t-PA or DNAse 1 alone.

[0065] Our findings are consistent with recent reports showing that NETs are important components of AIS thrombi. 6The latter study found that the amount of NETs was significantly higher in AIS thrombi of cardiac origin 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 show that NETs contribute to the scaffolding of thrombi, regardless of the origin of the thrombus. This particular architecture 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 showing that DNAse 1 can also help enhance t-PA-induced lysis of human coronary artery and AIS thrombi. Notably, we show herein that treatment with DNAse 1 alone has no thrombolytic effect ex vivo, indicating 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 performed to achieve successful recanalization.In this perspective, NETs increase the challenges for successful thrombus removal during EVT, as they may be involved in interactions between the thrombus and the arterial wall or between the thrombus and the EVT device.

[0067] Interestingly, previous experimental studies have already evaluated the impact of DNAse 1 injection in an ischemia-reperfusion model. In a mouse model of cerebral ischemia-reperfusion, DNAse 1 alone was found to significantly reduce infarct volume compared to vehicle. 8 In a rat myocardial ischemia-reperfusion model, DNAse 1 alone was ineffective, but co-administration of DNAse 1 and t-PA significantly reduced infarct size. 9These discrepant results may be explained by the endogenous expression of t-PA in brain capillaries, which would favor the effects of DNAse 1 alone in the brain. 10 Thus, 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 result from evaluation of the endothelial contribution to t-PA resistance. For example, the protein C pathway plays a critical role in coagulation and inflammation. Indeed, previous studies have shown that increased soluble endothelial protein C receptor is associated with thrombolysis resistance in AIS patients with large vessel occlusion. 11

[0069] Ultimately, this evidence supports a future "pharmacological cocktail" for the treatment of AIS that includes therapies targeting embedded thrombus in large vessels, activated endothelium, and downstream microvascular thrombosis. 12

[0070] conclusion NETs form a scaffold that is responsible, at least in part, for thrombus t-PA resistance.Our data support the notion that DNAse 1 infusion may have a synergistic effect to improve the efficacy of IV t-PA-induced thrombolysis in AIS.

[0071] [Table 1]

[0072] Table I. Clinical characteristics of acute ischemic stroke patients (n=108) whose thrombi were collected and used for this study. SD: standard deviation; LAA: large vessel atherosclerosis; CE: cardioembolism; 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 are made to the state of the art to which this invention pertains, the disclosures of which are incorporated herein by reference into this disclosure. [Table 2]

Claims

1. A combination comprising t-PA and DNAse for use in the treatment of AIS in a patient in need thereof.

2. The combination of claim 1, wherein the t-PA is recombinant t-PA.

3. A combination according to claim 1 or claim 2, wherein the t-PA is a modified form of native t-PA which retains the enzymatic or fibrinolytic activity of native t-PA.

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

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

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

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

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

9. A combination according to any one of claims 1 to 8, wherein the DNAse is dornase or pulmozyme.

10. A method of treating acute ischemic stroke (AIS) in a patient in need of treatment comprising administering to the patient a therapeutically effective combination of t-PA and DNAse, wherein administration of the combination results in enhanced therapeutic effectiveness compared to 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 pharmacologic effective amount of t-PA in combination with a DNAse.

12. A method for achieving recanalization of an occluded intracranial artery in a patient suffering from 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 the t-PA is recombinant t-PA.

14. The method according to any one of claims 10 to 13, wherein the t-PA is a modified form of native t-PA which retains the enzymatic or fibrinolytic activity of native t-PA.

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

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

17. The method according to any one of claims 10 to 16, wherein the 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 dornase or pulmozyme.

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