WO2021028927A1 - Combination of alpha-1-antitrypsin and steroids and uses therefor - Google Patents

Abstract

The present invention relates to a medicament comprising a combination of AAT and steroids. The present invention further relates to combinations of AAT with steroids for treating an inflammatory disease or tissue injury.

Description

COMBINATION OF ALPHA-l-ANTITRYPSIN AND STEROIDS AND USES

THEREFOR

FIELD OF THE INVENTION

The present invention relates to a medicament comprising a combination of Alpha- 1 antitrypsin (AAT) and steroids. The present invention farther relates to combinations of AAT with steroids for treating an inflammatory disease and tissue injury.

BACKGROUND OF THE INVENTION

Alpha- 1 antitrypsin (AAT) is a prototypical member of the serpin superfamily of serine protease inhibitors. hAAT is synthesized and secreted into the bloodstream mainly by hepatocytes. In addition, it is expressed and secreted by epithelial cells of the respiratory and intestinal systems, as well as by various immune cells, such as macrophages and neutrophils. Genetic deficiency in hAAT primarily manifests as non smoking pulmonary emphysema, most probably as a consequence of a protease anti protease imbalances in the lung and unrestrained tissue degradation. Patients with hAAT deficiency are treated with plasma-derived affinity-purified hAAT, a regimen directed at minimizing the rates of pulmonary emphysema.

Historically, the major function of hAAT has been attributed to its straight-forward inhibition of neutrophil elastase, proteinase-3 and cathepsin G, rendering it a relatively upstream anti-inflammatory agent. However, hAAT also exhibits immunomodulatory activities; indeed, its anti-apoptotic capabilities, facilitation of immune tolerance in vivo and ability to expedite wound repair are untypical in agents that merely block inflammation. These complex actions appear to surpass the activity of protease inhibition, and there are suggestions that hAAT may act as a highly conserved, context- sensitive and elaborate binding protein, such that may directly mitigate the effects of several DAMP molecules. This concept is consistent with its appropriate physiological rise in the circulation during infection, healthy pregnancies and advanced age from a steady-state of -1.3 mg/ml, to 4-6 mg/ml, while downplaying inflammation without suppressing the immune system. In this regard, outcomes of high concentrations of hAAT treatments (>1.3 mg/ml), are presumably representative of its role in acute phase responses, where it appears to help navigate innate immune cells through injured

Beneficial effects of hAAT therapy are observed across several animal models in the settings of autoimmunity, alloimmunity, and xenoimmunity. Its actions appear to be associated with reduced cellular infiltration and diminished pro-inflammatory responses, accompanied by the emergence of tolerogenic semi-mature dendritic cells (DCs) and expanding numbers of regulatory T cells (Tregs), thus setting it apart from classic immunosuppressive agents. Consistent outcomes were observed in disease models for collagen-induced arthritis, experimental autoimmune encephalomyelitis (EAE), and acute graft-versus-host disease (GvHD). As a result, treatment of patients who suffer from steroid-refractory acute GvHD with hAAT infusions has shown promising positive outcomes.

Common cytokine responses in hAAT-rich conditions include reduced levels of IL- 1b, TNFa and IL-6, and increased levels of anti-inflammatory IL-10 and IL-1 receptor antagonist (IL-IRa).

Although hAAT is anti-inflammatory as per treatment outcomes, hAAT promotes expression of inflammation-prompted molecules, such as IL-10, IL-IRa, VEGF, and CCR7. This is in marked contrast to corticosteroid-based therapies, where inhibition of inflammatory processes is more comprehensive. Corticosteroids interfere with nuclear translocation of NF-KB family of nuclear transcription factors; and diminish inflammation-triggered levels of IL-10 and IL-IRa. As a consequence, treatment with members of the corticosteroid family offers little specificity, and results in broad adverse effects that include poor wound healing and susceptibility to infections. Accordingly, corticosteroid therapy impairs epithelial monolayer gap repair.

Thus, there exists a need for additional therapeutics that are less toxic and with high potency.

SUMMARY OF THE INVENTION

As disclosed below, it has been found that providing a treatment which combines the effects of steroid treatments and AAT treatments is highly desirable and can be conducted without significantly disrupting the potency of the constituent pharmaceuticals. Furthermore, it has been found that in the presence of low dose steroids, AAT retains the anti-inflammatory activity shown with high dose steroids alone.

In one aspect of the invention, a composition of AAT, or physiologically functional derivative thereof, can advantageously be combined with a steroid, or a pharmaceutically acceptable salt, solvate, or physiologically functional derivative thereof, to provide an effective treatment. The composition can provide, in a single administration or multiple dosing regimen, the anti-inflammatory properties of the steroid and the anti-inflammatory (and/or other) properties of AAT, without any significant interference between the two, or adverse reactions.

It is believed that this combination has: a) the potential to decrease the dosage of corticosteroids to achieve the desired clinical response (e.g., the corticosteroid dose required is lower than found in other comparable medications or with corticosteroids used alone) b) an equal or greater clinical effect than corticosteroid therapy alone c) the potential to decrease the incidence of clinical exacerbations requiring corticosteroid therapy (e.g., less frequent exacerbations as compared to other modes of treatment) d) the potential to achieve the desired clinical effect (e.g., stabilize the disease) with a shorter duration and/or lower dosages of corticosteroid therapy e) the potential to decrease the incidence of adverse events resulting from corticosteroid therapy (e.g., bacterial infections).

According to certain embodiments the AAT may allow to taper down the steroid dose.

According to one aspect, the present invention provides a medicament comprising Alpha- 1 antitrypsin (AAT) for use in combination with a steroid, or a pharmaceutical salt thereof for treating inflammatory disease or tissue injury in a subject in need thereof.

According to certain embodiments, the medicament is in the form of a formulation selected from the group consisting of injectable liquid solution, nasal drops, eye drops, nasal sprays, nasal inhalation solutions, aerosol powders, nasal insufflation powders, pulmonary inhalation solutions, pulmonary aerosols, and pulmonary insufflation powders, oral administration, or a combination thereof. According to other embodiments, the AAT is selected from the group consisting of plasma-derived AAT and recombinant AAT.

According to certain embodiments, the steroid is selected from the group consisting of dexamethasone, flurandrenolide, methylprednisolone, methylprednisone, triamcinolone acetonide, prednisone, hydrocortisone, mometasone, budesonide, beclomethasone, fluticasone, clocortolone, corticorelin, corticotropin, cortisone, deflazacort, desonide, desoximetasone, diflorasone, difluprednate, bethamethasone, fludrocortisone, flunisolide, fluocinolone, fluorometholone, halcinonide, halobetasole, loteprednole, medrysone, paramethasone, prednicarbate, rimexolone and any other steroidal compound .

According to certain embodiments, the medicament is administered via parenteral administration. According to certain embodiments, the AAT is administered intravenously. According to certain embodiments, the AAT is administered by inhalation. According to certain embodiments, the steroid is administered via oral, parenteral, topical (e.g., dermal application, ocular administration etc), injectable or inhaled administration. According to certain embodiments the medicament is administered via inhalation.

According to another aspect, the present invention provides a method for treating an inflammatory disease or tissue injury in a subject in need thereof, the method comprising administering a therapeutically effective amount of AAT in combination with a therapeutically effective amount of a steroid.

According to certain embodiments, the AAT and the steroid are administered sequentially. According to certain embodiments, the AAT and the steroid are administered simultaneously.

According to certain embodiments, the AAT is administered intravenously. According to certain embodiments, the steroid is administered via oral administration. According to certain embodiments, the AAT and the steroid are administered by inhalation.

According to certain embodiments, the AAT and the steroid are administered in a dosage of about 30 mg to about 250 mg AAT/kg and 0.05-50 mg/Kg of steroid per dose. According to certain embodiments, the AAT and the Dexamethasone are administered in a dosage of 60-120 mg/Kg AAT and 0.01-20 mg/Kg Dexamethasone per dose.

According to certain embodiments, each dose comprises from about 40 mg AAT/kgBW to about 240 mg AAT/kgBW. According to certain embodiments, each dose comprises 40, 60, 80, 120, 180, or 240 mg AAT/kgBW.

According to certain embodiments, the doses are administered at intervals of from 1-4 days to 2 weeks. According to some embodiments, the intervals are selected from constant intervals and variable intervals. According to some embodiments, the doses contain the same amount of AAT and the steroid. According to some embodiments, the doses contain variable amounts of AAT and the steroid. According to some embodiments, the doses are administered at intervals of one week. According to some embodiments, the amount of AAT decreases from the first dose administered to the last dose administered. According to some embodiments, the steroid dose administered to the subject is reduced or ceased during or after completion of the AAT treatment. According to some embodiments, the steroid dose administered to the subject is reduced or ceased before the AAT treatment.

According to some embodiments, the duration of the treatment is in the range from 1 day, 2 weeks, to 74 weeks. According to other embodiments, the duration of the treatment is in the range from 8 weeks to 44 weeks. According to yet additional embodiments, the duration of the treatment is in the range from 6 weeks to 16 weeks. According to certain exemplary embodiments, the duration of the treatment is selected from the group consisting of 2, 4, 6, 8, 16, 44, and 74 weeks. According to some embodiments, the administration is carried out for a plurality of months. According to certain embodiments, the present invention employs a long-term multiple-dose regimen. Each possibility represents a separate embodiment of the present invention.

According to some embodiments, the inflammatory disease or tissue injury is a pulmonary disease selected from the group consisting of alpha- 1 antitrypsin deficiency (AATD), small airway disease, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, asthma, pneumonia, parenchymatic and fibrotic lung diseases or disorders (including lung involvement such as vasculitis (Wegener disease, etc.) and/or collagen disease (RA, etc.), interstitial pulmonary fibrosis, re-inflammation, acute respiratory distress syndrome (ARDS), and sarcoidosis.

According to some embodiments, the inflammatory disease or tissue injury is selected from the group consisting of graft-versus-host disease (GVHD), ischemia- reperfusion injury, ischemia/reperfusion injury following cardiac transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Wegener’s disease, Crohn's disease, ulcerative colitis, psoriasis, type I and/or type P diabetes, dermatitis, pneumonia, sepsis, wound healing, systemic lupus erythematosus, and multiple sclerosis.

According to another aspect, the present invention provides a method for treating an exacerbation of inflammatory disease, which comprises administering to a subject in need thereof a therapeutically effective amount of Alpha- 1 antitrypsin (AAT) in combination with a therapeutically effective amount of a steroid.

According to certain embodiments, the AAT alleviates imbalanced proteinase/antiproteinase ratios that develop during the exacerbation period.

According to certain embodiments, the medicament is administered at the time of an exacerbation episode. According to certain embodiments, the medicament is administered at the time of remission. According to other embodiments, the medicament is administered at the time of an exacerbation episode and continued during remission.

According to certain embodiments, the subject is human.

Any route of administration as is known in the art to be suitable for AAT and steroid administration can be used according to the teachings of the present invention.

Other objects, features and advantages of the present invention will become clear from the following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGs. 1A-C present immunocyte responses to inflammatory stimuli in the presence of AAT or DEX. Cells that were preincubated overnight in round-bottom 96- well plates (in triplicates) with AAT (indicated concentrations) or DEX (10 pg/ml) were exposed to various stimuli. Twenty-four-hour supernatants obtained from: (A) LPS- stimulated RAW 264.7 cells (lxlO⁵ cells/well), (B) Resiquimod (R848)- stimulated RAW 264.7 cells (lxlO⁵ cells/well), and (C) LPS-stimulated THP-1 cells (48-hr-PMA- primed; 2xl0⁴ cells/well), * p < 0.05, ** p < 0.01.

FIGs. 2A-C show the requirement of NF-KB nuclear translocation for induction of IL-IRa by AAT. (A) p65-dependent kinetics ofIL-IRa production. Murine peritoneal macrophages (lxl0⁵/well) were treated with AAT (0.5 mg/ml) one hour prior to stimulation with LPS (10 ng/ml). Left, 24-hr supernatant IL-IRa; right, 6-hr IL-IRa transcripts. Mean ± SEM; * p < 0.05 and, **p < 0.01 per time point. (B) p65-GFP protein localization. Raw 264.7 cells transfected with a vector that expresses p65-GFP fusion protein (2xl0⁶ cells per 60-mm confocal dish in triplicates), were treated with AAT (0.5 mg/ml) one hour prior to stimulation with LPS (10 ng/ml). Plates were imaged 0.5 hours later by confocal microscope. CT, no stimulation added. Representative images out of 3 independent experimental repeats. (C) p65 localization. RAW 264.7 cells (3xl0⁶ cells/well in 6-well plates) were treated with AAT (0.5 mg/ml) one hour prior to stimulation with LPS (10 ng/ml). Western blot analysis of fractionated cells c, cytosolic fraction; n, nuclear fraction. Representative blots out of 6 independent experiments.

FIGs. 3A-B demonstrate DEX-related activities under AAT-enriched conditions. (A) ConA-elicited T cell clumping and expansion in vitro. Mouse splenic lymphocytes (2xl0⁵ cells/well in round-bottom 96- well plates in 13-plicate) were treated with ConA (1 mg/ml), in the presence of DEX (0.3 mg/ml) and AAT (0.5 mg/ml). Seventy-two- hour photomicrographs of representative wells; cell viability depicted by XTT uptake assay. (B) T cell-dependent DTH response in vivo. OXA-primed mice (n = 3 per group) were re-challenged with OXA and then treated daily with DEX (10 mg/kg) and AAT (60 mg/kg) for 3 days. Peripheral blood FACS analysis, gated to CD3⁺ cells. CT, mice with no re-exposure to OXA.

FIGs. 4A-B demonstrate combined treatment, AAT and DEX. (A) RAW 264.7 responses to LPS. RAW 264.7 cells (lxlO⁵ cells/well) were preincubated overnight in round-bottom 96-well plates (in triplicates) with AAT (indicated concentrations) and DEX (indicated concentrations), then exposed to LPS (5 ng/ml). Twenty-four-hour supernatants were harvested and assayed. Data from control cells in the absence of added LPS are not shown. Representative data out of 2 independent experiments. (B) Epithelial gap repair. Monolayers of human epithelial cells (A549) were scratched and incubated in the presence of AAT (0.5 mg/ml) and DEX (40 pg/ml). Representative images 0 and 24 hours from scratch. Dashed lines, gap borders. Bottom, gap area at 24 hours (percent of value at time 0). CT, no added treatment. Data are representative of 3 independent experiments. Mean ± SEM; ns, non-significant, * p < 0.05.

FIGs. 5A-C demonstrate the infiltration and activation of peritoneal ThG-elicited macrophages. C57BL/6 mice were treated with a single dose of DEX and AAT at the indicated doses, or with a combination of DEX and AAT. One hour later, mice were stimulated with ThG (3%). Peritoneal lavage was collected 48 hours after stimulation and cells were examined by flow cytometry. Total number of ThG-elicited macrophages (A), Total number of CD40High Macrophages (B), and total number of CD80High Macrophages (C).

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses specific combinations of Alpha- 1 antitrypsin (AAT) with steroids for treating an inflammatory disease or tissue injury.

Definitions

As used herein, the term “Alpha- 1 Antitrypsin” (AAT) refers to a glycoprotein that in nature is produced by the liver and lung or intestinal epithelial cells and secreted into the circulatory system. AAT belongs to the Serine Proteinase Inhibitor (Serpin) family of proteolytic inhibitors. This glycoprotein consists of a single polypeptide chain containing one cysteine residue and 12-13% of the total molecular weight of carbohydrates. AAT has three N-glycosylation sites at asparagine residues 46, 83 and 247, which are occupied by mixtures of complex bi- and triantennary glycans. This gives rise to multiple AAT isoforms, having isoelectric points in the range of 4.0 to 5.0. The glycan monosaccharides include N-acetylglucosamine, mannose, galactose, fucose, and sialic acid. AAT serves as a pseudo-substrate for elastase; elastase attacks the reactive center loop of the AAT molecule by cleaving the bond between the methionine358 - serine359 residues to form an AAT-elastase complex. This complex is rapidly removed from the blood circulation. AAT is also referred to as “alpha- 1 Proteinase Inhibitor” (API). The term “glycoprotein” as used herein refers to a protein or peptide covalently linked to a carbohydrate. The carbohydrate may be monomeric or composed of oligosaccharides. It is to be explicitly understood that any AAT as is or will be known in the art, including plasma-derived AAT, and recombinant AAT, derivatives or analogs thereof can be used according to the teachings of the present invention.

As used herein, the term “Glucocorticoids” refers to small molecule steroids that bind to glucocorticoid receptors and are utilized in anti-inflammatory and immunosuppressive therapies. However, due to the ubiquitous expression of glucocorticoid receptors in many cell types, glucocorticoid treatments are compromised by toxicities to most organ systems.

Examples of steroids of the present invention are disclosed in Table 1:

The term "combination therapy", as used herein, refers to the administration of two or more therapeutic substances, e.g., AAT and steroids. The steroids may be administered concomitant with, prior to, or following the administration of AAT. The use of AAT in combination with steroids is intended to embrace administration of each agent in a distinct manner in a regimen that will provide beneficial effects of the drug combination.

The term "simultaneous administration" as used herein in relation to the administration of medicaments refers to the administration of medicaments such that the individual medicaments are present within a subject at the same time. In addition to the concomitant administration of medicaments (via the same or alternative routes), simultaneous administration may include the administration of the medicaments (via the same or an alternative route) at different times.

As used herein, "remission, cure, or resolution rate" refers to the percentage of patients who are cured or obtain remission or complete resolution of a condition in response to a given treatment.

As used herein, "response rate" refers to the percentage of patients who respond positively (e.g., reduced severity or frequency of one or more symptoms) to a given treatment.

The terms "treat" and "treating" include alleviating, ameliorating, halting, restraining, slowing or reversing the progression, or reducing the severity of the pathological conditions described above.

The term "dosage" as used herein refers to the amount, frequency, and duration of AAT and/or steroid given to a subject during a therapeutic period.

The term "dose" as used herein, refers to the amount of AAT and/or steroid given to a subject in a single administration.

The terms "multiple-variable dosage" and “multiple dosage” are used herein interchangeably and include different doses of AAT and/or steroid administration to a subject and/or variable frequency of administration of the AAT and/or steroid for therapeutic treatment. "Multiple dose regimen" or "multiple-variable dose regimen" describe a therapy schedule, which is based on administering different amounts of AAT and/or steroid at various time points throughout the course of therapy.

The term "treatment phase" or "maintenance phase", as used herein, refers to a period of therapy comprising administration of AAT and/or steroid to a subject in order to maintain a desired therapeutic effect.

The terms “treatment dose” or "treatment dose portion" as used herein refer to a dose of AAT and/or steroid administered to a subject to maintain or continue a desired therapeutic effect during the treatment phase.

As used herein, the terms "exacerbation" "exacerbation period" and "exacerbation episode" are used interchangeably to describe an increase in the severity of symptoms during the course of a disease, which is mostly associated with a worsening of quality of life. By definition, exacerbations are worsening and/or increase in severity and/or magnitude of the disease symptoms.

As used herein the term "about" refers to the designated value ± 10%.

The term "simultaneous administration," as used herein, means that the AAT and the steroids are administered with a time separation of no more than about 15 minute(s), such as no more than about any of 10, 5, or 1 minutes.

The term "sequential administration" as used herein means that the AAT and the steroids are administered with a time separation of more than about 15 minutes, such as more than about any of 20, 30, 40, 50, 60, or more minutes. Either the AAT or the steroid treatment may be administered first.

The term "dry powder" refers to a powder composition that contains finely dispersed dry particles that are capable of being dispersed.

The particles of the dry powder composition have a particle size distribution that enables the particles to target the mucosal region of the intestine when delivered via tablets. The particle-size distribution (PSD) of a powder is a list of values or a mathematical function that defines the relative amount of particles present according to size. The powders of the invention are generally polydispersed (i.e., consist of a range of particle sizes). In particular embodiments, the term “particle size distribution” refers to the size distribution of particle system and represents the number of solid particles that fall into each of the various size ranges, given as a percentage of the total solids of all sizes in the sample of interest.

As used herein, the term "particle size distribution D90 value" is defined as the numerical value, expressed in microns, at which 90 percent of the particles have particle sizes which are less than or equal to that value. As used herein, the term "particle size distribution D50 value" is defined as the numerical value, expressed in microns, at which 50% of the particles have particle sizes that are less than or equal to a given value.

According to some embodiments, the average particle size is below 10 pm. In other embodiments, the average particle size is below 9, 8, or 7 pm. Each possibility represents a separate embodiment of the invention. In additional embodiments, the average particle size is between 1 tolO pm, 2 to 9 pm, 3 to 8 pm, 4 to 8 pm, or 4-8 pm. Each possibility represents a separate embodiment of the invention. The average particle size of the powder may be measured as mass mean diameter (MMD) by conventional techniques.

The term "dry" means that the particles of the powder have moisture content such that the powder is physically and chemically stable when stored at room temperature. According to some embodiments, the moisture content of the particles is below 10%, 8%, 6%, 4%, 2%, or 1% by weight. Each possibility represents a separate embodiment of the invention.

According to certain embodiments, the dry powder composition consists of AAT and at least one excipient selected from the group consisting of Trehalose, Glycine, Dipalmitoylphosphatidylcholine (DPPC), and Ectoin, wherein the AAT is at least 60% (w/w) of the composition and wherein at least 90% of the AAT is in a monomeric form.

According to certain embodiments, AAT and/or steroid are administered in the form of a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to a preparation of AAT and/or steroid with other chemical components such as pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of an active ingredient to an organism and enhance its stability and turnover.

Any available AAT as is known in the art, including plasma-derived AAT, and recombinant AAT, derivatives or analogs thereof, can be used according to the teachings of the present invention. According to certain exemplary embodiments, the AAT is produced by the method described in U.S. Patent No. 7,879,800 to the Applicant of the present invention.

The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopoeia or another generally recognized pharmacopoeia for use in animals, and more particularly in humans.

The term "carrier" refers to a diluent or vehicle that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included in this category. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, isotonic buffers and physiological pH and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

The pharmaceutical compositions of the invention can further comprise an excipient. Herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, trehalose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, lipids, phospholipids, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates, or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.

The pharmaceutical compositions of the present invention can be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, spray drying, or lyophilizing processes.

According to certain exemplary embodiments, pharmaceutical compositions, which contain AAT and/or steroid as an active ingredient, are prepared as injectable, either as liquid solutions or suspensions, however, solid forms, which can be suspended or solubilized prior to injection, can also be prepared. According to yet additional embodiments, the AAT and/or steroid -containing pharmaceutical composition is formulated in a form suitable for subcutaneous administration. From the patient point of view, multiple injections are not a favorable treatment, and thus this mode of treatment may be replaced by slow and/or controlled release subcutaneous administration. Any other forms of slow and/or controlled release are also explicitly encompassed within the scope of the present invention.

The compositions can also take the form of emulsions, tablets, capsules, gels, syrups, slurries, powders, creams, depots, sustained-release formulations, and the like.

Methods of introduction of a pharmaceutical composition comprising AAT and/or steroid include, but are not limited to, intravenous, subcutaneous, intramuscular, intraperitoneal, oral, topical, intradermal, transdermal, intranasal, epidural, ophthalmic, vaginal, and rectal routes. The pharmaceutical compositions can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial linings (e.g., oral mucosa, rectal, and intestinal mucosa, etc.), and may be administered together with other therapeutically active agents. The administration may be locahzed, or may be systemic.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, typically in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be transversed are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active ingredients with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries as desired, to obtain tablets or dragee cores. Suitable excipients may be fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, and sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in a conventional manner.

Capsules and cartridges of, for example, gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with, optionally, an added preservative. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions. Suitable hpophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredients, to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use.

The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin.

According to certain exemplary embodiments, the AAT and/or steroid- containing pharmaceutical composition used according to the teachings of the present invention is a ready-to-use solution. According to further exemplary embodiments the AAT-containing pharmaceutical composition is marketed under the trade name Glassia^®.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES Materials & Methods

Cell systems

Three sources of immune cells were employed: murine macrophage cell line RAW 264.7, human monocyte cell line ΊΉR-1, and human peripheral-blood mononuclear cells (PBMC) derived from samples of whole blood obtained from healthy volunteers. Primary human cell collection was approved by the Magen David Adorn (Red Cross) in Israel. A549 Adeno Carcinoma alveolar epithelial cells were used for the gap repair assay.

Stimuli and inhibitors

Clinical-grade plasma-derived hAAT, liquid formulation (Glassia^®, Kamada Ltd., Israel). Dexamethasone (DEX; Cat# 4902; Sigma-Aldrich, Israel). Lipopolysaccharide (LPS; Cat# L2880, Sigma-Aldrich). Resiquimod (R848, Cat# tlrl-r848, InvivoGen Europe, Toulouse France).

RAW 264.7 cell stimulation assay

RAW 264.7 cells (10⁵/well) were seeded in round-bottom 96-well plates and cultured in RPMI 1640 medium supplemented with 5% FCS (Biological Industries, Kibbutz Beit Haemek, Israel). Cells were incubated overnight with indicated concentrations of hAAT or DEX (10 pg/ml) and then stimulated with LPS (5 ng/ml, Sigma-Aldrich). Supernatants were collected after 24 hours. Levels of mouse IL-lRa were determined by specific ELISA (Mouse IL-lra/IL-lF3 DuoSet, Cat# DY480, R&D systems, Minneapolis, USA), according to the manufacturer’s instructions. Cytokines analyzed included: KC, G-CSF, GM-CSF, lL-10, IL-12p70, CXCL-10, IL-Ib, IL-6, IL- 17A, IFNy, and TNFa; concentrations were determined by magnetic Luminex assay (LXSAMSM-11, R&D Systems). All measurements were performed in triplicate.

THP-1 cell stimulation assay

THP-1 cells (2xl0⁴/well) were cultured in RPMI 1640 medium supplemented with 10% FCS and incubated for 48 hours in the presence of PMA (80 nM, Cat# SC-3576A; Santa Cruz), as described elsewhere. Cells were then washed and incubated with indicated concentrations of hAAT or DEX (10 pg/ml) for 4 hours, followed by stimulation with LPS (100 ng/ml). Supernatants were collected 24 hours later. The levels of G-CSF, GM-CSF, lL-10, IL-12p70, IL-Ib, IL-6, IL-8 and TNFa were determined by magnetic multiplex (MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay, Cat # HCYTOMAG-60K-08), according to manufacturer’s instruction. All samples were tested in triplicate.

PBMC stimulation assay PBMCs were isolated from whole blood using SepMate™-50 tubes (Cat# 15450, STC Inc.). PBMC (2.5xl0⁵/well) and were treated with indicated concentrations of hAAT or DEX (10 pg/ml) for 2 hours, before stimulation with LPS (10 ng/ml). Supernatants were collected 24 hours later. TNFa levels were determined by specific ELISA (Cat# ELH-TNFA-1, RayBiotech, GA, USA), according to the manufacturer’s instruction. All samples were tested in triplicate. qRT-PCR

Total RNA was extracted from cultured cells using Tri-Reagent (Bio-Tri Reagent, Bio-Lab, Jerusalem, Israel) according to the manufacturer’s instructions. RNA was quantified using a NanoDrop device (Wilmington, DL, USA). Concentration- normalized RNA samples were reverse-transcribed using qScript microRNA cDNA Synthesis Kit (QuantaBio, Beverly, MA, USA). Transcript levels were analyzed using a qPCR system (StepOnePlus™ Real-Time PCR System, Thermo Fisher Scientific, MA, USA). qPCR reactions (performed in triplicates for each target gene) were set at a final volume of 10 mΐ containing: 5 mΐ Fast SYBR™ Green Master Mix (Thermo Fisher Scientific), 12.5 ng cDNA, 0.5 mM forward and reverse primers, UPW (Biological Industries). Relative quantification of transcript levels was performed using the delta- delta Ct method, 18s was used as reference gene. Variation of Ct in reference gene across all samples of a given assay was < 1 cycle. The efficiency of all primer pairs was between 95%-l 10% using a 5-point standard curve. p65-GFP fusion protein, transfection and cellular distribution study

The p65-GFP fusion protein vector was a kind gift from Gopas J. The Cignal NF- KB GPP generic response element reporter kit was obtained from Qiagen (Valencia, CA, USA). Transfection reagents Lipofectamine® 3000, 2000 (Life Technologies, Carlsbad, CA, USA) and TransIT-LTl (Mirus Bio, Madison, WI, USA) were used according to the manufacturer's instructions while applying the forward transfection approach. Briefly, cells were allowed to reach 70-90% confluence, and were then incubated with lipid-DNA complexes in serum-free media for 6 hours before the culture media was switched back to standard growth media. Efficacy was evaluated 24-48 hours after transfection using a fluorescence microscope. For cellular distribution studies, RAW 264.7 cells were cultured (2xl0⁶ per plate) in confocal dishes (35x10 mm, Holed-Coverglass, SPL Lifesciences, Korea), then transiently transfected with p65-GFP fusion protein vector. Cultures were treated with hAAT (0.5 mg/ml) one hour before stimulation with LPS (10 ng/ml). After 30 minutes, cells were fixed and permeabilized using 4% PFA (SantaCruz) and Triton X-100 (SantaCruz). Cells were visualized by confocal microscope (Olympus FV1000).

Western Blot analysis

Cytosolic and nuclear fractions were generated using NE-PER™ extraction reagents (ThermoFisher) according to the manufacturer’s instructions. Protein concentrations were determined using BCA protein assay (SantaCruz). Aliquots of 20 pg protein were separated on 7.5-18% SDS-PAGE and blotted onto a PVDF or nitrocellulose membranes (Bio-Rad, CA, USA). Transferred membranes were incubated in 5% BSA Tris-Buffered Saline/Tween (TBS/T) solution for 45 minutes, then submerged in primary antibody solution overnight. Antibodies included anti-p65 (poly6226, Biolegend, San Diego, CA, USA), anti-phosphorylated-p65 (49.Ser 311, SantaCruz), anti-P-actin (Poly6221, Biolegend) and anti-LaminA (Millipore, Billerica, MA, USA). For detection of primary antibodies, blots were incubated with horseradish- peroxidase-conjugated anti-rabbit or anti-mouse antibodies. Immobilized antibodies were detected by ECL (Cyanagen, Bologna, Italy). For Western Blot membrane re probing, membranes were incubated with stripping solution (5% (v/v) acetic acid) for 45 minutes at room temperature and tested for residual signal.

Lymphocyte stimulation assay

Mouse splenocytes were isolated using routine procedures. Briefly, aseptically removed C57BL/6 spleens were pressed through a 70- pm nylon mesh in RPMI 1640 medium supplemented with L-glutamine and sodium bicarbonate (Sigma-Aldrich). The cell suspension was placed on density gradient Histopaque-1077 (Sigma-Aldrich) and then centrifuged at 400 g for 30 min. Interface cells were collected and washed three times with the RPMI 1640 medium and centrifuged at 250 g for 10 min. Viability of cells was evaluated by trypan blue exclusion (Sigma-Aldrich) and was confirmed as > 95%. Cells were suspended in RPMI 1640 medium containing 10% FCS, 50 units/ml penicillin and 50 pg/ml streptomycin, and dispensed into round-bottom 96-well plates (lxlO⁶ per well). Cells were stimulated with concavalin A (ConA, 1 pg/ml; Sigma- Aldrich) for 72 hours then imaged through light microscope. XTT assay (Sigma- Aldrich) was performed as instructed by the manufacturer.

Delayed-type hypersensitivity (DTH) mouse model

Mice (C57BL/6) were sensitized by topical application of 2% oxazolone (OXA, 4- ethoxymethylene-2-phenyl-2-oxazoline-5-l, Sigma-Aldrich) in acetone/olive oil (4:1 vol/vol) on shaved abdomen (50 mΐ). Five days later, mice were challenged with topical application of 10 mΐ of 1% OXA on each side of the right ear, and treated with DEX or PBS, with or without hAAT, for 3 days. Peripheral blood was collected for flow cytometric analysis of CD3⁺ lymphocytes and the intracellular proliferation marker, Ki- 67. For staining, samples underwent red blood cell (RBC) lysis using RBC lysis buffer (Sigma-Aldrich), and were then washed with FACS buffer (PBS containing 1% BSA, 0.1% sodium azide and 2 mM EDTA, pH 7.4), and incubated with Fc_yRII/IH blocker (Biolegend). Intracellular staining for Ki-67 was performed using the eBioscience staining kit (according to the manufacturer’s instructions) and APC-anti-Ki-67 antibody (Biolegend). CD3 was stained using FITC-anti-CD3 antibody (BD Biosciences, NJ, USA). Analysis was performed using BD Canto P. Data were analyzed by How Jo (Version 10.1r5, FLOWJO LLC, Ashland, OR, USA).

A549 epithelial gap repair assay

A549 cells (0.2x10⁶/well in 24- well plates in triplicate) were allowed to reach full confluence while cultured in RPMI 1640 culture medium (Biological Industries) and 5% FCS. A gap was then mechanically created by scratching the cell layer with a 200 mΐ non-conical plastic pipette tip. Monolayers were washed 3 times with HANK’s balanced salt solution (HBSS, Biological Industries) at 37°C and the medium was exchanged for either 0% FCS media ( negative control), 10% FCS media {positive control ) or 5% FCS media in the presence of either plasma-purified hAAT (0.5 mg/ml) or DEX (40 pg/ml, referred to as ‘time O’). Images of the gap area were captured at 0 and 24 h after scratching using Nikon bright field microscope. Image analysis was performed with ImageJ software (NIH, USA). The extent of gap repair was expressed as the percentage of gap area from time 0. Example 1 Stimulated immunocytes: Anti-inflammatory responses in the presence of hAAT or DEX

Representative pro-inflammatory and anti-inflammatory cytokines, as produced by three immunocyte sources: mouse cell line RAW 264.7 (Fig. 1A and IB), and human cell line THP-1 (Fig. 1C), stimulated by LPS (Fig. 1A, 1C) or R848 (Fig. IB). Cells were pre-treated with hAAT at indicated concentrations, or with dexamethasone (DEX, 10 pg/ml). As shown, RAW 264.7 cells responded to LPS stimulation by releasing several-fold greater levels of TNFa and IL-6 (Fig. 1A). Pre-treatment with DEX, significantly reduced the stimulated levels of TNFa (3,494 pg/ml compared to 633 pg/ml, mean), and IL-6 1 (120.0 to 13.2 pg/ml). However, when cells were pre-treated with hAAT, the inhibition of stimulated TNFa and IL-6 was less extreme (1,954 pg/ml TNF and 31.4 pg/ml IL-6) than with DEX. In both examples, the decline in cytokine levels was evident already at 0.25 mg/ml hAAT and was relativelyconstant across 1 mg/ml, with the largest inhibition in the presence of 4 mg/ml hAAT.

Consistent with these findings, DEX treatment resulted in a 98% decline in the levels of the Thl-related chemokine CXCL10 (from 15,848 to 384 pg/ml), while exposure to hAAT caused a concentration-dependent decrease in CXCL10, that reached a maximum effect of approximately 60% inhibition of stimulated levels (6,050 pg/ml). Interestingly, the rise in inducible anti-inflammatory products was unique to hAAT: stimulated IL-10 levels rose to as much as threefold (from 30.9 pg/ml with LPS to 85.9 pg/ml with LPS/hAAT); a bell-shaped response pattern emerged, in which the optimal concentration of hAAT for upregulating LPS-stimulated IL-10 release was 1-4 mg/ml. Concomitantly, hAAT treatment increased stimulated IL-lRa levels by more than threefold (from 53,102 to 185,241 pg/ml). Importantly, in the case of IL-lRa, DEX abolished the effect of LPS stimulation altogether, resulting in flat background levels. Production of LPS-stimulated G-CSF crudely represents an aspect of the effects of hAAT on circulating immune cells; with an inverse bell-shaped response where higher G-CSF concentrations were observed in cell cultures treated with 0.25 and 4 mg/ml hAAT. Concentrations of hAAT in the range of 0.5-2 mg/ml inhibited the LPS stimulation of G-CSF by 90% (from 39,995 to 3,611 pg/ml). Parallel treatment with dexamethasone decreased the G-CSF concentration to below detection levels. A trend similar to LPS stimulation was observed upon exposing RAW 264.7 cells to the Thl -polarizing TLR7 agonist, resiquimod (R848) (Fig. IB). As expected, cells responded with an abrupt increase in the levels of stimulated TNFa, IL-6, and IL-10 (3.8-fold, 6.8-fold, and 2.8-fold respectively compared to control conditions,). With the addition of DEX, TNFa declined half-way to control levels, and IL-6 to below control levels. DEX also reduced the levels of induced IL-10, albeit without reaching statistical significance. The decline in these two inflammatory cytokines was comparable to the effect of hAAT on R848-stimulated cells: The levels of stimulated TNFa were reduced only at 0.5 mg/ml hAAT and higher, reaching to as low as twice control levels at 4 mg/ml hAAT. There was a marked inhibition of stimulated IL-6 at all tested hAAT concentrations, reaching with levels of inhibition comparable to those seen with DEX- treated R848-stimulated cells. In contrast, R848-elicited IL-10 levels were significantly increased in hAAT-treated cells, from a concentration of 0.25 mg/ml hAAT and higher. The maximum response was observed at 2 mg/ml hAAT (2.3-fold greater than stimulated levels, or 6.5-fold greater than resting conditions). Treatment of cells with DEX did not elevate IL-10 levels.

Outcomes obtained with human monocyte ΊΉR-1 cells were consistent with those obtained with RAW 264.7 cells with respect to the striking difference between DEX and hAAT treatments (Fig.lC). hAAT concentrations below 3 mg/ml did not affect stimulated cytokine levels (not shown), and the impact of 3 mg/ml hAAT appeared to be overall ineffective in blunting inflammatory responses in this cell line. However, a concentration of 5 mg/ml hAAT significantly reduced all the tested inflammatory markers except IL-Ib. DEX treatment reduced the levels of stimulation of TFNa, IL-6, IL-12, G-CSF, IL-Ib, and GM-CSF to near or below control conditions. Interestingly, hAAT (3 and 5 mg/ml) increased LPS-stimulated IL-10 levels (2.27-fold increase from LPS-stimulated levels and 7.4-fold from resting conditions, at 5 mg/ml). This phenomenon was absent with DEX treatment, which significantly decreased LPS- stimulated IL-10 levels in human THP-1 cells.

Example 2 hAAT-mediated outcomes require intact NF-KB subunit p65 activity

Induction of IL-IRa requires inflammation and, more specifically, a functional nuclear DNA-binding p65 subunit. This raises an apparent conflict with the anti- inflammatory attributes of hAAT, in that hAAT decreases inflammation, but increases IL-lRa expression. We used JSH23, an inhibitor of p65 nuclear translocation and DNA binding to examine the impact of hAAT (0.5 mg/ml) on LPS-induced IL-lRa levels (Fig. 2A). In accordance with previous observations, while LPS did induce IL-lRa production (left, dashed black line; 6.28-fold increase from control conditions at 24 hours), LPS plus hAAT produced an even greater increase in IL-lRa (left, solid black line; 8.40-fold from control conditions). As expected, the addition of JSH23 (grey), reduced the stimulations induced by both LPS and LPS+hAAT. With respect to the levels of IL-lRa transcripts (Fig. 2A, right), a predictable 336 ± 13-fold increase in relative IL-lRa transcript levels was observed 6 hours after LPS stimulation (dashed black line). Interestingly, in the presence of hAAT, the IL-lRa transcript levels reached a maximum of 270 ± 29-fold above control expression levels earlier, as soon as 1 hour after LPS stimulation, after which they declined steadily. As with the protein levels, treatment with JSH23 abrogated the LPS induction of IL-lRa transcript, whether in the presence or absence of hAAT.

The finding that an intact NF-kB pathway is required for hAAT anti-inflammatory activity and that this occurs earlier than with LPS alone, motivated us to directly analyze p65 cellular distribution under hAAT-enriched conditions (Fig. 2B and 2C). As shown in a cell-by-cell analysis, a p65 (NF-KB3)-GFP fusion construct was transiently transfected into RAW 264.7 cells, to permit the detection of fluorescently-labeled p65. Cells stimulated for 30 minutes with LPS (10 ng/ml), exhibited a typical conversion from a diffuse cytoplasmic pattern of p65 to a more condensed distribution with extensive nuclear localization. In the presence of hAAT, LPS-stimulated cells exhibited mixed responses: some cells displayed partial nuclear translocation, some cells appeared to better resemble resting unstimulated cells. Unexpectedly, cells incubated in the presence of hAAT alone, without added LPS, displayed an apparent aggregation of p65 outside the nucleus. Our observations point towards an altered dynamic of NF-KB signaling in hAAT-treated cell cultures. In parallel experiments (Fig. 2C), RAW 264.7 cells were pre-treated with hAAT (0.5 mg/ml) for 1 hour prior to stimulation with LPS (10 ng/ml) and the levels of phosphor-p65 were determined by Western Blot analysis in fractionated samples: cytosolic (c) and nuclear ( n ). As expected, unstimulated cells exhibited only low level phosphorylation, and the bulk of p65 was present in the cytosolic fraction. However after 30 minutes stimulation with LPS, p65 phosphorylation was maximal and present primarily in the nuclear fraction. Unexpectedly, cell lysates from cultures stimulated with LPS in the presence of hAAT displayed a mixed content of total and phosphorylated p65 in both cytosolic and nuclear fractions and the nuclear fraction of cells incubated in the presence of hAAT alone, without added LPS, also contained phosphorylated p65.

Example 3: AAT does not interfere with the intended immunosuppressive activities of DEX

Before considering the possibility of combining DEX and hAAT, we wanted to verify that hAAT does not interfere with the intended functions of DEX, including inducible T cell clumping and proliferation in vitro and in vivo (Fig. 3). As shown in Figure 3A, splenic mouse lymphocytes were stimulated with concavalin A (ConA) in the presence of inhibitory concentrations of DEX, with or without added hAAT (0.5 mg/ml). Culture wells were imaged 72 h later. As expected, ConA activated T cell clumping and proliferation, which were reduced by the addition of DEX. Exposure to hAAT, did not affect the DEX-mediated inhibition of the ConA-induced T cell responses. In accordance with previous findings, hAAT alone did not interfere with ConA-mediated T cell activation (not shown). The number of T cells was evaluated by mitochondrial XTT assay (not shown), according to which ConA induced T cell proliferation, and DEX reduced the stimulated T cell population size 6.41 ± 0.471-fold compared to ConA while a combination of DEX and hAAT had no additional effect. Vehicle alone (EtOH, not shown) did not interfere with T cell responses.

In the whole animal (Fig. 3B), DEX activity was assessed by the classic delayed- type hypersensitivity (DTH) model, typically used for identifying T cell responses in an authentic immune reaction. Mice were challenged with direct application of Oxazolone (OXA) on shaved abdominal skin, and then treated with DEX or PBS, with or without hAAT, for 3 days. Mice were then challenged again with OXA, this time on the surface of their right ear, and peripheral blood was collected for flow cytometric analysis of CD3⁺ T cell proliferation using the proliferation marker, Ki67. Mice exposed only to abdominal skin OXA served as controls (CT); mice exposed in addition to ear skin OXA exhibited an increase in T cell proliferation (3.067 ± 0.176-fold from CT). Treatment of OXA-challenged mice with DEX inhibited T cell proliferation and this was not affected by exposure to hAAT.

Example 4: Concomitant treatment with hAAT and DEX

Stimulation of RAW 264.7 cells with LPS after pre-treatment with various concentrations of both hAAT and DEX. Fig. 4A revealed that in the absence of hAAT (frontmost bars), DEX essentially completely inhibited the LPS stimulation of IL-6 from the lowest concentration tested. Similarly DEX also inhibited the LPS stimulation of IL-lRa. In this case, the inhibition could be seen to be concentration-dependent although there was almost complete inhibition at concentrations that are considered to represent a therapeutic trough (DEX 1-10 pg). In contrast, in the absence of DEX ( leftmost bars), hAAT exhibited a concentration-dependent inhibition of LPS stimulated IL-6, but a concentration-dependent increase in the levels of IL-lRa. Mid-range hAAT concentrations would represent steady-state plasma levels in otherwise healthy individuals, while the lower range reflects the levels seen in individuals with genetic hAAT deficiency, and the upper range is similar to the physiological levels seen in an uninterrupted acute phase response, advanced healthy pregnancy, or late age.

When combined with DEX, hAAT appears to lose the ability to stimulate IL-lRa release {each row, advancing from front to back). Even at a concentration of 2 mg/ml hAAT, DEX reduced the IL-lRa output in a concentration-dependent manner. At 5 pg/ml DEX, IL-lRa production was abolished at all the concentrations of hAAT tested.

Repair of an epithelial monolayer gap requires an intact inflammatory response and is impaired in the presence of corticosteroids. For this reason, we were interested in comparing gap repair in the presence of hAAT and dexamethasone. A549 monolayers were subjected to scratch injury under fasting conditions (0% FCS, negative controls), fully-nourished conditions (10% FCS, positive control) or intermediate settings (5% FCS, CT), the latter in the presence or absence of hAAT (0.5 mg/ml) or dexamethasone (40 pg/ml). As shown in Figure 4B, 24 hours after monolayer disruption, dexamethasone-treated monolayers displayed a larger gap remaining post-monolayer disruption, than seen in fully-nourished controls (75.5 ± 7.9% open area compared to 52.5 ± 1.9% in fully-nourished conditions). In contrast, hAAT-treated monolayers displayed a significantly smaller remainder gap area after the same time (35.7 ± 2.8%), indicating a superior repair capacity to that displayed by dexamethasone alone, indeed, an expedited repair compared to a disrupted healing, despite the fact that both agents possess anti-inflammatory properties.

Example 5: The effect of a combination treatment with A AT and Dexamethasone on activation of macrophages in a peritonitis model in mice

Peritonitis is the inflammation of abdominal membranes. Macrophages are known to play a significant role in the initiation and amplification of the inflammatory process during peritonitis. Corticosteroids such as DEX have been shown to reduce the total number of macrophages in the peritoneal cavity of mice, thus diminishing the inflammatory process. However, despite the positive anti-inflammatory effect, treatment with corticosteroids is associated with a wide range of side effects, affecting nearly every system of the body.

In the current study, peritonitis is induced by intraperitoneal injection of thioglycolate, which has long been used to induce peritonitis and recruit macrophages to the peritoneal cavity. The dose of DEX used for the study of peritonitis in mice varies from 0.1 mg/kg to 10 mg/kg. For the current study a dose of 5 mg/kg was used.

The number of peritoneal macrophages in this study was assessed by FACS analysis of the CD45⁺F480⁺GrT cell population. Activated macrophages exhibit increased levels of CD40, CD80 and MHC P.

Aim

To test the effect of a combination treatment of AAT and dexamethasone on the activation of peritoneal macrophages in a model of peritonitis.

Design

Eight-to-10 weeks old C57BL/6 mice, 4 animals per group, were used for the experiments. The animals were treated either with single-dose DEX (5 to 0.5 mg/kg i.p; West-ward pharmaceuticals, NDC 0641-0367-21, lot 028363), single-dose pdAAT (60 and 120 mg/kg i.p; Kamada, batch AJ7190616) or a combination of DEX and pdAAT. The control group was injected with same volume of NaCl 0.9% which is the vehicle of the AAT. Each drug was injected with a separate syringe. One hour after treatment, mice were stimulated with 2mL Thioglycolate 10% solution i.p (Fluka analytical, batch 071M0106). Peritoneal lavage was collected 48 hours after stimulation by i.p. injection of 10 mL of cold PBS and collection of the fluid using a 23-gauge needle. AAT doses were 60 mg/kg and 120 mg/kg doses (alone and in combination with DEX). Evaluation of several doses of DEX and its effect within the combination (5 mg/kg, 1 mg/kg and 0.5 mg/kg) were tested. Treatment groups and the number of mice in each group for the current experiment are shown in Table 2.

Table 2: Treatment groups

Analysis and results

Peritoneal macrophages can be identified as the CD45⁺F480⁺Grl cell population. Once the macrophages are activated, they express high levels of MHC II (a molecule that is required for antigen presentation) and high levels of CD40 and CD80 (co-stimulatory molecules that contribute to cell activation). Thus, the peritoneal lavage can be analyzed for activation profile of peritoneal macrophages by staining the cells with CD40, CD80 and MHC P. A BD FACS Canto P system was used for flow cytometry analysis. Following peritoneal lavage, the cells were washed with FACS buffer (PBS, 1% bovine serum albumin, 0.1% sodium azide, and 2 mM ethylenediaminetetraacetic acid at pH 7.4). Then, lxlO⁶ cells per sample, from the peritoneal cavity were incubated with FcyR blocker (BioLegend) and stained with the following antibodies: anti-CD45-PE, anti-F480-Pacific Blue, anti-Grl-APC, anti-MHC P-FITC, anti-CD80-PE/Cy7, and anti-CD40-APC Cy7. All antibodies were from BioLegend and were diluted according to the manufacturer’s protocol. Data was analyzed by How Jo software.

As shown in Figures 5A-C, similar anti-inflammatoiy effect (less infiltration and activation of macrophages) was obtained with one tenth of the DEX amount in combination with AAT. These results may support reduction of side effects of DEX with the same anti-inflammatory outcome.

Example 6; Comparison of the therapeutic efficacy of DEX at clinical dose vs. low dose of DEX and AAT

Study outline

Allogeneic model

Either minor mismatch: C3H.SW (H-2^b) donors into C57BL/6 (H-2^b) recipients or major mismatch: C57BL/6 (H-2^b) donors into Balb/C (H-2^d) recipients

Total body irradiation at day -1 followed by BM + splenic T-cells transplantation at day 0.

Animals are treated starting on day 5 (when clinical signs usually start to appear in mice) every other day for two weeks post transplantation (days 5-17 PT).

Drug dose & route of administration The study includes six groups according to Table 3.

Table 3: Study groups, treatments and doses

As shown in Table 4, for GVHD induction, all animals will undergo Radiation at day -1 followed by BM & splenic T-cells transplantation at day 0:

Allogeneic transplantation for groups 1-5 and syngeneic transplantation for group 6, as a positive control (no disease).

Table 4: Time line of GVHD induction

Treatment of groups 1-4 are performed as indicated in Table 5. Starting from day 5 post transplantation, groups 1-4 will receive treatment with AAT and /or Dex as indicated in Table 3.

Table 5: AAT and Dex treatments schedule

Animals are observed every other day for body weight and clinical score. The clinical score is according to weight loss, activity, posture, fur-texture and skin integrity. Maximal GVHD clinical score is 8.

On day 45 all animals will be sacrificed and samples of large and small intestine will be collected and fixed by formaldehyde. H&E Histology is performed and IHC is used to determine immune cells infiltration and cytokine levels.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A medicament comprising Alpha- 1 antitrypsin (AAT) for use in combination with a steroid, or a pharmaceutical salt thereof for treating inflammatory disease or tissue injury in a subject in need thereof.

2. The medicament of claim 1, wherein the medicament is in the form of a formulation selected from the group consisting of injectable liquid solution, nasal drops, eye drops, nasal sprays, nasal inhalation solutions, aerosol powders, nasal insufflation powders, pulmonary inhalation solutions, pulmonary aerosols and pulmonary insufflation powders, oral administration, or a combination thereof.

3. The medicament of claim 1, wherein the AAT is selected from the group consisting of plasma-derived AAT and recombinant AAT.

4. The medicament of claim 1, wherein the steroid is selected from the group consisting of dexamethasone flurandrenolide, methylprednisolone, methylprednisone, budesonide, beclomethasone, fluticasone, clocortolone, corticorelin, corticotropin, cortisone, deflazacort, desonide, desoximetasone, diflorasone, difluprednate, bethamethasone, fludrocortisone, flunisolide, fluocinolone, fluorometholone, halcinonide, halobetasole, loteprednole, medrysone, paramethasone, prednicarbate, rimexolone triamcinolone acetonide, prednisone, hydrocortisone valerate, and mometasone furoate.

5. The medicament of claim 1, wherein the medicament is administered via parenteral administration.

6. The medicament of claim 1, wherein the medicament is administered via oral administration.

7. The medicament of claim 1, wherein the AAT is administered intravenously.

8. The medicament of claim 1, wherein the steroid is administered via oral administration.

9. The medicament of claim 1, wherein the medicament is administered via inhalation.

10. A method of treating an inflammatory disease or tissue injury in a subject in need thereof, the method comprising administering a therapeutically effective amount of AAT in combination with a therapeutically effective amount of a steroid.

11. The method according to claim 10, wherein the AAT and the steroid are administered sequentially.

12. The method according to claim 10, wherein the AAT and the steroid are administered simultaneously.

13. The method according to claim 10, wherein the AAT is administered intravenously.

14. The method according to claim 10, wherein the AAT and the steroid are administered by inhalation.

15. The method according to claim 10, wherein the steroid is administered via oral administration.

16. The method according to claim 10, wherein the AAT and the steroid are administered in a dosage of about 30 mg to about 250 mg AAT/kgBW and 0.05-50 mg/Kg of steroid per dose.

17. The method according to claim 16, wherein each dose comprises from about 40 mg AAT/kgBW to about 240 mg AAT/kgBW.

18. The method according to claim 17, wherein each dose comprises 40, 60, 80, 120, 180 or 240 mg AAT/kgBW.

19. The method according to claim 16, wherein the doses are administered at intervals of from 1-4 days to 2 weeks.

20. The method according to claim 19, wherein the intervals are selected from constant intervals and variable intervals.

21. The method according to claim 10, wherein the doses contain the same amount of AAT and the steroid.

22. The method according to claim 10, wherein the doses contain variable amounts of AAT and the steroid.

23. The method according to claim 10, wherein the doses are administered at intervals of one week.

24. The method according to claims 10, wherein the amount of AAT decreases from the first dose administered to the last dose administered.

25. The method according to claim 10, wherein the administration is carried out for a plurality of months.

26. The method according to claim 10, wherein the steroid dose administered to the subject is reduced or ceased during or after completion of the AAT treatment.

27. The method according to any one of claims 1 or 10, wherein the inflammatory disease or tissue injury is a pulmonary disease selected from the group consisting of alpha- 1 antitrypsin deficiency (AATD), small airway disease, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, asthma, pneumonia, parenchymatic and fibrotic lung diseases or disorders, interstitial pulmonary fibrosis, re-inflammation, acute respiratory distress syndrome (ARDS), and sarcoidosis.

28. The method according to any one of claims 1 or claim 10, wherein the inflammatory disease or tissue injury is selected from the group consisting of graft-versus-host disease (GVHD), ischemia-reperfusion injury, ischemia/reperfusion injury following cardiac transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Wegener’s disease, Crohn's disease, ulcerative colitis, psoriasis, type I and/or type P diabetes, dermatitis, pneumonia, sepsis, wound healing, systemic lupus erythematosus, and multiple sclerosis.