U.S. patent application number 17/397298 was filed with the patent office on 2022-06-30 for ceramide levels in the treatment and prevention of infections.
The applicant listed for this patent is ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI, YEDA RESEARCH AND DEVELOPMENT CO. LTD. AT THE WEIZMANN INSTITUTE OF SCIENCE. Invention is credited to Anthony Futerman, Erich Gulbins, Yael Pewzner-Jung, Edward H. Schuchman.
Application Number | 20220202919 17/397298 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-30 |
United States Patent
Application |
20220202919 |
Kind Code |
A1 |
Schuchman; Edward H. ; et
al. |
June 30, 2022 |
CERAMIDE LEVELS IN THE TREATMENT AND PREVENTION OF INFECTIONS
Abstract
The present invention relates to a method for treating or
preventing pathogenic infections in a subject having Cystic
Fibrosis, COPD, and/or an open wound. This method involves
selecting a subject having Cystic Fibrosis, COPD, and/or an open
wound and administering to the selected subject a ceramidase 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.
Inventors: |
Schuchman; Edward H.;
(Haworth, NJ) ; Gulbins; Erich; (Essen, DE)
; Futerman; Anthony; (Rehovot, IL) ; Pewzner-Jung;
Yael; (Rehovot, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
YEDA RESEARCH AND DEVELOPMENT CO. LTD. AT THE WEIZMANN INSTITUTE OF
SCIENCE |
New York
Rehovot |
NY |
US
IL |
|
|
Appl. No.: |
17/397298 |
Filed: |
August 9, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16192241 |
Nov 15, 2018 |
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17397298 |
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15286327 |
Oct 5, 2016 |
10159724 |
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16192241 |
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14404881 |
Dec 1, 2014 |
9492514 |
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PCT/US2013/043608 |
May 31, 2013 |
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15286327 |
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61654519 |
Jun 1, 2012 |
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International
Class: |
A61K 38/50 20060101
A61K038/50; A61K 9/12 20060101 A61K009/12; A61K 9/00 20060101
A61K009/00; A61K 45/06 20060101 A61K045/06 |
Claims
1.-20. (canceled)
21. A method for treating a pathogenic infection in a subject, said
method comprising: selecting a subject having a pathogenic
infection and administering to said selected subject a recombinant
acid ceramidase under conditions effective to reduce ceramide and
to treat said pathogenic infection in said selected subject.
22. The method of claim 21, wherein said pathogenic infection
comprises a bacterial pathogen, a viral pathogen, or a fungal
pathogen.
23. The method of claim 21, wherein the subject has a disease
condition which predisposes the subject to pathogenic
infections.
24. The method of claim 23, wherein the disease condition is
selected from the group consisting of respiratory disease, lung
disease, 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, pneumocystosis, pneumothorax, pulmonary
actinomycosis, pulmonary alveolar proteinosis, pulmonary anthracis,
pulmonary arteriovenous malformation, pulmonary edema, pulmonary
embolus, pulmonary histiocytosis X (eosinophilic granuloma),
pulmonary nocardiosis, pulmonary tuberculosis, pulmonary
veno-occlusive disease, and rheumatoid lung disease.
25. The method of claim 21, wherein the recombinant acid ceramidase
comprises an acid ceramidase precursor protein.
26. The method of claim 21, wherein the recombinant acid ceramidase
comprises an acid ceramidase precursor protein and an active acid
ceramidase.
27. The method of claim 21, wherein the recombinant acid ceramidase
comprises UniProt Q13510, UniProt Q9H715, UniProt Q96AS2, OMIM
228000, NCBI Gene 427, NCBI RefSeq NP_808592, NCBI RefSeq
NP_004306, NCBI RefSeq NM_177924, NCBI RefSeq NM_004315, NCBI
UniGene 427, NCBI Accession Q13510, NCBI Accession AAC73009, or a
combination thereof.
28. The method of claim 21, wherein said selecting is based on
ceramide level in lung epithelium, nasal epithelium, mucus, or
isolated cells.
29. The method of claim 21, wherein said administering is carried
out under conditions effective to normalize ceramide levels in the
subjects' respiratory epithelia, mucus, or cells.
30. The method of claim 21, wherein the recombinant acid ceramidase
comprises an active acid ceramidase protein.
31. The method of claim 21, wherein one or more additional agents
that reduce ceramide levels are administered in combination with
said recombinant acid ceramidase.
32. The method of claim 31, wherein said one or more additional
agents are selected from the group consisting of one or more
additional ceramide reducing agents, one or more acid
sphingomyelinase inhibitors, one or more agents to reduce
infection, and combinations thereof.
33. The method of claim 31, wherein said one or more additional
agents is one or more agents to reduce infection and is selected
from the group consisting of antibiotics, reagents that block
binding of pathogens to lung epithelium, reagents to reduce mucus
viscosity, chaperone reagents to enhance missing protein function,
and combinations thereof.
34. The method of claim 21, wherein said recombinant acid
ceramidase is administered simultaneously, separately, or
sequentially with said one or more additional agents.
35. The method of claim 21, wherein said administering is oral,
intranasal, intraperitoneal, intravenous, subcutaneous, or by
aerosol inhalation.
36. The method of claim 21, wherein said administering is by
topical administration.
37. The method of claim 21, wherein said administering is by
aerosol inhalation.
38. The method of claim 21, wherein the ceramidase is administered
in an amount from 0.001 mg/kg to 500 mg/kg.
39. The method of claim 21, wherein said recombinant acid
ceramidase is administered prior to onset of infection.
40. The method of claim 21, wherein said recombinant acid
ceramidase is administered after onset of infection.
41. The method of claim 21, wherein the subject has Cystic
Fibrosis.
42. The method of claim 21, wherein the subject has COPD.
43. The method of claim 21, wherein the subject has an open wound.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/192,241, filed Nov. 15, 2018, which is a
continuation of U.S. patent application Ser. No. 15/286,327, issued
as U.S. Pat. No. 10,159,724 on Dec. 25, 2018, which is a
continuation of U.S. patent application Ser. No. 14/404,881, issued
as U.S. Pat. No. 9,492,514 on Nov. 15, 2016, which is a national
stage application under 35 U.S.C. .sctn. 371 of PCT Application No.
PCT/US2013/043608, filed May 31, 2013, which claims benefit of U.S.
Provisional Patent Application Ser. No. 61/654,519, filed Jun. 1,
2012, which are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to normalizing ceramide levels
to prevent and/or treat pathogenic infections in a subject with
cystic fibrosis, Chronic Obstructive Lung Disease (COPD), and/or an
open wound.
BACKGROUND OF THE INVENTION
[0003] Cystic Fibrosis ("CF") is the most common autosomal
recessive disorder in Europe and the USA, impacting one in every
2500 children born in Western Countries. It is a disease caused by
mutations of the CF transmembrane conductance regulator protein
("CFTR"). While this genetic mutation leads to several respiratory,
reproductive and gastrointestinal complications, the primary cause
of morbidity and mortality in these subjects results from the
destructive effects of chronic pulmonary colonization with
Pseudomonas aeruginosa ("P. aeruginosa"). Medical records indicate
approximately 80% of subjects with CF will host P. aeruginosa by
the age of 25. See Cystic Fibrosis Foundation Subject Registry:
Annual Data Report (2010). In addition to the increased
susceptibility to P. aeruginosa, CF lungs are characterized by
chronic inflammation and progressive fibrosis. At present, the
molecular mechanisms that mediate the hallmarks of CF disease,
i.e., infection susceptibility, inflammation, and fibrosis, require
definition.
[0004] P. aeruginosa infection of epithelial cells is initiated by
contact of the pathogen with the cell surface. Several binding
molecules for P. aeruginosa have been identified, including CFTR,
Fibronectin, .alpha.5.beta.1-integrin and glycolipids including
asialo-GM1. See Pier et. al., "Role Of Mutant CFTR In
Hypersusceptibility Of Cystic Fibrosis Subjects To Lung
Infections," Science. 271, 64-67 (1996); 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); 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); deBentzmann et. al., "Pseudomonas aeruginosa Adherence To
Remodeling Respiratory Epithelium," Eur. Respir. J. 9 pp. 2145-2150
(1996); Roger et. al., "Fibronectin And .alpha.5.beta.1-integrin
Mediate Binding Of Pseudomonas aeruginosa To Repairing Airway
Epithelium," Eur. Respir. J. 13 pp. 1301-1309 (1999); 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); 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).
[0005] Therefore, identification of epithelial receptors for P.
aeruginosa that are specifically altered in CF and involved in the
high infection susceptibility of these subjects to P. aeruginosa,
is an important consideration in the development of new strategies
for CF--and concomitant pathogenic infection--prophylaxis and
treatment. Such molecules would be ideal targets to prevent the
initial contact of the pathogen with bronchial epithelial cells in
CF subjects and, thus, to prevent the infection very early.
[0006] Current methods of treating and preventing disease
pathogenesis in subjects afflicted with a disease or condition
raise toxicity and efficacy concerns.
[0007] The present invention is directed to overcoming these
deficiencies in the art by, for example, by correcting the abnormal
expression of primary binding molecules of bacterial pathogens to
bronchial epithelial cells in vivo through a unique membrane lipid
mediated mechanism.
SUMMARY OF THE INVENTION
[0008] One aspect of the present invention is directed to a method
for treating or preventing pathogenic infections in a subject
having Cystic Fibrosis, Chronic Obstructive Lung Disease (COPD),
and/or an open wound. This method involves selecting a subject
having Cystic Fibrosis, COPD, and/or an open wound and
administering to the selected subject a ceramidase under conditions
effective to reduce ceramide and to treat or prevent the pathogenic
infection in the selected subject.
[0009] Another aspect of the present invention relates to
administering to the subject a ceramidase in combination with other
agents to reduce ceramide or reduce infections. Such agents may
include but not be limited to antibiotics, reagents to reduce mucus
viscosity, chaperone agents to enhance the function of the Cystic
Fibrosis transmembrane protein (CFTR), or acid sphingomyeliase
inhibitors.
[0010] Another aspect of the present invention relates to selecting
said subject based on the level of ceramide in their cells, tissues
or fluids, and/or the level of an endogenous ceramidase enzyme.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIGS. 1A and 1B show paraffin embedded sections of mouse
lungs. FIG. 1A is a representative section of wild type mice,
CerS2-deficient mice, and Cystic Fibrosis mice lung stained with
Cy3-anti-ceramide antibodies and then analyzed by confocal
microscopy.
[0012] FIG. 1B is a representative section of wild type mice,
CerS2-deficient mice, and Cystic Fibrosis mice lung stained with
the same antibody 2 hours after the mouse received a single
inhalation of acid ceramidase ("AC"). Results are representative of
at least 6 mice per group.
[0013] FIG. 2 demonstrates that recombinant acid ceramidase
prevents P. aeuriginosa infection in the lungs of mice accumulating
ceramide. Two mouse models were used. One (CerS2-/-) is a genetic
knockout for a ceramide producing enzyme, ceramide synthase 2.
Another (Cftr-/-) has a mutation in the Cystic Fibrosis
transmembrane protein gene and are a model of Cystic Fibrosis. The
wild type, CerS2-/- or Cftr-/- mice either received a single
inhalation of saline (light grey) or acid ceramidase (dark grey),
and then were infected with P. aeruginosa. Two hours later they
were sacrificed and the titer of P. aeruginosa remaining in the
mouse lungs was determined.
[0014] FIG. 3 shows results of mice that inhaled 100 .mu.g AC in
0.8 mL of 0.9% NaCl 30 to 45 minutes before intranasal infection
with 1.times.10.sup.8 colony-forming units (CFU) of P. aeruginosa
strain 762 or ATCC 27853. The 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 allowed to grow
overnight. CFUs on the LB plates were counted to determine the
number of P. aeruginosa bacteria in the lung. Shown are
means.+-.s.d. of four independent experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0015] In practicing 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 explained in, e.g., 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, N.Y.
(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., respectively, all of
which are hereby incorporated by reference in their entirety.
Methods to detect and measure levels of polypeptide gene expression
products, i.e., gene translation level, are well-known in the art
and include the use of polypeptide detection methods such as
antibody detection and quantification techniques. See also,
Strachan & Read, Human Molecular Genetics, Second Edition.
(John Wiley and Sons, Inc., New York (1999), which is hereby
incorporated by reference in its entirety.
[0016] It is to be appreciated that certain aspects, modes,
embodiments, variations and features of the present invention are
described below in various levels of detail in order to provide a
substantial understanding of the present technology. The
definitions of certain terms as used in this specification are
provided below. Unless defined otherwise, all technical and
scientific terms used herein generally have the same meaning as
commonly understood by one of ordinary skill in the art to which
this invention belongs.
[0017] Underlying disease conditions can predispose a subject to
acute and/or chronic pathogenic infections. As used herein a
"disease condition" refers to a pathological disease or condition
of any kind or origin, which a subject harbors. Accordingly,
disease conditions include the subject matter identified by the
following diseases and/or terms including, but not limited to,
e.g., a respiratory disease, lung disease, 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, pneumocystosis, pneumothorax, pulmonary
actinomycosis, pulmonary alveolar proteinosis, pulmonary anthracis,
pulmonary arteriovenous malformation, pulmonary edema, pulmonary
embolus, pulmonary histiocytosis X (eosinophilic granuloma),
pulmonary nocardiosis, pulmonary tuberculosis, pulmonary
veno-occlusive disease, rheumatoid lung disease, and/or an open
wound. Such diseases typically manifest an increased susceptibility
of a subject for pathogenic infection, i.e., compared to subjects
not afflicted with a disease condition.
[0018] For example, subjects suffering from CF, COPD, and/or an
open wound, may possess a high susceptibility for acquiring acute
and/or chronic pathogenic infections, such as, e.g., bacterial,
viral, fungal, protozoan, and/or prionic pathogenic infections.
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-typable, 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.,
Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus B,
Group A beta hemolytic Streptococcus, Streptococcus mutans,
Treponema pallidum, Vibrio cholerae, and Yersinia pestis. In some
embodiments, the pathogenic infection is a Pseudomonas infection.
In some embodiments, the Pseudomonas infection is a Pseudomonas
aeruginosa infection.
[0019] Viral pathogens include, without limitation, RNA viruses,
DNA viruses, adenovirdiae (e.g., mastadenovirus and aviadeno
virus), herpesviridae (e.g., herpes simplex virus 1, herpes simplex
virus 2, herpes simplex virus 5, and herpes simplex virus 6),
leviviridae (e.g., levivirus, enterobacteria phage MS2,
allolevirus), poxyiridae (e.g., chordopoxyirinae, parapoxvirus,
avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus,
molluscipox virus, and entomopoxyirinae), papovaviridae (e.g.,
polyomavirus and papillomavirus), paramyxoviridae (e.g.,
paramyxovirus, parainfluenza virus 1, mobillivirus such as measles
virus, rubulavirus (such as mumps virus), pneumonoviridae (e.g.,
pneumovirus, human respiratory syncytial virus), metapneumovirus
(e.g., avian pneumovirus and human metapneumo virus),
picornaviridae (e.g., enterovirus, rhinovirus, hepatovirus such as
human hepatitis A virus, cardiovirus, and apthovirus), reoviridae
(e.g., orthoreo virus, orbivirus, rotavirus, cypo virus, fijivirus,
phytoreo virus, and oryzavirus), retroviridae (e.g., mammalian type
B retroviruses, mammalian type C retroviruses, avian type C
retroviruses, type D retrovirus group, BLV-HTLV retroviruses,
lentivirus (such as human immunodeficiency virus 1 and human
immunodeficiency virus 2; and spuma virus), flaviviridae (e.g.,
hepatitis C virus), hepadnaviridae (e.g., hepatitis B virus),
togaviridae (e.g., alphavirus--such as sindbis virus and rubivirus,
such as rubella virus), rhabdoviridae (e.g., vesiculovirus,
lyssavirus, ephemera virus, cytorhabdovirus, and
necleorhabdovirus), arenaviridae (e.g., arenavirus, lymphocytic
choriomeningitis virus, Ippy virus, and lassa virus), and
coronaviridae (e.g., coronavirus and torovirus), Cytomegalovirus
(mononucleosis), Dengue virus (dengue fever, shock syndrome),
Epstein-Barr virus (mononucleosis, Burkitt's lymphoma), Human
T-cell lymphotropic virus type 1 (T-cell leukemia), Influenza A, B,
and C (respiratory disease), Japanese encephalitis virus
(pneumonia, encephalopathy), Poliovirus (paralysis), Rhinovirus
(common cold), Rubella virus (fetal malformations), Vaccinia virus
(generalized infection), Yellow fever virus (jaundice, renal and
hepatic failure), and Varicella zoster virus (chickenpox).
[0020] Pathogenic fungi include, without limitation, the genera
Aspergillus (e.g., Aspergillus fumigates), Blastomyces, Candida
(e.g., Candida albicans), Coccidiodes, Cryptococcus, Histoplasma,
Phycomyces, Tinea corporis, Tinea unguis, Sporothrix schenckii, and
Pneumocystis carinii. Pathogenic protozoan include, without
limitation, Trypanosome spp., Leishmania spp., Plasmodium spp.,
Entamoeba spp., and Giardia spp. such as Giardia lamblia
[0021] Because the molecular mechanisms which precipitate increased
susceptibility to a pathogenic infection in subjects afflicted with
a disease condition are not well understood, elucidating the
pathology of, for example, P. aeruginosa, with respect to cellular
attachment and internalization, are important considerations for
preventing and treating subjects prone to acquiring such
infections.
[0022] Integrins are receptor molecules which function to
coordinate cellular processes relating to, e.g., attachment and
adhesion. Integrins, however, are not characteristically expressed
at the luminal surface of normal, healthy bronchial epithelial
cells, and epithelial cell-layer tight junctions prevent the
contact of bronchial pathogens with the basolateral pole of
epithelial cells--where integrins typically reside.
[0023] The term "elevated levels" or "higher levels" as used herein
refers to levels of a measurable marker, molecule, or protein, such
as, for example, ceramide, that are higher than what would normally
be observed in a comparable sample from control or normal subjects,
i.e., a reference value or control levels or normalized levels. In
some embodiments, "control levels", i.e., normal levels, refer to a
range of that would normally be expected to be observed in a sample
from a subject that does not have a disease condition. A control
level may be used as a "reference level" for comparative purposes,
as further detailed, infra. "Elevated levels" therefore refer to
levels that are above the range of control levels. The ranges
accepted as "elevated levels" or "control levels" are dependent on
a number of factors. The skilled artisan is capable of considering
the relevant factors and establishing appropriate reference ranges
for "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 ranges that
are "normal" or "control" levels and ranges that are "elevated" or
"higher" compared to the control range or level.
[0024] Ceramidases are enzymes capable of hydrolyzing ceramide into
fatty acids and a sphingoid base (sphingosine), which is involved
in cellular proliferation and intracellular signal transduction.
The ceramidase, after enzymatic activation, facilitates ceramide
hydrolysis into individual fatty acid and sphingosine components.
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 hereby incorporated by
reference in their entirety. There is no de novo pathway for cells
to generate sphingosine, and it is therefore only generated by
ceramide hydrolysis pursuant to the enzymatic action of a
ceramidase.
[0025] One aspect of the present invention is directed to a method
for treating or preventing pathogenic infections in a subject
having Cystic Fibrosis, COPD, and/or an open wound. This method of
the present invention involves selecting a subject having Cystic
Fibrosis, COPD, and/or an open wound and administering to the
selected subject a ceramidase under conditions effective to reduce
ceramide and to treat or prevent the pathogenic infection in the
selected subject.
[0026] As described herein, an "open wound" refers to a type of
injury in which an epithelial layer, i.e., skin, is torn, cut,
and/or punctured. In some embodiments, an open wound refers to a
sharp injury which damages the dermis of the skin and concomitantly
increases the chance of acquiring an infection. The term "open
wound" also encompasses burns.
[0027] The methods of the present invention further involve
selecting the subject based on elevated ceramide levels compared to
a reference level for a subject not having Cystic Fibrosis, COPD,
and/or an open wound. As used herein, the term "reference level"
refers to a level of a substance, e.g., ceramide, which may be of
interest for comparative purposes. In some embodiments, a reference
level may be the level or concentration of a protein expressed as
an average of the level or concentration from samples of a control
population of healthy (disease-free and/or pathogen-free) subjects.
In other embodiments, the reference level may be the level in the
same subject at a different time, e.g., before the present
invention is employed, such as the level determined prior to the
subject developing a disease, disease condition, and/or pathogenic
infection, prior to initiating therapy, such as, for example,
ceramidase therapy, or earlier in the therapy.
[0028] Exemplary methods of comparing ceramide levels between a
subject and a reference level include, but are not limited to,
comparing differences in detected ceramide levels, based on results
of one or more protein assays as further described, infra. In some
embodiments, ceramide levels are higher in the presence of a
disease condition as described herein. A subject with, or a sample
possessing, a lower detected ceramide level compared to a reference
level would indicate that the subject may not require ceramidase
therapy and/or the subject may not have a disease condition as
described herein.
[0029] The method of the present invention further relates to
selecting a subject based on ceramide level in lung epithelium,
nasal epithelium, mucus, and/or cells isolated from an open wound
site. In some embodiments, administering is carried out under
conditions effective to normalize ceramide levels in the subjects'
respiratory epithelia, mucus, or cells at an open wound site. In
some embodiments, the ceramidase is an acid ceramidase ("AC"), such
as, but not limited to, the AC's listed in Table 1 below.
[0030] Acid ceramidase (N-acylsphingosine deacylase, I.U.B.M.B.
Enzyme No. EC 3.5.1.23) is one particular ceramidase responsible
for 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. The
protein has been purified from several sources, and the 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.
[0031] As described above, AC is a ceramidase which 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 hereby incorporated by reference in its entirety. Mature
AC is a .about.50 kDa protein composed of an .alpha.-subunit
(.about.13 kDa) and a .beta.-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
hereby incorporated 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 hereby
incorporated by reference in its entirety), which is the product of
the Asah1 gene (NCBI UniGene GeneID No. 427, which is hereby
incorporated by reference in its entirety).
[0032] AC function and/or activity, moreover, is directly related
to surrounding pH. In fact, it is normally found within lysosomes
with an acidic pH of .about.4.5, and in the absence of AC activity
in patients with Farber Lipogranulomatosis ceramides accumulate in
lysosomes.
[0033] In addition, recent studies have shown that an increase in
intracellular compartment pH reduces 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), which is hereby
incorporated by reference in its entirety. In some respects, these
results mimic Farber's disease, which is caused by a deficiency of
AC and results in an accumulation of ceramide. See He et al.,
"Purification And Characterization Of Recombinant, Human Acid
Ceramidase," J. Biol. Chem. 278, 32978-32986 (2003), which is
hereby incorporated by reference in its entirety. Furthermore, at a
pH of 5.9, AC has been shown to possess a reverse
activity--producing ceramide instead of consuming it. See id. This
activity in concert with impaired Asm function--at increasing
vesicular 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), which is hereby
incorporated by reference in its entirety.
[0034] Other studies have shown that CFTR deficiency in alveolar
macrophages result 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. As such, the present invention surprisingly functions to
prevent and/or treat pathogenic infections in CF subjects at least
because AC would not be expected to decrease elevated ceramide
levels in CF subjects possessing increased lysosomal pH. Moreover,
AC would not be expected to function on the accumulating ceramide
in the lung epithelial cell membrane.
[0035] The AC's that can be used in the context of the present
invention include, without limitation, those set forth in Table 1
below. In all aspects of the present invention, the AC can be
homologous (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.
TABLE-US-00001 TABLE 1 Exemplary Acid Ceramidase Family Members
Homo sapiens UniProt Q13510, Q9H715, Q96AS2 OMIM 228000 NCBI Gene
427 NCBI RefSeq NP_808592, NP_004306 NCBI RefSeq NM_177924,
NM_004315 NCBI UniGene 427 NCBI Accession Q13510, AAC73009 Mus
musculus UniProt Q9WV54, Q3U8A7, Q78P93 NCBI Gene 11886 NCBI RefSeq
NP_062708 NCBI RefSeq NM_019734 NCBI UniGene 11886 NCBI Accession
AK151208, AK034204 Gallus gallus UniProt Q5ZK58 NCBI Gene 422727
NCBI RefSeq NP_001006453 NCBI RefSeq NM_001006453 NCBI UniGene
422727 NCBI Accession CAG31885, AJ720226 Pan troglodytes NCBI Gene
464022 NCBI RefSeq XP_519629 NCBI RefSeq XM_519629 NCBI UniGene
464022 Caenorhabditis elegans UniProt O45686 IntAct O45686 NCBI
Gene 173120 NCBI RefSeq NP_493173 NCBI RefSeq NM_060772 NCBI
UniGene 173120 NCBI Accession O45686, CAB05556 Danio rerio UniProt
Q5XJR7 NCBI Gene 450068 NCBI RefSeq NP_001006088 NCBI RefSeq
NM_001006088 NCBI UniGene 450068 NCBI Accession AAH83231, CB360968
Rattus norvegicus UniProt Q6P7S1, Q9EQJ6 NCBI Gene 84431 NCBI
RefSeq NP_445859 NCBI RefSeq NM_053407 NCBI UniGene 84431 NCBI
Accession AAH61540, AF214647
[0036] In some embodiments, determining the level of ceramide
and/or AC concentration and/or activity is carried out prior to
treatment. Assays suitable for determining ceramide concentrations
and/or ceramidase levels or activity are readily apparent to the
skilled artisan. Suitable methods include, for example, activity
assays (see Eliyahu et al., "Acid Ceramidase is a Novel Factor
Required for Early Embryo Survival," FASEB J. 21(7):1403-9 (2007),
which is hereby incorporated by reference in its entirety, and well
known techniques, such as, western blotting to determine the
relative amount of ceramidase protein and/or activity present in
the sample (where a higher amount of ceramidase protein correlates
to a higher ceramidase activity level). See Eliyahu et al., "Acid
Ceramidase is a Novel Factor Required for Early Embryo Survival,"
FASEB J. 21(7):1403-9 (2007), which is hereby incorporated by
reference in its entirety.
[0037] As used herein, the term "assay" refers to an assay for
detecting the presence or absence of ceramide and/or ceramidase, in
a given sample of a bodily fluid. Also included are quantitative
assays, which measure the amount of a substance in a sample. As
used herein, the term "sample" is used in its broadest sense. In
one sense, it is meant to include a specimen or culture obtained
from biological samples. The bodily fluid sample is selected from
the group consisting of serum, synovial fluid, cerebrospinal fluid,
and peritoneal fluid. Of particular interest are samples that are
serum. Those skilled in the art will recognize that plasma or whole
blood or a sub-fraction of whole blood may also be used. Biological
fluid samples may be obtained from animals (including humans) and
include blood products, such as plasma, serum and the like. In some
embodiments, the sample contains a level of ceramide or ceramidase,
which can be readily ascertained by the methods described herein
and those well known in the art.
[0038] Immunoassays, in their most simple and direct sense, are
binding assays involving binding between antibodies and antigen.
Many types and formats of immunoassays are known and all are
suitable for detecting, e.g., ceramide levels. Examples of
immunoassays are enzyme linked immunosorbent assays ("ELISAs"),
enzyme linked immunospot assay ("ELISPOT"), radioimmunoassays
("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 their entirety, radioimmune precipitation assays ("RIPA"),
immunobead capture assays, dot blotting, gel-shift assays, flow
cytometry, immunohistochemistry, fluorescence microscopy, protein
arrays, multiplexed bead arrays, magnetic capture, in vivo imaging,
fluorescence resonance energy transfer ("FRET"), and fluorescence
recovery/localization after photobleaching ("FRAP/FLAP"). The steps
of various useful immunodetection methods have been described in
the scientific literature, such as, e.g., 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 incorporated herein by reference in its
entirety.
[0039] In general, immunoassays involve contacting a sample
suspected of containing a molecule or protein of interest (such as,
ceramide and/or ceramidase) with an antibody to the molecule or
protein of interest, under conditions effective to allow the
formation of immunocomplexes. In this regard, the skilled artisan
will be able to assess the presence and or level of specific
molecules or proteins of interest in a given sample.
[0040] Immunoassays can include methods for detecting or
quantifying the amount of a molecule or protein of interest in a
sample, which methods generally involve the detection or
quantitation of any immune complexes formed during the binding
process. In general, the detection of immunocomplex formation is
well known in the art and can be achieved through the application
of numerous approaches. These methods are generally based upon the
detection of a label or marker, such as any radioactive,
fluorescent, biological or enzymatic tags or any other known label.
See, for example, U.S. Pat. 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.
[0041] One particularly effective non-specific assay used to detect
total proteins 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 hereby
incorporated by reference in its entirety.
[0042] 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. The solution is added to 100 mL of 85% H.sub.3PO.sub.4,
and diluted to 200 mL with water, resulting in a dark red. The
final reagent concentrations of the assay are 0.5 mg/mL Coomassie
Blue G, 25% methanol, and 42.5% H.sub.3PO.sub.4. The assay reagent
of the Bradford assay is prepared by diluting 1 part dye stock with
4 parts distilled H.sub.2O. The resulting color should be brown
with a pH of 1.1. A series of protein standards are prepared in the
same buffer as the samples to be assayed, using bovine serum
albumin ("BSA") with concentrations of 0, 250, 500, 750, and 1500
.mu.g/mL for a standard assay. The absorbance is read at 595 nm for
standard assay procedure and 450 nm for a micro assay (Dynex
Technologies, Chantilly, Va.), and the ratio of the absorbances,
595 nm over 450 nm, was used for standard curve calculations. See
Zor, et al., "Linearization of the Bradford Protein Assay Increases
Its Sensitivity: Theoretical and Experimental Studies," Anal.
Biochem. 236:302-308 (1996), which is hereby incorporated by
reference in its entirety.
[0043] In some embodiments, the methods of the present invention
are carried out by administering an AC precursor protein, which is
then converted into an active acid ceramidase protein by the cell.
In particular, the AC precursor protein undergoes autoproteolytic
cleavage into the active form (composed of .alpha.- and
.beta.-subunits). This is promoted by the intracellular
environment, and based on highly conserved sequences at the
cleavage site of AC precursor proteins across species, is expected
to occur in most, if not all, cell types. Suitable acid ceramidase
precursor proteins include those set forth in Table 1, supra. As
will be apparent to the skilled artisan, the precursor protein
could optionally be contained in a culture medium to which the cell
is exposed. Embodiments in which the precursor protein is taken up
by the host subject or cell of interest and converted into active
acid ceramidase is thus contemplated.
[0044] Yet another approach for administering proteins or
polypeptide agents of the present invention, e.g., AC, involves
preparation of chimeric proteins according to U.S. Pat. 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 the polypeptide agent (e.g., AC, AC precursor
protein). The ligand domain is specific for receptors located on a
target cell. Thus, when the chimeric protein is delivered to the
subject, cell, and/or culture medium, the chimeric protein will be
internalized.
[0045] Depending on the level or activity of a substance, e.g.,
ceramide and/or ceramidase, one or more additional agents may be
administered in combination with the ceramidase, e.g., AC, in
accordance with the methods of the present invention. In some
embodiments, the one or more additional agents are selected from
the group consisting of one or more additional ceramide reducing
agents, one or more acid sphingomyelinase inhibitors, one or more
agents to reduce infection, and combinations thereof. Suitable
agents to reduce infection include antibiotics (e.g., inhaled
Tobramycin, TOBI), reagents that block binding of pathogens to lung
epithelium, reagents to reduce mucus viscosity (e.g., Dornase alfa,
Pulmozyme), chaperone reagents to enhance missing protein function
(e.g., Ivacaftor, Kalydeco), and combinations thereof. In some
embodiments, the ceramidase, e.g., AC, is administered
simultaneously, separately, or sequentially with the one or more
additional agents.
[0046] As used herein, the term "simultaneous" therapeutic use
refers to the administration of at least two active ingredients by
the same route and at the same time or at substantially the same
time. As used herein, the term "separate" therapeutic use refers to
an administration of at least two active ingredients at the same
time or at substantially the same time by different routes. As used
herein, the term "sequential" therapeutic use refers to
administration of at least two active ingredients at different
times, the administration route being identical or different. More
particularly, sequential use refers to the whole administration of
one of the active ingredients before administration of the other or
others commences. It is thus possible to administer one of the
active ingredients over several minutes, hours, or days before
administering the other active ingredient or ingredients. There is
no simultaneous treatment in this case.
[0047] Administration can be accomplished either via systemic
administration to the subject or via targeted administration to
affected tissues, organs, and/or cells. The therapeutic agent
(i.e., AC, AC precursor protein, nucleic acid encoding AC/AC
precursor protein) may be administered to a non-targeted area along
with one or more agents that facilitate migration of the
therapeutic agent to (and/or uptake by) a targeted tissue, organ,
or cell. Additionally and/or alternatively, the therapeutic agent
itself can be modified to facilitate its transport to (and uptake
by) the desired tissue, organ, or cell, as will be apparent to one
of ordinary skill in the art.
[0048] Any suitable approach for delivery of the agents can be
utilized to practice this aspect of the present invention.
Typically, the therapeutic agent will be administered to a patient
in a vehicle that delivers the therapeutic agent(s) to the target
cell, tissue, or organ. Exemplary routes of administration include,
without limitation, by intratracheal inoculation, aspiration,
airway instillation, aerosolization, nebulization, intranasal
instillation, oral or nasogastric instillation, intraperitoneal
injection, intravascular injection, topically, transdermally,
parenterally, subcutaneously, intravenous injection, intra-arterial
injection (such as via the pulmonary artery), intramuscular
injection, intrapleural instillation, intraventricularly,
intralesionally, by application to mucous membranes (such as that
of the nose, throat, bronchial tubes, genitals, and/or anus), or
implantation of a sustained release vehicle.
[0049] In some embodiments, a ceramidase, e.g., AC, is administered
orally, topically, intranasally, intraperitoneally, intravenously,
subcutaneously, or by aerosol inhalation. In some embodiments, a
ceramidase, e.g., AC, is administered via aerosol inhalation. In
some embodiments, the ceramidase and/or additional agents can be
incorporated into pharmaceutical compositions suitable for
administration, as described herein.
[0050] The agents of the present invention, e.g., AC, may be orally
administered, for example, with an inert diluent, or with an
assimilable 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 with the food of the diet. For oral
therapeutic administration, these active compounds may be
incorporated with excipients and used in the form of tablets,
capsules, elixirs, suspensions, syrups, and the like. Such
compositions and preparations should contain at least 0.1% of the
agent. The percentage of the agent in these compositions may, of
course, be varied and may conveniently be between about 2% to about
60% of the weight of the unit. The amount of the agent in such
therapeutically useful compositions is such that a suitable dosage
will be obtained.
[0051] The tablets, capsules, and the like may also contain a
binder such as gum tragacanth, acacia, corn starch, or gelatin;
excipients such as dicalcium phosphate; a disintegrating agent such
as corn starch, potato starch, or alginic acid; a lubricant such as
magnesium stearate; and a sweetening agent such as sucrose,
lactose, or saccharin. When the dosage unit form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a fatty oil.
[0052] The agents, e.g., AC, may also be administered parenterally.
Solutions or suspensions of the agent can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof in oils. Illustrative oils are those
of petroleum, animal, vegetable, or synthetic origin, for example,
peanut oil, soybean oil, or mineral oil. In general, water, saline,
aqueous dextrose and related sugar solutions, and glycols such as
propylene glycol or polyethylene glycol, are preferred liquid
carriers, particularly for injectable solutions. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0053] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol, and liquid polyethylene glycol),
suitable mixtures thereof, and vegetable oils.
[0054] The agents, e.g., AC, according the present invention may
also be administered directly to the airways in the form of an
aerosol. For use as aerosols, the compounds of the present
invention in solution or suspension may be packaged in a
pressurized aerosol container together with suitable propellants,
for example, hydrocarbon propellants like propane, butane, or
isobutane with conventional adjuvants. The materials of the present
invention also may be administered in a non-pressurized form.
[0055] Exemplary delivery devices include, without limitation,
nebulizers, atomizers, liposomes (including both active and passive
drug delivery techniques) (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. Pat. No. 5,653,996 to Hsu; U.S.
Pat. No. 5,643,599 to Lee et al.; U.S. Pat. No. 5,885,613 to
Holland et al.; U.S. Pat. No. 5,631,237 to Dzau & Kaneda; and
U.S. Pat. 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 hereby incorporated by
reference in its entirety), transdermal patches, implants,
implantable or injectable protein depot compositions, and syringes.
Other delivery systems which are known to those of skill in the art
can also be employed to achieve the desired delivery of the
therapeutic agent to the desired organ, tissue, or cells.
[0056] Administration can be carried out as frequently as required
and for a duration that is suitable to provide effective
prophylaxis or efficacy against a pathogen. For example,
administration can be carried out with a single sustained-release
dosage formulation or with multiple daily doses.
[0057] The amount to be administered will, of course, vary
depending upon the treatment regimen. Generally, an agent is
administered to achieve an amount effective for improving
pathogenic clearance. Thus, a therapeutically effective amount can
be an amount which is capable of at least partially preventing
and/or treating a pathogenic infection. This includes, without
limitation, delaying the onset of infection. The dose required to
obtain an effective amount may vary depending on the agent,
formulation, and individual to whom the agent is administered.
[0058] Dosage, toxicity and therapeutic efficacy of the agents or
compositions of the present invention can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. Compounds which exhibit
high therapeutic indices may be desirable. While compositions that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compositions to the site
of affected tissue in order to minimize potential damage to
uninfected cells and, thereby, reduce side effects.
[0059] As such, the ceramidase is administered in a therapeutically
effective amount, in some embodiments. As used herein, the terms
"therapeutically effective amount", "effective amount", or
"pharmaceutically effective amount" of an agent, protein, compound,
and/or composition, is a quantity sufficient to achieve a desired
therapeutic and/or prophylactic effect, e.g., an amount which
results in the prevention of, or a decrease in, the symptoms
associated with a disease that is being treated.
[0060] The effective amount of an agent or composition of the
present invention administered to the subject will depend on the
type and severity of the disease and on the characteristics of the
individual, such as general health, age, sex, body weight and
tolerance to drugs. It will also depend on the degree, severity and
type of disease. The skilled artisan will be able to determine
appropriate dosages depending on these and other factors. The
compositions of the present invention can also be administered in
combination with one or more additional therapeutic compounds.
[0061] Typically, the therapeutic agent will be administered as a
pharmaceutical formulation that includes the therapeutic agent and
any pharmaceutically acceptable adjuvants, carriers, excipients,
and/or stabilizers, and can be in solid or liquid form, such as
tablets, capsules, powders, solutions, suspensions, or emulsions.
The compositions preferably contain from about 0.01 to about 99
weight percent, more preferably from about 2 to about 60 weight
percent, of therapeutic agent together with the adjuvants, carriers
and/or excipients. In some embodiments, an effective amount ranges
from about 0.001 mg/kg to about 500 mg/kg body weight of the
subject. In some 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.
[0062] The agents, e.g., AC, of the present invention can be
administered at various times. Ceramidase, e.g., AC, is
administered prior to the onset of infection in some embodiments.
In other embodiments, the ceramidase, e.g., AC, is administered
after the onset of infection. Further still, the ceramidase, e.g.,
AC, may be administered prior to and after the onset of infection
according to some embodiments of the present invention.
[0063] Another aspect of the present invention relates to methods
of monitoring the effectiveness of a therapy in a subject having a
pathogenic infection and an underlying disease condition. The
method includes selecting a subject, providing a baseline ceramide
level in a bodily fluid sample from the selected subject before the
therapy, and treating the pathogenic infection with the therapy,
which, for example, can be the therapeutic administration of a
ceramidase such as, e.g., AC. The method further includes detecting
a post-therapy ceramide level in a bodily fluid sample from the
selected subject following the therapy, comparing the baseline
ceramide level with the post-therapy ceramidase level, and
identifying whether the therapy has been effective based on the
comparing and/or the pathology of the pathogenic infection.
[0064] In another aspect of the present invention, a kit or reagent
system for using or administering the agents of the present
invention. Such kits will contain a reagent combination including
the particular elements required to conduct an assay according to
the methods disclosed herein. The reagent system is presented in a
commercially packaged form, as a composition or admixture where the
compatibility of the reagents will allow, in a test device
configuration, or more typically as a test kit, i.e., a packaged
combination of one or more containers, devices, or the like holding
the necessary reagents, and preferably including written
instructions for the performance of assays. The kit may be adapted
for any configuration of an assay and may include compositions for
performing any of the various assay formats described herein.
[0065] Reagents useful for the disclosed methods can be stored in
solution or can be lyophilized. When lyophilized, some or all of
the reagents can be readily stored in microtiter plate wells for
easy use after reconstitution. It is contemplated that any method
for lyophilizing reagents known in the art would be suitable for
preparing dried down reagents useful for the disclosed methods.
[0066] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
EXAMPLES
Example 1--Mice
[0067] B6.129P2(CF/3)-Cftr.sup.TgH(neoim)Hgu ("CF.sup.MHH")
congenic mice were produced through the inbreeding of the original
Cftr.sup.TgH(neoim)Hgu mutant mouse, which was generated by
insertional mutagenesis in exon 10 of the Cftr gene. See
Charizopoulou et. al., "Instability Of The Insertional Mutation In
Cftr.sup.TgH(neoim)Hgu Cystic Fibrosis Mouse Model," BMC Genet. 5
p. 6 (2004), which is hereby incorporated by reference in its
entirety. This congenic Cftr.sup.MHH strain was then backcrossed
into the B6 background. These mice still express low levels of
CFTR, and can thus be fed a standard mouse diet. They exhibit
normal development, but also display pulmonary pathology typical
for CF. They are herein referred to 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), which is hereby
incorporated by reference in its entirety. Syngenic B6 mice were
used as controls.
[0068] For some experiments, Cftr.sup.tm1Unc-Tg.sup.(FABPCFTR) mice
("Cftr.sup.-/-", purchased from The Jackson Laboratory, Bar Harbor,
Me.) were backcrossed for more than 10 generations with C57BL/6
mice. The mice are completely deficient in Cftr in all organs
except the intestine, where they express human CFTR which is under
the control of a fatty acid binding protein ("FABP") promoter. The
transgene prevents intestinal obstruction and enables feeding with
a normal diet. Again, B6 mice were used as controls. No major
differences were observed in experiments which employed both the
Cftr.sup.-/- and Cftr.sup.MHH strains.
[0069] 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
live beyond .about.16 months and accumulate C16 ceramide in most
tissues (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 hereby
incorporated by reference in its entirety).
[0070] The mice were housed and bred within isolated cages in the
vivarium of the University Hospital, University of Duisburg-Essen,
Germany. They were repeatedly evaluated for a panel of common
murine pathogens according to the 2002 recommendations of the
Federation of European Laboratory Animal Science Associations. The
mice were free of all pathogens. Procedures performed on the
animals were approved by the Bezirksregierung Duesseldorf,
Duesseldorf, Germany.
Example 2--Antibodies and Reagents
[0071] All ceramide stainings were performed using the monoclonal
mouse anti-ceramide antibody clone S58-9 (Glycobiotech) that was
visualized with Cy3-donkey-anti-mouse IgM F(ab).sub.2 fragments
(Jackson #715-166-020) or Cy5-coupled 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 media as previously described. He et al., J Biol. Chem.
278:32978-86 (2003), which is hereby incorporated by reference in
its entirety.
Example 3--Bacteria
[0072] The laboratory strain American Type Culture Collection 27853
P. aeruginosa and the previously described clinical P. aeruginosa
isolate ("762") were used. Bacteria were plated from frozen stocks
on fresh Tryptic Soy Agar plates (TSA; Becton Dickinson), grown at
37.degree. C. for 14-16 hours and resuspended in 40 mL of
37.degree. C. warmed Tryptic Soy Broth (Becton Dickinson) to an
optic density of 0.225 at 550 nm. The bacterial suspension was then
incubated at 37.degree. C. for 1 hr with 125 rpm shaking to attain
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 hereby incorporated by reference in its entirety. Bacteria were
then washed twice and resuspended in warmed RPMI-1640 medium
(Invitrogen) supplemented with 10 mM HEPES (RPMI+HEPES). The final
concentration of bacteria was quantified by photospectrometry.
Example 4--In Vivo Immunohistochemistry
[0073] For immunohistochemical evaluation of murine bronchial
epithelial cells, mice were sacrificed by cervical dislocation and
immediately perfused via the right heart with ice cold normal
saline for two minutes at low pressure. This was followed by
cardiac perfusion with 4% PBS-buffered PFA for 10-15 minutes. After
this initial clearance of blood and fixation, the lungs were
removed and further fixed in 4% PFA for 24-36 hrs. The tissue was
serially dehydrated using an ethanol to xylol gradient and then
embedded in paraffin.
[0074] The samples were then sectioned at 7 .mu.m, dewaxed,
re-hydrated, and treated with Pepsin (Invitrogen) for 15 min at
37.degree. C. They were then washed with water and PBS and blocked
for 10 min at room temperature with PBS and 0.05% Tween 20 (Sigma)
and 1% FCS. The samples were then consecutively stained with
primary antibodies in H/S+1% FCS at room temperature for 45 min.
Samples were washed between the stainings twice with PBS+0.05%
Tween 20 and once with PBS. The tissue was secondarily labeled with
fluorescent-coupled secondary antibodies in H/S+1% FCS in the dark
for 30 minutes. Tissue was again washed twice with PBS+0.05% Tween
20, once with PBS and finally embedded in Mowiol. Samples were
evaluated using a confocal microscope as described below.
Example 5--Inhalation and In Vivo Infection
[0075] P. aeruginosa was prepared as described above and
resuspended in RPMI-1640 plus 10 mM HEPES to a final concentration
of 1.times.10.sup.8 CFU in 20 .mu.L medium. They were then
inoculated using a plastic-coated 30-gauge needle, which was
inserted 2 mm into the nose. Bacterial numbers were quantified in
mouse lungs 2 hrs after infection. Mice were sacrificed and the
lungs were removed, homogenized, and lysed in 5 mg/mL Saponin to
release intracellular bacteria. The samples were then washed in
sterile PBS, diluted, and plated in duplicate on TSA plates for 12
hours. Bacterial numbers were counted and represent the number of
the bacteria in whole lung samples. This mode of infection more
accurately evaluates mucociliary clearance than other pulmonary
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 hereby incorporated by
reference in their entirety.
Example 6--Statistics
[0076] Data are expressed as arithmetic means.+-.SD and performed
statistical analysis as indicated. Since all values were normally
distributed, one-way ANOVA was applied. Significances are indicated
in the figures with asterisks.
Example 7--Confocal Microscopy and Discussion
[0077] Samples were examined with a Leica TCS-SP5 confocal
microscope equipped with a 100.times. oil emersion lens, and images
were analyzed with Leica LCS software (Leica Microsystems). All
comparative samples were measured with identical settings.
[0078] Ceramide is increased in the lungs of CF subjects and mice
(FIG. 1A), and is an important factor in the susceptibility of CF
mice to P. 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 hereby incorporated by reference in its entirety. Previous
studies demonstrated that pharmacological inhibition of acid
sphingomyelinase or genetic heterozygosity of the acid
sphingomyelinase gene are sufficient to normalize ceramide levels
in mouse CF lungs. See Becker et. al., "Acid Sphingomyelinase
Inhibitors Normalize Pulmonary Ceramide And Inflammation In Cystic
Fibrosis," Am J Respir Cell Mot Biol. 42(6):716-24 (2010), which is
hereby incorporated by reference in its entirety.
[0079] In addition, CF.sup.MHHCftr-deficient mice inhaled acid
ceramidase, which hydrolyzes ceramide. This inhalation corrected
ceramide levels in the bronchial epithelial cells of CF mice (FIG.
1B). Inhalation of the solvent, i.e., 0.9% NaCl did not affect
ceramide levels.
[0080] In another example, Cftr-/-, CerS2 or normal mice were
inhaled with saline or acid ceramidase and then infected with P.
aeruginosa (FIG. 2). CF'Cftr-deficient and CerS2 mice accumulate
ceramide in their lungs relative to normal mice. Two hours after
inhalation they were sacrificed and the remaining bacteria in the
lungs was quantified. Wild-type mice cleared the P. aeruginosa
effectively, whereas Cftr-/- or CerS2-/- mice inhaled with saline
could not and had large numbers of remaining bacteria. In contrast,
Cftr-/- or CerS2-/- mice inhaled with acid ceramidase had very low
bacterial titers like normal mice.
[0081] The identification of irregularities in naive CF airways
provides a novel concept for the prevention of infection in CF
subjects. The present examples provide for several approaches to
thwarting infection and treating the leading cause of death for
subjects with CF.
Example 8--AC Inhalation Protects Against Pseudomonas
Infections
[0082] Mice were inhaled with 100 micrograms of recombinant acid
ceramidase (AC) in 0.8 mL of 0.9% NaCl 30 to 45 minutes before
intranasal infection with 1.times.10.sup.8 colony-forming units
(CFU) of P. aeruginosa strain 762 or ATCC 27853. The 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 allowed to grow overnight. CFUs on the LB plates were
counted to determine the number of P. aeruginosa bacteria in the
lung. Shown are means.+-.standard deviation of four independent
experiments.
[0083] A single inhalation of AC prevented infection of CF mice
with two different strains of P. aeruginosa (FIG. 3). Clinical
strain 762 was originally obtained from a urinary tract infection,
while strain ATCC 27853 is a laboratory strain. Inhalation of
saline alone was used as a control.
[0084] CF mice were inhaled with recombinant acid ceramidase (100
micrograms in 0.8 mL of 0.9% NaCl). Saline was used as a control.
In all cases, mice were inhaled 30-45 minutes before inhalation
with clinical Pseudomonas aeruginosa strain 762, and then
sacrificed 4 hours after. The 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 allowed to grow
overnight. CFUs on the LB plates were counted to determine the
number of P. aeruginosa bacteria in the lung.
[0085] Inhalation of CF mice with recombinant acid ceramidase
prevented infection with clinical strain 762 P. aeruginosa to a
similar degree (FIG. 3).
[0086] Although preferred embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be made without departing from the
spirit of the invention and these are, therefore, considered to be
within the scope of the invention as defined in the claims which
follow.
* * * * *