WO2016179365A1 - Nanocarriers for delivery of alpha-1-antitrypsin - Google Patents

Abstract

Provided is a composition, comprising an alpha-1-antitrypsin (A1AT) and an anti-inflammatory agent encapsulated in a nanocarrier that comprises on its surface an anti-oxidant agent.

Description

NANOC ARRIKRS FOR DELIVERY OF AUM I A-l -ANTITRY PSIN

FIELD OF THE INVENTION

Compositions and methods for administering alpha- 1 -antitrypsin (A1AT) to the lungs of a subject are disclosed that involve encapsulating Al AT within the core of a nanocarrier so that it may be protected until it is selectively taken up by cells of the respiratory tract. In some embodiments, an anti-oxidant, such as N-acetyl cysteine (NAC), can be conjugated to the nanocarrier surface to protect Al AT from intracellular oxidation. In some embodiments, the disclosed composition further comprises an anti-inflammatory agent, such as dexamethasone, loaded into the nanocarrier. For example, disclosed is a multi-drug loaded nanocarrier containing Al AT, NAC, and dexamethasone.

All documents cited or relied upon below are expressly incorporated herein by reference.

BACKGROUND

In recent years intravenous Al AT augmentation therapy has been employed to restore

Al AT in genetically-deficient patients (Z-Al AT). Restoration of physiological concentrations of Al AT in these patients is thought to restore protection of the lungs from neutrophil elastase, and hence slow the aggressive form of emphysema seen in these patients. This approach could undoubtedly benefit all emphysema patients, however, the high cost associated with intravenous therapy has prohibited its large-scale implementation. Despite these cost barriers, a strong rationale for augmenting non-genetic deficient emphysema patients (M-Al AT) has begun to attract attention. M-Al AT emphysema patients are not systemically deficient in Al AT, but have a local lung 'functional' deficiency induced by oxidative modification of Al AT by cigarette smoke exposure and reactive oxygen species (ROS) generated by the increased lung

inflammatory burden seen in these patients. This 'functional' deficiency renders patients prone to protease-mediated injury similar to that seen in Z-Al AT. Therefore, augmenting M-Al AT patients locally (directly into the lungs) may go a long way in restoring this protease / anti- protease imbalance induced by oxidative inactivation. In addition to Al AT canonical (anti- neutrophil elastase activity) functions numerous non-canonical functions have recently been described. Properties include inhibition of apoptosis, TNF-a biosynthesis, and immunomodulatory functions, such as antigen presentation and autoantibody levels, can all be modified directly by Al AT, and all play roles in the pathogenesis of emphysema. Therefore, given these powerful non-canonical anti-inflammatory functions of A1AT, augmentation of M- Al AT patients with locally delivered Al AT could potentially increase local functional Al AT levels, and further reduce inflammation via its non-canonical anti-inflammatory functions. The lungs of M-AIAT patients have a heavy oxidative burden, and the inhalation of large doses of Al AT could lead to the generation of oxidized Al AT, which is extremely pro-inflammatory. Therefore, while therapy has the potential to reduce inflammation, it also has the potential to potentiate the inflammatory response due to the oxidative microenvironment of the lungs of M- Al AT patients.

SUMMARY

Compositions and methods for administering A1AT to the lungs of a subject are disclosed that involve encapsulating Al AT within the core of a nanocarrier so that it may be protected until it is selectively taken up by cells of the respiratory tract.

In some embodiments, an anti-oxidant, such as N-acetyl cysteine (NAC), can be conjugated to the nanocarrier surface to protect Al AT from intracellular oxidation. Further, studies show that oxidative stress promotes steroid resistance, thus minimizing steroid efficacy in some patients. Therefore, in some embodiments, the disclosed composition further comprises an anti-inflammatory agent, such as dexamethasone, loaded into nanocarrier. For example, disclosed is a multi-drug loaded nanocarrier containing these three distinct therapeutic agents (e.g., Al AT, NAC, and dexamethasone).

In some embodiments, the nanocarrier is a micelle, liposome, or polymeric nanoparticle. For example, the nanocarrier can be pH sensitive, temperature sensitive, or a combination thereof.

Also disclosed are methods for alpha- 1 -antitrypsin (Al AT) augmentation in a subject that involve administering to the subject an effective amount of the disclosed nanocarrier

compositions. This technology has broad application to all acute and chronic lung diseases, transplantation, ischemia reperfusion injury, and autoimmune disorders. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

Figure 1 shows liposome conjugates encapsulated DAPI. LNC conjugated with the tracking dye DyLight 680 entered the cells (Fig. IB), but when DAPI, a nuclear stain, is successfully encapsulated into the core of LNC, very little of the membrane-permeant DAPI is able to escape from the LNC and enter into the nuclei of live cells (Figs. 1A, 1C).

Figure 2 is a schematic of liposomal formulation synthesis.

Figure 3 shows Western blots from supernatants collected from both the basolateral supernatants of endothelial cells cultured in the presence or absence of cigarette smoke extract (CS) and incubated with Al AT or Al AT plus NAC. Cells were apically treated with Al AT to analyze the impact of CS on Al AT migration to the basolateral side. Note that CS exposure reduces migration and that NAC restores Al AT trafficking. Representative image of n=3

Figure 4 shows 3T Liposomes improve Al AT transcytosis in Human Bronchial

Epithelial cells (NHBEs). HBMEs were treated apically with 20μΜ Al AT, 1 μΜ NAC, of 3T liposomes containing 20μΜ (Al AT, ΙμΜ NAC) in the presence of 7.5% Cigarette smoke extract media. Al AT concentrations were measured in basal culture media at different time points by western blot and results expressed as relative density. Note that Al AT transcytosis is improved by 3T liposomal delivery. Representative of n=3. Figure 5 shows mouse lungs from mice nebulized with free drug or 3TL. Lungs were removed at 6 and 24hrs and imaged with Maestro ex vivo imaging. Dylight 680 labeled 3TL constructs, 6 and 24 hrs post nebulization, show increased fluorescent signal in left and right lungs of 3TL as compared to the auto fluorescence seen in sham and free Dex/Al AT treated mice. Representative of n=2. DETAILED DESCRIPTION

Although intravenous augmentation therapy of Al AT seems ideal to combat emphysema progression, currently the treatment option benefits only a small subset of patients (1-2%).

Augmentation therapy is an expensive weekly treatment, administered at a dose of 60 mg/kg of A1AT, with a cost ranging from $60,000 to $150,000 per year. Furthermore, controversy exists over the best method of delivery. While Al AT is a serum protein, it is thought that direct instillation into the lungs, to the site of action, may be a better, safer, and easier route for self- administration. Should nebulization prove effective in Z-Al AT patients (clinical trials are ongoing), the adaptation of Al AT augmentation therapy to M-Al AT patients is not without risk. A nanocarrier is disclosed that incorporates three therapeutic compounds that have the potential to act individually and in combination to prevent oxidative stress, thereby improving delivery, biodistribution, up-take, and inhaled steroid function. The disclosed nanocarrier offers significant advantages over current inhaled steroids. By modulating the loading of these nanocarriers, the relative contribution of each of the three therapeutic compounds can be varied such that optimal treatment effects are seen in patients regardless of the underlying pathogenesis of emphysema. Blockade of three key therapeutic pathways with the disclosed "3T liposome" ("3TL") can substantially modify the lung microenvironment, and thus lead to improved outcomes.

Chronic obstructive pulmonary disease (COPD) is a major and increasing global health epidemic, which is predicted to become the third most common cause of by 2020. COPD is a chronic respiratory disease that is associated with an abnormal inflammatory response of the lung to noxious particles or gases. This leads to small airway disease and/or emphysema that causes airflow limitations that is not fully reversible. Several mechanisms have been postulated to contribute to the pathogenesis of emphysema. Perhaps the best characterized is the proteinase/ anti-protease hypothesis. This arose from observations that a genetic deficiency in Al AT, a major protease inhibitor in the lung, results in early onset of severe panlobular emphysema, and that the instillation of papain, and more recently porcine pancreatic elastase, results in the progressive airspace enlargement. These observations taken together with the fact that smokers have elevated levels of inflammatory cells within the lung led to the idea that proteases released by the neutrophils (specifically neutrophil elastase) overwhelm the local antiprotease defenses of the lung and promote tissue damage. Furthermore, cigarette smoke oxidatively inactivates Al AT causing a 'functional deficiency' thereby decreasing its antiprotease efficacy and promoting a protease/antiprotease imbalance. This initial description centered on the role of neutrophils and neutrophil elastases as the effector cell and protease, but has now been expanded to include macrophages and metalloproteinases. Another proposed mechanism is a result of another imbalance, which describes the generation of oxidants that overwhelm the lungs anti-oxidant defenses. Oxidants are generated in the airways by cigarette smoke or are released by infiltrating inflammatory cells. Oxidative stress can lead to cell dysfunction or cell death and can induce damage to the lung extracellular matrix. Moreover, oxidative stress influences the protease-antiprotease imbalance by activating proteases and inactivating antiproteases. Additionally, oxidants contribute to inflammation by activating F-KB and thus inducing the expression of pro-inflammatory genes and the up regulation of adhesion molecules that facilitate inflammatory cell adhesion and migration into the lung microenvironment. Therefore, it is clear that cigarette smoke and the chronic inflammatory response it evokes plays a critical role in both the protease/ antiproteinase and

oxidant/anti oxidant imbalances, and that both imbalances are intrinsically linked to the development of emphysema.

Data from both animal models of emphysema and from human subjects have postulated a role for an imbalance between apoptotic cell death and replenishment of structural cells that compose the alveolar walls. Numerous groups have linked apoptosis using human tissue samples to the development of emphysema. There is an increase in endothelial cell apoptosis in lung tissue sections from human emphysematous lungs, as well as increased numbers of epithelial, fibroblast and inflammatory cells. The mechanism of endothelial cell loss may be due to an overall decrease in vascular endothelial growth factor (VEGF) and its receptor VEGF receptor 2 (VEGFR2). A loss of these endothelial maintenance factors may promote apoptosis, loss of vascularity in the parenchymal wall, alveolar damage and emphysema. Furthermore, inhibition of VEGF signaling results in the development of emphysematous like changes. Linking this hypothesis to the protease/ antiproteinase hypothesis, Al AT prevents apoptosis by the direct inhibition of caspase 3 induced endothelial cell apoptosis. More recently an additional hypothesis has been proposed to address the question of why emphysema appears to progress in spite of cigarette smoking cessation. It has been postulated that emphysema may have an autoimmune disease phenotype. This was originally suggested based on histopathological observations of distinct T and B cell follicle formations in the lung, which correlated, with GOLD classification stages of disease. Emphysema is characterized by destruction of the extracellular matrix and damage to endothelial and epithelial cells, which may lead to the exposure of neoantigens that could be presented to antigen presenting cells and result in the production of anti-endothelial, epithelial or matrix antibodies. Further, with the use of elegant passive transfer experiments a role of autoreactive T cells has been described in driving the progression of emphysema.

With recent studies highlighting the non-canonical functions of Al AT, deficiency or 'functional' deficiency can predispose to the development/ progression of emphysema by all the described disease pathways. In addition conformational alterations of Al AT can themselves lead to lung inflammation, cellular oxidative stress, and pro-inflammatory cytokine signaling.

Therefore, therapeutic modulation of Al AT, either by replacement, or protection from oxidative modification, or both, have the potential to inhibit pathogenic mechanisms of disease in Z and M- Al AT patients.

Nanocarrier Compositions

A nanocarrier composition is disclosed that comprises an effective amount of an alpha- 1- antitrypsin, and further comprises an anti-inflammatory agent, and an anti-oxidant agent, or a combination thereof. In some embodiments, the Al AT and anti-inflammatory agent are encapsulated in the nanocarrier, and the anti-oxidant agent is fixed on the surface of the nanocarrier.

Alpha- 1 -antitrypsin

Alpha- 1 Antitrypsin or al -antitrypsin is obtainable from Sigma Aldrich and is a protease inhibitor belonging to the serpin superfamily. It is generally known as serum trypsin inhibitor. Alpha 1 -antitrypsin is also referred to as alpha- 1 proteinase inhibitor (AlPI) because it inhibits a wide variety of proteases. Disorders of this protein include Al AT deficiency, an autosomal codominant hereditary disorder in which a deficiency of Al AT leads to a chronic uninhibited tissue breakdown. This causes the degradation especially of lung tissue, and eventually leads to characteristic manifestations of pulmonary emphysema. Cigarette smoke can lead to oxidation of methionine 358 of Al AT (382 in the pre-processed form containing the 24 amino acid signal peptide), a residue essential for binding elastase; this is thought to be one of the primary mechanisms by which cigarette smoking (or second-hand smoke) can lead to emphysema. Because Al AT is expressed in the liver, certain mutations in the gene encoding the protein can cause misfolding and impaired secretion, which can lead to liver cirrhosis.

Over 100 different variants of al -antitrypsin have been described in various populations. North-Western Europeans are most at risk for carrying one of the most common mutant forms of Al AT, the Z mutation (Glu342Lys on MIA, rs28929474).

A1AT is a single-chain glycoprotein consisting of 394 amino acids in the mature form and exhibits a number of glycoforms. The A1AT used in the disclosed nanocarriers can be natural A1AT isolated from a subject, such as a human subject. In some embodiments, the Al AT is produced recombinantly by incorporating a nucleic acid encoding Al AT into an expression vector. An example human Al AT protein sequence is found in Accession No.

NP_000286, which is provided below (SEQ ID NO: 1):

MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNL AEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEI PEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEA FTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWER PFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNA TAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSI PPEVKFNKPF VFLMIEQNTKSPLFMGKVVNPTQK.

An example nucleic acid sequence encoding Al AT is found in Accession No.

NM_000295, which is provided below (SEQ ID NO:2):

1 acaatgactc ctttcggtaa gtgcagtgga agctgtacac tgcccaggca aagcgtccgg 61 gcagcgtagg cgggcgactc agatcccagc cagtggactt agcccctgtt tgctcctccg 121 ataactgggg tgaccttggt taatattcac cagcagcctc ccccgttgcc cctctggatc 181 cactgcttaa atacggacga ggacagggcc ctgtctcctc agcttcaggc accaccactg 241 acctgggaca gtgaatcgac aatgccgtct tctgtctcgt ggggcatcct cctgctggca 301 ggcctgtgct gcctggtccc tgtctccctg gctgaggatc cccagggaga tgctgcccag 361 aagacagata catcccacca tgatcaggat cacccaacct tcaacaagat cacccccaac 421 ctggctgagt tcgccttcag cctataccgc cagctggcac accagtccaa cagcaccaat 481 atcttcttct ccccagtgag catcgctaca gcctttgcaa tgctctccct ggggaccaag

541 gctgacactc acgatgaaat cctggagggc ctgaatttca acctcacgga gattccggag 601 gctcagatcc atgaaggctt ccaggaactc ctccgtaccc tcaaccagcc agacagccag 661 ctccagctga ccaccggcaa tggcctgttc ctcagcgagg gcctgaagct agtggataag 721 tttttggagg atgttaaaaa gttgtaccac tcagaagcct tcactgtcaa cttcggggac

781 accgaagagg ccaagaaaca gatcaacgat tacgtggaga agggtactca agggaaaatt 841 gtggatttgg tcaaggagct tgacagagac acagtttttg ctctggtgaa ttacatcttc

901 tttaaaggca aatgggagag accctttgaa gtcaaggaca ccgaggaaga ggacttccac 961 gtggaccagg tgaccaccgt gaaggtgcct atgatgaagc gtttaggcat gtttaacatc 1021 cagcactgta agaagctgtc cagctgggtg ctgctgatga aatacctggg caatgccacc 1081 gccatcttct tcctgcctga tgaggggaaa ctacagcacc tggaaaatga actcacccac 1141 gatatcatca ccaagttcct ggaaaatgaa gacagaaggt ctgccagctt acatttaccc 1201 aaactgtcca ttactggaac ctatgatctg aagagcgtcc tgggtcaact gggcatcact 1261 aaggtcttca gcaatggggc tgacctctcc ggggtcacag aggaggcacc cctgaagctc 1321 tccaaggccg tgcataaggc tgtgctgacc atcgacgaga aagggactga agctgctggg 1381 gccatgtttt tagaggccat acccatgtct atcccccccg aggtcaagtt caacaaaccc 1441 tttgtcttct taatgattga acaaaatacc aagtctcccc tcttcatggg aaaagtggtg

1501 aatcccaccc aaaaataact gcctctcgct cctcaacccc tcccctccat ccctggcccc 1561 ctccctggat cctcccatgt acagtgctgt gacattaaag aagggttgag

1621 cctgcatgtg actgtaaatc ctgtatgtgg gctccaggta catctggctg ggtaggcaca 1681 tttctctgag tctccctttg cttcgggccc cctgaactgt ctggtccctg cctgctgagg gttcatggag 1741 tgctgggctt gaatccaggg gggactgaat cctcagctta cggacctggg cccatctgtt 1801 tctggagggc tccagtcttc cttgtcctgt cttggagtcc ccaagaagga atcacagggg 1861 aggaaccaga taccagccat gaccccaggc tccaccaagc atcttcatgt ccccctgctc 1921 atcccccact cccccccacc cagagttgct catcctgcca gggctggctg tgcccacccc 1981 aaggctgccc tcctgggggc cccagaactg cctgatcgtg ccgtggccca gttttgtggc 2041 atctgcagca acacaagaga gaggacaatg tcctcctctt gacccgctgt cacctaacca 2101 gactcgggcc ctgcacctct caggcacttc tggaaaatga ctgaggcaga ttcttcctga 2161 agcccattct ccatggggca acaaggacac ctattctgtc cttgtccttc catcgctgcc 2221 ccagaaagcc tcacatatct ccgtttagaa tcaggtccct tctccccaga tgaagaggag 2281 ggtctctgct ttgttttctc tatctcctcc tcagacttga ccaggcccag caggccccag 2341 aagaccatta ccctatatcc cttctcctcc ctagtcacat ggccataggc ctgctgatgg 2401 ctcaggaagg ccattgcaag gactcctcag ctatgggaga ggaagcacat cacccattga 2461 cccccgcaac ccctcccttt cctcctctga gtcccgactg gggccacatg cagcctgact 2521 tctttgtgcc tgttgctgtc cctgcagtct tcagagggcc accgcagctc cagtgccacg 2581 gcaggaggct gttcctgaat agcccctgtg gtaagggcca ggagagtcct tccatcctcc 2641 aaggccctgc taaaggacac agcagccagg aagtcccctg ggcccctagc tgaaggacag 2701 cctgctccct ccgtctctac caggaatggc cttgtcctat ggaaggcact gccccatccc 2761 aaactaatct aggaatcact gtctaaccac tcactgtcat gaatgtgtac ttaaaggatg 2821 aggttgagtc ataccaaata gtgatttcga tagttcaaaa tggtgaaatt agcaattcta

2881 catgattcag tctaatcaat ggataccgac tgtttcccac acaagtctcc tgttctctta

2941 agcttactca ctgacagcct ttcactctcc acaaatacat taaagatatg gccatcacca 3001 agccccctag gatgacacca gacctgagag tctgaagacc tggatccaag ttctgacttt

3061 tccccctgac agctgtgtga ccttcgtgaa gtcgccaaac ctctctgagc cccagtcatt

3121 gctagtaaga cctgcctttg agttggtatg atgttcaagt tagataacaa aatgtttata

3181 cccattagaa cagagaataa atagaactac atttcttgca.

In some embodiments, the Al AT is a functional variant of a human Al AT having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: l . The A1AT can also be a functional fragment, e.g., containing at least 100, 110, 120, 130, 140, 141, 142, 143, 144, 145, 156, 147, 148, 149, 150, 151, 152, 153,

154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,

173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,

192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,

211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,

230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,

249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,

268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,

287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,

315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,

334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,

353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371,

372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 10 390,

391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,

410, 411, 412, 413, 414, 415, 417, or 418 consecutive amino acids from SEQ ID NO: l, or a variant thereof as discussed above. For example, amino acids 1-24 are a signal peptide that can be removed or replaced with an alternative signal peptide.

A1AT activity can be assessed by determining its elastase inhibitory activity 15 using standard enzyme reactivity assays. These assays investigate the ability of Al AT to impair elastase activity. Conformational alterations of Al AT would have a significantly reduced elastolytic activity score. Anti-inflammatory Agent

Anti-inflammatory agents include alclofenac, alclometasone dipropionate, algestone acetonide, alpha amylase, amcinafal, amcinafide, amfenac sodium, amiprilose hydrochloride, anakinra, anirolac, anitrazafen, apazone, balsalazide disodium, bendazac, benoxaprofen, benzydamine hydrochloride, bromelains, broperamole, budesonide, carprofen, cicloprofen, cintazone, cliprofen, clobetasol propionate, clobetasone butyrate, clopirac, cloticasone propionate, cormethasone acetate, cortodoxone, decanoate, deflazacort, delatestryl, depo- testosterone, desonide, desoximetasone, dexamethasone dipropionate, diclofenac potassium, diclofenac sodium, diflorasone diacetate, diflumidone sodium, diflunisal, difluprednate, diftalone, dimethyl sulfoxide, drocinonide, endrysone, enlimomab, enolicam sodium, epirizole, etodolac, etofenamate, felbinac, fenamole, fenbufen, fenclofenac, fenclorac, fendosal, fenpipalone, fentiazac, flazalone, fluazacort, flufenamic acid, flumizole, flunisolide acetate, flunixin, flunixin meglumine, fluocortin butyl, fluorometholone acetate, fluquazone,

flurbiprofen, fluretofen, fluticasone propionate, furaprofen, furobufen, halcinonide, halobetasol propionate, halopredone acetate, ibufenac, ibuprofen, ibuprofen aluminum, ibuprofen piconol, ilonidap, indomethacin, indomethacin sodium, indoprofen, indoxole, intrazole, isoflupredone acetate, isoxepac, isoxicam, ketoprofen, lofemizole hydrochloride, lomoxicam, loteprednol etabonate, meclofenamate sodium, meclofenamic acid, meclorisone dibutyrate, mefenamic acid, mesalamine, meseclazone, mesterolone, methandrostenolone, methenolone, methenolone acetate, methylprednisolone suleptanate, momiflumate, nabumetone, nandrolone, naproxen, naproxen sodium, naproxol, nimazone, olsalazine sodium, orgotein, orpanoxin, oxandrolane, oxaprozin, oxyphenbutazone, oxymetholone, paranyline hydrochloride, pentosan polysulfate sodium, phenbutazone sodium glycerate, pirfenidone, piroxicam, piroxicam cinnamate, piroxicam olamine, pirprofen, prednazate, prifelone, prodolic acid, proquazone, proxazole, proxazole citrate, rimexolone, romazant, salcolex, salnacedin, salsalate, sanguinarium chloride, seclazone, sermetacin, stanozolol, sudoxicam, sulindac, suprofen, talmetacin, talniflumate, talosalate, tebufelone, tenidap, tenidap sodium, tenoxicam, tesicam, tesimide, testosterone, testosterone blends, tetrydamine, tiopinac, tixocortol pivalate, tolmetin, tolmetin sodium, triclonide, triflumidate, zidometacin, and zomepirac sodium.

In particular embodiments, the anti-inflammatory agent comprises dexamethasone. Anti-oxidant agent

Although any suitable antioxidant may be employed, in specific aspects the antioxidant is N-acetyl cysteine (NAC). NAC is an antioxidant and a thiol donor.

Currently, NAC is clinically used for treatment of hepatotoxicity caused by

acetaminophen overdose, for treatment of chronic bronchitis and other pulmonary diseases complicated by the production of viscous mucus, and for treatment of reperfusion injury during cardio bypass surgery. Other endogenous antioxidant enzymes could also be conjugated.

Endogenous catalytic antioxidants have been used as models for the development of catalytic antioxidant mimetics. The three most prominent classes are the Superoxide dismutase (SOD), catalase, and GPx mimics. Most of these compounds contain a ligated transitional metal or selenium. They are generally broad-spectrum antioxidants that can scavenge 02^", H202, ONOO^", and a variety of lipid peroxides. The SOD and catalase mimic class include

macrocyclics, metallo-porphyrins, salens, and nitroxides. The GPx class includes selenium- and tellurium-based compounds.

Nanocarriers

The recent development in nanotechnologies has increased interest in the possible delivery of therapeutic agents to the lung microenvironment. The ability for therapeutics, such as vaccines, protein, and enzyme to be delivered directly to the lung via inhalation of nanosized particles offers therapeutic potential. The anatomy, breathing pattern, and particle size are the principle factors determining the pulmonary compartment to which a particle gains access. For nanosized particles, the main mechanism of transportation following aerosolization or direct nasal instillation is diffusion with only minimal contribution of gravitation or inerta, and therefore very little of the nanosized particle can be expected to be retained in the

tracheobronchial region but rather is expected to reach the alveoli, which is desirable in the therapy of emphysema. In order to design an efficient and effective drug carrier, these issues need to be addressed: (1) a tailored carrier surface capable of attaching biomolecules for targeted drug delivery; (2) a biocompatible coating which efficiently encapsulates the drug thereby reducing cytotoxicity; and (3) stimuli-induced (i.e., pH) disruption of the carrier agent for slow and controlled drug release to the desired environment. Liposomes, phospholipid vesicles composed of lipid bilayers enclosing an aqueous compartment, fit these criteria in a self- contained package. Hydrophilic molecules can be encapsulated in the aqueous spaces, and lipophilic molecules can be incorporated into the lipid bilayers, a region referred to as the corona. Liposomes are used for the selective delivery of antioxidants and other therapeutic drugs to different tissues in sufficient concentrations to be effective in ameliorating tissue injuries. The relative ease in incorporating hydrophilic and lipophilic therapeutic agents into liposomes, the possibility of directly delivering liposomes to an accessible body site, such as the lung, and the relative nonimmunogenicity and low toxicity of liposomes have rendered the liposomal system highly attractive for drug delivery. Liposomes have been developed for delivery because they are stable nanoparticles that can release their contents under specific intracellular circumstances (e.g. the low pH environment of the endosome can trigger release).

The nanocarrier can be any suitable vehicle for the delivery of active agents, including non-targeting and targeting. A variety of suitable nanocarriers are known in the art, and include for example micelles, solid nanoparticles, and liposomes.

In some embodiments, the nanocarrier can include a polymeric nanoparticle. For example, the nanocarrier can comprise one or more polymeric matrices. The nanocarrier can also include other nanomaterials and can be, for example, lipid-polymer nanoparticles. In some embodiments, a polymeric matrix can be surrounded by a coating layer (e.g., liposome, lipid monolayer, micelle, etc.). Examples of classes of nanocarriers that can be adapted (e.g., by incorporation of a suitable surface agent) to deliver immunosuppressive agents include (1) biodegradable nanoparticles, such as those described in U.S. Patent No. 5,543,158 to Gref et al., (2) polymeric nanoparticles such as those described in U.S. Patent No. 7,534,448 to Saltzman et al., (3) lithographically constructed nanoparticles, such as those described in U.S. Patent No. 8,420,124 to DeSimone et al., (4) nanoparticles such as those described in U.S. Patent

Application Publication No. 2010/0233251 to von Andrian et al., or (5) nanoparticles such as those described in U.S. Patent No. 7,364,919 to Penades et al.

To protect the Al AT from intracellular and extracellular oxidation, which would promote inflammation, the nanocarrier can be coated with polyethylene glycol (PEG) to slow clearance and prevent non-specific engulfment of the nanocarrier by resident immune cells. The PEG molecule forms a "conformational cloud" which is created by the highly flexible polymer chains having a large total number of possible conformations. The faster the rate of transition from one conformation to another, the longer the polymer stays statistically as a "conformational cloud" which avoids interactions with blood components along with protein interactions such as enzymatic degradation. This property of PEG ligand is known as stealth behavior which points to reduced interactions with the body. As a consequence, PEGylated carriers demonstrate less immunogenicity and antigenicity. In addition, a decrease in aggregation of erythrocytes and hemolysis is also observed. This also allows prolonged blood circulation. Another way to achieve the similar stealth properties of PEG is by the use of high molecular weight Hyaluronan- grafted liposomes (HA-liposomes) thereby providing a hydrophilic shield and promoting long circulation. Also, Polyoxazolines (POx) are increasingly studied as polymeric building blocks since it was proved that biocompatibility and stealth behavior of POx are similar to that of poly(ethylene glycol) (PEG). Although POx have a lot of advantages, they suffer an important drawback of preparing high molecular weight polyoxazolines, with low polydispersity indexes.

For example, the nanocarrier can be a micelle or liposome that comprises N-palmitoyl homocysteine (PHC). Other pH sensitive lipids include:

1) N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminium (DOBAQ)

2) l,2-dipalmitoyl-sn-glycero-3 -succinate (DGS)

These lipids can be used instead of PHC (N-palmitoyl homocysteine) in combination with PEG-PE amine. These molecules are zwitterionic in nature and are affected by pH changes of cellular milieu.

In some embodiments, liposome can be created with a mPEG-Hz- CHEMS. mPEG-Hz- CHEMS has a pH sensitive hydrazone linkage which breaks at around endosomal pH

(approximately pH5.5). pH sensitive nanocarriers are known in the art. See, for example, U.S. Patent Application Publication No. 2004/0234597 to Shefer et al. and U.S. Patent Application Publication No. 2010/0303850 to Lipford et al. Suitable pH sensitive nanocarriers can be formed from materials that are pH sensitive provided that the resulting nanocarriers provide for delivery of the immunosuppressive agent at the desired pH. For example, suitable pH sensitive nanocarriers include nanocarriers that provide for the release of one or more encapsulated immunosuppressive agents at a threshold pH of about 6.8 or less (e.g., about 6.5 or less, about 6 or less, or about 5.5 or less).

Such synthetic nanocarriers are well known in the art and include polyketal nanocarriers, pH sensitive liposomes, pH sensitive micelles, polymeric nanoparticles derived from amphiphilic block copolymers, and core-shell materials formed from a core material (e.g., a hydrophobic or hydrophilic core material such as a polymer) and a pH sensitive shell {see for example, U.S. Patent Application Publication No. 2004/0234597 to Shefer et al.).

In some embodiments, the pH sensitive nanocarrier can be a core-shell nanoparticle comprising a hydrophobic core material (e.g., a wax, a fat material such as a lipid, or a hydrophobic polymer) surrounded by a pH sensitive shell material.

In some embodiments, the pH sensitive nanocarrier can be a nanoparticle or micelle formed from an amphiphilic material, such as an amphiphilic block copolymer derived from a hydrophilic polymer segment and a hydrophobic polymer segment. By way of example, the pH sensitive nanocarrier can be a nanoparticle or micelle formed an amphiphilic block copolymer derived from a poly(alkylene oxide) segment (e.g., a polyethylene glycol (PEG) segment) and an aliphatic polyester segment. The aliphatic polyester segment can be a biodegradable aliphatic polyester, such as poly(lactic acid), poly(glycolic acid), or poly(lactic acid-co-glycolic acid).

In some embodiments, the pH sensitive nanocarrier can be a nanoparticle or micelle or liposome formed from amphiphilic molecule comprising a hydrophilic polymer segment (e.g., a poly(alkylene oxide) segment such as a PEG segment) and a lipid moiety. The lipid moiety can be conjugated to a terminus of the PEG segment, so as to afford a suitable amphiphile. Suitable lipid moieties are known in the art, and include, for example, mono-, di and triglycerides (e.g., glyceryl monostearate or glyceryl tristearate), phospholipids, sphingolipids, cholesterol and steroid derivatives, terpenes and vitamins. In some embodiments, the lipid moiety can be a phospholipid. Suitable phospholipids include, but are not limited to, phosphatidic acids, phosphatidylcholines with both saturated and unsaturated lipids, phosphatidyl ethanolamines, phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, lysophosphatidyl derivatives, cardiolipin, and beta-acyl-y-alkyl phospholipids. In certain embodiments, the pH sensitive nanocarrier can be a nanoparticle or micelle formed from amphiphilic molecule comprising a hydrophilic polymer segment (e.g., a poly(alkylene oxide) segment such as a PEG segment) and a phospholipid moiety.

In some embodiments, the targeted nanocarrier has a mean diameter of 5 nm to 100 nm, including 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 nm, to optimize vascular permeability and penetration into tissue and cells. The preferred and suitable size of the targeted nanocarrier depends on the biological system employed. From previous studies, it was observed that for a spherical particle (aspect ratio - 1 : 1), nanoparticle uptake in mammalian cells was highest at 50 nm and lowest at 100 nm. In addition, smaller particles less than 30 nm showed higher uptake than particles higher than 70 nm. In addition with its multifunctional character (large surface area due to small size, surface can be tailored with different functionalities), the nanocarrier behaves like a stealth agent and can evade immune response from the host system due to surface modifications including pegylation.

In some embodiments, the nanocarrier is conjugated with a near-infrared fluorophore, such as DyLight 680, Dylight 755, or IR-800. These fluorophores aid in noninvasive in vivo imaging for the detection of the graft site and monitoring of drug release. In some embodiments, the imaging reporter can be gadolinium, iron oxide, or radioisotopes to monitor delivery of the nanocarrier. In some embodiments, the imaging reporter is an enzyme, such as luciferase or beta- galactosidase.

Pharmaceutical Compositions

The disclosed nanocarrier compositions can be used therapeutically in combination with a pharmaceutically acceptable carrier. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art. Pharmaceutical compositions can include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.

Pharmaceutical compositions can also include one or more active ingredients such as

antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

The pharmaceutical composition can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example,

antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Some of the compositions can be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

The compounds or pharmaceutically acceptable salts thereof may be formulated as aerosols for topical application, such as by inhalation. Formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose.

The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the disorder's symptoms are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

Methods

Also disclosed are methods for alpha- 1 -antitrypsin augmentation in a subject that involve administering to the subject an effective amount of the disclosed nanocarrier compositions. This technology has broad application to all acute and chronic lung diseases, transplantation, ischemia reperfusion injury, and autoimmune disorders.

The herein disclosed compositions, including pharmaceutical composition, may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. The compositions may be administered orally, parenterally (e.g., intravenously), by

intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally,

ophthalmically, vaginally, rectally, intranasally, topically or the like, including topical intranasal administration or administration by inhalant, such ultrasonic nebulization. Definitions

The term "subject" refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term "patient" refers to a subject under the treatment of a clinician, e.g., physician. The term "therapeutically effective" refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

The term "carrier" means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. The term "prevent" or "suppress" refers to a treatment that forestalls or slows the onset of a disease or condition or reduced the severity of the disease or condition. Thus, if a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent or suppress that disease in a subject who has yet to suffer some or all of the symptoms.

The term "inhibit" refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

The terms "peptide," "protein," and "polypeptide" are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

The term "protein domain" refers to a portion of a protein, portions of a protein, or an entire protein showing structural integrity; this determination may be based on amino acid composition of a portion of a protein, portions of a protein, or the entire protein.

The term "nucleic acid" refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3 ' position of one nucleotide to the 5' end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

The term "variant" refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid substitutions (i.e. a degenerate variant), substitutions within the wobble position of each codon (i.e. DNA and RNA) encoding an amino acid, amino acids added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90%), or 95% homology to a reference sequence.

The term "specifically binds", as used herein, when referring to a polypeptide (including antibodies) or receptor, refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologies. Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody), a specified ligand or antibody "specifically binds" to its particular "target" (e.g. an antibody specifically binds to an endothelial antigen) when it does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism. Generally, a first molecule that "specifically binds" a second molecule has an affinity constant (Ka) greater than about 105 M^"1 (e.g., 10⁶ M^"1, 10⁷ M^"1, 10⁸ M^~ 10⁹ M^"1, 10¹⁰ M^"1, 10¹¹ M^"1, and 1012 M^"1 or more) with that second molecule.

The term "residue" as used herein refers to an amino acid that is incorporated into a polypeptide. The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino, acids.

The term "position," with respect to an amino acid residue in a polypeptide, refers to a number corresponding to the numerical place that residue holds in the polypeptide. By convention, residues are counted from the amino terminus to the carboxyl terminus of the polypeptide. A "fusion protein" refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein may be formed by the chemical coupling of the constituent polypeptides or it may be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone.

The term "specifically deliver" as used herein refers to the preferential association of a molecule with a cell or tissue bearing a particular target molecule or marker and not to cells or tissues lacking that target molecule. It is, of course, recognized that a certain degree of nonspecific interaction may occur between a molecule and a non- target cell or tissue. Nevertheless, specific delivery, may be distinguished as mediated through specific recognition of the target molecule. Typically specific delivery results in a much stronger association between the delivered molecule and cells bearing the target molecule than between the delivered molecule and cells lacking the target molecule. As used herein, "peptidomimetic" means a mimetic of a peptide which includes some alteration of the normal peptide chemistry. Peptidomimetics typically enhance some property of the original peptide, such as increase stability, increased efficacy, enhanced delivery, increased half life, etc. Methods of making peptidomimetics based upon a known polypeptide sequence is described, for example, in U.S. Patent Nos. 5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involve the incorporation of a non-amino acid residue with non-amide linkages at a given position. One embodiment of the present invention is a peptidomimetic wherein the compound has a bond, a peptide backbone or an amino acid component replaced with a suitable mimic. Some non-limiting examples of unnatural amino acids which may be suitable amino acid mimics include 3 -alanine, L-a-amino butyric acid, L-y-amino butyric acid, L-a-amino isobutyric acid, L-E-amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L- glutamic acid, N-E-Boc-N-a-CBZ-L-lysine, N-E-Boc-N-a-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-a-Boc-N-oCBZ-L-ornithine, N-o-Boc-N-a-CBZ-L- ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.

The term "percent (%) sequence identity" or "homology" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

EXAMPLES

Anti-inflammatories are commonly used for therapy in COPD patients. Unfortunately there is a low response rate for COPD patients following long-term usage, which is due to steroid resistance. Moreover, the lungs of COPD patients have high levels of oxidative stress, making any therapy given exposed to potential oxidative modification, possibly rendering the drug ineffective or in the case of Al AT, pro-inflammatory. Liposomal-based therapies have been shown to be successful in anticancer therapies but few studies have applied them to

inflammatory diseases. Therefore, liposomes have been developed that encompass A1AT to combat the enhanced protease activity within the lungs of Al AT deficient or functionally deficient patients, a steroid (dexamethasone) to help dampen the inflammatory response, and antioxidant (NAC) to protect against intracellular ROS and ROS mediated lung injury.

Example 1

Anti-oxidant liposomal constructs containing A1AT, dexamethasone, and NAC ("3TL") Lipid-core nanocapsule (LNC) formulations were synthesized using either standard lipid biofilm hydration or solvent injection methods with dexamethasone in the corona and the nuclear stain DAPI in the core of the LNC, to demonstrate the ability to deliver proteins loaded into the core of the LNC directly into the cell. As shown in Figure 1, LNC conjugated with the tracking dye DyLight 680 entered the cells (Fig. IB). But when DAPI, a nuclear stain, is successfully encapsulated into the core of LNC, very little of the membrane-permeant DAPI is able to escape from the LNC and enter into the nuclei of live cells (Figs. 1A, 1C). Free DAPI, in contrast, is able to traverse the cell membranes and travels into the cell's nuclei more easily and with 100% penetrance.

Liposomes are synthesized using either standard lipid biofilm hydration or solvent injection methods to contain the three therapeutic anti-inflammatory, anti-oxidant components: A1AT, NAC, and dexamethasone ("3TL") (Fig. 2). The LNC are composed of PEG-PE amine (l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000]), HSPC (L-a-phosphatidylcholine, hydrogenated (Soy)), DC-Cholesterol (3β-[Ν-(Ν',Ν'- dimethylaminoethane)-carbamoyl]-cholesterol hydrochloride) and PHC (N-palmitoyl homocysteine (ammonium salt)). PHC, a pH sensitive lipid is used to assist in the rupture of the 3TL at acidic pH (-5-6) to ensure the delivery of the cargo inside the 3TL. Fluorescent labeling of 3TL with DyLight 680 assists in verifying their cell targeting capacities and facilitate their detection in fluorescence-based applications such as immunofluorescence microscopy or flow cytometry. NAC is covalently coupled on the outer surface and a combination of Al AT (core) and dexamethasone (shell) is encapsulated within the liposome. Several single component LNC formulations are used as controls: empty LNC, NAC-conjugated empty LNC, Al AT- encapsulated LNC, dexamethasone-encapsulated LNC. In addition, several dual combination controls are prepared to compare to 3TL: Al AT and dexamethasone-encapsulated LNC, NAC- conjugated Al AT-encapsulated LNC, and NAC-conjugated dexamethasone-encapsulated LNC. Liposome formulations are compared with free drugs individually and in combination.

LNC of varying molar ratio bilayer compositions (HSPC to PHC) are synthesized using either standard lipid biofilm hydration or solvent injection methods to examine the size of the LNC and the efficiency of drug loading as well as the impact of composition on liposomal rupture. Mean particle size and charge are determined by dynamic light scattering (DLS) and zeta-potential. The phospholipid composition is determined with a phosphate assay and dexamethasone concentration is determined by high performance liquid chromatography (HPLC) on the organic phase after extraction of the liposomal preparation with chloroform. The aqueous phase after extraction is used to determine the liposomal Al AT concentration by HPLC. pH stability assays examine rupture capabilities of the LNC under physiologic conditions in vitro.

Next, the efficiency of surface conjugation of the LNC with NAC and DyLight 20 680 is examined. DLS, UV-Vis spectroscopy, CBQCA quantification assay, and cryo- TEM are used. DLS and MALDI-MS are used together to characterize the final size of the 3TL and to determine the effective concentration of drugs encapsulated within the 3TL. pH stability assays examine rupture capabilities of the LNC under physiologic conditions in vitro.

3TL uptake kinetics, cellular localization, and cellular viability in vitro are tested after incubation in normal human bronchial epithelial cells (NHBE), small airway epithelial cells (SAEC), alveolar epithelial cells and macrophages. Fluorescence measurements of DyLight 680 are performed by confocal microscopy and flow cytometry. Cell viability is measured with a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Fluorescence of challenged cells are assessed relative to control cells. Three different concentrations (based on NAC) of the 3TL are tested to examine the impact of LNC on oxidative stress by oxiselect ROS/RNS assays, glucocorticoid receptor expression and MALDI mass analysis. If NAC is shown to have an effect on cellular uptake, the optimal number of NAC conjugated to the surface is modified as too few NAC may not achieve sufficient protective effect to the target cells, whereas too many NAC may have a deleterious effect, decreasing the protective, anti-oxidant response.

Example 2

Payload stability, mode of action and therapeutic efficacy of liposomal constructs in human primary cell culture models, and in vivo delivery and biodistribution

Al AT transmigration across endothelial (circulation, systemic delivery) (Fig.3) and epithelial barriers (lung delivery) is impaired under conditions of oxidative stress, specifically cigarette smoke exposure, and oxidative stress leads to conformational modification of A1AT. These data suggest that current Al AT augmentation may be suboptimal and may fail to reach its target site (i.e. lung), due to impaired transcytosis, by both epithelial cells (nebulized route), and endothelial cells (i.v route). This problem is illustrated in Figure 3. Human endothelial cells were oxidatively stressed in transwells with cigarette smoke extract (CS) and incubated with Al AT or Al AT plus NAC to analysis Al AT transcytosis. Western blots from supernatants collected from both the apical or basolateral supernatants demonstrated that CS reduces transcytosis of Al AT, and that co-administration of NAC improves migration. Further recent studies demonstrate that administration of Al AT with high density lipoproteins potentiate the delivery of Al AT in a model of elastase induced emphysema. A first generation 3T liposome that contains Al AT in the core, Dexamethasone in the corona, and NAC coating the surface of the liposome was successfully constructed. Using this construct, Al AT transmigration studies demonstrated that liposomal encapsulation promotes improved epithelial uptake and basolateral release as compared to free drugs in CS media (Fig. 4). Taken together these data support use of 3T (NAC/DEX/A1 AT) liposomes as delivery devices that improve Al AT transcytosis, deliver steroids, and reduce oxidative stress, which can potentially induce steroid resistance, Al AT conformational alterations and transcytosis. These are important properties for administration by either i.v. or nebulized route. These properties are characterized. Primary normal lung epithelial cells (NHBEs) are cultured in a transwell cell culture model. Cells are exposed to air control (ACS) media or cigarette smoke extract (CSE) media for varying times as determined for each experimental end-point. All assays are compared against free drug controls. For instance, cell culture experiments include the following groups; 1. A1AT, 2. NAC, 3. A1AT + NAC, 4. DEX, 5. DEX + NAC, 6. DEX + A1AT, 7. DEX + A1AT + NAC, versus 3T (DEX/NAC/AIAT), 2TD (DEX/AIAT), and 2TN (NAC/A1AT). Single constructs cannot be constructed due to biomaterial instabilities. Doses of incorporated drugs and free drugs are determined from the literature and from prior experiments. Doses of ΙμΜ-100 μΜ for dexamethasone and NAC have been used and these dose ranges have been successfully incorporated in the 3T liposomes. These constructs and combinations are used to dissect the contribution of each component in cigarette smoked inflammation and drug trafficking.

Controversy exists over the route of administration in patients for Al AT augmentation therapy. Currently, Al AT is administered by intravenous route, but clinical trails are on-going investigating the efficacy of nebulized formulations. This route may be particularly useful in M- Al AT patients given that they have normal circulating levels of Al AT. Elegant work has demonstrated that Al AT transcytosis is impaired as a consequence of endothelial oxidative stress and controversy exists as to whether apical application of Al AT to epithelial cells (route of exposure following nebulization) leads to basolateral release.

2T/3T Liposomes have been constructed with Dy Light 680 on their outer surface for tracking proposes by confocal image analysis or in vivo maestro imaging. Times course analysis and co-localization staining demonstrate processing and retention of the therapy within the cells.

Presence of Al AT in the apical and basolateral supernatants, and cells of the culture system is measured by western blot analysis. Western blot analysis of Al AT conformations, using conformational specific antibodies to oxidized and polymerized Al AT is performed to determine whether free NAC, or liposome encapsulation with and without NAC protects Al AT from conformational alterations caused by CSE exposure or intracellular oxidative stress. The function of epithelial secreted (following transcytosis) and liposomal encapsulated Al AT is confirmed with standard neutrophil elastolytic assays. The impact of NAC therapy is determined by measuring intra and extracellular oxidative stress with OXISELECT fluorescent ROS assay kits. CS exposure can increase cellular ROS, and NAC can decrease this response.

Culture supernatants and cell lysates are probed with western blot and ELIS A techniques to determine Dex concentrations. Oxidative stress modifies glucocorticoid receptor expression. The impact of NAC on the surface of 3T liposomes on GR expression and localization is determined using western blot analysis of lysate and nuclear fractions.

In addition to localization studies, the effect of the liposomal treatment on the key inflammatory cytokines and genes linked to cigarette smoke related inflammation, including IL- 6, IL-8 TNF-a, IFN-γ, NF-κΒ and AP-1 is investigated.

Preliminary in vivo studies have been performed and demonstrate the feasibility of delivering 3TL liposomes to the lungs of mice via direct lung nebulization. This is likely the route of preferred administration clinically. Important ex-vivo imaging studies revealed localization to the distill airway, the zone most effected in COPD patients. Cell specific localization and drug release dynamics of 3TL constructs will be determined using two experimental groups: drug release experiments, where drug concentrations will be measured by ELISA techniques, and MALDI imaging, and drug biodistribution experiments, by cell specific confocal microscopy imaging. Mice are nebulized with 2T and 3T liposomes and combination free drugs by EMKA Technologies double chamber plethysmograph nebulization chamber.

For drug release experiments, mice are harvested at 6, 12, 24, and 48 hrs post

nebulization, and lungs lavaged and assayed for Dex, NAC, and Al AT concentrations and elastolytic activity using ELISA/Western/colorimetric techniques and compared between groups. For biodistribution, mice are imaged using the in vivo Maestro whole body imager at 6, 12, 24, 48 hrs. A sub group is sacrificed at 6 and 24 hrs to look at liposome localization at the tissue/cell level using confocal microscopy.

Preliminary feasibility studies have been performed for the Maestro imaging studies and nebulization. Mice were nebulized with 3T DyLight 680 labeled liposomes, and either free Al AT or PBS. Constructs were delivered to left and right lungs, and to all lung lobes. Results are shown in Figure 5.

The invention is further described by the following numbered paragraphs:

1. A composition, comprising an alpha- 1 -antitrypsin (A1AT) and an anti-inflammatory agent encapsulated in a nanocarrier that comprises on its surface an anti-oxidant agent. 2. The composition of paragraph 1, wherein the anti-inflammatory agent comprises dexamethasone.

3. The composition of paragraph 1, wherein the anti-oxidant agent comprises N-acetyl cysteine (NAC).

4. The composition of any one of paragraph 1, wherein the nanocarrier comprises a micelle, liposome, or polymeric nanoparticle.

5. The composition of paragraph 4, wherein the nanocarrier has a mean diameter of 5 nm to 100 nm.

6. The composition of any one of paragraph 1, wherein the nanocarrier is pH sensitive, temperature sensitive, or a combination thereof. 7. The composition of paragraph 6, wherein the nanocarrier is a micelle comprising N- palmitoyl homocysteine (PHC).

8. The composition of any one of paragraph 1, wherein the nanocarrier is a micelle comprising amino-polyethylene glycol-phosphatidylethanolamine (PEG-PE- Amine).

9. A method for alpha- 1 -antitrypsin (A1AT) augmentation in a subject, comprising administering to the subject an effective amount of composition comprising the composition of paragraph 1. 10. A method of treating an inflammatory disease, comprising the step of comprising administering to a subject in need thereof an effective amount of the composition according to paragraph 1.

11. The method according to paragraph 10, wherein the inflammatory disease is COPD.

12. The method according to paragraph 10, wherein the composition is administered by nebulization.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A composition, comprising an alpha- 1 -antitrypsin (A1AT) and an anti-inflammatory agent encapsulated in a nanocarrier that comprises on its surface an anti-oxidant agent.

2. The composition of claim 1, wherein the anti -inflammatory agent comprises

dexamethasone.

3. The composition of claim 1, wherein the anti-oxidant agent comprises N-acetyl cysteine (NAC).

4. The composition of any one of claim 1, wherein the nanocarrier comprises a micelle, liposome, or polymeric nanoparticle.

5. The composition of claim 4, wherein the nanocarrier has a mean diameter of 5 nm to 100 nm.

6. The composition of any one of claim 1, wherein the nanocarrier is pH sensitive, temperature sensitive, or a combination thereof.

7. The composition of claim 6, wherein the nanocarrier is a micelle comprising N-palmitoyl homocysteine (PHC).

8. The composition of any one of claim 1, wherein the nanocarrier is a micelle comprising amino-polyethylene glycol-phosphatidylethanolamine (PEG-PE- Amine).

9. A method for alpha- 1 -antitrypsin (A1AT) augmentation in a subject, comprising administering to the subject an effective amount of composition comprising the composition of claim 1.

10. A method of treating an inflammatory disease, comprising the step of administering to a subject in need thereof an effective amount of the composition according to claim 1.

11. The method according to claim 10, wherein the inflammatory disease is COPD.

12. The method according to claim 10, wherein the composition is administered by nebulization.