FIELD OF THE INVENTION [0002] This invention relates to a dry protein formulation, and in particular to a dry formulation of alpha 1-antitrypsin (AAT), particularly unglycosylated AAT.

BACKGROUND OF THE INVENTION [0003] Alpha 1-antitrypsin (AAT, al-proteinase inhibitor, al-PI), a 52 kDal glycoprotein, is the serine protease inhibitor found at highest concentration in plasma.

[0004] In the lung, AAT plays a prominent role in protecting tissue from the destructive activity of neutrophil elastase. Hereditary deficiency of AAT is associated with slowly progressive emphysema that typically manifests in the third to fourth decades of life (hereditary emphysema). Congenital AAT deficiency is also associated with early onset hepatic cirrhosis.

[0005] PROLASTIN (Bayer Corp. ) is a sterile, lyophilized preparation of native, glycosylated, human AAT purified from pooled plasma of normal human donors (plasma- derived, pAAT), and is approved for intravenous administration in treatment of patients with congenital deficiency of AAT having clinically demonstrable panacinar emphysema.

[0006] When reconstituted as directed, at 1 g AAT functional activity per 40 mL sterile water, the liquid composition comprises 2 20 mg/ml AAT.

[0007] In addition to AAT, PROLASTIN contains detectable levels of several proteins that copurify from plasma with AAT, including plasmin inhibitor, al- antichymotrypsin, Cl esterase inhibitor, antithrombin III, haptoglobin, albumin, a-lipoprotein, and IgA. PROLASTIN product insert (Bayer Corp. ) ; Coan et al., Vox. Sang.

48: 333-342 (1985). When reconstituted, the liquid composition also comprises 100-210 mEq/L Na, 60-180 mEq/L Cl, 15-25 gM sodium phosphate, <5 ppm PEG and <0. 1% sucrose. The product insert directs that the lyophilized formulation be stored at temperatures not to exceed 25°C.

[0008] ARALASTTM (Alpha Therapeutic Corporation, distributed by Baxter Healthcare Corp. ) is a sterile lyophilized preparation of native human glycosylated AAT purified from pooled human plasma (pAAT) which has also been approved for systemic therapy of hereditary emphysema.

[0009] When reconstituted as directed for intravenous administration, the ARALASTTM liquid composition comprises a 16 mg/mL AAT. The reconstituted liquid further contains albumin at no more than 5 mg/ml, polyethylene glycol at no more than 112 Ug/ml, polysorbate 80 at no more than 50 gg/ml, sodium at no more than 230 mEq/L, tri-n-butyl phosphate at no more than 1.0 Ug/ml, and zinc at no more than 3 ppm.

[0010] The ARALASTTM product insert directs that the lyophilized formulation be stored at 2-8°C, but notes that ARALAST may be removed from refrigeration and stored at temperatures not to exceed 25°C if used within one month thereafter.

[0011] Given the presence--or potential presence--of copurified IgA, both PROLASTIN and ARALAST Tm are specifically contraindicated in patients having antibodies to IgA.

[0012] PROLASTIN is heat-treated to reduce transmission of blood-borne infectious agents; ARALASTTM is treated with a solvent detergent mixture to inactivate enveloped viral agents and then nanofiltered to reduce the risk of transmission of non-enveloped viral agents. Despite such treatments, there remains a risk of transmitting disease, including diseases caused by protein infectious agents.

[0013] Lyophilized native, glycosylated, AAT purified from human plasma (pAAT) is available for research use from Athens Research & Technology (Athens, Georgia, USA; Prod.

No. 16-16-011609) and EMD Biosciences (San Diego, California, USA; Cat. No. 178251); in both cases, the product is lyophilized from 30 mM sodium phosphate buffer, pH 6.5, containing 300 mM NaCl.

[0014] U. S. Patent Nos. 5,780, 014 and 5,993, 783 describe a dry powder formulation of AAT intended for administration by inhalation. Inhalation can, in theory, effect both topical delivery directly to lung tissues and, upon subsequent absorption, systemic distribution. AAT purified from human plasma is exemplified.

[0015] Various drying techniques are suggested, including lyophilization, spray drying, agglomeration, spray coating, extrusion and combinations thereof to produce particles having a preferred diameter for delivery into lung alveolar spaces of 1-5 ßm. In preferred embodiments, particles produced by spray drying are further agglomerated into aggregates having diameters in the range of 200-500 gm. The larger agglomerates assist delivery from unit dosage blister packs into a proprietary

inhalation apparatuses. Edwards et al., Annu. Rev. Biomed.

Eng. 4: 93-107 (2002).

[0016] In preferred embodiments, the AAT compositions are said to be substantially pure, but may in other embodiments comprise carriers that serve as bulking agents, stabilizing agents, dispersing agents, and/or buffers.

Suitable pharmaceutically acceptable excipients or bulking agents are said to include carbohydrates, including monosaccharides such as galactose, D-mannose, and sorbose, disaccharides such as lactose and trehalose, cyclodextrins such as 2-hydroxypropyl-a-cyclodextrin, and polysaccharides such as raffinose, maltodextrins, and dextrans, and alditols. Preferred carbohydrate excipients are said to include lactose, trehalose, raffinose, maltodextrins and mannitol.

[0017] U. S. Pat. Nos. 6,258, 341,6, 309,671, and 6,589, 560 and corresponding European patent no.

EP 0 941 067 B1 describe powdered, dispersible compositions of proteins, including dry powdered AAT, that are said to have stable dispersibility over time. The compositions comprise a pharmaceutically-acceptable glassy matrix that confers upon the composition a characteristic glass transition temperature higher than the recommended storage temperature, preferably at least 10°C higher than the storage temperature.

[0018] The glass forming matrices include, inter alia, carbohydrates, carbohydrate derivatives, carbohydrate polymers, synthetic organic polymers, organic carboxylic salts, proteins, polypeptides, peptides, and high molecular weight polysaccharides. Salts of organic acids such as lactic acid, ascorbic acid, maleic acid, oxalic acid, malonic acid, malic acid, succinic acid, citric acid,

gluconic acid and glutamic acid are said to be particularly useful glass formers.

[0019] These four related patents exemplify a powdered composition of human AAT, prepared by spray drying a solution comprising, per ml of deionized water, 4.99 mg of glycosylated AAT derived from human plasma, 0.455 mg sodium citrate, and 0.082 mg citric acid. The spray-dried powder is said to provide a delivered dose by inhalation that is unchanged after 13 months dry storage at ambient temperature.

[0020] Although the dry compositions described above are particularly described as useful in the treatment of emphysema and related lung disorders, AAT has also been proposed as a treatment for a wide variety of other disorders having an inflammatory etiology, including, inter alia, psoriasis, U. S. Pat. No. 5,190, 917; mast cell tumors, U. S. Pat. No. 5,492, 889; allergic rhinitis, U. S. Pat. No.

5,166, 134; otitis, U. S. Pat. No. 6,174, 859; lupus erythematosus, U. S. Pat. No. 6,537, 968; chronic dermatitis, U. S. Pat. No. 5,290, 762, and others.

[0021] The large number of disorders for which AAT has been proposed as a therapeutic agent, coupled with the general disadvantages of agents derived from pooled human plasma, including the presence of copurifying protein contaminants and the risk of transmitting infectious agents, has motivated the development of recombinant AAT and engineered AAT muteins. Recombinant AAT has been produced, for example, in yeast, Rosenberg et al., Nature 312: 77-80 (1984) (rAAT); and in plants, Terashima et al., Appl. Microbiol. Biotechnol. 52: 516-23 (1999) and Huang et al., Biotechnol. Prog. 17: 126-33 (2001).

[0022] rAAT produced in Saccharomyces cerevisiae is a 395 amino acid unglycosylated protein of 44 kDal having an

amino acid sequence identical to human plasma AAT with the exception of an N-acetylmethionine residue at the amino terminus.

[0023] Although production of rAAT in yeast provides certain advantages in terms of yield, cost, and ease of purification, the stabilization of unglycosylated rAAT presents particular problems relative to the natural protein. Travis et al., J. Biol. Chem. 260: 4384-4389 (1985) describe a comparison of heat stabilities of yeast- derived rAAT with natural plasma-derived AAT. The half- life of non-glycosylated rAAT, with respect to its activity in response to thermal stress, is considerably less than that of its natural glycosylated counterpart.

[0024] Vemuri et al. , in Chapter 9 of Stability and Characterization of Protein and Peptide Drugs: Case Histories, ed. Wang and Pearlman, Plenum Press, New York (1993) (ISBN: 0306443651), describe formulations of rAAT, primarily in liquid form. Stability, e. g. at pH 7.5, is enhanced by increasing the salt content. However, salt is often disfavored for a lyophilized formulation because of the reduced glass transition temperature.

[0025] Vemuri et al. , PDA J. Pharm. Sci. Technol.

48: 241-246 (1994) describe tests of various cryoprotectants, including lactose, sucrose, and polyvinyl- pyrrolidone, on freezing, lyophilization, and storage of lyophilized rAAT formulations. None of the tested protectants enhanced stability, but the control lyophilisate was said to retain activity and purity during a 12 month stability period.

[0026] Despite the early efforts of Vermuri et al., there remains a need in the art for dry formulations that are suitable for stable storage of rAAT, and optionally for stable storage of native glycosylated AAT; that optionally

permit subsequent use directly in the dry state, as for example for inhalation therapy; and that optionally permit reconstitution in clinically acceptable diluents, such as water, and use immediately thereafter in the liquid state, such as for nebulization (see, e. g., EP 0 289 336 B1) or intravenous administration.

SUMMARY OF THE INVENTION [0027] The present invention is based on the discovery of a dry formulation of unglycosylated AAT, notably rAAT, that has good stability, e. g. of up to 2 years or more at 5°C or below.

[0028] Surprisingly, this stability is achieved without addition of free sugars, and without losing other desirable properties, such as the ability rapidly to dissolve the dry composition to form a clear resultant solution. The content of excipients, especially any that could potentially promote microbial growth, can be minimized, and non-approved or non-compendial chemicals can be avoided.

The formulation is amenable to preparation by a convenient lyophilization cycle, and can also be prepared by, inter alia, spray drying.

[0029] In typical embodiments, the composition demonstrates at least about 80% of initial serine protease inhibitory activity, preferably at least about 90% of initial serine protease inhibitory activity, upon rehydration following storage under conditions that are, or are equivalent to, 50°C for 3 months; in some embodiments, the formulation retains at least about 80% of initial serine protease inhibitory activity, preferably at least about 90%, upon rehydration following storage for two years at temperatures not to exceed 5°C.

[0030] Accordingly, in a first aspect, the invention provides a dry composition of unglycosylated AAT.

[0031] The composition comprises unglycosylated alpha 1- antitrypsin (AAT) and at least one halide salt.

[0032] In various embodiments, the unglycosylated alpha 1-antitrypsin (AAT) is human AAT, and can be a recombinant human AAT expressed in and purified from Saccharomyces cerevisiae, notably rAAT. In other embodiments, the unglycosylated AAT of the dry compositions of the invention are AAT muteins, truncates or fragments having serine protease inhibitory activity.

[0033] In certain of the dry compositions of the present invention, more than about 90% (w/w) of the composition's protein is unglycosylated AAT. In some, more than about 95% (w/w) of the composition's protein is AAT, even more than about 99% (w/w) of the composition's protein is AAT.

[0034] In some embodiments of the dry composition of the invention, the halide salt is present at a level of at least about 10 micromoles per 100 mg AAT. In various embodiments, the halide salt can be present at a level of least about 50 micromoles per 100 mg AAT, at least about 100 micromoles per 100 mg AAT, even at least about 200 micromoles per 100 mg AAT, and, typically, no more than about 2000 millimoles per 100 mg AAT, 1500 millimoles per 100 mg AAT, or 1000 millimoles per 100 mg AAT.

[0035] In certain embodiments, the halide salt is a chloride salt, typically NaCl.

[0036] The presence of at least one halide salt provides surprising stabilty to the unglycosylated protein during drying and in the dried state. Accordingly, in certain preferred embodiments, the dry composition can be substantially free of carbohydrate.

[0037] In embodiments that are presently preferred, the unglycosylated alpha 1-antitrypsin (AAT) is rAAT and the halide salt is NaCl.

[0038] In some embodiments, NaCl is present at a level of at least about 10 micromoles per 100 mg rAAT, at least about 100 micromoles per 100 mg rAAT, and typically at a level of no more than about 1000 millimoles per 100 mg rAAT. In certain embodiments, NaCl is present at a level of no more than about 500 millimoles per 100 mg rAAT. In one embodiment, NaCl is present at a level of about 200 micromoles per 100 mg rAAT.

[0039] The compositions of the present invention can, in some embodiments, further comprise a reducing agent, such as dithiothreitol, cysteine, glutathione, or N-acetyl cysteine (NAC).

[0040] For example, in some embodiments the dry composition of the present invention comprises NAC at a level of at least about 1 micromole per 100 mg AAT; in some embodiments NAC is present at a level of at least about 10 micromoles per 100 mg AAT. Typically, NAC is present at a level of no more than about 1000 micromoles per 100 mg AAT.

[0041] In various embodiments, the dry composition can further comprise phosphate, and/or citrate.

[0042] In various embodiments, citrate is present at a level of at least about 1 micromole per 100 mg AAT, and in others, at a level of at least about 10 micromoles per 100 mg AAT. In various citrate-containing embodiments, citrate is present at a level of no more than about 100 micromoles per 100 mg AAT.

[0043] The dry composition can, in certain embodiments, contain less than about 2% residual moisture. In various embodiments, the dry composition contains less than about

1% residual moisture, even less than about 0.5% residual moisture.

[0044] The dry composition of the present invention can, in certain embodiments, be dried from a liquid composition having pH less than about 7.5, on occasion from a liquid composition having pH less than about 7.0, and may, in certain embodiments, be dried from a liquid composition have pH of about 6.8 0.2.

[0045] In another aspect, the invention provides a method of preparing a dry composition of unglycosylated AAT.

[0046] The method comprises drying a liquid composition comprising unglycosylated AAT and at least one halide salt sufficiently to achieve a residual moisture content of less than about 5%, often less than about 4%, 3%, even 2% or lower.

[0047] In certain embodiments, the unglycosylated AAT is human AAT, such as rAAT.

[0048] The unglycosylated AAT can be present in the liquid composition at a concentration, prior to drying, of at least about 10 mg/ml, at least about 25 mg/ml, at least about 50 mg/ml, and typically at a concentration less than about 200 mg/ml, in some embodiments less than about 100 mg/ml or 75 mg/ml.

[0049] Often, the halide salt is present in the liquid composition at a concentration, prior to drying, of at least about 10 mM, at times at a concentration of at least about 50 mM, in some embodiments even at a concentration of at least about 100 mM. Typically, the halide salt is present in the liquid composition at a concentration, prior to drying, of no more than about 500 mM, at times no more than about 250 mM, and in some embodiments at a concentration of no more than about 100 mM.

[0050] In certain embodiments of the preparative methods of the present invention, the halide salt is NaCl.

[0051] In various embodiments, the pH of the liquid composition is buffered, as, e. g. , by a phosphate and/or a citrate buffer. In some embodiments, the liquid composition is buffered to a pH at least as high as 6.0, at times at least about 6.5, even at least about 6.8, 6.9, or 7.0. In some embodiments, the liquid composition is buffered to a pH no higher than about 7.5, in some embodiments no higher than about 7.0, and in certain embodiments, to a pH of about 6. 80. 2.

[0052] In various embodiments, the liquid composition may further comprise a reducing agent, such as dithiothreitol, cysteine, glutathione, or N-acetyl cysteine (NAC).

[0053] Because halide salts alone appear adequately to stabilize the unglycosylated AAT during and after drying, in various embodiments of the preparative methods of the present invention the liquid composition lacks detectable sugars, such as sugars covalently attached to the AAT and free sugars.

[0054] In embodiments of the invention in which the unglycosylated AAT is recombinantly produced, the liquid composition can be free from human serum proteins other than AAT.

[0055] Drying can be performed, inter alia, by lyophilization or by spray drying.

[0056] In another aspect, the invention provides a method of treatment using the dry compositions of the present invention. The method comprises administering an effective amount of the dry composition of the invention to a subject, such as a human patient, in need thereof. In

some embodiments, the dry composition is administered by inhalation.

[0057] In a related aspect, the invention provides methods of treatment using unglycosylated AAT in liquid formulation, the liquid formulation obtained by rehydrating the dry compositions of the present invention using a pharmaceutically acceptable diluent. In some embodiments, the solution is administered intravenously.

BRIEF DESCRIPTION OF THE DRAWINGS [0058] The above and other objects and advantages of the present invention will be apparent upon consideration of the following detailed description taken in conjunction with the accompanying drawings, in which like characters refer to like parts throughout, and in which: [0059] FIG. 1 presents FTIR spectra of liquid and solid rAAT in a formulation (917-1) according to the present invention; [0060] FIG. 2 presents FTIR spectral scans of unformulated rAAT (917-11) in the liquid and solid states; [0061] FIG. 3 shows FTIR spectra of rAAT in solid state formulations containing different levels of salt (917-1,3, 4); [0062] FIG. 4 shows the secondary structure of rAAT in a sugar-based formulation (1008-1) and in a salt-based formulation (1008-2) of the invention; and [0063] FIG. 5 illustrates the reversibility of the secondary structure of rAAT to its original structure upon reconstitution of protein lyophilized from a formulation having 100 mM NaCl, and 3 mM L-met.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS [0064] In a first aspect, the invention provides a dry composition of unglycosylated alpha 1-antitrypsin (AAT).

[0065] The composition comprises unglycosylated alpha 1- antitrypsin (AAT) and at least one halide salt.

[0066] The unglycosylated AAT, in typical embodiments, is human AAT.

[0067] Human AAT embodiments include, for example, proteins having serine protease inhibitory activity and the sequence of the human M1V AAT allele, the human M1A AAT allele, the human M2 AAT allele, or the human M3 allele, either with, or in certain embodiments, without the N- terminal signal sequence.

[0068] In other embodiments, human AAT is a protein having serine protease inhibitory activity and a primary amino acid sequence that varies from one of the common alleles described above.

[0069] Human AAT variants can differ from the allelic sequences described above by virtue of the insertion, deletion, and/or substitution of one or more amino acid residues within the sequence. Amino acid sequence variants generally will be at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the allelic sequences described above, as determined by the percent identity reported by the BLAST 2 SEQUENCES tool using the blastp program with default parameters (Matrix: BLOSUM62; open gap penalty: 11; extension gap penalty: 1; gap x dropoff : 50; expect: 10.0 ; wordsize: 3). See Tatiana et al.,"Blast 2 sequences-a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett.

174: 247-250 (1999), incorporated herein by reference in its entirety; the tool is available for use at the NCBI web site.

[0070] In typical embodiments, a human AAT variant of the compositions of the present invention will be more closely related to a known human AAT allelic sequence than to an AAT ortholog from any other species, as determined by using the entire human AAT variant protein sequence as query against the NCBI's protein databases using the BLASTP tool available at the NCBI web site.

[0071] In some embodiments, the human AAT is a truncate or a fragment that comprises the active (that is, serine protease inhibitory) portion of the human AAT or human AAT variants described above. Functionally active portions of AAT are known in the art, Schasteen et al., Mol. Immunol.

28: 17-26 (1991), and truncates and fragments of AAT can readily be assessed for serine protease inhibitory activity by routine use of standard, such as the assays described below.

[0072] The AAT of the compositions of the present invention, including human AAT and functionally active portions thereof, is unglycosylated.

[0073] By"unglycosylated"is intended that the AAT protein of the composition of the present invention lacks the pattern of N-linked carbohydrates of human AAT as obtained from plasma. In typical embodiments, the AAT protein is devoid of all post-translationally added sugar moieties. In other embodiments, the AAT protein contains sugar moieties, but has no more than about 50%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% even no more than about 1% the sugar content, on a weight/weight basis, of human AAT as obtained from human plasma.

[0074] Unglycosylated AAT can be obtained by recombinant expression in a host that does not glycosylate the expressed protein, or by treatment of recombinantly expressed or natural protein with glycosylases.

[0075] The unglycosylated AAT can possess other types of derivatizations.

[0076] In one embodiment, for example, the unglycosylated AAT is rAAT, a human AAT recombinantly expressed in Saccharomyces cerevisiae. rAAT is a 395 amino acid unglycosylated protein having an amino acid sequence identical to human plasma AAT with the exception of an N- acetylmethionine residue at the amino terminus. Rosenberg et al., Nature 312: 77-80 (1984), incorporated herein by reference in its entirety.

[0077] In another embodiment, the unglycosylated AAT is rAAT-val, an oxidation-stable unglycosylated AAT produced in Saccharomyces cerevisiae having an N-acetylmethionine residue at the amino terminus and sequence differing from rAAT by substitution of a valine for methionine at residue 358.

[0078] Whether the unglycosylated AAT of the dry composition of the present invention is produced by expression in a host cell that cannot or does not glycosylate the expressed AAT, or is produced by removal of carbohydrate from AAT expressed recombinantly in a host cell competent for post-translational glycosylation, or is produced by removal of carbohydrate from AAT purified from natural sources, the composition will, in typical embodiments, have less than 10%, more preferably less than 5%, most preferably less than 1%, and in some embodiments, less than 0.5%, 0.4%, 0. 3%, 0.2%, even less than 0.1% (as a weight percentage of protein in the composition) of any one of plasmin inhibitor, a1-antichymotrypsin, C1 esterase inhibitor, antithrombin III, haptoglobin, albumin, a- lipoprotein, and IgA. In preferred embodiments, the dry composition of the present invention will completely lack one or more of plasmin inhibitor, ul-antichymotrypsin,

Cl esterase inhibitor, antithrombin III, haptoglobin, albumin, a-lipoprotein, or IgA [0079] In preferred embodiments, the composition contains less than 10%, 5%, most preferably less than 1%, and in some embodiments, less than 0.5%, 0. 4%, 0.3%, 0.2%, even in some embodiments less than 0. 1% (as a cumulative weight percentage of protein in the composition) of a2- plasmin inhibitor, al-antichymotrypsin, Cl esterase inhibitor, antithrombin III, haptoglobin, albumin, a- lipoprotein, and IgA. In particularly preferred embodiments, the composition completely lacks each of a2- plasmin inhibitor, al-antichymotrypsin, Cl esterase inhibitor, antithrombin III, haptoglobin, albumin, a- lipoprotein, and IgA.

[0080] In particularly useful embodiments, the weight percentage of protein of human plasma proteins other than AAT in the composition is less than 10%, 5%, most preferably less than 1%, and in some embodiments, less than 0. 5%, 0.4%, 0.3%, 0.2%, even in some embodiments less than 0.1%. In particularly preferred embodiments, the composition completely lacks human plasma proteins other than AAT.

[0081] As a weight percentage of protein, the compositions of the present invention typically comprise more than 90%, 91%, 92%, 93%, 94%, even more than 95%, 96%, 97%, 98%, 99%, and in some embodiments, even more than 99. 5%, unglycosylated AAT, or active portion thereof. In some of these embodiments, the unglycosylated AAT is human, and in embodiments that are presently preferred, rAAT.

[0082] The dry compositions of the present invention comprise unglycosylated AAT (or active portions thereof), and at least one halide salt.

[0083] The dry composition may comprise, for example, 0.1 to 2000 milliequivalents halide salt per 100 mg of unglycosylated AAT (such as human AAT, particularly rAAT), 50-500 milliequivalents, even 100-200 milliequivalents halide salt per 100 mg of unglycosylated AAT (such as human AAT, particularly rAAT).

[0084] In typical embodiments of the dry composition of the present invention, the halide salt is present at a level of at least about 10 micromoles per 100 mg AAT, at least about 50 micromoles per 100 mg AAT, at least about 100 micromoles per 100 mg AAT, even at least about 200 micromoles per 100 mg AAT. In some embodiments, the halide salt is present at a level of about 200 micromoles per 100 mg AAT.

[0085] Typically, the halide salt is present at a level that does not exceed about 2000 millimoles per 100 mg AAT, 1500 millimoles per 100 mg AAT, no more than about 1000 millimoles per 100 mg AAT, no more than about 100 millimoles per 100 mg AAT, no more than about 10 millimoles per 100 mg AAT, even no more than about 1 millimole per 100 mg AAT.

[0086] In certain embodiments, the halide salt is present in the dry composition at a level of about 100- 500 micromoles per 100 mg AAT.

[0087] The halide salt can, for example, be a chloride salt, a bromide salt, or an iodide salt. The cation can, e. g., be Na+, K+, or other monovalent or divalent cation.

[0088] In certain embodiments, the halide salt is NaCl.

In a variety of such embodiments, the dry composition of the present invention comprises rAAT and NaCl.

[0089] In certain of these embodiments, the NaCl is present at a level of at least about 10 micromoles per 100 mg rAAT, 50 micromoles per 100 mg rAAT, even at least about

100 micromoles per 100 mg rAAT. In some of the embodiments, NaCl is present at a level of no more than about 1000 millimoles per 100 mg rAAT, no more than about 500 millimoles per 100 mg rAAT, even no more than about 250 millimoles per 100 mg rAAT. In various embodiments, NaCl is present at a level of about 200 micromoles per 100 mg rAAT.

[0090] The dry composition of the present invention may comprise components additional to unglycosylated AAT and at least one species of halide salt.

[0091] In some embodiments, the composition further comprises at least one reducing agent. The at least one reducing agent may, for example, be selected from the group consisting of dithiothreitol, cysteine, glutathione, and N- acetyl cysteine (NAC).

[0092] In certain particularly preferred embodiments, the reducing agent is NAC.

[0093] NAC may be present, e. g. , at a level of at least about 1 micromole per 100 mg AAT, at least about 10 micromoles per 100 mg AAT, even at least about 100 micromoles per 100 mg AAT. NAC is typically present in such embodiments at a level of no more than about 1000 micromoles per 100 mg AAT, no more than about 500 micromoles per 100 mg AAT, even no more than about 100 micromoles per 100 mg AAT.

[0094] The composition may, in addition or in the alternative, further comprise an antioxidant, such as ascorbic acid or L-Met, e. g. in an amount of up to 10 mM on reconstitution to 50 mg/ml AAT.

[0095] The composition may, in addition or in the alternative, further comprise agents useful in buffering the pH of the solution from which the composition is dried,

and in buffering the pH of the solution into which the composition is optionally rehydrated.

[0096] The amount of buffer may be such that, on rehydration of the composition in deionized water to 50 mg/ml AAT, the reconstituted solution has a pH of at least about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 even 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or higher. In some embodiments, the amount of buffer is such that, on rehydration of the composition in deionized water to 50 mg/ml AAT, the reconstituted solution has a pH of no more than about 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, even no more than about 6.9, 6.8, or even no more than about 6.7, 6.6, or 6.5.

[0097] For example, the composition may further comprise phosphate, citrate, histidine or combinations thereof, in some embodiments in an amount of 5-50 mM, preferably 10-20 mM on reconstitution to 50 mg/ml AAT.

[0098] In some embodiments, the composition further comprises organic acids (and salts of organic acids) other than citric acid, including one or more of lactic acid, ascorbic acid, maleic acid, oxalic acid, malonic acid, malic acid, succinic acid, gluconic acid and glutamic acid.

[0099] In some embodiments, the composition may further comprise, in addition to unglycosylated AAT and at least one species of halide salt, one or more detergents or other surface active agents. In particular, dry compositions of the present invention intended for administration by inhalation may comprise one or more surfactants, such as polyoxyethylene sorbitan oleate or combinations of natural or synthetic lung surfactants, such as those described, e. g. , in U. S. Pat. Nos. 4,312, 860; 5,110, 806; 5,260, 284; 5,861, 481; 6,129, 934; and 6,770, 619, the disclosures of

which are incorporated herein by reference in their entireties.

[0100] The compositions of the present invention may further comprise chelating agents (e. g. EDTA), preferably of a type and in an amount that does not significantly chelate either the cation or anion of the halide salt of the composition.

[0101] Presently preferred embodiments will typically not comprise free sugars (that is, sugars that are not covalently linked to the AAT of the composition).

[0102] Thus, typical embodiments of the dry composition of the present invention have less than 0.5%, 0.4%, 0.3%, 0.2%, even less than 0.1%, 0.05%, even less than about 0.01%, on a weight percentage basis, of any one or more of monosaccharides such as galactose, D-mannose, and sorbose; disaccharides such as lactose and trehalose; cyclodextrins such as 2-hydroxypropyl-ß-cyclodextrin ; and polysaccharides such as raffinose, maltodextrins, and dextrans; polyhydric polymers such as polyethylene glycol; and alditols, such as inositol, ribitol, galactitol, and ribitol. In presently preferred embodiments, the dry composition completely lacks free sugars.

[0103] In some embodiments in which the unglycosylated AAT completely lacks sugar moieties, the dry composition will have less than about 0. 5%, 0.4%, 0.3%, 0.2%, even less than 0.1%, 0.05%, even less than about 0.01%, on a weight percentage basis, of any sugar or carbohydrate. In some embodiments, the composition will completely lack detectable sugar or carbohydrate.

[0104] Although the compositions of the present invention may comprise one or more agents additional to unglycosylated AAT and at least one species of halide salt, presently preferred embodiments will typically contain as

few such additional components as necessary to achieve the desired physical form, including residual moisture content, particle size, ease of rehydration, and stability.

[0105] As further described below, the dry compositions of the present invention are conveniently prepared by a variety of known techniques, including lyophilization and spray drying.

[0106] By whatever means prepared, the composition will, in typical embodiments, comprise less than about 5% residual moisture, as assessed, e. g., by the Karl Fischer coulometry method, conveniently performed on an anhydrous methanol extract of the dry composition.

[0107] In various embodiments, the dry composition comprises less than about 4.5%, 4%, 3.5%, 3%, 2.5%, 2.0%, 1.5%, 1% residual moisture. In presently preferred embodiments, the dry composition comprises less than about 0. 9%, 0.8%, 0.7%, 0.6%, 0. 5% and even, in some embodiments, less than about 0.4% residual moisture.

[0108] Preferred size of particles in the dry composition will vary depending upon the proposed use of the dry composition.

[0109] For example, for use directly in inhalation therapy, the particles are usefully less than about 5 ßm MMAD (mass median aerodynamic diameter); MMAD refers to the particle size distribution of the particles of a dispersible powder when so dispersed as an aerosol. MMAD determinations are usefully made using a cascade impactor.

In other such embodiments, the particles are usefully less than about 4 Am MMAD, 3 gm MMAD, and can even be less than about 2 ßm MMAD. In various such embodiments, the particles can be at least about 1 Am MMAD, 2 Am MMAD, even at least 3 Am MMAD or more. Typically, the particles will

range from 1 ßm to 5 Am MMAD for use directly in inhalation therapy.

[0110] In various inhalation embodiments, the particles can usefully be less than about 5 ßm MMD (mass median diameter); MMD refers to the particle size distribution of the particles in bulk powder. MMD determinations can usefully be made using centrifugal sedimentation techniques (e. g., using the Horiba Particle Size Analyzer--Model CAPA-700). In other such embodiments, the particles are usefully less than about 4 Am MMD, 3 ßm MMD, and can even be less than about 2 Mm MMD. In various such embodiments, the particles can be at least about 1 Am MMD, 2 ßm MMD, even at least 3 Am MMD or more. Typically, the particles will range from 1 ym to 5 Am MMD for use directly in inhalation therapy.

[0111] Particles may be further agglomerated to facilitate delivery to an inhaler, as described, e. g., in U. S. Patent Nos. 5,780, 014 and 5,993, 783, the disclosures of which are incorporated herein by reference in their entireties. In such embodiments, the agglomerates are dispersible into component particles having particle diameters (MMAD) of about 1 Am-about 5 Am.

[0112] In embodiments intended to be rehydrated before use--as, for example, by addition of a clinically acceptable sterile diluent, such as sterile water, saline, dextrose solution, D5 normal saline, or Ringer's solution--the particle size range may be greater than that in embodiments intended for direct inhalation.

[0113] In such embodiments, for example, the particles can usefully have MMD of up to 10 Am, 20 Am, 30 Am, 40 Am, even up to 50 gm or more.

[0114] In embodiments of the dry composition of the present invention intended for rehydration before use--

as, for example, for intravenous administration, nebulization, or for further incorporation into an aqueous vehicle, such as a topical gel--the dry composition will be readily dissolvable.

[0115] For example, in some embodiments at least 80% of the solids will dissolve at room temperature within 1 hour of addition of sufficient deionized water to achieve a nominal AAT concentration of 50 mg/ml; in other embodiments, at least 81%, 82%, 83%, 84%, 85% and even as much as 86%, 87%, 88%, 89% even as much as 90% of solids will dissolve within 1 hour of addition of a volume of deionized water sufficient to achieve a nominal concentration of 50 mg/ml AAT. In presently preferred embodiments, at least 91%, 92%, 93%, 94%, even as much as 95%, 96% or more of the solids will dissolve within 1 hour of addition of sufficient deionized water to achieve a nominal concentration of 50 mg/ml AAT.

[0116] The compositions of the present invention are stable; surprisingly so for unglycosylated AAT, particularly in embodiments lacking free sugars.

[0117] In typical embodiments, the dry composition of the present invention demonstrates at least about 80%, 81%, 82%, 83%, 84%, even at least about 85%, 86%, 87%, 88%, and 89% retention of initial serine protease inhibitory activity upon rehydration following storage under conditions that are, or are equivalent to, 50°C for 3 months. In preferred embodiments, the dry composition of the present invention retains at least 90%, even at least 91%, 92%, 93%, 94%, and in the most preferred embodiments, even at least about 95% or more of the initial serine protease inhibitory activity upon rehydration following storage under conditions that are, or are equivalent to, 50°C for 3 months.

[0118] Percent retention of initial activity (stability) can be assessed using assays known to those skilled in the art, such as in vitro elastase inhibition assays, for example using a colorimetric substrate. See, e. g., the porcine pancreatic elastase inhibition assay reported by Beatty et al., J. Biol. Chem. 255: 3931 (1980), the disclosure of which is incorporated herein by reference in its entirety.

[0119] In various embodiments, the dry composition of the present invention retains at least about 80%, 81%, 82%, 83%, 84%, even at least about 85%, 86%, 87%, 88%, and 89% of initial serine protease inhibitory activity, upon rehydration following storage under conditions that are, or are equivalent to, 2 years at s 5°C. In preferred embodiments, the dry composition of the present invention demonstrates at least 90%, even at least 91%, 92%, 93%, 94%, and in the most preferred embodiments, even at least about 95% or more of the initial serine protease inhibitory activity upon rehydration following storage under conditions that are, or are equivalent to, 2 years at s 5°C.

[0120] In preferred embodiments, the dry composition retains at least about 90% of initial serine protease inhibitory activity for more than 2 years at s 5°C, even as much as 3 years at s 5°C.

[0121] Equivalence to the given conditions will be understood by one of ordinary skill in the art (e. g., based on the Arrhenius equation).

[0122] The dry compositions of the present invention, in preferred embodiments, demonstrate low levels of AAT denaturation. Denaturation is monitored by evaluation of aggregate formation, and the non-denatured AAT usefully

reported as % monomer using a size exclusion chromatography (SEC) HPLC method.

[0123] In typical embodiments, the AAT remains about 80%, 81%, 82%, 83%, 84%, even at least about 85%, 86%, 87%, 88%, and 89% monomeric following storage under conditions that are, or are equivalent to, 50°C for 3 months. In preferred embodiments of the dry composition of the present invention, the AAT remains at least about 90%, even at least 91%, 92%, 93%, 94%, and in the most preferred embodiments, even at least about 95% or more monomeric following storage under conditions that are, or are equivalent to, 50°C for 3 months.

[0124] In typical embodiments, the AAT remains about 80%, 81%, 82%, 83%, 84%, even at least about 85%, 86%, 87%, 88%, and 89% monomeric following storage under conditions that are, or are equivalent to, 5°C for 2 years. In preferred embodiments of the dry composition of the present invention, the AAT remains at least about 90%, even at least 91%, 92%, 93%, 94%, and in the most preferred embodiments, even at least about 95% or more monomeric following storage under conditions that are, or are equivalent to, 5°C for 2 years.

[0125] In another aspect, the invention provides methods of preparing the dry compositions of the present invention.

[0126] The method comprises drying a liquid composition comprising unglycosylated AAT and at least one species of halide salt sufficiently to achieve a residual moisture content of less than about 2%.

[0127] In typical embodiments, the liquid composition is dried sufficiently to achieve a residual moisture content of no more than about 2.0%, 1. 9%, 1. 8%, 1.7%, 1.6%, 1.5%, 1. 4%, 1. 3%, 1. 2%, 1.1%, and even no more than about 1.0%, with presently preferred embodiments having even less

residual moisture. Such embodiments may have a residual moisture content of no more than about 0.9%, 0. 8%, 0.7%, 0. 6%, even as low as no more than about 0.5%, or even lower.

[0128] As would be understood, the unglycosylated AAT present in the liquid composition can be any of the species that are described above as being present in the resulting dried composition, including unglycosylated human AAT and rAAT.

[0129] In typical embodiments of the preparatory methods of the present invention, the unglycosylated AAT is present in the liquid composition at a concentration of at least 10 mg/ml, at least 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, even at least about 50 mg/ml, or even higher. In certain embodiments of the preparatory methods of the present invention, the unglycosylated AAT is present in the liquid composition at a concentration of at least 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml even 100 mg/ml or more.

[0130] Typically, the unglycosylated AAT is present in the liquid composition at a concentration of less than about 200 mg/ml, more typically at a concentration of 100 mg/ml or less, even 75 mg/ml or less.

[0131] In certain particularly useful embodiments, the liquid composition from which the composition is dried comprises unglycosylated AAT at a concentration of 30-70 mg/ml, more preferably 40-60 mg/ml, most preferably about 50 mg/ml.

[0132] The at least one species of halide salt can be any of the halide salts described above as present in the resulting dried compositions of the present invention, including NaCl.

[0133] In typical embodiments, the at least one halide salt is present in the liquid composition at a

concentration of at least about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, even at least about 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and even higher, including 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM or more. In various embodiments, the halide salt is present in the initial solution at a concentration of no more than about 300 mM, typically no more than about 250 mM, 200 mM, 150 mM, 100 mM, or even lower. In certain particularly useful embodiments, the solution from which the composition is dried comprises halide salt at a concentration of 50-150 mM, more preferably 75-125 mM, most preferably at a concentration of about 100 mM.

[0134] In some embodiments, the at least one halide salt is present in the liquid composition at a concentration of at least 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, even at least about 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM or more.

[0135] Typically, the at least one halide salt is present in the liquid composition at a concentration of no more than about 500 mM, 450 mM, 400 mM, 350 mM, even no more than about 300 mM. In some embodiments, the at least one halide salt is present in the liquid composition at a concentration of no more than about 250 mM, 200 mM, 150 mM, even in some instances no more than about 100 mM.

[0136] In some embodiments, the pH of the liquid composition is buffered, and the dry compositions are prepared by drying a solution comprising unglycosylated AAT and at least one halide salt buffered to a pH of at least 6.5, 6.6, 6.7, 6.8, 6.9, even at least 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5. In various embodiments, the solution to be dried is buffered to a pH of no more than about 7.5, 7.4, 7.3, 7.2, 7.1, even no more than about 7.0, 6.9, 6.8, 6.7,

6.6, or 6.5. In presently preferred embodiments, the composition of the present invention is dried from a solution buffered to a pH of 6.6-7. 0, more preferably 6. 7 - 6. 9, most preferably 6.8 0.1.

[0137] The buffer can, in some embodiments, be a phosphate buffer, a citrate buffer, a histidine buffer, or combinations thereof.

[0138] As would be understood from the description of the dry compositions set forth above, the liquid composition from which the dry composition is prepared may further comprise a reducing agent, such as a reducing agent is selected from the group consisting of dithiothreitol, cysteine, glutathione, and N-acetyl cysteine (NAC), and/or an antioxidant.

[0139] As would also be understood from the description of the dry compositions of the invention hereinabove, in typical embodiments the liquid composition lacks detectable sugars, including free sugars and/or sugars covalently pending from the AAT protein, and is free from human serum proteins other than AAT, such as plasmin inhibitor, al- antichymotrypsin, Cl esterase inhibitor, antithrombin III, haptoglobin, albumin, a-lipoprotein, and IgA.

[0140] In embodiments in which recombinantly expressed unglycosylated AAT, such as rAAT, is used, the liquid composition to be dried in the preparatory methods of the present invention does not require viral inactivation, either by heating, e. g., at 60°C or 65°C, or by treatment with solvent detergent mixtures and nanofiltration.

Neither does the solution obtained therefrom after drying and optional subsequent rehydration.

[0141] The liquid composition may be dried by methods well known in the art, such as: lyophilization ; spray drying; lyophilization followed by milling to micronize the

lyophilisate; atomization onto a cold surface, followed by sublimation and collection of the micronized powder; evaporative drying of a non-frozen solution in a vacuum oven or centrifugal evaporator maintained at temperatures where the solution does not freeze, followed by milling; spray coating; spray freeze-drying; fluidized bed technology; super critical fluid drying; agglomeration; extrusion, and combinations thereof. See, e. g., Maa et al. , "Biopharmaceutical powders: particle formation and formulation considerations,"Curr. Pharm. Biotechnol.

1: 283-302 (2000), the disclosure of which is incorporated herein by reference in its entirety.

[0142] Lyophilization may usefully be used in the preparatory methods of the present invention, particularly for embodiments of the dry composition intended for rehydration before use--as, for example, for intravenous administration, for nebulization, or for further incorporation into an aqueous vehicle, such as a topical gel.

[0143] Protein lyophilization methods are known in the art. See, e. g., Carpenter et al.,"Rational design of stable lyophilized protein formulations: theory and practice,"Pharm. Biotechnol. 13: 109-133 (2002); Carpenter et al. ,"Rational design of stable lyophilized protein formulations: some practical advice,"Pharm Res. 14 (8): 969- <BR> <BR> 75 (1997); Rey et al. (eds. ), Freeze-Drying/Lyophilization of Pharmaceutical and Biological Products (Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs), Marcel Dekker (2nd rev. & ex. edition, 2004) (ISBN : 0824748689), the disclosures of which are incorporated herein by reference in their entireties.

[0144] Lyophilization apparatuses are commercially available.

[0145] For example, lyophilization can be performed using a Genesis Pilot-scale Virtis 12XL lyophilizer equipped with three stoppering shelves and an external condenser. Parameters such as ramp rate, shelf- temperature, time, and vacuum can be programmed into the cycle run and the product temperatures recorded using four available thermocouples by the Wizard control system software provided by the Virtis Company.

[0146] The lyophilization cycle can usefully be one typically used for amorphous formulations, as shown below in Table A.

Table A Exemplary Lyophilization Cycle Step Temperature Time (min) Ramp/Hold Freezing +5°C 60 Hold -45°C 100 Ramp -45°C 240 Hold Drying at lOOmT chamber pressure-45°C 60 Hold -15°C 240 Ramp - 15°C 1150 Hold +40°C 240 Ramp +40°C 600 Hold [0147] Such a lyophilization cycle is a conservative cycle with primary drying performed at a shelf temperature of-15° C that maintains the product temperature around - 30°C, well below the collapse temperature of typical formulations of the present invention (eutectic melting temperature of NaCl). In this cycle, moisture content is minimized by raising the secondary drying temperature to 40°C for a period of 10 hours.

[0148] Production scale lyophilization apparatuses are also available commercially.

[0149] Spray drying may also usefully be used in the preparatory methods of the present invention, particularly for embodiments of the dry composition intended for

direct--that is, dry--administration of unglycosylated AAT by inhalation, and for bulk preparation of unglycosylated AAT.

[0150] Spray-drying consists of a three-step process which results in dry particle formation. The process begins by atomizing a liquid feed into a spray of fine droplets using compressed air, followed by heating media in order to dry the droplets by evaporating the moisture content of the droplets. The final particles, in the form of dry powder, are collected as product; the gas and the excess fine dust are exhausted. These steps are carried out using three components: the atomizer in shape of a nozzle; the drying chamber; and the collecting system known as cyclone and pot.

[0151] Spray drying apparatuses are available commercially. For example, spray drying in the methods of the present invention can usefully be performed using a Buchi spray drier B-191 or B-290 (Brinkmann Instruments, Inc.).

[0152] Spray drying may optionally be followed by further drying, and optionally by agglomeration. Spray drying may usefully be performed, e. g., as described in U. S. Pat. Nos. 5,780, 014; 6,258, 341; 6,309, 671, and 6,589, 560, and European patent no. EP 0 941 067 B1, the disclosures of which are incorporated herein by reference in their entireties, or according to the protocols set forth in Example 5, hereinbelow.

[0153] Spray coating may also be performed in the preparatory methods of the present invention, and may be performed using standard techniques. See, e. g., Maa et al.,"Spray-coating for biopharmaceutical powder formulations : beyond the conventional scale and its

application,"Pharm Res. 21 (3): 515-23 (2004), incorporated herein by reference in its entirety.

[0154] The dry composition, or, after rehydration in a clinically acceptable diluent, the resulting solution, is suitable for administration to a subject, such as a human patient, in need thereof.

[0155] Accordingly, in a further aspect, the invention provides methods of treatment.

[0156] In one series of embodiments, the method comprises administering an effective amount of the dry composition of the present invention to a subject, such as a human patient, in need thereof. In another series of embodiments, the method comprises dissolving the dry composition of the present invention using a pharmaceutically acceptable diluent to create a solution, and then administering an effective amount of the solution to a subject, such as a human patient, in need thereof.

[0157] Suitable routes of administration include, but are not limited to, inhalation, intranasal, topical, sub- cutaneous, intramuscular, and intravenous delivery.

[0158] Inhalation--delivery of an effective amount of unglycosylated AAT directly to the air-proximal luminal surface of lung alveoli--may be effected by nebulization, dry powder inhaler, or metered dose inhaler, as is well known in the art. For delivery devices and methods, see, e. g., U. S. Patent Nos. 4,137, 914; 4,174, 712; 4,524, 769; 4,667, 688; 5,672, 581; 5,709, 202; 5,780, 014; 5,672, 581; 5,915, 378; 5,993, 783; 5,997, 848; 6,123, 068; 6,123, 936; 6,258, 341; 6,397, 838; 6,681, 767; and 6,796, 303, the disclosures of which are incorporated herein by reference in their entireties.

[0159] Inhalation delivery is appropriate for diseases in which protease activity--such as serine protease

activity--in the lungs contributes to disease etiology, as, e. g., in emphysema, either hereditary or acquired.

[0160] Depending on severity of disease, the daily dose of unglycosylated AAT by inhalation can be from about 1 mg to about 650 mg, or from about 5 mg to about 200 mg, or from about 5 mg to about 100 mg, or from about 10 mg to about 80 mg, or from about 25 mg to about 70 mg, or about 65 mg, in liquid solution or dry composition (such as a dry powder composition), to treat or to prevent (e. g., eliminate the appearance, increase the time to appearance, delay or slow the development, and/or decrease the number and severity of clinical or other manifestations) lung diseases, such as emphysema. In one embodiment, the daily dose of unglycosylated AAT is about 65 mg.

[0161] In typical embodiments, the individual will administer unglycosylated AAT until a cumulative dosage is reached that achieves the desired result. As will be appreciated by the skilled artisan, the size of a single dose of unglycosylated AAT to be delivered by pulmonary administration depends on the form of unglycosylated AAT used (i. e. , liquid or dry), the likely volume to be inhaled, and in the case of a liquid, the solubility of the AAT.

[0162] Dose frequency may be from once daily (QD), twice daily (BID), three times daily (TID), four times daily, six times daily, eight times daily, or more than eight times daily. In some embodiments, dosing will be less frequent than QD.

[0163] In various embodiments, therapy with AAT will be combined with administration of one or more additional agents that act additively or synergistically. In some of these embodiments, the one or more additional agents may be

included with AAT in a composition for pulmonary delivery by inhalation.

[0164] The composition of the present invention may, in other therapeutic embodiments, be administered topically.

[0165] Topical delivery can usefully be accomplished by incorporating the dry composition of the present invention--either directly, or following rehydration in an aqueous solution--within a pharmaceutically acceptable gel, ointment, cream, lotion, or mousse vehicle.

Compositions and topical vehicles useful in these embodiments are described, e. g., in copending international application no. PCT/US2004/015907, the disclosure of which is incorporated herein by reference in its entirety.

[0166] Topical delivery can be used, e. g., to treat skin lesions in which serine protease activity contributes to disease etiology, including various forms of dermatitis, including atopic dermatitis.

[0167] Topical delivery to the external ear canal can be used to treat otitis media with compromise of the tympanic membrane, including otitis in which Pseudomonas aeruginosa is present ; topical delivery to the eye can be used to treat opthalmic inflammation and infection.

[0168] Intravenous delivery can be used to treat hereditary emphysema using the dosage regimen already established for IV treatment using any one of PROLASTIN, ARALAST, and ZEMAIRA" (Aventis Behring LLC).

[0169] The following Examples are intended by way of illustration, not by way of limitation. The following abbreviations (not described above) are used in the Examples--NaPi : sodium phosphate; Tw80. Tween 80; FTIR : Fourier transform infrared spectroscopy; His : Histidine.

EXAMPLE 1 Conformational Stability of Lyophilized Formulations of rAAT [0170] In order to assess the conformational stability of rAAT in the dried state, FTIR spectra were collected on the formulations presented in Table 1.

Table 1 Sample [AAT] NaPi His NaCl Citrate NAC L-met ID mg/ml P mM MM mM MM mM mM 917-1 so 7 20 0 175 5 2. 5 3 917-3 50 7 20 0 100 5 2. 5 3 917-4 50 7 20 0 50 5 2. 5 3 917-11 50 7. 4 20 00000 [0171] It has previously been shown that retention of native structure in the solid state can be predictive of long-term storage stability for dried proteins (Carpenter et al.,"Rational design of stable lyophilized protein formulations: theory and practice,"Pharm. Biotechnol.

13: 109-133 (2002)).

[0172] FIG. 1 shows the FTIR of liquid and solid rAAT in Formulation 917-1. Note that the amide I region (1700-1600 cm-1) is sensitive to changes in secondary structure and that all peaks in the second derivative spectra are negative. Each peak in the amide I region corresponds to a different secondary structural type. There are clearly perturbations of the rAAT conformation before and after lyophilization. The peak near 1655 cm-1 corresponds to a- helical structure, the band near 1635 cm-1 corresponds to ß-sheet structure, and the 1688 cm-1 peak arises from extended ß-strands or ß-sheets. Random coil structure is assigned to bands near 1644 cm-1.

[0173] The liquid sample, representing the native conformation, displays a significant amount of A-sheet and

a-helical structure. Upon lyophilization without stabilizers (formulation 917-11), there is significant structural perturbation as shown in FIG. 2. The a-helix band is almost completely lost, while there are marked increases in bands above 1680 cm-1, corresponding to extended and loop structures.

[0174] FIG. 3 shows the effect of salt on rAAT structure in the solid state. Formulations 917-1, -3 and-4 contain 175 mM, 100 mM and 50 mM NaCl, respectively, in addition to 20 mM sodium phosphate, 5 mM citrate, 2.5 mM NAC, and 3 mM L-Met. Formulations 3 and 4, which have the lower salt concentrations, appear to have the greatest degree of structural perturbation and all three formulations are less perturbed than when no stabilizers are present.

Overall, it appears that lyophilization produces some structural perturbation compared to the native conformation. The extent of the changes is minimized by the addition of excipients, including salt.

[0175] It appears that a NaCl concentration above 50 mM produces a more native-like structure, with a 50-100 mM optimum. The result is unanticipated, since sugars are usually required or used to maintain native protein structure in the dried state.

[0176] Conformational stability of these formulations was also assessed by FTIR in order to elucidate any subtle differences between the rAAT structure in the dried state.

[0177] FIG. 4 shows the FTIR spectra of rAAT formulated in a sugar-based formulation (1008-1) and in a salt-based formulation (1008-2). The secondary structure of rAAT in both these formulations is superimposable. The fact that salt can accomplish the same degree of stabilization with protein at high concentrations is remarkable and

surprising. Upon reconstitution, the original rAAT secondary structure is retained, as shown in FIG. 5.

EXAMPLE 2 Stability Analyses of rAAT Lyophilisates [0178] Based on the surprising observations of conformational stability of lyophilized rAAT formulations containing high levels of NaCl, stability analyses were done in order to evaluate systematically whether addition of common stabilizers in lyophilized protein formulations enhances the conformational stability and acute stability (3 month storage at 60°C) of rAAT.

[0179] Sugars are commonly used in protein formulations to stabilize the molecule, presumably by substituting for the H-bonding following removal of the water molecules around the protein during lyophilization. Sugars also offer an amorphous environment in the dry state that promotes conformational stability of the protein, and they effectively replace the water of hydration removed during drying.

[0180] Surfactants are also often employed in protein formulations to reduce surface adsorption that may damage the protein. Since a possible administration route for rAAT is pulmonary delivery via aerosolization, the effect of surfactant is especially of interest. Therefore, the role of polyoxyethylene sorbitans, such as polysorbate 80 (Tween 80), at various concentrations was also evaluated.

Table 2 NaPi Trehalose Sucrose Tw80 NaCl L-met NAC Citrate MM mM MM mM mM 1008-1 7. 4 10 5 0 0. 02 0 5 0 0 1008-2 6. 810000100 300 1008-3 6. 8 10 0 0 0. 02 100 3 0 0 1008-4 6. 8 10 2. 5 0 0 100 3 0 0 1008-5 6. 8102. 500. 02 100 3 0 0 1008-6 6. 8 10 2.5 0 0 100 3 0 0 1008-7 6. 8 10 0 2. 5 0. 02 100 3 0 0 1008-8 7. 4 10 0 0 0 100 3 0 0 1008-9 6. 810000100 32. 5 5 1008-10 810010100 300 1008-116. 81002. 50100 3 0 0 1008-126. 810050100 300

[0181] The lyophilized formulations were evaluated for short-term stability (at 1 and 3 months) under accelerated storage conditions at 60°C. It should be noted that this storage temperature is particularly harsh for evaluating protein stability and may bias the results towards the trehalose-based formulations that have a particularly high glass-transition temperature (Tg). The rationale for choosing this temperature was based on previous stability studies that assessed rAAT stability over shorter time frames. The activity and percent monomer recovered were determined for up to 3 months storage at 60°C, as shown in Tables 3 and 4, respectively.

Table 3 Specific Activity of rAAT (IU/mg) Pre-lyo Lyo Moisture Lyo Lyo (1 month (1 month (3 months RT) 60°C) 60°C) Sample ID Liquid control 3.75 1008-1 3. 6 3.45 0. 4% 3. 42 2. 4 1008-2 3. 69 2. 83 1. 4% 2. 89 2 1008-3 4. 02 3. 22 3. 21 2 1008-4 3. 57 3. 47 0. 6% 3. 31 2. 6 1008-5 3. 67 3. 21 3. 24 2. 7 Table 3 Specific Activity of rAAT (IU/mg) Pre-lyo Lyo Moisture Lyo Lyo (1 month (1 month (3 months RT) 60°C) 60°C) 1008-6 3. 7 3. 10 3. 43 2. 5 1008-7 3. 89 3. 08 3. 22 2. 6 1008-8 3. 43 3. 15 3. 09 2. 3 1008-9 3. 57 3. 16 3. 23 2. 5 1008-10 3. 38 3. 16 3. 12 2. 7 1008-114. 043. 280. 4% 3. 2 2. 7 1008-12 3. 31 3. 31 3. 18 3. 0 Table 4 Percent Monomer by Size Exclusion (SEC) HPLC Pre-lyo Lyo Lyo Lyo (1 month RT) (1 month 60°C) (3 month 60°C) Sample ID liquid control 97.6 1008-1 98. 2 97. 02 96. 7 74. 71 1008-2 97. 4 95. 65 96. 7 65. 57 1008-3 97. 4 96. 03 94. 89 60. 62 1008-4 97. 4 96. 04 96. 3 85. 85 1008-5 97. 4 96. 36 96. 07 86. 94 1008-6 97. 3 96. 73 96. 16 83. 2 1008-7 97. 4 96. 33 95. 29 86. 76 1008-8 97. 5 96. 3 94. 26 65. 46 1008-9 97. 1 96. 03 93. 12 74. 32 1008-10 97. 2 96. 3 94. 35 84. 29 1008-11 97. 3 95. 72 95. 03 82. 83 1008-12 97. 2 96. 19 95. 96 5. 41

[0182] No significant differences were observed in any of the formulations tested after 1 month, suggesting that both sugar-containing and sugar-free formulations offer comparable stability.

[0183] The stability data after storage for 3 months at 60°C display more variable results. It appears that formulations containing both sugar and salt have a better stability profile than those containing either sugar or salt alone. These data are consistent with FTIR studies that show a high degree of retention of secondary structure in these types of formulations. The low specific activity seen in formulation 1008-2 may be due to the moisture content in that formulation, which is almost 1% higher than

that determined in the other selected formulations. This suggests that stable lyophilized rAAT formulations should preferably have a moisture content below 1 %.

[0184] These results suggest that rAAT is a relatively stable protein in the presence of halide salts and may not require exogenous free sugars for stabilization in the lyophilized state.

EXAMPLE 3 Accelerated Stability Testing of Final Formulation Candidates [0185] A six month study including extensive characterization and stability assessment of the following formulations was performed.

Table 5 Formulation Description of Lyophilization Candidates mg/ml mM mM % MM mM mM % Treh Form. rAAT PH NaPi NaCl alos L-met NAC Citrate Tween80 e 1 50 7.4 10 02. 5 0 0 0. 1 2 50 6.8 10 100 0 0 0 0 0 3 50 6.8 20 100 0 0 5 1 0 4 50 6.8 10 100 0 3 0 0 0 [0186] Tables 6 and 7 summarize the biochemical characterization of these formulations at-70°C, 5°C, 25°C, 40°C and 50°C for up to 6 months.

Table 6 % Initial Activity of rAAT at stated month % Initial Activity at month: 0 1 2 3 4.5 6 - 70°C 1 4308C 100 77 80 85 87 73 2 4304B FB NaPi 100 95 100 95 90 105 3, 4304B FB NAC 100 94 94 94 90 86 4 4304B FB L-Meth 100 98 94 92 89 103 5°C 0 1 2 3 4.5 6 1 4308C 100 77 - 83 87 75 Table 6 % Initial Activity of rAAT at stated month ,. % Initial Activity at month : 0 1 2 3 4. 5 6 2 4304B FB NaPi 100 93 91 90 106 3 4304B FB NAC 100101969292 4 4304B FB L-Meth 100 94 91 89 102 25°C 0 1 2 3 4.5 6 1 4308C 100 79 - 88 83 76 2 4304B FB NaPi 100 100-96 93 106 3 4304B FB NAC 100101969082 4 4304B FB L-Meth 100 98-93 89 97 40°C 0 1 2 3 4. 5 6 1 4308C 100 79 81 84 84 71 2 4304B FB NaPi 100 98 106 91 91 100 3 4304B FB NAC 100 98 95 95 90 77 4 4304B FB L-Meth 100 89 91 89 86 96 50°C 0 1 2 3 4.5 6 1 4308C 100 74 79 78 77 61 2 4304B FB NaPi 100 103 105 90 86 91 3 4304B FB NAC 100 100 95 90 85 75 4 4304B FB L-Meth 100 97 95 86 84 Table 7 Percent Monomer by SEC at stated month %Monomer (280nm) at month@ Formulation 0 1 2 3 4. 5 6 - 70°C 1 4308C 97. 2 96.0 76.0 98.4 94.0 93.0 2 4304B FB NaPi 99.2 99.0 95.7 98.8 97. 4 97.7 3 4304B FB NAC 99. 7 99.2 96.2 98.7 97.5 97.9 4 4304B FB L-Meth 98. 9 99.7 95.3 98.9 97.6 97.6 RS-002 99. 5 99.6 99.4 99. 3 99.0 99.2 5°C 0 1 2 3 4. 5 6 1 4308C 97. 2 94. 6 94. 3 93.5 92.8 2 4304B FB NaPi 99. 2 98.9 98.6 97.5 97.5 3 4304B FB NAC 99. 7 99. 7 98. 6 97.5 97.7 4 4304B FB L-Meth 98. 9 99. 7 98. 6 97.1 97.5 RS-002 99. 5 99. 6-99. 3 99.0 99.2 25°C 0 1 2 3 4. 5 6 1 4308C 97. 2 96. 9 92. 4 90.4 88.7 2 4304B FB NaPi 99.2 98. 7-98. 1 96. 3 96.4 3 4304B FB NAC 99. 7 98. 8 - 98.2 96.8 93.5 4 4304B FB L-Meth 98. 9 99. 7 98. 2 96.2 96.5 RS-002 99. 5 99. 6 99. 3 99.0 99.2 40°C 0 1 2 3 4.5 6 1 4308C 97. 2 88.2 68.7 86.7 84.4 82.0 2 4304B FB NaPi 99.2 97.9 90.5 96.4 93.9 92.5 3 4304B FB NAC 99. 7 98.3 89.5 97.0 94.5 94.5 4 4304B FB L-Meth 98. 9 98.1 92.6 96.5 94.0 92.7 RS-002 99. 5 99.6 99.4 99.3 99.0 99.2 50°C 0 1 2 3 4.5 6 1 4308C 97. 2 80.5 60.2 77.5 75.2 69.4 2 4304B FB NaPi 99.2 96.5 84.5 92.9 89.2 85.0 3 4304B FB NAC 99. 7 96.5 90.8 94.1 89.6 88.4 4 4304B FB L-Meth 98. 9 96.4 84.9 93.7 89.2 85.8 RS-002 99. 5 99.6 99.4 99.3 99.0 99.2

[0187] A preliminary analysis of the 6-month data set was performed and Table 8 summarizes the predicted shelf lives at four storage temperatures: 5°C, 25°C, 40°C and 50°C. The analysis was performed using the percent monomer data, as this assay has less variability than the activity assay (1% vs. 10% coefficient of variation) and is one of the stability-indicating assays.

[0188] The criterion for desirable shelf-life was predetermined to be 90% of the starting value.

[0189] The results suggest that the salt-based formulations are more stable than the trehalose-based formulation. Differences between the salt-based formulations are minor and difficult to distinguish, suggesting that the additional excipients (NAC, citrate, and L-Met) play a lesser role than NaCl in stabilizing the lyophilized product.

Table 8 Predicted Shelf Lives of Formulations from Arrhenius Plots Formulation 90% Monomer Predicted Shelf Life 1 16 Months 234 Months 3 2 6 Months 4 29 Months EXAMPLE 4 Additional Real-Time Stability Studies [0190] Formulation 3 was analyzed after 15 months of storage at 5°C by SEC for % monomer content. The product remains stable with a % monomer value of 98.8 0. 01 (n=6).

EXAMPLE 5 Spray Dried formulations [0191] Recombinant, yeast-produced, unglycosylated human AAT (rAAT) was spray-dried from various formulations and under various conditions using a Buchi B-191 laboratory scale spray dryer fitted with a two-fluid nozzle. The activity of the resulting dry powder was assayed to evaluate the rAAT potency after drying.

[0192] Table 9 presents the formulations of rAAT solutions (pre-drying) ; Table 10 presents the spray drying parameters; Table 11 presents the data from these experiments.

Table 9 Formulation pH [rAAT] NaPi NaCl NAC Citrate L-Met mg/mL Mm Mm mm Mm Mm ARV-8 6. 8 10 10 100 0 0 3 ARV-9 6. 8 1010100 2. 5 5 3 ARV-13 6. 8 10 10 100 5 1 Table 10 Spray Drying Parameters used for rAAT Run reed Composition Spray Drying Conditions rAAT Solid Temp Feed Atom Air Formulation (ml) Diluent mg Additive Content Inlet/Outlet Flow Pressure (ml) (%) (mg (%)) % w/v °C (ml/min) (Bar) (mg) 1 ARV-8 (5) Water 500 None 2.2 110/75 2 6 (20) (93) (545.14) 2 ARV-8 (2.5) Buffer 250 None 2.7 110/77 1.8 5.5 (10) (73) (342. 76) 3 ARV-8 (2.5) Buffer 250 None 2. 0 110/77 1. 8 5. 5 (22.5) (50) (498.94) 4 ARV-8 (2.5) Buffer 250 None 2. 0 80/63 1 7 (22.5) (50) (498.94) 7 ARV-13* (25) 0 250 None 1. 98 80/62 1 7 (51) (494.15) 8 ARV-132 (25) 0 250 None 1. 98 110/77 1. 8 5. 5 (51) (494.15) 9 ARV-9 (25) Buffer 250 None 2.0 80.62 1 7 (22.5) (49) (508.6) Table 10 Spray Drying Parameters used for rAAT Run Feed Composition Spray Drying Conditions Solin rAAT Temp Feed Atom Air Diluent Additive Content Formulation (ml) Inlet/Outlet Flow Pressure (ml) (mg (%)) % w/v (%) °C (ml/min) (Bar) (mg) (22. 5) (49) (508.6) 10 ARV-9 (25) Buffer 250 None 2. 0 (508.6) 1. 8 5. 5 (22.5) (49) 11 ARV-9 (25) External Buffer 250 None 2. 6 80/64 1 2 Nozzle Configuration (12.5) (64) (391.05) 12 ARV-9 (2.5) Buffer 250 None 2.6 80/63 1 1 (12.5) (64) (391.31) 15 ARV-8 (25) 0 250 None 2 100/77 1.8 5. (50) (498.9) 16 ARV-13 (25) 0 250 None 1. 98 105/75 1. 8 5. 5 (51) (498.2) 1 7 ARV-13 (25) 250 None 1. 98 80/64 7 (51) (498.2) 18 ARV-8 0 250 280/64 1 7 (50) (498.9) Scaled ARV-13 Buffer (50) None 1.98 80/61 1 7 Up log run** 2. 5g ARV-13 (337) Buffer (3370) None 1.98 80/61 1 7 AAT (already (50) added) Table 11 Formulation Spray Dry Temp Conditions Specific Activity Inlet/Outlet °C/°C U/mg AVR-8 Retain 3. 3 ARV-8 110/77 2. 2 ARV-8 80/63 2.9 AVR-9 Retain 4.5 ARV-9 80/62 4. 0 ARV-9 110/77 4. 4 AVR-13 Retain 4. 9 ARV-13 80/62 2. 9 ARV-13 110/77 4. 3

[0193] All references cited throughout the specification, and the references cited respectively

therein, are hereby expressly incorporated by reference herein.

[0194] While the foregoing invention has been described in some detail by way of illustration and example, it would be understood by those skilled in the art that various changes may be made, equivalents substituted, and embodiments combined without departing from the true spirit and scope of the invention. All such modifications and equivalents are within the scope of the present invention as defined by the following claims and their equivalents.