WO2002102317A2 - Traitement de troubles respiratoires associes a une bronchoconstriction, a l'aide d'un aerosol d'acide hyaluronique - Google Patents

Traitement de troubles respiratoires associes a une bronchoconstriction, a l'aide d'un aerosol d'acide hyaluronique Download PDF

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WO2002102317A2
WO2002102317A2 PCT/US2002/019269 US0219269W WO02102317A2 WO 2002102317 A2 WO2002102317 A2 WO 2002102317A2 US 0219269 W US0219269 W US 0219269W WO 02102317 A2 WO02102317 A2 WO 02102317A2
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polysaccharide
molecular weight
lung
hyaluronic acid
airway
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PCT/US2002/019269
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English (en)
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WO2002102317A3 (fr
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William M. Abraham
Mario Scuri
Rosanna Forteza
Jing-Wen Kuo
Paul Milhalko
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Exhale Therapeutics, Inc.
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Priority to AU2002315330A priority Critical patent/AU2002315330A1/en
Publication of WO2002102317A2 publication Critical patent/WO2002102317A2/fr
Publication of WO2002102317A3 publication Critical patent/WO2002102317A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides

Definitions

  • the present invention relates to formulations and methods for treating respiratory conditions associated with bronchoconstriction and/or airway hyperreactivity. More particularly, the disclosed therapeutic methods involve administering aerosolized hyaluronic acid (“HA”) in amounts sufficient to interact with CD44 and/or receptors for hyaluronic acid-mediated motility (“RHAMM”) disposed on airway epithelium, such that HA binds and thereby inhibits the enzymatic activity of tissue kallikreins (TKs) released in response to a variety of inflammatory stimuli.
  • HA aerosolized hyaluronic acid
  • RHAMM hyaluronic acid-mediated motility
  • Respiratory tract disorders are a widespread problem in the United States and throughout the world. Respiratory tract disorders fall into a number of major categories, including inflammatory conditions, infections, cancer, trauma, embolism, and inherited diseases. Lung damage may also be due to physical trauma and exposure to toxins.
  • Inflammatory conditions of the respiratory tract include asthma, chronic obstructive pulmonary disease, sarcoidosis, and pulmonary fibrosis.
  • Lung infections include pneumonia (bacterial, viral, fungal, or tuberculin) and viral infections.
  • Cancers in the lung may be primary lung cancer, lymphomas, or metastases from other cancerous organs. Trauma to the lung includes lung contusion, barotrauma, and pneumothorax. Embolisms to the lung can consist of air, bacteria, fungi, and blood clots.
  • Inherited lung diseases include cystic fibrosis, and alpha one antitrypsin deficiency.
  • Toxins that can injure the lung include acidic stomach contents (e.g. aspiration pneumonia), inhaled smoke, and inhaled hot air (e.g. from a fire scene).
  • Tissue kallikreins are a family of serine proteases secreted by salivary glands (Schenkels LC et al. 1995 Crit Rev. Oral Biol. Med. 6:161-175; Berg T ef al. 1990 A a Physiol. Scand. 139:29-37; Anderson
  • TK has been identified as the major kininogenase in the airways (Schenkels LC ef al. 1995 Crit. Rev. Oral Biol. Med. 6:161-175).
  • TK activity is increased in human nasal and bronchoalveolar lavages (BALF) after antigen challenge (Christiansen S et al. 1992 Am. Rev. Resp. Dis. 145:900-905; Christiansen S et al. 1987 J. Clin. Invest. 79:188-197; Baumgarten CR ef al. 1986 J. Immunology 137:1323- 1328).
  • BALF human nasal and bronchoalveolar lavages
  • Bronchoconstriction and/or airway hyperreactivity caused by a wide range of inflammatory stimuli such as allergen, metabisulfite, ozone, and bacterial supernatant are associated with increased levels of immunoreactive kinins and increased TK activity in BALF of allergic sheep (Abraham WM ef al. 1994 Am. J. Resp. Crit. Care Med. 149:A533; Forteza R et al. 1994 Am. J. Resp. Crit. Care Med. 149(4):A158; Forteza R ef al. 1994 Am. J. Resp. Crit. Care Med. 149:687-693; Mansour E ef al. 1992 J. Appl. Physiol.
  • HA hyaluronic acid
  • HA hyaluronic acid
  • HA is a large linear polymer with a molecular mass from about 2 x 10 5 to about 10 x 10 6 daltons formed by a repeating disaccharide structure of glucuronic acid and N-acetylglucosamine.
  • HA is present in all vertebrates and some strains of streptococci (De Angelis PL ef al. 1993 J. Biol. Chem. 268:19181-19184) and is abundant in virtually all biologic fluids. Its biological actions include cell-cell and cell-matrix signaling, regulation of cell migration and proliferation as well a providing the fundamental biochemical properties of many tissues (Fraser JR ef al. 1997 J. Intern. Med. 242:27-33). In the lung HA accumulates as part of the fibroproliferative response to injury and tissue remodeling. These actions are mediated by the binding to two major cell surface receptors: CD44 and RHAMM (receptor for hyaluronic acid-mediated motility).
  • CD44 binding stimulates signaling via Rac (Oliferenko S ef al. 2000 J. Cell Biol. 148:1159-1164; Bourguignon LY ef al. 2000 J. Biol. Chem. 275:1829-1838), and Ras (Fitzgerald KA ef al. 2000 J. Immunol. 164:2053-2063).
  • RHAMM is also thought to signal via Ras but, unlike CD44, it is present both on the cell surface and intracellularly (Zhang S. ef al. 1998 J. Biol. Chem. 273:11342-11348; Hofmann M ef al. 1998 J. Cell. Sci. 111:1673-1684; Fieber C ef al. 1999 Gene 226:41-50). Accordingly there is a need for a treatment wherein inhaled HA is administered in amounts sufficient to inhibit the increases in TK activity resulting from various inflammatory stimuli, thus treating and/or preventing bronchoconstriction and/or airway hyperreactivity.
  • a method for treating or preventing respiratory conditions associated with tissue kallikrein-induced bronchoconstriction and/or airway hyperreactivity.
  • the method comprises administering to a mammal in need thereof an amount of an aerosolized formulation comprising a polysaccharide capable of binding to CD44 and/or RHAMM cell surface receptors at a location along the airway epithelium.
  • the amount of polysaccharide is sufficient to sequester tissue kallikrein to the location along the airway epithelium, wherein the enzymatic activity of the tissue kallikrein is inhibited, thereby treating or preventing the respiratory condition.
  • the polysaccharide is a glycosaminoglycan, selected from the group consisting of hyaluronic acid, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, heparan sulfate and heparin.
  • the polysaccharide is hyaluronic acid.
  • the aerosolized formulation further comprises a step of preparing a liquid formulation comprising the polysaccharide, wherein the concentration of the polysaccharide is less than about 5 mg/ml and the molecular weight of the polysaccharide is less than about 1.5 x 10 6 Daltons. The formulation is then aerosolized to form a breathable mist such that the particle size of the polysaccharide is less than about 10 microns. The formulation is then delivered in an effective amount by inhalation of the breathable mist.
  • the molecular weight of the polysaccharide is less than about 587,000 Daltons. More preferably, the molecular weight of the polysaccharide is less than about 220,000 Daltons, and most preferably, the molecular weight of the polysaccharide is about 150,000 Daltons.
  • the breathable mist is formed by a nebulizer.
  • the nebulizer is operated at a pressure of at least about 15 psi.
  • the nebulizer operates at a pressure of at least about 30 psi.
  • the polysaccharide is chemically modified.
  • the modification may comprise cross-linking.
  • the modification comprises addition of a functional group selected from the group consisting of sulfate group, carboxyl group, lipophilic side chain, acetyl group, and ester.
  • the location along the airway epithelium is a ciliated border of the airway epithelium.
  • the amount of polysaccharide is in a range of about 10 ⁇ g/kg body weight/day to about 10 mg/kg body weight/day.
  • the method may further comprise the step of monitoring tissue kallikrein activity via bronchoalveolar lavage or airway resistivity.
  • a method for treating or preventing respiratory conditions associated with tissue kallikrein-induced bronchoconstriction and/or airway hyperreactivity. The method comprises administering to a mammal in need thereof an aerosolized formulation comprising hyaluronic acid in an amount sufficient to bind to RHAMM cell surface receptors at a ciliated border of an airway epithelium and sequester tissue kallikrein to the ciliated border, thereby treating or preventing the respiratory condition.
  • a method for preventing acute bronchoconstriction due to an induction of neutrophil elastase.
  • the method comprises administering to a mammal at least four hours prior to the induction an aerosolized formulation comprising hyaluronic acid at a concentration of at least 0.1% (w/v) with an average molecular weight of 150,000 daltons.
  • the hyaluronic acid may be administered at least eight hours prior to the induction at a concentration of at least 0.5% (w/v).
  • Figure 1 shows the effect of low molecular weight hyaluronic acid (LMW-HA) on elastase-induced bronchoconstriction. Elastase-induced bronchoconstriction was short-lived and reached its peak immediately after challenge to resolve within 30 minutes. LMW-HA 0.2% completely blocked this response whereas LMW- HA 0.1% and 0.05% showed a differential protection indicating a dose-related effect. Values are expressed as mean ⁇ SE for 6 sheep. *P ⁇ 0.001 vs elastase and LMW-HA 0.1 and 0.05%. +P ⁇ 0.001 vs elastase and LMW-HA 0.2 and 0.05%.
  • LMW-HA low molecular weight hyaluronic acid
  • Figure 2 shows the effect of high molecular weight hyaluronic acid (HMW-HA) on elastase-induced bronchoconstriction. Elastase-induced bronchoconstriction was short-lived and reached its peak immediately after challenge to resolve within 30 minutes. HMW-HA 0.05% completely blocked this response whereas HMW-HA 0.01% showed a partial protection and HMW-HA 0.005% was ineffective against the elastase-induced airway response indicating a dose-related effect. Values are expressed as mean ⁇ SE for 6 sheep. *P ⁇ 0.001 vs elastase and HMW-HA 0.01 and 0.005%. +P ⁇ 0.001 vs elastase and HMW-HA 0.05 and 0.005%.
  • HMW-HA high molecular weight hyaluronic acid
  • Figure 3 shows the dose-dependent and molecular weight-dependent effect of hyaluronic acid on elastase-induced bronchial response.
  • the percent protection against elastase-induced bronchoconstriction is plotted against different concentrations of either LMW-HA or HMW-HA (on a logarithmic scale). Both molecular weights of HA show a dose-related effect.
  • HMW-HA achieved an almost complete degree of protection at a much lower concentration than LMW-HA, indicating a molecular weight-dependent effect. Values in the figure are expressed as mean ⁇ SE for 6 sheep.
  • FIG. 4 shows the effect of HMW-HA on elastase-induced TK activity in sheep BALF.
  • Elastase challenge caused a significant increase in TK activity in sheep BALF.
  • This increase was inhibited by pretreatment with HMW-HA 0.05%, a dose that proved effective in blocking the elastase-induced bronchoconstriction.
  • the lower dose of HMW-HA (0.005%), which didn't affect the elastase-induced airway responses, however did't block the elastase-induced increase in sheep BALF TK activity.
  • FIG. 5 shows staining for bronchial tissue kallikrein (TK), airway lactoperoxidase (LPO) and HA in airway epithelial cells (DIC images). Cultured airway epithelial cells are shown in panels A-C. Control cells exposed to pre-immune serum (A) do not show any non-specific labeling. Cells stained for LPO (B) or TK (C) using specific antibodies and DAB revealed specific labeling along cilia. D-l.
  • TK tissue kallikrein
  • LPO airway lactoperoxidase
  • FIG. 6 shows immunohistochemistry and immunocytochemistry for RHAMM (CD168) in airway epithelial cells. Labeling for RHAMM using a specific antibody (R36) and NBT/BCIP reveals its presence in the apical portion of ciliated cells including the cilia themselves (A), while pre-immune serum shows no non-specific staining (B).
  • RHAMM is also expressed on the surface of cultured, non-permeabilized airway epithelial cells (C). All bars are 10 ⁇ m.
  • C non-permeabilized airway epithelial cells
  • specific primers for RHAMM bolded in D
  • ovine airway epithelial cDNA library PCR reactions yielded a 249 by cDNA fragment (nucleotide sequence shown in D) with a deduced amino-acid sequence that was 91% and 81% identical to the human and the mouse sequence, respectively.
  • FIG. 7 shows HA-induced CBF increase is blocked by anti-RHAMM antibodies. Tracings show continuous recordings of ciliary beat frequency ("CBF") in primary cultures of ovine tracheal epithelial cells in response to exogenous HA (50 ⁇ g/ml). All cells respond to 20 ⁇ M ATP with a statistically indistinguishable transient increase in CBF.
  • CBF ciliary beat frequency
  • A/B Cells incubated with a non-specific control IgG (before and during the experiment) respond to HA with an increase in CBF. There are two types of responses: (A) a transient, but continuous increase in CBF, and (B) an oscillatory response.
  • (C) reveals that the CBF response to HA is blocked using a functionally blocking anti-RHAMM antibody.
  • Figure 8 shows the effect of HA on TK and albumin movement by the mucociliary transport system.
  • Figure 9 shows the effect of 0.1% HA pretreatment on human neutrophil elastase-induced bronchoconstriction in sheep.
  • Figure 10 shows the effect of 0.5% HA pretreatment on human neutrophil elastase-induced bronchoconstriction in sheep.
  • Figure 11 shows tissue distribution 3 H-HA clearance.
  • Figure 12 shows the time course of 3 H-HA clearance from lung tissue and lavage fluid.
  • Embodiment Enzymes such as lactoperoxidase and tissue kallikrein (TK), which are secreted onto epithelial surfaces, play a vital role in innate mucosal defense.
  • TK tissue kallikrein
  • one aspect of the present invention relates to the observation that the enzymes of the airway mucosa bind to surface-associated glycosaminoglycans (GAG's; e.g., HA), providing an apical enzyme pool "ready for use" independent of secretion.
  • GAG's surface-associated glycosaminoglycans
  • polysaccharide formulations disclosed herein may be useful in treating and/or preventing a variety of pulmonary conditions and disorders, including for example emphysema, as detailed in U.S. Patent No. 5,633,003 to Cantor and U.S. patent application No. 09/079,209; the disclosures of which are incorporated herein in their entirety by reference thereto.
  • other therapeutic indications for polysaccharide administration to the lung includes: stabilizing the lung matrix (tissue which contains the alveolar sacs and bronchii) by forming a polymer network within the lung matrix; placing a polysaccharide barrier on the matrix fibers of the lung to reduce or eliminate future degradation of the lung fibers, or to protect the fibers from noxious agents while they undergo repair; providing a polysaccharide coating of the lung matrix, surface, bronchioles, and/or alveoli that enhances the moisture content, lubrication, or elastic recoil of the lung; replacing HA in conditions where HA is diminished (e.g.
  • aging, emphysema providing a bulking agent in the lung to reinforce delicate anatomic structures such as alveolar walls (e.g. blebs); providing a lubricant between the internal & external pleura; providing a viscoelastic agent to facilitate elastic lung recoil; providing a dressing to facilitate healing of injured lung tissue; reducing and/or preventing inflammation due to infection, cancer, irritation, allergy, etc.; treating bronchospasm; lubricating and/or loosening mucous; binding to cell receptors to influence cell activity in the lung, such as ciliary cell beating, cell attachment (or adhesion), or cell migration.
  • a bulking agent in the lung to reinforce delicate anatomic structures such as alveolar walls (e.g. blebs)
  • providing a lubricant between the internal & external pleura providing a viscoelastic agent to facilitate elastic lung recoil
  • providing a dressing to facilitate healing of injured lung tissue; reducing and/or preventing inflammation due to
  • Binding in the context of the present invention includes both covalent and non-covalent binding.
  • the binding may be either high or low affinity.
  • the binding may be temporary such that the binding is a coating sufficient to provide a temporary interation.
  • binding forces include, but are not limited to, ionic and covalent bonds, hydrogen binding, electrostatic forces, dipole interactions, or Van der Waals forces. Binding can be defined empirically by those skilled in the art by fluorescence microscopy, following - conjugation of the compound with a fluorescent dye, as discussed in greater detail below.
  • the polysaccharide or carbohydrate moiety may be administered alone or in combination with other polysaccharides or carbohydrate moieties, with or without a suitable carrier.
  • suitable carriers include, but are not limited to, carriers like saline solution, DMSO, alcohol, or water. It may be composed of naturally occurring, chemically modified, or artificially synthesized compounds which are wholly or partially composed of polysaccharides or other carbohydrate moieties, and which are capable of binding to elastic fibers.
  • the amount of the polysaccharide or carbohydrate moiety administered daily may vary from about 1 ⁇ g/kg to about 1 mg/kg of body weight, depending on the site and route of administration. More preferably, the dose is in a range of from about 50 ⁇ g/kg body weight/day to about 500 ⁇ g/kg body weight/day. Most preferably, the dose is in a range of from about 100 ⁇ g/kg body weight/day to about 300 ⁇ g/kg body weight/day. For example, a 50 minute exposure to an aerosol containing a 0.1% solution of bovine tracheal hyaluronic acid (HA) in water (1 mg/ml) was effective in coating hamster lung elastic fibers with HA.
  • HA bovine tracheal hyaluronic acid
  • a method for using a formulation comprising a polysaccharide to treat and/or prevent a respiratory disorder comprises the steps of selecting formulation parameters, which include the molecular weight, the concentration and the viscosity of polysaccharide, such that when aerosolized, the formulation yields a droplet size adapted for delivery to the lungs. The formulation is then aerosolized to form an aerosol, and delivered to the lungs.
  • Another aspect of the invention relates to a method for delivering to the lung alveoli, also referred to as the respiratory zone or deep lung, a polysaccharide or derivative thereof.
  • the method comprises selecting a preparation of the polysaccharide or derivative having a molecular weight sufficient to provide a desired therapeutic profile. Then, preparing a delivery formulation comprising the selected preparation of polysaccharide or derivative at a concentration which when aerosolized yields a particle size suitable for delivery to the deep lung. The delivery formulation is then aerosolized to form an aerosol, and delivered to the deep lung.
  • formulation parameters are selected.
  • Another aspect of the invention relates to a method of treating and/or preventing respiratory disorders by the use of hyaluronic acid, its derivatives, other polysaccharides, and other polysaccharides, either alone or in conjunction with pharmaceuticals, delivered by nebulization or instillation, etc., to the lung tissues.
  • Another aspect of the invention relates to a method for delivering to a selected target site in a lung, a polysaccharide or derivative thereof.
  • the method comprises the steps of preparing a formulation comprising the polysaccharide or derivative at a molecular weight and concentration adapted to yield a desired rheological profile for effective mass transfer during aerosolization or nebulization; and selecting a delivery apparatus and operation parameters, such that when aerosolized, the formulation yields a median droplet size of less than 10 microns, preferably less than 5 microns and most preferably between .05 - 5 microns, with the size range of approximately 2 - 5 microns being adapted for delivery to conducting airways, or the size range of approximately 0.5 - 2 microns being adapted for delivery to the deep lung or respiratory zone.
  • Another aspect of the invention relates to a formulation comprising HA, other polysaccharides and derivatives thereof having a molecular weight, a concentration and a viscosity that are selected to provide a desired therapeutic profile, and to be deliverable by aerosolization to the deep lung for the treatment of a respiratory disorder.
  • Another aspect of the invention relates to a formulation comprising HA conjugated with a second active agent, wherein the formulation has a molecular weight, a concentration and a viscosity that are selected to be deliverable in aerosol form to an alveolus for the treatment of a respiratory disorder.
  • Another aspect of the invention relates to a formulation comprising a polysaccharide and a second agent, wherein the formulation is adapted to be delivered to a lung and also adapted to provide systemic delivery of the second agent.
  • the biocompatible polymers useful in the present invention include without limitation, natural and synthetic, native and modified, anionic or acidic saccharides, disaccharides, oligosaccharides, polysaccharides and in particular, the glycosaminoglycans (GAGs) or acid mucopolysaccharides, which include both non-sulfated (e.g., HA and chondroitin) and sulfated forms (e.g., chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin sulfate, and keratan sulfate).
  • non-sulfated e.g., HA and chondroitin
  • sulfated forms e.g., chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin sulfate, and keratan sulfate.
  • This class of acid mucopolysaccharides can be defined more generally as any polysaccharide having a repeating unit of a dissacharide composed of a hexosamine, e.g., N-acetylated glucosamine, and a uronic acid, e.g., D-glucuronic acid, with or without a sulfate group.
  • a hexosamine e.g., N-acetylated glucosamine
  • a uronic acid e.g., D-glucuronic acid
  • polysaccharides may be obtained via any variety of 5 methods in the prior art such as bacterial fermentation, via processing from animal or plant tissue, or via chemical synthesis.
  • the formulation of the material will enable delivery of the polysaccharides into the lung via aerosol, dry powder delivery, or direct instillation in such a fashion as to adequately cover target, or susceptible, or diseased tissue.
  • the concentration, molecular weight, and viscosity will be such that the material can be dispersed throughout the target site(s) within the lungs, and allow for a desired dosing
  • the material is preferably free from impurities or bacteria that may render it unsafe for human use.
  • HA is one of the GAGs naturally present in the matrix of human lung. It plays a number of roles, including acting as a lubricant, and interacting with various cells and molecules in the lung environment. It is secreted by mesothelial cells in response to congestive heart failure, acute respiratory distress syndrome
  • HA means hyaluronic acid and any of its hyaluronate salts, including, for example, sodium hyaluronate (the sodium salt), potassium hyaluronate, magnesium hyaluronate, and calcium hyaluronate.
  • HA is a polymer consisting of simple, repeating disaccharide units. These repeating disaccharide units consist of glucuronic acid and N-acetyl glycosamine. It is made by connective tissue cells of all animals,
  • HA 20 is present in large amounts in such tissues as the vitreous humor of the eye, the synovial fluids of joints, and the rooster comb of chickens.
  • One method of isolating HA is to process tissue such as rooster combs.
  • This invention can utilize HA isolated and purified from natural sources, as described in the prior art; HA isolated from natural sources can be obtained from commercial suppliers, such as Biomatrix, Anika Therapeutics, ICN, and Pharmacia.
  • HA is via fermentation of bacteria, such as streptococci.
  • the bacteria are incubated in a sugar rich broth, and excrete HA into the broth.
  • HA is then isolated from the broth and impurities are removed.
  • the molecular weight of HA produced via fermentation may be altered by the sugars placed in the fermentation broth.
  • This invention can utilize HA produced by bacterial fermentation as described in the prior art; HA produced via fermentation can be obtained from companies such as Bayer,
  • HA has a molecular weight in the range of 5*10 4 up to 1*10 7 Daltons. HA is soluble in water and can form highly viscous aqueous solutions. Its molecular weight may be reduced via a number of cutting processes such as exposure to acid, heat (e.g. autoclave, microwave, dry heat), or ultrasonic waves. HA obtained from either animal tissue (e.g. rooster combs) or bacterial fermentation may contain contaminant proteins. Inhalation of protein contaminants may induce an allergic reaction in certain patients, causing bronchoconstriction, edema, and influx of inflammatory cells to the lung.
  • heat e.g. autoclave, microwave, dry heat
  • ultrasonic waves e.g. autoclave, microwave, dry heat
  • HA obtained from either animal tissue (e.g. rooster combs) or bacterial fermentation may contain contaminant proteins. Inhalation of protein contaminants may induce an allergic reaction in certain patients, causing bronchoconstriction,
  • the HA of the invention have a protein content of less than 5%, more preferably less than 2%, and most preferably from 0% to undetectable levels.
  • HA preparations may also contain endotoxin contaminants.
  • the HA of the invention have an endotoxin concentration of less than 0.07 EU/mg, and preferably less than 0.01 /EU/mg, and most preferably from 0% to undetectable levels.
  • the polysaccharides may serve as medium for bacterial growth. To insure that delivery of polysaccharides to the lung does not induce pneumonia, the material should be sterile. Other physiologic parameters of the polysaccharides for use in the lung include pH between 4.0 to
  • a liquid formulation of polysaccharides is used.
  • the liquid may be aerosolized for inhalation as a mist via an aerosolization device such as a nebulizer, atomizer, or inhaler.
  • the formulation is a dry powder which individuals would mix at home or the hospital with saline or water before instillation to an aerosol device. The device would produce an aerosol for inhalation by the patient.
  • a dry powder formulation could also be delivered in powder form by an aerosol device, such as air gun powered aerosol chamber. Companies which produce dry powder delivery devices include Dura Delivery Systems (the "Dryhaler"), Inhale Therapeutics, and Glaxo Wellcome (Diskhaler).
  • the respiratory system consists generally of three components: the tracheal/pharyngeal, the bronchial and the alveolar. It is known that particles of 10-50 microns migrate to the tracheal/pharyngeal component. Particles of about 5-10 microns migrate to the bronchial component, and particles of 0.5 to 5 microns migrate to the alveolar component. Particles less than 0.5 microns in size are not retained.
  • the mass median aerodynamic diameter (MMAD) is predictive of where in the lung a given particle will end up. The MMAD is usually expressed in microns.
  • a related parameter is the geometric standard deviation (GSD).
  • GSD geometric standard deviation
  • a GSD of 1 is equal to a normal distribution. A GSD of less than one indicates a narrow size dispersion and a GSD of more than 1 indicates a broad size dispersion.
  • Elastin is a cationic protein. Consequently, introducing negatively charged groups, ions or substitutions can enhance the electostatic forces between the polysaccharide and the elastic fibers. For example, sulfate groups could be added to make the compound more negatively charged.
  • Another means of adding sulfate groups to HA involves reaction with NH 2 after deacetylation of N- acetyl.
  • the sulfation is completed in two steps, (a) deacetylation of N-acetylglucosamine moeity of HA by its reaction with anhydrous hydrazine at elevated temperature, followed by (b) treatment of the derived product with trimethylamine-sulfur trioxide.
  • a) deacetylation of N-acetylglucosamine moeity of HA by its reaction with anhydrous hydrazine at elevated temperature
  • treatment of the derived product with trimethylamine-sulfur trioxide.
  • carboxyl groups can be added to polysaccharides to increase their negative charge, thereby improving their binding to elastin in the lung matrix.
  • the following reactions are provided to illustrate carboxylation schemes reactions for HA:
  • the 6-hydroxyl of the N-acetylglucosamine can be a target for further modification to introduce an additional carboxyl group, for example, reaction of dry HA with sodium chloroacetate.
  • the hydroxyl functional groups of HA are esterified by converting the carboxyl functional groups of HA into a tertiary ammonium or tertiary phosphonium salt in the presence of water and aprotic solvent and then treating the solution with succinic anhydride, as disclosed in U.S. Patent No. 6,017,901, entitled "Heavy metal salts of succinic acid hemiesters with hyaluronic acid or hyaluronic acid esters, a process for their preparation and relative pharmaceutical compositions.
  • dianhydrides such as ethylenediamine tetraacetic acid dianhydride (EDTAA) can be used. This reaction produces crosslinked HA.
  • free pendant carboxyl groups from the anhydride may exist after the reaction of dianhydrides and HA, as described in U.S. Patent No. 5,690,961, entitled "Acidic polysaccharides crosslinked with polycarboxylic acids and their uses”.
  • Lipophilic side chains can also be attached to polysaccharides to increase the binding strength between the polysaccharide and elastin.
  • Polar functional groups such as carboxyl and hydroxyl groups impart hydrophilicity.
  • the introduction of lipophilic moieties to the polysaccharide can improve their affinity for elastin fibers, because elastin has a composition that is rich in amino acids with aliphatic side chains.
  • the following reaction schemes are provided with respect to HA:
  • a method of manufacturing acetylhyaluronate comprises the steps of suspending hyaluronic acid powder in an acetic anhydride solvent and then adding concentrated sulfuric acid thereto to effect acetylation.
  • the maximum degree of substitution is four, since there are four hydroxyl groups in each dissacharide unit of HA. Practically, only partial acetylation occurs.
  • the degree of substitution determines the lipophilicity (thus hydrophobicity) of the modified HA. The more lipophilic, the higher the affinity of HA derivatives to the lipophilic moiety of elastin fibers. See e.g., U.S. Patent No.
  • HA can react with alkylhalide, such as propyl iodide to form the ester function from the carboxyl group.
  • alkylhalide such as propyl iodide
  • the HA derivatives are less water-soluble and more lipophilic, proportional to the increase of degree of derivatization, as described in European Patent Application No. 86305233.8.
  • a molecular weight of the polysaccharide or derivative is selected to produce a desired physiologic effect or molecular interaction, i.e., a desired therapeutic profile.
  • the polysaccharides and their derivatives are polymers of repeating units and as a result, may be isolated, purified, synthesized, and/or commercially obtained in a wide range of molecular weights.
  • the physiologic effects and molecular interactions of the polymers vary with molecular weight.
  • the physical delivery of the polymers to a selected target site within the lung also varies with polymer size (molecular weight). Different therapeutic profiles would be desirable for different clinical indications, and can be individually developed and optimized without undue experimentation by a physician skilled in the art, using the teachings disclosed herein.
  • a high molecular weight preparation of polysaccharide would be desirable in order to provide effective binding to and coating of elastin fibers.
  • a high molecular weight polysaccharide derivative, modified to enhance its affinity for elastin would be preferred.
  • High molecular weight preparations are also preferred for depot of drugs, where the large polymer may be a better excipient, a better carrier and better for addressing large airway diseases.
  • lower molecular weight preparations may be better for loosening sputum, penetrating to the deep lung tissues, and traversing alveolar-epithelial barrier.
  • the physician will have to balance the desired therapeutic profile against physical restraints on delivery into the deep lungs.
  • a polymer preparation in accordance with the present invention may have a molecular weight that resides in the lung for between 0.5 hour and one week, preferably between 1 hour and one day, and more preferably between 4 and 16 hours. Most preferably, a GAG will remain associated with the lung matrix for at least 6 hours. This would allow for dosing four or less time a day.
  • molecular weights of HA preparations for between 25,000 Daltons and 2,000,000 Daltons can be used to provide lung duration times, water retention, elastic recoil, and matrix coverage, consistent with the above.
  • the relationship between polysaccharide concentration, molecular weight and viscosity is discussed in greater detail below.
  • a preparation of HA having a molecular weight of greater than 2,000,000 Daltons was used, it produced a solution that was excessively viscous.
  • these properties must be balanced against excessive viscosity, particularly at lower deployment temperatures (e.g., jet nebulizers that cool the solutions significantly during expansion).
  • HA it has been observed for HA, that it was preferred to use a preparation having a molecular weight of less than about 1.5 x 10 6 Daltons, more preferably less than 500 kD, more preferably still, less than about 220 kD, and most preferably less than about 150 kD.
  • the concentration of the glycosaminoglycan solution also influences duration times, water retention, elastic recoil, and matrix coverage, and formulation viscosity. Viscosity increases with increasing concentration. Viscosity increases with decreasing temperature. Concentrations of HA are preferably between about 0.05 mg/L and 5 mg/L at ambient temperature (20° to 25° C). The preferred concentration is less than 5 mg/L, more preferably less than 2 mg/L, and more preferably less than 1 mg/L. The preferred concentration is above 0.05mg/L, more preferably over 0.5 mg/L. The concentration of a selected molecular weight preparation may be adjusted to yield a selected viscosity, depending on the temperature.
  • the viscosity or thickness of the material is related to the combination of concentration and molecular weight. Viscosity increases with increasing molecular weight if concentration remains constant. Likewise, viscosity increases with increasing concentration if molecular weight remains constant. Viscosity can be measured by a viscometer (one such device is manufactured by the company Brookfield), and is expressed in units of centipoises (abbreviation: cps).
  • the material must be transferred from the delivery device (e.g. via an aerosolization device) into the respiratory tract, down to the distal bronchi and alveoli, from where it can diffuse into the extracellular lung matrix.
  • the delivery formulation should have physical characteristics which avoid clogging of the aerosol device and clumping of aerosolized particles. It should be noted that a viscous material, delivered slowly, may not cause clogging or plugging, whereas a less viscous material may, if delivered quickly.
  • Formulations of specific molecular weight, concentration and viscosity are preferably produced by adding a volume of sterile delivery solvent (e.g., water or saline) to an amount of sterile, medical grade polysaccharide powder. More preferably, unit dose vials containing a pre-weighed dose of polysaccharide may be dissolved just prior to use by injection of sterile solvent into the sealed vial. The powdered polysaccharide is then mixed in the solvent until dissolved. Alternatively, polysaccharide of a certain concentration can be prepared by diluting liquid polysaccharide with sterile solvent.
  • sterile delivery solvent e.g., water or saline
  • Formulation temperatures of between about 0° to about 100° C, preferably between about 4° and 60° C and more preferably between about 15° and 37° C may be used in accordance with the present invention; however, the viscosity of a given molecular weight and concentration of a polysaccharide varies with temperature.
  • the user can determine empirically the viscosity with a viscometer, and adjust the concentration accordingly to yield a viscosity adapted for delivery by the desired delivery mechanism (e.g., nebulizer, aerosolizer, inhaler etc.) to the selected target site in the lungs.
  • the viscosity is preferably below about 1,000 cps, more preferably below about 100 cps, and most preferably below about 50 cps.
  • Particle size is preferably below about 10 microns in diameter. More preferably, the particle size is between 2 and 5 microns.
  • the relationship between particle size in microns and fluorescence-labeled polysaccharide molecular weight and concentration can be measured as the Mass Median Aerodynamic Diameter using a Cascade Impactor (see data in Examples below).
  • the numbers on the x-axis represent sieve sizes in microns and the numbers on the y-axis represent fluorescence (i.e., amount of polysaccharide) which impacts on the particular sieve (i.e., median particle size is too large to fit through the pores).
  • fluorescence i.e., amount of polysaccharide
  • a humidified variation of the Cascade Impactor can also be used to more closely reflect pulmonary delivery, because the polymers of the present invention may be hydroscopic and therefore absorb water and swell in size.
  • Raabe et al. reported a survey of particle size access to various airways in small laboratory animals using inhaled monodisperse aerosol particles. Raabe et al., Ann. Occup. Hyg. 1988, 32:53-63; incorporated herein by reference thereto. Similar analysis may be performed to inform the clinician as to the desirable particle size for delivery to a target site within the lung.
  • Particle size in accordance with a preferred mode of the present invention may be between about 2 microns and about 5 microns, thereby being adapted for delivery into the lung alveoli. Larger size particles are not as efficiently delivered through the distal bronchioles, whereas much smaller sizes tend to be exhaled before contacting the alveolar lining. Thus, whereas the therapeutic profile (e.g., duration, water retention, elastic recoil and matrix coverage) tend to increase with increasing molecular weight, the relative deliverability (i.e., frequency of particles within the 2-5 micron range) tends to decrease with increasing molecular weight.
  • the therapeutic profile e.g., duration, water retention, elastic recoil and matrix coverage
  • the relative deliverability i.e., frequency of particles within the 2-5 micron range
  • the glycosaminoglycan In order to produce an aerosol which can be inhaled by human beings for distribution throughout the lung, the glycosaminoglycan must be aerosolized into appropriate droplet sizes as detailed above, preferably between about 2-5 microns in diameter. Some droplets larger than 5 microns in diameter may deposit in the nebulizer tubing or mask, mouth, pharynx, or laryngeal region. Droplets less than 2 microns in diameter tend not to be deposited in the respiratory tract, but are exhaled and lost. Droplet sizes of 2-5 microns can be achieved by selection of appropriate aerosol devices, solution concentration, compound molecular weight, and additives, in accordance with the teachings herein.
  • Additives such as surfactants, soaps, Vitamin E, and alcohol may be added to avoid clumping of droplets after they are produced, and to facilitate generation of small particles from an aerosol device.
  • One embodiment of the invention includes glycosaminoglycans in combination with one or more of these additives.
  • a method of selecting breathable formulations for delivery to the lung by aerosol is to screen multiple formulations for those formulations which will produce droplets of less than 10 microns in diameter, more preferable less than 6 microns, most preferably 2-5 microns. Formulations which produce droplets larger than 10 microns are not suitable for delivery into the lung. Particle size distribution of the aerosolized mist for each formulation is measured with a device such as a Malvern Laser or a Cascade Impactor (as used to generate the data shown in Figures 1A-L).
  • This invention includes all molecular weight and concentration combinations of polysaccharides that can be aerosolized into droplet sizes of under 10 microns, and more preferably between about 2-5 microns.
  • One embodiment of the invention involves use of an aerosol-generating device to produce an inhalable mist.
  • One class of device to generate polysaccharide aerosols is a spray atomizer.
  • Another class of device to generate polysaccharide aerosols is a nebulizer. Nebulizers are designed to produce droplets under 10 microns.
  • nebulizers may be used to aerosolize polysaccharides for delivery to the lung: 1) compressed air nebulizers (examples of these include the AeroEclipse, Pari L.C., the Parijet and the Whisper Jet) and 2) ultrasonic nebulizers.
  • Compressed air nebulizers generate droplets by shattering a liquid stream with fast moving air.
  • One mode of the invention involves use of a compressed air nebulizer to aerosolize polysaccharide solutions into droplets under 10 microns in size.
  • Ultrasonic nebulizers use a piezoelectric transducer to transform electrical current into mechanical oscillations, which produces aerosol droplets from a liquid solution. Droplets produced by ultrasonic nebulizers are carried off by a flow of air.
  • Another mode of the invention involves the use of an ultrasonic nebulizer to aerosolize polysaccharide solutions into droplets less than 10 microns in size.
  • Another mode of this invention is use of a hand-held inhaler to generate polysaccharide aerosols.
  • This portable device will permit an individual to administer a single dose of mist, rather than a continuous "cloud" of mist into the patient's mouth.
  • Individuals with bronchoconstrictive diseases such as asthma, allergies, or COPD often carry these hand-held inhalers (e.g., MDI and DPI) in their pocket or purse for use to alleviate a sudden attack of shortness of breath.
  • These devices contain bronchodilator medication such as albuterol or atrovent. They would also be a convenient way to deliver glycosaminoglycan to patients.
  • nebulizer For treatment via nebulizer, patients would inhale the aerosolized polysaccharide solution via continuous nebulization, similar to the way patients with acute attacks of asthma or emphysema are treated with aerosolized bronchodilators.
  • the aerosol may be delivered through tubing or a mask to the patient's mouth for inhalation into the lungs. Treatment time may last 30 minutes or less.
  • the mouth is preferably used for inhalation (rather than the nose) to avoid "wasted" nasal deposition.
  • the volumetric flow rate (L/min) of the nebulizer preferably does not exceed two times the patient's minute ventilation, although this can be varied depending on the polysaccharide formulation and the clinical status of the patient. This is because the average inspiratory rate is about twice the minute ventilation when exhalation and inhalation each represent about half of the breathing cycle.
  • a nebulizer with a volumetric flow rate of under 15 L/min is employed.
  • Nebulizer related factors for compressed air nebulizers include air pressure, air flow, and air jet diameter.
  • Nebulizer related factors for ultrasonic nebulizers include ultrasound frequency, and rate/volume of air flow.
  • a compressed air nebulizer with specific air pressure, air flow, and hole diameter settings is used to generate droplets of a specific polysaccharide formulation under 10 microns.
  • an ultrasonic nebulizer with specific frequency and hole diameter settings is employed to generate droplets of a specific polysaccharide formulation under 10 microns.
  • nebulizer and formulation include solution use rate (ml/min), aerosol mass output (mg/L), and nebulizer "hold up” (retained) volume (ml). The interaction among these factors will be appreciated by those of skill in the art. Aerosolized polysaccharide could be delivered from nebulizer to a patient's respiratory tract via face mask, nonrebreather, nasal cannula, nasal covering, "blow by" mask, endotracheal tube, and Ambu bag. All of these connections between the patient and nebulizer are considered to fall within the scope of the present invention.
  • another embodiment of the invention involves delivery of aerosolized polysaccharides under positive pressure ventilation.
  • a commonly used ventilatory assist device is CPAP: Continuous Positive Airway Pressure.
  • CPAP Continuous Positive Airway Pressure
  • a breathing mask is sealed around the mouth of a patient. The patient is then administered oxygen through the mask at a certain pressure to facilitate inspiration. Delivery of polysaccharides through a CPAP mask might enhance delivery of material to the deep airways.
  • the polysaccharide could be delivered while the patient is being ventilated with positive end expiratory pressure (PEEP).
  • PEEP positive end expiratory pressure
  • Another mode of the invention is to deliver aerosolized polysaccharides with a device that delivers material when the patient generates a certain level of negative inspiratory pressure.
  • Another mode of the invention is to deliver polysaccharides in conjunction with ventilation through an endotracheal tube.
  • One benefit of this embodiment is to protect against oxygen toxicity in patients ventilated with high concentrations of oxygen.
  • the viscoelastic properties of polysaccharides should protect the lungs from ventilator associated barotrauma that results in the complication of pneumothorax.
  • this invention is a nontoxic therapy, which exerts its beneficial effects in respiratory disease by its physical presence in the lung
  • the formulation of this invention should allow for the polysaccharide to remain in the lung continuously.
  • the half-life of HA injected in the pleural (potential space between the lung and the chest wall) of rabbits has been shown to range between 8 and 15 hours. The half- life is longer if more HA is injected.
  • Commonly inhaled medications for emphysema are used from one to three times a day. More frequent dosing requirements present a compliance issue with patients.
  • One aspect of this invention involves a formulation of polysaccharide that resides in the lung for 6 hours to be given 4 times per day, or preferably for 8 hours, to be given three times per day.
  • a more preferable embodiment is a formulation that remains in the lung for 12 hours, which will be administered twice a day.
  • a more preferable embodiment is a formulation that remains in the lung for 24 hours, which will be administered once a day.
  • a radiolabel such as tritium, C 14 , Thallium, or Technecium.
  • a direct assay for the particular polymer could be employed.
  • One radiometric assay for HA uses 125 l-labeled HABP (HA binding protein); this assay is commercially available from Pharmacia ("Pharmacia HA Test"). Material is delivered to the lungs and monitored over time by use of a scintillation counter (e.g. gamma camera). Alternatively, a group of animals (e.g. rats) is given radiolabeled-glycosaminoglycan in the lungs and then serially sacrificed over time.
  • a scintillation counter e.g. gamma camera
  • Excised lung tissue is examined for radioactivity, and duration time or half-life is determined.
  • the invention encompasses protecting the lungs with aerosol polysaccharide
  • the invention also encompasses application of polysaccharide by aerosol delivery to other tissues, including for example, exposed tissues during surgery, sinus passageways, burns, and mucous membranes.
  • pulmonary resistance the proximal end of the endotracheal tube was connected to a pneumotachograph (Fleisch; Dyna Sciences, Blue Bell, PA). The signals of flow and transpulmonary pressure were recorded on an oscilloscope recorder, which was linked to a computer, for on- line calculation of RL.
  • Respiratory volume was obtained by digital integration of the flow signal and was used, together with transpulmonary pressure and flow, at isovolumetric points to derive R (as described by Von Neergaad K ef al. 1927 Z. Klin. Med. 105:51-82), as previously described (Forteza R ef al. 1996 Am. J. Resp. Crit. Care Med. 154:36-42; incorporated herein in its entirety by reference). Analysis of 5-10 breaths was used for the determination of RL. Aerosols
  • Aerosols were generated using a disposable medical nebulizer (Raindrop; Puritan Bennett, Lenexa, KS).
  • the output from the nebulizer generated an aerosol with mass median aerodynamic diameter of 3.2 ⁇ m (geometric SD 1.9) as determined by an Andersen cascade impactor.
  • the output of the nebulizer was directed into a plastic T-piece, which was interconnected to the inspiratory port of a Harvard piston ventilator (Harvard Apparatus, Natick, MA) with the animal's tracheal tube.
  • a dosimeter system consisting of a solenoid valve and a source of compressed air (20 psi) was used.
  • the solenoid valve was activated for I second at the beginning of the inspiratory cycle of the ventilator. Aerosols were delivered at a tidal volume of 500 ml and a rate of 20 breaths/min.
  • Agent Porcine pancreatic elastase (PPE) was purchased from Sigma Aldrich Co. (St. Louis, MO), dissolved in phosphate buffered saline (PBS; pH 7.4) to a stock concentration of 5 mg/ml. Aliquots of 500 ⁇ g were kept at -20° C, dissolved in 3 ml PBS (pH 7.4) the day of the experiment and delivered as an aerosol (20 breaths/min x 20 min). LMW-HA from pig trachea (avg.
  • the distal tip of a specially designed 80 cm fiberoptic bronchoscope was wedged into a randomly selected subsegmental bronchus.
  • Lung lavage was performed by slow infusion and gentle aspiration of 60 ml of PBS (pH 7.4 at 37° C) in two different airway segments (30 ml each), using a 30 ml syringe attached to the working channel of the instrument.
  • the effluent was filtered through a double layer of gauze and transferred into a tube. All tubes were placed immediately on ice and then centrifuged at 250 x g at 4° C for 15 minutes. The supernatant was recentrifuged at 3000 x g at 4° C for 15 minutes, saved and frozen at -80° C for subsequent analysis.
  • BALF bronchoalveolar lavage fluid
  • HA hyaluronic acid
  • BALF TK activity was measured at baseline and 30 minutes after challenging the animals with inhaled elastase (PPE 500 ⁇ g). The same procedure was repeated after pretreatment with HMW-HA at concentrations of 0.05, and 0.005%. Statistics.
  • FIG. 2 Aerosolization of lower doses of HMW-HA (0.01, 0.005%), again, showed a dose-dependent effect (Figure 2).
  • inhaled HA prevents the elastase-induced bronchoconstriction in a dose-dependent and molecular weight-dependent fashion.
  • This protection is associated with inhibition of TK activity in BALF of allergic sheep.
  • inhaled elastase increased lung TK activity and caused bronchoconstriction via the formation of bradykinin (Scuri M ef al. 2000 J. Appl. Physiol. 89(4):1397-1402; incorporated herein in its entirety by reference thereto).
  • bradykinin Scuri M ef al. 2000 J. Appl. Physiol. 89(4):1397-1402; incorporated herein in its entirety by reference thereto.
  • bronchial TK bound to HA thereby reducing its activity in vitro (Forteza R ef al. 1999 Am. J. Resp. Cell Mol. Biol.
  • HA is a long polymer and, although the TK binding site has not yet been characterized, it is conceivable that a heavier and thus longer HA molecule, carries more binding sites for TK. Thus, it is possible that HMW-HA can bind more TK molecules at any given concentration and so provide better protection against the elastase-induced airway responses.
  • LMW-HA causes the induction of inflammatory factors via a CD44-mediated mechanism (Noble PW ef al. 1998 In: The chemistry, biology and medical applications of hyaluronan and its derivatives. London, Portland Press, pgs. 219-225). In this study, however, no inflammatory- response was observed in the animals that received LMW-HA. This observation is consistent with data from Lackie ef al. (Lackie P ef al. 1997 Am. J. Resp. Cell Mol. Biol. 16(1):14-22), who showed that CD44-mediated actions in the airways are associated with repair rather than with inflammatory processes.
  • HMW-HA was used at two different concentrations: one that was effective in blocking the elastase-induced bronchoconstriction (0.05%) and one that was ineffective (0.005%).
  • the mediator data supported the functional ones with 0.05% HMW-HA suppressing the increase in TK while 0.005% HMW-HA failed to do so.
  • GAGs glycosaminoglycans
  • proteases and protease inhibitors can regulate their functions by different mechanisms including but not limited to: (1) enzyme immobilization, leading to the restriction of its range of action; (2) stearically blocking its activity; (3) providing a reservoir for delayed release; or (4) protecting it from proteolytic degradation (see e.g., Ying QL ef al. 1997 Am. J. Physiol. 272(3 Pt 1)1533-541).
  • GAGs-proteinase interactions could be similar for TK-HA interactions. Forteza ef al.
  • HA binding to TK blocks its enzymatic activity (Forteza R ef al. 1999 Am. J. Resp. Cell Mol. Biol. 21 :666-674). HA is elevated in BALF of asthmatic patients (Vignola AM ef al. 1998 Am. J. Resp. Crit. Care Med. 157(2):403-409) indicating that its turnover is altered in these subjects.
  • human neutrophil elastase causes the release of TK from primary cultures of ovine tracheal gland cells as already shown in studies conducted in this laboratory (Forteza R ef al. 1997 Am. J. Resp. Crit. Care Med. 155(4):A357).
  • inhaled elastase caused bronchoconstriction in allergic sheep via a bradykinin-mediated mechanism (Scuri M ef al. 2000 J. Appl. Physiol. 89(4): 1397- 1402).
  • Antigen challenge also increased free elastase activity in BALF of allergic sheep (O'Riordan TG et al. 1997 Am. J. Resp. Crit. Care Med. 155:1522-1528) which, in turn, stimulated TK release.
  • Other stimuli such as cell products (Trahir JF ef al. 1989 Histochem. Cytochem. 37:309-314; Sommerhoff SP et al. 1990 J. Clin. Invest.
  • TK is also thought to be a mediator in rhinitis and asthma.
  • Bronchial TK was resistant in vitro to inhibition by most of the serine protease inhibitors present in BALF, suggesting that there was no effective inhibition for TK in the airways.
  • HA does plays an important role in the regulation of bronchial TK activity by binding to it, thus preventing its biologic actions.
  • This disclosure adds new evidence that, in vivo, exogenous HA can restore the physiologic HA-TK interaction, thus preventing the elastase-induced bronchoconstriction and increase in BALF kinins. It is likely that this anti-elastase effect of HA is based on enzymatic inhibition of TK. This represents the first observation of a functional protection of HA in the airways.
  • Cantor ef al. (Cantor JO et al. 1999 Connect. Tiss. Resp. Tech. 40(2)97-104) showed that HA prevents the elastase-induced emphysema in hamsters. This effect, however, depends on a mechanic property of HA, which forms a protective coating on the elastin in the lung, thus limiting its degradation by elastase released from neutrophil and/or macrophages.
  • the results of these studies suggest that the protective effect of HA against elastase-induced lung injury is not related to an enzymatic interaction with TK, and thereby does not interfere with its regulation.
  • neutrophil elastase can cause release of TK from tracheal gland cells in allergic mammals.
  • the increase in lung TK activity mediates the bronchoconstrictor response to inhaled elastase via the formation of bradykinin.
  • HA bound airway TK thereby reducing its activity in vitro.
  • pulmonary resistance (RL) was measured in allergic sheep before and after inhalation of elastase alone (500 ⁇ g) and after pretreatment with either inhaled low molecular weight HA (LMW-HA; 3 ml) or high molecular weight HA (HMW-HA; 3 ml) at different concentrations.
  • LMW-HA low molecular weight HA
  • HMW-HA high molecular weight HA
  • HMW-HA 0.05% but not by 0.005% HMW-HA, which was also ineffective in blocking the elastase-induced bronchoconstriction.
  • HMW-HA blocks the elastase-induced bronchoconstriction in a dose-dependent and molecular weight-dependent fashion.
  • Airway LPO was purified as described (Salathe M ef al. 1997 Am. J. Resp. Cell Mol. Biol. 17:97-105) and shown to stimulate bacterial clearance of the airways (Gerson C ef al. 2000 Am. J. Resp. Cell Mol. Biol. 22:665-671).
  • Bronchial TK mediates allergic bronchoconstriction and thereby limits the inhalation of noxious substances. Both enzymes are secreted from airway submucosal gland cells. It has been commonly believed that proteins are rapidly cleared by the mucociliary apparatus after secretion. Therefore, secretion has been postulated to be the main determinant of enzyme availability (and activity) on mucosal surfaces.
  • polyclonal rabbit anti-human urinary kallikrein serum (Calbiochem) has previously been demonstrated to recognize specifically bronchial TK.
  • Antiserum to purified sheep airway LPO was made in rabbits (Covance, Hazelton, PA). Specificity was determined by Western blotting with purified sheep and bovine LPO as well as canine and human MPO.
  • Rabbit anti-chicken IgG used as a control in the CBF experiments, was from Cappel (Organon Teknika Corporation). Sheep trachea and cell cultures were fixed with acid formalin and processed according to standard procedures for immunohistochemistry and immunocytochemistry.
  • Hyaluronidase digestion was accomplished with hyaluronidase (50 U/ml at pH 5.5; Seikagaku) in a cocktail of protease inhibitors (pepstatin 10 ⁇ g/ml, aprotinin and leupeptin 10 ng/ml) in 50 mM Tris buffered saline, pH 5.5, at 37° C overnight.
  • protease inhibitors pepstatin 10 ⁇ g/ml, aprotinin and leupeptin 10 ng/ml
  • the results shown in Figure 5 indicate that the ciliated border of the epithelium was labeled. Digestion with hyaluronidase eliminated the apical staining for HA as well as LPO and TK ( Figure 5).
  • Airway LPO was also binding to HA, as determined by non-denaturing agarose gel electrophoresis. However, analysis of the airway LPO amino-acid sequence did not reveal the presence of known HA-binding motifs. Instead, LPO probably binds to HA because of its alkaline pi by ionic interaction. In fact, HA may act as a cation exchanger and may be able to bind several other cations to the epithelial surface. Among those could be a variety of cationic antimicrobial substances, for example those studied by Cole ef al. (Cole AM ef al. 1999 Infect. Immun. 67:3267-75) .
  • HA binding inhibits the activity of TK. This is important because TK activity can lead to bronchoconstriction, only useful during exposure to certain stimuli.
  • Airway LPO should be active at all times because it contributes to host defense against bacteria. In fact, measurements of airway LPO activity in vitro according to published methods revealed that the enzyme did not change its activity whether or not HA was present over a large concentration range.
  • HA-binding receptor expressed at the apical surface of the epithelium is involved in mediating the interaction between HA and TK.
  • CD44 a common extracellular HA receptor
  • CD168 a common extracellular HA receptor
  • Immunohistochemistry revealed specific staining for RHAMM in the apical portion of ciliated cells, but no staining in goblet cells ( Figure 6). This suggests a role for RHAMM in ciliated cells.
  • an ovine tracheal cDNA library and primers for RHAMM were used (Figure 6), which were designed according to consensus regions of the published sequences.
  • An ovine mucosa cDNA library was used as a template with a specific 5' oligonucleotide and a 3-fold degenerate 3' primer, both designed from consensus RHAMM sequences ( Figure 6).
  • the FailSafeTM PCR system (Epicentre Technologies, Madison, Wl) was used with annealing at 52° C. PCR yielded a band of expected size (249 bp).
  • RHAMM 2000 J. Aerosol Med. 231-237 was mediated by RHAMM, primary cultures of ovine airway epithelial cells were used as described (Salathe M ef al. 1995 J. Cell Sci. 108:431-440). Using anti-RHAMM antibody and fixed, non-permeabilized cells, the expression of RHAMM could also be shown to occur on the surface of cultured ciliated cells ( Figure 6). These results were confirmed by adding anti-RHAMM antibody to live, cultured cells before fixation. The expression of RHAMM increased during the time in culture (18% of all ciliated cells stained positive on day 3 after plating, 57% on day 5, 65% on day 8, and 76% on day 11).
  • HA serves a dual role in the airway epithelium by binding enzymes to the ciliary border and by simultaneously stimulating ciliary beat frequency through interactions with RHAMM.
  • recombinant TK was labeled with fluorescein and both airway LPO and albumin were labeled with rhodamine.
  • recombinant TK gift kindly provided by Dr. Cliff Wright from Amgen Pharmaceuticals
  • purified airway LPO and bovine serum albumin (Sigma) were labeled with fluorescein or rhodamine isothiocyanate according to published methods.
  • the products were purified on Sephadex G50, concentrated to 1 mg/ml in PBS and applied in equimolar amounts to the mucosal surface of a trachea obtained from a freshly sacrificed sheep, opened by cutting through the membranous portion and kept in a humidified chamber at 37° C.
  • the movement of the applied fluorescent substances was monitored using a broad spectrum UV-illuminator and a digital camera every 10 minutes for a total of 30 minutes.
  • HA was removed from the surface by 5 lU/ml hyaluronidase (Worthington, active at pH 7.4). Tracheas from freshly sacrificed sheep were opened by cutting through their posterior membranous portions and kept in a humidified chamber at 37° C.
  • TK and albumin were applied together (as a mixture) onto the same region of the surface epithelium and the migration of the fluorescence measured over a 30 minute period.
  • TK was not transported after application whereas albumin moved forward over the whole 30 minute period.
  • albumin moved forward over the whole 30 minute period.
  • the two substances separated which was indicated by a change of the original orange fluorescence (mixture) into a clearly defined green (TK) and red (albumin) band (Figure 8).
  • rhodamine-labeled airway LPO was used with the same result (not shown).
  • HA serves a previously unrecognized pivotal role in mucosal host defense. It stimulates ciliary beating (through its interaction with RHAMM) and thereby the clearance of foreign material from mucosal surfaces, but simultaneously it retains and regulates enzymes important for homeostasis at the apical mucosal surface. Therefore, the common belief that constitutive and stimulated secretion onto the mucosal surface determines enzyme availability has to be revisited.
  • the new paradigm shown here provides an apical enzyme pool "ready for use” and protected from ciliary clearance. It is likely that this paradigm may also apply to other mucosal surfaces such as the ones found in the mouth or gut. Thus, this apical enzyme pool will have to be considered in enzymatic reactions at the mucosal surface, be it in health or disease.
  • FIG. 9 shows the prevention of resistance in the lungs (RL) (bronchoconstriction) with aerosol delivery of a formulation comprising 0.1% HA (average molecular weight of 150,000 daltons), pretreated 0.5, 4 and 8 hours before challenge with neutrophil elastase (HNE).
  • HNE neutrophil elastase
  • Figures 11 and 12 show prolonged half-life in the lungs following aerosol delivery of HA (0.15 mg/kg).
  • Samples solutions of HA were prepared with varying concentration for a series of different molecular weights. Molecular weights above 200,000 Dalton was measured by intrinsic viscosity and calculated by the Mark-Houwink Equation. Alternatively, molecular weight was measured by HPLC or Light Scattering analysis. By varying the concentration for a given molecular weight of HA, a range of different viscosities were achieved. These solutions were tested in commercially available nebulizers and the mass median aerodynamic diameter (MMAD) in microns and the geometric standard deviation (GSD) were determined for each tested sample.
  • MMAD mass median aerodynamic diameter
  • GSD geometric standard deviation
  • HA HA-containing HA
  • Concentrations were varied from 0.5 to 2.0 mg/ml at a molecular weight of 150,000, determined by HPLC and light scattering (Table 6). A range of viscosities from 1.72 to 3.04 centistoke were achieved. These solutions were tested in Whisper nebulizers and the mass median aerodynamic diameter (MMAD) in microns and the geometric standard deviation (GSD) were determined for each tested sample.
  • MMAD mass median aerodynamic diameter
  • GSD geometric standard deviation
  • the nebulizer droplet size distributions tended to be bimodal with one mode for sizes larger than about 2 ⁇ m in aerodynamic diameter and one mode smaller than about 0.5 ⁇ m. Both of these modes are relatively effectively deposited in the lung airways during inhalation and the balance between these modes determines the effective regional deposition of aerosol between the conducting airways and the deep lung. These bimodal size distributions are a result of the complex interaction of evaporation phenomena for aerosols from aqueous solutions. Small droplets have higher vapor pressure than larger droplets by virtue of their surface curvature so that small droplets tend to evaporate and larger droplets grow under saturated water vapor conditions.
  • evaporation is inhibited by the HA in solutions so that the smaller droplets do not completely evaporate and may actually have a higher HA concentration per droplet volume than found in the larger droplets.
  • the result is a bimodal distribution whose exact characteristics depends in part on the selected HA concentration.
  • Aerosol volumetric output concentration tends to be lower with concentrations of 5 mg/ml than for the lower concentrations (1 mg/ml and 2 mg/ml) all three nebulizers (Misty, Pari, and AeroEclipse). This does not mean that there is proportionately less HA generated at 5 mg/ml since the concentration in solution is much higher.
  • the total HA aerosolized is therefore 0.144 ml/min.
  • x 2 mg/ml 0.29 mg/min. of HA aerosol generated with the 2 mg/ml concentration.
  • the nebulizers acted differently in direct comparison tests.
  • the Misty nebulizer tended to yield undesirable large geometric standard deviations in all tests.
  • the AeroEclipse tended to give smaller droplet size standard deviations, a desirable characteristic.
  • nebulizer that allows auxiliary air to pass through the nebulization zone adding aerosol to that auxiliary air can significantly increase the aerosolization rate and the deposition of HA during a given time period of inhalation treatment.
  • AeroEclipse nebulizer demonstrate this advantageous use of auxiliary air. That auxiliary air is automatically drawn into the nebulizer from the room in response to the inhalation demand of a patient.
  • nebulizer and formulation must be compatible such that the process of producing a respirable aerosol affects no significant changes in HA molecular size or integrity. Examples of such formulation and nebulizer combinations are presented in Table 9. Table 9. Nebulizer and Formulation Compatibility

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Abstract

L'invention concerne un procédé de traitement et/ou de prévention de bronchoconstriction provoquée par une élastase neutrophile et par l'activité de la kallicréine tissulaire. Ce procédé consiste à administrer un aérosol d'acide hyaluronique en quantité suffisante pour que l'acide se lie à des récepteurs RHAMM (CD168) situées le long de la surface apicale de l'épithélium des voies aériennes. Comme l'acide hyaluronique se lie à la kallicréine tissulaire sécrétée et retient cette kallicréine tissulaire sécrétée, il permet de traiter et/ou de prévenir une bronchoconstriction provoquée par l'activité de la kallicréine.
PCT/US2002/019269 2001-06-15 2002-06-17 Traitement de troubles respiratoires associes a une bronchoconstriction, a l'aide d'un aerosol d'acide hyaluronique WO2002102317A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2847818A1 (fr) * 2002-11-28 2004-06-04 Agro Ind Rech S Et Dev Ard Composition pharmaceutique a base d'acide hyaluronique
ITMI20101440A1 (it) * 2010-07-30 2012-01-31 Eupharma Srl Formulazioni inalatorie in forma di soluzioni o polveri secche, per la rimozione delle secrezioni mucose dall'apparato respiratorio
US9717752B2 (en) 2012-05-15 2017-08-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Uses of antagonists of hyaluronan signaling
WO2019200274A1 (fr) * 2018-04-12 2019-10-17 MatRx Therapeutics Corporation Compositions et procédés de traitement de rupture de fibre élastique
IT202100001349A1 (it) * 2021-01-25 2022-07-25 Sofar Swiss Sa Composizione in forma di polvere secca per inalazione comprendente un acido ialuronico e una condroitina solfato, uso della composizione e dispositivo inalatore contenente la composizione
IT202100021602A1 (it) * 2021-08-09 2023-02-09 Sofar Spa Composizione in forma di polvere secca per inalazione per uso in un metodo di trattamento di un’infiammazione e/o di uno stress ossidativo dell’apparato respiratorio causata/o da inquinamento dell’aria

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WO2001093846A2 (fr) * 2000-05-23 2001-12-13 The Trustees Of Columbia University In The City Of New York Procede de traitement des troubles respiratoires associes a une lesion de la fibre elastique pulmonaire

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WO2001093846A2 (fr) * 2000-05-23 2001-12-13 The Trustees Of Columbia University In The City Of New York Procede de traitement des troubles respiratoires associes a une lesion de la fibre elastique pulmonaire

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Title
CANTOR ET AL.: 'Aerosolized hyaluronic acid decreases alveolar injury induced by human neurophil elastase' ST. LUKE'S ROOSEVELT INSTITUTE FOR HEALTH SCIENCES 1997, NEW YORK, pages 471 - 475, XP001034167 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2847818A1 (fr) * 2002-11-28 2004-06-04 Agro Ind Rech S Et Dev Ard Composition pharmaceutique a base d'acide hyaluronique
WO2004050187A1 (fr) * 2002-11-28 2004-06-17 Agro Industrie Recherches Et Developpements (A.R.D.) Utilisation d'acide hyaluronique pour les affections respiratoires des voies aeriennes superieures.
ITMI20101440A1 (it) * 2010-07-30 2012-01-31 Eupharma Srl Formulazioni inalatorie in forma di soluzioni o polveri secche, per la rimozione delle secrezioni mucose dall'apparato respiratorio
US9717752B2 (en) 2012-05-15 2017-08-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Uses of antagonists of hyaluronan signaling
WO2019200274A1 (fr) * 2018-04-12 2019-10-17 MatRx Therapeutics Corporation Compositions et procédés de traitement de rupture de fibre élastique
US10933084B2 (en) 2018-04-12 2021-03-02 MatRx Therapeutics Corporation Compositions and methods for treating elastic fiber breakdown
IT202100001349A1 (it) * 2021-01-25 2022-07-25 Sofar Swiss Sa Composizione in forma di polvere secca per inalazione comprendente un acido ialuronico e una condroitina solfato, uso della composizione e dispositivo inalatore contenente la composizione
WO2022157744A1 (fr) * 2021-01-25 2022-07-28 Sofar Swiss Sa Composition sous forme de poudre sèche pour inhalation comprenant un acide hyaluronique et un sulfate de chondroïtine, utilisation de la composition et dispositif d'inhalation contenant la composition
IT202100021602A1 (it) * 2021-08-09 2023-02-09 Sofar Spa Composizione in forma di polvere secca per inalazione per uso in un metodo di trattamento di un’infiammazione e/o di uno stress ossidativo dell’apparato respiratorio causata/o da inquinamento dell’aria
WO2023017370A1 (fr) * 2021-08-09 2023-02-16 Sofar S.P.A. Composition sous forme de poudre sèche pour inhalation destinée à être utilisée dans une méthode topique de traitement de l'inflammation et/ou du stress oxydatif du système respiratoire provoquée par la pollution atmosphérique

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