Anti-thrombin in stroke

Abstract

The present invention relates to the use of antithrombin in the treatment of stroke.

Description

Technical Field

[0001] The present invention relates to the use of antithrombin in treating stroke. Background Art

[0002] Stroke is one of the most common non-traumatic brain injury categories, and occurs when the blood supply to a part of the brain is interrupted or reduced, preventing the brain tissue from getting oxygen and nutrients. Neurodegenerative processes begin almost immediately, and brain cells begin to die within minutes. Ischemic stroke occurs when the blood vessels (arteries) that supply blood to a brain area become blocked by a blood clot. Hemorrhagic stroke occurs when an artery in the brain leaks or ruptures. Of the two, ischemic stroke is the most common form of stroke.

[0003] The prevalence of stroke in modern society places a huge burden on healthcare infrastructure and expenditures. Currently, recombinant tissue plasminogen activator (rtPA) is the only FDA-approved therapeutic agent for ischemic stroke. The main function of rtPA is to dissolve blood clots and promote reperfusion. An alternative method to reestablish blocked blood flow is through surgical intervention.

[0004] rtPA therapy has a narrow therapeutic time window, and therefore its broad applicability is limited. Furthermore, although restoration of perfusion of ischemic tissue by rtPA is important, the cascade of necrosis, apoptosis, and inflammation begins within minutes of severe oxygen deprivation. Increasingly, it is hypothesized that genetically programmed neuronal cell death during post-ischemic tissue inflammation (which can last days to weeks) contributes significantly to the final pathology due to delays in patient treatment / evaluation. Thus, permanent neural and neuronal damage can occur before patients are even evaluated.

[0005] There are many reports in the prior art about molecules that exert neuroprotective effects to limit damage caused by conditions such as stroke and traumatic injuries to the brain and spinal cord. One such example is U.S. Patent Publication No. 2016008437 in the name of Grifols Worldwide Operations Ltd, which discloses that apo-transferrin exerts neuroprotective effects by regulating the activity of hypoxia inducible factor (HIF) in a rat stroke model. The inventors observed a neuroprotective effect of apo-transferrin, which was manifested by a reduction in the volume of the infarct area in rats treated with apo-transferrin compared to control rats.

[0006] It is immediately apparent that the severe and debilitating effects of neurodegeneration associated with brain injury require considerable concern and attention. Therefore, alternative treatments focused on this need are highly desirable. Summary of the invention

[0007] When used herein with reference to the present invention, the words "comprises / comprising" and the words "having / containing" are used to specify the presence of stated features, integers, steps or components, but do not exclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0008] Those skilled in the art will appreciate that the specific embodiments disclosed herein should not be read in isolation, and that this specification is intended to be read in combination with the disclosed embodiments, rather than individually. Therefore, each embodiment may be used as a basis for modifying or limiting other embodiments disclosed herein.

[0009] Concentration, amount and other numerical data can be represented or presented in range format herein.It should be understood that such range format is used only for convenience and simplicity, and therefore should be flexibly interpreted as not only including the numerical value clearly recorded as range limit, but also including all single numerical values ​​or sub-ranges included in the range, as each numerical value and sub-range are clearly recorded.As an example, the numerical range of "10 to 100" should be interpreted as not only including the value of 10 to 100 clearly recorded, but also including single values ​​and sub-ranges in the specified range.Therefore, included in the numerical range is a single value, such as 10,11,12,13 ... 97,98,99,100, and sub-ranges, such as 10 to 40, 25 to 40 and 50 to 60, etc. The same principle applies to the range of only recording a numerical value, such as "at least 10".In addition, no matter how the breadth of the described feature or range is, such an explanation should be applicable.

[0010] Treatment of the present invention

[0011] In a first aspect, the invention provides a pharmaceutical composition comprising antithrombin for treating stroke in a patient in need thereof, wherein the antithrombin is administered in a dose sufficient to: increase the patient's plasma antithrombin activity to greater than about 120%; or

[0012] The patient's plasma antithrombin concentration is increased to greater than about 1.2 IU / mL.

[0013] In one embodiment, the patient suffers from ischemic stroke. For example, the patient may suffer from focal cerebral ischemic stroke.

[0014] In some embodiments, antithrombin can be used with a dosage sufficient to increase the patient's plasma antithrombin activity to greater than about 120%. For example, antithrombin can be used with a dosage sufficient to increase the patient's plasma antithrombin activity to greater than about 120% but less than about 200%. For example, antithrombin can be used with a dosage sufficient to increase the patient's plasma antithrombin activity to greater than about 130%. For example, antithrombin can be used with a dosage sufficient to increase the patient's plasma antithrombin activity to greater than about 130% but less than about 200%. For example, antithrombin can be used with a dosage sufficient to increase the patient's plasma antithrombin activity to greater than about 140%. For example, antithrombin can be used with a dosage sufficient to increase the patient's plasma antithrombin activity to greater than about 140% but less than about 200%. For example, antithrombin can be used with a dosage sufficient to increase the patient's plasma antithrombin activity to greater than about 150%. For example, antithrombin can be used with a dosage that is enough to increase the patient's plasma antithrombin activity to greater than about 150% but less than about 200%. For example, antithrombin can be used with a dosage that is enough to increase the patient's plasma antithrombin activity to greater than about 160%. For example, antithrombin can be used with a dosage that is enough to increase the patient's plasma antithrombin activity to greater than about 160% but less than about 200%.

[0015] Antithrombin activity is measured using standard techniques known to those skilled in the art. For example, patient plasma can be mixed with heparin and thrombin, and the degree of thrombin inhibition (%) is proportional to the functional antithrombin activity of the patient sample. Thrombin activity is measured by the conversion rate of a chromogenic substrate, and the signal obtained (if any) is used to calculate the antithrombin activity.

[0016] In other embodiments, antithrombin can be used with the dosage that is enough to increase the plasma antithrombin concentration of the patient to be greater than about 1.2IU / mL. For example, antithrombin can be used with the dosage that is enough to increase the plasma antithrombin concentration of the patient to be greater than about 1.2IU / mL but less than about 2.0IU / mL. For example, antithrombin can be used with the dosage that is enough to increase the plasma antithrombin concentration of the patient to be greater than about 1.3IU / mL. For example, antithrombin can be used with the dosage that is enough to increase the plasma antithrombin concentration of the patient to be greater than about 1.3IU / mL but less than about 2.0IU / mL. For example, antithrombin can be used with the dosage that is enough to increase the plasma antithrombin concentration of the patient to be greater than about 1.4IU / mL. For example, antithrombin can be used with the dosage that is enough to increase the plasma antithrombin concentration of the patient to be greater than about 1.4IU / mL. For example, antithrombin can be used with the dosage that is enough to increase the plasma antithrombin concentration of the patient to be greater than about 2.0IU / mL. For example, antithrombin can be used with a dosage that is enough to increase the plasma antithrombin concentration of the patient to be greater than about 1.5IU / mL. For example, antithrombin can be used with a dosage that is enough to increase the plasma antithrombin concentration of the patient to be greater than about 1.5IU / mL but less than about 2.0IU / mL. For example, antithrombin can be used with a dosage that is enough to increase the plasma antithrombin concentration of the patient to be greater than about 1.6IU / mL. For example, antithrombin can be used with a dosage that is enough to increase the plasma antithrombin concentration of the patient to be greater than about 1.6IU / mL.

[0017] Antithrombin concentration can be measured using standard techniques that are common knowledge to those skilled in the art. For example, as part of a conventional chromogenic immunoassay or ELISA, patient plasma can be contacted with an antibody specific for antithrombin and the antithrombin concentration can be determined based on a control sample of known concentration.

[0018] In certain embodiments, antithrombin can be used with a dosage that is enough to increase the plasma antithrombin level of the patient to greater than about 140 μ g / mL. For example, antithrombin can be used with a dosage that is enough to increase the plasma antithrombin level of the patient to greater than about 140 μ g / mL but less than about 267 μ g / mL. For example, antithrombin can be used with a dosage that is enough to increase the plasma antithrombin level of the patient to greater than about 160 μ g / mL. For example, antithrombin can be used with a dosage that is enough to increase the plasma antithrombin level of the patient to greater than about 160 μ g / mL but less than about 267 μ g / mL. For example, antithrombin can be used with a dosage that is enough to increase the plasma antithrombin level of the patient to greater than about 180 μ g / mL. For example, antithrombin can be used with a dosage that is enough to increase the plasma antithrombin level of the patient to greater than about 180 μ g / mL but less than about 267 μ g / mL. For example, antithrombin can be used with a dosage that is enough to increase the plasma antithrombin level of the patient to be greater than about 200 μ g / mL. For example, antithrombin can be used with a dosage that is enough to increase the plasma antithrombin level of the patient to be greater than about 200 μ g / mL but less than about 267 μ g / mL. For example, antithrombin can be used with a dosage that is enough to increase the plasma antithrombin level of the patient to be greater than about 220 μ g / mL. For example, antithrombin can be used with a dosage that is enough to increase the plasma antithrombin level of the patient to be greater than about 220 μ g / mL but less than about 267 μ g / mL.

[0019] In certain embodiments, antithrombin may be administered to a patient in multiple doses.

[0020] Advantageously, the compositions of the invention administer antithrombin to a patient at a concentration effective to mitigate the effects of stroke without the attendant increase in bleeding risk that may be associated with AT supplementation.

[0021] Those skilled in the art will appreciate that the specific embodiments disclosed in paragraphs

[0010] to

[0018] should not be read in isolation, and the present specification is intended to disclose these embodiments in combination with other embodiments rather than being disclosed alone.

[0022] Antithrombin

[0023] Antithrombin is a single-chain glycoprotein (68kD) serine protease inhibitor based on plasma, and its main function is to mainly maintain the balance of the coagulation system by inactivating multiple coagulation factors (such as FXa). In addition, the enhancement of antithrombin activity is the mechanism by which its anticoagulant effect is achieved by heparin. In addition to its effect as an anticoagulant, antithrombin has anti-inflammatory properties. Among its many names, antithrombin is also commonly referred to as antithrombin III, AT or ATIII; All of them are applicable to this specification.

[0024] The present invention includes within its scope all wild-type mammalian antithrombins and functionally active mutants of antithrombins. The present invention also includes within its scope both cleaved and latent forms of wild-type antithrombins. Particularly preferred are human antithrombins, such as both latent and cleaved forms of human antithrombins. For example, human antithrombin (UniProtKB Seq. No. P01008) having the amino acid sequence shown in SEQ ID NO:1.

[0025] The antithrombin used in the present invention can be plasma-derived or recombinant. Antithrombin derived from plasma is purified from the plasma of a blood donor by methods well known to those skilled in the art. Suitable commercially available preparations of antithrombin derived from human plasma that illustrate materials that can be used in the present invention include but are not limited to the preparation sold under the trade name THROMBATE III.

[0026] Illustrative recombinant forms of antithrombin that can be used in the present invention include, but are not limited to, ATα and ATγ.

[0027] The present specification includes within its scope recombinant derivatives of antithrombin, which differ from the wild-type amino acid sequence of the human protein summarized in SEQ ID NO: 1 by one or more substitutions, one or more deletions, or one or more insertions, which do not substantially change the structure or hydrophilic properties of the recombinant protein relative to the wild-type protein. Recombinant variants of antithrombin within the scope of the present invention may additionally comprise at least one post-translational modification, such as pegylation, glycosylation, polysialylation, or a combination thereof.

[0028] In one embodiment, the present invention contemplates recombinant variants of antithrombin having one or more conservative substitutions relative to the wild-type protein in SEQ ID NO: 1. A "conservative substitution" is a substitution in which an amino acid is replaced with another amino acid having similar properties such that one skilled in the art of peptide chemistry would expect the secondary structure and hydrophilicity of the polypeptide to remain essentially unchanged. Generally, changes within the following amino acid groups represent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

[0029] For example, a recombinant antithrombin within the scope of the present invention may have at least 90%, 95%, 96%, 97%, 98% or 99% homology to the wild-type human antithrombin protein outlined in SEQ ID NO:1.

[0030] The skilled person will appreciate that recombinant proteins can be obtained using standard techniques known in the field of protein expression, production and purification. The nucleic acid sequence of the target recombinant protein can be inserted into any expression vector suitable for expression in a selected host cell (e.g., mammalian cells, insect cells, plant cells, yeast and bacteria).

[0031] The term "expression vector" as used herein refers to an entity capable of introducing a protein expression construct into a host cell. Some expression vectors also replicate in host cells, which improves protein expression of protein expression constructs. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be connected. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC), fosmids, phages and phagemids. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (for example, a vector with a replication origin that plays a role in the host cell). Other vectors can be integrated into the genome of the host cell after being introduced into the host cell, and therefore replicate together with the host genome. In addition, some preferred vectors can direct the expression of genes effectively connected thereto.

[0032] Suitable bacterial cells include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas spp., Streptomyces spp., and Staphylococcus spp. Suitable yeast cells include Saccharomyces spp., Pichia spp., and Kuyveromyces spp. Suitable insect cells include those derived from Bombyx mori, Mamestra brassicae, Spodoptera frugiperda, Trichoplusia ni, and Drosophila melanogaster. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, COS, MDCK, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0, CRL703O, HsS78Bst, human hepatocellular carcinoma cells (e.g., Hep G2), 293 cells transformed by human adenovirus (e.g., HEK293), PER.C6, mouse L-929 cells, HaK hamster cell line, mouse 3T3 cells derived from Swiss, Balb-c or NIH mice, and CV-1 cell line cells.

[0033] The present invention also contemplates the use of wild-type and recombinant antithrombin proteins, which are conjugated or fused to any other protein, protein fragment, protein domain, peptide, small molecule or other chemical entity. For example, suitable fusion or conjugation partners include serum albumin (e.g., cattle, rabbit or human), keyhole limpet hemocyanin, immunoglobulin molecules (including the Fc domain of immunoglobulin), thyroglobulin, ovalbumin, tetanus toxoid, toxoids from other pathogenic bacteria, attenuated toxin derivatives, cytokines, chemokines, glucagon-like peptide-1, exendin-4, XTEN or a combination thereof.

[0034] In one embodiment, the antithrombin useful in the present invention can be fused with an immunoglobulin Fc domain. For example, the immunoglobulin Fc domain can include at least a portion of an immunoglobulin constant heavy chain domain. The immunoglobulin constant heavy chain domain is preferably an Fc fragment, which includes CH2 and CH3 domains, and optionally at least a portion of a hinge region. The immunoglobulin Fc domain can be an IgG, IgM, IgD, IgA or IgE immunoglobulin Fc domain, or a modified immunoglobulin Fc domain derived therefrom. Preferably, the immunoglobulin Fc domain includes at least a portion of a constant IgG immunoglobulin Fc domain. The IgG immunoglobulin Fc domain can be selected from IgG1, IgG2, IgG3 or IgG4 Fc domains, or its modified Fc domains.

[0035] Those skilled in the art will appreciate that the specific embodiments disclosed in paragraphs

[0020] to

[0031] should not be read in isolation, and that the specification is intended to disclose these embodiments in combination with other embodiments rather than individually. For example, unless the context suggests otherwise, each of the embodiments disclosed in paragraphs

[0020] to

[0031] should be understood to be expressly combined with each of the embodiments in paragraphs

[0010] to

[0019] , or with any permutation of two or more embodiments disclosed therein.

[0036] Dosing and administration

[0037] In one embodiment, the pharmaceutical composition of the invention comprising antithrombin can be delivered to a patient by a route of administration selected from the group consisting of intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intrapulmonary, intranasal, transdermal, transmucosal, oral, vaginal, and rectal.

[0038] In certain embodiments, the pharmaceutical composition of the present invention comprising antithrombin is administered to the patient parenterally (e.g., intravenously or subcutaneously). In another embodiment, the pharmaceutical composition of the present invention comprising antithrombin is administered to the patient by a route of administration selected from intrapulmonary or intranasal, such as using a nebulizer or inhaler device.

[0039] Antithrombin can be administered to a patient as part of a multiple dosing regimen, wherein for each administration, antithrombin is administered to the patient using the same route of administration. In one embodiment, each dose of antithrombin is administered to the patient parenterally. For example, each dose of antithrombin can be administered intravenously to the patient.

[0040] The present invention also provides a dosage regimen incorporating the combined modes of administration. For example, the initial dose may be administered parenterally (e.g., intravenously or subcutaneously), and the subsequent dose may be delivered by a route of administration selected from intrapulmonary or intranasal. In an alternative embodiment, the first 1 to 5 doses may be administered parenterally (e.g., intravenously or subcutaneously), and the subsequent dose may be administered by a route of administration selected from intrapulmonary or intranasal.

[0041] Alternatively, the initial dose can be delivered by a route of administration selected from intrapulmonary or intranasal, and subsequent doses can be administered parenterally (e.g., intravenously or subcutaneously). In another embodiment, the first 1 to 5 doses can be delivered by a route of administration selected from intrapulmonary or intranasal, and subsequent doses can be administered parenterally (e.g., intravenously or subcutaneously).

[0042] The multiple administration period may include about 5 to about 30 administrations, until the total cumulative dose. For example, the multiple administration period may include about 5 to about 25 administrations, until the total cumulative dose. In one embodiment, the multiple administration period may include about 5 to about 20 administrations, until the total cumulative dose. In another embodiment, the multiple administration period may include about 5 to about 15 administrations, until the total cumulative dose. In another embodiment, the multiple administration period may include about 4 to about 20 administrations, until the total cumulative dose. In one embodiment, the multiple administration period may include about 2 to about 20 administrations, until the total cumulative dose. In another embodiment, the multiple administration period may include about 3 to about 10 administrations, until the total cumulative dose. In another embodiment, the multiple administration period may include about 4 to about 10 administrations, until the total cumulative dose. In one embodiment, the multiple administration period may include about 5 to about 10 administrations, until the total cumulative dose.

[0043] The multiple administration period can extend from about 1 to about 30 weeks. For example, the multiple administration period can extend from about 1 to about 20 weeks. In one embodiment, the multiple administration period can extend from about 1 to about 10 weeks. In some embodiments, the multiple administration period can extend from about 1 to about 5 weeks. In one embodiment, the multiple administration period can extend for less than about 5 weeks. In another embodiment, the multiple administration period can extend for less than about 3 weeks.

[0044] Multiple doses in batches can be administered at equidistant intervals of about 1 day to about 30 days. For example, multiple doses in batches can be administered at equidistant intervals of about 1 day to about 20 days. In some embodiments, multiple doses in batches can be administered at equidistant intervals of about 1 day to about 15 days. In one embodiment, multiple doses in batches can be administered at equidistant intervals of about 1 day to about 10 days. In another embodiment, multiple doses in batches can be administered at equidistant intervals of about 1 day to about 7 days.

[0045] Alternatively, multiple doses can be administered at unequal intervals from about 1 day to about 30 days in batches. For example, multiple doses can be administered at unequal intervals from about 1 day to about 20 days in batches. In some embodiments, multiple doses can be administered at unequal intervals from about 1 day to about 15 days in batches. In one embodiment, multiple doses can be administered at unequal intervals from about 1 day to about 10 days in batches. In another embodiment, multiple doses can be administered at unequal intervals from about 1 day to about 7 days in batches.

[0046] The term "unequal intervals" used herein should mean that at least one of the multiple doses in batches is administered at an interval different from that of the other doses. For example, the term should be interpreted as including such a dosing regimen in which the first 5 doses are administered at an interval of 1 day, and the subsequent 5 doses are administered at an interval of 7 days.

[0047] Those skilled in the art will appreciate that the specific embodiments disclosed in paragraphs

[0033] to

[0042] should not be read in isolation, and that the specification is intended to disclose these embodiments in combination with other embodiments rather than individually. For example, unless the context suggests otherwise, each of the embodiments disclosed in paragraphs

[0033] to

[0042] should be understood to be expressly combined with each of the embodiments in paragraphs

[0010] to

[0032] , or with any permutation of two or more embodiments disclosed therein.

[0048] Pharmaceutical composition of the present invention

[0049] The pharmaceutical composition of the present invention may also optionally comprise at least one pharmaceutically acceptable carrier. The at least one pharmaceutically acceptable carrier may be selected from adjuvants and carriers. The at least one pharmaceutically acceptable carrier includes any and all solvents, diluents, other liquid carriers, dispersing aids, suspending aids, surfactants, isotonic agents, thickeners, emulsifiers and preservatives suitable for the desired specific dosage form.

[0050] Suitable carriers are Remington: The Science and Practice of Pharmacy, 21st Edition, 2005, ed.DBTroy,LippincottWilliams&Wilkins,Philadelphia,andEncyclopediaofPharmac euticalTechnology,eds.J.SwarbrickandJ.C.Boylan,1988-1999,MarcelDekker,NewYork The invention is described in the literature, the contents of which are incorporated herein by reference. Some preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glycols, dextrose solutions, buffer solutions (e.g., phosphates, glycine, sorbic acid, and potassium sorbate), and human serum albumin. Depending on the route of administration, liposomes and non-aqueous carriers, such as glyceride mixtures of saturated vegetable fatty acids and fixed oils (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil) may also be used.

[0051] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. For systemic use, the pharmaceutical composition of the present invention can be formulated to be administered by a conventional route selected from the group consisting of intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, intracranial, intrapulmonary, intranasal, intraspinal, intrathecal, transdermal, transmucosal, oral, vaginal, and rectal.

[0052] In one embodiment, parenteral administration is the route of administration of choice. The pharmaceutical composition can be packaged in an ampoule, disposable syringe, sealed bag, or multiple dose vial made of glass or plastic. In one embodiment, administration as intravenous injection is the route of administration of choice. The preparation can be administered continuously by infusion or by push injection.

[0053] The pharmaceutical composition that is suitable for injectable use comprises sterile aqueous solution (when having water solubility) or dispersion, and the sterile powder for preparing sterile injectable solution or dispersion immediately.For intravenous administration, suitable carrier comprises physiological saline, antibacterial water, CREMOPHOR EL or phosphate buffered saline (phosphate buffered saline, PBS).In some embodiments, carrier can be solvent or dispersion medium, and it comprises for example water, ethanol, polyol (for example glycerol, propylene glycol and liquid polyethylene glycol etc.) and its suitable mixture.In all cases, composition must be sterile and should be the fluid that exists with the degree that reaches easy injection.

[0054] Preventing the growth of microorganisms can be achieved by a variety of antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it will be preferred to include isotonic agents in the composition, such as sugars (e.g., mannitol, sorbitol, etc.), polyols, or sodium chloride. In some embodiments, the composition of the present invention may include stabilizers, such as amino acids; e.g., alanine, glycine, and combinations thereof. Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption (e.g., aluminum monostearate or gelatin) in the composition.

[0055] Sterile injectable solutions of the pharmaceutical compositions of the invention can be prepared by incorporating the active molecule in the desired amount in an appropriate solvent with one or a combination of ingredients as discussed above, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze drying, which provide a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof.

[0056] In one embodiment, antithrombin can constitute at least 20 weight % of the total protein content of the injectable pharmaceutical composition of the present invention.For example, antithrombin can constitute greater than or equal to about 30 weight %, 40 weight %, 50 weight %, 60 weight %, 70 weight %, 75 weight %, 80 weight %, 85 weight %, 90 weight %, 93 weight %, 95 weight %, 96 weight %, 97 weight %, 98 weight % or 99 weight % of the total protein content of the injectable pharmaceutical composition of the present invention.

[0057] The injectable pharmaceutical compositions of the present invention may be presented in unit dosage unit form, ie, as physically discrete units intended as single dosages for the subject to be treated.

[0058] In one embodiment, the invention provides aerosolized administration of antithrombin. Aerosols can be suspensions of fine solid particles or droplets in any pharmaceutically acceptable propellant gas. In one embodiment, antithrombin can be formulated as a dry powder suitable for use or aerosolization in an inhalation device.

[0059] Those skilled in the art will appreciate that the specific embodiments disclosed in paragraphs

[0044] to

[0053] should not be read in isolation, and that the specification is intended to disclose these embodiments in combination with other embodiments rather than individually. For example, unless the context implies otherwise, each of the embodiments disclosed in paragraphs

[0044] to

[0053] should be understood to be expressly combined with each of the embodiments in paragraphs

[0010] to

[0043] , or with any permutation of two or more embodiments disclosed therein.

[0060] Combination therapy

[0061] The present invention also contemplates the use of supplementary active compounds and molecules in combination with antithrombin.Supplementary active compounds and molecules may be co-formulated with antithrombin in unit dosage form, ie, co-formulated as physically discrete units intended as single dosages for the subject to be treated.

[0062] Alternatively, the supplementary active compounds and molecules may be present as a kit-of-parts, and:

[0063] · Administered separately from antithrombin in a staged or sequential dosing pattern; or

[0064] • Simultaneous co-administration from different dosage forms.

[0065] For example, the present invention contemplates the use of other serum or plasma-based proteins in combination with antithrombin. Serum or plasma proteins within the scope of the present invention include those purified from a suitable plasma source (e.g., human plasma), and those prepared using recombinant manufacturing techniques. For example, serum or plasma proteins may be selected from albumin (e.g., ALBUTEIN), alpha-1 antitrypsin (e.g., PROLASTIN), polyclonal immunoglobulins (IgG, IgA, and combinations thereof), multispecific immunoglobulins (IgM), C1 esterase inhibitors (e.g., BERINERT), thyroxine transporter, transferrin, lactoferrin, and combinations thereof.

[0066] Exemplary polyclonal immunoglobulins within the scope of the present invention include commercially available polyclonal IgG preparations such as FLEBOGAMMADIF 5% and 10%, GAMUNEX-C 10%, BIVIGAM 10%, GAMMAGARD liquid 10%, and the like.

[0067] Exemplary multispecific immunoglobulins (IgM) within the scope of the present invention include commercially available immunoglobulin preparations containing multispecific IgM, such as PENTAGLOBIN or TRIMODULIN.

[0068] In one embodiment, the serum or plasma protein may be selected from albumin, alpha-1 antitrypsin, transferrin, lactoferrin, and combinations thereof. For example, the serum or plasma protein may be selected from alpha-1 antitrypsin, transferrin, lactoferrin, and combinations thereof.

[0069] In a specific embodiment, a therapeutically effective amount of a protein selected from transferrin, lactoferrin, and combinations thereof is administered to the patient in addition to antithrombin. In one embodiment, a therapeutically effective amount of transferrin is administered to the patient in addition to antithrombin.

[0070] As used herein, the term "transferrin" shall include transferrin at all iron saturation levels, including forms commonly referred to as holo-transferrin and apo-transferrin. "Apo-transferrin" means transferrin with an iron saturation of less than about 5%. "Hollo-transferrin" means transferrin with an iron saturation of about 95% or more.

[0071] Similarly, the term "lactoferrin" as used herein shall include lactoferrin at all iron saturation levels, including forms commonly referred to as holo-lactoferrin and apolactoferrin. "Apolactoferrin" means lactoferrin with an iron saturation of less than about 5%. "Holactoferrin" means lactoferrin with an iron saturation of about 95% or more.

[0072] In one embodiment, about combination therapy of the present invention, antithrombin can constitute at least 20 weight % of the total protein content used in combination therapy of the present invention.For example, antithrombin can constitute greater than or equal to about 30 weight %, 40 weight %, 50 weight %, 60 weight %, 70 weight %, 75 weight %, 80 weight %, 85 weight %, 90 weight %, 93 weight %, 95 weight %, 96 weight %, 97 weight %, 98 weight % or 99 weight % of the total protein content used in combination therapy of the present invention.

[0073] Those skilled in the art will appreciate that the specific embodiments disclosed in paragraphs

[0055] to

[0064] should not be read in isolation, and that the specification is intended to disclose these embodiments in combination with other embodiments rather than individually. For example, unless the context suggests otherwise, each of the embodiments disclosed in paragraphs

[0055] to

[0064] should be understood to be expressly combined with each of the embodiments in paragraphs

[0010] to

[0054] , or with any permutation of two or more embodiments disclosed therein. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Further features and advantages of the present invention are more clearly apparent from the accompanying drawings, in which:

[0075] Figures 1 to 4 shows the effect of different doses of antithrombin administered by the intraperitoneal route in a mouse model of middle cerebral artery occlusion; and

[0076] Figure 5 Correlation of the dose of antithrombin administered by the intraperitoneal route with plasma antithrombin activity and the effect of Figures 1 to 4 beneficial effects of the variables measured in the study. DETAILED DESCRIPTION

[0077] It will be apparent to those skilled in the art that the embodiments disclosed below represent only typical examples and that other arrangements and methods capable of reproducing the present invention are possible and are included in the present invention.

[0078] Example 1: Evaluation of the effect of antithrombin III (ATIII) in a mouse model of middle cerebral artery occlusion

[0079] C57 / BL6 mice of 8 to 10 weeks of age and weighing about 20 g were placed under isoflurane anesthesia and occluded the left common carotid artery (CCA). Subsequently, permanent sutures were performed around the external common carotid artery (ECA). Then, 6.0 silicon-coated monofilament sutures were introduced into the ECA. An occluder was introduced to occlude the beginning of the middle cerebral artery (MCA). The occlusion was performed for 60 minutes. During the operation, the mice were temperature controlled. Afterwards (within two hours after occlusion), the animals were evaluated by neuroscore analysis. The cutoff value used in the study was a neuroscore ≥ 4.

[0080] Five different groups were evaluated: sham, MCAO, MCAO+ATIII (incorporating 3 groups with different ATIII doses). In the group with MCAO mice, initially n=8 animals / group were included in the study. The sham group was n=5 animals. For appropriate control, the same volume of vehicle solution and ATIII was administered to sham and MCAO mice, respectively. The same volume of ATIII appropriate dilution was administered to the different ATIII treatment groups.

[0081] 3 hours after MCAO, different doses of ATIII were intraperitoneally administered to mice (250, 500 and 750 IU / Kg). The administration was then repeated 24 hours and 48 hours after MCAO. 54 hours after MCAO (sacrifice), mice were evaluated by neuroscore analysis, plasma samples were collected, and the brain was perfused.

[0082] Plasma samples were obtained by cardiac puncture at sacrifice for analysis of plasma ATIII activity. Table 1 shows the 7-point neurological scoring scale used to evaluate the extent of brain pathology after MCAO.

[0083] Table 1

[0084]

[0085] The brain was prepared as follows for analysis. Mice were perfused transcardially with ice-cold heparinized (heparin 2.5 IU / ml) saline to remove blood from the brain. The fresh brain was removed, and the entire left hemisphere brain block was placed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at +4°C for 24 hours, and frozen in 30% sucrose in 0.1 M PB on a oscillator at +4°C for 2 to 3 days. The brain was then dried, placed on a vial stopper, and frozen on liquid nitrogen, and the brain block was subsequently stored at -80°C until frozen sections were obtained. Lesion volume was measured by MAP2 staining. In order to quantify apoptotic cell death, immunofluorescence for cleaved caspase-3 (Cell Signaling Technology) was measured. In order to study neutrophil infiltration, immunofluorescence was performed using anti-neutrophil antibody NIMP-R14 (Abcam). In both cases, quantification was performed using stereological analysis.

[0086] Neuroscore evaluation after MCAO determined whether MCAO-mediated brain lesions were induced in mice; before ATIII treatment, there were no differences in neuroscore values ​​between groups. Four animals showed no neuroscore deficits (cutoff value>4), and the final distribution was:

[0087] Sham operation - 5 animals;

[0088] MCAO—7 animals;

[0089] MCAO+250IU / Kg ATIII-7 animals;

[0090] MCAO+500IU / Kg ATIII-7 animals;

[0091] MCAO + 750IU / Kg ATIII - 7 animals

[0092] Neurological scores were assessed in a blinded manner at 54 hours. Figure 1 The groups were compared using ANOVA and Bonferroni post hoc analysis (***p<0.001 relative to the MCAO-vehicle group). Figure 1 It can be clearly seen that the MCAO+250IU / Kg group showed no difference compared to the MCAO group (MCAO: 4.29±0.31; MCAO+250ATIII: 4.14±0.28). Interestingly, the administration of 500 and 750IU / Kg of ATIII resulted in statistically significantly improved neurological score values ​​compared to the MCAO group (MCAO+500ATIII: 2.29±0.18; MCAO+750ATIII: 2.14±0.15). The sham group showed a neurological score value of 0.

[0093] Lesion size / volume was measured in different groups. Figure 2 The groups were compared using ANOVA and Bonferroni post hoc analysis (***p<0.001 relative to the MCAO-vehicle group). Lesion volumes are expressed as % relative to infarct volume. Figure 2 As can be seen in the MCAO group, the lesion size showed a 53±2.5% volume increase. Administration of ATIII resulted in a statistically significant reduction in the volume lesions induced by MCAO (MCAO+500ATIII: 29±2.8%; MCAO+750ATIII: 25±2.8%). In contrast, administration of 250 IU / Kg of ATIII did not reduce the volume of the lesions (MCAO+250ATIII: 48±2.4%).

[0094] Effect of ATIII on caspase-3 levels Figure 3 . Groups were compared using ANOVA and Bonferroni post hoc analysis (P value ***p<0.001 relative to MCAO-vehicle). MCAO-induced lesions resulted in a substantial increase in caspase-3 activation. However, administration of ATIII at 500 and 750 IU / Kg significantly reduced caspase-3 activation. No differences were detected between the MCAO group and MCAO+ATIII 250 mg / Kg. Therefore, ATIII may mediate its effects on neurological improvement and volume lesion reduction through anti-apoptotic effects.

[0095] Effect of ATIII on neutrophil infiltration Figure 4 . Groups were compared using ANOVA and Bonferroni post hoc analysis (P value *p<0.05 relative to MCAO-vehicle). MCAO-induced lesions resulted in increased neutrophil infiltration. Administration of ATIII at a dose of only 750 IU / Kg significantly reduced neutrophil infiltration. No differences were detected between the MCAO group and MCAO+ATIII 250 and 500 IU / Kg. Therefore, ATIII can mediate its effects on neurological improvement and volume lesion reduction by maintaining blood-brain barrier permeability.

[0096] Example 2: Correlation of antithrombin III (ATIII) administered by intraperitoneal route with plasma ATIII activity

[0097] Plasma samples were obtained by cardiac puncture at sacrifice for analysis of plasma ATIII activity. Table 2 below lists the correlation of plasma ATIII activity for each intraperitoneal (IP) dose administration.

[0098] Table 2

[0099] IP Dosage Plasma ATIII activity 250IU / Kg 110% to 130% 500IU / Kg 130% to 160% 750IU / Kg 125% to 145%

[0100] Figure 5 Repainted Figures 1 to 4 Each biological variable measured in as a function of plasma ATIII activity. It is very clear from the figure that increasing plasma ATIII activity has a beneficial effect on improving the effects of manipulation in MCAO mice.

[0101] sequence

[0102] The sequences mentioned in the preceding text are summarized below in fasta format. In the event that there are discrepancies between the sequences listed herein and the corresponding sequences in the attached sequence listing, for the purpose of error correction, the sequences listed herein shall prevail.

[0103] SEQ ID No.1 Antithrombin

[0104]

Claims

1. A pharmaceutical composition comprising antithrombin for treating stroke, wherein the antithrombin is administered in an amount sufficient to: increasing the patient's plasma antithrombin activity to greater than about 120%; or The patient's plasma antithrombin concentration is increased to greater than about 1.2 IU / mL.

2. The composition of claim 1, wherein the stroke is selected from ischemic stroke and focal cerebral ischemic stroke.

3. The composition of claim 1 or 2, wherein the antithrombin is administered at a dose sufficient to increase the patient's plasma antithrombin activity by greater than about 120% but less than about 200%.

4. The composition of claim 1 or 2, wherein the antithrombin is administered at a dose sufficient to increase the patient's plasma antithrombin concentration to greater than about 1.2 IU / mL but less than about 2.0 IU / mL.

5. The composition of claim 1 or 2, wherein the antithrombin is administered at a dose sufficient to increase the patient's plasma antithrombin level to greater than about 140 μg / mL but less than about 267 μg / mL.

6. The composition of any preceding claim, wherein the antithrombin is administered in multiple doses as part of a multiple dosing regimen.

7. The composition of claim 6, wherein the antithrombin is administered at a dose sufficient to increase the patient's plasma antithrombin activity to greater than about 120% at at least one time point during the multiple dosing periods.

8. The composition of claim 6, wherein administration of the antithrombin increases the patient's plasma antithrombin concentration to greater than about 1.2 IU / mL at at least one time point during the multiple dosing periods.

9. The composition of claim 6, wherein administration of the antithrombin increases the patient's plasma antithrombin level to greater than about 140 μg / mL at at least one time point during the multiple dosing periods.

10. The composition of claims 6 to 9, wherein the multiple dosing regimen comprises from about 2 to about 30 administrations up to the total cumulative dose.

11. The composition of claim 10, wherein the multiple dosing regimen comprises from about 5 to about 20 administrations up to the total cumulative dose.

12. The composition of claims 6 to 11, wherein the multiple dosing regimen extends over a period of about 1 to about 30 weeks.

13. The composition of claims 6 to 12, wherein the multiple dosing regimen extends over a period of about 1 to about 10 weeks.

14. The composition of claims 6 to 13, wherein the multiple dosing regimen comprises divided doses administered at equally spaced intervals of about 1 day to about 30 days.

15. The composition of claims 6 to 13, wherein the multiple dosing regimen comprises divided doses administered at equally spaced intervals of about 1 day to about 10 days.

16. The composition of claims 6 to 13, wherein the multiple dosing regimen comprises divided doses administered at intervals varying from about 1 day to about 30 days.

17. The composition of claims 6 to 13, wherein the multiple dosing regimen comprises divided doses administered at intervals varying from about 1 day to about 10 days.

18. The composition of any preceding claim, wherein the antithrombin is administered by a route of administration selected from the group consisting of intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intrapulmonary, intranasal, and combinations thereof.

19. The composition of any preceding claim, wherein the antithrombin is administered as part of a combination therapy with at least one other plasma protein selected from: Albumin, alpha-1 antitrypsin, transferrin, lactoferrin, polyclonal immunoglobulin, multispecific immunoglobulin, C1 esterase inhibitor, thyroxine transporter, and combinations thereof.

20. The composition of claim 19, wherein the antithrombin is administered as part of a combination therapy with transferrin.

21. The composition of claim 20, wherein the iron saturation of the transferrin is about 30% or less.

22. The composition of claims 19 to 21, wherein the antithrombin and the plasma protein are administered as a single dosage form.

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