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1. WO2020157752 - COMPOSITION D'AÉROSOL PHARMACEUTIQUE

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[ EN ]

PHARMACEUTICAL AEROSOL COMPOSITION

FIELD OF THE INVENTION

The present invention relates to an aqueous solution of alpha 1 -antitrypsin (AAT), and to methods for treating pulmonary diseases by using the aqueous solution of A AT.

BACKGROUND OF THE INVENTION

AAT is a heavily glycosylated plasma protein of 52 kDa in size. AAT is produced by the liver and secreted into the circulation, and is also produced locally by lung epithelial cells. Circulating levels of AAT increase during acute phase response. This increase is due to the presence of IL-1 and IL-6 responsive elements inside the promoter region of the AAT encoding gene. AAT functions as a serine protease inhibitor that primarily targets elastase, trypsin and proteinase-3, three inflammatory and immune cell-derived enzymes that are involved in protease-activated receptor (PAR) activation and the onset and progression of inflammation (Vergnolle N. 2009. Pharmacol Ther 123(3):292-309). AAT induces the production and release of anti-inflammatory mediators such as IL-10 and IL- 1-receptor antagonist (IL-IRa) (Lewis E C et al. 2008. Proc Natl Acad Sci USA. 105(42): 16236-41).

In individuals with AAT deficiency, (serum level below the normal range 80 mg/dl), there is an excess of neutrophil elastase that increases the breakdown of elastin leading to the airway destruction. This manifests clinically as chronic obstructive pulmonary disease (COPD) with emphysema and/or chronic bronchitis.

International application W02005/027821 to the applicant of the present invention teaches a novel composition of purified, stable, active alpha- 1 antitrypsin (AAT) for intravenous administration and inhalation, a process for its preparation, and its use for treating pulmonary disease, including pulmonary emphysema and CF associated lung disease or disorder. The contents of W02005/027821 are incorporated herein by reference in their entirety.

AAT is currently administered intravenously by using intravenous formulations indicated for augmentation therapy in patients having congenital deficiency of AAT with clinically evident emphysema.

Although the use of augmentation therapy restores physiological levels of AAT to patient’s plasma, and may protect the remaining structure of lung parenchyma, there still remains unmet needs in disease management. Only a small fraction of plasma AAT reaches the lung, raising uncertainty regarding dose-efficiency and cost effectiveness of therapy, particularly as the use of intravenous replacement requires relatively large amounts of the protein.

A formulation for efficient administration by inhalation is highly desired and not yet commercially available due to problems in achieving a suitable quantity, dispersion and activity of the protein. Therefore, it would be desirable to achieve an improved modality and administer therapeutically effective amounts of an aqueous solution of AAT via inhalation.

SUMMARY OF THE INVENTION

The present invention provides an aqueous solution of alpha 1-antitrypsin (AAT), derivative or analog thereof. Particularly, the present invention provides methods for treating pulmonary diseases, including pulmonary diseases associated with alpha- 1 antitrypsin (AAT) deficiency and CF by administering the aqueous solution of AAT via inhalation.

The present invention discloses for the first time characterization of the nebulized AAT in order to evaluate the feasibility of the technology to aerosolize liquid formulations of delicate pharmaceutical proteins.

According to one aspect, the present invention provides an aqueous solution of alpha 1 -antitrypsin (AAT) wherein when a therapeutic dose is administered by a nebulizer to generate an inhaled aerosol composition; said aerosol composition comprises AAT which retained at least about 96% specific activity after nebulization. According to certain embodiments, said aerosol composition comprises AAT which retained at least about 98% specific activity after nebulization.

According to certain embodiments, said aerosol composition comprises not more than 500 non-soluble sub-visible proteinaceous particles from about 5 micron to about 100 microns. According to certain embodiments, said aerosol composition comprises not more than 100 non-soluble sub-visible proteinaceous particles from about 5 micron to about 100 microns.

According to certain embodiments, the nebulizer is based on a vibrating membrane technology. According to certain embodiments, the nebulizer is an eFlow nebulizer.

According to certain embodiments, said aerosol composition comprises a less than 50% increase in the percentage of high molecular weight species after nebulization.

According to certain embodiments, said aerosol composition comprises a less than 30% increase in the percentage of high molecular weight species after nebulization.

According to certain embodiments, the temperature in the reservoir during nebulization does not exceed about 47 degrees C to about 55 degrees C.

According to certain embodiments, the nebulization time is between about 5 to 15 minutes.

According to another aspect, the present invention provides a method for treating pulmonary diseases, which comprises administering to a subject in need thereof a therapeutically effective amount of the aqueous solution of AAT.

According to certain embodiments, the pulmonary disease is 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, and sarcoidosis.

According to other embodiments, the aqueous solution of AAT is absorbed by lung tissues of the subject.

According to certain embodiments, the AAT is naturally occurring AAT purified from an unpurified mixture of proteins by a process comprising of chromatography on a plurality of ion exchange resins, comprising a first anion exchange resin followed by a cation and a second anion exchange resins.

According to certain embodiments, the AAT is recombinant or transgenic AAT.

According to certain embodiments, the subject is human.

According to certain embodiments, the method results in: reduced hospitalization; reduced intensive care or mechanical ventilation need; reduced healthcare utilization or burden; reduced absences from school or work; decreased antibiotic need; decreased steroid need; decreased relapse frequency; and decreased morbidity.

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-B demonstrate representative images of the sub-visible particles before (A) and after (B) nebulization as detected by Micro-Flow Imaging (MFI).

FIGs. 2A-B demonstrate representative proteinaceous aggregate Raman spectra (A) versus the reference protein spectrum and proteinaceous aggregate images (B).

FIG. 3 demonstrates the correlation between reservoir temperature and nebulization time.

FIG. 4 demonstrates the temperature measured in the medication reservoir, close to the membrane, during Nebulization. Five devices were tested after usage for 21 runs with AAT.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses an aqueous solution of alpha 1 -antitrypsin (AAT) and methods for treating pulmonary diseases by using the aqueous solution of AAT.

The route of inhalation for the prevention or treatment of respiratory diseases has several advantages over other routes of administration, specifically IV administration: Inhalation delivery is directed to the target site, such that there is negligible systemic absorption and side effects are minimized; it requires lower therapeutic doses, and thus there is greater product availability; it provides quick relief of symptoms and expected good tolerance; it is a more convenient form for patients thus better compliance is expected; and it reduces treatment costs as a result of efficient utilization of an expensive drug using stable, purified AAT with a highly efficient nebulizer such as the eFlow.

Definitions

As used herein, the term“Alpha- 1 Antitrypsin” (AAT) refers to a glycoprotein

that in nature is produced by the liver and lung 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 methionine358 - serine359 residues to form an AAT-elastase complex. This complex is rapidly removed from the blood circulation and the lung airways. 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, derivative or analog thereof as is or will be known in the art, including plasma-derived AAT, recombinant AAT and transgenic AAT can be used according to the teachings of the present invention.

As used herein, the term "aggregate" refers to agglomeration or oligomerization of two or more individual molecules of the protein of interest to form, for example, dimers, trimers, tetramers, oligomers and other high molecular weight species. Protein aggregates can be soluble or insoluble. The term "aggregates" refers to a chunk of protein material which contains high molecular weight oligomers. The molecular aggregates can be measured by HPLC.

The term "subject," as used herein, refers to any animal, individual, or patient to which the methods described herein are performed. Generally, the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus, other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and non-human primates (including monkeys, chimpanzees, orangutans, and gorillas) are included within the definition of subject.

A "subject in need thereof," as used herein, refers to a subject having or at risk of developing a pulmonary disease. A subject in need thereof may have or be at risk of developing respiratory disease or disorder that is associated with pulmonary disease.

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.

"Acute" as used herein means arising suddenly and manifesting intense severity. With relation to delivery or exposure, "acute" refers to a relatively short duration.

"Chronic" as used herein means lasting a long time, sometimes also meaning having a low intensity. With regard to delivery or exposure, "chronic" means for a prolonged period or long-term.

As used herein, the terms "exacerbation" "exacerbation period" and "exacerbation episode" are used interchangeably to describe an increase in the severity of symptoms during a course of a disease, which is mostly associated with a worsening of quality of life. Exacerbations are quite frequent in patients with chronic lung diseases in general and in AAT deficient patients in particular. By definition, exacerbations are acute worsening and/or increase in severity and/or magnitude of the pulmonary disease symptoms that may require additional therapy.

The terms "prevent" or "preventing" includes alleviating, ameliorating, halting, restraining, slowing, delaying, or reversing the progression, or reducing the severity of pathological conditions described above, or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing risk of developing a disease, disorder, or condition.

"Amelioration" or "ameliorate" or "ameliorating" refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

The terms "pulmonary delivery" and "respiratory delivery" refer to delivery of AAT to a subject by inhalation/nebulization through the mouth and into the lungs.

"Pulmonary administration" means administration topical to the surface of the

respiratory tract. Pulmonary administration includes nebulization, inhalation, or insufflation of powders or aerosols, by mouth and/or nose.

"Inhalation" refers to a method of administration of a compound that delivers an effective amount of the compound so administered or delivered to the tissues of the lungs or lower respiratory tract by inhalation of the compound by the subject, thereby drawing the compound into the lung. As used herein, "administration" is synonymous with "delivery".

The phrases "pulmonary administration," "respiratory administration," "pulmonary delivery," and "respiratory delivery" are synonymous as used herein and refer to the administration and or delivery of AAT to a subject by inhalation through the mouth and or nose and into the lungs and lower respiratory tract.

"Nebulization time" refers to the time needed to deliver a therapeutic dose of the drug formulation.

"Fibrosis" refers to the formation of fibrous tissue. Excess fibrosis in an organ or tissue can lead to a thickening of the affected area and scar formation. Fibrosis can lead to organ or tissue damage and a decrease in the function of the organ or tissue. An example of fibrosis includes, but is not limited to, pulmonary fibrosis (fibrosis of the lung).

As used herein, the terms "cystic fibrosis" or "CF" refer to an inherited autosomal recessive disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel.

The term "emphysema," as is used herein, refers to a pathological condition of the lungs in which there is a decrease in respiratory function and often breathlessness due to an abnormal increase in the size of the air spaces, caused by irreversible expansion of the alveoli and/or by the destruction of alveolar walls by neutrophil elastase. Emphysema is a pathological condition of the lungs marked by an abnormal increase in the size of the air spaces, resulting in strenuous breathing and an increased susceptibility to infection. It can be caused by irreversible expansion of the alveoli or by the destruction of alveolar walls. Due to the damage caused to lung tissue, elasticity of the tissue is lost, leading to trapped air in the air sacs and to impairment in the exchange of oxygen and carbon dioxide. In light of the walls breakdown, the airway support is lost, leading to obstruction in the airflow. Emphysema and chronic bronchitis frequently co-exist together to comprise chronic obstructive pulmonary disease.

As used herein, the term "chronic obstructive pulmonary disease" abbreviated "COPD", refers to a disease state characterized by airflow limitation that is not fully reversible. The airflow limitation is usually both progressive and associated with an abnormal inflammatory response of the lungs to noxious particles or gases. COPD is the fourth leading cause of death in America, claiming the lives of 120,000 Americans in 2002, with smoking being a primary risk factor. A diagnosis of COPD exacerbation is considered when there is increased dyspnea, increased sputum volume, and increased sputum purulence. Severity of an exacerbation can be quantified by assessing the magnitude of these three symptoms (Dewan NA 2002. Chest 122:1118-1121).

"Bronchiectasis," as used herein, refers to the abnormal and irreversible dilation of the proximal medium-sized bronchi (>2 mm in diameter) caused by destruction of the muscular and elastic components of the bronchial walls. It can be congenital or acquired. Bronchiectasis can be caused by the bacteria Streptococcus pneumoniae , Haemophilus influenzae, Staphylococus aureus, and Moraxella catarrhalis, and the atypical pneumonias Legionella pneumonia, Chlamydia pneumoniae, Mycoplasma pneumoniae, and pneumonia caused by Pseudomonas aeruginosa.

"Asthma," as used herein, refers to a chronic respiratory disease, often arising from an allergy that is characterized by sudden recurring attacks of labored breathing, chest constriction, and coughing. In a typical asthmatic reaction, IgE antibodies predominantly attach to mast cells that lie in the lung interstitium in close association with the bronchioles and small bronchi. An antigen entering the airway will thus react with the mast cell-antibody complex, causing release of several substances, including, but not limited to interleukin cytokines, chemokines, and arachidonic acid-derived mediators, resulting in bronchoconstriction, airway hyperreactivity, excessive mucus secretion, and airway inflammation.

"Pneumonia" as used herein, refers to an acute infection of one or more functional elements of the lung, including alveolar spaces and interstitial tissue. Generally, pneumonia can result from acute lung disease, lung inflammatory disease, or any perturbations in lung function due to factors such as inflammation or coagulation.

"Mycobacterial infection," as used herein, refers to the pulmonary infection caused by various species of Mycobacterium. "Tuberculosis" or "TB" is one example of an airborne, chronic Mycobacterium tuberculosis infection.

The term“eFlow nebulizer” refers to the nebulizer disclosed in international application WO 01/34232. The term "inhalation nebulizer" refers to a nebulizer comprising the basic elements of the eFlow nebulizer and any equivalent nebulizer. The terms "pulmonary delivery" and "respiratory delivery" refer to delivery of API to a patient by inhalation through the mouth and into the lungs.

The term "dry powder" refers to a powder composition that contains finely dispersed dry particles that are capable of being dispersed in an inhalation device and subsequently inhaled by a subject.

The particles of the dry powder composition have a particle size distribution that enables the particles to target the alveolar region of the lung when delivered via inhalation. 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.

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

The term "dose" as used herein, refers to an amount of AAT which is 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 administration to a subject and/or variable frequency of administration of the AAT for therapeutic treatment. "Multiple dose regimen" or "multiple-variable dose regimen" describe a therapy schedule which is based on administering different amounts of AAT at various time points throughout the course of therapy. In one embodiment, the invention describes a multiple- variable dosage method of treatment.

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 additional lung treatment 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.

"Maintenance therapy" as used herein, refers to the regular, periodic administration of AAT to maintain a sufficient level of A1PI in a subject's lungs or circulatory system to have a therapeutic effect on the subject.

"Augmentation therapy," as used herein, refers to supplementing, replacing, or increasing deficient in vivo quantities or concentrations of a biomolecule, such as AAT, to have a therapeutic effect on a subject.

"Recombinant AAT" as used herein, refers to AAT that is the product of recombinant DNA or transgenic technology. The phrase, "recombinant AAT," also includes functional fragments of AAT, chimeric proteins comprising AAT or functional fragments thereof, fusion proteins or fragments of AAT, homologues obtained by analogous substitution of one or more amino acids of AAT, and species homologues. For example, the gene coding for AAT can be inserted into a mammalian gene encoding a milk whey protein in such a way that the DNA sequence is expressed in the mammary gland as described in, e.g., U.S. Pat. No. 5,322,775, which is herein incorporated by reference for its teaching of a method of producing a proteinaceous compound. "Recombinant AAT," also refers to AAT proteins synthesized chemically by methods known in the art such as, e.g., solid-phase peptide synthesis. Amino acid and nucleotide sequences for AAT and/or production of recombinant AAT are described by, e.g., U.S. Pat. Nos. 4,711,848; 4,732,973; 4,931,373; 5,079,336; 5,134,119; 5,218,091; 6,072,029; and Wright et al., Biotechnology 9: 830 (1991); and Archibald et al., Proc. Natl. Acad. Sci. (USA), 87: 5178 (1990), are each herein incorporated by reference for its teaching of AAT sequences, recombinant AAT, and/or recombinant expression of AAT.

Preparation of AAT

According to one aspect of the present invention a purified stable composition of AAT is provided. Preferably, a liquid composition of purified, stable AAT is provided. International application WO 2005/027821, to the applicant of the present invention, provides pharmaceutical compositions comprising purified, stable, active AAT in a

form of a ready to use sterile solution. WO 2005/027821 also provides process, which combines removal of contaminating substances (i.e., lipids, lipoproteins and other proteins) and separation of active from inactive AAT by sequential chromatography steps. The process disclosed in that invention is highly suitable for a large-scale production of AAT, in the range of tens of kilograms or more. The mixture of proteins from which the AAT is purified is preferably Cohn Fraction IV-1 paste, but can include other Cohn Fractions, separately or in combination; human blood plasma; plasma fractions; or any protein preparation containing AAT. For instance, the process is applicable to purification of recombinant human AAT from the milk of transgenic animals.

In that application, the mixture of proteins comprising AAT is dispersed in an aqueous medium, preferably water, at a ratio of about 13 to about 35 liter per about 1 kg of source material, preferably Cohn Fraction IV- 1 paste. The pH of the dispersion is adjusted to a pH range of from about 8.0 to about 9.5. The pH adjustment stabilizes the AAT and promotes the dissolution of the AAT in the dispersion, thereby increasing the production yield. Dispersion may take place at an elevated temperature of between 30°C and 40°C for further increase in AAT solubility.

A particular advantage of that process is the elimination of contaminants or by-products that otherwise compromise the efficiency of AAT purification processes. In particular, Cohn Fraction IV- 1 paste preparations contain a significant amount of the lipoprotein Apo A-l, which has the effect of compromising column flow and capacity during purification. Other non-desired proteins such as albumin and transferrin are also present in the paste preparation. Removing a portion of such contaminants according to the invention disclosed in WO 2005/-27821 is performed by two steps: (a) removing contaminating lipids and lipoproteins by lipid removal agent and (b) precipitating a portion of contaminating protein from the AAT-containing aqueous dispersion. The removal of contaminating proteins, without loss of AAT, enables a significant reduction in equipment scale, e.g., column size.

The precipitate that forms can be separated by conventional means such as centrifugation or filtration, and is then discarded. The supernatant is ready for further purification, for example an anion exchange resin. The AAT is then eluted from the

column. The solution is treated to reduce its water content and change the ionic composition by conventional means such as by diafiltration, ultrafiltration, lyophilization, etc., or combinations thereof.

According to one embodiment, the AAT-containing effluent obtained after the first anion exchange chromatography is concentrated by ultrafiltration. The retentate is then diafiltered against pure water to reach conductivity within the range of from about 3.5 to about 4.5 mS/cm.

To further purify the AAT-containing solution obtained after the first anion exchange chromatography, the solution is loaded on a cation exchange resin with the same type of buffer used for the anion-exchange step, having appropriate pH and conductivity such as to allow the AAT to pass and be washed off with the buffer flow through, while contaminating substances are retained on the cation exchange resin. The AAT-containing solution obtained after the cation exchange chromatography can be treated to reduce its water content. According to one embodiment, the solution is concentrated by ultrafiltration.

The ion-exchange chromatography is also used to separate active AAT from inactive AAT. That invention further comprises methods for separating active AAT from other contaminating substances, including solvent/detergent compounds used for viral inactivation. Such separation is achieved by the second anion exchange chromatography. The AAT eluted from the second anion exchange chromatography step is therefore not only highly active, but also highly pure. Throughout the process of that invention only one type of buffer is used, with adjustment of pH and conductivity as required throughout the various process steps. According to one embodiment, the buffer is any suitable acid/salt combination that provides an acceptable buffer capacity over the ranges of pH required throughout the process. According to preferred embodiments, the process uses a buffer other than citrate-based buffer. According to yet other embodiments, the buffer anion is acetate. According to one embodiment, the process of that invention further comprises viral removal and/or viral inactivation steps. Methods for viral removal and inactivation are known in the art.

One method for viral removal is filtration, preferably nanofiltration, removing both enveloped and non-enveloped viruses. According to one embodiment, the viral removal step comprises filtration. According to another embodiment, the vims removal step is performed after the cation exchange chromatography. Typically, the cation exchange flow-through solution containing AAT is concentrated, and then nanofiltered. According to one embodiment, the method of viral inactivation employed comprises a solvent/detergent (S/D) treatment. The viral inactivation step is preferably performed prior to loading the solution on the second anion exchange resin. According to one embodiment, the detergent used is polysorbate and the solvent is Tri-n-Butyl-Phosphate (TnBP). According to another embodiment, the polysorbate is polysorbate 80. According to one embodiment Polysorbate 80 may be added from about 0.8% to about 1.3% volume per weight (v/w) of the resulting mixture and TnBP may be added from about 0.2% to about 0.4% weight per weight of the resulting mixture. The solution containing active, purified AAT obtained after the second anion exchange chromatography can be further processed to obtain a pharmaceutical composition for therapeutic, diagnostic, or other uses. To prepare the product for therapeutic administration, the process further comprises the steps of changing the ionic composition of the solution containing purified, active AAT to contain a physiologically compatible ion and sterilizing the resulted solution.

The purified AAT obtained by the process of that invention is highly stable. According to one embodiment, the pharmaceutical composition comprises at least 90% pure, preferably 95% pure, more preferably 99% pure AAT. According to another embodiment, at least 90% of the AAT is in its active form.

According to some embodiments, highly dispersible dry powder compositions are used, comprising high concentration of active alpha- 1 antitrypsin (AAT) and specific excipients, suitable for pulmonary delivery of AAT. The dry powder compositions disclosed herein comprise according to some embodiments AAT molecules in their monomeric form, having low aggregation level. The AAT dry powder compositions exhibit an exceptional stability and low aggregation properties, and thus are highly suitable for use with inhalation devices as well as in other dry-powder dosage forms.

Pharmaceutical Compositions and Methods of Treatment

The term "pharmaceutical composition" is intended to be used herein in its

broader sense to include preparations containing a protein composition in accordance with this invention used for therapeutic purposes. The pharmaceutical composition intended for therapeutic use should contain a therapeutic amount of AAT, i.e., that amount necessary for preventative or curative health measures.

As used herein, the term "therapeutically effective amount" refers to an amount of a protein or protein formulation or composition which is effective to treat a condition in a living organism to which it is administered over some period of time. Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g. by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more acceptable diluents or carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Proper formulation is dependent on the route of administration chosen. According to certain currently preferred embodiments, the pharmaceutical compositions of the present invention are formulated in a form suitable for inhalation.

The AAT-containing pharmaceutical compositions disclosed in W02005/027821 to the Applicant of the present invention is advantageous over hitherto known AAT-containing preparations, as the AAT is highly stable also when the composition is kept in a liquid form. Therefore, it is not necessary to lyophilize the AAT preparation for stable storage in the form of a powder. Subsequently, there is no need to reinstate the powder to a liquid before use for parenteral administration or for inhalation. According to certain currently preferred embodiments, AAT in a ready-to-use liquid formulation is used with the methods of the present invention. It has been estimated that only 2% of the intravenously administered AAT dose reaches the lung (Hubbard and Crystal, 1990. Lung 168 Suppl:565-78, 1990). This is a major disadvantage in treating pulmonary diseases in general, and in treating exacerbation episodes in particular.

Therefore, administration of AAT by the inhalation route may be more beneficial

as it reaches the lower respiratory tract directly. The inhalation route also requires lower therapeutic doses of AAT and thus the scarce supply of human plasma-derived AAT, currently being the only source for AAT, would be available for the treatment of more patients. This route of administration may be also more effective in neutralizing neutrophil elastase, and in correcting the imbalance between proteinase and anti-proteinases in the lung tissues, and is thus highly suitable for treating pulmonary diseases at periods of exacerbation. In addition, administration by inhalation is simpler and less stressful for the patient than the intravenous route and would reduce the burden on the local health care system (by requiring less clinical input). Formulations of pharmaceutical compositions for administration by the route of inhalation are known in the art, as well as inhaler systems and devices. In general, for administration by inhalation, the active ingredients are delivered in the form of an aerosol spray from a pressurized metered dose inhaler with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, or carbon dioxide. The active ingredient in the aerosol spray may be in a powder form administered using a dry powder inhaler, or in aqueous liquid aerosol form using a nebulizer.

Powder inhalers are designed to be used until a given charge of active material is exhausted from the device. The charge loaded into the device will be formulated accordingly to contain the proper inhalation dose amount of AAT for delivery in a single administration. (See generally, Remington's Pharmaceutical Sciences, 18th Ed. 1990, Mack Publishing Co., Easton, Pa., Chapter 92 for information relating to aerosol administration).

Nebulizers for liquid aerosol delivery may be categorized as jet nebulizers operated by a pressurized flow of air using a portable compressor or central air supply in a hospital, ultrasonic nebulizers incorporating a piezo-crystal to provide the energy for generating the aerosol out of an ultrasonic fountain, and electronic nebulizers based on the principle of a perforated vibrating membrane.

Any of a variety of powder inhalers and nebulizers as are known in the art can be used for AAT administration according to the teachings of the present invention. For example, U.S. Pat. No. 6,655,379 discloses methods and devices for delivering an active agent formulation to the lung of a human patient. The active agent formulation

may be in dry powder form, it may be nebulized, or it may be in admixture with a propellant. According to the teaching of that patent, the active agent formulation, particularly insulin, is delivered to a patient at an inspiratory flow rate of less than 17 liters per minute.

Methods regarding the delivery of AAT formulations using nebulizers are discussed, for example, in U.S. Pat. Nos. 5,093,316, 5,618,786 and 5,780,440. The Applicant of the present invention and co-workers disclosed the use of eFlow nebulizer, disclosed in International Patent Application WO 01/34232, for AAT delivery to the lung. The eFlow nebulizer provides an increased amount of aerosol during inhalation while minimizing both aerosol losses during exhalation and the residual drug in the nebulizer reservoir. The nebulizer includes an aerosol generator that atomizes the liquid through a vibrating diaphragm into particle sizes that are efficiently delivered to the lungs.

The operating conditions for delivery of a suitable inhalation dose will vary according to the type of mechanical device employed. For some aerosol delivery systems, such as nebulizers, the frequency of administration and operating period will be dictated chiefly by the amount of the active composition (AAT according to the present invention) per unit volume in the aerosol. Typically, the higher the concentration of the protein in the nebulizer solution the shorter the operating period. Some devices such as metered dose inhalers may produce higher aerosol concentrations than others and thus will be operated for shorter periods to give the desired result. According to certain embodiments, the methods of the present invention employ a nebulizer comprising a ready-to-use inhalation solution comprising a therapeutically effective amount of AAT.

According to currently certain preferred embodiments, the ready-to-use liquid pharmaceutical composition is packed in pre-sterilized unit dose vials containing 0.25 ml- 10 ml, preferably 0.25 ml to 5 ml, commonly used for ready to use inhalation solutions. The vial can be made of glass or polymeric materials or the liquid can be filled into polyethylene or any other suitable polymer vials, manufactured for instance by a blow fill seal process.

According to other preferred embodiments, at least 60% of the nebulized dose is

dissolved in droplets having a diameter of 5 mm or less. Such droplet size enhances the AAT delivery to the alveolar regions, where its activity is most required. According to certain embodiments, at least 50%, preferably 60%, and more preferably 70% or more of the loaded nominal dose of AAT can be delivered to the subject. According to the teaching of the present invention, AAT is administered at the early stages of various pulmonary diseases. As described hereinabove, the pulmonary disease may be associated with an inherited deficiency in AAT and in such cases, patients typically receive intravenous augmentation therapy of AAT. Thus, according to certain embodiments, the method of the present invention comprises administering to a subject in need thereof a therapeutic amount of AAT via inhalation in combination with administering the AAT intravenously.

Typically, the inhaled AAT is administered for relatively short periods of time. According to certain embodiments, the inhalation time is between about 5-15 minutes, preferably about 10 minutes. The AAT may be administered once a week or administration can be repeated at least twice a week, each day, or even twice a day.

The AAT protein is an acute phase reactant protein and, as such, its synthesis is amplified during episodes of inflammation or stress (Sandhaus RA. Alpha 1-Antitrypsin deficiency *6: New and emerging treatments for alpha 1-antitrypsin deficiency. Thorax 59:904-909, 2004), which particularly occurs during exacerbations. AAT deficient patients risk severe lung damage during exacerbation periods, due to the inability to mount an effective acute phase AAT elevation. During acute exacerbation periods, such a shortage of AAT may also occur in normal individuals, resulting in the excess of neutrophil elastase leading to destruction of lung tissues. Addition of a therapeutically significant amount of AAT directly to the lung tissue as disclosed by the present invention satisfies the clinical need for a treatment that provides an adequate answer to the patient's condition and prevents the potential accelerated decline in the disease state due to the exacerbation.

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

Example 1: Characterization of Sub- Visible Particles in AAT post-Nebulization

Alpha- 1 Antitrypsin (AAT) via inhalation is on an ongoing process of development for the treatment of pulmonary diseases. The delivery device used for the nebulization of the AAT formulation is an investigational eFlow nebulizer based on the vibrating membrane technology, manufactured by PARI Pharma GmbH (Munich, Germany). A major aim of this study was a thorough characterization of the nebulized AAT in order to evaluate the feasibility of the technology to aerosolize liquid formulations of delicate pharmaceutical proteins.

METHODS

MFI - Micro-Flow Imaging (Protein Simple). Each sample was analyzed in triplicate without dilution. Results are reported as cumulative particle concentrations for particles > 2 mm, > 5 mm, > 10 mm, and > 25 mm. In addition to reporting cumulative concentrations, post-nebulization samples were classified with an image filter to determine populations of ‘dark’ particles compared to‘translucent’ (protein suspect) particles. The classification was performed by MVAS 1.4 software.

Morphologi G3SE-ID - Fused silica membrane filters with 10 mm pores were used to isolate particles greater than 10 mm in size. 1 mL of sample was removed from the tube in 250 mL aliquots and filtered onto a 10 mm pore fused silica filter. 2 mL of 0.22 mm-filtered PW in aliquots of 250 mL was used to rinse the filter to remove water-soluble materials. The filter was allowed to dry before experiments were conducted.

All samples were tested on the G3SE-ID with a 10x magnification automated imaging method. After particle images were collected, non-sample areas were removed from the analysis and automated Raman targeting of particles greater than 25 mm was undertaken for all samples. Raman spectra were collected for 1 second by 150 coadditions (2.5 minutes) for each particle, and manual supplementation of poor-quality spectra was used to improve the analysis. Background correction against a clean fused silica substrate was used for the spectra, and experimental spectra were identified using a manually constructed library.

RESULTS

First it was noticed that the amount of sub-visible particles increased after nebulization and MFI analysis indicated that their appearance was altered during nebulization as shown in Figures 1A-B. AAT fresh and aged lots (1 and 35 month from production, respectively) were nebulized and the aerosol was captured and analyzed. The amount of sub-visible particles before nebulization was higher for the aged AAT lot compared with the fresh AAT lot (see Table 1). Post-nebulization both lots reached similar levels of sub-visible particles, so that only the fresh samples showed an increase.

Table 1: The amount of sub-visible particles in a fresh AAT lot, an aged AAT lot

Particles identification by Raman (G3SE-ID), indicated a composition of mainly cellulose and proteins, with some polyester, polyethylene, talc, carbon, carbonate, rubber and others (not identified by Raman). The dark particles were found to correlate with non- proteinaceous particles, such as cellulose while the proteinaceous aggregate remain translucent post-nebulization, as shown in Figures 2A-B.

Based on the ability to distinguish between proteinaceous aggregates and environmental particles by light transparency the MFI images were further analyzed, differentiating between proteinaceous aggregates (transparent) and extrinsic particles (dark). The image classification coupled with Morphologi G3-ID analyses would predict that up to 95% of particles present post-nebulization are related to extrinsic material rather than proteinaceous particulates (Table 2).

Table 2: Classification of Transparent (protein-like) and Dark (Extrinsic) Particles in Pre- and Post- Nebulized Samples


The > 2 mih is provided for information only, as image classification below 5 mm is not accurate due to technical limitations.

The use of the MFI-based method with the power to distinguish proteinaceous from non-pro teinaceous particles enabled an in-depth analysis of the nebulization of AAT via an investigational eFlow nebulizer system, revealing consistent amount of sub- visible proteinaceous particles, and supporting the safety assessment of the nebulized product.

Example 2: Characterization of the nebulization duration and the temperature in the reservoir during nebulization

AAT via inhalation has been administered as a 2% solution formulated with 20 mM phosphate buffer and 0.7% sodium chloride.

The delivery device used for the nebulization of the AAT formulation is an eFlow nebulizer handset used in combination with an eBase Controller (type 678) manufactured by PARI Pharma GmbH. The eFlow handset uses an included monthly disposable compartment - an aerosol head, which holds the membrane that vibrates during nebulization in order to convert the liquid AAT into an aerosol.

Easycare cleaning aid is a back wash system that was introduced in order to reduce possible clogging of the membrane pores (aerosol head) which could lead to an increase in nebulization time. The pores have a conus shape with a very narrow end of about 2-5 microns and they may block after use. The easycare provides an easy way for the patient to clean the pores with saline in a reverse mode by connecting this element to the eFlow device.

Procedure

AAT samples were nebulized, while measuring nebulization time and temperature in the sample reservoir.

Cleaning at first use before start of tests and regularly after each test the nebulizers were cleaned according to the following procedure:

The nebulizer was disassembled and placed in a beaker containing 500 mL warm tap water (~40°C) and some clear liquid dish soap (2 mL of a 5% aqueous solution of “Dish soap”). The beaker was put on a laboratory shaker for 5 min. The parts were then rinsed several times with warm tap water until all detergent was removed. Afterwards, they were rinsed twice with purified water.

Easycare treatment was performed with 5 mL saline once in every 3 cycles for the fresh membrane. Disinfection was done at the end of every workday (every 2-3 cycles) by boiling all parts in distilled water for 5 min.

Sampling and Methods:

The temperature of the reservoir was measured by a thermocouple and monitored automatically. Temperature data were recorded using the Ahlborn ALMEMO® 710 data logger with a sampling interval of 1 data point every 10 seconds. The temperature sensor was a NiCr - Ni thermocouple that was placed in the reservoir. The temperature was recorded at a point very close to the membrane.

RESULTS

AAT lot AN4120318, 3 months age at testing, was nebulized three times using a fresh membrane (1 to 3 cycles) and the aerosol was collected. The temperature was monitored during nebulization by a thermocouple at the reservoir which was placed in proximity to the membrane. Membranes were cleaned according to the instructions for use after every nebulization run. The reservoir temperature and nebulization time correlation is presented in Figure 3.

According to Figure 3, the temperature increased moderately during most of the nebulization and then rapidly close to the end of nebulization when the reservoir is almost empty.

The maximal temperature and nebulization time are summarized in Table 3.

Table 3: Nebulization Time and Maximum Temperature Recorded in the

Reservoir

5


As shown in Table 3, the nebulization duration increased slowly from 7 minutes

(buffer run) to 11 minutes and the maximum temperature varied between 47.3-50.6 °C.

Example 3: AAT activity before and after nebulization

AAT activity was measured by elastase inhibition before and after nebulization and was normalized either to the content of proteins (OD280) in the sample (specific activity) or to 5 the content of antigenic AAT (antigenic specific activity) as summarized in Table 4 and Table 5 for fresh and old AAT lots, respectively. The results are presented separately for each aerosol head (AH 1-3) by cycle (nebulization run).

Table 4: Specific Activity and Specific antigenic activity pre- and post- Nebulization - Fresh AAT lot

5

Table 5: Specific Activity and Specific antigenic activity pre- and post- Nebulization - Old AAT lot

As shown in Tables 4 & 5, AAT specific activity remains constant pre- and post- nebulization throughout the 36 membrane cycles for both fresh and old AAT lots, without a specific trend.

Example 4: Analysis of AAT polymeric forms before and after nebulization

AAT samples were tested before and after nebulization for molecular size 5 distribution (by SE-HPLC). High molecular weight species- polymeric forms (dimers and oligomers), monomers and fragments were analyzed proportionally, as summarized in Table 6 and Table 7 for fresh and old AAT lots, respectively. Dimers and oligomers were calculated together as polymeric forms.

Table 6: Molecular Size Distribution pre- and post-Nebulization

Fresh AAT lot

Table 7: Molecular Size Distribution pre- and post-Nebulization

Old AAT lot

The portion of fragments was below limit of detection before as well as after nebulization.

As shown in Tables 6 & 7, the fresh AAT lot started with lower portion of polymeric forms compared with the older lot as expected (1.39% vs 4.26% respectively). The results indicated that the nebulization had a slight effect of monomer decrease with increase of the polymeric forms, as they are calculated proportionally. The difference was in average of 0.39% for the fresh AAT lot and 0.25% for the AAT old lot. This difference before and after nebulization (0.39% vs. 0.25%) is small and does not indicate meaningful difference between fresh and old AAT lots.

Example 5: Monitoring temperature in the reservoir during nebulization

AAT via inhalation has been administered as a 2% solution formulated with 20 mM phosphate buffer and 0.7% sodium chloride.

The delivery device used for the nebulization of the AAT formulation is an eFlow nebulizer handset used in combination with an eBase Controller (type 678) manufactured by PARI Pharma GmbH. The eFlow handset uses a monthly disposable compartment -an aerosol head, which holds the membrane that vibrates during nebulization in order to convert the liquid AAT into an aerosol.

Procedure

AAT samples were nebulized by 5 devices for 21 cycles to mimic 21 treatment. The temperature in the sample reservoir was measured on the 21st run, while measuring nebulization time.

Sampling and Methods:

The temperature of the reservoir was measured by a thermocouple and monitored automatically. Temperature data were recorded using the Ahlborn ALMEMO® 710 data logger with a sampling interval of 1 data point every 10 seconds. The temperature sensor was a NiCr - Ni thermocouple that was placed in the reservoir. The temperature was recorded at a point very close to the membrane.

RESULTS

AAT was nebulized by 5 devices and the temperature was monitored during nebulization by a thermocouple at the reservoir which was placed in proximity to the membrane. Membranes were cleaned according to the instructions for use after every nebulization run. The reservoir temperature and nebulization time correlation is presented in Figure 4.

According to Figure 4, the temperature increased moderately during most of the nebulization and then rapidly close to the end of nebulization when the reservoir is almost empty. The temperature did not accede 41 °C, which correlates with high activity results po st-nebulization .

The maximal temperature and nebulization time are summarized in Table 8.

Table 8: Nebulization Time and Maximum Temperature Recorded in the

Reservoir


As shown in Table 8, the nebulization duration was similar between devices and remained up to 10 minutes and the maximum temperature varied between 35.7-40.8 °C.

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.