Ceramide levels in treatment and prevention of infections

Abstract

To provide a method for treating or preventing a pathogenic infection in a subject having cystic fibrosis, COPD, and / or an open wound.SOLUTION: This method involves selecting a subject having cystic fibrosis, COPD, and / or an open wound, and administering a ceramidase to the selected subject under conditions effective to reduce ceramide and to treat or prevent the pathogenic infection. The method also involves the use of a ceramidase in combination with other drugs to reduce infection, reduce ceramide, or improve lung function in cystic fibrosis, COPD, and / or open wound patients.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 654,519, filed Jun. 1, 2012, which is hereby incorporated by reference in its entirety.

[0002] The present invention relates to normalizing ceramide levels to prevent and / or treat pathogen infections in subjects having cystic fibrosis, chronic obstructive pulmonary disease (COPD), and / or open wounds.

Background Art

[0003] Cystic fibrosis (“CF”) is the most common autosomal recessive disorder in Europe and the United States, affecting 1 in 2,500 children born in Western European countries. It is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator protein (“CFTR”). This genetic mutation results in several respiratory, genital, and gastrointestinal complications, and the first cause of morbidity and mortality in these subjects results from the destructive effects of chronic lung colonization by Pseudomonas aeruginosa (P. aeruginosa). Medical records indicate that approximately 80% of CF patients are infected with P. aeruginosa by the age of 25. See Cystic Fibrosis Foundation Subject Registry: Annual Data Report (2010) (Non-Patent Document 1). In addition to increased susceptibility to P. aeruginosa, the CF lung is characterized by chronic inflammation and progressive fibrosis. Currently, there is a need to interpret the molecular mechanisms that mediate the characteristics of the CF disease, namely, infection susceptibility, inflammation, and fibrosis.

[0004] Pseudomonas aeruginosa infection of epithelial cells is initiated by contact of the pathogen with the cell surface. Several binding molecules for Pseudomonas aeruginosa have been identified, including CFTR, fibronectin, α5β1-integrin, and glycolipids including asialo-GM1. Pier et.al., "Role Of Mutant CFTR In Hypersusceptibility Of Cystic Fibrosis Subjects To Lung Infections," Science.271,64-67(1996)(Non-Patent Document 2); Schroeder et.al., "CFTR Is A Pattern Recognition Molecule That Extracts Pseudomonas aeruginosa LPS From The Outer Membrane Into Epithelial Cells And Activates NF-kappa B Translocation," Proc.Natl.Acad.Sci.U.S.A.99,pp.6907-6912(2002)(Non-Patent Document 3); deBentzmann et.al., "Asialo GM1 Is A Receptor For Pseudomonas aeruginosa Adherence To Regenerating Respiratory Epithelial Cells," Infect.Immun.64(5)pp.1582-1588(1996)(Non-Patent Document 4); deBentzmann et.al., "Pseudomonas aeruginosa Adherence To Remodeling Respiratory Epithelium," Eur.Respir.J.9 pp.2145-2150(1996)(Non-Patent Document 5); Roger et.al., "Fibronectin And α5β1-integrin Mediate Binding Of Pseudomonas aeruginosa To Repairing Airway Epithelium," Eur.Respir.J.13 pp.1301-1309(1999)(Non-Patent Document 6); Saiman et.al., "Pseudomonas aeruginosa Pili Bind To AsialoGM1 Which Is Increased On The Surface Of Cystic Fibrosis Epithelial Cells," J.Clin.Invest.92 pp.1875-1880(1993)(Non-Patent Document 7); and Davies et.al., "Reduction In The Adherence Of Pseudomonas aeruginosa To Native Cystic Fibrosis Epithelium With Anti-AsialoGM1 Antibody And Neuraminidase Inhibition," Eur.Respir.J;13 pp.565-570(1999)(Non-Patent Document 8). See also.

[0005] Therefore, the identification of Pseudomonas aeruginosa epithelial receptors that are specifically altered in CF and contribute to the high susceptibility of these subjects to Pseudomonas aeruginosa infection is an important consideration in the development of new strategies for the prevention and treatment of CF and concomitant pathogen infections. Such molecules would be ideal targets for interfering with the initial contact of pathogens with bronchial epithelial cells in CF subjects and thus for interfering with infection at a very early stage.

[0006] Current methods for treating and preventing the etiology of diseases in subjects suffering from a disease or condition raise concerns about toxicity and efficacy.

[0007] The present invention is directed to overcoming these deficiencies in the art, for example, by modifying in vivo the abnormal expression of the major binding molecule of bacterial pathogens to bronchial epithelial cells through a unique membrane lipid-mediated mechanism.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

[0009] One aspect of the present invention is directed to a method for treating or preventing infection of a pathogen in a subject having cystic fibrosis, chronic obstructive pulmonary disease (COPD), and / or an open wound. This method includes the steps of selecting a subject having cystic fibrosis, COPD, and / or an open wound, and administering ceramidase to the selected subject under effective conditions for reducing ceramide and for treating or preventing infection of the pathogen in the selected subject.

[0010] Another aspect of the invention relates to administering ceramidase to a subject in combination with other agents to reduce ceramide or to reduce infection. Such agents may include, but are not limited to, antibiotics, reagents for reducing the viscosity of mucus, chaperone agents for enhancing the function of cystic fibrosis transmembrane conductance regulator (CFTR), or acid sphingomyelinase inhibitors.

[0011] Another aspect of the invention relates to selecting a subject based on the level of ceramide and / or the level of endogenous ceramidase enzyme in the cells, tissues, or body fluids of the subject. [Invention 1001] A method for treating or preventing infection by a pathogen in a subject having cystic fibrosis, COPD, and / or an open wound, comprising: selecting a subject having cystic fibrosis, COPD, and / or an open wound, and administering ceramidase to the selected subject under effective conditions for reducing ceramide and treating or preventing infection by the pathogen in the selected subject. A method comprising the steps. [Invention 1002] The method of Invention 1001, wherein the subject is selected based on an elevated ceramide level compared to a reference level of a subject not having cystic fibrosis, COPD, and / or an open wound. [Invention 1003] The method of Invention 1001, wherein the selecting step is based on the level of ceramide in cells isolated from the lung epithelium, nasal epithelium, mucus, and / or the open wound site. [Invention 1004] The method of Invention 1001, wherein the administering step is performed under effective conditions for normalizing the level of ceramide in cells of the respiratory epithelium, mucus, or open wound site of the subject. [Invention 1005] The method of Invention 1001, wherein the ceramidase is acid ceramidase. [Invention 1006] The method of the present invention 1001, wherein one or more additional agents that reduce the ceramide level are administered in combination with the ceramidase. [The present invention 1007] The method of the present invention 1006, wherein the one or more additional agents are selected from the group consisting of one or more additional ceramide-reducing agents, one or more acidic sphingomyelinase inhibitors, one or more agents for reducing infection, and combinations thereof. [The present invention 1008] The method of the present invention 1007, wherein the one or more additional agents are one or more agents for reducing infection and are selected from the group consisting of antibiotics, reagents that block the binding of pathogens to the lung epithelium, reagents for reducing the viscosity of mucus, chaperone reagents for enhancing lost protein function, and combinations thereof. [The present invention 1009] The method of the present invention 1007, wherein the ceramidase is administered simultaneously with, separately from, or sequentially with the one or more additional agents. [The present invention 1010] The method of the present invention 1001, wherein the administering step is by oral, topical, intranasal, intraperitoneal, intravenous, subcutaneous, or aerosol inhalation. [The present invention 1011] The method of the present invention 1010, wherein the administering step is by aerosol inhalation. [The present invention 1012] The method of the present invention 1001, wherein the ceramidase is administered in an amount of 0.001 mg / kg to 500 mg / kg. [The present invention 1013] The method of the present invention 1001, wherein the infection by the pathogen is a viral, fungal, prion, or bacterial infection. [The present invention 1014] The method of the present invention 1013, wherein the infection by the pathogen is a Pseudomonas infection. [The present invention 1015] The method of the present invention 1014, wherein the Pseudomonas infection is a Pseudomonas aeruginosa infection. [The present invention 1016] The method of the present invention 1001, wherein the ceramidase is administered before the onset of infection. [The present invention 1017] The method of the present invention 1001, wherein the ceramidase is administered after the onset of infection. [The present invention 1018] The method of the present invention 1001, wherein the selected subject has cystic fibrosis. [The present invention 1019] The method of the present invention 1001, wherein the selected subject has COPD. [The present invention 1020] The method of the present invention 1001, wherein the selected subject has an open wound.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0013] In the practice of the present invention, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA are used. These techniques are well known and are described, for example, in Current Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)); DNA Cloning: A Practical Approach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis, Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds. (1985); Transcription and Translation, Hames & Higgins, Eds. (1984); Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the series, Meth. Enzymol., (Academic Press, Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring Harbor Laboratory, New York (1987)); and Meth. Enzymol., Vols. 154 and 155, Wu & Grossman, and Wu, Eds., all of which are incorporated herein by reference in their entirety.Methods for detecting and measuring the level of a gene expression product of a polypeptide, i.e., the gene translation level, are well known in the art and include the use of polypeptide detection methods such as techniques for the detection and quantification of antibodies. See also Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., New York (1999)), which is incorporated herein by reference in its entirety.

[0014] To provide a substantial understanding of the present technology, it should be recognized that certain aspects, modes, embodiments, variations, and features of the present invention are described below at various levels of detail. Definitions of certain terms as used herein are provided below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains.

[0015] Underlying disease states can render a subject more susceptible to acute and / or chronic pathogen infections. As used herein, "disease state" refers to any type or origin of disease or condition caused by a pathogen that a subject has. Thus, disease states include, for example, but are not limited to, the following diseases and / or subjects identified by the following terms: respiratory diseases, lung diseases, cystic fibrosis ("CF"), chronic obstructive pulmonary disease ("COPD"), emphysema, asthma, pulmonary fibrosis, chronic bronchitis, pneumonia, pulmonary hypertension, lung cancer, sarcoidosis, necrotizing pneumonia, asbestosis, aspergilloma, aspergillosis, acute invasive atelectasis, eosinophilic pneumonia, pleural effusion, pneumoconiosis, pneumocystis, pneumothorax, pulmonary actinomycosis, alveolar proteinosis, pulmonary anthrax, pulmonary arteriovenous malformation, pulmonary edema, pulmonary embolism, pulmonary histiocytosis X (eosinophilic granuloma), pulmonary nocardiosis, pulmonary tuberculosis, pulmonary veno-occlusive disease, rheumatic lung disease, and / or open wounds. Such diseases typically result in an increased susceptibility of the subject to pathogen infection, i.e., an increase compared to a subject not suffering from the disease state.

[0016] For example, a subject suffering from, e.g., CF, COPD, and / or an open wound may have a high susceptibility to acute and / or chronic pathogen infections such as infections by bacterial, viral, fungal, protozoal, and / or prion pathogens. Bacterial pathogens include, without limitation, Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, Corynebacterium dipththeriae, Escherichia coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, Haemophilus influenzae type B and non-typeable, Helicobacter pylori, Legionella pneumophila, Listeria monocytogenes, Mycobacterium spp., Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus spp., Pseudomonas aeruginosa, Rickettsia, Salmonella spp., Shigella spp., Staphylococcus spp., Staphylococcus aureus, Streptococcus spp.) include Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus B, group A β-hemolytic Streptococcus, Streptococcus mutans, Treponema pallidum, Vibrio cholerae, and Yersinia pestis. In certain embodiments, the pathogen infection is a Pseudomonas infection. In certain embodiments, the Pseudomonas infection is a Pseudomonas aeruginosa infection.

[0017] Viral pathogens include, without limitation, RNA viruses, DNA viruses, the Adenoviridae (e.g., Mastadenovirus and Aviadenovirus), the Herpesviridae (e.g., Herpes simplex virus 1, Herpes simplex virus 2, Herpes simplex virus 5, and Herpes simplex virus 6), the Leviviridae (e.g., Levivirus, Enterobacteria phage MS2, Allolevirus), the Poxviridae (e.g., the Chordopoxyirinae subfamily, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Molluscipoxvirus, and the Entomopoxyirinae subfamily), the Papovaviridae (e.g., Polyomavirus and Papillomavirus), the Paramyxoviridae (e.g., Paramyxovirus, Parainfluenza virus 1), Mobiliviruses such as Measles virus, Rubulavirus (such as Mumps virus), the Pneumoviridae (e.g., Pneumovirus, Human respiratory syncytial virus), Metapneumovirus (e.g., Avian pneumovirus and Human metapneumovirus), the Picornaviridae (e.g., Enterovirus, Rhinovirus, Hepatovirus such as Human hepatitis A virus, Cardiovirus, and Aphthovirus), the Reoviridae (e.g., Orthoreovirus, Orbivirus, Rotavirus, Cypovirus, Fiji virus, Phytoreovirus, and Oryzavirus), the Retroviridae (e.g., Mammalian type B retrovirus, Mammalian type C retrovirus, Avian type C retrovirus, D-type retrovirus group, BLV-HTLV retrovirus), Lentivirus (Human immunodeficiency virus 1 and Human immunodeficiency virus 2;and Flaviviridae (e.g., hepatitis C virus), Hepadnaviridae (e.g., hepatitis B virus), Togaviridae (e.g., alphaviruses such as Sindbis virus and rubiviruses such as rubella virus), Rhabdoviridae (e.g., vesiculovirus, lyssavirus, ephemerovirus, cytorhabdovirus, and nucleorhabdovirus), Arenaviridae (e.g., arenavirus, lymphocytic choriomeningitis virus, ipi virus, and Lassa virus), and Coronaviridae (e.g., coronavirus and torovirus), cytomegalovirus (mononucleosis), dengue virus (dengue fever, shock syndrome), Epstein - Barr virus (mononucleosis, Burkitt lymphoma), human T - cell lymphotropic virus type 1 (T - cell leukemia), influenza A, B, and C (respiratory disease), Japanese encephalitis virus (pneumonia, brain damage), poliovirus (paralysis), rhinovirus (common cold), rubella virus (congenital abnormalities in the fetus), vaccinia virus (systemic infection), yellow fever virus (jaundice, renal failure, and liver failure), and varicella - zoster virus (varicella virus).;

[0018] Pathogenic fungi include, without limitation, the genus Aspergillus (e.g., Aspergillus fumigates), the genus Blastomyces, the genus Candida (e.g., Candida albicans), the genus Coccidiodes, the genus Cryptococcus, the genus Histoplasma, the genus Phycomyces, Tinea corporis, Tinea unguis, Sporothrix schenckii, and Pneumocystis carinii. Pathogenic protozoa include, without limitation, species of the genus Trypanosome, species of the genus Leishmania, species of the genus Plasmodium, species of the genus Entamoeba, and species of the genus Giardia such as Giardia lamblia.

[0019] Since the molecular mechanisms that induce an increased susceptibility to pathogen infection in subjects suffering from a disease state are not well understood, elucidating the pathology of, for example, Pseudomonas aeruginosa with respect to cell adhesion and internalization is an important consideration for preventing and treating subjects prone to such infections.

[0020] Integrins are receptor molecules that function, for example, to coordinate cell processes related to attachment and adhesion. However, integrins are not characteristically expressed on the luminal surface of normal, healthy bronchial epithelial cells, and the tight junctions of the epithelial cell layer impede contact of bronchial pathogens with the basolateral poles of the epithelial cells where integrins are typically present.

[0021] As used herein, the terms "elevated level" or "higher level" refer to a measurable marker, molecule, or protein, such as ceramide, that is observed normally in a comparable sample from a control or normal subject, i.e., higher than a reference value or control level or normalization level. In certain embodiments, the "control level," i.e., the normal level, refers to the range that is normally expected to be observed in a sample from a subject without a disease state. The control level may be used as a "reference level" for comparison purposes, as further detailed below. Thus, "elevated level" refers to a level above the range of the control level. The range accepted as an "elevated level" or "control level" depends on a number of factors. One of ordinary skill in the art can consider the relevant factors and establish an appropriate reference range for the "control values" and "elevated values" of the present invention. For example, a series of samples from control subjects and subjects diagnosed with CF can be used to establish a range that is a "normal" or "control" level, and a range that is "elevated" or "high" compared to the control range or level.

[0022] Ceramidase is an enzyme capable of hydrolyzing ceramide into fatty acid and sphingoid base (sphingosine), and it is involved in cell proliferation and intracellular signal transduction. Ceramidase facilitates the hydrolysis of ceramide into individual fatty acid and sphingosine components after enzyme activation. See Gatt, "Enzymic Hydrolysis and Synthesis of Ceramide," J.Biol.Chem.238:3131-3(1963); Gatt, "Enzymatic Hydrolysis of Sphingolipids.1.Hydrolysis and Synthesis of Ceramides by an Enzyme from Rat Brain," J.Biol.Chem.241:3724-31(1966); Hassler & Bell, "Ceramidase:Enzymology and Metabolic Roles," Adv.Lip.Res.26:49-57(1993), all of which are incorporated by reference in their entirety. There is no de novo pathway for cells to produce sphingosine, and thus it is only produced by the hydrolysis of ceramide following the enzymic action of ceramidase.

[0023] One aspect of the present invention is directed to a method for treating or preventing infection by a pathogen in a subject having cystic fibrosis, COPD, and / or an open wound. This method of the present invention includes the steps of selecting a subject having cystic fibrosis, COPD, and / or an open wound, and administering ceramidase to the selected subject under effective conditions for reducing ceramide and treating or preventing infection by the pathogen in the selected subject.

[0024] As described herein, "open wound" refers to a type of injury in which the epithelial layer, i.e., the skin, is torn, cut, and / or punctured. In certain embodiments, an open wound refers to a sharp instrument injury that damages the dermis of the skin and at the same time increases the likelihood of infection. The term "open wound" also encompasses burns.

[0025] The method of the present invention further includes the step of selecting a subject based on elevated ceramide levels as compared to a reference level for a subject having cystic fibrosis, COPD, and / or an open wound. As used herein, the term "reference level" refers to the level of a substance that can be of interest for comparison purposes, e.g., the level of ceramide. In certain embodiments, the reference level may be the level or concentration of a protein expressed as the average of the levels or concentrations from samples of a control population of healthy (disease-free and / or pathogen-free) subjects. In other embodiments, the reference level may be a level determined at a different time, e.g., prior to the subject developing a disease, disease state, and / or pathogen infection, such as prior to initiating treatment such as ceraminidase treatment, or at an earlier stage in the treatment, e.g., the level in the same subject prior to the use of the present invention.

[0026] Exemplary methods of comparing the ceramide level between a subject and a reference level include, without limitation, comparing the detected difference in ceramide levels based on the results of one or more protein assays as further described below. In certain embodiments, the ceramide level is higher in the presence of a disease state as described herein. A subject, or a sample thereof, having a detected ceramide level lower than the reference level may indicate that the subject may not require ceraminidase treatment and / or that the subject does not have a disease state as described above.

[0027] The method of the present invention further relates to selecting a subject based on the ceramide level in cells isolated from lung epithelium, nasal epithelium, mucus, and / or an open wound site. In certain embodiments, the administering step is performed under conditions effective to normalize the ceramide level in cells in the respiratory epithelium, mucus, or open wound site of the subject. In certain embodiments, the ceraminidase is an acidic ceraminidase (such as "AC") including, but not limited to, the acidic ceraminidases (such as "AC") listed in Table 1 below.

[0028] Acid ceramidase (N-acylsphingosine deacylase, I.U.B.M.B. enzyme number EC 3.5.1.23) is one specific ceramidase that causes the catabolism of ceramide. Due to its involvement in the human genetic disorder Farber lipogranulomatosis, AC is one of the most extensively studied members of the ceramidase enzyme family. This protein has been purified from several sources, and human and mouse cDNAs and genes have been obtained. See Bernardo et al., "Purification, Characterization, and Biosynthesis of Human Acid Ceramidase," J. Biol. Chem. 270:11098-102 (1995); Koch et al., "Molecular Cloning and Characterization of a Full-length Complementary DNA Encoding Human Acid Ceramidase. Identification of the First Molecular Lesion Causing Farber Disease," J. Biol. Chem. 2711:33110-5 (1996); Li et al., "Cloning and Characterization of the Full-length cDNA and Genomic Sequences Encoding Murine Acid Ceramidase," Genomics 50:267-74 (1998); Li et al., "The Human Acid Ceramidase Gene (ASAH): Chromosomal Location, Mutation Analysis, and Expression," Genomics 62:223-31 (1999), all of which are hereby incorporated by reference in their entirety.

[0029] As described above, AC is a ceramidase that catalyzes the hydrolysis of ceramide to sphingosine and free fatty acid. See Bernardo et al., "Purification, Characterization, and Biosynthesis of Human Acid Ceramidase," J. Biol. Chem. 270(19):11098-102(1995), which is incorporated herein by reference in its entirety. Mature AC is a ~50 kDa protein composed of an α subunit (about 13 kDa) and a β subunit (about 40 kDa). See Bernardo et al., "Purification, Characterization, and Biosynthesis of Human Acid Ceramidase," J. Biol. Chem. 270(19):11098-102(1995), which is incorporated herein by reference in its entirety. It is produced through cleavage of the AC precursor protein (see Ferlinz et al., "Human Acid Ceramidase: Processing, Glycosylation, and Lysosomal Targeting," J. Biol. Chem. 276(38):35352-60(2001), which is incorporated herein by reference in its entirety), and this protein is the product of the Asah1 gene (NCBI UniGene GeneID number 427, which is incorporated herein by reference in its entirety).

[0030] The function and / or activity of AC is further directly related to the surrounding pH. Indeed, it is normally found in lysosomes, which have an acidic pH of about 4.5, and in the absence of AC activity in patients with Farber lipogranulomatosis, ceramide accumulates in lysosomes.

[0031] In addition, recent studies have shown that an increase in intracellular compartment pH can reduce AC activity / function by up to 90%. See Teichgraber et al., "Ceramide Accumulation Mediate Inflammation, Cell Death And Infection Susceptibility In Cystic Fibrosis," Nat Med. 14(4), pp. 382-391 (2008). This is incorporated by reference in its entirety. In some respects, these results are similar to Fabry disease, which is caused by a deficiency in AC and results in the accumulation of ceramide. See He et al., "Purification And Characterization Of Recombinant, Human Acid Ceramidase," J. Biol. Chem. 278, 32978-32986 (2003). This is incorporated by reference in its entirety. Furthermore, at pH 5.9, AC has been shown to possess reverse activity, producing ceramide instead of consuming it. See ibid. This activity, combined with impaired Asm function at increased alveolar pH levels, can result in a net accumulation of ceramide. See Teichgraber et al., "Ceramide Accumulation Mediate Inflammation, Cell Death And Infection Susceptibility In Cystic Fibrosis," Nat Med. 14(4), pp. 382-391 (2008). This is incorporated by reference in its entirety.

[0032] Other studies have shown that CFTR deficiency in alveolar macrophages results in a lysosomal pH shift from pH 4.5 to at least pH 5.9. See Di et al., "CFTR Regulates Phagosome Acidification In Macrophages And Alters Bactericidal Activity," Nat. Cell Biol. 8, 933-944 (2006), which is hereby incorporated by reference in its entirety. However, the present invention surprisingly functions to prevent and / or treat pathogen infections in CF patients, at least because it was not expected that AC would reduce elevated ceramide levels in CF patients with increased lysosomal pH. Furthermore, AC was not expected to function against the accumulation of ceramide in the pulmonary epithelial cell membrane.

[0033] ACs that can be used in the context of the present invention include, but are not limited to, those shown in Table 1 below. In all aspects of the present invention, the AC can be autologous (i.e., derived from the same species) or heterologous (i.e., derived from a different species) to the tissue, cells, and / or subject being treated.

[0034] (Table 1) Members of the exemplary acid ceramidase family TIFF2025102904000002.tif107150

[0035] In one embodiment, determining the level of the concentration and / or activity of ceramide and / or AC is performed prior to treatment. Assays suitable for determining ceramide concentration and / or the level or activity of ceramidase will be readily apparent to those of ordinary skill in the art. Suitable methods include, for example, activity assays (see, e.g., Eliyahu et al., "Acid Ceramidase is a Novel Factor Required for Early Embryo Survival," FASEB J. 21(7):1403-9(2007), which is incorporated by reference in its entirety), and well-known techniques such as Western blotting for determining the relative amount of ceramidase protein and / or activity present in a sample (wherein a greater amount of ceramidase protein correlates with a higher level of ceramidase activity). See Eliyahu et al., "Acid Ceramidase is a Novel Factor Required for Early Embryo Survival," FASEB J. 21(7):1403-9(2007), which is incorporated by reference in its entirety.

[0036] As used herein, the term "assay" refers to an assay for detecting the presence or absence of ceramides and / or ceramidases in a sample of a given body fluid. Also included are quantitative assays for measuring the amount of a substance in a sample. As used herein, the term "sample" is used in its broadest sense. In one meaning, this means including a sample or culture obtained from a biological sample. A body fluid sample is selected from the group consisting of serum, synovial fluid, cerebrospinal fluid, and ascites. Of particular interest is a sample that is serum. One of ordinary skill in the art will recognize that plasma or whole blood or a fractionated whole blood may also be used. A sample of a biological fluid may be obtained from an animal (including a human), and this includes blood preparations such as plasma, serum, etc. In certain embodiments, the sample contains a certain level of ceramides or ceramidases, which can be readily confirmed by the methods described herein and methods well known in the art.

[0037] An immunoassay is, in its most simple and direct sense, a binding assay involving the binding between an antibody and an antigen. Many types and formats of immunoassays are known, and all are suitable, for example, for the detection of ceramide levels. Examples of immunoassays are enzyme-linked immunosorbent assay ("ELISA"), enzyme-linked immunospot assay ("ELISPOT"), radioimmunoassay ("RIA") (see Ferlinz et al., "Human Acid Ceramidase: Processing, Glycosylation, and Lysosomal Targeting," J. Biol. Chem. 276(38):35352-60(2001), which is hereby incorporated by reference in its entirety), radioimmunoprecipitation assay ("RIPA"), immunobead capture assay, dot blotting, gel shift assay, flow cytometry, immunohistochemistry, fluorescence microscopy, protein array, multiplex bead array, magnetic capture, in vivo imaging, fluorescence resonance energy transfer ("FRET"), and fluorescence recovery after photobleaching / localization ("FRAP / FLAP"). Steps of various useful immunodetection methods are described in scientific literature such as Maggio et al., Enzyme-Immunoassay (1987) and Nakamura, et al., "Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of Experimental Immunology," Vol. 1: Immunochemistry, 27.1-27.20(1986), each of which is hereby incorporated by reference in its entirety herein.

[0038] Generally, an immunoassay involves contacting a sample suspected of containing a molecule or protein of interest (such as ceramide and / or ceramidase) with an antibody against the molecule or protein of interest under conditions effective to permit the formation of immune complexes. In this regard, one of ordinary skill in the art is able to assess the presence and / or level of a particular molecule or protein of interest in a given sample.

[0039] An immunoassay can include a method for detecting or quantifying the amount of a molecule or protein of interest in a sample, which generally involves detecting or quantifying any immune complexes formed during the binding process. Generally, the detection of immune complex formation is well known in the art and can be achieved through the application of numerous approaches. These methods generally are based on the detection of a label or marker such as radioactivity, fluorescence, either a biological or enzyme tag, or any other known label. See, for example, U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each of which is incorporated herein by reference in its entirety.

[0040] One particularly effective non-specific assay used to detect total protein is the Bradford protein assay. See Bradford, M.M., "A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Proteins Utilizing the Principle of Protein-Dye Binding," Anal. Biochem. 72:248-254 (1976), which is incorporated herein by reference in its entirety.

[0041] The Bradford protein assay uses a dye stock of Coomassie Blue G (C.I. # 42655) (100 mg), which is dissolved in 50 mL of methanol. This solution is added to 100 mL of 85% H3PO4 and diluted to 200 mL with water to yield a dark red color. The final reagent concentrations for the assay are 0.5 mg / mL Coomassie Blue G, 25% methanol, and 42.5% H3PO4. The assay reagent for the Bradford assay is prepared by diluting the dye stock at a ratio of 1 part dye stock to 4 parts distilled water. The resulting color should be brown at pH 1.1. A series of protein standards are prepared in the same buffer as the samples to be assayed using bovine serum albumin ("BSA") at concentrations of 0, 250, 500, 750, and 1500 μg / mL for the standard assay. Absorbance is read at 595 nm for the standard assay procedure and at 450 nm for the microassay (Dynex Technologies, Chantilly, VA), and the ratio of absorbance at 595 nm / 450 nm was used for the calculation of the standard curve. See Zor, et al., "Linearization of the Bradford Protein Assay Increases Its Sensitivity: Theoretical and Experimental Studies," Anal. Biochem. 236:302-308 (1996). This is hereby incorporated by reference in its entirety.

[0042] In certain embodiments, the method of the invention is carried out by administering an AC precursor protein, which is then converted by the cell into the active acid ceramidase protein. In particular, the AC precursor protein undergoes auto-proteolytic cleavage to become active (composed of an α subunit and a β subunit). This is facilitated by the intracellular environment and is predicted to occur in most, if not all, cell types based on the sequence of the cleavage site of the highly conserved AC precursor protein across species. Suitable acid ceramidase precursor proteins include those shown in Table 1 above. As will be apparent to those skilled in the art, this precursor protein can optionally be contained in the culture medium to which the cells are exposed. Accordingly, embodiments are contemplated in which the precursor protein is taken up by the host subject or cell of interest and converted into the active acid ceramidase.

[0043] Yet another approach for administering the protein or polypeptide agent of the invention, e.g., AC, involves the preparation of a chimeric protein according to U.S. Patent No. 5,817,789 to Heartlein et al., which is hereby incorporated by reference in its entirety. The chimeric protein can include a ligand domain and a polypeptide agent (e.g., AC, AC precursor protein). This ligand domain is specific for a receptor located on the target cell. Thus, when the chimeric protein is delivered to the subject, cell, and / or culture medium, the chimeric protein is internalized.

[0044] Depending on the level or activity of a substance, such as ceramide, and / or ceramidase, one or more additional agents may be administered in accordance with the methods of the invention in combination with a ceramidase, such as AC. In certain embodiments, the one or more additional agents are selected from the group consisting of one or more additional ceramide reducing agents, one or more sphingomyelinase inhibitors, one or more agents for reducing infection, and combinations thereof. Agents suitable for reducing infection include antibiotics (e.g., inhaled tobramycin, TOBI), reagents that block the binding of pathogens to the lung epithelium, reagents that reduce the viscosity of mucus (e.g., dornase alfa, Pulmozyme), and chaperone reagents for enhancing lost protein function (e.g., Ivacaftor, Kalydeco, and combinations thereof). In certain embodiments, a ceramidase, such as AC, is administered simultaneously with, separately from, or sequentially to the one or more additional agents.

[0045] As used herein, the term "simultaneous" therapeutic use refers to the administration of at least two active ingredients simultaneously or substantially simultaneously by the same route. As used herein, the term "separate" therapeutic use refers to the administration of at least two active ingredients simultaneously or substantially simultaneously by different routes. As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, where the route of administration is the same or different. More specifically, sequential use refers to the administration of one active ingredient in its entirety before the start of one or more other administrations. Thus, it is possible to administer one active ingredient over a period of minutes, hours, or days before administering one or more other active ingredients. In this case, there is no simultaneous treatment.

[0046] Administration can be achieved either via systemic administration to the subject or via targeted administration to the affected tissue, organ, and / or cell. The therapeutic agent (i.e., AC, AC precursor protein, nucleic acid encoding AC / AC precursor protein) may be administered to non-targeted areas together with one or more agents that facilitate movement of the therapeutic agent (and / or uptake of the therapeutic agent by the tissue, organ, or cell) to the targeted tissue, organ, or cell. Further, and / or alternatively, as will be apparent to those skilled in the art, the therapeutic agent itself can be modified to facilitate its transport (and uptake by the tissue, organ, or cell) to the desired tissue, organ, or cell.

[0047] Any suitable approach for agent delivery can be utilized to practice this aspect of the invention. Typically, the therapeutic agent(s) is / are administered to the patient in a vehicle that delivers the therapeutic agent(s) to the target cell, tissue, or organ. Exemplary routes of administration include, but are not limited to, intratracheal inoculation, aspiration, airway instillation, aerosolization, nebulization, intranasal instillation, oral or nasogastric instillation, intraperitoneal injection, intravascular injection, topical, transdermal, parenteral, subcutaneous, intravenous injection, intraarterial injection (such as via the pulmonary artery), intramuscular injection, intrapleural infusion, intracerebroventricular, intralesional, application to mucosa (such as nasal, pharyngeal, bronchial, genital, and / or anal mucosa), or by implantation of a sustained release vehicle.

[0048] In certain embodiments, the ceramidase, e.g., AC, is administered orally, topically, intranasally, intraperitoneally, intravenously, subcutaneously, or by aerosol inhalation. In certain embodiments, the ceramidase, e.g., AC, is administered via aerosol inhalation. In certain embodiments, the ceramidase and / or further agent(s) can be incorporated into a pharmaceutical composition suitable for administration as described herein.

[0049] The agents of the present invention, for example, AC, may be administered orally, for example, together with an inert diluent or with an absorbable edible carrier, or they may be enclosed in hard or soft shell capsules, or they may be compressed into tablets, or they may be incorporated directly into the food of daily diet. For oral therapeutic administration, these active compounds may be incorporated into excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, etc. Such compositions and preparations should contain at least 0.1% of the agent. Of course, the percentage of the agent in these compositions may vary and may conveniently be between about 2% and about 60% of the unit weight. The amount of the agent in such therapeutically useful compositions is such that an appropriate dosage is obtained.

[0050] Tablets, capsules, etc. may also contain binders such as tragacanth gum, acacia, corn starch, or gelatin; excipients such as dipotassium phosphate; disintegrants such as corn starch, potato starch, or alginic acid; lubricants such as magnesium stearate; and sweetening agents such as sucrose, lactose, or saccharin. When the dosage unit form is a capsule, it may contain, in addition to the materials of the above types, a liquid carrier such as a fatty oil.

[0051] The agents, for example, AC, may also be administered parenterally. Solutions or suspensions of this agent can be prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Exemplary oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, physiological saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, especially for injectable solutions. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0052] Suitable pharmaceutical forms for injection purposes include sterile aqueous solutions or suspensions for the immediate preparation of sterile injection solutions or suspensions, and sterile powders. In all cases, this form must be sterile and must have a fluidity to the extent that easy syringe passage is present. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures of these, and vegetable oils.

[0053] The agent according to the present invention, for example, AC may also be administered directly to the respiratory tract in the form of an aerosol. For use as an aerosol, the compound of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with a suitable propellant, such as a hydrocarbon propellant like propane, butane, or isobutane, along with conventional adjuvants. The material of the present invention may also be administered in a non-pressurized form.

[0054] Exemplary delivery devices include, without limitation, nebulizers, atomizers, liposomes (including both active drug delivery and passive drug delivery technologies) (Wang & Huang, "pH-Sensitive Immunoliposomes Mediate Target-cell-specific Delivery and Controlled Expression of a Foreign Gene in Mouse," Proc. Nat'l Acad. Sci. USA 84:7851-5 (1987); Bangham et al., "Diffusion of Univalent Ions Across the Lamellae of Swollen Phospholipids," J. Mol. Biol. 13:238-52 (1965); U.S. Patent No. 5,653,996 to Hsu; U.S. Patent No. 5,643,599 to Lee et al.; U.S. Patent No. 5,885,613 to Holland et al.; U.S. Patent No. 5,631,237 to Dzau & Kaneda; and U.S. Patent No. 5,059,421 to Loughrey et al.; Wolff et al., "The Use of Monoclonal Anti-Thy1 IgG1 for the Targeting of Liposomes to AKR-A Cells in Vitro and in Vivo," Biochim. Biophys. Acta 802:259-73 (1984), each of which is incorporated by reference in its entirety), transdermal patches, implants, implantable or injectable protein depot compositions, and syringes. Other delivery systems known to those of skill in the art can also be utilized to achieve the desired delivery of a therapeutic agent to a desired organ, tissue, or cell.

[0055] Administration can be carried out at the frequency required and for a period of time appropriate to provide effective prophylaxis or efficacy against the pathogen. For example, administration can be carried out using a single sustained release dosage formulation or multiple daily doses.

[0056] The amount administered will, of course, vary depending on the treatment plan. Generally, the agent is administered to achieve an amount effective to improve clearance of the pathogen. Thus, a therapeutically effective amount can be an amount that is capable of at least partially preventing and / or treating infection by the pathogen. This can include, without limitation, delaying the onset of infection. The dosage required to obtain an effective amount can vary depending on the agent, the formulation, and the individual to whom the agent is administered.

[0057] The dosage, toxicity, and therapeutic efficacy of the agent or composition of the present invention can be determined, for example, by standard pharmacological procedures in cell culture or experimental animals in order to determine the LD 50 (the dosage lethal to 50% of the population) and the ED 50 (the dosage therapeutically effective in 50% of the population). The dosage ratio between the toxic effect and the therapeutic effect is the therapeutic index, which can be expressed as the ratio of LD 50 / ED 50 . Compounds exhibiting a high therapeutic index may be desirable. Compositions exhibiting toxic side effects may be used, but care should be taken to design a delivery system that targets such compositions to the site of the affected tissue in order to minimize potential damage to uninfected cells and thereby reduce side effects.

[0058] As such, ceramidase is administered in a therapeutically effective amount in certain embodiments. As used herein, the terms "therapeutically effective amount", "effective amount", or "pharmaceutically effective amount" of an agent, protein, compound, and / or composition are amounts sufficient to achieve the desired therapeutic and / or prophylactic effect, e.g., an amount that results in the prevention, or a reduction, of symptoms associated with the disease being treated.

[0059] The effective amount of the agent or composition of the present invention to be administered to a subject depends on the type and severity of the disease, as well as individual characteristics such as general health, age, sex, body weight, and tolerance to the drug. This also depends on the degree, severity, and type of the disease. Those skilled in the art can determine an appropriate dosage depending on these and other factors. The composition of the present invention can also be administered in combination with one or more additional therapeutic compounds.

[0060] Typically, this therapeutic agent is administered as a pharmaceutical formulation comprising the therapeutic agent and any pharmaceutically acceptable adjuvant, carrier, excipient, and / or stabilizer, and can be in a solid dosage form such as tablets, capsules, powders, solutions, suspensions, or emulsions, or a liquid dosage form. These compositions preferably contain from about 0.01 to about 99 weight percent, more preferably from about 2 to about 60 weight percent, of the therapeutic agent, together with an adjuvant, carrier, and / or excipient. In certain embodiments, the effective amount ranges from about 0.001 mg / kg of subject body weight to about 500 mg / kg of subject body weight. In certain embodiments, the effective amount of the agent ranges from about 0.05 mg / kg to about 30 mg / kg, from about 0.1 mg / kg to about 30 mg / kg, from about 1 mg / kg to about 25 mg / kg, from about 1 mg / kg to about 20 mg / kg, or from about 1 or 2 mg / kg to about 15 mg / kg.

[0061] The agent of the present invention, for example, AC, can be administered at various time points. The ceramidase, for example, AC, is administered prior to the onset of infection in certain embodiments. In other embodiments, the ceramidase, for example, AC, is administered after the onset of infection. Still further, the ceramidase, for example, AC, may be administered both prior to and after the onset of infection according to certain embodiments of the present invention.

[0062] Another aspect of the invention relates to a method of monitoring the effectiveness of treatment in a subject having an infection by a pathogen and an underlying disease state. The method includes the steps of selecting a subject, providing a baseline ceramide level in a body fluid sample from the selected subject prior to treatment, and treating the infection by the pathogen with a therapy, for example, the therapy can be a therapeutic administration of a ceramidase such as, for example, AC. The method further includes the steps of detecting a post-treatment ceramide level in a body fluid sample from the selected subject after treatment, comparing the baseline ceramide level to the post-treatment ceramidase level, and determining whether the treatment was effective based on this comparison and / or the pathology of the infection by the pathogen.

[0063] A kit or reagent system for use or administration of the therapy of the invention in another aspect of the invention. Such a kit contains a combination of reagents including the specific elements required to perform an assay according to the methods disclosed herein. The reagent system is provided in a commercial package type as a composition or mixture, where the compatibility of the reagents is allowed to hold the required reagents in a test device arrangement or, more typically, as a test kit, i.e., in a packaged combination of one or more containers, devices, etc., and preferably includes instructions written for the performance of the assay. The kit may be adapted for any configuration of the assay and may include compositions for performing any of the various assays described herein.

[0064] Reagents useful for the disclosed methods can be stored in solution or lyophilized. When lyophilized, some or all of the reagents can be easily stored in microtiter plate wells for easy use after reconstitution. Any method known in the art for lyophilizing reagents is contemplated to be suitable for preparing the dried-down reagents useful for the disclosed methods.

[0065] Although the present invention has been generally described herein, the invention will be more readily understood through reference to the following examples, which are provided for purposes of illustration and are not intended to limit the invention unless otherwise specified.

Example

[0066] Example 1 - Mouse B6.129P2(CF / 3)-Cftr TgH(neoim)Hgu ("CF" MHH ) congenic mice were generated by insertional mutagenesis in exon 10 of the Cftr gene from the original Cftr TgH(neoim)Hgu mutant mice through inbreeding. See Charizopoulou et.al., "Instability Of The Insertional Mutation In Cftr TgH(neoim)Hgu Cystic Fibrosis Mouse Model," BMC Genet.5 p.6(2004). This is incorporated herein by reference in its entirety. Subsequently, this congenic Cftr MHH strain was backcrossed to the B6 background. These mice still expressed low levels of CFTR and thus could be fed a standard mouse diet. They showed normal development but also exhibited lung pathology typical of CF. These are referred to herein as "CF" mice. See Teichgraber et.al., "Ceramide Accumulation Mediate Inflammation,Cell Death And Infection Susceptibility In Cystic Fibrosis," Nat Med.14(4),pp.382-391(2008);Wolbeling et.al., "Head-out Spirometery Accurately Monitors the Course of Pseudomonas aeruginosa Lung Infection in Mice," Respiration 80:340-6(2010). This is incorporated herein by reference in its entirety. Congenic B6 mice were used as controls.

[0067] For one experiment, Cftr tm1Unc -Tg (FABPCFTR) mice (purchased from The Jackson Laboratory, Bar Harbor, ME, designated as "Cftr - / - ") were backcrossed more than 10 generations to C57BL / 6 mice. In all organs other than the intestine that express human CFTR under the control of the fatty acid binding protein ("FABP") promoter, the mice are completely deficient in Cftr. This transgene prevents intestinal obstruction and allows for a normal diet. Again, B6 mice were used as controls. Cftr - / - strains and Cftr MHH No significant differences were observed in experiments using both strains.

[0068] For other experiments, mice deficient in the enzyme ceramide synthase 2 were used. These mice (CerS2- / -) were generated by disruption of the first intron of the CerS2 mouse gene. They do not survive beyond about 16 months and accumulate C16 ceramide in many tissues (see Pewzner-Jung et al., "A Critical Role of Ceramide Synthase 2 in Liver Homeostasis I. Alterations in The Lipid Metabolic Pathway," J Biol. Chem. 285:10902 (2010), which is incorporated herein by reference in its entirety).

[0069] The mice were housed and bred in isolated cages in the animal facility of the University Hospital, University of Duisburg-Essen, Germany. They were repeatedly evaluated for a panel of common mouse pathogens according to the 2002 recommendations of the Federation of European Laboratory Animal Science Associations. The mice were free of any pathogens. The procedures performed on the animals were approved by the Bezirksregierung Duesseldorf, Duesseldorf, Germany.

[0070] Example 2 - Antibodies and Reagents All ceramide staining was performed using the monoclonal mouse anti-ceramide antibody clone S58-9 (Glycobiotech), which was visualized using Cy3 donkey anti-mouse IgM F(ab)2 fragment (Jackson #715-166-020) or Cy5-conjugated donkey anti-mouse IgM antibody (Jackson #715-176-020). Recombinant human acid ceramidase was produced in Chinese hamster ovary (“CHO”) cells and purified from the medium as previously described. He et al., J Biol.Chem. 278:32978-86 (2003), which is incorporated herein by reference in its entirety.

[0071] Example 3 - Bacteria The laboratory strain of Pseudomonas aeruginosa American Type Culture Collection 27853 and the previously described clinical isolate of Pseudomonas aeruginosa (“762”) were used. Bacteria were plated from frozen stocks on fresh tryptic soy agar plates (TSA; Becton Dickinson) and grown at 37°C for 14 - 16 hours and resuspended in 40 mL of pre-warmed tryptic soy broth (Becton Dickinson) at 37°C to an optical density of 0.225 at 550 nm. The bacterial suspension was then incubated at 37°C for 1 hour with shaking at 125 rpm to obtain bacteria in the early logarithmic growth phase. See Grassme et.al., "Host Defense Against Pseudomonas aeruginosa Requires Ceramide-Rich Membrane Rafts," Nat Med.9(3):322-330(2003), which is incorporated herein by reference in its entirety. The bacteria were then washed twice and resuspended in pre-warmed RPMI-1640 medium (Invitrogen) supplemented with 10 mM HEPES (RPMI+HEPES). The final concentration of bacteria was quantified by spectrophotometer.

[0072] Example 4 - In Vivo Immunohistochemistry For immunohistochemical evaluation of mouse bronchial epithelial cells, mice were sacrificed by cervical dislocation and immediately perfused via the right heart at low pressure with ice-cold saline for 2 minutes. This was followed by perfusion of the heart with 4% PBS-buffered PFA for 10 - 15 minutes. After this initial blood clearance and fixation, the lungs were removed and further fixed in 4% PFA for 24 - 36 hours. The tissue was then dehydrated successively using an ethanol to xylene gradient and then embedded in paraffin.

[0073] Next, the sample was sectioned into 7 μm slices, dewaxed, rehydrated, and treated with pepsin (Invitrogen) at 37 °C for 15 minutes. The sample was then washed with water and PBS, and blocked with PBS, 0.05% Tween 20 (Sigma), and 1% FCS for 10 minutes at room temperature. The sample was then continuously stained with the primary antibody in H / S + 1% FCS for 45 minutes at room temperature. The sample was washed twice with PBS + 0.05% Tween 20 and once with PBS during the staining. The tissue was secondarily labeled with the fluorescent-conjugated secondary antibody in H / S + 1% FCS for 30 minutes in the dark. The tissue was washed again twice with PBS + 0.05% Tween 20 and once with PBS, and finally embedded in Mowiol. The sample was evaluated using a confocal microscope as described below.

[0074] Example 5 - Inhalation and In Vivo Infection Pseudomonas aeruginosa was prepared as described above and adjusted to 1×10 in 20 μL of medium in RPMI-1640 plus 10 mM HEPES 8Resuspended in the final concentration of CFU. Then, these were inoculated using a plastic-coated 30-gauge needle inserted 2 mm into the nose. The number of bacteria in the mouse lungs was quantified 2 hours after infection. The mice were sacrificed, the lungs were removed, homogenized, and lysed in 5 mg / mL saponin to release intracellular bacteria. Then, the samples were washed with sterile PBS, diluted, and plated in duplicate on TSA plates for 12 hours. The number of bacteria was counted, showing the number of bacteria in the whole lung samples. This mode of infection more accurately assesses mucociliary clearance than other lung infection models such as intratracheal infection. See Teichgraber et.al., "Ceramide Accumulation Mediate Inflammation,Cell Death And Infection Susceptibility In Cystic Fibrosis," Nat Med.14(4):382-391(2008);Zhang et.al., "Kinase Suppressor Of Ras-1 Protects Against Pulmonary Pseudomonas aeruginosa Infections," Nat Med.17(3):341-346(2011), which are incorporated by reference in their entirety.

[0075] Example 6 - Statistics Data were expressed as arithmetic mean ± SD, and statistical analysis was performed as indicated. Since all values were normally distributed, one-way ANOVA was applied. Significance is indicated by asterisks in the figures.

[0076] Example 7 - Confocal Microscopy and Discussion Samples were examined using a Leica TCS-SP5 confocal microscope equipped with a 100× oil immersion lens, and images were analyzed using Leica LCS software (Leica Microsystems). All comparative samples were measured using the same settings.

[0077] Ceramide is increased in CF subjects and in the lungs of mice (Figure 1A) and is an important factor in the susceptibility of CF mice to Pseudomonas aeruginosa infection. See Grassme et.al., "CFTR-dependent Susceptibility Of The Cystic Fibrosis-Host To Pseudomonas aeruginosa," Int J Med Microbiol. 300(8):578-83(2010), which is incorporated herein by reference in its entirety. Previous studies have demonstrated that pharmacological inhibition of acid sphingomyelinase or genetic heterozygosity of the acid sphingomyelinase gene is sufficient to normalize ceramide levels in the CF mouse lung. See Becker et.al., "Acid Sphingomyelinase Inhibitors Normalize Pulmonary Ceramide And Inflammation In Cystic Fibrosis," Am J Respir Cell Mol Biol. 42(6):716-24(2010), which is incorporated herein by reference in its entirety.

[0078] In addition, CF MHH Cftr-deficient mice inhaled acid ceramidase, which hydrolyzes ceramide. This inhalation modified ceramide levels in the bronchial epithelial cells of CF mice (Figure 1B). Inhalation of the vehicle, i.e., 0.9% NaCl, had no effect on ceramide levels.

[0079] In another example, Cftr- / - or normal mice were infected with Pseudomonas aeruginosa after inhaling saline or acid ceramidase (Figure 2). Compared to normal mice, CF MHHCftr-deficient mice and CerS2 mice accumulate ceramide in their lungs. Two hours after inhalation, they were sacrificed and the bacteria remaining in the lungs were quantified. While wild-type mice effectively cleared Pseudomonas aeruginosa, Cftr− / − mice or CerS2− / − mice that inhaled saline were unable to clear and had a large amount of residual bacteria. In contrast, Cftr− / − mice or CerS2− / − mice that inhaled acidic ceramidase had very low bacterial titers, similar to normal mice.

[0080] Identification of irregularities in untreated CF airways provides a novel concept for the prevention of infection in CF subjects. This example provides several approaches for preventing infection and treating a major cause of death in subjects with CF.

[0081] Example 8 - AC inhalation protects against Pseudomonas infection Mice were inhaled with 100 micrograms of recombinant acidic ceramidase (AC) in 0.8 mL of 0.9% NaCl 30 - 45 minutes prior to intranasal infection with 1×10 8 colony forming units (CFU) of Pseudomonas aeruginosa strain 762 or ATCC 27853. Lungs were removed 4 hours after infection, homogenized, lysed in 5 mg / mL saponin for 10 minutes, and washed. Aliquots were plated on LB plates and grown overnight. CFUs on the LB plates were counted to determine the number of Pseudomonas aeruginosa bacteria in the lungs. Mean ± standard deviation of 4 independent experiments are shown.

[0082] Single inhalation of AC prevented infection of CF mice with two different strains of P. aeurginosa (Figure 3). Clinical strain 762 was originally obtained from a urinary tract infection, whereas the ATCC 27853 strain is a laboratory strain. Inhalation of saline alone was used as a control.

[0083] CF mice were inhaled with recombinant acid ceramidase (100 micrograms in 0.8 mL of 0.9% NaCl). Physiological saline was used as a control. In all cases, the mice were inhaled 30 - 45 minutes before inhalation with the clinical Pseudomonas aeruginosa strain 762 and then sacrificed 4 hours later. Lungs were removed 4 hours after infection, homogenized, lysed in 5 mg / mL saponin for 10 minutes, and then washed. Aliquots were plated on LB plates and grown overnight. CFUs on the LB plates were counted to determine the bacterial count of Pseudomonas aeruginosa in the lungs.

[0084] Inhalation of recombinant acid ceramidase in CF mice prevented infection with the clinical strain 762 Pseudomonas aeruginosa to a similar extent (Figure 3).

[0085] Preferred embodiments have been shown and described in detail herein, but various modifications, additions, substitutions, etc. can be made without departing from the spirit of the invention, and therefore, these are considered to be within the scope of the invention as defined in the following claims, which will be apparent to those skilled in the relevant art.

Claims

1. A method for treating or preventing infection by a pathogen in a subject having cystic fibrosis, COPD, and / or an open wound, comprising: selecting a subject having cystic fibrosis, COPD, and / or an open wound; and administering ceramidase to the selected subject under conditions effective to reduce ceramide and to treat or prevent infection by the pathogen. A method comprising the above steps.

2. The method according to claim 1, wherein the subject is selected based on an elevated ceramide level compared to a reference level of a subject not having cystic fibrosis, COPD, and / or an open wound.

3. The method according to claim 1, wherein the selecting step is based on ceramide levels in cells isolated from lung epithelium, nasal epithelium, mucus, and / or the open wound site.

4. The method according to claim 1, wherein the administering step is performed under conditions effective to normalize ceramide levels in cells of the respiratory epithelium, mucus, or open wound site of the subject.

5. The method according to claim 1, wherein the ceramidase is acidic ceramidase.

6. The method according to claim 1, wherein one or more additional agents that reduce ceramide levels are administered in combination with the ceramidase.

7. The method according to claim 6, wherein the one or more additional agents are selected from the group consisting of one or more additional ceramide reducing agents, one or more acidic sphingomyelinase inhibitors, one or more agents for reducing infection, and combinations thereof.

8. The method according to claim 7, wherein the one or more additional agents are one or more agents for reducing infection and are selected from the group consisting of antibiotics, reagents that block the binding of the pathogen to the lung epithelium, reagents that reduce the viscosity of mucus, chaperone reagents that enhance lost protein function, and combinations thereof.

9. The method according to claim 7, wherein the ceramidase is administered simultaneously with, separately from, or sequentially to the one or more additional agents.

10. The method according to claim 1, wherein the administering step is by oral, topical, intranasal, intraperitoneal, intravenous, subcutaneous, or aerosol inhalation.

11. The method according to claim 10, wherein the administering step is by aerosol inhalation.

12. The method according to claim 1, wherein the ceramidase is administered in an amount of 0.001 mg / kg to 500 mg / kg.

13. The method according to claim 1, wherein the infection by the pathogen is a viral, fungal, prion, or bacterial infection.

14. The method according to claim 13, wherein the infection by the pathogen is a Pseudomonas infection.

15. The method according to claim 14, wherein the Pseudomonas infection is a Pseudomonas aeruginosa infection.

16. The method according to claim 1, wherein the ceramidase is administered before the onset of the infection.

17. The method according to claim 1, wherein the ceramidase is administered after the onset of the infection.

18. The method according to claim 1, wherein the selected subject has cystic fibrosis.

19. The method according to claim 1, wherein the selected subject has COPD.

20. The method according to claim 1, wherein the selected subject has an open wound.

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