Note: Descriptions are shown in the official language in which they were submitted.
CA 02696833 2010-01-11
1
DISEASE TREA-TMENT VIA ANTIMICROBIAL PEPTIDES OR THEIR
INHIBITORS
CROSS-REFERENCE TO RELATED APPLICATIONS
100011 This application claims priority to each of Israel application serial
nos. 184611
filed July 15, 2007 and 187627 filed November 26, 2007, the disclosures of
which are
incorporated by reference herein in their entireties.
FIELD OF THE INVENTION
[0002] The invention relates to the field of mammalian antimicrobial peptides
(AMPs)
and their use in the treatment of disease.
BACKGROUND OF T'HE INVENTION
[0003] The present invention relates to methods of treating diseases using
anti-
antimicrobial peptide (AMP) and/or AMP-like molecule (AML) and in particular
cathelicidin type AMPs, and to methods of identifying compounds capable of
regulating,
decreasing or increasing activities/levels of AMPs/AMLs so as to enable
treatment of
diseases. More particularly, the present invention relates to methods of
treating diseases by
using cathelicidin or catlielicidin fragments or cathelicidin analogs or
compounds capable of
regulating the levels/activity of cathelicidin, such diseases including
dysregulated cell
proliferation/differentiat:ion leading to bone loss or degradation,
osteoporosis osteoarthritis,
or to other autoimmune diseases such as multiple sclerosis, arthritis,
psoriasis, and to
malignancies such as carcinomas, which are associated with inflammation, to
metabolic
diseases, obesity, insulin resistance, diabetes type 2, diabetes type 1 and
related diseases.
Also, the present invention relates to methods of identifying compounds
capable of
regulating levels of cathelicidin or other AMPs or to increasing or to
decreasing
activity/levels of AMPs so as to enable treatment of diseases including
autoimmune and
inflammatory diseases such as, multiple sclerosis, arthritis, metabolic
disorders such as
diabetes, obesity and malignant diseases such as carcinomas, which are
associated with
inflammation, dysregulaited cell proliferation/differentiation, angiogenesis
and/or metastasis.
[0004] Both inhibiting endogenous cathelicidin based peptides or other AMPs as
well as
the use of such catheliciclin based peptides or analogs of cathelicidin
peptides are effective
modes of treatments for disease. As was demonstrated in WO 2004-056307 filed
by the
present inventors and incorporated herein, cathelicidins are immune regulators
and are over
expressed locally in autoimmune diseases. They are also expressed systemically
through
CA 02696833 2010-01-11
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bone marrow such that tiormal plasma concentrations average around 1.2 ug/ml
to 1.5ug/ml
(Journal of Immunological Methods 206_1997.53-59). Regulation of their
expression is
essential for homeostasis. AMPs are involved is skewing dendritic cell
activation between
Th 1 and Th2 inflammatory processes via Toll-like receptors and therefore are
involved in
homeostasis (J Immunol.. 2004 Jan 15;172(2):1146-56). Controlling or
maintaining such
homeostasis is performed by either increasing or decreasing of level/activity
between the
various AMPs.
100051 Cathelicidins. are mainly expressed by Vitamin D3 (calcitriol), via
vitamin D3
receptor elements (VDRE) and Vitamin D3 itself has a modulating influence on
cathelicidin
expression both as an agonist via calcitriol/VDRE and by a negative feedback
mechanisms
(Marshall T BioEssays 30:173-182, 2008). This VDRE/cathelicidin pathway is
unique to
humans and furry/haired. animals such as rodents for example whose skin is
less exposed to
sunlight do not possess this pathway. As shown in data included in this
invention for the first
time relative to prior art, cathelicidin forms a major immune regulator for
diseases which are
known to be also regulated by vitamin D3. These include amongst others bone
loss in
Periodontitis (which is associated with low vitamin D and low cathelicidin),
Obesity, Type 2
Diabetes mellitus type 1 and type 2 (which is associated with low vitamin D
and Toll like
receptor 4, which cathelicidin inhibits), Atherosclerosis ( low vitamin D
association),
Hypertension ( low vitarnin D association), Asthma and Allergy ( low vitamin D
association), Osteoporosis and Ostepenia ( low vitamin D association),
Multiple Sclerosis (
low vitamin D associatian), Rheumatoid arthritis (low vitamin D association),
Autoimmune
Diseases such as Crohn, Type 1 Diabetes ( low vitamin D association),
Schizophrenia (low
vitamin D association), Tvluscle wasting disease including age associated
muscle wasting
(low vitamin D association as well as beta defensin overexpression), Cancer (
low vitamin
D association as well as Cathelicidin and beta defensin overexpression),
Depression ( low
vitamin D association), Skin inflammation including Psoriasis ( treated with
vitamin D
analogues), Tubeculosis and Influenza ( low vitamin D association), Chronic
Pain ( low
vitamin D association), Osteoartheritis (low vitamin D association), The
Common Cold and
other known diseases (The Breast Journal, Volume 14 Number 3, 2008 255-260,
Photochem
Photobiol. 2008 Mar-Apr;84(2):356-65) associated with vitamin D3, commonly
known as
the "Sunshine vitamin" and inappropriately called a vitamin but is in fact a
hormone. Data
as presented in this inverition indicate a common pathway of disease
regulation between
cathelicidin and vitamin D3. For this reason, the inventor reasons that
diseases such as
CA 02696833 2010-01-11
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schizophrenia and depression which cannot be modeled suitably by animals are
also
regulated by cathelicidiri.
[0006] Diseases, such as malignant, autoimmune, and allergic diseases, which
are
associated with biological processes such as inflammation, dysregulated cell
proliferation/differentiation, and dysregulated cell
proliferation/differentiation balance
include a vast range of highly debilitating and/or lethal pathologies of great
economic
impact, for which no satisfactory treatment methods are presently available.
For example
autoimmune diseases represent diseases of major clinical and economic impact.
These
include major diseases such as psoriasis, rheumatoid arthritis, type I
diabetes, inflammatory
bowel diseases, and multiple sclerosis for which no satisfactory treatment
methods are
available. Similarly, malignant diseases, such as skin carcinoma, breast
carcinoma, colon
carcinoma, head and neck carcinoma, hepatic carcinoma, lung carcinoma, renal
cell
carcinoma, urinary bladder carcinoma, and the like, represent numerous lethal
diseases for
which no satisfactory treatment methods are available.
[0007] There is an urgent and long-felt need for optimal methods of treating
such
diseases which are associated with inflammation, dysregulated cell/tissue
proliferation/differentiation and autoimmunity.
[0008] The epithelial lining of the skin, gastrointestinal tract and bronchial
tree produces
a number of peptides with antimicrobial activities termed antimicrobial
peptides (AMPs),
which appear to be involved in both innate host defense and adaptive immune
responses
(Yang D. et al., 2001. Cell Mol Life Sci. 58:978-89). AMPs are cationic
peptides which
display antimicrobial activity at physiological concentrations under
conditions prevailing in
the tissues of origin. AMP synthesis and release is regulated by microbial
signals,
developmental and differentiation signals, cytokines and in some cases
neuroendocrine
signals in a tissue-specific manner. Their mode of action is unknown, however
the leading
theory claims that permeabilization of target membranes is the crucial step in
AMP-mediated
antimicrobial activity and cytotoxicity. AMPs are classified into two major
groups in
humans; cathelicidins and defensins. AMPs appear to have common
characteristics that
enable them to affect mammalian cells in a way that does not necessarily
function through a
ligand-receptor pathway, and that, being small, and highly ionic or
hydrophobic or
structurally amphiphilic, AMPs can bind mammalian cell membranes. They are
able to
penetrate through the cell membrane to the cytoplasm. For example, it was
shown that
granulysin penetrates an<i damages human cell membranes dependent upon
negative charge
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(J. Immunol., 2001, 167:350-356). At high concentrations they are cytotoxic to
cells, they
tear through the membrane causing lysis or apoptosis. Likewise they are able
to change the
charge density of the inner membrane by the very fact that they have charge,
are small and
are distributed around the cell membrane from the outer surface of the
membrane.
[0009] Cathelicidins contain a conserved "cathelin" precursor domain. Their
organization includes an. N-terminal signal peptide, a highly conserved
prosequence, and a
structurally variable cationic peptide at the C-terminus. The prosequence
resembles cathelin,
a protein originally isolated from porcine neutrophils as an inhibitor of
cathepsin L (hence,
the name cathelin). The 37 amino acid-long human cathelicidin, LL-37/hCAP18
has a
hydrophobic N-terminal domain in an a-helical conformation, particularly in
the presence of
negatively charged lipids. In a step essential for its activation, LL-37 is
enzymatically
cleaved from the C-terminus of hCAP18 precursor via enzymes such as neutrophil
elastase
and proteinase 3. LL-37 functions in synergy with other AMPs, and can directly
activate
host cells. Inappropriate cleavage of the cathelicidin hCAP18 pro-peptide by
endogenous
proteases can produce pro-inflammatory fragments of the cathelicidin (Nat Med.
2007
Aug; 13(8):975-80). At the same time, correct cleavage via appropriate
endogenous protease
processing will produce the anti-inflammatory cathelicidin analogs and
peptides. Thus, a
method for regulating inadequate processing of cathelicidin is required as
well as a method
of using the anti-inflamnnatory analogs or fragments to the cathelicidin
peptides or pro-
peptide is described and exemplified below.
1000101 The ability of cathelicidins such as LL-37 to both kill bacteria and
regulate
immune responses is a characteristic of numerous AMPs. The peptide can
influence host
immune responses via a variety of cellular interactions, for example, it has
been suggest to
possibly function as a chemoattractant by binding to formyl-peptide-receptor-
like-1 (FPRL-
1). LL-37 can recruit mast cells, and then be produced by the mast cell to
kill bacteria.
1000111 AMPs exert their effects either individually or as the resultant
effect of multiple
AMPs. For example, in the menstrual cycle there is a monthly cycle-dependent
expression
of various AMPs (King A.E. et al., 2003. J. Reprod. Immunol. 59:1-16). For
example, there
is higher expression during the menstrual cycle of beta-defensin-2 in the
menstrual stage,
beta-defensin-4 in the proliferative stage, beta-defensin-3 in the early
secretory stage, beta-
defensin-1 in the mid secretory stage, and beta-defensin-3 in the late
secretory stage. It has
been suggested that mairitaining the balance between the AMPs is essential for
normal
proliferation, differentiation and in the specific example of menstrual cycle
for
CA 02696833 2010-01-11
development.In light of the apparent roles of AMPs and most importantly of
cathelicidin as
was demonstrated in this and the former patent application (number WO 2004-
056307) of
the current inventor, cathelicidin is associated with inflammation,
dysregulated cell
proliferation/differentiation, dysregulated cell proliferation/differentiation
balance,
angiogenesis metastasis, and/or epithelial wounds, the inventor hypothesized
that an optimal
strategy for treating sucli diseases would be via methods involving decreasing
the
levels/activity of such AMPs/AMLs, and/or via methods involving administering
such
AMPs/AMLs or enhancing their expression.
[00012] The prior art approaches relating to such methods involve the previous
application of the inventors in WO 2004-056307 which show that cathelicidin is
an immune
regulator in-vivo and therefore poses a target in treating autoimmune
diseases.
[000131 The current application provides in-vivo data for specific diseases
such as
metabolic diseases and low grade inflammatory diseases, obesity, insulin
resistance, diabetes
type 2, type 1 diabetes, insulin related diabetes, osteoporosis,
periodontitis, osteoarthritis,
arthritic diseases, rheumatologic diseases such as rheumatoid arthritis,
ankylosing
spondylitis, gout and systemic lupus erythematosus, as well as multiple
sclerosis,
neurological and central nervous system diseases as well as osteoporosis.
In particular, the current invention shows in-vivo the use of cathelicidin or
cathelicidin analogs in the treatment of said diseases.
SUMMARY OF THE 1[NVENTION
[00014] According to one aspect of the present invention there is provided a
method of
treating a medical condition, such as a disease, in a subject in need of
treatment thereof, the
method comprising providing to the subject a therapeutically effective amount
of a
compound in particular a cathelicidin peptide or fragment analog thereof,
being capable of
treating the disease in the subject in need thereof or of regulating, or
increasing or decreasing
an activity and/or level of an antimicrobial peptide (AMP) and/or AMP-like
molecule,
thereby treating the disease in the subject in need thereof.
[00015] According to further features in preferred embodiments of the
invention
described below, administering the compound to the subject is effected by
exposing a
location of the subject to a carrier which includes the compound at a
concentration selected
from a range of about 50 nanograms per milliliter to about 2 milligram per
milliliter.
[00016] According to still further features in the described preferred
embodiments,
administering the compound to the subject is effected by administering to the
subject a
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plurality of doses of the compound selected from a range of 2 doses to 30
doses, wherein
each inter dose interval of the plurality of doses is selected from a range of
about 2.4 hours
to about 30 days.
[00017] According to still further features in the described preferred
embodiments,
administering the compound to the subject is effected via a route selected
from the group
consisting of the topical, intravenous, intranasal, transdermal, intradermal,
oral, buccal,
parenteral, rectal and inlialation route.
1000181 According to still further features in the described preferred
embodiments, the
disease is associated with a biological process in a cell and/or tissue,
wherein the biological
process is selected from the group consisting of growth, differentiation,
autoimmunity or
inflammation.
[00019] According to still further features in the described preferred
embodiments, the
subject is human.
[00020] According to another aspect of the present invention there is provided
an article
of manufacture comprising packaging material and a pharmaceutical composition,
the article
of manufacture being identified for treatment of a disease being associated
with a biological
process in a cell and/or tissue, the biological process being selected from
the group
consisting of growth, differentiation or diseases associated with inflammation
or
autoimmunity; the pharnlaceutical composition comprising a pharmaceutically
acceptable
carrier and, as an active iingredient, a compound being capable of regulating
an activity
and/or level of an antimicrobial peptide (AMP) and/or AMP-like molecule.
[00021] According to further features in preferred embodiments of the
invention
described below, the pharmaceutically acceptable carrier is selected so as to
enable
administration of the pharmaceutical composition via a route selected from the
group
consisting of the topical, intranasal, transdermal, intradermal, intravenous,
oral, buccal,
parenteral, rectal and inhalation route.
[00022] According to still further features in the described preferred
embodiments, the
pharmaceutical composition is formulated as a solution, suspension, emulsion
or gel.
1000231 According to still further features in the described preferred
embodiments, the
pharmaceutical composition is composed so as to enable exposure of a cell
and/or tissue of a
subject having the disease to the compound at a concentration selected from a
range of about
50 nanograms per milliliter to about 1 milligram per milliliter.
[00024] According to still further features in the described preferred
embodiments, the
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pharmaceutical composiition is further identified for administration to a
subject of a plurality
of doses of the pharmaceutical composition selected from a range of 2 doses to
30 doses,
wherein each inter dose interval of the plurality of doses is selected from a
range of about 2.4
hours to about 30 days
[000251 According to still further features in the described preferred
embodiments, the
cell and/or tissue is selected from the group consisting of skin cells, bone
cells beta cells and
synovial tissue.
[00026] According to still further features in the described preferred
embodiments, the
disease is selected from the group consisting of an autoimmune disease, a bone
resorption
disease, a neurological c'lisease, a metabolic disease including diabetes,
obesity, and a
diabetes related disease.
1000271 According to yet another aspect of the present invention there is
provided a
method of regulating a biological process in a cell and/or tissue, the method
comprising
exposing the cell and/or tissue to a compound in particular a cathelicidin
peptide or its
analog, being capable of'regulating the biological process in the cell and/or
tissue or of
increasing or decreasing an activity and/or level of an antimicrobial peptide
(AMP) and/or
AMP-like molecule, thereby regulating the biological process in the cell
and/or tissue.
[00028] According to further features in preferred embodiments of the
invention
described below, exposing the cell and/or tissue to the compound (such as for
example, a
cathelicidin peptide or its analog) effected by providing said compound to a
subject.
[00029] According to still further features in the described preferred
embodiments, the
providing to the subject the compound is effected by administering the
compound to the
subject and/or by expressing the compound in the subject.
[00030] According to still further features in the described preferred
embodiments, the
exposing the cell and/or tissue to the compound is effected by exposing the
cell and/or tissue
to the compound at a coiicentration selected from a range of about 50
nanograms per
milliliter to about one milligram per milliliter.
[00031] According to still further features in the described preferred
embodiments, the
cell and/or tissue is bone or nerve tissue or synovial tissue, wherein the
exposing the cell
and/or tissue to the compound (such as for example, a cathelicidin peptide or
its analog) is
effected by exposing the cell and/or tissue to the compound at a concentration
selected from
a range of about 0.4 mici-ogram per milliliter to about 100 micrograms per
milliliter.
[00032] According to still another aspect of the present invention there is
provided a
CA 02696833 2010-01-11
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method of identifying a compound being capable of regulating a biological
process in a cell
and/or tissue, the method comprising: (a) exposing the cell and/or tissue to a
test compound
which is: (i) capable of <iecreasing an activity and/or level of an
antimicrobial peptide (AMP)
and/or AMP-like molecule, and/or (ii) the AMP and/or AMP-like molecule; and
(b)
evaluating a capacity of the test compound to regulate the biological process
in the cell
and/or tissue, thereby identifying the compound being capable of regulating
the biological
process in the cell and/or tissue.
1000331 According to still further features in the described preferred
embodiments, the
cell and/or tissue is a cultured cell and/or tissue.
1000341 According to still further features in the described preferred
embodiments, the
cell and/or tissue is derived from a human.
[00035] According to still further features in the described preferred
embodiments, the
exposing the cell and/or tissue to the test compound is effected by providing
the test
compound to a subject.
1000361 According to still further features in the described preferred
embodiments, the
exposing the cell and/or tissue to the test compound is effected by exposing
the cell and/or
tissue to a cell which produces the test compound.
[00037] According to still further features in the described preferred
embodiments, the
cell which produces the test compound is a B-cell hybridoma.
[00038] According to still further features in the described preferred
embodiments, the
providing the test compciund to the subject is effected by administering the
test compound to
the subject and/or by expressing the test compound in the subject.
[00039] According to still further features in the described preferred
embodiments,
administering the test compound to the subject is effected via a route
selected from the group
consisting of the topical, intranasal, intravenous, transdermal, intradermal,
oral, buccal,
parenteral, rectal and inhalation route.
[00040] According to still further features in the described preferred
embodiments, the
test compound is selected from the group consisting of: (a) a molecule capable
of binding the
AMP and/or AMP-like molecule; (b) an enzyme capable of cleaving the AMP and/or
AMP-
like molecule; (c) an siRNA molecule capable of inducing degradation of an
mRNA
encoding the AMP and/ar AMP-like molecule; (d) a DNAzyme capable of cleaving
an
mRNA or DNA encoding the AMP and/or AMP-like molecule; (e) an antisense
polynucleotide capable of hybridizing with an mRNA encoding the AMP and/or AMP-
like
CA 02696833 2010-01-11
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molecule; (f) a ribozyme capable of cleaving an mRNA encoding the AMP and/or
AMP-like
molecule; (g) a non-functional analog of at least a functional portion of the
AMP and/or
AMP-like molecule; (h) a molecule capable of inhibiting activation or ligand
binding of the
AMP and/or AMP-like imolecule; and (i) a triplex-forming oligonucleotide
capable of
hybridizing with a DNA. encoding the AMP and/or AMP-like molecule.
[00041] According ta still further features in the described preferred
embodiments, the
molecule capable of binding the AMP and/or AMP-like molecule is an antibody or
an
antibody fragment.
[00042] According to still further features in the described preferred
embodiments, the
antibody fragment is selected from the group consisting of a single-chain Fv,
an Fab, an
Fab', and an F(ab')2.
[00043] According to still further features in the described preferred
embodiments, the
AMP and/or AMP-like inolecule is selected from the group consisting of a
defensin, a
cathelicidin, a cationic peptide, a hydrophobic peptide, a human AMP and a
human AMP-
like molecule.
[00044] According to still further features in the described preferred
embodiments, the
AMP is any one of the cathelicidin and/or cathelicidin fragments listed below
as SEQ. ID
NOS. 1-59.
[00045] According to still further features in the described preferred
embodiments, the
cell and/or tissue is selected from the group consisting of an synovial cell
and/or tissue, a
nerve cell and/or tissue, a beta cell and/or tissue, an osteoblast, osteocyte
or osteoclast cell
and/or tissue and an endothelial cell and/or tissue.
1000461 According to still further features in the described preferred
embodiments, the
biological process is selected from the group consisting of growth,
differentiation, and
associated with an inflammatory disease or autoimmunity.
[00047] According to a further aspect of the present invention there is
provided a method
of treating a disease in a subject, such as a mammal, for example, a human
pateitn, in need
thereof, the method comprising providing to the subject a therapeutically
effective amount of
an antimicrobial peptide (AMP) and/or AMP-like molecule (and in particular a
cathelicidin,
active fragment thereof or active cathelicidin analog of the cathelicidin or
the fragment
thereof), thereby treating; the disease in the subject in need thereof.
[00048] According to further features in preferred embodiments of the
invention
described below, administering the AMP and/or AMP-like molecule to the subject
is
CA 02696833 2010-01-11
effected by exposing a location of the subject to a carrier which includes the
AMP and/or
AMP-like molecule at a concentration selected from a range of about 2
nanograms per
milliliter to about 10 micrograms per milliliter.
1000491 According ta still further features in the described preferred
embodiments,
administering the AMP and/or AMP-like molecule to the subject is effected via
a route
selected from the group consisting of the topical, intranasal, transdermal,
intradermal, oral,
buccal, intravenous, parenteral, rectal and inhalation route.
1000501 According to still further features in the described preferred
embodiments, the
subject is human.
1000511 According to yet a further aspect of the present invention there is
provided an
article of manufacture comprising packaging material and a pharmaceutical
composition, the
article of manufacture being identified for treatment of a disease being
associated with a
biological process in a cell and/or tissue, the biological process being
selected from the
group consisting of growth, differentiation, or inflammation associated with a
disease; the
pharmaceutical composition comprising a pharmaceutically acceptable carrier
and, as an
active ingredient, an antimicrobial peptide (AMP) and/or AMP-like molecule.
[000521 According to further features in preferred embodiments of the
invention
described below, the pharmaceutically acceptable carrier is selected so as to
enable
administration of the pharmaceutical composition via a route selected from the
group
consisting of the topical, intranasal, transdermal, intravenous, intradermal,
oral, buccal,
parenteral, rectal and inhalation route. The pharmaceutically acceptable
carrier may, for
example, be of the sort of carriers known in the art for the delivery of
therapeutic peptides.
The pharmaceutically acceptable carrier may, for example, be other than water
alone or other
than water altogether.
[00053] According to still further features in the described preferred
embodiments, the
pharmaceutical composition is formulated as a solution, suspension, emulsion
or gel.
[000541 According to still further features in the described preferred
embodiments, the
pharmaceutical composition is composed so as to enable exposure of a cell
and/or tissue of a
subject having the disease to the compound at a concentration selected from a
range of about
2 nanograms per milliliter to about 10 micrograms per milliliter.
1000551 According to a further aspect of the present invention there is
provided a method
of treating an autoimmune disease, chronic inflammatory disease, an
inflammatory disease, a
cancer, the method comprising of delivering the AMP or analog thereof, in
particular a
CA 02696833 2010-01-11
11
cathelicidin AMP to a human subject or mammal, thereby regulating the
biological process
in the subject.
1000561 According to still a further aspect of the present invention there is
provided a
method of regulating a biological process in a cell and/or tissue, the method
comprising
exposing the cell and/or tissue to an antimicrobial peptide (AMP) and/or AMP-
like
molecule, thereby regulating the biological process in the cell and/or tissue.
1000571 According to further features in preferred embodiments of the
invention
described below, exposing the cell and/or tissue to the AMP and/or AMP-like
molecule is
effected by providing the AMP and/or AMP-like molecule to a subject.
[00058] According to still further features in the described preferred
embodiments, the
providing to the subject the AMP and/or AMP-like molecule is effected by
administering the
AMP and/or AMP-like molecule to the subject and/or by expressing the AMP
and/or AMP-
like molecule in the subject.
[00059] According to still further features in the described preferred
embodiments, the
exposing the cell and/or tissue to the AMP and/or AMP-like molecule is
effected by
exposing the cell and/or tissue to the AMP and/or AMP-like molecule at a
concentration
selected from a range of about 2 nanograms per milliliter to about 10
micrograms per
milliliter or from about 1.0 micrograms per milliliter to about 30 micrograms
per milliliter.
[00060] According to still further features in the described preferred
embodiments, the
AMP and/or AMP-like rnolecule is selected from the group consisting of and LL-
37 or
analogs of LL-37 or other cathelicidins and cathelicidin fragments or analogs
as listed below.
[00061] According to still further features in the described preferred
embodiments, the
cell and/or tissue is derived from a human.
[00062] The present invention successfully addresses the shortcomings of the
presently
known configurations by providing: (i) a method of treating a disease which is
associated
with a biological process in a cell/tissue such as growth, differentiation,
inflammation,
metastasis and/or angiogenesis by using a compound which is capable of
regulating
levels/activity of an AM:P and/or an AMP-like molecule, of decreasing
olevels/activity of an
AMP and/or an AMP-like molecule; and/or by using an AMP and/or an AMP-like
molecule
or by increasing levels/activity of an AMP and/or an AMP-like molecule; (ii)
an article of
manufacture including such a compound and being labeled for treatment of such
a disease;
and (iii) a method of identifying such a compound.
[00063] Unless othervvise defined, all technical and scientific terms used
herein have the
CA 02696833 2010-01-11
12
same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs. Although methods and materials similar or equivalent to
those described
herein can be used in the; practice or testing of the present invention,
suitable methods and
materials are described below. All publications, patent applications, patents,
and other
references mentioned herein are incorporated by reference in their entirety.
In case of
conflict, the patent specification, including definitions, will control. In
addition, the
materials, methods, and examples are illustrative only and not intended to be
limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[00064] The invention is herein described, by way of example only, with
reference to the
accompanying drawings. With specific reference now to the drawings in detail,
it is stressed
that the particulars shown are by way of example and for purposes of
illustrative discussion
of the preferred embodirnents of the present invention only, and are presented
in the cause of
providing what is believed to be the most useful and readily understood
description of the
principles and conceptual aspects of the invention. In this regard, no attempt
is made to
show structural details of the invention in more detail than is necessary for
a fundamental
understanding of the invention, the description taken with the drawings making
apparent to
those skilled in the art how the several forms of the invention may be
embodied in practice.
[00065] FIG. 1 is a graph depicting incidence of arthritis in mouse model of
collagen
induced arthritis. Treatment using cathelicidin 34a.a. mCRAMP peptide
(experimental
group) at a concentratiori of 1.5mg/kg. Subsequently on days 2 and 4 post
immunization, the
dose was reduced to 1.0 mg/kg. Starting with day 7 and through day 72, a dose
of 0.8 mg/kg
was used. All treatments were performed 3 times per week, on a Monday,
Wednesday, and
Friday schedule, and the peptide or control vehicle was administered
intraperitoneally for
each treatment, rotating :injection areas. All mice were weighed at the
beginning of the
experiment in order to calculate dosage administered. At day 49, the mice were
again
weighed (average of 1.6 gm increase) and dosages were adjusted accordingly.
[00066] Starting on day 11, all mice were examined 3 times per week for
incidence and
severity of arthritis and each arthritic limb was assigned a numerical score
based on the
degree of inflammation observed according to the scale below.
[00067] Erythema or inild swelling to the tarsals, metatarsal, foot, digits,
ankylosis or
ankle joint in any one of the four legs marks incidence of arthritis. As can
be seen, incidence
rate is greater in the control group.
[00068] Further analysis of incidence rate of inflamed paws in all mice is
shown in FIG.
CA 02696833 2010-01-11
13
6.
[00069] The sequence of mCRAMP is: gllrkggekigeklkkigqkiknffqklvpqpeq.
[00070] FIG. 2 is a graph depicting Severity of Arthritis - The severity of
arthritis was
analyzed on the basis of degree of inflammation scored as follows and the
number of
affected limbs. 0 -No evidence of erythema and swelling, 1 -Erythema & mild
swelling
confined to the tarsals oir ankle joint, 2-Erythema & mild swelling extending
from the ankle
to the tarsals, 3-Erytherr.ia & moderate swelling extending from the ankle to
metatarsal joints,
4-Erythema & severe smielling encompass the ankle, foot, and digits, or
ankylosis of the
joint.
[00071] As seen in the FIG. 2, differences between the two groups were clearly
observed
when analyzed as mean Severity Score/Mouse. While these data are weighted
somewhat by
the differences in arthritis incidence, the differences in the severity appear
to be even greater
than the differences in iricidence. Data in FIG. 2 is from the same experiment
described in
FIG. 1.
[000721 FIG. 3 is a graph depicting the number of Arthritic Limbs/Mouse.
Similar to the
Severity/Mouse score as in FIG. 2, the number of Arthritic Limbs/Mouse was
also generally
lower in the experimental group, although the appearance of arthritic limbs
followed similar
kinetics as the control group, but at a delayed incidence.
[00073] Data in FIG. 3 is from the same experiment described in FIG. 1 and
FIG. 2.
[00074] FIG. 4 is a graph showing a follow-up of clinical score from the day
of incidence
of arthritis until day 19 after incidence. This follow-up is required since
each arthritic mouse
develops an incidence oi' inflammation on any one of four paws at a varying
number of days
since the beginning of the experiment. Therefore in order to determine the
significance level
between the groups it is necessary to run a follow-up test statistic. Data in
FIG. 4 is from the
same experiment described in FIG. 1 and FIG. 2.
[00075] FIG. 5 is a graph showing the sum of the severity index of clinical
score in all
mice of control versus treatment group during the time in days since
immunization.
[00076] A clear trend is shown of greater severity of disease in the control
group from day
27 onwards. Data in FIG. 5 is from the same experiment described in FIG. 1 and
FIG. 2.
[000771 FIG. 6 is a graph showing the incidence of arthritic paws in treatment
versus
control groups. Any one mouse may be included in this data up to four times
corresponding
to four different paws in any one mouse. A trend line is computed for each of
the treatment
and control groups using Microsoft excel technology. Data in FIG. 6 is from
the same
CA 02696833 2010-01-11
14
experiment described.in FIG. 1 and FIG. 2.
[00078] FIG. 7 shows a table listing the results of the mouse Experimental
Autoimmune
Encephalitis (EAE) model.
[00079] C57BL/6 (Bfi) mice were purchased from Harlan (Jerusalem, Israel).
Female, 9
week old mice were used in the experiment. The mice were housed in the
specific-pathogen
free (SPF) animal facility of the Hebrew University and all experiments were
approved by
the institutional animal care and use committee (IACUC).
1000801 MOGB35-55B peptide (MEVGWYRSPFSRVVHLYRNGK) 1.25mg/ml in PBS
was emulsified in complete Freund's adjuvant (CFA) supplemented with 400 g M.
tuberculosis (Mt) H37RA (Difco). Mice were immunized s.c. in the flank with
250 g
MOGB35-55B/CFA using a 25G needle. 200ng Pertussis Toxin (Sigma) was injected
i.v. at
the time of immunization and 48h later. EAE was scored on a scale of 0-6: 0,
no impairment;
1, limp tail; 2, limp tail and hind limb paresis; 3, >1 hind limb paralysis;
4, full hind limb and
hind body paralysis; 5, hind body paralysis and front limb paresis; 6, death.
Mice were
treated with the cathelicidin peptide supplied by Biosight Ltd. Karmiel,
Israel diluted in PBS,
vs. PBS as a control. Ca.thelicidin (GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ)
was diluted in sterile PBS and divided to aliquots kept at -20oC such that
each aliquot was
thawed once for use. Mice were treated by intraperitoneal (i.p.) injection of
roughly 200ul
volume (adjusted for weight) 3 times a week (Sun-Tues-Thurs) starting the day
of
immunization with MOG/CFA and through day 48. Clinical EAE scores were
evaluated
through day 60. Dosage of Cathelicidin injections (IP) was 2mg/Kg and 0.2
mg/Kg. There
were six mice in each group (total of 18 mice).
[00081] Of particular note is the fact that all the mice who developed EAE
eventually died
by day 50 while none of the mice in either of the treatment groups died even
by day 60.
[00082] There is a clear significant difference in average clinical score and
in Average
score at first peak of disease.
[00083] The lower dose of peptide, 0.2mg/Kg was more protective than the
higher
2mg/Kg dose.
[00084] FIG. 8 shows a graph of the average clinical score for each day after
immunization for the three groups in the EAE experiment as described in FIG.
7.
FIG. 9 shows photogaplis taken on day 60 of the three remaining healthy mice
in the control
group, all six remaining live mice in the low dose group, and two examples of
EAE affected
mice having paralyzed hind legs and tail.
CA 02696833 2010-01-11
FIG. 10 shows a Western blot analysis of 4 different scFv developed that bind
LL-37.
[00085] FIG. 11 shows the inhibitory effect of scFv on LL-37 in bacteria
killing assays. In
order to find out the concentration of LL37 at which 50% of the bacteria could
be killed
(called "IC50"). Basically the activity protocol follows the ability of the
antibody to block
the antimicrobial activity of LL-37. The bacteria used were Pseudomonas that
was isolated
from a wound. The growth medium was LB. LL-37 was added at a concentration of
100
microgram/ml (the final volume or the reaction is 50 microliter). Blocking
antibodies at 1 or
5 microliter of antibody (= 1:50 or 1:10 dilutions respectively. Low antibody
levels ensure a
non- specific effect. Concentration of bacteria was estimated by optical
density (OD)
reading at 490.
[00086] FIG. 12 shows the percentage change in glucose levels two hours
following an
LPS injection in treatment vs. control. LPS was administered to C57BL/6 mice
at 0.2 mg/kg.
Mice were bled approxitnately 2 h after LPS injection (T = 0). Changes in
glucose levels
were examined.
[00087] FIG. 13 shows the average weight gain in male DBA/1 mice about ten
weeks of
age being fed on a normal no-high fat diet for 21 days. Two groups of mice, 10
in each
group were weighed. The control increased in weight at an average of 0.0536
gms per day
whereas the treatment (cathelicidin mCRAMP at 0.8 ug/ml) increased at 0.0488
per day.
[00088] FIG. 14 shows a similar experiment as in Fig. 13 only that the mice
were given
0.4 ug/ml and were weighed 3 times a week while being fed a high fat diet of
60%Kcal.
[00089] FIG. 15 shows a list of mouse paws selected for analysis for bone
resortion,
deformation, immunohistology and osteoclast analysis and counting. Mouse paws
were
obtained from experimetit in example 1.
[00090] FIG. 16 to FIG. 19 shows the histology analysis showing a beneficial
effect of
cathelicidin on bone witli reduced bone resorption in treatment group even the
inflamed
treatment group had less osteoclast than the non-inflamed control. Staining
was done with
H&E and for tartrate-resistant acid phosphatase (TRAP).
[00091] FIG. 20 shows the effect of human beta defensin 2 given for a duration
of 7
weeks on human psoriatic skin. Inhibition by dominant negative peptide
analogues or
fragments is suggested as a viable mode of treatment for this disease
[00092] FIG. 21 shows a histogram depicting significant BETATC beta cell line
proliferation brought about by cathelicidin LL-37. Murine beta cell line were
treated for 3.5
days with LL-37 at 2 mic,rogram/ml (red/dark bars) and compared to PBS control
(red/dark
CA 02696833 2010-01-11
16
bars). Cell proliferation was estimated by measuring [3(H)]-thymidine
incorporation and
was expressed as percent of control untreated cells. A representative
experiment is shown.
DETAILED DESCRIP'TION
[00093] The present invention provides methods of using compounds capable of
increasing activities/levels of antimicrobial peptides (AMP)/antimicrobial
peptide-like
molecules (AMLs) and/or of decreasing activities/levels of antimicrobial
peptides,
(AMP)/antimicrobial peptide-like molecules (AMLs) and/or of using AMPs/AMLs or
analogs or fragments thereof for regulating in cells/tissues biological
processes such as
growth, differentiation, growth/differentiation balance, of methods of using
such molecules
for treating diseases associated with such biological processes and/or which
are amenable to
treatment via regulation of such biological processes; for treating
autoimmunity,
inflammation, metastasis and angiogenesis; of articles of manufacture which
include such
molecules and which are labeled as being for use in treating such diseases;
and of methods of
identifying such compounds capable of regulating or increasing or decreasing
activities/levels of AMPs/AMLs and/or of identifying such AMPs/AMLs.
Specifically, the
present invention can be used to optimally treat a vast range of diseases
associated with such
biological processes, including inflammatory diseases/diseases associated with
cellular
proliferation/differentiat:ion imbalance, autoimmune and inflammatory diseases
such as
multiple sclerosis, arthritis, obesity, insulin resistance, osteoporosis,
periodontitis, and other
diseases associated with autoimmunity and/or cellular
proliferation/differentiation
imbalance.
1000941 Specifically the AMP involved is endogenous cathelicidin or its
analogs or
fragments thereof.
1000951 The principles and operation of the present invention may be better
understood
with reference to the drawings and accompanying descriptions.
[00096] Before explaining at least one embodiment of the invention in detail,
it is to be
understood that the inveiition is not limited in its application to the
details set forth in the
following description or exemplified by the Examples. The invention is capable
of other
embodiments or of being practiced or carried out in various ways. Also, it is
to be
understood that the phraseology and terminology employed herein is for the
purpose of
description and should n-ot be regarded as limiting.
[00097] Diseases which are associated with autoimmunity, inflammation, and
dysregulated cell/tissue proliferation/differentiation, dysregulated
cell/tissue
CA 02696833 2010-01-11
17
proliferation/differentiation balance, include a multitude of diseases which
are of great
medical and/or economic impact and for which no satisfactory treatment methods
are
available. While conceiving the present invention, the present inventors have
hypothesized
that AMPs/AMLs are involved in the pathogenesis of such diseases, and/or and
hence that
methods of regulating or increasing or decreasing activities/levels and/or
administering such
molecules and in particular, cathelicidins or analogs or fragments of
cathelicidin could be
used for treating such diseases.
[00098] The prior art approach relating to such methods involves the invention
of the
current inventors in WO 2004-056307 incorporated by reference herein.
[00099] The prior art approach, however, relates to autoimmune diseases in
general and
provides in-vivo data on psoriasis showing that cathelicidin is indeed an
immune regulator in
psoriasis. The present irivention therefore, a continuation in part to the
previous invention of
the current inventors, shows in-vivo data for various specific diseases
associated with
autoimmunity, inflammation including low grade inflammation found in metabolic
diseases
as well as bone cell diffe;rentiation/proliferation leading to bone
degradation.
[000100] While reducing the present invention to practice, it was also
uncovered that
cathelicidin and therefore AMPs could be used to significantly regulate growth
of cultured
mouse beta-cells.
[000101] Hence, in sharp contrast to prior art techniques, the method
according to the
present invention enables use of compounds capable of regulating by either
increasing or
decreasing levels/activity of cathelicidin or of other AMPs/AMLs, and/or the
use of such
cathelicidins or analogs or fragments thereof or other AMPs/AMLs for
regulating biological
processes such as growth, differentiation, inflammation, and for the treatment
of numerous
specific diseases such as for example type 1 diabetes and other diseases such
as those which
are associated with inflammation, dysregulated cell
proliferation/differentiation, chronic
inflammatory diseases aiid autoimmune diseases.
[000102] Thus, the present invention provides a method of regulating a
biological process
in a cell and/or tissue associated with a disease. The method is effected by
exposing the cell
and/or tissue to: an AMF' and in particular a cathelicidin peptide, an analog
of a cathelicidin
peptide, an analog of a cathelicidin peptide that has been designed to be more
stable in-vivo
so as not to break down into pro-inflammatory fragments of itself, a
cathelicidin analog
functioning as a dominant negative or a cathelicidin peptide that competes on
binding to
cognate receptors with an AMP without inducing disease, a compound being
capable of
CA 02696833 2010-01-11
18
decreasing or increasing an activity and/or level of an antimicrobial peptide
(AMP) and/or
AMP-like molecule (AML).
[000103] The method can be used to regulate in a cell/tissue a biological
process such as
growth, differentiation, autoimmunity and inflammation. By virtue of enabling
regulation of
such a biological process in a cell/tissue, the method can be used for
treating a disease which
is associated with such a. biological process, and can be used for identifying
the regulator, as
described in further detail herein below. Diseases associated with such
biological processes
include, for example, autoimmune diseases, diseases associated with
dysregulated cell/tissue
growth/proliferation balance, wound-associated diseases, and tumors.
[000104] As used hereiin, the term "regulator" refers to the compound which is
capable of
decreasing an activity and/or level of an AMP/A.ML, increasing an activity
and/or level of an
A.MP/A.ML, and/or to an AMP which is used for practicing any aspect of the
present
invention.
10001051 As used herein, the phrases "the compound", "compound of the present
invention", and "AMP/AML inhibitor" interchangeably refer to the compound
which is
capable of regulating, decreasing or increasing an activity/level of an
AMP/A.ML.
[000106] Any of various types of AMP/AML or AMP/AML inhibitors may be employed
according to the teachings of the present invention for regulating the
biological process,
depending on the application and purpose.
[000107] As used herein, the term "AMP" includes any cathelicidin, and/or
including any
naturally occurring variant of such a molecule, such as a natural
mutant/polymorphic
variant/allele of such a molecule, or any synthetic variant of such a
molecule.
[000108] As used herein, the term "AML" includes any molecule having a
biological
activity which is substantially similar to that of a cathelicidin, includes
any molecule which
substantially promotes the biological activity of a cathelicidin, and/or
includes any molecule
which is substantially structurally homologous to a cathelicidin. In such
case, homology
implied may, for example, vary between 50% to 60%, 60% to 70%, 70% to 80%, 80%
to
90%, 90% to 99%, 90%-100%, or at least 95%.
[000109] The method may be effected using a single regulator of the present
invention, or
using any combination of multiple regulators of the present invention.
[000110] The AMP/AML inhibitor may be: a molecule capable of binding the
AMP/AML;
an enzyme capable of cleaving the A.MP/A.ML; an siRNA molecule capable of
inducing
degradation of an mRNA, encoding the AMP/AML; a DNAzyme capable of cleaving an
CA 02696833 2010-01-11
19
mRNA or DNA encodirig the AMP/AML; an antisense polynucleotide capable of
hybridizing with an mRNA encoding the AMP/AML; a ribozyme capable of cleaving
an
mRNA encoding the AT/IP/AML; a non-functional analog of at least a functional
portion of
the AMP/AML; a molecule capable of inhibiting activation or ligand binding of
the
AMP/AML; and a triplex-forming oligonucleotide capable of hybridizing with a
DNA
encoding the AMP/AMIL.
[000111] Ample guidance for obtaining and utilizing such AMP/AML inhibitors is
provided herein below and in the literature of the art (for example, refer to
U.S. Patent
Application Pub. No. 20030044907 which is incorporated herein by reference).
[000112] The AMP/AML inhibitor may be any small molecule, AMP/AML dominant
negative, or polypeptide that competes with the AMPs for cognate cell
receptors without
inducing disease. For example, the AMP/AML inhibitor may be a topological
analog of an
AMP/AML that has been engineered to remain anti microbial yet lose its
chemoattracting
ability. Engineering of (lisulfide bridges to dissect antimicrobial and
chemotactic activities
of AMPs/AMLs such as human beta-defensin-3 can be performed as previously
described
(Wu Z. et al., 2003. Proc. Natl. Acad. Sci. U. S. A. 100:8880-5).
10001131 The term "dominant negative mutant" as used herein refers to a
polypeptide or a
nucleic acid coding region sequence which has been changed with regard to at
least one
position in the sequence., relative to the corresponding wild type native
version at a position
which changes an amino acid residue position at an active site required for
biological and/or
pharmacological activity of the native peptide. Accordingly, dominant negative
mutants or
fragments of the cathelicidin peptide as listed below and contemplated herein
include, but
are not limited to, polypeptide species which manifest any change
(substitution and/or
deletion) with regard to at least one amino acid of the AMP or cathelicidin
peptide.
Dominant negative mutant embodiments of the invention are moreover nucleic
acids which
encode peptides, as well as the peptides themselves, which comprise fragments
of the AMP
or more specifically of the cathelicidin hCAP-18 and are listed as in (SEQ. ID
NOS: 1-59).
[000114] The AMP/AML inhibitor may be a synthetic antibody mimic in which
multiple
peptide loops are attached to a molecular scaffold (described in U.S. Patent
No. 5,770,380).
[000115] Such an AMP/AML mimic can be obtained, for example, by molecule
imprinting. This technique may be performed by preparing a polymer by cross-
linking a
monomer around a "template molecule" (the AMP/AML). This template molecule is
removed after the polymerization of the monomer and its size, shape and
chemical functions
CA 02696833 2010-01-11
are recorded in the polymer. The sites of the removed template molecule are
named "imprint
sites". These sites allow the recognition of the template molecule or close
structural
molecules. Molecularly imprinted polymers can serve as artificial binding
mimics as do
natural antibodies.
[000116] The molecule capable of inhibiting activation or ligand binding of
the AMP/AML
may advantageously inhibit binding of a receptor expressed on cell, such as a
leukocyte,
which binds the AMP/AML to inhibit a biological process mediated by binding of
the
AMP/AML to the recepitor. Examples of such AMPs/AMLs and cognate receptors
thereof
are shown in Table 1.
CA 02696833 2010-01-11
21
Table 1
AMPs/AMLs and cognate cell receptors, and diseases associated with interaction
therebetween
AMP/AML Receptor Receptor-expressing Disease
cells
Psoriasis, rheumatoid
arthritis (RA), atopic
dermatitis, contact
Monocyte, dendritic dermatitis, chronic
cell, T cell, neutrophils, hepatitis, inflammatory
LL-37 EGFR, F'PRLI eosinophils, leukocytes, bowel disease (1BD),
epithelial cell, allergy, B cell
endothelial cells malignancies,
hepatocellular carcinoma,
pancreatic
adenocarcinoma and
others
beta-defensin- Tol11-like Dendritic cells
2 receptor- 4
beta-defensin- Toll-like
2 receptor-2
Psoriasis, RA, atopic
dermatitis, contact
beta defensin- CC-chemokine dermatitis, chronic
1 receptor-6 Hematopoietic cells, hepatitis, IBD, allergy, B
beta defensin- (CCR6) dendritic cells, cell malignancies,
2 hepatocellular carcinoma,
pancreatic
adenocarcinoma and more
defensin-5 Intestinal mucosa Crohn's disease
L1 and calcitonin IBD, allergy,
adrenomedulli receptor-like gastric epithelial cells hepatocellular carcinoma,
n receptor ( CRLR) and more
10001171 Of particular note is that cathelicidin antimicrobial peptides block
dendritic cell
TLR4 activation (J Immunol. 2007 Feb 1;178(3):1829-34) and therefore
cathelicidins would
act as inhibitors to beta-defensin activation of TLR4.
[000118] Further examples of receptors of AMPs/AMLs such as chemokines, the
cells in
which such receptors are expressed, and the diseases in which the interaction
between such
AMPs/AMLs and such receptors are involved are provided in D'Ambrosio et al.,
2003. J.
Immunol. Methods 273 3 - 13.
[000119] The activity of LL-37 (Weiner, DJ. et al., 2003. Am.J.Respir.Cell
Mol.Biol.
CA 02696833 2010-01-11
22
28:738-745), defensin-3, lactoferrin and IL-8 (Perks, B. et al., 2000.
Am.J.Respir.Crit Care
Med. 162:1767-1772) is inhibited by F-actin, further inhibitors therefore the
AMP/AML
inhibitor may be F-actin . F-actin forms bundles in the presence of the
polycationic
interleukin IL-8, therefore F-actin is an inhibitor of downstream elements of
the ligand-
receptor connectivity of both LL-37 and interleukin IL-8. LL-37 and defensin-3
are
inhibited by gelsolin, therefore the AMP/AML inhibitor may be gelsolin.
Serpins and their
analogs or fragments are inactivators of AMP by formation of complexes with
AMP
(Panyutich, AV. et al., 1995. Am.J.Respir.Cell Mol.Biol. 12:351-357; alpha-1
antichymotrypsin, the antimicrobial proteins alpha PI, SLPI and elafin are
serpins that form
complexes with other AMPs) thereby reducing specific types of inflammation
(Hiemstra, PS,
2002. Biochem.Soc.Trans. 30:116-120), therefore the AMP/AML inhibitor may be
serpins
and their analogs or fragments. The AMP/AML inhibitor may be SIC, a secreted
protein of
streptococcus pyogenes that inactivates antibacterial peptides.
[000120] Other AMP inhibitors or specifically cathelicidin inhibitors include,
Alpha 2-
Macroglobulin-Proteinase Complexes (Patrik Nyberg et al THE JOURNAL OF
BIOLOGICAL CHEMISTRY Vol. 279, No. 51, Issue of December 17, pp. 52820-52823,
2004), aureolysin production by S. aureus and taphylococcus and aureus-Derived
Proteinases (Antimicrobial agents and chemotherapy, Dec. 2004, p. 4673-4679),
Elastolytic Cathepsins (Journal of Immunology, 2003, 170: 931-937), SufA - a
novel
subtilisin-like serine proteinase of Finegoldia magna (Microbiology (2007),
153, 4208-
4218).
[000121] Preferably, the molecule capable of binding the AMP/AML is an
antibody or an
antibody fragment.
[000122] Alternately, the molecule capable of binding the AMP/AML may be any
of
various type of molecule, including non-immunoglobulin peptides and
polypeptides,
[000123] Preferably, the antibody fragment is selected from the group
consisting of a
single-chain Fv, an Fab, an Fab', and an F(ab')2.
[000124] As used herein, the term "antibody" refers to a substantially intact
antibody
molecule. The antibody may, for example, be an IgG, IgA or IgM. Antibodies
used
according to the invention may be monoclonal antibodies or polyclonal
antibodies. The
antibodies may, for exaniple, be non-human, human or humanized antibodies.
[000125] As used herein, the phrase "antibody fragment" refers to a functional
fragment of
an antibody that is capable of binding to an AMP/AML.
CA 02696833 2010-01-11
23
[000126] Suitable antilbody fragments for practicing the present invention
include a
complementarity-determining region (CDR) of an immunoglobulin light chain
(referred to
herein as "light chain"), a CDR of an immunoglobulin heavy chain (referred to
herein as
"heavy chain"), a variable region of a light chain, a variable region of a
heavy chain, a light
chain, a heavy chain, an Fd fragment, and antibody fragments comprising
essentially whole
variable regions of both light and heavy chains such as an Fv, a single chain
Fv, an Fab, an
Fab', and an F(ab')z.
[000127] Functional antibody fragments comprising whole or essentially whole
variable
regions of both light and heavy chains are defined as follows:
(i) Fv, defined as a genetically engineered fragment consisting of the
variable
region of the light chain and the variable region of the heavy chain expressed
as two chains;
(ii) single chain Fv ("scFv"), a genetically engineered single chain molecule
including the variable region of the light chain and the variable region of
the heavy chain,
linked by a suitable polypeptide linker.
(iii) Fab, a fragment of an antibody molecule containing a monovalent antigen-
binding portion of an antibody molecule which can be obtained by treating
whole antibody
with the enzyme papain to yield the intact light chain and the Fd fragment of
the heavy chain
which consists of the vai-iable and CH1 domains thereof;
(iv) Fab', a fragment of an antibody molecule containing a monovalent antigen-
binding portion of an antibody molecule which can be obtained by treating
whole antibody
with the enzyme pepsin, followed by reduction (two Fab' fragments are obtained
per
antibody molecule); and
(v) F(ab' )2, a fragment of an antibody molecule containing a monovalent
antigen-
binding portion of an antibody molecule which can be obtained by treating
whole antibody
with the enzyme pepsin (i.e., a dimer of Fab' fragments held together by two
disulfide
bonds).
[0001281 Methods of generating antibodies (i.e., monoclonal and polyclonal)
are well
known in the art. Antibodies may be generated via any one of several methods
known in the
art, which methods can employ induction of in-vivo production of antibody
molecules,
screening of immunoglolbulin libraries (Orlandi D.R. et al., 1989. Proc. Natl.
Acad. Sci. U. S.
A. 86:3833-3837; Winte:r G. et al., 1991. Nature 349:293-299) or generation of
monoclonal
antibody molecules by continuous cell lines in culture. These include, but are
not limited to,
the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-
Barr virus
CA 02696833 2010-01-11
24
(EBV)-hybridoma technique (Kohler G. et al., 1975. Nature 256:495-497; Kozbor
D. et al.,
1985. J. Immunol. Methods 81:31-42; Cote RJ. et al., 1983. Proc. Natl. Acad.
Sci. U. S. A.
80:2026-2030; Cole SP. et al., 1984. Mol. Cell. Biol. 62:109-120).
[000129] In cases whe:re target antigens are too small to elicit an adequate
immunogenic
response when generatirig antibodies in-vivo, such antigens (haptens) can be
coupled to
antigenically neutral carriers such as keyhole limpet hemocyanin (KLH) or
serum albumin
[e.g., bovine serum albumin (BSA)] carriers (see, for example, US. Pat. Nos.
5,189,178 and
5,239,078]. Coupling a hapten to a carrier can be effected using methods well
known in the
art. For example, direct coupling to amino groups can be effected and
optionally followed
by reduction of the imino linkage formed. Alternatively, the carrier can be
coupled using
condensing agents such as dicyclohexyl carbodiimide or other carbodiimide
dehydrating
agents. Linker compourids can also be used to effect the coupling; both
homobifunctional
and heterobifunctional linkers are available from Pierce Chemical Company,
Rockford, Ill.
The resulting immunogenic complex can then be injected into suitable mammalian
subjects
such as mice, rabbits, and the like. Suitable protocols involve repeated
injection of the
immunogen in the preserice of adjuvants according to a schedule which boosts
production of
antibodies in the serum. The titers of the immune serum can readily be
measured using
immunoassay procedures which are well known in the art.
[000130] The antisera obtained can be used directly or monoclonal antibodies
may be
obtained as described hereinabove.
[000131] Antibody fragments can be obtained using methods well known in the
art. [(see,
for example, Harlow ancl Lane, "Antibodies: A Laboratory Manual", Cold Spring
Harbor
Laboratory, New York, (1988)]. For example, antibody fragments according to
the present
invention can be prepared by proteolytic hydrolysis of the antibody or by
expression in E.
coli or mammalian cells (e.g., Chinese hamster ovary cell culture or other
protein expression
systems) of DNA encoding the fragment.
[000132] Alternatively, antibody fragments can be obtained by pepsin or papain
digestion
of whole antibodies by conventional methods. As described hereinabove, an
(Fab')2
antibody fragments can be produced by enzyrnatic cleavage of antibodies with
pepsin to
provide a 5S fragment. 'This fragment can be further cleaved using a thiol
reducing agent,
and optionally a blocking group for the sulfhydryl groups resulting from
cleavage of
disulfide linkages to produce 3.5S Fab' monovalent fragments. Alternatively,
enzymatic
cleavage using pepsin produces two monovalent Fab' fragments and an Fc
fragment directly.
CA 02696833 2010-01-11
Ample guidance for practicing such methods is provided in the literature of
the art (for
example, refer to: Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647; Porter,
RR., 1959.
Biochem. J. 73:119-126). Other methods of cleaving antibodies, such as
separation of heavy
chains to form monovalent light-heavy chain fragments, further cleavage of
fragments, or
other enzymatic, chemical, or genetic techniques may also be used, so long as
the fragments
bind to the antigen that i,s recognized by the intact antibody.
[0001331 As described. hereinabove, an Fv is composed of paired heavy chain
variable and
light chain variable domains. This association may be noncovalent (see, for
example, Inbar
et al., 1972. Proc. Natl. Acad. Sci. USA. 69:2659-62). Alternatively, as
described
hereinabove, the variable domains can be linked to generate a single chain Fv
by an
intermolecular disulfide bond, or alternately, such chains may be cross-linked
by chemicals
such as glutaraldehyde.
[0001341 Preferably, the Fv is a single chain Fv.
[0001351 Single chain Fv's are prepared by constructing a structural gene
comprising DNA
sequences encoding the heavy chain variable and light chain variable domains
connected by
an oligonucleotide encoding a peptide linker. The structural gene is inserted
into an
expression vector, whicli is subsequently introduced into a host cell such as
E. coli. The
recombinant host cells synthesize a single polypeptide chain with a linker
peptide bridging
the two variable domains. Ample guidance for producing single chain Fv's is
provided in
the literature of the art (for example, refer to: Whitlow and Filpula, 1991.
Methods 2:97-105;
Bird et al., 1988. Science 242:423-426; Pack et al., 1993. Bio/Technology
11:1271-77; and
Ladner et al., U.S. Pat. No. 4,946,778).
10001361 Isolated complementarity determining region peptides can be obtained
by
constructing genes encociing the complementarity determining region of an
antibody of
interest. Such genes may be prepared, for example, by RT-PCR of mRNA of an
antibody-
producing cell. Ample guidance for practicing such methods is provided in the
literature of
the art (for example, refer to Larrick and Fry, 1991. Methods 2:106-10).
[0001371 It will be appreciated that for human therapy or diagnostics,
humanized
antibodies are preferably used. Humanized forms of non human (e.g., murine)
antibodies are
genetically engineered chimeric antibodies or antibody fragments having-
preferably
minimal-portions derived from non human antibodies. Humanized antibodies
include
antibodies in which complementary determining regions of a human antibody
(recipient
antibody) are replaced by residues from a complementarity determining region
of a non
CA 02696833 2010-01-11
26
human species (donor antibody) such as mouse, rat or rabbit having the desired
functionality.
In some instances, Fv framework residues of the human antibody are replaced by
corresponding non human residues. Humanized antibodies may also comprise
residues
which are found neither in the recipient antibody nor in the imported
complementarity
determining region or framework sequences. In general, the humanized antibody
will
comprise substantially all of at least one, and typically two, variable
domains, in which all or
substantially all of the complementarity determining regions correspond to
those of a non
human antibody and all, or substantially all, of the framework regions
correspond to those of
a relevant human consensus sequence. Humanized antibodies optimally also
include at least
a portion of an antibody constant region, such as an Fc region, typically
derived from a
human antibody (see, for example, Jones et al., 1986. Nature 321:522-525;
Riechmann et al.,
1988. Nature 332:323-3:29; and Presta, 1992. Curr. Op. Struct. Biol. 2:593-
596).
[000138] Methods for humanizing non human antibodies are well known in the
art.
Generally, a humanized antibody has one or more amino acid residues introduced
into it
from a source which is r.ion human. These non human amino acid residues are
often referred
to as imported residues which are typically taken from an imported variable
domain.
Humanization can be essentially performed as described (see, for example:
Jones et al.,
1986. Nature 321:522-525; Riechmann et al., 1988. Nature 332:323-327;
Verhoeyen et al.,
1988. Science 239:1534-1536; U.S. Pat. No. 4,816,567) by substituting human
complementarity determining regions with corresponding rodent complementarity
determining regions. Accordingly, such humanized antibodies are chimeric
antibodies,
wherein substantially less than an intact human variable domain has been
substituted by the
corresponding sequence from a non human species. In practice, humanized
antibodies may
be typically human antibodies in which some complementarity determining region
residues
and possibly some frame;work residues are substituted by residues from
analogous sites in
rodent antibodies.
[000139] Thus, for example, antibodies used in the treatments of the invention
may be
humanized antibodies against LL-37 or against an epitope of hCAP- 18.
[000140] Human antibodies can also be produced using various techniques known
in the
art, including phage display libraries [see, for example, Hoogenboom and
Winter, 1991. J.
Mol. Biol. 227:38 1; Marks et al., 1991. J. Mol. Biol. 222:58 1; Cole et al.,
"Monoclonal
Antibodies and Cancer T'herapy", Alan R. Liss, pp. 77 (1985); Boerner et al.,
1991. J.
Immunol. 147:86-95). Humanized antibodies can also be made by introducing
sequences
CA 02696833 2010-01-11
27
encoding human immun,oglobulin loci into transgenic animals, e.g., into mice
in which the
endogenous immunoglobulin genes have been partially or completely inactivated.
Upon
antigenic challenge, hur.nan antibody production is observed in such animals
which closely
resembles that seen in humans in all respects, including gene rearrangement,
chain assembly,
and antibody repertoire. Ample guidance for practicing such an approach is
provided in the
literature of the art (for example, refer to: U.S. Pat. Nos. 5,545,807,
5,545,806, 5,569,825,
5,625,126, 5,633,425, and 5,661,016; Marks et al., 1992. Bio/Technology 10:779-
783;
Lonberg et al., 1994. Nature 368:856-859; Morrison, 1994. Nature 368:812-13;
Fishwild et
al., 1996. Nature Biotechnology 14:845-51; Neuberger, 1996. Nature
Biotechnology 14:826;
Lonberg and Huszar, 1995. Intern. Rev. Immunol. 13:65-93; Kellermann, SA. et
al., 2002.
Curr. Op. Biotechnol. 13:593-597).
[000141] Once antiboclies are obtained, they may be tested for activity, for
example via
ELISA.
[000142] Suitable antibodies may in many cases be purchased ready for use from
commercial suppliers, such as Pharmingen, Dako, Becton-Dickinson, Sigma-
Aldrich, and
the like. Algae can be used to industrially mass-produce antibodies (Proc Natl
Acad Sci U S
A. 2003, 100:438-42).
[000143] As described hereinabove, the AMP/AML inhibitor may be a small
interfering
RNA (siRNA) molecule. RNA interference is a two step process. the first step,
which is
termed as the initiation step, input dsRNA is digested into 21-23 nucleotide
(nt) small
interfering RNAs (siRNA), probably by the action of Dicer, a member of the
RNase III
family of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA
(introduced
directly or via a transgerie or a virus) in an ATP-dependent manner.
Successive cleavage
events degrade the RNA to 19-21 bp duplexes (siRNA), each with 2-nucleotide 3'
overhangs
[Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002);
and
Bernstein Nature 409:363-366 (2001)].
[000144] In the effector step, the siRNA duplexes bind to a nuclease complex
to from the
RNA-induced silencing complex (RISC). An ATP-dependent unwinding of the siRNA
duplex is required for activation of the RISC. The active RISC then targets
the homologous
transcript by base pairing interactions and cleaves the mRNA into 12
nucleotide fragments
from the 3' terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics
and
Development 12:225-23.2 (2002); Hammond et al. (2001) Nat. Rev. Gen. 2:110-119
(2001);
and Sharp Genes. Dev. 15:485-90 (2001)]. Although the mechanism of cleavage is
still to be
CA 02696833 2010-01-11
28
elucidated, research indicates that each RISC contains a single siRNA and an
RNase
[Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)].
[000145] Because of the remarkable potency of RNAi, an amplification step
within the
RNAi pathway has been. suggested. Amplification could occur by copying of the
input
dsRNAs which would generate more siRNAs, or by replication of the siRNAs
formed.
Alternatively or additiorially, amplification could be effected by multiple
turnover events of
the RISC [Hammond et al. Nat. Rev. Gen. 2:110-119 (2001), Sharp Genes. Dev.
15:485-90
(2001); Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232
(2002)].
For more information on RNAi see the following reviews Tuschl ChemBiochem.
2:239-245
(2001); Cullen Nat. Immunol. 3:597-599 (2002); and Brantl Biochem. Biophys.
Act.
1575:15-25 (2002).
[000146] Synthesis of RNAi molecules suitable for use with the present
invention can be
effected as follows. First, the AMP/AML mRNA sequence is scanned downstream of
the
AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the
3'
adjacent 19 nucleotides -is recorded as potential siRNA target sites.
Preferably, siRNA target
sites are selected from the open reading frame, as untranslated regions (UTRs)
are richer in
regulatory protein binding sites. UTR-binding proteins and/or translation
initiation
complexes may interfere: with binding of the siRNA endonuclease complex
[Tuschl
ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed
at
untranslated regions may also be effective, as demonstrated for GAPDH wherein
siRNA
directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and
completely abolished protein level (www.ambion.com/techlib/tn/91/912.html).
[000147] As used hereiin the term "about" refers to plus or minus 10%.
Wherever the term
"about" occurs, it shoulcl be understood that the invention also provides
corresponding
embodiments wherein the degree of variation is plus or minus 5%.
[000148] Second, potential target sites are compared to an appropriate genomic
database
(e.g., human, mouse, rat etc.) using any sequence alignment software, such as
the BLAST
software available from the NCBI server (www.ncbi.nlm.nih.gov/BLAST/).
Putative target
sites which exhibit significant homology to other coding sequences are
filtered out.
10001491 Qualifying target sequences are selected as template for siRNA
synthesis.
Preferred sequences are ithose including low G/C content as these have proven
to be more
effective in mediating gene silencing as compared to those with G/C content
higher than 55
%. Several target sites are preferably selected along the length of the target
gene for
CA 02696833 2010-01-11
29
evaluation. For better evaluation of the selected siRNAs, a negative control
is preferably
used in conjunction. Negative control siRNA preferably include the same
nucleotide
composition as the siRlNfAs but lack significant homology to the genome. Thus,
a scrambled
nucleotide sequence of the siRNA is preferably used, provided it does not
display any
significant homology to any other gene.
[000150] As described hereinabove, the AMP/AML inhibitor may be a DNAzyme
molecule capable of specifically cleaving an mRNA transcript or DNA sequence
of the
AMP/AML. DNAzymes are single-stranded polynucleotides which are capable of
cleaving
both single and double stranded target sequences (Breaker, R.R. and Joyce, G.
Chemistry
and Biology 1995;2:655; Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA
1997;943:4262). A general model (the "10-23" model) for the DNAzyme has been
proposed. "10-23" DNAzymes have a catalytic domain of 15 deoxyribonucleotides,
flanked
by two substrate-recognition domains of seven to nine deoxyribonucleotides
each. This type
of DNAzyme can effectiively cleave its substrate RNA at purine:pyrimidine
junctions
(Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 199; for rev of
DNAzymes see
Khachigian, LM [Curr C>pin Mol Ther 4:119-21 (2002)].
[000151] Examples of construction and amplification of synthetic, engineered
DNAzymes
recognizing single and double-stranded target cleavage sites have been
disclosed in U.S. Pat.
No. 6,326,174 to Joyce et al.
[000152] As described hereinabove, the AMP/AML inhibitor may be an antisense
polynucleotide capable of specifically hybridizing with an mRNA transcript
encoding the
AMP/AML.
[000153] Design of antisense molecules which can be used to efficiently
decrease
levels/activity of an AMP/AML must be effected while considering two aspects
important to
the antisense approach. The first aspect is delivery of the oligonucleotide
into the cytoplasm
of the appropriate cells, while the second aspect is design of an
oligonucleotide which
specifically binds the designated mRNA within cells in a way which inhibits
translation
thereof.
10001541 The prior art teaches of a number of delivery strategies which can be
used to
efficiently deliver oligonucleotides into a wide variety of cell types [see,
for example, Luft J
Mol Med 76: 75-6 (1998); Kronenwett et al. Blood 91: 852-62 (1998); Rajur et
al. Bioconjug
Chem 8: 935-40 (1997); Lavigne et al. Biochem Biophys Res Commun 237: 566-71
(1997)
and Aoki et al. (1997) Biiochem Biophys Res Commun 231: 540-5 (1997)].
CA 02696833 2010-01-11
10001551 In addition, algorithms for identifying those sequences with the
highest predicted
binding affinity for theii- target mRNA based on a thermodynamic cycle that
accounts for the
energetics of structural alterations in both the target mRNA and the
oligonucleotide are also
available [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9 (1999)].
10001561 Such algorithms have been successfully used to implement an antisense
approach
in cells. For example, the algorithm developed by Walton et al. enabled
scientists to
successfully design antisense oligonucleotides for rabbit beta-globin (RBG)
and mouse
tumor necrosis factor-alpha (TNF alpha) transcripts. The same research group
has more
recently reported that the antisense activity of rationally selected
oligonucleotides against
three model target mRNAs (human lactate dehydrogenase A and B and rat gp130)
in cell
culture as evaluated by a kinetic PCR technique proved effective in almost all
cases,
including tests against three different targets in two cell types with
phosphodiester and
phosphorothioate oligonucleotide chemistries.
[000157] In addition, several approaches for designing and predicting
efficiency of specific
oligonucleotides using an in vitro system were also published (Matveeva et
al., Nature
Biotechnology 16: 1374 - 1375 (1998)].
[000158] Several clinical trials have demonstrated safety, feasibility and
activity of
antisense oligonucleotides. For example, antisense oligonucleotides suitable
for the
treatment of cancer have been successfully used [Holmund et al., Curr Opin Mol
Ther 1:372-
85 (1999)], while treatment of hematological malignancies via antisense
oligonucleotides
targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been
shown to be
tolerated by patients [Gerwitz Curr Opin Mol Ther 1:297-306 (1999)].
10001591 More recently, antisense-mediated suppression of human heparanase
gene
expression has been reported to inhibit pleural dissemination of human cancer
cells in a
mouse model [Uno et al., Cancer Res 61:7855-60 (2001)].
10001601 Thus, the current consensus is that recent developments in the field
of antisense
technology which, as described above, have led to the generation of highly
accurate
antisense design algorithms and a wide variety of oligonucleotide delivery
systems, enable
an ordinarily skilled artisan to design and implement antisense approaches
suitable for
downregulating expression of known sequences without having to resort to undue
trial and
error experimentation.
[000161] As described hereinabove, the AMP/AML inhibitor may be a ribozyme
molecule
capable of specifically cleaving an mRNA transcript encoding the AMP/AML.
Ribozymes
CA 02696833 2010-01-11
31
are being increasingly used for the sequence-specific inhibition of gene
expression by the
cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin
Biotechnol.
9:486-96 (1998)]. The possibility of designing ribozymes to cleave any
specific target RNA
has rendered them valuable tools in both basic research and therapeutic
applications. In the
therapeutics area, ribozymes have been exploited to target viral RNAs in
infectious diseases,
dominant oncogenes in cancers and specific somatic mutations in genetic
disorders [Welch
et al., Clin Diagn Virol. 10:163-71 (1998)]. Most notably, several ribozyme
gene therapy
protocols for HIV patients are already in Phase 1 trials. More recently,
ribozymes have been
used for transgenic anirrial research, gene target validation and pathway
elucidation. Several
ribozymes are in various stages of clinical trials. ANGIOZYME was the first
chemically
synthesized ribozyme to be studied in human clinical trials. ANGIOZYME
specifically
inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor
receptor), a key
component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well
as other
firms have demonstratecl the importance of anti-angiogenesis therapeutics in
animal models.
HEPTAZYME, a ribozyme designed to selectively destroy Hepatitis C Virus (HCV)
RNA,
was found effective in decreasing Hepatitis C viral RNA in cell culture assays
(Ribozyme
Pharmaceuticals, Incorporated - WEB home page).
[000162] As described hereinabove, the AMP/AML inhibitor may be a triplex
forming
oligonucleotides (TFOs). TFOs can be used for regulating the expression of an
AMP/AML
gene in cells. Recent stLidies have shown that TFOs can be designed which can
recognize
and bind to polypurine/polypyrimidine regions in double-stranded helical DNA
in a
sequence-specific manner. These recognition rules are outlined by Maher 111,
L. J., et al.,
Science,1989;245:725-730; Moser, H. E., et al., Science,1987;238:645-630;
Beal, P. A., et
al, Science,1992;251:1360-1363; Cooney, M., et al., Science,1988;241:456-459;
and Hogan,
M. E., et al., EP Publication 375408. Modification of the oligonucleotides,
such as the
introduction of intercalators and backbone substitutions, and optimization of
binding
conditions (pH and cation concentration) have aided in overcoming inherent
obstacles to
TFO activity such as charge repulsion and instability, and it was recently
shown that
synthetic oligonucleotides can be targeted to specific sequences (for a recent
review see
Seidman and Glazer, J Clin Invest 2003;112:487-94).
[000163] In general, the triplex-forming oligonucleotide has the sequence
correspondence:
oligo, 3'--A G G T; duplex, 5'--A G C T; and duplex, 3'--T C G A.
[000164] However, it has been shown that the A-AT and G-GC triplets have the
greatest
CA 02696833 2010-01-11
32
triple helical stability (Reither and Jeltsch, BMC Biochem, 2002, Sept12,
Epub). The same
authors have demonstrated that TFOs designed according to the A-AT and G-GC
rule do not
form non-specific triplexes, indicating that the triplex formation is indeed
sequence specific.
[000165] Thus for any given sequence in the AMP/AML gene a triplex forming
sequence
may be devised. Triplex-forming oligonucleotides preferably are at least 15,
more
preferably 25, still more preferably 30 or more nucleotides in length, up to
50 or 100 bp.
[000166] Transfection of cells (for example, via cationic liposomes) with
TFOs, and
formation of the triple helical structure with the target DNA induces steric
and functional
changes, blocking transcription initiation and elongation, allowing the
introduction of
desired sequence changes in the endogenous DNA and resulting in the specific
downregulation of gene expression. Examples of such suppression of gene
expression in
cells treated with TFOs include knockout of episomal supFGl and endogenous
HPRT genes
in mammalian cells (Vasquez et al., Nucl Acids Res. 1999;27:1176-81, and Puri,
et al, J Biol
Chem, 2001;276:28991-98), and the sequence- and target specific downregulation
of
expression of the Ets2 transcription factor, important in prostate cancer
etiology (Carbone, et
al, Nucl Acid Res. 2003;;31:833-43), and the pro-inflammatory ICAM-1 gene
(Besch et al, J
Biol Chem, 2002;277:32473-79). In addition, Vuyisich and Beal have recently
shown that
sequence specific TFOs can bind to dsRNA, inhibiting activity of dsRNA-
dependent
enzymes such as RNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res
2000;28:2369-74).
[000167] Additionally, TFOs designed according to the abovementioned
principles can
induce directed mutagenesis capable of effecting DNA repair, thus providing
both
downregulation and upregulation of expression of endogenous genes (Seidman and
Glazer, J
Clin Invest 2003;112:487-94). Detailed description of the design, synthesis
and
administration of effective TFOs can be found in U.S. Patent Application Nos.
2003 017068
and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 to
Emanuele et al,
and U.S. Pat. No. 5,721,138 to Lawn.
[000168] Techniques for administering such molecules to a cell or cellular
structure are
routinely practiced by the ordinarily skilled artisan, and ample guidance is
provided in the
literature of the art for such administration (refer, for example, to the
references relevant to
such molecules cited hereinabove and to U.S. Patent Application No.
2003/0044907 which is
incorporated herein by reference).
[000169] As described hereinabove, the method of regulating the biological
process of the
CA 02696833 2010-01-11
33
present invention comprises the step of exposing the cell/tissue to the
regulator.
[000170] Exposing the: cell/tissue to the regulator may be effected in various
ways
depending on the application and purpose. In cases where the cell/tissue form
part of a
human or an animal subject, exposing the cell/tissue to the regulator is
preferably effected by
providing the regulator to the subject.
[000171] Administering the regulator to a subject may be effected via any
suitable route
facilitating exposure of the cell/tissue with the regulator, including a route
selected from the
group consisting of the topical, intravenous, intranasal, transdermal,
intradermal, oral,
buccal, parenteral, rectal and inhalation route.
[000172] Preferably, siubcutaneous and/or local injection of the regulator in
saline solution
is used for treating a disease such as arthritis.
[000173] Preferably, oral delivery in combination with aspirin or with NSAID
of the
regulator (such as for example cathelicidin or its analog or fragments or
analogs of its
fragments) in tablet form is used for treating a disease such as arthritis or
other inflammatory
diseases regularly treated by NSAID or aspirins such as for example
atherosclerosis,
osteoarthritis, or rheumatic diseases.
[000174] Preferably, topical application of the regulator in lipid or saline
solution, or in a
cream on the skin is used for treating a cutaneous disease such as a psoriasis
legion.
[000175] Preferably, for treating respiratory diseases such as cystic fibrosis
and asthma or
COPD, the regulator is dissolved in a solution or provided in an inhalable
powder form and
administered using an inhaler.
10001761 Alternately, the cells may be exposed to regulator by expressing the
regulator in
the human or animal. In cases where the cell/tissue is a cultured cell/tissue,
exposing the
regulator to the cell/tissue is preferably effected by providing the regulator
to the cell/tissue
in-vitro using standard tiissue culture methods. Preferably, providing the
regulator to the
cell/tissue in-vitro is effected as described in the Examples section which
follows.
[000177] The regulator can be expressed in a subject by directly administering
to the
subject a nucleic acid construct configured so as to suitably express the
regulator in-vivo.
Alternatively, a nucleic acid construct for expressing the regulator may be
introduced into a
suitable cell ex-vivo via an appropriate gene delivery vehicle/method
(transfection,
transduction, homologous recombination, etc.), and using a suitable genetic
expression
system as needed. The modified cells may be expanded in culture and
administered to the
subject where they will produce the regulator in-vivo. To enable cellular
expression of the
CA 02696833 2010-01-11
34
regulator, a nucleic acid construct which encodes the regulator preferably
includes at least
one cis acting regulatory element, most preferably a promoter which is active
in the specific
cell population transforr.ned. The nucleic acid construct can further include
an enhancer,
which can be adjacent o:r distant to the promoter sequence and can function in
up regulating
the transcription there from.
[000178] Suitable in vivo nucleic acid transfer techniques include
transfection with viral or
non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus,
or adeno-
associated virus (AAV) and lipid-based systems, polylysine based systems and
dendrimers.
Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA,
DOPE, and
DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)]. The
most preferred
constructs for use in gene therapy are viruses, most preferably adenoviruses,
AAV,
lentiviruses, or retroviruses. A viral construct such as a retroviral
construct includes at least
one transcriptional promoter/enhancer or locus-defining element(s), or other
elements that
control gene expression by other means such as alternate splicing, nuclear RNA
export, or
post-translational modifiication of messenger. Such vector constructs also
include a
packaging signal, long terminal repeats (LTRs) or portions thereof, and
positive and negative
strand primer binding sites appropriate to the virus used, unless it is
already present in the
viral construct. The construct may include a signal that directs
polyadenylation, as well as
one or more restriction sites and a translation termination sequence. By way
of example,
such a constructs will typically include a 5' LTR, a tRNA binding site, a
packaging signal, an
origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
10001791 The various aspects of the present invention may be practiced by
using,
increasing or by decreasing the activity/level, of any of various types of
AMPs/AMLs,
depending on the application and purpose. For example in the experimental data
use of
cathelicidin peptide or its analog or its fragment is shown for treating
disease.
[000180] Preferably, the AMP/AML is a cationic and/or hydrophobic peptide.
[000181] As used hereiin, the term "peptide" (with the exception of the term
in the context
of the phrases "antimicrobial peptide" or "antimicrobial-like peptide", refers
to a polypeptide
which is composed of less than 51 amino acid residue.
[000182] Alteratively, a cathelidin may be more that 51 a.a. such as for
example hCAP-18
which contains and includes the pro-region of the LL-37 peptide.
[000183] Preferably, the AMP/AML is a cathelicidin.
Preferably, the catheliciclin is LL-37.
CA 02696833 2010-01-11
[000184] Preferably, the AMP/AML is of human origin. Alternately, it may be of
non-
human origin, in which case it is preferably of mammalian origin.
[000185] Numerous examples of AMPs/AMLs which may be used, and/or whose
activity/levels may be decreased, for practicing the various aspects of the
present invention
are described in further detail herein below.
[000186] The method imay be practiced so as to regulate the biological process
in any of
various cells/tissues of the present invention.
10001871 The cell/tissue is preferably from bone, synovial fluid or beta
cells.
[000188] The method may be used to regulate the biological process in any of
various
types of cells/tissues involved in disease included in the present invention.
[000189] The method may be affected by exposing the cell/tissue to the
regulator at any of
various concentrations, depending on the application and purpose.
[000190] Preferably, when using an AMP/AML inhibitor of the present invention
for
regulating the biological process, exposing the cell/tissue to the AMP/AML
inhibitor is
effected by exposing the cell/tissue to the AMP/AML inhibitor at a
concentration selected
from a range of about 50 nanograms per milliliter to about one milligram per
milliliter.
[000191] Exposing the cell/tissue to the AMP/AML inhibitor may advantageously
be
effected, depending on the application and purpose, by exposing the
cell/tissue to the
AMP/AML inhibitor at a concentration selected from a range of about 50 ng/ml
to about 100
micrograms/ml, from a range of about 100 micrograms/ml to about 200
micrograms/ml,
from a range of about 200 micrograms/ml to about 300 micrograms/ml, from a
range of
about 300 micrograms/nil to about 400 micrograms/ml, from a range of about 400
micrograms/mi to about 500 micrograms/ml, from a range of about 500
micrograms/ml to
about 600 micrograms/ml, from a range of about 600 micrograms/ml to about 700
micrograms/ml, from a range of about 700 micrograms/ml to about 800
micrograms/ml,
from a range of about 800 micrograms/ml to about 900 micrograms/ml, and from a
range of
about 900 micrograms/ml to about 1 mg/ml.
Preferably, when using an AMP/AML of the present invention for regulating the
biological
process, exposing the ce][l/tissue to the AMP/AML is effected by exposing the
cell/tissue to
the AMP/AML at a concentration selected from a range of about 2 ng/ml to about
50
micrograms/ml or from `50 micrograms/ml to 100 micrograms/ml. Exposing the
cell/tissue
to the AMP/AML and in particular to cathelicidin may advantageously be
effected,
depending on inhibitor at a concentration selected from a range of about 2
ng/ml to about 1
CA 02696833 2010-01-11
36
micrograms/ml, from a range of about 1 micrograms/ml to about 2 micrograms/ml,
from a
range of about 2 micrograms/ml to about 3 micrograms/ml, from a range of about
3
micrograms/ml to about 4 micrograms/ml, from a range of about 4 micrograms/ml
to about 5
micrograms/ml, from a range of about 5 micrograms/ml to about 6 micrograms/ml,
from a
range of about 6 microg,rams/ml to about 7 micrograms/ml, from a range of
about 7
micrograms/ml to about 8 micrograms/ml, from a range of about 8 micrograms/ml
to about 9
micrograms/ml, from a range of about 9 micrograms/ml to about 10 mg/ml, from a
range of
about 10 micrograms/m]. to about 11 micrograms/ml, from a range of about 11
micrograms/ml to about 12 mg/ml, from a range of about 12 micrograms/ml to
about 13
micrograms/ml, from a range of about 13 micrograms/ml to about 17 mg/ml, from
a range
of about 17 micrograms/ml to about 20 micrograms/ml, from a range of about 20
micrograms/ml to about 25 mg/ml.
[000192] The method can be used to regulate in the cell/tissue a biological
process such as
growth, differentiation, inflammation or autoimmunity.
[000193] For regulating growth in an epithelial, skin and/or gastrointestinal
cell/tissue, the
regulator may advantageously be an AMP/AML inhibitor of the present invention
and/or an
AMP/AML (in particular, a cathelicidin or a cathelicidin analog) of the
present invention.
[000194] As used herein, the phrase "cathelicidin inhibitor" refers to a
compound of the
present invention which is capable of decreasing an activity and/or level of a
cathelicidin.
[000195] As described hereinabove, the present invention can be used for
regulating
biological processes sucli as growth, differentiation, inflammation, chronic
inflammation and
autoimmunity. It will be appreciated that such biological processes are
associated with the
pathogenesis of numerous diseases, and that regulation of such biological
processes
according to the teachings of the present invention can be used for treating
such diseases.
[000196] As used herein, the term "disease" refers to any medical disease,
disorder,
condition, or syndrome, or to any undesired and/or abnormal physiological
morphological,
cosmetic and/or physical state and/or condition.
10001971 Herein, the term "treating" includes abrogating, substantially
inhibiting, slowing
or reversing the progression of a disease, substantially ameliorating clinical
symptoms of a
disease or substantially preventing the appearance of clinical symptoms of a
disease.
10001981 The method can be used for treating any of various diseases.
[000199] In particular, the method can be used for treating any of various
diseases which
are associated with: (i) ir.iflammation; (ii) dysregulation of
growth/differentiation of a
CA 02696833 2010-01-11
37
cell/tissue; (iv) dysregulation of growtb/differentiation balance in bone (v)
dysregulation of
growth/differentiation balance in beta cell function and insulin production
(iii) dysregulation
of growth/differentiatioti balance of a cell/tissue; and (iv) autoimmunity.
[000200] Examples of such diseases, and others, which are amenable to
treatment via the
present invention are listed hereinbelow.
10002011 One of ordinary skill in the art, such as a physician, most
preferably a physician
specialized in the disease, will possess the necessary expertise for treating
a disease
according to the teachings of the present invention.
[000202] As used herein, the phrase "subject in need thereof' refers to a
subject having the
disease.
[000203) Preferably, the subject is a mammal, most preferably a human.
10002041 By virtue of demonstrably enabling regulation of growth of a
pancreatic beta cell
cell/tissue, the method described above for inducing or inhibiting such growth
is particularly
suitable for treating any of various diseases associated with
dysregulated/diminished growth
of insulin producing cells, and hence can be used for treating any of various
diseases
associated with insulin d.epletion. Such diseases notably include diabetes
mellitus type 1 or
insulin dependant diabetes or type 2 diabetes, and other metabolic diseases
including low
grade inflammatory diseases. In addition, the method of cell growth regulation
as required in
the treatment of cancer is also demonstrated.
[000205] By virtue of demonstrably enabling inhibition of Experimental
Autoimmune
Encephalitis (EAE) inflammation in an in-vivo mouse model, the method
described above
for inhibiting such inflammation is particularly suitable for treating any of
various diseases
associated with such inflammation. Such diseases notably include inflammatory
or
autoimmune diseases, such as neurodegenerative disease, central nervous system
diseases,
multiple sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia
gravis, motor
neuropathy, Guillain-Barre syndrome, autoimmune neuropathy, Lambert-Eaton
myasthenic
syndrome, paraneoplastic neurological disease, paraneoplastic cerebellar
atrophy, non-
paraneoplastic stiff man syndrome, progressive cerebellar atrophy, Rasmussen's
encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la
Tourette syndrome,
autoimmune polyendocrinopathy, dysimmune neuropathy, acquired neuromyotonia,
arthrogryposis multiplex, optic neuritis, spongiform encephalopathy, migraine,
headache,
cluster headache, and stii:f-man syndromeautoimmune diseases.
10002061 By virtue of demonstrably enabling inhibition of Collagen induced
arthritis (CIA)
CA 02696833 2010-01-11
38
inflammation in an in-vivo mouse model for rheumatic diseases including
rheumatoid
arthritis, the method described above for inhibiting such inflammation is
particularly suitable
for treating any of various diseases associated with such inflammation or
autoimmunity and
particularly in rheumatic diseases. Such diseases notably include inflammatory
or
autoimmune diseases, such as arthritis, rheumatic diseases and connective
tissue/inflammatory diseases include arthritis, rheumatoid arthritis, pyogenic
arthritis, mixed
connective tissue disease, cholesteatoma, lupus, relapsing polychondritis,
autoimmune
myositis, primary Sjogren's syndrome, smooth muscle autoimmune disease,
myositis,
tendinitis, a ligament inflammation, chondritis, a joint inflammation, a
synovial
inflammation, carpal tunnel syndrome, osteoarthritis, ankylosing spondylitis,
a skeletal
inflammation, an autoinimune ear disease, osteoporosis, fibromyalgia,
periodontitis, and an
autoimmune disease of the inner ear, Diseases diagnosed or managed by the
rheumatologist
include, Rheumatic diseases such as systemic Lupus Erythematosus, scleroderma
(systemic
sclerosis), dermatomyositis, polymyositis, polymyalgia rheumatica,
osteoarthritis, septic
arthritis, sarcoidosis, gout, pseudogout spondyloarthropathies, ankylosing
spondylitis,
reactive arthritis, psoriatic arthropathy, enteropathic spondylitis, reactive
arthropathy
vasculitis, polyarteritis nodosa, Henoch-Schonlein purpura, serum sickness,
Wegener's
granulomatosis, giant cell arteritis, temporal arteritis, Takayasu's
arteritis, Behget's
syndrome, Kawasaki's disease (mucocutaneous lymph node syndrome), Buerger's
disease
(thromboangiitis obliterans), Juvenile Idiopathic Arthritis(JIA).
[000207] By virtue of demonstrably enabling inhibition of weight gain and
nutritional and
Metabolic Diseases and low grade inflammation in an in-vivo mouse model that
uses a high
fat diet and LPS injections (IP) in order to induce disease, the method
described above for
inhibiting such inflammation is particularly suitable for treating any of
various diseases
associated with such inflammation. Such diseases notably include inflammatory
or
autoimmune diseases, such as obesity, diabetes, insulin resistance, type 2
diabetes,
Phenylketonuria (PKU), Metabolic syndrome, Sodium metabolism disorders,
Calcium
metabolism disorders, Hypercalcemia, Hypocalcemia, Potassium metabolism
disorders,
Hyperkalemia, Hypokalemia, Phosphate metabolism disorders, Magnesium
metabolism
disorders, Acid-Base metabolism disorders, atherosclerosis, cardio vascular
diseases
including; Aneurysm, Angina, Arrhythmia, Atherosclerosis, Cardiomyopathy,
Cerebrovascular Accident (Stroke), Cerebrovascular disease, Congenital heart
disease,
Congestive Heart Failure, Myocarditis, Valve Disease, Coronary Artery Disease,
Dilated
CA 02696833 2010-01-11
39
cardiomyopathy, Diastolic dysfunction, Endocarditis, High Blood Pressure
(Hypertension),
Hypertrophic cardiomyopathy, Mitral valve prolapse, Myocardial infarction
(Heart Attack),
Venous Thromboembolism.
[000208] By virtue of demonstrably enabling prevention of insulin resistance
and
Metabolic Diseases and low grade inflammation in an in-vivo mouse model that
uses LPS
injections (IP) in order to induce disease, the method described above for
inhibiting such
inflammation is particularly suitable for treating any of various diseases
associated with such
inflammation. Such diseases notably include inflammatory or autoimmune
diseases, such as
Fatigue, Intestinal bloating, Sleepiness, Weight gain, Increased blood
triglyceride levels,
Increased blood pressure, depression, Hyperglycemia, hyperglycaemia, or high
blood sugar,
diabetes mellitus, type 1 diabetes, type 2 diabetes, Polycystic ovarian
syndrome (PCOS),
Hypertension, Dyslipidemia that includes high triglyceride levels,
glandular/inflammatory
diseases that include type B insulin resistance, Schmidt's syndrome, Cushing's
syndrome,
thyrotoxicosis, benign prostatic hyperplasia, pancreatic disease, Hashimoto's
thyroiditis,
idiopathic adrenal atrophy, Graves' disease, androgenic alopecia, thyroid
disease, thyroiditis,
spontaneous autoimmune thyroiditis, idiopathic myxedema, ovarian autoimmunity,
autoimmune anti-sperm infertility, autoimmune prostatitis, Addison's disease,
and Type I
autoimmune polyglandular syndrome,
[000209] By virtue of demonstrably enabling prevention of synovial pathology
and bone
resorption and degradation or deformation and low grade inflammation in an in-
vivo mouse
model, the method described above for inhibiting such inflammation is
particularly suitable
for treating any of various diseases associated with such inflammation. Such
diseases
notably include inflammatory or autoimmune diseases, such as osteoporosis,
Osteogenesis
imperfecta, Paget's disease, Osteochondroma, Osteomalacia, Osteomyelitis,
Osteopetroses,
Renal Osteodystrophy, tJnicameral Bone Spurs, Bone Tumor, Craniosynostosis,
Enchondroma, Fibrous Dysplasia, Giant Cell Tumor of Bone, Infectious
Arthritis,
Osteomyelitis, Klippel-Feil Syndrome, Limb Length Discrepancy, Osteochondritis
Dissecans, periodontitis, bone loss in periodontitis, connective
tissue/inflammatory diseases
which include arthritis, rheumatoid arthritis, pyogenic arthritis, mixed
connective tissue
disease, cholesteatoma, relapsing polychondritis, autoimmune myositis, primary
Sjogren's
syndrome, smooth muscle autoimmune disease, myositis, tendinitis, a ligament
inflammation, chondritis, a joint inflammation, a synovial inflammation,
carpal tunnel
syndrome, osteoarthritis, ankylosing spondylitis, a skeletal inflammation, an
autoimmune ear
CA 02696833 2010-01-11
disease, osteoporosis, fibromyalgia, periodontitis, and an autoimmune disease
of the inner
ear.
[000210] By virtue of demonstrably enabling improvement of psoriasis and/or
skin
inflammation in an in-vivo model, the method described above for inhibiting
such
inflammation is particularly suitable for treating any of various diseases
associated with such
inflammation or for wound healing. Such diseases notably include inflammatory
or
autoimmune diseases, such as cutaneous/inflammatory diseases include
psoriasis, rosacea,
dandruff, pemphigus vulgaris, lichen planus, atopic dermatitis, excema,
scleroderma,
dermatomyositis, alopecia, blepharitis, skin carcinoma, melanoma, squamous
cell carcinoma,
acne vulgaris, erythema toxicum neonatorum, folliculitis, skin wrinkles,
autoimmune bullous
skin disease, bullous pemphigoid, pemphigus foliaceus, dermatitis, and drug
eruption.
[000211] For treating the disease, the regulator such as for example an
AMP/AML or the
cathelicidin peptide or cathelicidin peptide analog or a cathelicidin protein
or a cathelicidin
dominant negative analog or a cathelicidin fragment or inhibitor may be
administered via
any of various suitable regimens.
[0002121 Depending on the application and purpose, each inter dose interval of
the
plurality of doses may advantageously be selected from a range of about 2.4
hours to about 3
days, from a range of about 3 days to about 6 days, from a range of about 6
days to about 9
days, from a range of about 9 days to about 12 days, from a range of about 12
days to about
15 days, from a range of about 15 days to about 18 days, from a range of about
18 days to
about 21 days, from a range of about 21 days to about 24 days, from a range of
about 24
days to about 27 days, or from a range of about 27 days to about 30 days.
[0002131 Preferably, the inter dose interval of the plurality of doses is
about I day. This
bearing in mind that the half-life of LL-37 peptide in blood of humans is
approximately 3.4
days during which the peptide is usually protected from degradation by LDL and
HDL.
(Infection and immunity, Nov. 1999, p. 6084-6089).
[0002141 If administered orally, the cathelicidin analog or peptide can be
delivered to the
gastro intestinal tract (GIT). Peptides are usually delivered by injection or
infusion due to
their limited bioavailability and stability when delivered by other routs. The
major problems
to overcome in the development of oral peptide delivery are enzymatic
degradation and
denatuartion in the GIT environment and their poor penetration through
physiological
barriers (E.C. Lavelle et al. Vaccine 15 (1997), pp. 1070-1078). However,
microencapsulation technologies using synthetic polyesters such as poly(L-
lactide) (L.PLA)
CA 02696833 2010-01-11
41
and copolymers such as poly(D,L-lactide co-glycolide) (PLG), used for the
delivery of drugs
to humans and are now being considered for the delivery of oral vaccines and
peptides (M.
Manocha et al Vaccine 23 (2005) (48-49), pp. 5599-5617). The objective is an
oral
formulations for the delivery of a peptidic agent that needs to be delivered
and act in the
GIT. This objective is achieved by encapsulation of the peptide into
nanoparticles made of
PLA or other pharmaceutically acceptable carriers. These nanoparticles will
protect the
peptide from deterioration in the GIT and will allow adhesion and penetration
to the GI
mucosal tissue and being released in its active form. Such formulations should
be in a
nanometer scale where the peptide is fully protected when in the GI fluid but
being able to
release the peptide within the GI mucosal tissue after absorption.
[000215] Two types of systems has been developed, encapsulation in PLA based
polymer
using the Liposphere technology and stereointeraction of the peptide with
stereoregular PLA.
The first system is based on the formation of PLA nanoparticles coated with
phospholipids
by emulsion evaporatioti method. The second system is based on a physical
interaction
between the peptide and a common biodegradable stereoregular PLA. In both
methods, PLA
based polymers are used. These polymers are FDA approved for the delivery of
drugs as
well as peptides and proteins. In this technology, the peptide is dissolved in
a safe solvent
along with the polymer (D-PLA) which upon mixing the solution, a precipitate
of the
peptide-PLA sterocomplex is formed. The precipitate can be of nanometer scale
and contain
a high load of the peptide. The peptide is released from the streocomplex as a
result of
hydrolysis of the PLA chain that intertwined along the peptide chain. This
system has been
investigated extensively for the injectable delivery of insulin, somatostatin
and LHRH for
extended release of weeks after injection. (Macromol Biosci. 2006 Dec
8;6(12):1019-25, J
Control Release 2005 Oct 20;107(3):474-83, Biomaterials. 2002 Nov;23(22):4389-
96,
Macromol Biosci. 2006 Dec 8;6(12):977-90)
[000216] As is described in the examples section which follows, in vivo mouse
models
show by implication that administering 3 doses per week of a cathelicidin
regulator of the
present invention to the subject (IP) with an inter dose interval of about 2.5
days can be used
for effectively treating a disease such as Collagen Induced arthritis or EAE
or multiple
sclerosis or obesity of insulin resistance in a human subject.
10002171 Disease treatment may be effected via polytherapy by administration
of the
regulator in conjunction with Vitamin D3, calcitriol analogs, or peptide
inhibitors such as
protease inhibitors, the serpin serine proteinase inhibitory components (alpha-
1 PI) and alpha
CA 02696833 2010-01-11
42
-1 antichymotrypsin (Panyutich, AV. etal., 1995. Am.J.Respir.Cell Mol.Biol.
12:351-357),
BAPTA-AM (an intracellular Ca(2+) chelating agent), pertussis toxin and U-
73122 (a
phospholipase C inhibitor; Niyonsaba, F. et al., 2001. Eur.J.Immunol. 31:1066-
1075), T-cell
targeted therapies, monoclonal antibody against chemokine tumor necrosis
factor and
cytokine targeted therapies, fibroblast growth factor inhibitors. For example,
topical
treatments may advantageously include cell proliferation regulators such as
retinoid -
vitamin A - analog which modulates or changes the cellular differentiation of
the epidermis.
Such polytherapy may be effected using anti-inflammatory drugs/treatments as a
precautionary measure against relapse of psoriasis or other auto-immune
disease. Such
drugs/treatments include tazarotene, methotrexate, acitretin, bexarotene,
ploralem, etretinate,
corticosteroid creams and ointments, synthetic vitamin D3, IL-10, IL-4 and IL-
1RA
(receptor antagonist).
[000218] A cathelicidin inhibitor/vitamin D combination is particularly
claimed as a
treatment modality for cancer. This is because whereas vitamin D pathway can
skew cancer
cells into a desired differenciating state, cathelicidin, which is also
expressed via the vitamin
D pathway, skews cancer cells into the undesired proliferative state. Thus the
maximum
desired differentiating non-proliferating pathway is achieved.
[000219] To enable treatment of the disease, the regulator is preferably
included as an
active ingredient in a pharmaceutical composition which includes a suitable
carrier and
which is suitably packaged and labeled for treatment of the disease.
[000220] The regulator according to the present invention can be administered
to a subject
per se, or in a pharmaceutical composition where it is mixed with suitable
carriers or
excipients.
[000221] As used herein a "pharmaceutical composition" refers to a preparation
of one or
more of the active ingredients described herein with other chemical components
such as
physiologically suitable carriers and excipients. The purpose of a
pharmaceutical
composition is to facilitate administration of active ingredients to an
organism.
[000222] Herein the term "active ingredients" refers to the regulator of the
present
invention accountable for the biological effect.
[000223] Hereinafter, the phrases "physiologically acceptable carrier" and
"pharmaceutically acceptable carrier" which may be interchangeably used refer
to a carrier
or a diluent that does not cause significant irritation to an organism and
does not abrogate the
biological activity and properties of the administered active ingredients. An
adjuvant is
CA 02696833 2010-01-11
43
included under these phrases.
10002241 Herein the term "excipient" refers to an inert substance added to a
pharmaceutical composition to further facilitate administration of an active
ingredient.
Examples, without limitation, of excipients include calcium carbonate, calcium
phosphate,
various sugars and types of starch, cellulose derivatives, gelatin, vegetable
oils and
polyethylene glycols.
[000225] Techniques for formulation and administration of drugs may be found
in
"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest
edition,
which is incorporated herein by reference.
[000226] Suitable routes of administration may, for example, include oral,
rectal,
transmucosal, transnasal, intestinal or parenteral delivery, including
intramuscular,
subcutaneous and intramedullary injections as well as intrathecal, direct
intraventricular,
intravenous, inrtaperitoneal, intranasal, or intraocular injections.
10002271 Alternately, one may administer the pharmaceutical composition in a
local rather
than systemic manner, for example, via injection of the pharmaceutical
composition directly
into a tissue region of a patient.
[000228] Pharmaceutical compositions of the present invention may be
manufactured by
processes well known in the art, e.g., by means of conventional mixing,
dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping
or
lyophilizing processes.
[000229] Pharmaceutical compositions for use in accordance with the present
invention
thus may be formulated in conventional manner using one or more
physiologically
acceptable carriers comprising excipients and auxiliaries, which facilitate
processing of the
active ingredients into preparations which, can be used pharmaceutically.
Proper
formulation is dependent upon the route of administration chosen.
[000230] For injection, the active ingredients of the pharmaceutical
composition may be
formulated in aqueous solutions, preferably in physiologically compatible
buffers such as
Hank's solution, Ringer's solution, or physiological salt buffer. For
transmucosal
administration, penetrants appropriate to the barrier to be permeated are used
in the
formulation. Such penetrants are generally known in the art.
[000231] For oral administration, the pharmaceutical composition can be
formulated as
described above using PLA based polymers or can be formulated readily by
combining the
active ingredients with pharmaceutically acceptable carriers well known in the
art. Such
CA 02696833 2010-01-11
44
carriers enable the pharmaceutical composition to be formulated as tablets,
pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral
ingestion by a
patient. Pharmacological preparations for oral use can be made using a solid
excipient,
optionally grinding the resulting mixture, and processing the mixture of
granules, after
adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
Suitable excipients
are, in particular, fillers such as sugars, including lactose, sucrose,
mannitol, or sorbitol;
cellulose preparations such as, for example, maize starch, wheat starch, rice
starch, potato
starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-
cellulose, sodium
carbomethylcellulose; and/or physiologically acceptable polymers such as
polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added,
such as cross-
linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as
sodium alginate.
[000232] Dragee cores are provided with suitable coatings. For this purpose,
concentrated
sugar solutions may be used which may optionally contain gum arabic, talc,
polyvinyl
pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and
suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be
added to the
tablets or dragee coatings for identification or to characterize different
combinations of
active ingredient doses.
[000233] Pharmaceutical compositions which can be used orally, include push-
fit capsules
made of gelatin as well as soft, sealed capsules made of gelatin and a
plasticizer, such as
glycerol or sorbitol. The push-fit capsules may contain the active ingredients
in admixture
with filler such as lactose, binders such as starches, lubricants such as talc
or magnesium
stearate and, optionally, stabilizers. In soft capsules, the active
ingredients may be dissolved
or suspended in suitable liquids, such as fatty oils, liquid paraffin, or
liquid polyethylene
glycols. In addition, stabilizers may be added. All formulations for oral
administration
should be in dosages suitable for the chosen route of administration.
[000234] For buccal administration, the compositions may take the form of
tablets or
lozenges formulated in conventional manner.
10002351 For administration by nasal inhalation, the active ingredients for
use according to
the present invention are conveniently delivered in the form of an aerosol
spray presentation
from a pressurized pack or a nebulizer with the use of a suitable propellant,
e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or
carbon
dioxide. In the case of a pressurized aerosol, the dosage unit may be
determined by
providing a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for
CA 02696833 2010-01-11
use in a dispenser may be formulated containing a powder mix of the active
ingredients and
a suitable powder base such as lactose or starch.
10002361 The pharmaceutical composition described herein may be formulated for
parenteral administration, e.g., by bolus injection or continuous infusion.
Formulations for
injection may be presented in unit dosage form, e.g., in ampoules or in
multidose containers
with optionally, an added preservative. The compositions may be suspensions,
solutions or
emulsions in oily or aqueous vehicles, and may contain formulatory agents such
as
suspending, stabilizing and/or dispersing agents.
[000237] Pharmaceutical compositions for parenteral administration include
aqueous
solutions of the active preparation in water-soluble form. Additionally,
suspensions of the
active ingredients may be prepared as appropriate oily or water based
injection suspensions.
Suitable lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic
fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection
suspensions may contain substances, which increase the viscosity of the
suspension, such as
sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the
suspension may also
contain suitable stabilizers or agents which increase the solubility of the
active ingredients to
allow for the preparation of highly concentrated solutions. Cream solutions
can include any
lipids or organic alcohols or chemicals including for example benzyl alcohol,
macrogol,
hexylene glycol, carbomer, ascorbic acid, butyl hydroxyainisole, butyl
hydroxytoluene,
disodium edentate, water, trometamol, poxoamer.
10002381 Alternatively, the active ingredients may be in powder form for
constitution with
a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before
use.
[000239) The pharmaceutical composition of the present invention may also be
formulated
in rectal compositions such as suppositories or retention enemas, using, e.g.,
conventional
suppository bases such as cocoa butter or other glycerides.
[000240] It should be understood that by administering a peptide of the
invention it is
meant administering a peptide of the invention or a pharmaceutically
acceptable salt thereof
or other pharmaceutically acceptable form thereof.
10002411 Pharmaceutical compositions suitable for use in context of the
present invention
include compositions wherein the active ingredients are contained in an amount
effective to
achieve the intended purpose. More specifically, a therapeutically effective
amount means
an amount of active ingredients (regulator of the present invention) effective
to prevent,
alleviate or ameliorate symptoms of a disorder (e.g., psoriasis or a
carcinoma) or prolong the
CA 02696833 2010-01-11
46
survival of the subject being treated.
[000242] Determination of a therapeutically effective amount is well within
the capability
of those skilled in the art, especially in light of the detailed disclosure
provided herein.
10002431 For any preparation used in the methods of the invention, the
therapeutically
effective amount or dose can be estimated initially from in vitro and cell
culture assays. For
example, a dose can be formulated in animal models to achieve a desired
concentration or
titer. Such information can be used to more accurately determine useful doses
in humans.
[000244] Toxicity and therapeutic efficacy of the active ingredients described
herein can be
determined by standard pharmaceutical procedures in vitro, in cell cultures or
experimental
animals. The data obtained from these in vitro and cell culture assays and
animal studies can
be used in formulating a range of dosage for use in human. The dosage may vary
depending
upon the dosage form employed and the route of administration utilized. The
exact
formulation, route of administration and dosage can be chosen by the
individual physician in
view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The
Pharmacological Basis
of Therapeutics", Ch. I p.1).
[000245J Dosage amount and interval may be adjusted individually to provide
plasma or
brain levels of the active ingredients which are sufficient to achieve a
desired therapeutic
effect (minimal effective concentration, MEC). The MEC will vary for each
preparation, but
can be estimated from in vitro data. Dosages necessary to achieve the MEC will
depend on
individual characteristics and route of administration. Detection assays can
be used to
determine plasma concentrations.
10002461 Depending on the severity and responsiveness of the condition to be
treated,
dosing can be of a single or a plurality of administrations, with course of
treatment lasting
from several days to several weeks or until cure is effected or diminution of
the disease state
is achieved.
[000247] The amount of a composition to be administered will, of course, be
dependent on
the subject being treated, the severity of the affliction, the manner of
administration, the
judgment of the prescribing physician, etc.
[0002481 Compositions of the present invention may, if desired, be presented
in a pack or
dispenser device, such as an FDA approved kit, which may contain one or more
unit dosage
forms containing the active ingredients. The pack may, for example, comprise
metal or
plastic foil, such as a blister pack. The pack or dispenser device may be
accompanied by
instructions for administration. The pack or dispenser may also be
accommodated by a
CA 02696833 2010-01-11
47
notice associated with the container in a form prescribed by a governmental
agency
regulating the manufacture, use or sale of pharmaceuticals, which notice is
reflective of
approval by the agency of the form of the compositions or human or veterinary
administration. Such notice, for example, may be of labeling approved by the
U.S. Food and
Drug Administration for prescription drugs or of an approved product insert.
Compositions
comprising a preparation of the invention formulated in a compatible
pharmaceutical carrier
may also be prepared, placed in an appropriate container, and labeled for
treatment of an
indicated condition, as if further detailed above.
[000249] Thus, the present invention provides an article of manufacture which
comprises
packaging material identified for treatment of the disease, and a
pharmaceutical composition
which includes a pharmaceutically acceptable carrier and, as an active
ingredient, the
regulator.
10002501 Preferably, the pharmaceutical composition is formulated as a
solution,
suspension, emulsion or gel.
10002511 Preferably, the pharmaceutically acceptable carrier is selected so as
to enable
administration of the pharmaceutical composition via a route selected from the
group
consisting of the topical, intravenous, intranasal, transdermal, intradermal,
oral, buccal,
parenteral, rectal and inhalation route.
[000252] Preferably, the pharmaceutical composition is composed so as to
enable exposure
of an affected cell/tissue of the subject having the disease, to the regulator
at a suitable
concentration, as described hereinabove, for treating the disease.
10002531 Preferably, the pharmaceutical composition is further identified for
administration to the subject according to a suitable regimen, as described
hereinabove.
[000254] Thus, the present invention provides a method of identifying a
compound capable
of treating autoimmune or inflammatory diseases by using a method of
regulating the
biological process in a cell/tissue involved in disease. The method is
effected in a first step
by exposing the cell/tissue to a test compound which is: a compound capable of
decreasing
or increasing an activity and/or level of an antimicrobial peptide (AMP)
and/or AMP-like
molecule (AML); and/or which is itself the AMP and/or AML. In a second step,
the method
is effected by evaluating a capacity of the test compound to regulate the
biological process in
the cell and/or tissue. In particular, the method involves the regulation of
the AMP
cathelicidin or by using the cathelicidin peptide or its analog or fragments.
10002551 It will be appreciated that the method of identifying the compound
can be used
CA 02696833 2010-01-11
48
for screening a plurality of compounds so as to identify a compound having a
desired
capacity for regulating a biological process.
[000256] The method is preferably used to identify a compound capable of
regulating a
biological process as described hereinabove with respect to the method of the
present
invention of regulating a biological process.
[000257] Preferably, the test compound is a regulator as described hereinabove
with
respect to the method of the present invention of regulating a biological
process.
[0002581 The method is preferably used to identify a compound capable of
regulating the
biological process in the cell/tissue as described hereinabove with respect to
the method of
the present invention of regulating a biological process, and as described in
the Examples
section which follows. As is described hereinabove with respect to the method
of regulating
the biological process, and in the Examples section which follows, the method
is preferably
employed for identifying a compound which is capable of: inducing growth in an
epithelial,
bone cells, osteoclasts or osteoblasts, nerve, synovial cell/tissue, beta
cell, skin,
keratinocytic and/or gastrointestinal cell/tissue; inhibiting growth in a
tumor, epithelial, skin,
keratinocytic and/or bone cells, osteoclasts or osteoblasts, nerve, synovial
cell/tissue, beta
cell, gastrointestinal cell/tissue; inhibiting angiogenesis/endothelial
cell/tissue growth;
inhibiting metastasis in a tumor cell/tissue; correcting dysregulated balance
of
proliferation/differentiation in an epithelial, keratinocytic and/or skin
cell/tissue; and/or
inhibiting inflammation in an epithelia], keratinocytic an/or skin
cell/tissue.
[000259] The identification method may advantageously be performed using high-
throughput methodology. Ample guidance for practicing relevant high-throughput
methods
is provided in the literature of the art (refer, for example, to U.S. Patent
Application No.
20030044907).
[000260] The test compound may be exposed to the cell/tissue in any of various
ways.
Preferably, the test compound is exposed to the cell/tissue in-vitro as
described in the
Examples section which follows. Alternately, the test compound may be exposed
to the
cell/tissue by exposing the test compound to a cultured cell/tissue.
10002611 Preferably, the cell which produces the test compound is a B-cell
hybridoma.
Alternately, the cell which produces the test compound may be of any of
various types,
depending on the application and purpose.
[000262] It will be appreciated that a B-cell hybridoma is an antibody
producing cell, and
hence that exposing the cell/tissue to a B-cell hybridoma can be used for
identifying a B-cell
CA 02696833 2010-01-11
49
hybridoma which expresses an antibody which is capable of regulating the
biological
process.
10002631 Exposing the cell/tissue to the test compound may be effected by
providing the
test compound to a subject which includes the cell/tissue (in-vivo model).
Preferably
providing the test compound to the test subject is effected as described
hereinabove with
respect to providing the regulator to a subject.
[000264] The identification method may be effected by exposing the test
compound to:
lesions of any of various diseases associated with bone resorption, CNS
disease including
multiple sclerosis, arthritis, fat cells of the obeseepithelial wounds
included in the present
invention; a lesion in an animal model as included in the examples of this
invention; or a
lesion in a human having the associated disease; a human biopsy of a normal or
pathological
involved lesion maintained in an organotypic culture containing plasma and
lymphocytes of
patients suffering from the disease having and not having polymorphism on AMPs
or their
genes and promoters or that are induced to express an AMP (and in particular a
cathelicidin)using known technologies such as transfection (Graham FL Virology
52 (2):
456-67m, Bacchetti S Proc Natl Acad Sci U S A 74 (4): 1590-4); andlor to a
cell/tissue of a
disease in which the disease inductive isoforms are ApoE4 and the non
inductive isoform is
ApoE3.
[000265] The identification method may be effected by exposing the test
compound to a
human lesion biopsy grafted onto an animal (xenograft model), whereby the
biopsy is taken
with informed consent. The biopsy may be transplanted onto an immunodeficient
mouse
(for example, NIHS-bg-nu-xid or BNX). For establishing such a xenograft model,
PBMCs
may be isolated from the blood obtained from the biopsy donor and activated
(for example,
using a superantigen), and the animals injected with the activated PBMCs.
Ample guidance
for practicing the identification method using such animal models is provided
in Examples
6-8 of the Examples section below and in the literature of the art (refer, for
example, to U.S.
Patent Application No. 20030044907).
10002661 Evaluation of the regulation of the biological processes encompassed
by the
identification method may be effected using any of various suitable methods
known to the
ordinarily skilled artisan. Preferably, such evaluation is performed, where
relevant, as
described in the Examples section which follows.
[000267] Evaluating regulation of the biological process may be effected using
quantitative
evaluation or lessional thickness when using an in-vivo model, cell count or
histological
CA 02696833 2010-01-11
evaluation.
[000268] Preferably, data obtained from the evaluation is processed using
statistical
analysis and ANOVA for maximum informativity.
[000269] According to one embodiment, exemplified by Example 3, the
identification
method may involve exposing the test compound to cultured microbes/bacteria
and
evaluating regulation of the biological process is effected by measuring
survival of the
microbes/bacteria. This may be effected by a colony-forming unit assay
performed with
Staphylococcus aureus (isolated from clinical sample), GAS (NZ131), and
enteroinvasive
Escherichia coli 029 as described (Porter et al, 1997). Before analysis, the
concentration of
the bacteria in culture will be determined by plating different bacterial
dilutions. The
protocol may be performed as follows. Cells are washed twice with 10 mM sodium
phosphate buffer (20 mM NaH2P04=H20, 20 mM Na2HP04=7H20) and diluted to a
concentration of 2,000,000 cells per milliliter (S. aureus, GAS) or 200,000
cells per
milliliter (E. coli) in phosphate buffer. S. aureus and E. coli are incubated
for 4 hours at 37
degrees centigrade with various concentrations of an AMP/AML in the presence
of various
concentrations of the test compound to be examined, in 50 microliters of
buffer in 96 well
round bottom tissue culture plates (Costar 3799, Corning inc., NY). GAS are
incubated for 1
hour due to the poor ability of GAS to grow in such buffers. After incubation,
the cells are
diluted from l Ox to 100,000x , and each of 20 ml of those solutions are
plated in triplicate on
tryptic soy broth (for S. aureus) and Todd Hewitt broth (for GAS and E. coli),
and the mean
number of colonies is determined. The number of cfu per ml is calculated, and
the blocking
activity of the examined test compounds to block the bactericidal activities
of the
AMP/AML will be calculated as follows: (cell survival after AMP/AML
incubation)/(cell
survival after incubation without AMP/AML)x 100, which represents the
percentage of cells
that are alive, as compared to those which are not (cell survival after
AMP/AML+test
compound incubation)/(cell survival after incubation with test compound
alone)x 100.
[000270] All compounds identified will be screened for one or all of the
following effects:
their ability to inhibit or regulate the antimicrobial activity of the AMP
(cathelicidin in
particular) or to which they were raised against; their ability to affect the
proliferation or
differentiation or other cellular processes of cultured cells of the affected
target tissue,
originally isolated from normal or diseased individuals or models, for example
HaCaT,
primary human or murine keratinocytes or fibroblasts for screening for
psoriasis; nerve cells,
bone cells or osteoclasts or osteoblasts and the effects of the inhibitors on
activation of the
CA 02696833 2010-01-11
51
immune system.
[000271] Identified compounds may be further screened for their effects on
organotypic
cocultures and animal models so as to identify inhibitors that will be able to
effectively
inhibit a desired biological effect or combination of biological effects. This
may include,
where suitable, identifying compounds that will inhibit or regulate the
effects of
AMPs/AMLs on proliferation/differentiation balance but which maintain their
antibacterial/antimicrobial activity.
10002721 The test compound or regulator may be any of various type of
molecule, such as a
small synthetic/non-polypeptidic molecule.
[000273] The test compound or regulator may advantageously be a peptide, a
protein or a
glycosylated protein.
[000274] Test compounds and regulators of the present invention of any of
various suitable
types may be obtained from a commercial chemical library such as, for example,
one held by
a large chemical company such as Merck, Glaxo Welcome, Bristol Meyers Squib,
Monsanto/Searle, Eli Lilly, Novartis, Pharmacia UpJohn, and the like. Test
compounds and
regulators of the present invention of any of various suitable types may also
be ordered via
the World Wide Web (Internet) via companies such as Chemcyclopedia
(http://www.mediabrains.com/client/chemcyclop/BGl /search.asp). Alternatively,
test
compounds and regulators of the present invention of any of various suitable
types may be
synthesized de novo using standard chemical and/or biological synthesis
techniques. Ample
guidance for synthesis of molecules suitable for use as test compounds or
regulators of the
present invention of any of various suitable types is provided in the
literature of the art. For
biological synthesis of molecules, such as polypeptides and nucleic acids,
refer, for example
to: Sambrook et al., infra; and associated references in the Examples section
which follows.
For guidance regarding chemical synthesis of molecules, refer, for example to
the extensive
guidelines provided by The American Chemical Society
(http://www.chemistry.org/portal/Chemistry). One of ordinary skill in the art,
such as, for
example, a chemist, will possess the required expertise for chemical synthesis
of suitable test
compounds.
[000275] In designing small molecules capable of binding the AMP/AML, several
features,
such as structures of antibody, receptors, ligands, and relevant biochemical
and biological
data may be considered. Such features may include de novo folding design using
energy
minimization and molecular dynamics, and comparative modeling followed by
energy
CA 02696833 2010-01-11
52
minimization and molecular dynamics. These two approaches differ only in
developing the
trial or initial structures. The folding patterns are studied using energy
minimization and
molecular dynamics.
[000276] Cathelicidins or cathelicidin peptides include the hCAP-18 pro-
peptide and its
following fragments and/or analogs of these fragments sequences (SEQ ID NOs: 1-
59) listed
in order as follows:
10002771 SEQ ID 1: fdiscdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes
[000278] SEQ ID 2: discdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes
[000279] SEQ ID 3: iscdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes
[000280] SEQ ID 4: scdkdnkrfallgdffrkskekigkeflcrivqrikdflrnlvprtes
[0002811 SEQ ID 5: cdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes
[000282] SEQ ID 6: dkdnkrfallgdffrkskekigkefkrivqrikdflmlvprtes
[000283] SEQ ID 7: kdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes
[000284] SEQ ID 8: dnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes
[000285] SEQ ID 9: nkrfallgdffrkskekigkefkrivqrikdflrnlvprtes
[000286] SEQ ID 10: krfallgdffrkskekigkefkrivqrikdflrnlvprtes
[000287] SEQ ID 11: rfallgdffrkskekigkefkrivqrikdflrnlvprtes
10002881 SEQ ID 12: fallgdffrkskekigkefkrivqrikdflrnlvprtes
[000289] SEQ ID 13: allgdffrkskekigkefkrivqrikdflrnlvprtes
[000290] SEQ ID 14: llgdffrkskekigkefkrivqrikdflrnlvprtes
[000291] SEQ ID 15: lgdffrkskekigkefkrivqrikdflrnlvprtes
[000292] SEQ ID 16: gdffrkskekigkefkrivqrikdflrnlvprtes
[000293] SEQ ID 17: dffrkskekigkefkrivqrikdflmlvprtes
10002941 SEQ ID 18: ffrkskekigkefkrivqrikdflmlvprtes
[000295] SEQ ID 19: frkskekigkefkrivqrikdflrnlvprtes
[000296] SEQ ID 20: rkskekigkefkrivqrikdflrnlvprtes
[000297] SEQ ID 21: kskekigkefkrivqrikdflrnlvprtes
[000298] SEQ ID 22: skekigkefkrivqrikdflrnlvprtes
[000299] SEQ ID 23:11 gdffrkskekigkeflcrivqrikdflrnlvprte
10003001 SEQ ID 24: Ilgdffrkskekigkefkrivqrikdflrnlvprt
[000301] SEQ ID 25: llgdffrkskekigkefkrivqrikdflrnlvpr
[000302] SEQ ID 26: llgdffrkskekigkefkrivqrikdflrnlvp
[000303] SEQ ID 27: llgdffrkskekigkefkrivqrikdflrnlv
CA 02696833 2010-01-11
53
[000304] SEQ ID 28: llgdffrkskekigkefkrivqrikdflrnl
10003051 SEQ ID 29: llgdffrkskekigkefkrivqrikdflrn
[000306] SEQ ID 30: llgdffrkskekigkeflcrivqrikdflr
[000307] SEQ ID 31: llgdffrkskekigkefkrivqrikdfl
10003081 SEQ ID 32: llgdffrkskekigkefkrivqrikdf
[000309] SEQ ID 33: llgdffrkskekigkefkrivqrikd
10003101 SEQ ID 34: llgdffrkskekigkefkrivqrik
[000311] SEQ ID 35: llgdffrkskekigkefkrivqri
10003121 SEQ ID 36: llgdffrkskekigkefkrivqr
[000313] SEQ ID 37: llgdffrkskekigkefkrivq
[000314] SEQ ID 38: llgdffrkskekigkefkriv
10003151 SEQ ID 39: llgdffrkskekigkefkri
[0003161 SEQ ID 40: efkriv
[000317] SEQ ID 41: kefkrivq
[000318] SEQ ID 42: gkefkrivqr
[000319] SEQ ID 43: igkefkrivqri
[000320] SEQ ID 44: kigkefkrivqrik
[000321] SEQ ID 45: ekigkefkrivqrikd
[000322] SEQ ID 46: kekigkefkrivqrikdf
10003231 SEQ ID 47: skekigkefkrivqrikdfl
[000324] SEQ ID 48: skekigkefkrivqrikdflrnlvprtes
10003251 SEQ ID 49: kskekigkefkrivqrikdflr
[000326] SEQ ID 50: rkskekigkefkrivqrikdflrn
[000327] SEQ ID 51: frkskekigkefkrivqrikdflrnl
[0003281 SEQ ID 52: ffrkskekigkefkrivqrikdflrnlv
[000329] SEQ ID 53: dffrkskekigkefkrivqrikdflrnlvp
10003301 SEQ ID 54: gdffrkskekigkeflcrivqrikdflrnlvpr
[000331] SEQ ID 55: lgdffrkskekigkefkrivqrikdflrnlvprt
[000332] In accordance with the instant invention, AMPs, compositions
comprising the
same, and methods of use thereof are provided. In a particular embodiment, the
antimicrobial
peptide has at least 90% homology with amino acid sequence fallgdffrksk.Xi
(SEQ ID NO:
56), wherein Xi is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12,
13, 14, 15, or 16 amino acids. The amino acid sequence of the peptides may
also be in
CA 02696833 2010-01-11
54
reverse orientation. In another embodiment, the antimicrobial peptide has at
least 90%
homology with amino acid sequence Xligkefkrivq2 (SEQ ID NO: 57),
wherein X1
is 0, l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids and
X2 is 0, 1, 2, 3, 4, 5,
6, 7, or 8 amino acids. In yet another embodiment, the antimicrobial peptide
has at least 90%
homology with amino acid sequence XI ffrkskekigkX2 (SEQ ID NO: 57), wherein X,
is 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids and X2 is 0,
1, 2, 3, 4, 5, 6, 7, or
8 amino acids. In yet another embodiment, the antimicrobial peptide has at
least 90%
homology with amino acid sequence Xlvqrikdflrn X2 (SEQ ID NO: 58) where X2 is
0, 1, 2,
3, 4, 5, 6,7 amino acids and X1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17,
18,or 19 amino acids. In yet another embodiment, the antimicrobial peptide has
at least 90%
homology with amino acid sequence XI gkefla-ivqrikdflrnX2 (SEQ ID NO: 59)
where X2 is 0,
1, 2, 3, 4, 5, 6,7 amino acids and X, is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17,
18 or 19 amino acids.
[000333] In another embodiment, the antimicrobial peptide has at least 60%,
70%, 80%,
90% or 95% homology (or identity) with amino acids in cathelicidin peptides
from mammals
as described in (Curr Issues Mol Biol. 2005 Ju1;7(2):179-96) namely:
10003341 RL-37: RLGNFFRKVKEKIGGGLKKVGQKIKDFLGNLVPRTAS Rhesus
monkey, CAP18: GLRKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY Rabbit,
CRAMP: GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE Mouse, rCRAMP:
GLVRKGGEKFGEKLRKIGQKIKEFFQKLALEIEQ rat, CAP 11:
(GLRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI) Guinea,
10003351 Canine cathelicdin:
KKIDRLKELITTGGQKIGEKIItRIGQRIKDFFKNLQPREEKS,
[000336] Bac5: RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPPFRPPLGPFP-NH2 Cow,
[000337] Bac7:
RRIRPRPPRLPRPRPRPLPFPRPGPRPIPRPLPFPRPGPRPIPRPLPFPRPGPRPIPRPL Cow
, BMAP-27: GRFKRFRKKFKKLFKKLSPVIPLLHL-NH2 Cow,
10003381 BMAP-28: GGLRSLGRKILRAWKKYGPIIVPIIRI-NH2 Cow,
[000339] BMAP-34: GLFRRLRDSIRRGQQKILEKARRIGERIKDIFR-NHZ Cow,
[000340] Indolicidin: ILPWKWPWWPWRR-NH2 Cow, Dodecapeptide:
RLCRIVVIRVCR Cow, Water buffalo cath GLPWILLRWLFFR-NH2 Water buffalo,
OADode: RYCRIIFLRVCR Sheep, SMAP-29: RGLRRLGRKIAHGVKKYGPTVLRIIRIA-
NH2 Sheep, SMAP-34: GLFGRLRDSLQRGGQKILEKAERIWCKIKDIFR-NH2 Sheep,
CA 02696833 2010-01-11
[000341] OaBac5 RFRPPIRRPPIRPPFRPPFRPPVRPPIRPPFRPPFRPPIGPFP-NH2
Sheep, OaBac6:
RRLRPRHQHFPSERPWPKPLPLPLPRPGPRPWPKPLPLPLPRPGLRPWPKPL Sheep,
OaBac7.5:
RRLRPRRPRLPRPRPRPRPRPRSLPLPRPQPRRIPRPILLP WRPPRPIPRPQIQPIPRW L
Sheep, OaBac 11:
RRLRPRRPRLPRPRPRPRPRPRSLPLPRPKPRPIPRPLPLPRPRPKPIPRPLPLPRPRPRRI
PRPLPLPRPRPRPIPRPLPLPQPQPSPIPRPL
[000342] Sheep,
[000343] ChBac5: RFRPPIRRPPIRPPFNPPFRPPVRPPFRPPFRPPFRPPIGPFP-NH2
Goat, eCATH-1: KRFGRLAKSFLRMRILLPRRKILLAS, eCATH-2:
KRRHWFPLSFQEFLEQLRRFRDQLPFP Horse, eCATH-3
KRFHSVGSLIQRHQQMIRDKSEATRHGIRIITRPKLLLAS, PR-39:
RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFP-NH2 Pig,
AFPPPNVPGPRFPPPNFPGPRFPPPNFPGPRFPPPNFPGPRFPPPNFPGPPFPPPIFPGPWF
PPPPPFRPPPFGPPRFP- NH2 Pig,
[000344] Prophenin-2:
AFPPPNVPGPRFPPPNVPGPRFPPPNFPGPRFPPPNFPGPRFPPPNFPGPPFPPPIFPGPW
FPPPPPFRPPPFGPPRFP- NH2 Pig,
[000345] Protegrin-1: RGGRLCYCRRRFCVCVGR-NH2 Pig,
[000346] RGGRLCYCRRRFCICV-NH2 Pig,
[000347] Protegrin-3: RGGGLCYCRRRFCVCVGR-NH2 Pig,
[0003481 Protegrin-4: RGGRLCYCRGWICFCVGR-NH2 Pig,
10003491 Protegrin-5: RGGRLCYCRPRFCVCVGR-NH2 Pig,
[000350] PMAP-23: RHDLLWRVRRPQKPKFVTVWVR Pig,
[000351] PMAP-36: GRFRRLRKKTRKRLKKIGKVLKWIPPIVGSIPLGC-NH2 Pig,
[000352] PMAP-37: GLLSRLRDFLSDRGRRLGEKIERIGQKIKDLSEFFQS
[000353] chCATH-B1: (Proc Natl Acad Sci U S A. 2007 Sep 18;104(38):15063-8)
chicken,
[000354] Canine cathelicidin (K9CATH): (Dev Comp Immunol. 2007;31(12):1278-
96),
[000355] Fowlicidin-3: (FEBS J. 2007 Jan;274(2):418-28.), (J Biol Chem. 2006
Feb
3;281(5):2858-67), (Immunogenetics. 2004 Jun;56(3):170-7.) chicken,
[000356] CMAP27: (Vet Immunol Immunopathol. 2005 Jul 15;106(3-4):321-7),
CA 02696833 2010-01-11
56
[000357] Fish (cathelicidin from Atlantic cod and Atlantic salmon) Maier VH et
al. Mol
Immunol. 2008 Jul 7.
[000358] Peptides with enhanced LPS neutralization and reduced pro-
inflammatory
activity are also included. Such peptides, for example 18-mer LLKKK or GKE and
P60,
P60.4, P60.4-Ac, CAP11 (cationic antibacterial polypeptide of 11 kDa),CAP18,
GSLL-39,
SMAP-29, and others as well as methods of discovering such peptides are
disclosed in
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY Sept. 2002, p. 972-
982 (18-mer LLKKK),ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2006,
p. 2983-2989 Vol. 50, No. 9 (GKE) , Laryngoscope. 2008 May;118(5):816-20,
Inflamm
Res. 2004 Nov;53(11):609-22, Antimicrob Agents Chemother. 2005 Ju1;49(7):2845-
50,
Protein Expr Purif. 2004 Sep;37(l):229-35, Int J Antimicrob Agents. 2004
Jun;23(6):606-12,
Am J Respir Crit Care Med. 2004 Jan 15;169(2):187-94, Eur J Biochem. 2002
Feb;269(4):1181-9, Surgery. 1995 Jun;117(6):656-62, Prog Clin Biol Res.
1995;392:317-26,
peptides27(2006)649-660(P60,P60.4,P60.4-Ac),and P18asin Biotechnol
Lett (2008) 30:1183-1187 and are all incorporated by reference herein. In
particularly, such
peptides having LPS neutralizing activity are included for the treatment of
metabolic
diseases, obesity and insulin resistance as is demonstrated with cathelicidin
in example 4
below.
[000359] Treatment using analogs of these above peptides, peptide fragments
and proteins
can be formed by modification as described below so as to make the analog
(modified
cathelicidin peptide or modified peptide fragment) more stable in blood (as
described below)
while preventing their degradation into their pro-inflammatory fragments by
extracellular
endogenous protease. Methods of evaluating and discovering suitable analogs or
fragments
of cathelicidin and other AMPs can, for example, be through the use of assays
as disclosed in
example 9 below.
[000360] Many different analogs of the above fragments can be made as for
example as
described in United States Patent 4242256. There Compounds being analogs of a
dipeptide
in which the nitrogen atom of the linking amide group of the dipeptide is
replaced by
trivalent group and in which, optionally, the carbonyl function of this
linking group is
replaced by the divalent group --CH2 -- are of value in the synthesis of
isosterically
modified peptides.
[000361] As used herein, the term "peptide" includes native peptides (either
degradation
products, synthetically synthesized peptides or recombinant peptides) and
peptidomimetics
CA 02696833 2010-01-11
57
(typically, synthetically synthesized peptides), such as peptoids and
semipeptoids which are
peptide analogs, which may have, for example, modifications rendering the
peptides more
stable while in a body or more capable of penetrating into target cells. Such
modifications
include, but are not limited to N terminus modification, C terminus
modification, peptide
bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S=O, 0=C-
NH,
CH2-O, CH2-CH2, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue
modification. Methods for preparing peptidomimetic compounds are well known in
the art
and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd.,
Chapter
17.2, F. Choplin Pergamon Press (1992).
[000362] Peptide bonds (-CO-NH-) within the peptide may be substituted, for
example, by
N-methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)H-C-O-O-C(R)-N-),
ketomethylen
bonds (-CO-CH2-), a-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g.,
methyl,
carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds
(-CS-
NH-), olefinic double bonds (-CH=CH-), retro amide bonds (-NH-CO-), peptide
derivatives
(-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on
the carbon
atom.
[000363] These modifications can occur at any of the bonds along the peptide
chain and
even at several (2-3) at the same time.
[000364] Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted
for synthetic
non-natural acid such as TIC, naphthylelanine (Nol), ring-methylated
derivatives of Phe,
halogenated derivatives of Phe or o-methyl-Tyr.
10003651 In addition to the above, the peptides of the present invention may
also include
one or more modified amino acids or one or more non-amino acid monomers (e.g.
fatty
acids, complex carbohydrates etc).
[000366] As used herein in the specification and in the claims section below
the term
"amino acid" or "amino acids" is understood to include the 20 naturally
occurring amino
acids; those amino acids often modified post-translationally in vivo,
including, for example,
hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino
acids
including, but not limited to, 2-aminoadipic acid, hydroxylysine,
isodesmosine, nor-valine,
nor-leucine and ornithine. Furthermore, the term "amino acid" includes both D-
and L-
amino acids.
[000367] The peptides of the present invention can be utilized in a linear or
cyclic form.
[000368] A peptide can be either synthesized in a cyclic form, or configured
so as to
CA 02696833 2010-01-11
58
assume a cyclic structure under suitable conditions.
[0003691 For example, a peptide according to the teachings of the present
invention can
include at least two cysteine residues flanking the core peptide sequence. In
this case,
cyclization can be generated via formation of S-S bonds between the two Cys
residues.
Side-chain to side chain cyclization can also be generated via formation of an
interaction
bond of the formula -(-CH2-)n-S-CH-2-C-, wherein n = 1 or 2, which is
possible, for
example, through incorporation of Cys or homoCys and reaction of its free SH
group with,
e.g., bromoacetylated Lys, Orn, Dab or Dap. Furthermore, cyclization can be
obtained, for
example, through amide bond formation, e.g., by incorporating Glu, Asp, Lys,
Om, di-amino
butyric (Dab) acid, di-aminopropionic (Dap) acid at various positions in the
chain (-CO-NH
or -NH-CO bonds). Backbone to backbone cyclization can also be obtained
through
incorporation of modified amino acids of the formulas H-N((CH2)n-COOH)-C(R)H-
COOH
or H-N((CH2)n-COOH)-C(R)H-NH2, wherein n = 1-4, and further wherein R is any
natural
or non-natural side chain of an amino acid.
10003701 Depending on the application and purpose, any of various AMPs/AMLs or
combinations of different AMPs/AML may be employed and/or regulated so as to
practice
the various embodiments of the present invention. Numerous examples of
AMPs/AMLs
suitable for use in the present invention are listed on the InternetlWorld
Wide Web at
http://www.bbcm.units.it/-tossi/pagl.htm, and are described hereinbelow.
[000371] Examples of AMPs/AMLs include defensins, cathelicidins, and
thrombocidins
(alternately termed "platelet microbicidal proteins [PMPs]").
[000372] Examples of defensins include alpha-defensins, beta-defensins, and
neutrophil
defensins.
[000373] Examples of alpha-defensins include alpha-defensin-1 to -6 (Mol
Immunol. 2003
Nov;40(7):463-7; J Clin Invest. 1985 Oct;76(4):1427-35).
[000374] Examples of beta-defensins include beta-defensin-1 (Genomics. 1997
Aug
1;43(3):316-20; Biochem Biophys Res Commun. 2002 Feb 15;291(1):17-22; FEBS
Lett.
1995 Jul 17;368(2):331-5; Paulsen F et al., J Pathol. 2002 Nov;198(3):369-77),
beta-
defensin-2 (Biochemistry. 2001 Apr 3;40(13):3810-6; Gene. 1998 Nov
19;222(2):237-44;
Paulsen F et al., J Pathol. 2002 Nov;198(3):369-77), beta-defensin-3 (Cell
Tissue Res. 2001
Nov;306(2):257-64; J Biol Chem. 2002 Mar 8;277(10):8279-89. Epub 2001 Dec 11;
J Biol
Chem. 2001 Feb 23;276(8):5707-13; Paulsen F et al., J Pathol. 2002
Nov;198(3):369-77),
beta-defensin-4 (J Immunol. 2002 Sep 1;169(5):2516-23), beta-defensin-5 (Am J
Pathol.
CA 02696833 2010-01-11
59
1998 May;152(5):1247-58; J Biol Chem. 1992 Nov 15;267(32):23216-25), and beta-
defensin-6 (FEBS Lett. 1993 Jan 4;315(2):187-92; Crit Care Med. 2002
Feb;30(2):428-34).
[000375] Beta-defensins include those encoded by five conserved beta-defensin
gene
clusters identified using a computational search strategy (Schutte BC. et al.,
2002. Proc Nati
Acad Sci U S A. Feb 19; 99(4):2129-33).
[000376] Examples of neutrophil defensins include neutrophil alpha-defensins
and
neutrophil beta-defensins.
[000377] Examples of neutrophil alpha-defensins include neutrophil alpha-
defensin-
1//human neutrophil peptide (HNP)-1 (J Clin Invest. 1985 Oct;76(4):1436-9;
Paulsen F et
al., J Pathol. 2002 Nov;198(3):369-77), neutrophil alpha-defensin-2/HNP-2 (J
Clin Invest.
1985 Oct;76(4):1436-9; Paulsen F et al., J Pathol. 2002 Nov;198(3):369-77),
neutrophil
alpha-defensin-3/HNP-3 (J Clin Invest. 1985 Oct;76(4):1436-9; Paulsen F et
al., J Pathol.
2002 Nov;l98(3):369-77), neutrophil alpha-defensin-4/HNP-4 (Mol hnmunol. 2003
Nov;40(7):463-7), human defensin-5 (HD-5; D.E. Jones and C.L. Bevins, J. Biol.
Chem. 267
(1992), pp. 23216-23225; J Biol Chem. 1992 Nov 15;267(32):23216-25; Mol
Immunol.
2003 Nov;40(7):469-75; Quayle AJ et al., Am. J. Pathol. 1998, 152:1247-1258;
FEBS Lett.
1993 Jan 4;315(2):187-92; D.E. Jones and C.L. Bevins, FEBS Lett. 315 (1993);
Paulsen F et
al., J Pathol. 2002 Nov;198(3):369-77), and human defensin-6 (HD-6; Mol
Immunol. 2003
Nov;40(7):463-7), human defensin-5 (HD-5; D.E. Jones and C.L. Bevins, J. Biol.
Chem. 267
(1992), pp. 23216-23225; J Biol Chem. 1992 Nov 15;267(32):23216-25; Mol
Immunol.
2003 Nov;40(7):469-75; Quayle AJ et al., Am. J. Pathol. 1998, 152:1247-1258;
FEBS Lett.
1993 Jan 4;315(2):187-92; D.E. Jones and C.L. Bevins, FEBS Lett. 315 (1993);
Paulsen F et
al., J Pathol. 2002 Nov;198(3):369-77).
[000378] Examples of cathelicidins include LL-37/hCAP18 (LL-37) in humans
(Curr Drug
Targets Inflamm Allergy. 2003 Sep;2(3):224-31; Eur J Biochem. 1996 Jun
1;238(2):325-32;
Paulsen F et al., J Pathol. 2002 Nov;198(3):369-77). LL-37 is a 37 amino acid
residue
peptide corresponding to amino acid residue coordinates 134-170 of its
precursor
hCAPl8/human cathelicidin antimicrobial peptide protein (GenBank: ACCESSION
NP_004336; VERSION NP_004336.2 GI:39753970; REFSEQ: accession NM004345.3).
The proliferation and angiogenesis pathway of LL-37 can be inhibited using
pertussis toxin,
an inhibitor of G-protein coupled receptors (Koczulla, R. et al., 2003.
J.Clin.Invest
111:1665-1672). Similar AMPs/AMLs are listed in the following patent
applications: US
2003120037, US 200309626, US20020141620, US20020507, CA 2383172, US
CA 02696833 2010-01-11
20020072495 and are incorporated by reference herein. The human antibacterial
cathelicidin
precursor hCAP-18, is synthesized in myelocytes and metamyelocytes and
localizes to
specific granules in neutrophils (Blood. 1997 Oct 1;90(7):2796-803).
[000379] Examples of AMP-like molecules include chemokines or fragments
thereof.
[000380] Examples of such chemokines include CC chemokines and CXC chemokines.
Considerable overlap of chemokine and AMP functions has been demonstrated
(Cole et al.,
2001. J. Immunol. 167:623), and certain chemokines and defensins have actually
been
shown to bind to the same chemokine receptor, CCR6. Defensins and certain
chemokines
strikingly share similar characteristics, including size, disulfide bonding,
interferon (IFN)
inducibility, cationic charge, and more. Relevant similarities between
chemokines and
AMPs are described in the literature (refer, for example, to Durr and Peschel,
2002. Infection
and Immunity 70:6515). As such various chemokines and antibodies specific for
such
chemokines may be employed in various applications of the present invention.
[000381] Examples of such CC chemokines include CCL1, CCL5/RANTES (Infect
Immun. 2002 Dec;70(12):6524-33; Eur J Biochem 1996 Apr 1;237(1):86-92), CCL8,
CCL11, CCL17, CCL18, CCL19, CCL20/activation-regulated chemokine
(LARC)/macrophage inflammatory protein-3alpha (MIP-3alpha)/Exodus-1/Scya20
(Yang D
et al., Journal of Leukocyte Biology Volume 74, September 2003;74(3):448-55),
CCL21,
CCL22, CCL25, CCL27/CTACK, and CCL28 (J Biol Chem. 2000 Ju121;275(29):22313-
23;
J Immunol. 2003 Feb 1;170(3):1452-61). CCL chemokines are described in Yang D
et al.,
Journal of Leukocyte Biology Volume 74, September 2003;74(3):448-55.
[000382] Examples of such CXC chemokines include CXCL1, CXCL2, CXCL3, CXCL4
(PF-4), CXCL7/NAP-2, CXCL8/IL-8, CXCL9 (MIG; Yang D et al., Journal of
Leukocyte
Biology Volume 74, September 2003;74(3):448-55), CXCL10/IP-10 (The Journal of
Immunology, 2001, 167: 623-627), CXCL11/ IP-9/I-TAC (The Journal of
Immunology,
2001, 167: 623-627), CXCL12/SDF-1 (Yang D et al., Journal of Leukocyte Biology
Volume
74, September 2003;74(3):448-55), CXCL13, CXCL14, connective tissue activating
peptide
3 (CTAP-3; Infect Immun. 2002 Dec;70(12):6524-33; Eur J Biochem 1996 Apr
1;237(1):86-
92), and CTAP-3 precursor platelet basic protein. CXC chemokines are described
in Yang D
et al., Journal of Leukocyte Biology Volume 74, September 2003;74(3):448-55.
10003831 Examples of fibrinopeptides include fibrinopeptide-A (Infect Immun.
2002
Dec;70(12):6524-33; Eur J Biochem 1996 Apr 1;237(l):86-92), fibrinopeptide-B
(Infect
Immun. 2002 Dec;70(12):6524-33; Eur J Biochem 1996 Apr 1;237(l):86-92).
CA 02696833 2010-01-11
61
[000384] Examples of AMPs/AM]Ls further include XCLI (Yang D et al., Journal
of
I.eukocyte Biology Volume 74, September 2003;74(3):448-55), MIP-lbeta (Yang D
et al.,
Journal of Leukocyte Biology Volume 74, September 2003;74(3):448-55).
[000385] Further examples of AMPs/AMLs include adrenomedullin (Regul Pept.
2003 Apr
15;112(1-3):147-52; J Biol Chem 1998 Jul 3;273(27):16730-8), alpha-melanocyte
stimulating hormone (Cutuli M et al., J Leukoc Biol. 2000 Feb;67(2):233-9;
Neuroimmunomodulation-2002-2003;10(4):208-16), an angiogenin (Nature
Immunology,
March 2003), angiogenin-4 (Nature Immunology, March 2003), antibacterial
peptides
B/enkelytin (Neuroimmunol 2000 Sep 22;109(2):228-35), antileukoprotease (ALP;
Biochem
Biophys Res Commun. 1998 Jul 30;248(3):904-9; Am J Respir Crit Care Med 1999
Jul;160(1):283-90), a lymphokine-activated killer cell-derived antimicrobial
peptide, a
platelet-derived antimicrobial peptide, antimicrobial peptide PR39, an
apolipoprotein, an
apolipoprotein-C, apolipoprotein-C2 (Hypertens Pregnancy 2002;21(3):199-204;
Peptides.
2000 Mar;21(3):327-30), apolipoprotein-C3 (Hypertens Pregnancy 2002;21(3):199-
204;
Peptides. 2000 Mar;21(3):327-30), an apolipoprotein-E (Hypertens Pregnancy
2002;21(3):199-204; Peptides. 2000 Mar;21(3):327-30), apolipoprotein-E2 (Brain
Res 1997
Feb 21;749(1):135-8; Biochemistry 2002 Oct 1;41(39):11820-3; Eur J Clin Chem
Clin
Biochem 1997 Aug;35(8):581-9), a bactericidal/permeability-increasing protein
(Paulsen F
et al., J Pathol. 2002 Nov; 198(3):369-77; Mol Microbiol 1995 Aug 17:523-31; J
Biol Chem
1987 Nov 5;262(31):14891-4), a bone morphogenetic protein (BMP), BMP-2/4, BMP-
5,
buforin, calcitermin (FEBS Lett. 2001 Aug 24;504(1-2):5-10), a cathepsin,
cathepsin B,
cathepsin G, cathepsin K, a lysosomal cathepsin, a chromogranin (Blood 2002
Jul
15;100(2):553-9), chromogranin A (Blood 2002 Jul 15;100(2):553-9),
chromogranin B
(Blood 2002 Jul 15;100(2):553-9), chymase (Immunology 2002 Apr;105(4):375-90),
connective tissue activating peptide-3, cystatin (APMIS. 2003 Nov;l l
l(11):1004-1010; Biol
Chem Hoppe Seyler 1988 May;369 Suppl:l91-7), DCD-1 (J Immunol Methods. 2002
Dec
1;270(1):53-62), dermicidin (Nat Immunol. 2001 Dec;2(12):1133-7), elastase-
specific
inhibitor/SKALP (skin-derived antileucoproteinase)/elafin (Biochem Soc Trans.
2002
Apr;30(2):111-5; J Invest Dermatol 2002 Jul;119(1):50-5), eNAP-1, eosinophil
cationic
protein (Peptides. 2003 Apr;24(4):523-30; J Immunol 2002 Mar 168:2356-64; Eur
J
Biochem 1996 Apr 1;237(1):86-92; Peptides. 2003 Apr;24(4):523-30; J Exp Med
1989 Jul
1;170(1):163-76), ESC42, ESkine, FALL-39 (Proc Natl Acad Sci U S A. 1995 Jan
3;92(1):195-9), Fas ligand (FasL; Berthou C etal., J Immunol. 1997 Dec
1;159(11):5293-
CA 02696833 2010-01-11
62
300), fractalkine, a glycosaminoglycan, granulysin (Reprod Biol Endocrinol.
2003 Nov 28; J
Immunol. 2003 Mar 15;170(6):3154-61; Cancer Immunol Immunother. 2002
Jan;50(11):604-14. Epub 2001 Nov; Expert Opin Investig Drugs. 2001
Feb;10(2):321-9),
granzyme B (Berthou C et al., J Immunol. 1997 Dec 1;159(11):5293-300), HAX-1,
heparin
binding protein/CAP37 (Paulsen F et al., J Pathol. 2002 Nov;198(3):369-77; J
Clin Invest
1990 May;85(5):1468-76), a hepcidin (J Biol Chem. 2001 Mar 16;276(11):7806-10.
Epub
2000 Dec 11; Eur J Biochem 2002 Apr 269:2232-7), an HE2, HE2alpha (Biol
Reprod. 2002
Sep;67(3):804-13), an HE2alpha C-terminal fragment (Biol Reprod. 2002
Sep;67(3):804-
13), HE2betal (Biol Reprod. 2002 Sep;67(3):804-13), an HE2-gene derived
transcript,
histatin (Antimicrob Agents Chemother 2001 Dec 45:3437-44; Biochem Cell Biol.
1998;76(2-3):247-56), a histone, histone H2A, histone H-2b (Peptides. 2003
Apr;24(4):523-
30; J Immunol 2002 Mar 168:2356-64; Eur J Biochem 1996 Apr 1;237(1):86-92),
HMG-17,
HtpG, an HtpG homolog, HS 1 binding protein, interleukin-8, lactoferrin (Eur J
Nucl Med.
2000 Mar;27(3):292-301; Paulsen F et al., J Pathol. 2002 Nov;198(3):369-77; J
Mammary
Gland Biol Neoplasia 1996 Jul;1(3):285-95), a lymphokine-activated killer
(LAK) cell AMP
(Hua Xi Yi Ke Da Xue Xue Bao 2002 Jan;33(1):87-90), lysozyme (Paulsen F et
al., J Pathol.
2002 Nov;198(3):369-77; Anat Embryol (Berl) 2002 Jul;205(4):315-23), a
macrophage
inflammatory protein (MIP), MIP-lalpha, MIP-lbeta, MIP-3alpha, a mast cell
granule serine
proteinase (Immunology 2002 Apr;105(4):375-90), a matrix metalloproteinase
(MMP),
MMP-2, MMP-7 (Paulsen F et al., J Pathol. 2002 Nov;198(3):369-77), migration
inhibitory
factor (J Immunol. 1998 Sep 1;161(5):2383-90; Scand J Infect Dis.
2003;35(9):573-6),
MMP-9, MRP8 (Behring Inst Mitt. 1992 Apr;(91):126-37), MRP14 (Behring Inst
Mitt. 1992
Apr;(91):126-37), neutrophil gelatinase-associated lipocalin (NGAL; Exp
Dermatol. 2002
Dec;l 1(6):584-91; Mol Cell. 2002 Nov;10(5):1033-43), neutrophil lysozyme (Int
J
Antimicrob Agents. 1999 Sep;13(1):47-51), an opioid peptide, perforin (Berthou
C et al., J
Immunol. 1997 Dec 1;159(11):5293-300), phospholipase A(2) (PLA(2); Peptides.
2003
Apr;24(4):523-30; J Exp Med 1989 Jul 1;170(l):163-76), platelet basic protein
(Infect
Immun. 2002 Dec;70(12):6524-33; Eur J Biochem 1996 Apr 1;237(1):86-92),
platelet factor-
4, psoriasin (J Histochem Cytochem. 2003 May;51(5):675-85; Glaser R et al., J
Invest
Dermatol 117: 768(abstr 015)), retrocyclin (Proc Natl Acad Sci U S A 2002 Feb
19;99(4):1813-8), secretory leukocyte proteinase inhibitor (SLPI; Shugars DC
et al.,
Gerontology. 2001 Sep-Oct;47(5):246-53; Biochem Soc Trans. 2002 Apr;30(2):111-
5; J
Invest Dermatol 2002 Jul;119(1):50-5), secretory phospholipase A(2) (Peptides.
2003
CA 02696833 2010-01-11
63
Apr;24(4):523-30; J Immunol 2002 Mar 168:2356-64; Eur J Biochem 1996 Apr
1;237(1):86-
92; Paulsen F et al., J Pathol. 2002 Nov;198(3):369-77), substance P, an S100
calcium-
binding protein, S100A7, S100A8, S100A9, a thymosin, thymosin beta-4 (Infect
Immun.
2002 Dec;70(12):6524-33; Eur J Biochem 1996 Apr 1;237(l):86-92; Infect Immun.
2002
Dec;70(12):6524-33; Eur J Biochem 1996 Apr 1;237(1):86-92), thyinus and
activation-
regulated chemokine (TARC), TL1A, tryptase (Immunology 2002 Apr;105(4):375-
90),
ubiquicidin (Eur J Nucl Med. 2000 Mar;27(3):292-301; Hiemstra PS, van den
Barselaar MT
et al., J Leukocyte Biol 1999; 66: 423-428; J Nucl Med 2001 May 42:788-94),
and
urokinase-type plasminogen activator.
[000386] The AMP/AML may any one of 28 potential candidates for defensin like
peptides
which were computationally discovered. (Am J Respir Cell Mol Biol. 2003
Jul;29(1):71-80).
[000387] As described hereinabove, the present invention can be used to treat
any of
various diseases which are associated with: (i) a tumor; (ii) inflammation;
(iii) a wound; (iv)
autoimmunity; (v) dysregulation of growth/differentiation of a cell/tissue;
(vi) dysregulation
of growth/differentiation balance of a cell/tissue; and/or (vii) angiogenesis.
10003881 Examples of diseases which can be treated according to the present
invention are
listed in International Pub. No. WO 2004-056307.
[000389] Examples of diseases which can be treated according to the present
invention are
also as follows.
[000390] Examples of tumors include a skin tumor, Osteosarcoma, Ewing's
sarcoma,
Chondrosarcoma, Malignant fibrous histiocytoma, Fibrosarcoma, Chordoma ,
osteoid
osteoma, osteoblastoma, osteochondroma, enchondroma, chondromyxoid fibroma,
and giant
cell tumor, lymphoma and multiple myeloma, a keratinocytic tumor, a
gastrointestinal
tumor, a carcinoma, a melanoma, a squamous cell tumor, oral squamous cell
carcinoma,
lymphoma, a malignant tumor, a benign tumor, a solid tumor, a metastatic tumor
and a non-
solid tumor.
10003911 The concentration of human beta-defensin-2 in oral squamous cell
carcinoma is
much higher than in normal oral epithelium (Sawaki, K. et al., 2002.
Anticancer Res.
22:2103-2107). There is a genetic link between proliferation of cells and
cancer.
Impairment of regulation of proliferation and differentiation lead to cancer
development. A
developing tumor needs help from neighboring cells in order to become
cancerous.
Overexpression or overactivity of cytokines is involved in orchestrating these
processes.
Continuous assault by chronic inflammation contributes to the transformation
of cells as
CA 02696833 2010-01-11
64
well. Angiogenesis is an important process for cancer development. AMPs are
inductors of
angiogenesis (Koczulla, R. et al., 2003. J.Clin.Invest 111:1665-1672).
Therefore inhibiting
differentiation and proliferation as well as angiogenesis by antagonists to
AMPs and
cytokines can be used to treat cancer. Urokinase-type plasminogen activator
(uPA), has
antimicrobial properties (Gyetko, MR. et al., 2002. J.Immunol. 168:801-809)
and is
involved in metastatic spreading of malignant cells. The in vitro and in vivo
findings
suggest that alpha-defensins are frequent peptide constituents of malignant
epithelial cells in
renal cell carcinoma with a possible direct influence on tumor proliferation
(Muller, CA. et
al., 2002. Am.J.Pathol. 160:1311-1324). Certain anti-angiogenic compounds were
found to
have potent anticancer property in vivo experimental studies. Therefore
inhibition of
angiogenic AMPs such as LL-37 is one form of treatment for cancer. Matrix
metalloproteinases (MMPs) are known to play an important role in extracellular
matrix
remodeling during the process of tumor invasion and metastasis. Overexpression
of MMP-2
and MMP-9 proteins was observed in a large percentage of ESCC tumors,
respectively
localized in tumor cell cytoplasm and stromal elements (J Cancer Res Clin
Oncol. 2003 Oct
16).
[0003921 BMP-2/4 and BMP-5 but not BMPR-IA might be involved in the metastasis
of
oral carcinoma cells (Overexpression of BMP-2/4, -5 and BMPR-IA associated
with
malignancy of oral epithelium Oral Oncol. 2001, 37:225-33.)
[000393] Examples of diseases include an idiopathic/inflammatory disease, a
chronic/inflammatory disease, an acute/inflammatory disease, an inflammatory
cutaneous
disease, an inflammatory gastrointestinal disease, a tumor associated with
inflammation, an
allergic disease, an autoimmune disease, an infectious disease, a malignant
disease, a
transplantation related disease, an inflammatory degenerative disease, an
injury associated
with inflammation, a disease associated with a hypersensitivity, an
inflammatory
cardiovascular disease, an inflammatory glandular disease, an inflammatory
hepatic disease,
an inflammatory neurological disease, an inflammatory musculo-skeletal
disease, an
inflammatory renal disease, an inflammatory reproductive disease, an
inflammatory systemic
disease, an inflammatory connective tissue disease, an inflammatory
neurodegenerative
disease, necrosis, an inflammatory disease associated with an implant, an
inflammatory
hematological disease, an inflammatory eye disease, an inflammatory
respiratory disease.
[000394] Examples of cutaneous/inflammatory diseases include psoriasis,
dandruff,
pemphigus vulgaris, lichen planus, atopic dermatitis, excema, scleroderma,
dermatomyositis,
CA 02696833 2010-01-11
alopecia, blepharitis, skin carcinoma, melanoma, squamous cell carcinoma, acne
vulgaris,
erythema toxicum neonatorum, folliculitis, skin wrinkles, autoimmune bullous
skin disease,
bullous pemphigoid, pemphigus foliaceus, dermatitis, and drug eruption.
[000395] Examples of gastrointestinal/inflammatory diseases include Crohn's
disease,
chronic autoimmune gastritis, autoimmune atrophic gastritis, primary
sclerosing cholangitis,
autoimmune achlorhydra, colitis, ileitis, chronic inflammatory intestinal
disease,
inflammatory bowel syndrome, chronic inflammatory bowel disease, celiac
disease, an
eating disorder, gallstones and a gastrointestinal ulcer.
10003961 Crohn's disease is an inflammatory bowel disease. Since the bowel is
exposed to
the outer environment, the importance of AMPs as part of its defense and
normal cellular
regulation is important, as in skin, and the activity of the AMPs plays an
important role in
the normal physiology as well as pathological conditions in these tissues.
Abnormalities in
the expression and/or activity of the AMPs will contribute to pathologies in
these tissues.
Paneth cells (a specific type of cell in the intestine) are required to help
promote normal
vessel formation in cooperation with bacteria - mice absent Paneth cells were
incapable of
appropriate blood vessel formation. Of note, colonization by one particular
type of bacteria
commonly found in normal mouse and human intestine, called Bacteroides
thetaiotaomicron,
or B. thetaiotaomicron, stimulated blood vessel development as efficiently as
implantation
of a whole microbial society. The conclusion, B. thetaiotaomicron and Paneth
cells work
together to stimulate postnatal blood vessel formation. The ability of AMPs to
act as
chemoattractants for cells of the innate- and adaptive-immune system plays an
important role
in perpetuating chronic inflammation in the gastrointestinal tract (Cunliffe,
RN, Mahida,
YR., 2003. J Leukoc Biol. Oct 2 [Epub ahead of print]). The AMP LL-37, beta-
defensins,
human alpha-defensins, beta-defensins (including HD5), HN-6, lysozyme and
secretory
PLA2, TL1A, are expressed in Paneth cells and intestine, secretory epithelial
cells in the
small intestine (Ghosh, D. et al., 2002. Nat.Immunol. 3:583-590; Fellermann,
K. et al., 2003.
Eur. J. Gastroenterol. Hepatol. 15:627-634). Where alpha-defensins are
overexpressed, they
are chemoattract naive T and immature dendritic cells and dendritic cells and
monocytes
(Yang, D. et al., 2000. J. Leukoc. Biol. 68:9-14; Risso, A., 2000. J. Leukoc.
Biol. 68:785-
792; Territo, MC. et al., 1989. J.Clin.Invest 84:2017-2020). Human alpha-
defensins as well
as other AMPs contribute to local intestinal host defense as part of innate
immunity and may
be of major relevance in microbial infection and chronic inflammatory bowel
disease
(Wehkamp, J. et al., 2002. Dig. Dis. Sci. 47:1349-1355). The alpha-defensins
convert an
CA 02696833 2010-01-11
66
acute inflammation to a chronic inflammation by downregulating human
polymorphonuclear
leukocyte chemotaxis, for example, alpha-defensin-1/human neutrophil protein-
1, acts as an
antichemotactic agent for human polymorphonuclear leukocytes). It is known
that chronic
inflammation is commonly characterized by the presence of increased cell
proliferation and
connective tissue than exudate with the presence of lymphocytes and plasma
cells rather than
polymorphonuclear leukocytes. Thus, suitable regulation of such AMPs/AMLs can
be used
to treat diseases such as inflammatory bowel disease, Crohn's disease and
ulcerative colitis.
[000397] Gastritis is an inflammatory condition of the stomach. There are two
main forms
of gastritis, A and B. Gastritis type A is considered to develop in an
autoimmune process.
In both types there is a role for infectious agents such as Helicobacter
pylori. AMPs are
involved in both processes. Defensins are involved in pathogenesis of
gastritis (Bajaj-Elliott,
M. et al., 2002. Gut 51:356-361). Thus, suitable regulation of such AMPs/AMLs
can be
used to treat diseases such as gastritis.
[000398] Examples of allergic/inflammatory diseases include asthma, hives,
urticaria, a
pollen allergy, a dust mite allergy, a venom allergy, a cosmetics allergy, a
latex allergy, a
chemical allergy, a drug allergy, an insect bite allergy, an animal dander
allergy, a stinging
plant allergy, a poison ivy allergy, anaphylactic shock, anaphylaxis, atopic
allergy and a food
allergy.
[000399] Examples of hypersensitivity include Type I hypersensitivity, Type II
hypersensitivity, Type III hypersensitivity, Type IV hypersensitivity,
immediate
hypersensitivity, antibody mediated hypersensitivity, immune complex mediated
hypersensitivity, T lymphocyte mediated hypersensitivity, delayed type
hypersensitivity,
helper T lymphocyte mediated hypersensitivity, cytotoxic T lymphocyte mediated
hypersensitivity, TH1 lymphocyte mediated hypersensitivity, and TH2 lymphocyte
mediated
hypersensitivity.
[000400] Examples of cardiovascular/inflammatory and/or inflammatory
hematological
diseases include atherosclerosis, Takayasu's arteritis, polyarteritis nodosa,
Raynaud's
phenomenon, temporal arteritis, inflammatory anemia, inflammatory lymphopenia,
pernicious anemia, occlusive disease, myocardial infarction, thrombosis,
Wegener's
granulomatosis, lymphoma, leukemia, Kawasaki syndrome, anti-factor VIII
autoimmune
disease, necrotizing small vessel vasculitis, microscopic polyangiitis, Churg
and Strauss
syndrome, pauci-immune focal necrotizing glomerulonephritis, crescentic
glomerulonephritis, antiphospholipid syndrome, antibody induced heart failure,
CA 02696833 2010-01-11
67
thrombocytopenic purpura, autoimmune hemolytic anemia, cardiac autoimmunity,
Chagas'
disease, iron-deficiency anemia, and anti-helper T lymphocyte autoimmunity.
[000401] Inflammation is part of the pathological process leading to the
development of
atherosclerosis. Chlamydia pneumonia as well as other various microorganisms
serve as
potential etiological factors, linking inflammation and atherosclerosis.
Inflammation is a
predisposing factor as well as a consequence of several CNS pathologies.
Inflammation is
part of the pathophysiologic processes occurring after the onset of cerebral
ischemia in
ischemic stroke, as well as other CNS pathologies such as head injury and
subarachnoid
hemorrhage. In addition, inflammation in the CNS or in the periphery by itself
is considered
as a risk factor for the triggering the development of cerebral ischemia.
Endothelial cells
express and secrete AMPs. Cationic antimicrobial protein of 37 kDa (CAP37)
also termed
heparin binding protein, originally isolated from human neutrophils, is an
important
multifunctional inflammatory mediator is expressed within the vascular
endothelium
associated with atherosclerotic plaques (Lee, TD. et al., 2002. Am.J.Pathol.
160:841-848).
Human beta-defensin-2 is expressed by astrocytes and its expression is
increased in response
to cytokines and LPS (Hao, HN. et al., 2001. J.Neurochem. 77:1027-1035).
Therefore, AMP
regulation can be used for treatment or prevention of these conditions.
[000402] Anemia associated with inflammatory chronic diseases is one of the
body's
methods of fighting pathogens by reducing available inter cellular iron uptake
of pathogens.
Iron is absorbed by neutrophils. Sometimes chronic inflammation can occur
without the
presence of pathogens. Under chronic inflammatory conditions, cytokines induce
a
diversion of iron traffic leading to hypoferremia. Such as in chronic
bacterial endocarditis,
osteomyelitis, juvenile rheumatoid arthritis, rheumatic fever, Crohn's
disease, and ulcerative
colitis and Chronic renal failure. Transferrin bound iron transports to
monocytes causing
anemia. This "transport" is thought to be related to AMP activity. Cytokines
IL-1, IL-6 and
TNF-beta initiate defensin production and defensin initiate the cytokine
production, the
result being iron over absorption by monocytes. The regulation of iron
transport by
cytokines is a key mechanism in the pathogenesis of anemia of chronic disease
(Ludwiczek,
S. et al., 2003. Blood 101:4148-4154). Therefore, regulation of AMPs can be
used to
regulate iron level homeostasis. Hepcidin AMP is known to regulate iron
uptake, therefore
inhibiting hepcidin can be used to increase iron absorption (Nicolas, G. et
al., 2002. Blood
Cells Mol.Dis. 29:327-335). However, there are other AMPs indirectly involved
in iron
regulation such as defensin and LL-37. Since HNP-1 is a non-specific defensive
peptide
CA 02696833 2010-01-11
68
present in neutrophils, it plays an important role in the protection against
diseases such as
oral lichen planus, leukoplakia, and glossitis associated with iron deficiency
(Mizukawa, N.
et al., 1999. Oral Dis. 5:139-142). Likewise all cationic neutrophil derived
AMPs would
induce iron hypoferremia when over expressed. Therefore regulating of these
AMPs can be
used to treat such diseases.
[000403] Leukocyte SLPI (secretory leukocyte proteinase inhibitor (SLPI))
expression
seems to be up-regulated in active Wegner's granulomatosis, therefore
inhibiting its activity
can be used to treat diseases such as Wegener's granulomatosis and other types
of vasculitis
[000404] Examples of glandular/inflammatory diseases include type I diabetes,
type II
diabetes, type B insulin resistance, Schmidt's syndrome, Cushing's syndrome,
thyrotoxicosis, benign prostatic hyperplasia, pancreatic disease, Hashimoto's
thyroiditis,
idiopathic adrenal atrophy, Graves' disease, androgenic alopecia, thyroid
disease, thyroiditis,
spontaneous autoimmune thyroiditis, idiopathic myxedema, ovarian autoimmunity,
autoimmune anti-sperm infertility, autoimmune prostatitis, Addison's disease,
and Type I
autoimmune polyglandular syndrome.
[000405] Diabetes mellitus is a systemic disease with several major
complications affecting
both the quality and length of life. One of these complications is periodontal
disease
(periodontitis). Periodontitis is much more than a localized oral infection.
(lacopino, AM.,
2001. Ann.Periodontol. 6:125-137). When diabetes mellitus is under therapeutic
control,
periapical and other lesions heal as readily as in nondiabetics (Bender, IB,
Bender, AB. et
al., 2003. J.Endod. 29:383-389). Recent studies on diseases which involve
insulin
insensitivity (e.g. obesity, type 2 diabetes and atherosclerosis) also show
increased cytokine
production and markers of inflammation. Evidence at present favors chronic
inflammation
as a trigger for chronic insulin insensitivity, rather than the reverse
situation. (Grimble, RF.,
2002. Curr.Opin.Clin.Nutr.Metab Care 5:551-559). Recent human studies have
established a
relationship between high serum lipid levels and periodontitis. Possible
causes are a high
glucose levels (such as hyperglycemia of diabetics) with added LDL levels such
as in high
diabetic patients are prone to elevated low density lipoprotein cholesterol
and triglycerides
(LDL/TRG) even when blood glucose levels are well controlled, lead to LPS-like
bondings
that induce AMP overexpression. Thus, the present invention can be used to
treat diabetes
and diabetes related diseases such as periodontitis and diabetes associated
healing
deficiencies.
10004061 Proliferative retinopathy is one of the chronic complications of
diabetes. The
CA 02696833 2010-01-11
69
process includes the development of abnormal blood vessels that might lead to
retinal
detachment and blindness. LL37 and other AMPs are involved in angiogenesis
(Koczulla,
R. et al., 2003. J.Clin.Invest 111:1665-1672), therefore LL-37 regulation can
be used to
prevent the development of newly formed blood vessels and therefore for
preventing
diabetes related eye diseases.
10004071 Examples of hepatic inflammatory diseases include primary biliary
cirrhosis,
active chronic hepatitis, lupoid hepatitis, autoimmune hepatitis, and hepatic
cirrhosis.
[000408] Examples of neurological inflammatory diseases include
neurodegenerative
disease, multiple sclerosis, Alzheimer's disease, Parkinson's disease,
myasthenia gravis,
motor neuropathy, Guillain-Barre syndrome, autoimmune neuropathy, Lambert-
Eaton
myasthenic syndrome, paraneoplastic neurological disease, paraneoplastic
cerebellar
atrophy, non-paraneoplastic stiff man syndrome, progressive cerebellar
atrophy,
Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea,
Gilles de la
Tourette syndrome, autoimmune polyendocrinopathy, dysimmune neuropathy,
acquired
neuromyotonia, arthrogryposis multiplex, optic neuritis, spongiform
encephalopathy,
migraine, headache, cluster headache, and stiff-man syndrome.
[000409] With respect to multiple sclerosis (MS), defensins and lactoferrins
exist in
cerebrospinal fluid (CSF). These peptides have antimicrobial expression in
some diseases
like pneumonia and meningitis, which may trigger a pathway. It seems that
pathways to MS
are similar to rheumatoid arthritis where AMPs reside in the synovial fluid
surrounding the
joint. Peptides involved are amongst others: IP-10, defensins and
lactoferrins, CAP37.
10004101 Examples of connective tissue inflammatory diseases include
arthritis,
rheumatoid arthritis, pyogenic arthritis, mixed connective tissue disease,
cholesteatoma,
relapsing polychondritis, autoimmune myositis, primary Sjogren's syndrome,
smooth muscle
autoimmune disease, myositis, tendinitis, a ligament inflammation, chondritis,
a joint
inflammation, a synovial inflammation, carpal tunnel syndrome, osteoarthritis,
ankylosing
spondylitis, a skeletal inflammation, an autoimmune ear disease, osteoporosis,
fibromyalgia,
periodontitis, and an autoimmune disease of the inner ear.
[000411] With respect to diseases such as arthritis, AMPs are expressed and
produced in
healthy and inflamed human synovial membranes. Deposition of the AMPs
lysozyme,
lactoferrin, secretory phospholipase A(2) (sPA(2)), matrilysin (MMP7), human
neutrophil
alpha-defensin-1, -2, and -3, human beta-defensin- 1, and human beta-defensin-
2 was
determined by immunohistochemistry. Expression of mRNA for the AMPs
bactericidal
CA 02696833 2010-01-11
permeability-increasing protein (BPI), heparin binding protein, LL37, human
alpha-
defensin-5, human alpha-defensin-6, and human beta-defensin-1, -2, and -3 was
analyzed by
reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR revealed
CAP37 and
human beta-defensin-1 mRNA in samples of healthy synovial membrane.
Additionally,
human beta-defensin-3 and/or LL37 mRNA was detected in synovial membrane
samples
from patients with pyogenic arthritis (PA), osteoarthritis (OA) or rheumatoid
arthritis (RA).
Immunohistochemistry has identified lysozyme, lactoferrin, sPA(2), and MMP7 in
type A
synoviocytes of all samples. Human beta-defensin-1 was only present in type B
synoviocytes of some of the samples. Immunoreactive human beta-defensin-2
peptide was
only visible in some inflamed samples. HNPl-3 was detected in both healthy and
inflamed
synovial membranes. The data suggest that human synovial membranes produce a
broad
spectrum of AMPs. Under inflammatory conditions, the expression pattern
changes, with
induction of human beta-defensin-3 in PA (LL37 in RA; human beta-defensin-3
and LL37 in
OA) as well as down-regulation of human beta-defensin-1 (Paulsen, F. et al.,
2002. J.Pathol.
198:369-377; Cunliffe, RN, Mahida, YR., 2003. J Leukoc Biol. Oct 2 [Epub ahead
of print]).
Thus regulating one or more of these AMPs or their activity will inhibit the
pathological
process in a disease such as arthritis.
[000412] Microbial mixed keratin-biofilms in cholesteatomas are caused by AMPs
which
are overexpressed (Jung, HH. et al., 2003. Laryngoscope 113:432-435; Chole,
RA, Faddis,
BT., 2002 Arch.Otolaryngol.Head Neck Surg. 128:1129-1133), AMPs such as LL-37
or
other defensins or other AMPs are involved. Therefore, suitable regulation of
such AMPs
can be used for treating diseases such as cholesteatomas.
[0004131 Examples of inflammatory renal diseases include diabetic nephropathy.
10004141 Examples of inflammatory reproductive diseases include repeated fetal
loss,
ovarian cyst, or a menstruation associated disease.
10004151 Examples of inflammatory systemic diseases include systemic lupus
erythematosus, systemic sclerosis, septic shock, toxic shock syndrome,
Reiter's syndrome,
and cachexia.
10004161 Examples of inflammatory infectious diseases include candidiasis, a
fungal
infection, mycosis fungoides, a chronic infectious disease, a subacute
infectious disease, an
acute infectious disease, a viral disease, a bacterial disease, a protozoan
disease, a parasitic
disease, a mycoplasma disease, gangrene, sepsis, a prion disease, influenza,
tuberculosis,
bacterial pneumonia, malaria, acquired immunodeficiency syndrome, chronic
fatigue
CA 02696833 2010-01-11
71
syndrome, and severe acute respiratory syndrome.
[000417] Examples of transplantation related/inflammatory diseases include
graft rejection,
chronic graft rejection, subacute graft rejection, acute graft rejection
hyperacute graft
rejection, rejection of an implant and graft versus host disease.
[000418] Examples of implants include a prosthetic implant, a breast implant,
a silicone
implant, a dental implant, a penile implant, a cardiac implant, an artificial
joint, a bone
fracture repair device, a bone replacement implant, a drug delivery implant, a
catheter, a
pacemaker, an artificial heart, an artificial heart valve, a drug release
implant, an electrode,
and a respirator tube.
[000419] Examples of injuries associated with inflammation include a skin
wound, an
abrasion, a bruise, a cut, a puncture wound, a laceration, an impact wound, a
concussion, a
contusion, a thermal burn, frostbite, a chemical burn, a sunburn, a
desiccation, a radiation
burn, a radioactivity burn, a smoke inhalation, a torn muscle, a pulled
muscle, a torn tendon,
a pulled tendon, a pulled ligament, a torn ligament, a hyperextension, a torn
cartilage, a bone
fracture, a pinched nerve and a gunshot wound.
[000420] Examples of inflammatory respiratory diseases include asthma,
allergic asthma,
diffuse panbronchiolitis, emphysema, idiopathic pulmonary fibrosis, cystic
fibrosis,
influenza, sinusitis, sinusitis and chronic obstructive pulmonary disease.
10004211 Examples of inflammatory eye diseases include dry-eye disease,
phacogenic
uveitis, blepharitis and sympathetic ophthalmia.
[000422] Dry eye disease is a chronic inflammatory eye disease. Is
particularly an issue
for post-menopausal women, the elderly, and patients with systemic diseases
such as
Sjogren's syndrome, rheumatoid arthritis, lupus and diabetes (37% of people
with diabetes
suffer from the disease and 28% of adults having the disease). Defensins act
as chemokines
to T-cells (Stern, ME, et aL, 2002. Invest Ophthalmol.Vis.Sci. 43:2609-2614).
[000423] For identifying and classifying disease, a kit comprising a reagent
useful for
identifying the level of cathelicidin in blood for identifying diseases types
is included. The
kit is compartmentalized to receive one or more of (i) an oligonucleotide for
detection of a
cathelicidin or fragment thereof; or (ii) an antibody for detection of
cathelicidin or a
fragment thereof.
[000424] As described above, preventing binding of AMPs/AMLs to cognate
receptors by
using ananlogues of same AMPs that compete with binding to same receptors
without
inducing the disease may be used to inhibit a biological process mediated by
binding of the
CA 02696833 2010-01-11
72
AMP/AML to the receptor. Over 50 AMPs/AMLs and over 20 receptors thereof are
involved disease pathogenesis, therefore inhibiting correct target
combinations of ligand and
receptors is essential for treatment of such diseases. Examples of such
AMPs/AMLs and
cognate receptors thereof, and examples of the types of diseases which can be
treated using
this approach are shown in Table 1.
10004251 Ample guidance for practicing methods and techniques of the present
invention,
and for obtaining and utilizing materials employed for practicing the present
invention is
provided in the literature of the art (refer, for example, to U.S. Patent
Application No.
20030044907)
10004261 Thus, the present invention enables for the first time relative to
the prior art,
treatment of any of various diseases by AMPs in particular by cathelicidin.
The present
invention clearly shows how cathelicidin is associated with biological
processes in
cells/tissues such as dysregulated growth/differentiation, dysregulated
growth/differentiation
balance, inflammation, and angiogenesis and autoimmunity. Using AMPs/AMLs,
and/or
inhibitors of pro-inflammatory fragments thereof is needed for treatment of
disease.
Further, in addition to treatment with AMPs, such as cathelicidin, and
functional fragments
and analogs thereof, the invention also provides a new medical use for the
treatment of
obesity and/or excess body weight that includes the administration of a
therapeutically
effective amount of one or more LPS neutralizing compounds selected from the
group
consisting of: BPI (bactericidal/permeability-increasing protein) and
fragments and
variations thereof such as NeuprexTM (rBPI21, opebacan) a modified recombinant
fragment
of BPI and MycoprexTM (both Xoma Corporation); protegrins such as protegrin-1;
lactoferrins such as lactoferricin; Nisin(s) and their variants (Mol
Microbiol. 2008
Jul;69(l):218-30); Heliomicin and its variants (e.g., ETD151) (International
Journal of
Antimicrobial Agents 25 (2005) 448-452; Biochemistry. 2001 Oct 9;40(40):11995-
2003);
and magainin (Biochemistry. 2003 Oct 28;42(42):12251-9), Colistin (polymyxin
E),
Polymyxin b(polymyxin b sulfate), and polymyxin derivatives Antimicrob Agents
Chemother. 2008 Jun 30, Antiendotoxin antibody Curcumin and lipopolysaccharide
binding
peptides, Lipid A analogs, phospholipid emulsion, and ethyl pyruvate Curr Opin
Anaesthesiol. 2008 Apr;21(2):98-104. Therefore, with regards to treating
obesity and
overweight, the term "cathelicidin" will include any of the above LPS
neutralizing
antimicrobials.
[000427] .
CA 02696833 2010-01-11
73
[000428] It is expected that during the life of this patent many relevant drug
screening
techniques will be developed and the scope of the phrase "method of
identifying a
compound" is intended to include all such new technologies a priori.
[000429] Additional objects, advantages, and novel features of the present
invention will
become apparent to one ordinarily skilled in the art upon examination of the
following
examples, which are not intended to be limiting. Additionally, each of the
various
embodiments and aspects of the present invention as delineated hereinabove and
as claimed
in the claims section below finds experimental support in the following
examples.
10004301 EXAMPLES
[000431] Reference is now made to the following examples, which together with
the above
descriptions illustrate the invention in a non limiting fashion.
[000432] Generally, the nomenclature used herein and the laboratory procedures
utilized in
the present invention include molecular, biochemical, microbiological and
recombinant
DNA techniques. Such techniques are thoroughly explained in the literature.
See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989);
"Current
Protocols in Molecular Biology" Volumes 1-III Ausubel, R. M., ed. (1994);
Ausubel et al.,
"Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore,
Maryland
(1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons,
New York
(1988); Watson et al., "Recombinant DNA", Scientific American Books, New York;
Birren
et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold
Spring Harbor
Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828;
4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory
Handbook",
Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology"
Volumes I-III
Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical
Immunology" (8th Edition),
Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected
Methods in
Cellular Immunology", W. H. Freeman and Co., New York (1980); available
immunoassays
are extensively described in the patent and scientific literature, see, for
example, U.S. Pat.
Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517;
3,879,262;
3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;
5,011,771
and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic
Acid
Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription
and Translation"
Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney,
R. I., ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide
to
CA 02696833 2010-01-11
74
Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317,
Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic
Press,
San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and
Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which
are
incorporated by reference as if fully set forth herein. Other general
references are provided
throughout this document. The procedures therein are believed to be well known
in the art
and are provided for the convenience of the reader.
[000433] 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 this
invention belongs. Although methods and materials similar or equivalent to
those described
herein can be used in the practice or testing of the present invention,
suitable methods and
materials are described below.
[000434] In the examples that follow, all animal work was performed under
guidelines
approved by either the Hebrew University of Jerusalem Animal Care and Ethics
Committee
(EAE model, obesity model, insulin resistance and periodontitis model) and the
University
of Tennessee USA (CIA model and osteoporosis model).
[000435] Example 1
10004361 Use of cathelicidins for optimal treatment of arthritis and rheumatic
diseases
such as rheumatoid arthritis which are associated with inflammation,
autoimmunity.
[000437] Background:
10004381 Diseases associated with inflammation, autoimmunity and/or skin
cell/tissue
proliferation/differentiation imbalance include numerous diseases, such as
arthritis, for
which no optimal therapy exists. Cathelicidin hCAP- 18 pro-sequence and its
active form
(LL-37) is expressed in the bone marrow.
[000439] The present inventors have hypothesized that regulating such
AMPs/AMLs as
cathelicidin may be used for treating diseases such as arthritis. While
reducing the present
invention to practice, a method of using the 34a.a. cathelicidin (mCRAMP)
(GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ) for optimal treatment in an
Collagen induced arthritis mouse model of the human disease associated with
inflammation,
autoimmunity such as in rheumatoid arthritis, Sjogren's, scleroderma,
dermatomyositis,
Systemic Lupus Erythematosus, sarcoidosis was demonstrated for the first time,
as described
below, thereby overcoming the limitations of the prior art.
10004401 The present in-vivo experiment uses a mouse model of collagen induced
arthritis.
CA 02696833 2010-01-11
[0004411 Materials:
Antimicrobial peptides (AMPs): Synthetic peptides for the Mouse Cathelicidin
mCRAMP
34a.a. was purchased from Biosight Ltd (Israel). Chick native CII purchase
from Sigma
or Chondrex), al(II) chains or CB11 fragment of CII. 10 mM acetic acid, filter
sterilized
with 0.2-um filter Incomplete Freund's adjuvant (IFA; e.g., Difco).
Mycobacterium
tuberculosis (strain H37Ra; heat-killed; available by writing to Ministry of
Agriculture,
Fisheries, and Food, Central Veterinary Laboratory, Weybridge, Surrey, United
Kingdom).
DBAl1JLacJ mice (Jackson Labs)
[000442] Methods:
10004431 The Protocol for this model is described in the publication Nature
Protocols
(Brand DD et al. 2007; 2(5):1269-75).
[0004441 Briefly, Male DBA/1 mice, 9-11 weeks of age were used in these
experiments.
Mice were divided into two groups, experimental and control, and each mouse
was
immunized at the base of the tail with 50 l containing 100 gg of bovine CII
emulsified in
complete Freund's supplemented to 4 mg/ml of heat killed mycobacterium. Mice
were
anesthetized during the immunization by inhaled isofluorane. On the same day
as the
immunization, mice also received their first injection of the vehicle (150 mM
saline, control
group) or experimental peptide (experimental group) at a concentration of
1.5mg/kg.
Subsequently on days 2 and 4 post immunization, the dose was reduced to 1.0
mg/kg.
Starting with day 7 and through day 72, a dose of 0.8 mg/kg was used. All
treatments were
performed 3 times per week, on a Monday, Wednesday, and Friday schedule, and
the peptide
or control vehicle was administered intraperitoneally for each treatment,
rotating injection
areas. All mice were weighed at the beginning of the experiment in order to
calculate
dosage administered. Mice were weighed on days 0, 21 and 46. At day 4946, the
mice were
again weighed (average of 1.6 gm increase and control group had a
significantly greater
increase in weight than did treatment group) and dosages were adjusted
accordingly.
[000445] Starting on day 11, all mice were examined 3 times per week for
incidence and
severity of arthritis. and each arthritic limb was assigned a numerical score
based on the
degree of inflammation observed according to the scale below.
[000446] Severity scoring system is as described in the publication (Rosleniec
E et al.
Current protocols in immunology, 1997). Briefly, Score 0 No evidence of
erythema or
swelling. Score 1 Erythema and mild swelling confined to the tarsals or ankle
joint. Score 2
Erythema and mild swelling extended from the ankle to the tarsals. Score 3
Erythema and
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moderate swelling extended from the ankle to the metatarsal joints. Score 4
Erythema and
severe swelling encompass the ankle, foot and digits or ankylosis of the
joint.
In this experiment, two groups of mice (10 in treatment and 11 in control)
were compared.
The control group received saline in parallel with treatment group on same
days as the
treatment group received mCRAMP (0.8mg/Kg) intraperitoneuly 3 times a week.
10004471 Experimental Results and statistical analysis:
10004481 Results of statistical analysis: A very significant difference
(p=0.0037) between
treatment and control groups in the progression of arthritis (t-test
difference between mean
severity score from day one since incidence until day 19 since incidence).
[000449] Arthritis Incidence - In the control group, autoimmune arthritis
developed a rate
and incidence considerable normal for this strain of mouse (DBA/1). The
control group
achieved a 100% incidence by day 44. The incidence of arthritis in the
experimental group
(peptide treated) was somewhat lower than the control group, and the rate of
arthritis
development appeared to be delayed.
[000450) Likewise, the number of paws per mouse in the treatment group was
significantly
lower than control at a 95% confidence limit.
[000451] Severity of Arthritis - The severity of arthritis was analyzed on the
basis of
degree of inflammation (scored as described above) and the number of affected
limbs. As
seen in the figure below, differences between the two groups were clearly
observed when
analyzed as mean Severity Score/Mouse. While these data are weighted somewhat
by the
differences in arthritis incidence, the differences in the severity appear to
be even greater
than the differences in incidence.
[000452] Confidence limits were drawn using a 95% t-statistic on the residual
distributions
obtained by subtracting Actual-Expected readings. Expected readings were
obtained using a
linear regression model of the true data.
10004531 Weight loss is normally found in CIA and can therefore be a factor in
determining severity of disease. The weight gain of the control group was less
than that of
the treatment group. Therefore weight measurement were compared between days
21 and
day 46 and a Mann-Witney significance test showed that the weight difference
between the
two samples (control vs. treatment) marked as weight on day 46 divided by
weight on day
21, is marginally significant (P < 0.05, two-tailed test). Mean weight gain
(day 21 to 46) in
control was 2.1 % and in treatment group was 5.4%.
[000454] Results of the statistical analysis for arthritis paw severity and
incidence are
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shown in Figures 1 to 6.
[000455] Conclusion and discussion: The above-described results in FIGs 1-6
clearly
demonstrate for the first time relative to the prior art, treatment of a
disease using AMPs and
in particular, cathelicidin. Specifically, the above described results clearly
demonstrate for
the first time relative to the prior art optimal in-vivo treatment in a mouse
model for arthritis,
which is associated with inflammation and an autoimmune disease.
[000456] Cathelicidin significantly lowers incidence rate as well as severity
of arthritis in
model for Collagen induced arthritis (p=.025).
[000457] This experiment shows that intravenous or subcutaneous or IP
injection of
cathelicidin is a viable mode of treatment for arthritis, rheumatic diseases
and connective
tissue/inflammatory diseases include arthritis, rheumatoid arthritis, pyogenic
arthritis, mixed
connective tissue disease, cholesteatoma, relapsing polychondritis, autoimmune
myositis,
primary Sjogren's syndrome, smooth muscle autoimmune disease, myositis,
tendinitis, a
ligament inflammation, chondritis, a joint inflammation, a synovial
inflammation, carpal
tunnel syndrome, osteoarthritis, ankylosing spondylitis, a skeletal
inflammation, an
autoimmune ear disease, osteoporosis, fibromyalgia, periodontitis, and an
autoimmune
disease of the inner ear.
This experiment also shows that oral, intravenous or subcutaneous or IP
injection of
cathelicidin forms a viable mode of treatment for the related inflammatory
systemic diseases
include systemic lupus erythematosus, systemic sclerosis, septic shock, toxic
shock
syndrome, Reiter's syndrome, and cachexia.
[000458] Example 2
[000459] Cathelicidin for the treatment of Multiple sclerosis and CNS
inflammatory
disease
[000460] Multiple sclerosis (MS) is an immune-mediated demyelinating disease
of the
central nervous system (CNS) of unknown etiology.
[000461] Cathelicidin is expressed in the CNS. In this experiment delivery of
the
cathelicidin was made by injection (IP).
[000462] Materials and Methods:
[000463] Protocol for Myelin Oligodendrocyte Protein (MOG)-peptide induced EAE
in
C57BL/6 mice.
[000464] Mice.
C57BL/6 (B6) mice were purchased from Harlan (Jerusalem, Israel). Female, 9
week old
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mice were used in the experiment. The mice were housed in the specific-
pathogen free (SPF)
animal facility of the Hebrew University and all experiments were approved by
the
institutional animal care and use committee (IACUC).
[000465] Induction of EAE
[000466] Emulsion preparation: MOGB35-55B peptide
(MEVGWYRSPFSRVVHLYRNGK) 1.25mg/mi in PBS was emulsified in complete
Freund's adjuvant (CFA) supplemented with 400 g M. tuberculosis (Mt) H37RA
(Difco).
Mice were immunized s.c. in the flank with 250 g MOGB35-55B/CFA using a 25G
needle.
[000467] 200ng Pertussis Toxin (Sigma) was injected i.v, at the time of
immunization and
48h later.
[000468] EAE score
[000469] EAE was scored on a scale of 0-6: 0, no impairment; 1, limp tail; 2,
limp tail and
hind limb paresis; 3, >1 hind limb paralysis; 4, full hind limb and hind body
paralysis; 5,
hind body paralysis and front limb paresis; 6, death.
[000470] EAE treatment
[000471] Mice were treated with cathelicidin peptide of sequence
GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ was purchased and supplied by
Biosight Ltd of Karmiel, Israel and diluted in PBS, vs. PBS as a control. The
cathelicidin
was diluted in sterile PBS and divided to aliquots kept at -20 C such that
each aliquot was
thawed once for use. Mice were treated by intraperitoneal (i.p.) injection of
roughly 200u1
volume (adjusted for weight) 3 times a week (Sun-Tues-Thurs) starting the day
of
immunization with MOG/CFA and through day 48. Clinical EAE scores were
evaluated
through day 60.
10004721 Results:
[000473] Results are displayed in Figures 7, 8, 9,
10004741 A graph showing clinical score up to day 50 is shown as graph in FIG.
8.
[000475] Conclusion:
[000476] Cathelicidin peptide treatment lowered EAE severity and protected
mice from
fatal EAE observed at a late stage of the disease in control animals. The
lower dose of
peptide, 0.2mg/Kg was more protective than the higher 2mg/Kg dose.
[000477] Cathelicidin or its analogs or fragments can therefore be used as a
drug for the
treatment of neurological and CNS inflammatory diseases.
[000478] These include neurological/inflammatory diseases include
neurodegenerative
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disease, multiple sclerosis, Alzheimer's disease, Parkinson's disease,
myasthenia gravis,
motor neuropathy, Guillain-Barre syndrome, autoimmune neuropathy, Lambert-
Eaton
myasthenic syndrome, paraneoplastic neurological disease, paraneoplastic
cerebellar
atrophy, non-paraneoplastic stiff man syndrome, progressive cerebellar
atrophy,
Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea,
Gilles de la
Tourette syndrome, autoimmune polyendocrinopathy, dysimmune neuropathy,
acquired
neuromyotonia, arthrogryposis multiplex, optic neuritis, spongiform
encephalopathy,
migraine, headache, cluster headache, and stiff-man syndrome.
10004791 Example 3
[000480] Development of a fully humanized antibody to LL-37
[000481] A fully humanized antibody Single Chain Variable Fragment (scFv) to
the
cathelicidin LL-37 was developed using the two-hybrid system in yeast, a
technology as
described in US patent 6,610,472.
[000482] Briefly, a library of expression vectors was generated in yeast cells
through
homologous recombination; and the encoded proteins complexes with high binding
affinity
to their target molecule LL-37 was selected by high throughput screening in
vivo or in vitro.
Testing for ability to inhibit LL-37 in-vivo was performed by measuring the
ability of the
humanized antibody to inhibit bacterial killing by LL-37.
[000483] FIG. 10 shows a Western blot analysis of 4 different scFv developed
that bind
LL-37.
10004841 FIG. 11 shows the inhibitory effect of scFv on LL-37 in a bacterial
killing assays.
In order to find out the concentration of LL37 at which 50% of the bacteria
could be killed
(called "IC50"). Basically the activity protocol follows the ability of the
antibody to block
the antimicrobial activity of LL-37. The bacteria used was Pseudomonas that
was isolated
from a wound. The growth medium was LB. LL-3 7 was added at a concentration of
100
microgram/ml (the final volume or the reaction is 50 microliter). Blocking
antibodies at 1 or
microliter of antibody (= 1:50 or 1:10 dilutions respectively. Low antibody
levels ensure a
non- specific effect. A 2nd fraction from the elution with 100mM imidazole was
used.
[000485] The antibody and LL-37 mixture was incubated at room temperature for
30
minutes.
The bacteria were added (volume of 40 microliters). The mixture was incubated
shaking for
3 hours at 37 degrees. At that point LB was added to maintain the growth since
the volumes
we used were so small in order to grow the bacteria for longer incubation
times, the mixture
CA 02696833 2010-01-11
was further incubated for additional 2-3 hours. Concentration of bacteria was
estimated by
optical density (OD) reading at 490.
10004861 Example 4
[000487] Cathelicidin in the treatment, diabetes and related diseases
including
Hyperglycemia or Hypoglycemia, hypotension, hypertension,
glandular/inflammatory
diseases obesity, atherosclerosis and diabetes related diseases such as
periodontitis and
diabetes associated healing deficiencies or wounds.
[000488] Background:
[000489) TLR4 and CD14 are the receptor for LPS and play a critical role in
innate
immunity. Stimulation of TLR4 activates pro-inflammatory pathways and induces
cytokine
expression in a variety of cell types. Inflammatory pathways are activated in
tissues of obese
animals and humans and play an important role in obesity-associated insulin
resistance.
TLR4 and CD 14 are a molecular link among nutrition, lipids, and inflammation
and that the
innate immune system participates in the regulation of energy balance and
insulin resistance
in response to changes in the nutritional environment. (Hang Shi et al. The
Journal of
Clinical Investigation Volume 116 Number 11 November 2006)In a paper published
(Diabetes 56:1761-1772, 2007), It was shown that metabolic Endotoxemia
Initiates Obesity
and Insulin and it was suggested that lowering plasma LPS concentration could
be a potent
strategy for the control of metabolic diseases including insulin
resistance.Therefore, the
present experiment shows that insulin resistance and thereby glucose levels
can be controlled
using cathelicidin and is therefore a novel drug for the treatment of diabetes
and diabetes
related diseases. The in-vivo mouse model used is as described in Biochemical
and
Biophysical Research Communications 361 (2007) 140-145, " LPS-induced
biomarkers in
mice: A potential model for identifying insulin sensitizers".
Lipopolysaccharide (LPS)-
mediated inflammatory response may modulate pathways implicated in insulin
resistance (J
Clin Endocrinol Metab 85: 3770-3778, 2000).
[000490] Materials and Methods:
10004911 Two groups of 6 mice were used. One group was treated with PBS and
the other
group with the mCRAMP cathelicidin peptide at 0.4mg/Kg, both groups injected
(IP) three
times a week on Sunday Tuesday and Thursday. Both groups were fed on a high
fat diet of
60%Kcal fat diet (Research Diets Inc. New Brunswick USA) for a four week
period by
which time under normal circumstances they would be insulin resistant. At the
end of four
weeks blood glucose was determined using a glucometer on blood drawn by tail-
nicking of
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mice.
[000492] LPS was administered to C57BL/6 mice at 0.2 mg/kg. Mice were bled
approximately 2 h after LPS injection (T = 0). Changes in insulin dependent
(or non-insulin
dependent) sensitivity in regulating the glucose uptake were examined by
calculating the
linear slope of the fall or gain in glucose. Such a slope/gradient shows the
rate of decrease of
glucose over time.
[000493] mCRAMP sequence: GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ.
[0004941 In calculating the statistics, for each individual mouse, the glucose
level at time
T= 2hrs. was divided by the glucose level at time T=0 to obtain a ratio at
T=2hrs for each
mouse. An average was calculated for all the ratios and a students t-test was
performed.
[000495] Results:
10004961 A significant difference rate of change in glucose levels was noted
(students t-test
< 0.05). Two hours following LPS administration, average glucose levels in the
control mice
rose by 5.31 % whereas average glucose levels in the cathelicidin treated mice
came down to
90.05% of their initial level of 2 hours previously.
[000497] The treatment group was protected from insulin insensitivity thereby
leading to a
reduction of glucose levels during the two hour period as compared to the
control group. The
control group being insulin resistant due to the high fat diet remained at
high glucose level.
Cathelicidin protected the treatment group mice from insulin resistance
normally developed
as by the control group over the four week period of high fat diet. A graphic
representation
of the data is shown in FIG. 12.A graphic representation of the data is shown
in FIG. 12
[000498] Conclusion:
10004991 This experiment shows that intravenous or subcutaneous or IP
injection of
Cathelicidin, its analogs or fragments inhibits insulin resistance and
hyperglacemia, as well
as LPS induced disregulation of glucose levels in blood, and can therefore be
used for the
treatment of diseases such as metabolic diseases or a glandular/inflammatory
diseases
including: type I diabetes, type II diabetes, type B insulin resistance,
Schmidt's syndrome,
Cushing's syndrome, thyrotoxicosis, benign prostatic hyperplasia, pancreatic
disease,
Hashimoto's thyroiditis, idiopathic adrenal atrophy, Graves' disease,
androgenic alopecia,
thyroid disease, thyroiditis, spontaneous autoimmune thyroiditis, idiopathic
myxedema,
ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune
prostatitis, Addison's
disease, and Type I autoimmune polyglandular syndrome Diabetes mellitus and
Type II
diabetes, obesity, Hyperglycemia or Hypoglycemia, complications of diabetes
including skin
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ulcerations, and diabetes related eye diseases such as Proliferative
retinopathy
[000500] Example 5
10005011 Cathelicidin in the treatment of obesity and overweight as well as
related diseases
such as periodontitis and diabetes associated diseases and healing
deficiencies.
[000502] Background:
[000503] A high-fat diet chronically increased insulin resistance, obesity and
metabolic
diseases. Diabetes and obesity are two metabolic diseases characterized by
insulin resistance
and low-grade inflammation.
[000504] The present experiment shows that obesity can be controlled using
cathelicidin or
cathelicidin fragments or analogues and is therefore a novel drug for the
treatment of obesity
and obesity related diseases. The in-vivo mouse model used is as described in
(Diabetes
56:1761-1772, 2007).
[000505] Materials and Methods:
[000506] Two experiments were performed, one on a regular diet and one on a
high-fat
diet:
1000507] In the first experiment:
[000508] Briefly, mice were fed on a normal non-high-fat diet for 21 days and
their
average weight was monitored. Two groups of Male DBA/1 mice, 10 mice in each
group
(treatment & Control) with an average age of 10 weeks in each group.
[000509] Control group were injected with vehicle (150 mM saline) whilst the
experimental group were injected with the cathelicidin mCRAMP at a
concentration of
1.5mg/kg on day 0. Subsequently on days 2 and 4 , the dose was reduced to 1.0
mg/kg.
Starting with day 7 and through to day 21, a dose of 0.8 mg/kg was used. All
treatments
were performed 3 times per week, on a Monday, Wednesday, and Friday schedule,
and the
peptide or control vehicle was administered intraperitoneally for each
treatment, rotating
injection areas. Results shown in Figure 13.
[000510] mCRAMP sequence is: GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ
[000511] In the second experiment:
[000512] Briefly, Experimental obesity was induced in C57BL/6 mice by
maintaining them
on a 60%Kcal fat diet (Research Diets Inc. New Brunswick USA) for a six week
period.
[000513] The experiment contained 2 groups of 6 mice: Group 1: PBS, Group 2:
mCRAMP cathelicidin 0.2mg/Kg for 3 weeks and then 0.4mg/Kg for another 3
weeks. Mice
were treated by intraperitoneal injection of PBS vs. peptide (200 l per
injection) on Sunday,
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Tuesday, and Thursday of each week. Mice were weighed at baseline and three
times a week
on each day of treatment.
[0005141 Results:
10005151 In the first experiment using a non-high-fat diet, and as shown in
Figure 13, mice
in treatment group increased in weight at a rate of 0.0536 gm/day while in the
treatment the
weight gain was 0.0488 gm/day.
[000516] In the second experiment using a high-fat diet the average weight in
the control
was divided by the average weight in the treatment groups for each of the
readings (three per
week). A trend was seen in the graph plotted as seen in figure 14. This trend
is significant
when analyzing using a statistical test of linear regression and residual
analysis.
10005171 Continuing the treatment to day 50, a statistically significant
difference between
the two groups was noted by using a students t-test (<0.05) after comparing
the weights of
the two groups on day 50.
[000518] Therefore, treatment over long term using cathelicidin at a normally
endogenous
level would significantly reduce weight in obese mice.
[000519] Conclusion:
[000520] This experiment shows that intravenous or subcutaneous or IP
injection of
cathelicidin is a viable mode of treatment for obesity. At a 60%Kcal diet, a
dosage of
0.4mg/Kg three times a week was enough to significantly reduce weight in obese
mice and
prevent obesity.
[000521] Conclusion:
[000522] This experiment shows that intravenous or subcutaneous or IP
injection of
cathelicidin is a viable mode of treatment for obesity and that its effect is
dose dependant. At
a 60%Kcal diet, a dosage of 0.4mg/Kg three times a week was enough to
significantly
reduce weight in obese mice and prevent obesity.
10005231 Example 6
Use of Cathelicidin for treating osteoporosis, ankylosing spondylitis,
osteoarthritis and
periodontitis by preventing bone erosion or resorption.
[000524] Background: Vitamin D3, a commonly used medication for osteoporosis
also
induces the expression of cathelicidin through the calcitriol/VDRE. For this
reason
cathelicidin was studied on its effect on bone resorption, degradation or
formation.
10005251 Bone erosion or degradation in rheumatoid arthritis, periodontitis
and
osteoarthritis is a result of persistent chronic inflammation. Likewise, in
osteoporosis there
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exists an imbalance between bone resorption and bone formation. This imbalance
is due to
process by which osteoclast cell activity, the process that breaks down bone,
dominates
osteoblast cell activity, the process by which bone formation is performed.
[000526] Therefore the present experiment tested to see if there was any
difference in the
bone degradation, inflammation or resorption status between arthritic paws of
control versus
cathelicidin treated mice in the mouse model of collagen induced arthritis as
well as between
the treatment and control groups of LPS induced bone loss in periodontitis (J
Clin
Periodonto12004; 31: 596-603). Also tested and observed were non-inflamed
joints and
bone of control versus non-inflamed treatment joints and bone.
[000527] Cathelicidin was injected (IP) into treatment mice and compared with
control.
Histological samples of bone taken from the ankle joints of mice paws were
analyzed and
osteoclasts were counted using the H&E (hematoxylin and eosin stain)
Immunohistochemical staining and with TRAP staining technique (Acid
Phosphatase,
Leukocyte - Procedure No, 387 A from Sigma-Aldrich).
[000528] Eight groups were analyzed according to their inflammatory status:
[000529] Two groups: control and treatment groups had induced inflammation in
their
paws but their inflammation levels were similar.
[0005301 Two further groups: control and treatment groups had no inflammation
in their
paws.
[000531] Two further groups: control and treatment groups had induced
inflammation in
their paws but their inflammation levels were dissimilar.
[000532] Two further groups: control and treatment groups having induced low
grade
inflammation via LPS injections (IP) were studied for bone morphology
differences in
mandibles.
10005331 By this method, it was possible to observe bone degradation,
inflammation or
resorption by monitoring osteoclast and immune or inflammatory cell activity.
[000534] Materials and Methods:
10005351 Paws from the Collagen induced arthritis (CIA) experiment were
studied. In all
80 paws from 20 mice were available for study and of those, only 15 were
studied according
to their inflammation/ arthritic severity score. The protocol for induction of
the CIA is
reported in experiment 1 above.
[000536] Histology:
[000537] For the detection of TRAP+ cells in histological slides of joints,
amputated limbs
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were fixed in 1% paraformaldehyde for several weeks and washed with PBS. The
tissues
were decalcified by incubation in 0.5 M EDTA/PBS, pH 7.4, for 10 days, in
which the
EDTA solution was changed every day. Tissues were embedded in paraffin and 6
gm
sections were made. Deparaffinised, rehydrated sections were either stained
with
haematoxylin and eosin or preincubated for 2.5 hours at 37 C in a 12.5 mM
sodium tartrate
solution in 100 mM acetate buffer, pH 5.5.
[000538] Subsequently, sections were incubated for 1 hour at 37 C in acid
phosphatase
substrate solution (0.05% naphthol AS-BI phosphate 50 mM sodium tartrate,
0.16% p-
rosanilin, 0.16% NaNO2, 25% Michaelis' 0.14 M acetate/barbital buffer, pH 5.0,
in distilled
water). Sections were washed with distilled water, counterstained with 0.15%
Lightgreen SF
Yellowish in 0.2% acetic acid, incubated for 10 s in 1% acetic acid and dried
at 37 C. Red-
staining cells were considered to contain TRAP, and TRAP+ multinucleated cells
(three or
more nuclei) were regarded as osteoclasts.
[0005391 Paws having similar arthritic scores for equal lengths of time were
compared for
bone resorption and degradation in cathelicidin treatment group versus control
group. This
type of comparison rules out any influence of inflammation as a determinant of
bone
degradation or ankylosis leaving the differentiation status of osteoblasts and
osteoclasts as
the main influence.
[0005401 The materials and methods used for inducing arthritic bone
degredation are
described for the mouse model in example 1 above. The arthritic paws of grade
3 severity
index and above were obtained from this same experiment and placed in fixative
for
histology measurements.
[0005411 Experimental Results:
[000542] For observation of Osteoclasts and Inflammatory bone degradation
including
periodontitis, arthritis osteoarthritis, slides are stained with H&E and with
TRAP. Several
paws having similar severity index at equal duration were histologically
examined (see table
of FIG. 15. In addition, non-inflammed paws in cotrol and treatment groups
were also
studies (FIG. 15). Other paws having different severity index and durations
were also
studied. In the figures, LF = Left Front paw, RF = Right Front paw, LH = Left
Hind paw,
and RH = Right Hind paw.
[0005431 A clear difference in erosion and resorption between treatment and
control groups
was noted with less degradation and less resorption observed in treatment
group.
Degradation or deformation was mainly seen in control group. Likewise for mice
chosen as
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having no difference in severity index and duration of arthritic paws, there
was a similar
distinct difference in bone resorption/erosion between the two groups.
[000544] FIG. 16 is an example of histology slides between mouse 3(Right
Front) and
FIG. 17 shows the H&E staining of mouse 3 (Right Front) paws.
[000545] FIG. 18 shows the TRAP staining of control mouse 13 having no
inflammatory
sign in its paw yet still showing more osteoclasts that the inflamed mouse 3
shown above.
[000546] FIG.19 shows a TRAP staining of inflamed paw of control mouse 17-
Right front
paw clearly showing a marked increase in osteoclasts.
[000547] Clearly, the control mouse has a higher number of active osteclasts
as well as
higher resorption and degradation even though both mice have the same
inflammatory status.
[000548] Conclusion and discussion:
[0005491 Cathelicidin, inhibits bone erosion and deformation as found in
either
osteoporosis, ankylosing spondylitis, osteoarthritis and periodontitis and can
therefore be
used as a drug for treating these diseases.
[000550] In the present experiment, cathelicidin was delivered by IP
injections. Therefore,
it is obviously implied that the drug delivery of cathelicidin can be either
orally using a
vehicle carrier to the blood steam via the GI tract or by injection i.v. or
subcutaneous
injections.
[000551] The data convincingly shows that Cathelicidin is a suitable drug
candidate in the
treatment of osteoporosis, ankylosing spondylitis, osteoarthritis and
periodontitis,
Osteomyelitis, bone cancer, Osteogenesis imperfecta, Paget's disease,
Osteochondroma,
Osteomalacia, Osteomyelitis, Osteopetroses, Renal Osteodystrophy, Unicameral
Bone Spurs,
Bone Tumor, Craniosynostosis, Enchondroma, Fibrous Dysplasia, Giant Cell Tumor
of
Bone, Infectious Arthritis, Osteomyelitis, Klippel-Feil Syndrome, Limb Length
Discrepancy,
Osteochondritis Dissecans, and bone loss in periodontitis .
[000552] Example 7
Use of Cathelicidin analog and fragment for optimal treatment of diseases,
such as psoriasis,
which are associated with inflammation, autoimmunity and/or skin cell/tissue
proliferation/differentiation imbalance and wound healing.
[000553] Background: Diseases associated with inflammation, autoimmunity
and/or skin
cell/tissue proliferation/differentiation imbalance include numerous diseases,
such as
psoriasis and dandruff, for which no optimal therapy exists. Angiogenesis and
epithelialization common in psoriatic skin is enhanced by AMPs such as LL-37
(Koczulla,
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87
R. et al., 2003. J.Clin.Invest 111:1665-1672; Heilborn, JD. et al., 2003. J
Invest Dermatol
120:379-389). An optimal strategy for treating such diseases would be to
identify factors
involved in dysregulation of skin cell/tissue proliferation/differentiation,
and to use
compounds capable of inhibiting the activity of such factors to treat such
diseases.
10005541 Human tissue kallikreins are a family of 15 trypsin-like or
chymotrypsin-like
secreted serine proteases (K.LK1-KLK15). Multiple KLKs have been
quantitatively
identified in normal stratum corneum (SC) and sweat as candidate desquamation-
related
proteases. Aberrant human tissue kallikrein levels levels in the stratum
corneum and serum
of patients with psoriasis (British Journal of Dermatology 2007 156, pp875-
883). These
kallikreins are protease involved in the maturation process of cathelicidin LL-
37 from its
precursor hCAP- 18. Inappropriate balance between various proteases can be a
determining
factor as to whether cathelicidin is cleaved into its pro-inflammatory or to
its anti-
inflammatory fragments.
[000555] As was demonstrated by the present inventor in WO 2004-056307,
cathelicidin is
an immune regulator in-vivo and plays a major role in psoriasis and skin
inflammation.
Inhibiting or regulating its activity is essential for treatment of the
disease. Inhibition of
cathelicidin in skin inflammation was further demonstrated in psoriasis
(Nature 2007 Oct
4;449(7162):564-9) and in other skin inflammatory diseases such as rosacea
(Nat Med. 2007
Aug;13(8):975-80).
10005561 While reducing the present invention to practice, a method of using
dominant
negative cathelicidin peptide or fragments for optimal treatment in a human of
a disease
associated with inflammation, autoimmunity and/or skin cell/tissue
proliferation/differentiation imbalance, such as psoriasis, was demonstrated
for the first time,
as described below, thereby overcoming the limitations of the prior art.
[000557] Materials and Methods:
10005581 Antimicrobial peptides (AMPs): The antimicrobial peptides used were
used were
the fragment cathelicidin SK29:SKEKIGKEFKRIVQRIKDFLRNLVPRTES or
GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ the mouse cathelicidin CRAMP
(BIOSIGHT LTD, Karmiel, Israel),
[000559] LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES Catalogue No. 61302
LL-37 antimicrobial peptide human, AnaSpec, Inc. USA. , Human Beta-Defensin-2
peptide
was purchased from Sigma-Aldrich.
[000560] Human in-vivo psoriatic lesion treatment: Cathelicidin l0ug/ml or
human beta
CA 02696833 2010-01-11
88
defensin-2 diluted in PBS containing 0.1 % BSA, or buffer carrier was applied
to lesions in a
human subject in a blind trial.
[000561] Experimental Results:
10005621 Whereas the human beta defensin-2 showed a worsening of skin
psoriatic legion
over a seven week course of treatment, as exemplified in figure 20, the
cathelicidins LL-37
and SK29 showed a slight improvement over the course of 5 days treatment.
[000563] Conclusion and discussion:
[000564] Recently published material regarding rosacea has shown (Nat Med.
2007
Aug;13(8):975-80) that inappropriate cathelicidin processing by endogenous
protease is
responsible for the disease progression of rosacea. It may well be that
similar mechanisms
are in effect in other diseases such as psoriasis in which case dominant
negative peptide
inhibitors that compete with fragments of LL-37 whithout activating the
disease would form
viable modes of treatment for the disease.
[000565] There is no contradiction that both inhibiting LL-37 by an antibody
as was
demonstrated by the previous application of the inventors in WO 2004-056307
and in the
publication (Nature 2007 Oct 4;449(7162):564-9) and making use of LL-37 can be
similarly
effective. One possibility is that LL-37 may inhibit its own fragments formed
by
inappropriate endogenous protease action.
[000566] Example 8
Use of cathelicidin fragments or analogs for the treatment of diabetes
[000567] In-vitro studies on Beta-Cells
10005681 Background: No optimal therapy exists for treatment of type 1
diabetes. An
optimal strategy for treating such a disease would be to identify factors
involved in inducing
beta cell growth. While reducing the present invention to practice, a
significant role for
AMPs in driving beta cell proliferation was identified, and the capacity of
cathelicidin to
induce growth so as to enable optimal treatment of type 1 diabetes was
demonstrated, as
described below, thereby overcoming the limitations of the prior art.
10005691 Materials and Methods:
10005701 Antimicrobial peptides (AMPs): The antimicrobial peptides human
cathelicidin
LL-37: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES was obtained from
AnaSpec, USA (Catalogue numbers: 61302). Cathelicidin at 2ug/ml was added to
the plate
and compared with control.
[000571] Thymidine incorporation cell proliferation assay: Cell proliferation
was
CA 02696833 2010-01-11
89
evaluated by measuring [3(H)]-thymidine incorporation into DNA. Cells were
pulsed with
[3(H)]-thymidine (1 microcurie/mL, ICN, Irvine, CA) for 1 hour, at 37 degrees
centigrade.
After incubation, cells were washed 3 times with PBS, incubated for 15 minutes
at room
temperature in 5 % trichloroacetic acid and solubilized in 1 % triton X-100.
The
radioactivity incorporated into the cells was counted in the [3(H)]-window of
a Tricarb
liquid scintillation counter. Mean values were determined from measurements of
triplicate
samples under each experimental condition for each experiment. Thymidine
incorporation
was determined as number of disintegrations per minute (DPM) per mg of
protein.
[000572] Experimental Results:
[000573] Beta cells are significantly stimulated to proliferate by AMPs: In
order to
investigate the effects of AMPs on beta cell growth, mouse BETATC beta cell
line were
chronically treated with cathelicidin, and their proliferation was monitored.
As can be seen
in Figure 21, in all skin epithelial cells exposure to cathelicidin led to a
slight increase in cell
proliferation. This data clearly demonstrates that AMPs are involved in the
pathogenesis of
diabetes and in particular to type I diabetes with respect to cellular
hyperproliferation.
[000574] Conclusion: The above-described results clearly demonstrate that
AMPs, such as
cathelicidin or its fragments or its analogs, are involved in driving
proliferation of beta cells
and therefore can be used in the treatment of diabetes by enabling the
presence of an
increased number of insulin secreting beta cells.
[000575] Example 9 Chemotaxis assays.
[000576] Chemotaxis assays. Cells (e.g. neutrophils, monocytes, T cells,
HEK293; 25
microliters at a density of 1.0-3.0 x 106 cells/ml) in RPMI medium (Beit
Haemek)
containing 0.5% BSA (Sigma-Aldrich) are placed on the top of a 96-well ChemoTx
disposable chemotaxis apparatus with a 5 micron pore size (Neuroprobe).
Tenfold serial
dilutions of the tested reagent in RPMI medium with or without 0.5% BSA are
placed in the
bottom wells of the chamber. The apparatus is incubated for 60-600 min at 37
C in an
atmosphere of 5% carbon dioxide, and the cells migrating at each concentration
of
chemoattractant is counted with the use of an inverted microscope.
[000577] Cells (I X l 07/mL) are suspended in a buffer containing 0.25% BSA,
145 mM
NaCl, 5 mM KCI, 10 mM Na/MOPS, 1 mM CaC12, 1 mM MgC12, 10 mM glucose, 10 mM
HEPES (all from Sigma-Aldrich), pH 7.4, and incubated with 2 micromolar Fura-2-
AM
(Molecular Probes, Eugene, OR), for 40 min at room temperature. The cells are
washed
once, resuspended in the buffer containing 0.25% BSA, and are kept at room
temperature.
CA 02696833 2010-01-11
Just before use, aliquots of the cells (4X 105) are washed and resuspended in
2 ml buffer
containing 0.05% BSA in a stirred cuvette at 37 C. Measurement of
intracellular Ca2+
concentration and chemotaxis assays are performed as previously described
(Maghazachi,
AA. et al., 1997. FASEB J. 11:765-774)
10005781 It is appreciated that certain features of the invention, which are,
for clarity,
described in the context of separate embodiments, may also be provided in
combination in a
single embodiment. Conversely, various features of the invention, which are,
for brevity,
described in the context of a single embodiment, may also be provided
separately or in any
suitable subcombination.
[000579] Although the invention has been described in conjunction with
specific
embodiments thereof, it is evident that many alternatives, modifications and
variations will
be apparent to those skilled in the art. Accordingly, it is intended to
embrace all such
alternatives, modifications and variations that fall within the spirit and
broad scope of the
appended claims. All publications, patents, and patent applications and
sequences identified
by their accession numbers mentioned in this specification are herein
incorporated by
reference in their entireties into the specification, to the same extent as if
each individual
publication, patent, or patent application or sequence identified by its
accession number was
specifically and individually indicated to be incorporated herein by
reference. In addition,
citation or identification of any reference in this application shall not be
construed as an
admission that such reference is available as prior art to the present
invention.
