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WO2016179365A1 - Nanotransporteurs pour l'administration d'alpha-1-antitrypsine - Google Patents

Nanotransporteurs pour l'administration d'alpha-1-antitrypsine Download PDF

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Publication number
WO2016179365A1
WO2016179365A1 PCT/US2016/030932 US2016030932W WO2016179365A1 WO 2016179365 A1 WO2016179365 A1 WO 2016179365A1 US 2016030932 W US2016030932 W US 2016030932W WO 2016179365 A1 WO2016179365 A1 WO 2016179365A1
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WIPO (PCT)
Prior art keywords
composition
nanocarrier
nac
acid
inflammatory
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PCT/US2016/030932
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English (en)
Inventor
Carl Atkinson
Ann-Marie BROOME
Satish NADIG
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Musc Foundation For Research Development
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Publication of WO2016179365A1 publication Critical patent/WO2016179365A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers

Definitions

  • compositions and methods for administering alpha- 1 -antitrypsin (A1AT) to the lungs of a subject 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.
  • an anti-oxidant such as N-acetyl cysteine (NAC)
  • NAC N-acetyl cysteine
  • the disclosed composition further comprises an anti-inflammatory agent, such as dexamethasone, loaded into the nanocarrier.
  • an anti-inflammatory agent such as dexamethasone
  • 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
  • compositions and methods for administering A1AT to the lungs of a subject 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.
  • an anti-oxidant such as N-acetyl cysteine (NAC)
  • NAC N-acetyl cysteine
  • the disclosed composition further comprises an anti-inflammatory agent, such as dexamethasone, loaded into nanocarrier.
  • an anti-inflammatory agent such as dexamethasone
  • a multi-drug loaded nanocarrier containing these three distinct therapeutic agents (e.g., Al AT, NAC, and dexamethasone).
  • the nanocarrier is a micelle, liposome, or polymeric nanoparticle.
  • the nanocarrier can be pH sensitive, temperature sensitive, or a combination thereof.
  • 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.
  • 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.
  • 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
  • FIG. 4 shows 3T Liposomes improve Al AT transcytosis in Human Bronchial
  • HBMEs Epithelial cells
  • HBMEs were treated apically with 20 ⁇ Al AT, 1 ⁇ NAC, of 3T liposomes containing 20 ⁇ (Al AT, ⁇ NAC) in the presence of 7.5% Cigarette smoke extract media.
  • 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.
  • 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.
  • COPD chronic obstructive pulmonary disease
  • 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.
  • oxidative stress influences the protease-antiprotease imbalance by activating proteases and inactivating antiproteases.
  • 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
  • VEGF vascular endothelial growth factor
  • VAGFR2 vascular endothelial growth factor receptor 2
  • 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.
  • 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.
  • a nanocarrier composition 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.
  • 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 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.
  • AlPI alpha- 1 proteinase inhibitor
  • 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.
  • 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.
  • al -antitrypsin 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.
  • 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):
  • NM_000295 which is provided below (SEQ ID NO:2):
  • 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,
  • 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 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, di
  • 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,
  • the anti-inflammatory agent comprises dexamethasone.
  • Anti-oxidant agent comprises dexamethasone.
  • NAC N-acetyl cysteine
  • 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.
  • SOD Superoxide dismutase
  • catalase catalase
  • GPx mimics Endogenous catalytic antioxidants.
  • SOD Superoxide dismutase
  • catalase catalase
  • GPx mimics 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
  • the GPx class includes selenium- and tellurium-based compounds.
  • 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.
  • suitable nanocarriers are known in the art, and include for example micelles, solid nanoparticles, and liposomes.
  • the nanocarrier can include a polymeric nanoparticle.
  • the nanocarrier can comprise one or more polymeric matrices.
  • the nanocarrier can also include other nanomaterials and can be, for example, lipid-polymer nanoparticles.
  • a polymeric matrix can be surrounded by a coating layer (e.g., liposome, lipid monolayer, micelle, etc.).
  • 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.
  • the nanocarrier can be coated with polyethylene glycol (PEG) to slow clearance and prevent non-specific engulfment of the nanocarrier by resident immune cells.
  • PEG polyethylene glycol
  • 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.
  • the nanocarrier can be a micelle or liposome that comprises N-palmitoyl homocysteine (PHC).
  • PLC N-palmitoyl homocysteine
  • Other pH sensitive lipids include:
  • 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.
  • liposome can be created with a mPEG-Hz- CHEMS.
  • mPEG-Hz- CHEMS has a pH sensitive hydrazone linkage which breaks at around endosomal pH
  • 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.
  • 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.).
  • a core material e.g., a hydrophobic or hydrophilic core material such as a polymer
  • 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.
  • a hydrophobic core material e.g., a wax, a fat material such as a lipid, or a hydrophobic polymer
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • a hydrophilic polymer segment e.g., a poly(alkylene oxide) segment such as a PEG segment
  • a phospholipid moiety e.g., a poly(alkylene oxide) segment such as a PEG segment
  • 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.
  • 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.
  • the imaging reporter can be gadolinium, iron oxide, or radioisotopes to monitor delivery of the nanocarrier.
  • the imaging reporter is an enzyme, such as luciferase or beta- galactosidase.
  • 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.
  • compositions can also include one or more active ingredients such as
  • antimicrobial agents 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.
  • 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,
  • 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.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid,
  • 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.
  • 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.
  • This technology has broad application to all acute and chronic lung diseases, transplantation, ischemia reperfusion injury, and autoimmune disorders.
  • 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.
  • 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,
  • subject refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • 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.
  • 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.
  • 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.
  • a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
  • 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.
  • 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.
  • 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.
  • inhibitor 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.
  • peptide protein
  • 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.
  • 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.
  • 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).
  • 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.
  • a specified ligand or antibody 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.
  • a specified ligand or antibody 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.
  • a first molecule that "specifically binds" a second molecule has an affinity constant (Ka) greater than about 105 M “1 (e.g., 10 6 M “1 , 10 7 M “1 , 10 8 M ⁇ 10 9 M “1 , 10 10 M “1 , 10 11 M “1 , and 1012 M “1 or more) with that second molecule.
  • Ka affinity constant
  • amino acid 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.
  • 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.
  • 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.
  • 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.
  • percent (%) sequence identity 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.
  • 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
  • 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.
  • LNC Lipid-core nanocapsule
  • DAPI a nuclear stain
  • 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 (3 ⁇ -[ ⁇ -( ⁇ ', ⁇ '- dimethylaminoethane)-carbamoyl]-cholesterol hydrochloride) and PHC (N-palmitoyl homocysteine (ammonium salt)).
  • 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.
  • LNC of varying molar ratio bilayer compositions 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.
  • 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.
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • 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 ⁇ -100 ⁇ 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.
  • 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.
  • mice are harvested at 6, 12, 24, and 48 hrs post
  • 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.
  • 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.
  • A1AT alpha- 1 -antitrypsin
  • an anti-inflammatory agent encapsulated in a nanocarrier that comprises on its surface an anti-oxidant agent.
  • the anti-inflammatory agent comprises dexamethasone.
  • composition of paragraph 1, wherein the anti-oxidant agent comprises N-acetyl cysteine (NAC).
  • composition of any one of paragraph 1, wherein the nanocarrier comprises a micelle, liposome, or polymeric nanoparticle.
  • composition of paragraph 4 wherein the nanocarrier has a mean diameter of 5 nm to 100 nm.
  • composition of any one of paragraph 1, wherein the nanocarrier is pH sensitive, temperature sensitive, or a combination thereof.
  • the nanocarrier is a micelle comprising N- palmitoyl homocysteine (PHC).
  • composition of any one of paragraph 1, wherein the nanocarrier is a micelle comprising amino-polyethylene glycol-phosphatidylethanolamine (PEG-PE- Amine).
  • 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.
  • 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.

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Abstract

L'invention concerne une composition comprenant une alpha-1-antitrypsine (A1AT) et un agent anti-inflammatoire encapsulés dans un nanotransporteur dont la surface présente un agent antioxydant.
PCT/US2016/030932 2015-05-05 2016-05-05 Nanotransporteurs pour l'administration d'alpha-1-antitrypsine WO2016179365A1 (fr)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
US20120282328A1 (en) * 2006-10-24 2012-11-08 Aradigm Corporation Dual action, inhaled formulations providing both an immediate and sustained release profile
US20150010654A1 (en) * 2012-10-29 2015-01-08 The University Of North Carolina At Chapel Hill Methods and Compositions for Treating Mucosal Tissue Disorders

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120282328A1 (en) * 2006-10-24 2012-11-08 Aradigm Corporation Dual action, inhaled formulations providing both an immediate and sustained release profile
US20150010654A1 (en) * 2012-10-29 2015-01-08 The University Of North Carolina At Chapel Hill Methods and Compositions for Treating Mucosal Tissue Disorders

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NADIG, SN ET AL.: "Immunosuppressive nano-therapeutic micelles downregulate endothelial cell inflammation and immunogenicity.", RSC ADVANCES., vol. 5, no. 54, April 2015 (2015-04-01), pages 43553, XP055328208 *
PIROOZNIA, N ET AL.: "Encapsulation of Alpha-1 antitrypsin in PLGA nanoparticles: In Vitro characterization as an effective aerosol formulation in pulmonary diseases.", JOURNAL OF NANOBIOTECHNOLOGY., vol. 10, May 2012 (2012-05-01), pages 1 - 15, XP021121593 *

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