Antithrombin 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 the treatment of stroke.

Background Art

[0002] Stroke is one of the most common categories of non-traumatic brain injury and occurs when the blood supply to a part of the brain is blocked or reduced, such that brain tissue is deprived of oxygen and nutrients. A variety of neurodegenerative processes begin almost immediately and brain cells start to die within minutes. Ischemic stroke occurs when the blood vessels (arteries) that supply blood to an area of the brain are blocked by a blood clot. Hemorrhagic stroke occurs when an artery within the brain leaks or ruptures. Of these two, ischemic stroke is the most common form of stroke.

[0003] The spread of stroke in modern society places a significant burden on the healthcare infrastructure and healthcare expenditure. 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 for re-establishing blocked blood flow is by surgical intervention.

[0004] rtPA treatment has a narrow treatment time frame and as such its widespread applicability is limited. Furthermore, although it is important to restore perfusion to ischemic tissue via rtPA, a series of events of necrosis, apoptosis and inflammation begin within minutes of severe oxygen deprivation. Gradually, although it is gradual, genetically programmed neuronal death during post-ischemic tissue inflammation (which can last from days to weeks) is hypothesized to be a major contributor to the final pathology due to delays in patient treatment / assessment. As such, permanent nerve damage and neuronal injury can occur even before the patient even presents for evaluation.

[0005] There are numerous prior art reports on various molecules that exert a neuroprotective effect to limit damage caused by disorders such as stroke and traumatic injuries to the brain and spinal cord. One such example is US Patent Application Publication No. 2016008437 in the name of Grifols Worldwide Operations Ltd, which discloses apotransferrin as having a neuroprotective effect by regulating the activity of hypoxia-inducible factor (HIF) in a rat model of stroke. The inventors observed the neuroprotective effect of apotransferrin manifested in that the volume of the infarct area decreased in rats treated with apotransferrin compared to control rats.

[0006] It is clear that the severe and debilitating effects of neurodegeneration associated with brain injury require considerable attention and care, and alternative therapies focused on this need are highly desirable.

Best Mode for Carrying Out the Invention

[0007] The words “comprises / comprising” and the words “having / including” as used herein with respect to the present invention are used to specify the presence of the recited features, integers, steps or components, but do not preclude the presence or addition of one or more other features, integers, steps or components or groups thereof.

[0008] Those skilled in the art should understand that the specific embodiments disclosed herein should not be read in isolation, and this specification is intended to disclose the disclosed embodiments not individually but in combination with each other. Thus, each embodiment may serve as a basis for modifying or limiting other embodiments disclosed herein.

[0009] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. Such range formats are used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also all individual numerical values or sub-ranges that are included in the range as if each such numerical value and sub-range were explicitly recited. By way of illustration, a numerical range of "10 to 100" should be interpreted to include not only the explicitly recited values from 10 to 100 but also the individual values and sub-ranges within the indicated range. Thus, this numerical range includes, for example, individual values 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. This same principle applies to ranges in which only a single numerical value, such as "at least 10", is recited. Further, such interpretation applies regardless of whether the breadth or characteristics of the range are described.

[0010] Treatment of the present invention In a first aspect, the present invention is a pharmaceutical composition for use in treating stroke in a patient in need of stroke treatment, comprising antithrombin, wherein the antithrombin is administered in a sufficient dose to increase the patient plasma antithrombin activity by more than about 120%, or administered in a sufficient dose to increase the patient plasma antithrombin concentration by more than about 1.2 IU / mL to provide a pharmaceutical composition.

[0011] In one embodiment, the patient has an ischemic stroke. For example, the patient has a local cerebral ischemic stroke.

[0012] In some embodiments, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin activity to greater than about 120%. For example, antithrombin is administered in a dosage sufficient to increase the patient's plasma antithrombin activity to greater than about 120% and less than about 200%. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin activity to greater than about 130%. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin activity to greater than about 130% and less than about 200%. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin activity to greater than about 140%. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin activity to greater than about 140% and less than about 200%. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin activity to greater than about 150%. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin activity to greater than about 150% and less than about 200%. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin activity to greater than about 160%. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin activity to greater than about 160% and less than about 200%.

[0013] Antithrombin activity is measured using standard techniques generally known to those of skill in the art. For example, when patient plasma is mixed with heparin and thrombin, the degree of thrombin inhibition (%) is proportional to the functional antithrombin activity of the patient sample. Thrombin activity is measured by the rate of conversion of a chromogenic substrate, and the resulting signal (if any) is used to calculate the antithrombin activity.

[0014] In other embodiments, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin concentration to greater than about 1.2 IU / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin concentration to greater than about 1.2 IU / mL and less than about 2.0 IU / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin concentration to greater than about 1.3 IU / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin concentration to greater than about 1.3 IU / mL and less than about 2.0 IU / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin concentration to greater than about 1.4 IU / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin concentration to greater than about 1.4 IU / mL and less than about 2.0 IU / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin concentration to greater than about 1.5 IU / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin concentration to greater than about 1.5 IU / mL and less than about 2.0 IU / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin concentration to greater than about 1.6 IU / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin concentration to greater than about 1.6 IU / mL and less than about 2.0 IU / mL.

[0015] Antithrombin concentration can be measured using standard techniques generally known to those of skill 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 determined by comparison with control samples of known concentration.

[0016] In certain embodiments, antithrombin is administered to a patient in a dosage sufficient to increase the patient plasma antithrombin level to greater than about 140 μg / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin level to greater than about 140 μg / mL and less than about 267 μg / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin level to greater than about 160 μg / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin level to greater than about 160 μg / mL and less than about 267 μg / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin level to greater than about 180 μg / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin level to greater than about 180 μg / mL and less than about 267 μg / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin level to greater than about 200 μg / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin level to greater than about 200 μg / mL and less than about 267 μg / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin level to greater than about 220 μg / mL. For example, antithrombin is administered in a dosage sufficient to increase the patient plasma antithrombin level to greater than about 220 μg / mL and less than about 267 μg / mL.

[0017] In certain embodiments, antithrombin is administered to a patient in multiple doses.

[0018] Advantageously, the compositions of the invention administer antithrombin to a patient at an effective concentration to mitigate the effects of stroke without an associated increased risk of bleeding related to AT replacement.

[0019] One skilled in the art should understand that the specific embodiments disclosed in paragraphs

[0010] to

[0018] should not be read alone, and this specification is intended to disclose these embodiments not individually but in combination with other embodiments.

[0020] Antithrombin Antithrombin is a plasma-based serine protease inhibitor of a single-chain glycoprotein (68 kD), and mainly plays a major role in maintaining the balance of the coagulation system by inactivating several coagulation factors (for example, FXa, etc.). Furthermore, the enhancement of antithrombin activity is the mechanism by which heparin exerts its anticoagulant effect. In addition to its role as an anticoagulant, antithrombin has anti-inflammatory properties. Antithrombin has many names and is generally also called antithrombin III, AT, or ATIII, and all of them are applicable in this specification.

[0021] The present invention includes within its scope all mammalian-derived wild-type and functionally active variants of antithrombin. The present invention further includes within its scope both the cleaved and latent forms of wild-type antithrombin. Particularly preferred is human antithrombin, for example, both the latent and cleaved forms of human antithrombin. For example, human antithrombin having the amino acid sequence shown in SEQ ID NO: 1 (UniProtKB Seq. No. P01008).

[0022] The antithrombin utilized in the present invention is either plasma-derived or recombinant. Plasma-derived antithrombin is purified from the plasma of donors by processes well known to those skilled in the art. Suitable commercially available preparations of human plasma-derived antithrombin, which are examples of useful materials in the present invention, include, but are not limited to, preparations sold under the trade name THROMBATE III.

[0023] Exemplary recombinant forms of antithrombin useful in the present invention include, but are not limited to, AT alpha (ATryn®) and AT gamma.

[0024] This specification includes within its scope recombinant derivatives of antithrombin that differ from the wild-type amino acid sequence of the human protein outlined in SEQ ID NO: 1 by one or more substitutions, one or more deletions, or one or more insertions that do not substantially alter the structure or hydropathicity of the recombinant protein as compared to the wild-type protein. Recombinant variants of antithrombin within the scope of the present invention may include at least one post-translational modification such as pegylation, glycosylation, polysialylation, or combinations thereof.

[0025] In one embodiment, the present invention contemplates recombinant variants of antithrombin having one or more conservative substitutions as compared to the wild-type protein of SEQ ID NO: 1. A "conservative substitution" is one in which an amino acid is replaced with another amino acid having similar properties, such that a person skilled in peptide chemistry would predict that the secondary structure and hydropathicity of the polypeptide are not substantially altered. Generally, changes within the following groups of amino acids 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.

[0026] For example, recombinant antithrombin within the scope of the present invention has at least 90%, 95%, 96%, 97%, 98% or 99% homology with the wild-type human antithrombin protein outlined in SEQ ID NO: 1.

[0027] One of ordinary skill in the art will understand that recombinant proteins can be obtained using standard techniques well known in the art of protein expression, production, and purification. The nucleic acid sequence of the recombinant protein of interest 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).

[0028] As used herein, the term "expression vector" refers to an entity capable of introducing a protein expression construct into a host cell. Some expression vectors can also replicate in the host cell and increase protein expression by the protein expression construct. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments may be ligated. Other vectors include cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), fosmids, phages, and phagemids. Another type of vector is a viral vector, to which additional DNA segments may be ligated within the viral genome. Certain vectors can replicate autonomously in the introduced host cell (e.g., vectors having an origin of replication that functions in the host cell). Other vectors are integrated into the host cell genome upon introduction into the host cell and replicated with the host genome. Furthermore, certain preferred vectors are capable of inducing the expression of the genes to which they are operably linked.

[0029] Suitable bacterial cells include Escherichia coli ( Escherichia coli ), Bacillus subtilis ( Bacillus subtilis ), Salmonella typhimurium ( Salmonella typhimurium ), Pseudomonas spp. ( Pseudomonas spp.), Streptomyces spp. ( Streptomyces spp.), and Staphylococcus spp. ( Staphylococcus spp.). Suitable yeast cells include Saccharomyces spp. ( Saccharomyces spp.), Pichia spp. ( Pichia spp.), and Kluyveromyces spp. (Kluyveromyces including spp.). Insect cells include silkworms ( Bombyx mori ), Spodoptera litura ( Mamestra brassicae ), Trichoplusia ni ( Spodoptera frugiperda ), Antheraea pernyi ( Trichoplusia ni ), and Drosophila melanogaster ( 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, CRL7O3O, HsS78Bst, human hepatocellular carcinoma cells (e.g., Hep G2), human adenovirus-transformed 293 cells (e.g., HEK293), PER.C6, mouse L-929 cells, HaK hamster cell line, mouse 3T3 cells derived from Swiss mice, Balb-c mice or NIH mice, and CV-1 cell line cells.

[0030] The present invention also contemplates the use of wild-type or recombinant antithrombin proteins conjugated or fused to other proteins, protein fragments, protein domains, peptides, small molecules or other chemical substances. For example, suitable fusion partners or binding partners include serum albumin (e.g., bovine, rabbit or human), keyhole limpet hemocyanin, immunoglobulin molecules (including the Fc domain of immunoglobulins), thyroglobulin, ovalbumin, tetanus toxoid, toxoids from other pathogenic bacteria, attenuated toxin derivatives, cytokines, chemokines, glucagon-like peptide-1, exendin-4, XTEN, or combinations thereof.

[0031] In one embodiment, the antithrombin useful in the present invention is fused to an immunoglobulin Fc domain. For example, the immunoglobulin Fc domain includes at least a portion of a constant heavy chain immunoglobulin domain. The constant heavy chain immunoglobulin domain is preferably an Fc fragment that includes the CH2 domain and the CH3 domain, and optionally at least a part of the hinge region. The immunoglobulin Fc domain is an immunoglobulin Fc domain of IgG, IgM, IgD, IgA or IgE, 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 is selected from the Fc domains of IgG1, IgG2, IgG3 or IgG4, or modified Fc domains thereof.

[0032] Those skilled in the art should understand that the specific embodiments disclosed in paragraphs

[0020] to

[0031] should not be read alone, and this specification is intended to disclose these embodiments not individually but in combination with other embodiments. For example, unless the context implies otherwise, each embodiment disclosed in paragraphs

[0020] to

[0031] is to be read as being explicitly combined with each embodiment in paragraphs

[0010] to

[0019] , or with any rearrangement, with a combination of two or more of the embodiments disclosed in that paragraph.

[0033] Dosing and administration In one embodiment, the pharmaceutical composition of the present invention containing antithrombin can be delivered to a patient by an administration route selected from the group consisting of intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intralung, intranasal, transdermal, transmucosal, oral, vaginal and rectal.

[0034] In certain embodiments, the pharmaceutical composition comprising antithrombin of the present invention is administered to a patient by parenteral administration, such as intravenous administration or subcutaneous administration. In another embodiment, the pharmaceutical composition comprising antithrombin of the present invention is administered to a patient by an administration route selected from intralung or intranasal using, for example, a nebulizer or an inhaler device.

[0035] Antithrombin can be administered to a patient as part of a multiple-dose regimen, in which case antithrombin is administered to the patient using the same administration route for each dose. In one embodiment, each dose of antithrombin is administered to the patient by parenteral administration. For example, each dose of antithrombin is administered to the patient by intravenous administration.

[0036] The present invention also provides an administration regimen that combines multiple administration methods. For example, the first dose is administered by parenteral administration, such as intravenous administration or subcutaneous administration, and subsequent doses are administered by an administration route selected from intralung or intranasal. In an alternative embodiment, the first 1 to 5 doses are administered by parenteral administration, such as intravenous administration or subcutaneous administration, and subsequent doses are administered by an administration route selected from intralung or intranasal.

[0037] Alternatively, the first dose is delivered by an administration route selected from intralung or intranasal, and subsequent doses are administered by parenteral administration, such as intravenous administration or subcutaneous administration. In another embodiment, the first 1 to 5 doses are delivered by an administration route selected from intralung or intranasal, and subsequent doses are administered by parenteral administration, such as intravenous administration or subcutaneous administration.

[0038] The multiple dosing period includes about 5 to about 30 administrations until the total cumulative dose is reached. For example, the multiple dosing period includes about 5 to about 25 administrations until the total cumulative dose is reached. In one embodiment, the multiple dosing period includes about 5 to about 20 administrations until the total cumulative dose is reached. In another embodiment, the multiple dosing period includes about 5 to about 15 administrations until the total cumulative dose is reached. In yet another embodiment, the multiple dosing period includes about 4 to about 20 administrations until the total cumulative dose is reached. In one embodiment, the multiple dosing period includes about 2 to about 20 administrations until the total cumulative dose is reached. In another embodiment, the multiple dosing period includes about 3 to about 10 administrations until the total cumulative dose is reached. In yet another embodiment, the multiple dosing period includes about 4 to about 10 administrations until the total cumulative dose is reached. In one embodiment, the multiple dosing period includes about 5 to about 10 administrations until the total cumulative dose is reached.

[0039] The multiple dosing period spans a period of about 1 week to about 30 weeks. For example, the multiple dosing period spans a period of about 1 week to about 20 weeks. In one embodiment, the multiple dosing period spans a period of about 1 week to about 10 weeks. In some embodiments, the multiple dosing period spans a period of about 1 week to about 5 weeks. In one embodiment, the multiple dosing period spans a period of less than about 5 weeks. In yet another embodiment, the multiple dosing period spans a period of less than about 3 weeks.

[0040] The multiple doses are administered at equal intervals of about 1 day to about 30 days. For example, the multiple doses are administered at equal intervals of about 1 day to about 20 days. In some embodiments, the multiple doses are administered at equal intervals of about 1 day to about 15 days. In one embodiment, the multiple doses are administered at equal intervals of about 1 day to about 10 days. In a further embodiment, the multiple doses are administered at equal intervals of about 1 day to about 7 days.

[0041] Alternatively, the multi-dose is administered at unequal intervals of from about 1 day to about 30 days. For example, the multi-dose is administered at unequal intervals of from about 1 day to about 20 days. In some embodiments, the multi-dose is administered at unequal intervals of from about 1 day to about 15 days. In one embodiment, the multi-dose is administered at unequal intervals of from about 1 day to about 10 days. In a further embodiment, the multi-dose is administered at unequal intervals of from about 1 day to about 7 days.

[0042] As used herein, the term "unequal intervals (intervals that are not equal intervals)" is meant to mean that at least one of the multi-doses is administered at an interval different from that of the other doses. For example, this term is to be construed as including a dosing regimen in which the first 5 doses are administered at 1-day intervals and the subsequent 5 doses are administered at 7-day intervals.

[0043] One of ordinary skill in the art should understand that the specific embodiments disclosed in paragraphs

[0033] to

[0042] should not be read alone, and that this specification is not intended to disclose these embodiments individually, but rather in combination with other embodiments. For example, unless the context otherwise implies, each embodiment disclosed in paragraphs

[0033] to

[0042] is to be read as being explicitly combined with each embodiment in paragraphs

[0010] to

[0032] , or with any rearrangement, with two or more of the embodiments disclosed in that paragraph.

[0044] Pharmaceutical composition of the present invention The pharmaceutical composition of the present invention further comprises, if necessary, at least one pharmaceutically acceptable carrier. The at least one pharmaceutically acceptable carrier is selected from adjuvants and vehicles. The at least one pharmaceutically acceptable carrier includes any solvent, diluent, other liquid vehicle, dispersion aid, suspension aid, surfactant, isotonic agent, thickening agent, emulsifying agent and preservative suitable for the desired specific dosage form.

[0045] Suitable carriers are described in Remington: The Science and Practice of Pharmacy (21st Edition, 2005, edited by D.B. Troy, Lippincott Williams & Wilkins, Philadelphia), and Encyclopedia of Pharmaceutical Technology (edited by J. Swarbrick and J.C. Boylan, 1988 - 1999, Marcel Dekker, New York) (the contents of which are incorporated herein by reference). Preferred examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer's solution, glycols, dextrose solutions, buffered solutions (e.g., phosphates, glycine, sorbic acid and potassium sorbate, etc.), and human serum albumin. Depending on the route of administration, non-aqueous vehicles such as liposomes, and mixtures of glycerides 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, etc.) can also be used.

[0046] The pharmaceutical composition of the present invention is formulated to be compatible with the intended route of administration. In the case of systemic administration, the pharmaceutical composition of the present invention can be formulated for administration by conventional routes selected from the group consisting of intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, intracranial, intratracheal, intranasal, intraspinal, intrathecal, transdermal, transmucosal, oral, vaginal and rectal.

[0047] In one embodiment, parenteral administration is the selected route of administration. The pharmaceutical composition is enclosed in an ampoule, disposable syringe, sealed bag, or multi-dose vial made of glass or plastic. In one embodiment, administration as an intravenous injection is the selected route of administration. The formulation can be administered continuously by infusion or bolus injection.

[0048] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble), dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In the case of intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL, or phosphate buffered saline (PBS). In some embodiments, the carrier is, for example, a solvent or dispersion medium containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. In all cases, the composition must be sterile and should be fluid to the extent that it can be easily injected into a syringe.

[0049] Prevention of the growth of microorganisms can be achieved by various antibacterial and antifungal agents such as, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include in the composition isotonic agents such as, for example, saccharides (such as mannitol, sorbitol, etc.), polyalcohols or sodium chloride. In some embodiments, the compositions of the present invention include stabilizers such as, for example, amino acids (such as alanine, glycine, and combinations thereof). Prolonged absorption of injectable compositions is brought about by including in the composition agents that delay absorption (such as aluminum monostearate or gelatin).

[0050] The sterile injectable solution of the pharmaceutical composition of the present invention can be prepared by incorporating the required amount of the active molecule into a suitable solvent with components including one or a combination of the above-mentioned components, followed by filtration sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods include vacuum drying and freeze drying from which powders of the active ingredient and any additional desired components are obtained from a pre-sterile filtered solution.

[0051] In one embodiment, antithrombin constitutes at least 20% by weight ratio of the total protein content of the injectable pharmaceutical composition of the present invention. For example, antithrombin constitutes about 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more by weight ratio of the total protein content of the injectable pharmaceutical composition of the present invention.

[0052] The injectable pharmaceutical composition of the present invention can be provided in unit dosage form, that is, as a physically discrete unit intended for unit dosage for a subject to be treated.

[0053] In one embodiment, the present invention provides aerosolized administration of antithrombin. An aerosol is a suspension of fine solid particles or liquid droplets in any pharmaceutically acceptable propellant gas. In one embodiment, antithrombin is formulated as a dry powder suitable for use in an inhalation device or for spraying.

[0054] Those skilled in the art should understand that the specific embodiments disclosed in paragraphs

[0044] to

[0053] should not be read alone, and this specification is intended to disclose these embodiments not individually but in combination with other embodiments. For example, unless the context implies otherwise, each embodiment disclosed in paragraphs

[0044] to

[0053] is to be read as being explicitly combined with each embodiment in paragraphs

[0010] to

[0043] , or with any rearrangement, and with a combination consisting of two or more of the embodiments disclosed in that paragraph.

[0055] Combination therapy The present invention also contemplates the use of auxiliary active compounds and molecules in combination with antithrombin. The auxiliary active compounds and molecules can be formulated with antithrombin as unit dosage forms, i.e., as physically discrete units intended for unitary administration to a subject to be treated.

[0056] Alternatively, the supplementary active compounds and molecules are provided as a kit-of-parts and · are administered separately from antithrombin in a stepwise or continuous dosing pattern, or · are administered simultaneously from different dosage forms can be.

[0057] For example, the present invention contemplates administering other serum proteins or plasma-based proteins in combination with antithrombin. Serum proteins or plasma proteins within the scope of the present invention include those purified from a suitable plasma source such as human plasma and those prepared using recombinant manufacturing techniques. For example, the serum protein or plasma protein is selected from the group consisting of albumin (e.g., ALBUTEIN), alpha-1 antitrypsin (e.g., PROLASTIN), polyclonal immunoglobulins (IgG, IgA, and combinations thereof), multispecific immunoglobulins (IgM), C1 esterase inhibitor (e.g., BERINERT), transthyretin, transferrin, lactoferrin, and combinations thereof.

[0058] Exemplary polyclonal immunoglobulins within the scope of the present invention include commercially available polyclonal IgG formulations such as, for example, FLEBOGAMMA DIF 5%&10%, GAMUNEX-C 10%, BIVIGAM 10%, GAMMAGARD Liquid 10%.

[0059] Exemplary multispecific immunoglobulins (IgM) within the scope of the present invention include commercially available immunoglobulin formulations containing multispecific IgM, such as PENTAGLOBIN or TRIMODULIN, for example.

[0060] In one embodiment, the serum protein or plasma protein is selected from the group consisting of albumin, alpha-1 antitrypsin, transferrin, lactoferrin, and combinations thereof. For example, the serum protein or plasma protein is selected from the group consisting of alpha-1 antitrypsin, transferrin, lactoferrin, and combinations thereof.

[0061] In certain embodiments, 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.

[0062] As used herein, the term "transferrin" shall be taken to include transferrins at all levels of iron saturation, including forms commonly referred to as holotransferrin and apotransferrin. "Apotransferrin" means transferrin with an iron saturation of less than about 5%. "Holotransferrin" means transferrin with an iron saturation of about 95% or more.

[0063] Similarly, as used herein, the term "lactoferrin" shall be taken to include lactoferrins at all levels of iron saturation, including forms commonly referred to as hololactoferrin and apolactoferrin. "Apolactoferrin" means lactoferrin with an iron saturation of less than about 5%. "Hololactoferrin" means lactoferrin with an iron saturation of about 95% or more.

[0064] In one embodiment, regarding the combination therapy of the present invention, antithrombin constitutes at least 20% by weight ratio of the total protein content utilized in the combination therapy of the present invention. For example, antithrombin constitutes about 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more by weight ratio of the total protein content utilized in the combination therapy of the present invention.

[0065] Those skilled in the art should understand that the specific embodiments disclosed in paragraphs

[0055] to

[0064] should not be read alone, and this specification is not intended to disclose these embodiments individually, but in combination with other embodiments. For example, unless otherwise implied by the context, each embodiment disclosed in paragraphs

[0055] to

[0064] shall be read as being explicitly combined with each embodiment in paragraphs

[0010] to

[0054] , or with any rearrangement, and any rearrangement consisting of two or more of the embodiments disclosed in that paragraph.

Brief Description of the Drawings

[0066] Further features and advantages of the present invention will become more apparent in the accompanying drawings.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5-1

Figure 5-2

Figure 5-3

Figure 5-4

Example

[0067] Those skilled in the art will appreciate that the examples disclosed below are merely generalized examples and that other arrangements and methods capable of reproducing the present invention are possible and are clearly encompassed by the present invention.

[0068] Example 1: Evaluation of the effect of antithrombin III (ATIII) in a middle cerebral artery occlusion mouse model C57 / BL6 mice, 8-10 weeks old and weighing approximately 20 g, were placed under isoflurane anesthesia, and occlusion of the left common carotid artery (CCA) was performed. Subsequently, permanent suturing was performed around the external carotid artery (ECA). Next, a 6.0 silicon-coated monofilament suture was introduced into the ECA. An occluder was introduced to occlude the origin of the middle cerebral artery (MCA). The occlusion was performed for 60 minutes. During the procedure, the temperature regulation of the mice was operative. Thereafter (within 2 hours after occlusion), the animals were evaluated by neuroscore analysis. The cutoff value used in this study was a neuroscore of 4 or higher.

[0069] Five different groups were evaluated: sham (control), MCAO, MCAO + ATIII (including three groups with different ATIII doses). In the group of MCAO mice, initially n = 8 animals per group were included in the study. The sham group consisted of n = 5 animals. As appropriate controls, sham mice and MCAO mice were administered the same volume of vehicle solution and ATIII, respectively. In the different ATIII treatment groups, appropriate dilutions of the same amount of ATIII were administered.

[0070] Three hours after MCAO, mice were intraperitoneally administered various doses of ATIII (250, 500, and 750 IU / Kg). Subsequently, this administration was repeated at 24 and 48 hours after MCAO. At 54 hours after MCAO (sacrifice), mice were evaluated by neurological score analysis, plasma samples were collected, and the brains were perfused.

[0071] Plasma samples were obtained by cardiac puncture at sacrifice for analysis of plasma ATIII activity. Table 1 shows the 7 - point neurological score scale used to evaluate the extent of brain lesions after MCAO.

[0072]

Table 1

[0073] The brain was prepared for analysis as follows: Mice were perfused transcardially with ice-cold heparinized (2.5 IU / ml heparin) saline to remove blood from the brain. Fresh brains were removed, and the entire left hemisphere mass was placed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at +4 °C for 24 h and cryoprotected in 30% sucrose in 0.1 M PB on a shaker at +4 °C for 2 - 3 days. Then, the brains were dried, placed on vial corks, and frozen on liquid nitrogen. Subsequently, the blocks were stored at -80 °C until cryostat sections were obtained. Lesion volume was measured by MAP2 staining. To quantify apoptotic cell death, immunofluorescence against cleaved caspase-3 (Cell Signaling Technology) was measured. To examine neutrophil infiltration, immunofluorescence using the anti-neutrophil antibody NIMP-R14 (Abcam) was measured. In both cases, quantification was performed using stereological analysis.

[0074] In the neurological score evaluation after MCAO, it was demonstrated that MCAO-mediated brain lesions were induced in the mice: There was no difference in neurological score values between the groups before ATIII treatment. Four animals did not show neurological score deficits (a cut-off of >4), and the final distribution was as follows: Sham - 5 animals; MCAO - 7 animals; MCAO + 250 IU / Kg of ATIII - 7 animals; MCAO + 500 IU / Kg of ATIII - 7 animals; MCAO + 750 IU / Kg of ATIII - 7 animals.

[0075] The neurological score was evaluated in a blinded fashion 54 h later. The results are shown in Figure 1. The groups were compared using ANOVA with Bonferroni post hoc analysis ( ***p < 0.001, compared with the MCAO-vehicle group). From Figure 1, it is clear that the MCAO + 250 IU / Kg group showed no difference compared with the MCAO group (MCAO: 4.29 ± 0.31; MCAO + 250 ATIII: 4.14 ± 0.28). Interestingly, administration of 500 IU / Kg and 750 IU / Kg of ATIII significantly improved the neurological score statistically compared with the MCAO group (MCAO + 500 ATIII: 2.29 ± 0.18; MCAO + 750 ATIII: 2.14 ± 0.15). The sham group showed a neurological score of 0.

[0076] Lesion size / volume was measured in different groups. The results are shown in Figure 2. The groups were compared using ANOVA with Bonferroni post hoc analysis ( *** p < 0.001, compared with the MCAO-vehicle group). Lesion volume was expressed as a percentage of relative infarct volume. From Figure 2, it can be seen that the MCAO group showed a volume lesion size of 53 ± 2.5%. Administration of ATIII resulted in a statistically significant decrease in MCAO-induced volume lesions (MCAO + 500 ATIII: 29 ± 2.8%; MCAO + 750 ATIII: 25 ± 2.8%). In contrast, administration of 250 IU / Kg of ATIII did not reduce the volume of the lesions (MCAO + 250 ATIII: 48 ± 2.4%).

[0077] The effect of ATIII on caspase-3 levels is shown in Figure 3. The groups were compared using ANOVA with Bonferroni post hoc analysis (P value, *** p < 0.001, compared with MCAO-vehicle). MCAO-induced lesions resulted in a large increase in caspase-3 activation. However, administration of ATIII significantly decreased caspase-3 activation at 500 IU / Kg and 750 IU / Kg. No difference was detected between the MCAO group and MCAO + ATIII 250 mg / Kg. Therefore, ATIII may mediate its effects on neurological improvement and volume lesion reduction via an anti-apoptotic action.

[0078] The effect of ATIII on neutrophil infiltration is shown in Figure 4. The groups were compared using ANOVA with Bonferroni post - analysis (P - value, *** p < 0.05, compared to MCAO - vehicle). MCAO - induced lesions caused an increase in neutrophil infiltration. Administration of ATIII significantly decreased neutrophil infiltration, exclusively at a dose of 750 IU / Kg. No difference was detected between the MCAO group and MCAO + ATIII 250 IU / Kg and 500 IU / Kg. Thus, ATIII may mediate its effect on neurological improvement and volume lesion reduction through preservation of blood - brain barrier permeability.

[0079] Example 2: Correlation between intraperitoneally administered antithrombin III (ATIII) and plasma ATIII activity Plasma samples were obtained by cardiac puncture at the time of sacrifice for analysis of plasma ATIII activity. The correlation of plasma ATIII activity for each intraperitoneal (IP) dose administered is summarized in Table 2 below.

[0080]

Table 2

[0081] Figure 5 replots each of the biological variables measured in Figures 1 - 4 as a function of plasma ATIII activity. It is clear from the figure that an increase in plasma ATIII activity has a beneficial effect in improving the impact of MCAO mouse treatment.

[0082] Sequence The sequences shown in the foregoing text are outlined in fasta format below. In case of a discrepancy between the sequences described in this text and the corresponding sequences in the attached sequence listing, for the purpose of correcting errors, the sequences described in this text shall prevail.

[0083]

Table 3

Claims

1. A pharmaceutical composition containing antithrombin for use in the treatment of stroke, The aforementioned antithrombin, A pharmaceutical composition administered in a dose sufficient to increase the patient's plasma antithrombin activity by more than approximately 120%, or to increase the patient's plasma antithrombin concentration by more than approximately 1.2 IU / mL.

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

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

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

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

6. The composition according to claim 1, wherein antithrombin is administered multiple times as part of a multiple-dose regimen.

7. The composition according to claim 6, wherein antithrombin is administered in a dose sufficient to increase the patient's plasma antithrombin activity by more than about 120% at at least one time point during the multi-dose period.

8. The composition according to claim 6, wherein the antithrombin is administered such that the patient's plasma antithrombin concentration increases to more than about 1.2 IU / mL at at least one time point during the multi-dose period.

9. The composition according to claim 6, wherein the antithrombin is administered such that the patient's plasma antithrombin level increases to more than about 140 μg / mL at at least one time point during the multi-dose period.

10. The composition according to claim 6 or 7, wherein the multi-dose regimen comprises approximately 2 to approximately 30 doses until a total cumulative dose is reached.

11. The composition according to claim 10, wherein the multi-dose regimen comprises approximately 5 to approximately 20 doses until a total cumulative dose is reached.

12. The composition according to claim 6 or 7, wherein the multi-dose regimen extends over a period of approximately 1 week to approximately 30 weeks.

13. The composition according to claim 6 or 7, wherein the multi-dose regimen extends over a period of about one week to about ten weeks.

14. The composition according to claim 6 or 7, wherein the multi-dose regimen comprises multiple doses administered at equal intervals of approximately 1 to 30 days.

15. The composition according to claim 6 or 7, wherein the multi-dose regimen comprises multiple doses administered at equal intervals of approximately 1 to 10 days.

16. The composition according to claim 6 or 7, wherein the multi-dose regimen comprises multiple doses administered at unequal intervals of approximately 1 to 30 days.

17. The composition according to claim 6 or 7, wherein the multi-dose regimen comprises multiple doses administered at unequal intervals of approximately 1 to 10 days.

18. The composition according to claim 1, wherein 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 according to claim 1, wherein antithrombin is administered as part of a combination therapy with at least one other plasma protein selected from the group consisting of albumin, alpha-1 antitrypsin, transferrin, lactoferrin, polyclonal immunoglobulin, multispecific immunoglobulin, C1 esterase inhibitor, transthyretin, and combinations thereof.

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

21. The composition according to claim 20, wherein the transferrin has an iron saturation of about 30% or less.

22. The composition according to claim 19 or 20, wherein antithrombin and transferrin are administered as unit doses.

Images