U.S. patent application number 12/380435 was filed with the patent office on 2009-10-29 for use of galectin-7 to promote the re-epithelialization of wounds.
Invention is credited to Zhiyi Cao, Noorjahan Panjwani.
Application Number | 20090270320 12/380435 |
Document ID | / |
Family ID | 32072730 |
Filed Date | 2009-10-29 |
United States Patent
Application |
20090270320 |
Kind Code |
A1 |
Panjwani; Noorjahan ; et
al. |
October 29, 2009 |
Use of galectin-7 to promote the re-epithelialization of wounds
Abstract
Methods for the therapeutic treatment of epithelial wounds in
mammals comprising administering to a mammal afflicted with an
epithelial wound a therapeutically effective amount of a galectin-3
protein and/or a galectin-7 protein are provided. Pharmaceutical
compositions comprising a pharmaceutically suitable carrier or
diluent and as an active agent a galectin-3 protein and/or a
galectin-7 protein are also provided.
Inventors: |
Panjwani; Noorjahan;
(Medford, MA) ; Cao; Zhiyi; (Cambridge,
MA) |
Correspondence
Address: |
LAWSON & WEITZEN, LLP
88 BLACK FALCON AVE, SUITE 345
BOSTON
MA
02210
US
|
Family ID: |
32072730 |
Appl. No.: |
12/380435 |
Filed: |
February 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11211401 |
Aug 25, 2005 |
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12380435 |
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10133234 |
Apr 26, 2002 |
6967021 |
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11211401 |
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60286903 |
Apr 27, 2001 |
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Current U.S.
Class: |
514/9.4 |
Current CPC
Class: |
A61K 38/1709
20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 38/17 20060101
A61K038/17 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with Government support under grant
number EY-07088 from the National Institutes of Health.
Accordingly, the government may have certain rights in this
invention.
Claims
1. (canceled)
2. A method for the therapeutic treatment of epithelial wounds in
mammals comprising administering to a mammal afflicted with an
epithelial wound a therapeutically effective amount of a galectin-7
protein.
3. The method according to claim 2, wherein said epithelial wound
is a persistent epithelial defect or a recurrent epithelial
erosion.
4. The method according to claim 2, wherein said epithelial wound
is an epithelial lesion or an epithelial erosion.
5. The method according to claim 2, wherein said epithelial wound
is selected from the group consisting of ulcers, blisters, burns,
sores, boils, cuts, abrasions, and lacerations.
6. The method according to claim 2, wherein said epithelial wound
is a corneal wound.
7. The method according to claim 6, wherein said corneal wound is a
persistent corneal defect or a recent corneal erosion.
8. The method according to claim 6, wherein said corneal wound was
caused by excimer laser keratectomy.
9. The method according to claim 2, wherein said epithelial wound
is a skin wound or a wound to the gastrointestinal tract.
10-14. (canceled)
15. The method of claim 2, wherein the galectin-7 protein includes
the amino acid sequence of SEQ ID NO:2.
16. The method of claim 2, wherein the galectin-7 protein includes
an amino acid sequence that is substantially identical to the amino
acid sequence of SEQ ID NO:2.
17. The method of claim 2, wherein the galectin-7 protein includes
a galectin-7 galactoside-binding domain.
18-35. (canceled)
36. A method for the therapeutic treatment of epithelial wounds in
mammals comprising administering to a mammal afflicted with an
epithelial wound a therapeutically effective amount of a substance
that influences the expression of a galectin-7 protein.
37-38. (canceled)
Description
RELATED APPLICATIONS
[0001] The present application claims priority to provisional
application U.S. Ser. No. 60/286,903, filed Apr. 27, 2001 which is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] The repair of wounds in mammalian tissue (e.g., epithelial
defects, lesions, or erosions caused by disease, accidental injury,
surgical procedure, etc.) involves an orderly, controlled cellular
response. Three phases have been described in normal wound healing:
acute inflammatory phase, extracellular matrix and collagen
synthesis, and remodeling (Wound Repair by Peacock, W.B. Saunders,
Philadelphia, Pa., 1984). The sequence of the healing process is
initiated during an acute inflammatory phase with the deposition of
provisional tissue. This is followed by re-epithelialization,
collagen synthesis and deposition, fibroblast proliferation, and
neovascularization, all of which ultimately define the remodeling
phase (see, for example, Clark, J. Am. Acad Dermatol. 13:701,
1985). These events are known to be influenced by growth factors
and cytokines secreted by inflammatory cells and by epithelial
cells, endothelial cells, platelets, and fibroblasts localized at
the edges of the wound (see, for example, The Molecular and
Cellular Biology of Wound Repair (The Language of Science) Ed. by
Clark, Plenum Press, New York, N.Y., 1996; Hunt et al., in The
Surgical Wound Ed. by Dineen at al., Lea & Febiger,
Philadelphia, Pa., 1981; Nemeth et al., in Growth Factors and Other
Aspects of Wound Healing: Biological and Clinical Implications Ed.
by Barbul et al., A. R. Liss, New York, N.Y., 1988; and Assoian et
al., Nature 309:804, 1984). During re-epithelialization, cells at
the leading edge undergo a phenotypic conversion characterized by a
dramatic reorganization of the cytoskeleton, disruption of stable
intercellular adhesion, and redistribution of adhesion related
molecules. The breakage of the stable intercellular contacts is a
prerequisite for initiating re-epithelialization. Following
re-epithelialization, reversion to the epithelial phenotype,
including the reformation of stable intercellular contacts, must
occur if the function of the epithelium is to be fully restored.
The failure of epithelial cells to migrate over the wound surface
and failure of migrated epithelial cells to remain adherent to the
substratum are fundamental causes of debilitating clinical
conditions known as persistent epithelial defects (i.e., non
healing defects) and recurrent epithelial erosions
respectively.
[0004] Disorders of wound healing constitute a serious medical
problem for several different organ systems including the skin,
gastrointestinal tract, and cornea. For example, loss of cell-cell
adhesions within the epidermis produces life-threatening blistering
skin diseases known as pemphigus foliaceus and pemphigus vulgaris
(Cell Adhesion and Human Disease Ed. by Marsh et al., Ciba
Foundation Symposium, Vol. 189, John Wiley & Sons, New York,
N.Y., 1995). Persistent epithelial defects in the form of delayed
re-epithelialization are a characteristic of chronic skin wounds,
in particular venous stasis ulcers (Falanga et al., J. Dermatol.
Surg. Oncol. 19:764, 1993). Within the cornea, lack of epithelial
cell adhesion to the stroma and the basement membrane leads to
recurrent corneal erosions (Macaluso et al., in Cornea Ed. by
Krachmer, Mosby, St. Louis Mo., 1997). Persistent corneal
epithelial defects occur in a wide variety of clinical situations
such as in injuries caused by radiation, corneal abrasions or
lacerations, chemical burns of the cornea such as alkali and acid
burns, keratopathies, keratities and corneal dystrophies.
Persistent corneal epithelial defects carry a high risk of corneal
perforation and ulceration (Macaluso et al., supra).
[0005] Despite the need for more rapid healing of wounds, to date
there has been only limited success in accelerating wound healing
with pharmaceutical agents. In the case of corneal injuries, the
use of epidermal growth factor (Eiferman et al., Invest. Opthalmol.
Vis. Sci. (Suppl.) 28:52, 1987), fibronectin (Nishida et al., J.
Cell. Biol. 97:1653, 1983), collagenase inhibitors (Kenyon et al.,
Invest. Opthalmol. Vis. Sci. 18:570, 1979), topical steroids (Lass
et al., Arch. Opthalmol. 99:673, 1981), matrix metalloproteinase
inhibitors (Murphy et al., Biochemistry 30:8097, 1991), ascorbates
(Foster et al., Invest. Opthalmol. Vis. Sci. (Suppl.) 19:227,
1980), heparin (Aronson, Am. J. Opthalmol. 70:65, 1970), and
tetracyclines (Perry et al., Ophthalmology (Suppl.) 92:77, 1985)
does not always result in successful long-term management. For
example, topical application to a corneal injury of epidermal
growth factor (EGF) (Singh et al., Am. J. Opthalmol. 103:802, 1987)
or fibronectin (Tenn et al., Invest. Opthalmol. Vis. Sci. (Suppl.)
26:92, 1985), enhances epithelial wound healing but does not
prevent recurrent erosion and secondary breakdown of the corneal
epithelial surface.
[0006] Accordingly, there is a need in the art for additional
pharmaceutical agents and compositions that promote the healing of
wounds. In particular, there is a need for agents, compositions and
therapeutic methods that promote the re-epithelialization of
persistent epithelial defects and prevent recurrent epithelial
erosions.
SUMMARY OF THE INVENTION
[0007] In one aspect, the present invention provides methods for
the therapeutic treatment of epithelial injuries in mammalian
tissue involving administering to a mammal afflicted with an
epithelial injury a therapeutically effective amount of galectin-3,
galectin-7, or a combination of galectin-3 and galectin-7.
[0008] In another aspect, the present invention provides
pharmaceutical compositions that include a pharmaceutically
acceptable carrier or diluent and an amount of galectin-3 and/or
galectin-7 sufficient to promote the re-epithelialization of wounds
in injured mammalian tissues.
[0009] In general, it is believed that galectin-3 and/or galectin-7
will be clinically useful in promoting the healing of wounds
associated with any epithelial tissue including but not limited to
the skin epithelium; the corneal epithelium; the lining of the
gastrointestinal tract; the lung epithelium; and the inner surface
of kidney tubules, of blood vessels, of the uterus, of the vagina,
of the urethra, or of the respiratory tract. The present invention
encompasses the treatment of a variety of wounds that include but
are not limited to persistent epithelial defects and recurrent
epithelial erosions such as surgical wounds, excisional wounds,
blisters, ulcers, lesions, abrasions, erosions, lacerations, boils,
cuts, sores, and burns resulting from heat exposure or chemicals.
These wounds may be in normal individuals or those subject to
conditions which induce abnormal wound healing such as diabetes,
corneal dystrophies, uremia, malnutrition, vitamin deficiencies,
obesity, infection, immunosuppression and complications associated
with systemic treatment with steroids, radiation therapy,
non-steroidal anti-inflammatory drugs (NSAID), anti-neoplastic
drugs and anti-metabolites.
[0010] In certain embodiments, the present invention involves the
administration of pharmaceutical compositions that include
galectin-3 proteins with the amino acid sequence of human
galectin-3 as represented by SEQ ID NO:1 of the sequence listing.
In other embodiments, the present invention involves the
administration of pharmaceutical compositions that include
galectin-3 proteins with an amino acid sequence that is
substantially identical to the amino acid sequence of SEQ ID NO:1.
For example, in certain embodiments, the present invention involves
the administration of pharmaceutical compositions that include
galectin-3 proteins which contain accidentally or deliberately
induced alterations, such as deletions, additions, substitutions or
modifications of the amino acid residues of SEQ ID NO:1. In yet
other embodiments, the present invention involves the
administration of pharmaceutical compositions that include proteins
represented by fragments of the amino acid sequence SEQ ID NO:1 or
hybrid proteins that comprise these fragments. Fragments of SEQ ID
NO:1 preferably include a galectin-3 N-terminal domain and a
galectin-3 proline, glycine, and tyrosine-rich domain; a galectin-3
proline, glycine, and tyrosine-rich domain and a galectin-3
galactoside-binding domain; or a galectin-3 galactoside-binding
domain.
[0011] In certain other embodiments, the present invention involves
the administration of pharmaceutical compositions that include
galectin-7 proteins with the amino acid sequence of human
galectin-7 as represented by SEQ ID NO:2 of the sequence listing.
In other embodiments, the present invention involves the
administration of pharmaceutical compositions that include
galectin-7 proteins with an amino acid sequence that is
substantially identical to the amino acid sequence of SEQ ID NO:2.
For example, in certain embodiments, the present invention involves
the administration of pharmaceutical compositions that include
galectin-7 proteins which contain accidentally or deliberately
induced alterations, such as deletions, additions, substitutions or
modifications of the amino acid residues of SEQ ID NO:2. In yet
other embodiments, the present invention involves the
administration of pharmaceutical compositions that include
proteins, represented by fragments of the amino acid sequence SEQ
ID NO:2 or hybrid proteins that comprise these fragments. Preferred
fragments of SEQ ID NO:2 include a galectin-7 galactoside-binding
domain.
[0012] The present invention also encompasses the administration of
pharmaceutical compositions that include proteins represented by
the amino acid sequence of galectin-3 and/or galectin-7 taken from
any mammalian species including but not limited to bovine, canine,
feline, caprine, ovine, porcine, murine, and equine species.
[0013] In certain embodiments, the pharmaceutical compositions of
the present invention further include one or more additional
therapeutic agents. In certain embodiments, the additional
therapeutic agent or agents are selected from the group consisting
of growth factors, anti-inflammatory agents, vasopressor agents,
collagenase inhibitors, topical steroids, matrix metalloproteinase
inhibitors, ascorbates, angiotensin II, angiotensin III,
calreticulin, tetracyclines, fibronectin, collagen, thrombospondin,
transforming growth factors (TGF), keratinocyte growth factor
(KGF), fibroblast growth factor (FGF), insulin-like growth factors
(IGF), epidermal growth factor (EGF), platelet derived growth
factor (PDGF), neu differentiation factor (NDF), hepatocyte growth
factor (HGF), and hyaluronic acid.
[0014] The pharmaceutical compositions of the present invention can
be administered to humans and other mammals topically, orally,
rectally, parenterally, intracisternally, intravaginally,
intraperitoneally, bucally, ocularly, or nasally, depending on the
severity and location of the wound being treated. Administration
may be therapeutic or it may be prophylactic. Liquid dosage forms
for oral administration of an inventive pharmaceutical composition
include, but are not limited to, pharmaceutically acceptable
emulsions, microemulsions, solutions, suspensions, syrups and
elixirs. Solid dosage forms for oral administration include
capsules, tablets, pills, powders, and granules. Dosage forms for
topical or transdermal administration include ointments, pastes,
creams, lotions, gels, powders, solutions, sprays, inhalants, or
patches. Injectable preparations may be in the form of sterile
injectable aqueous or oleaginous suspensions. Compositions for
rectal or vaginal administration are preferably suppositories.
Prophylactic formulations may be present or applied to the site of
potential wounds, or to sources of wounds, such as contact lenses,
contact lens cleaning and rinsing solutions, containers for contact
lens storage or transport, devices for contact lens handling, eye
drops, surgical irrigation solutions, ear drops, eye patches, and
cosmetics for the eye area. The invention includes opthalmological
devices, surgical devices, audiological devices or products which
contain disclosed pharmaceutical compositions (e.g., gauze bandages
or strips).
DESCRIPTION OF THE DRAWING
[0015] FIG. 1 depicts the amino acid sequence and composition of
human galectin-3 (Accession No. BAA22164 in GenBank, SEQ ID
NO:1).
[0016] FIG. 2 depicts the amino acid sequence and composition of
human galectin-7 (Accession No. I55469 in GenBank, SEQ ID
NO:2).
[0017] FIG. 3 depicts a CLUSTAL W alignment of the amino acid
sequence of human galectin-3 (SEQ ID NO:1) with the amino acid
sequences of rabbit galectin-3 (Accession No. JC4300 in GenBank),
chicken galectin-3 (Accession No. AAB02856 in GenBank), and hamster
galectin-3 (Accession No. CAA55479 in GenBank). The first (upper)
sequence in the figure is amino acids 1 to 250 of human galectin-3
(SEQ ID NO:1), the second sequence in the figure is amino acids 1
to 245 of hamster galectin-3, the third sequence in the figure is
amino acids 1 to 242 of rabbit galectin-3, and the fourth (lower)
sequence in the figure is amino acids 1 to 262 of chicken
galectin-3.
[0018] FIG. 4 depicts a CLUSTAL W alignment of the amino acid
sequence of human galectin-7 (SEQ ID NO:2) with the amino acid
sequences of rat galectin-7 (Accession No. P97590 in GenBank) and
mouse galectin-7 (Accession No. O54974 in GenBank). The first
(upper) sequence in the figure is amino acids 1 to 136 of rat
galectin-7, the second sequence in the figure is amino acids 1 to
136 of mouse galectin-7, and the third (lower) sequence in the
figure is amino acids 1 to 136 of human galectin-7 (SEQ ID
NO:2).
[0019] FIG. 5 is a summary of the results of a PROSITE scan of
human galectin-3 (SEQ ID NO:1).
[0020] FIG. 6 is a summary of the results of a PROSITE scan of
human galectin-7 (SEQ ID NO:2).
[0021] FIG. 7 depicts an alignment of the galactoside-binding
domain of human galectin-3 with a consensus amino acid sequence
(PF00337) derived from a hidden Markov model (HMM) from PFAM. The
upper sequence is the consensus amino acid sequence (PF00337, SEQ
ID NO:3), while the lower amino acid sequence corresponds to amino
acids 117 to 247 of SEQ ID NO:1.
[0022] FIG. 8 depicts an alignment of the galactoside-binding
domain of human galectin-7 with a consensus amino acid sequence
(PF00337) derived from a hidden Markov model (HMM) from PFAM. The
upper sequence is the consensus amino acid sequence (PF00337, SEQ
ID NO:3), while the lower amino acid sequence corresponds to amino
acids 5 to 135 of SEQ ID NO:2.
[0023] FIG. 9 includes a series of photographs of corneas with 2 mm
abrasion or excimer laser wounds that were allowed to partially
heal in vivo and were then analyzed for galectin-3 immunoreactivity
in paraffin sections. (A), Hematoxylin and eosin staining of (i)
normal corneas and corneas immediately after (ii) abrasion and
(iii) excimer laser injury. (B), Immunohistochemical staining of
(i) normal gal3.sup.+/+ corneas and (ii) healing gal3.sup.+/+
corneas after excimer laser injury. Immunohistochemical staining of
(iii) normal gal3.sup.-/- corneas and (iv) healing gal3.sup.-/-
corneas after excimer laser injury. Dark color indicates positive
immunostaining. WE, wound edge; LE, leading edge of migrating
epithelium; arrows, epithelium; arrowheads, leukocytes/stromal
cells.
[0024] FIG. 10 is a graph illustrating the effect of .beta.-lactose
(Lac) and sucrose (Suc) on the healing rate of injured corneal
epithelium.
[0025] FIG. 11 is a series of graphs illustrating the healing rate
of injured corneal epithelium in wild type (gal-3.sup.+/+) and
galectin-3 deficient (gal-3.sup.-/-) mice injured by excimer laser
or alkali treatment and allowed to heal in vivo or in vitro.
[0026] FIG. 12 is a table depicting differences in gene expression
of galectin-7 and a selection of house keeping genes (GAPDH is
D-glyceraldehyde-3-phosphate dehydrogenase; RPS29 is ribosomal
protein S29; ODC is ornithine decarboxylase) between wild type
(gal-3.sup.+/+) and galectin-3 deficient (gal-3.sup.-/-) mice as
determined by cDNA microarray and semi-quantitative PCR.
[0027] FIG. 13 illustrates differences in the expression of
galectin-7 between wild type (gal-3.sup.+/+) and galectin-3
deficient (gal-3.sup.-/-) mice as determined by (A) western blot
analysis, (B) immunohistochemical analysis, and (C) using mouse
embryonic fibroblasts.
[0028] FIG. 14 is a graph illustrating the effect of exogenous
galectin-3 on the healing rate of injured corneal epithelium in (A)
galectin-3 deficient (gal-3.sup.-/-) mice and (B) wild type
(gal-3.sup.+/+) mice.
[0029] FIG. 15 is a graph illustrating the effect of .beta.-lactose
(Lac) and sucrose (Suc) on the healing rate of injured corneal
epithelium of wild type (gal-3.sup.+/+) mice in the presence of
exogenous galectin-3.
[0030] FIG. 16 includes (A) a graph illustrating the effect of
exogenous galectin-7 on the healing rate of injured corneal
epithelium in wild type (gal-3.sup.+/+), when used alone, with
.beta.-lactose (Lac), or with sucrose (Suc); and (B) a graph
comparing the effect of exogenous galectin-7 on the healing rate of
injured corneal epithelium in wild type (gal-3.sup.+/+) and
galectin-3 deficient (gal-3.sup.-/-) mice.
DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
[0031] The present application mentions various patents, scientific
articles, and other publications. The contents of each such item
are hereby incorporated by reference. In addition, the contents (as
of the filing date of the application) of all websites referred to
herein are incorporated by reference.
[0032] The present invention provides pharmaceutical compositions
comprising galectin-3 and/or galectin-7 useful for enhancing the
re-epithelialization of wounds in injured mammalian tissues. The
invention also provides methods for the therapeutic treatment of
epithelial injuries in mammalian tissue comprising administering to
a mammal afflicted with an epithelial injury a therapeutically
effective amount of galectin-3, galectin-7, or a combination of
galectin-3 and -7. When administering a combination of galectin-3
and -7, galectin-3 may be administered before, in conjunction with,
or after the administration of galectin-7.
[0033] The invention encompasses the finding that galectin-3 is
up-regulated in migrating corneal epithelial cells following injury
to the cornea (Example 1). The invention also includes the
discovery that the re-epithelialization of corneal transepithelial
excimer laser wounds and corneal alkali-burn wounds is
significantly slower in galectin-3-deficient mice compared to that
in wild type mice (Example 2). The invention further provides the
discovery that the expression of a number of injury-related genes
(e.g., tolloid-like protein and galectin-7) are abnormal in
galectin-3-deficient mice (Example 3). Additionally, the invention
demonstrates that exogenous galectin-3 and -7 promote the
re-epithelialization of corneal wounds (Examples 4 and 5,
respectively).
Galectins
[0034] Lectins are proteins that are defined by their ability to
bind carbohydrates specifically and to agglutinate cells (see, for
example, Sharon, Trends Biochem. Sci. 18:221, 1993). Lectins have
been shown to be involved in a wide variety of cellular functions
including cell-cell and cell-matrix interactions. Lectins are
widespread among plants, invertebrates and mammals. Animal lectins
have been grouped into four distinct families: 1) C-type lectins;
2) P-type lectins; 3) galectins (formerly termed S-type lectins);
and 4) pentraxins (see, for example, Barondes et al., J. Biol.
Chem. 269:20807, 1994).
[0035] All mammalian galectins that have been analyzed in detail
recognize .beta.-lactose and related .beta.-galactosides. While all
mammalian galectins share similar affinity for small
.beta.-galactosides, they show significant differences in binding
specificity for more complex glycoconjugates (Henrick et al.,
Glycobiology 8:45, 1998; Sato et al., J. Biol. Chem. 267:6983,
1992; and Seetharaman et al., J. Biol. Chem. 273:13047, 1998). In
addition to binding .beta.-galactoside sugars, galectins possess
hemagglutination activity. Laminin, a naturally occurring
glycoprotein containing numerous polylactosamine chains, has been
shown to be a natural ligand for certain galectins. Laminin is a
component of the basal laminae, the extracellular matrix which
underlies all epithelia and surrounds individual muscle, fat and
Schwann cells. Interactions between cells and the basal laminae are
known to influence the migration and/or differentiation of various
cell types during mammalian development. Galectins do not contain
traditional sequences that specify membrane translocation, but are
both secreted and located intracellularly. In addition to their
affinity for .beta.-galactoside sugars, members of the galectin
family share significant sequence similarity in the carbohydrate
recognition domain (CRD; also referred to as the
carbohydrate-binding domain), the relevant amino acid residues of
which have been determined by X-ray crystallography (Lobsanov et
al., J. Biol. Chem. 267:27034, 1993 and Seetharaman et al., supra).
Galectins have been implicated in a wide variety of biological
functions including cell adhesion (Cooper et al., J. Cell Biol.
115:1437, 1991), growth regulation (Wells et al., Cell 64:91,
1991), cell migration (Hughes, Curr. Opin. Struct. Biol. 2:687,
1992), neoplastic transformation (Raz et al., Int. J. Cancer
46:871, 1990) and immune responses (Offner et al., J. Neuroimmunol.
28:177, 1990). There are presently 12 characterized eukaryotic
members of the galectin family.
Galectin-3
[0036] Members of the galectin-3 family of proteins (previously
known as CBP-35, Mac-2, L-34, .epsilon.BP, and RL-29) typically
include between about 240 and 270 amino acids and have molecular
weights that range between about 25 and 29 kDa. Galectin-3 proteins
are generally composed of a short N-terminal domain, a C-terminal
domain which includes a galactoside-binding region, and an
intervening proline, glycine, and tyrosine-rich domain which
includes repeats of 7-10 conserved amino acids (Liu et al.,
Biochemistry 35:6073, 1996 and Cherayil et al., Proc. Natl. Acad.
Sci. USA, 87:7324, 1990). The tandem repeats are similar to those
found in the collagen gene superfamily. The number of repeats
varies between galectin-3 proteins and accounts for the differences
in size between galectin-3 proteins from different species. The
N-terminal domain of galectin-3 permits the protein to undergo
multimerization upon binding to surfaces containing glycoconjugate
ligands.
[0037] Galectin-3 is expressed in various inflammatory cells (e.g.,
activated macrophages, basophils, and mast cells) and in epithelia
and fibroblasts of various tissues (Perillo et al., J. Mol. Med.
76:402, 1998). It is found on the cell surface, within the
extracellular matrix (ECM), in the cytoplasm, and in the nucleus of
cells. On the cell surface or in the ECM galectin-3 is thought to
mediate cell-cell and cell-matrix interactions by binding to
complementary glycoconjugates containing polylactosamine chains
found in many ECM and cell surface molecules. Galectin-3 is thought
to inhibit cell-matrix adhesion by binding to laminin. In the
nucleus of cells galectin-3 may influence cell-matrix interactions
indirectly by influencing the expression of well-known cell
adhesion molecules (e.g., .alpha.6.beta.1 and .alpha. 4.beta.7
integrins, Warlfield et al., Invasion Metastasis 17:101, 1997 and
Matarrese et al., Int. J. Cancer 85:545, 2000) and cytokines (e.g.,
IL-1, Jeng et al., Immunol. Lett. 42:113, 1994). Galectin-3
expression is developmentally regulated in selected organs such as
the kidney and its expression level in pulmonary alveolar
epithelial cells and hepatocytes is up-regulated following injury.
Galectin-3 has been shown to concentrate in the nucleus of certain
cell types during proliferation. Expression of galectin-3 is
elevated in certain tumors, suggesting galectin-3 plays a role in
metastasis. Indeed, overexpression of galectin-3 in a weakly
metastatic cell line caused a significant increase in metastatic
potential (Raz et al., supra).
[0038] Human galectin-3 is 250 amino acids long and has an
approximate molecular weight of 26.1 kDa (SEQ ID NO:1, FIG. 1). As
illustrated in FIGS. 1, 3, 5, and 7, human galectin-3 contains the
following domains, signature sequences, or other structural
features (for general information regarding PS and PF prefix
identification numbers, refer to Sonnhammer et al., Protein 28:405,
1997): an N-terminal domain located at about amino acid residues 1
to 14 of SEQ ID NO:1; a proline, glycine, and tyrosine-rich domain
located at about amino acid residues 15 to 116 of SEQ ID NO:1; a
galactoside-binding domain located at about amino acid residues 117
to 247 of SEQ ID NO:1; a galaptin signature sequence (PROSITE No.
PS00309) located at about amino acids 181 to 200 of SEQ ID NO:1;
one potential N-glycosylation site (PROSITE No. PS00001) located at
about amino acids 4 to 7 of SEQ ID NO:1; two potential protein
kinase C phosphorylation sites (PROSITE No. PS00005) located at
about amino acids 137 to 139 and 194 to 196 of SEQ ID NO:1; two
potential casein kinase II phosphorylation sites (PROSITE No.
PS00006) located at about amino acids 6 to 9 and 175 to 178 of SEQ
ID NO: 1; and eight potential myristoylation sites (PROSITE No.
PS00008) located at about amino acids 24 to 29, 27 to 32, 34 to 39,
43 to 48, 52 to 57, 61 to 66, 65 to 70, and 68 to 73 of SEQ ID
NO:1.
[0039] As defined herein, a "galectin-3 protein" may include a
galectin-3 "N-terminal domain", a galectin-3 "proline, glycine, and
tyrosine-rich domain", and/or a galectin-3 "galactoside-binding
domain". These domains are further defined as follows.
[0040] As used herein, a galectin-3 "N-terminal domain" includes an
amino acid sequence of about 10-20 amino acids, preferably about 14
amino acids that shares at least about 60%, 70%, 80%, 90%, 95%,
99%, or 100% identity with amino acids 1 to 14 of SEQ ID NO:1. The
N-terminal domain can include an N-glycosylation site (PROSITE No.
PS00001) and/or a casein kinase II phosphorylation site (PROSITE
No. PS00006). The PROSITE N-glycosylation site has the consensus
sequence: N-{P}-[ST]-{P} and the PROSITE casein kinase II
phosphorylation site has the consensus sequence: [ST]-X(2)-[DE]. In
the above consensus sequences, and other motifs or signature
sequences described herein, the standard IUPAC one-letter code for
the amino acids is used. Each element in the pattern is separated
by a dash (-); square brackets ([ ]) indicate the particular
residues that are accepted at that position; X indicates that any
residue is accepted at that position; and numbers in parentheses ((
)) indicate the number of residues represented by the accompanying
amino acid. In certain embodiments, the N-terminal domain includes
amino acids L7 and L11 of SEQ ID NO:1. As shown in FIG. 3, these
amino acids are conserved across several mammalian species of
galectin-3 and may therefore play a catalytic and/or structural
role.
[0041] As used herein, a galectin-3 "proline, glycine, and
tyrosine-rich domain" includes an amino acid sequence of about 60
to 140 amino acids, more preferably about 80 to 120 amino acids, or
about 90 to 110 amino acids that shares at least about 60%, 70%,
80%, 90%, 95%, 99%, or 100% identity with amino acids 15 to 116 of
SEQ ID NO:1. The proline, glycine, and tyrosine-rich domain can
also include one, two, three, four, five, six, seven, or eight
N-myristoylation sites (PROSITE No. PS00008) which have the
consensus sequence: G-{EDRKHPFYW}-X(2)-[STAGCN]-{P}. In certain
embodiments, the proline, glycine, and tyrosine-rich domain
includes the following amino acids and regions of SEQ ID NO:1: G21,
P23, G27, N28, P30, G32, G34, P37, Y41-P46, G53, Y55-G57, P61, G62,
G66, P72, G73, G77, Y79-G81, P83, G87, Y89, P90, G99, Y101, P102,
P106, Y107, A109, L114, and V116. These amino acids and regions are
conserved across several mammalian species of galectin-3 and may
play a catalytic and/or structural role (see amino acids indicated
with a "*" in FIG. 3).
[0042] As used herein, a galectin-3 "galactoside-binding domain"
includes an amino acid sequence of about 80 to 180 amino acids
having a bit score for the alignment of the sequence to the
consensus sequence PF00337 from PFAM (SEQ ID NO:3) of at least 150.
Preferably, a galectin-3 galactoside-binding domain includes at
least about 100 to 160 amino acids, more preferably about 110 to
150 amino acids, or about 120 to 140 amino acids and has a bit
score for the alignment of the sequence to the consensus sequence
PF00337 from PFAM (SEQ ID NO:3) of at least 150, more preferably at
least 175, most preferably 200 or greater.
[0043] To calculate the bit score for the alignment of a particular
sequence to the consensus sequence PF00337 from PFAM, the sequence
of interest can be searched against the PFAM database of HMMs
(e.g., the PFAM database, release 2.1) using the default parameters
available at www.sanger.ac.uk/Software/Pfam. A description of the
PFAM database can be found in Sonnhammer et al., supra and a
detailed description of HMMs can be found, for example, in Gribskov
et al., Meth. Enzymol. 183:146, 1990 and Stultz et al., Protein
Sci. 2:305, 1993.
[0044] The galectin-3 galactoside-binding domain can further
include one, preferably two, protein kinase C phosphorylation sites
(PROSITE No. PS00005); a casein kinase II phosphorylation site
(PROSITE No. PS00006); and/or a galaptin signature sequence
(PROSITE No. PS00309). The protein kinase C phosphorylation site
has the following consensus sequence: [ST]-X-[RK]. The galaptin
signature sequence has the following consensus sequence:
W-[GEK]-X-[EQ]-X-[KRE]-X(3,6)-[PCTF]-[LIVMF]-[NQEGSKV]-X-[GH]-X-
(3)-[DENKHS]-[LIVMFC]. In certain embodiments, the galectin-3
galactoside-binding domain includes the following amino acids and
regions of SEQ ID NO:1: P117, Y118, L120-L122, G125, P128, R129,
L131-I134, G136-V138, N141, N143, R144, L147, F149, R151, G152,
D154, A156-F163, E165, R169-N174, N179-G182, E184-R186, F190-E193,
G195, P197-K199, Q201-L203, E205, D207-Q220, N222, R224, L228,
I231, I236, G238-I240, and L242-S244. These amino acids and regions
are conserved across several mammalian species of galectin-3 and
may play a catalytic and/or structural role (see amino acids
indicated with a "*" in FIG. 3).
[0045] Certain galectin-3 proteins of the present invention include
the amino acid sequence of human galectin-3 as represented by SEQ
ID NO:1. Other galectin-3 proteins of the present invention include
an amino acid sequence that is substantially identical to the amino
acid sequence of SEQ ID NO:1. The term "substantially identical" is
used herein to refer to a first amino acid that contains a
sufficient or minimum number of amino acid residues that are
identical to aligned amino acid residues in a second amino acid
sequence such that the first and second amino acid sequences can
have a common structural domain and/or common functional activity.
For example, amino acid sequences that contain a common structural
domain having at least about 60%, or 65% identity, preferably at
least 75% identity, more preferably at least 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1 are
termed substantially identical to the amino acid sequence of SEQ ID
NO:1. In particular, proteins which contain accidentally or
deliberately induced alterations, such as deletions, additions,
substitutions or modifications of certain amino acid residues of
SEQ ID NO:1 may fall within the definition of galectin-3 proteins
provided herein. It will also be appreciated that as defined
herein, galectin-3 proteins may include regions represented by the
amino acid sequence of galectin-3 taken from other mammalian
species including but not limited to bovine, canine, feline,
caprine, ovine, porcine, murine, and equine species.
[0046] Calculations of sequence identity between sequences are
performed as follows. To determine the percent identity of two
amino acid sequences, the sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in one or both of
a first and a second amino acid sequence for optimal alignment).
The amino acid residues at corresponding amino acid positions or
nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the proteins are identical at that position. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences, taking into account
the number of gaps, and the length of each gap, which need to be
introduced for optimal alignment of the two sequences.
[0047] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using an
alignment software program using the default parameters. Suitable
programs include, for example, CLUSTAL W by Thompson et al., Nuc.
Acids Research 22:4673, 1994 (www.ebi.ac.uk/clustalw), BL2SEQ by
Tatusova and Madden, FEMS Microbiol. Lett. 174:247, 1999
(www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html), SAGA by Notredame and
Higgins, Nuc. Acids Research 24:1515, 1996
(igs-server.cnrs-mrs.fr/.about.cnotred), and DIALIGN by Morgenstern
et al., Bioinformatics 14:290, 1998
(bibiserv.techfak.uni-bielefeld.de/dialign).
Galectin-7
[0048] Members of the galectin-7 family of proteins typically exist
as monomers that include between about 130 and 140 amino acids and
have molecular weights that range between about 15 and 16 kDa (see,
for example, Magnaldo et al., Develop. Biol. 168:259, 1995 and
Madsen et al., J. Biol. Chem. 270:5823, 1995). The expression of
galectin-7 has been associated with the onset of epithelial
stratification (Timmons et al., Int. J. Dev. Biol. 43:229, 1999).
Galectin-7 is thought to play a role in cell-matrix and cell-cell
interactions. Galectin-7 is found in areas of cell-cell contact
(e.g., in the upper layers of human epidermis); its expression is
sharply downregulated in anchorage independent keratinocytes and it
is absent in a malignant keratinocyte cell line. Galectin-7 may be
required for the maintenance of normal keratinocytes (see, Madsen
et al., supra).
[0049] Human galectin-7 includes 136 amino acids and has an
approximate molecular weight of 15.1 kDa (SEQ ID NO:2, FIG. 2). As
illustrated in FIGS. 2, 4, 6, and 8, human galectin-7 contains the
following domains, signature sequences, or other structural
features: a galactoside-binding domain located at about amino acid
residues 5 to 135 of SEQ ID NO:2; a galaptin signature sequence
(PROSITE No. PS00309) located at about amino acids 70 to 89 of SEQ
ID NO:2; one N-glycosylation site (PROSITE No. PS00001) located at
about amino acids 29 to 32 of SEQ ID NO:2; one protein kinase C
phosphorylation site (PROSITE No. PS00005) located at about amino
acids 132 to 134 of SEQ ID NO:2; one casein kinase II
phosphorylation site (PROSITE No. PS00006) located at about amino
acids 9 to 12 of SEQ ID NO:2; and two myristoylation sites (PROSITE
No. PS00008) located at about amino acids 13 to 18 and 44 to 49 of
SEQ ID NO:2.
[0050] As defined herein, a "galectin-7 protein" includes a
galectin-7 "galactoside-binding domain". This domain is further
defined as follows.
[0051] As used herein, a galectin-7 "galactoside-binding domain"
includes an amino acid sequence of about 80 to 180 amino acids
having a bit score for the alignment of the sequence to the
consensus sequence PF00337 from PFAM (SEQ ID NO:3) of at least 80.
Preferably, a galectin-7 galactoside-binding domain includes at
least about 100 to 160 amino acids, more preferably about 110 to
150 amino acids, or about 120 to 140 amino acids and has a bit
score for the alignment of the sequence to the consensus sequence
PF00337 from PFAM (SEQ ID NO:3) of at least 80, more preferably at
least 100, most preferably 120 or greater. The galectin-7
galactoside-binding domain can include one N-glycosylation site
(PROSITE No. PS00001); one protein kinase C phosphorylation site
(PROSITE No. PS00005); one casein kinase II phosphorylation site
(PROSITE No. PS00006); one or two myristoylation sites (PROSITE No.
PS00008); and/or a galaptin signature sequence (PROSITE No.
PS00309). In certain embodiments, the galectin-7
galactoside-binding domain includes the following amino acids and
regions of SEQ ID NO:2: M1, S2, H6, K7, L10, P11, G13, R15,
G17-V19, R21-G24, V26, P27, A30, R32-Q43, D46-N63, K65, Q67, G68,
W70-G76, G78, P80-L90, I92, G97-K99, V101, G103, D104, Y107, H109,
F110, H112, R113, P115, V119, R120, V122-L130, S132, I135, and
F136. These amino acids and regions are conserved across several
mammalian species of galectin-7 and may play a catalytic and/or
structural role (see amino acids indicated with a"*" in FIG.
4).
[0052] Certain galectin-7 proteins of the present invention include
the amino acid sequence of human galectin-7 as represented by SEQ
ID NO:2. Other galectin-7 proteins of the present invention include
an amino acid sequence that is substantially identical to the amino
acid sequence of SEQ ID NO:2. In particular, proteins which contain
accidentally or deliberately induced alterations, such as
deletions, additions, substitutions or modifications of certain
amino acid residues of SEQ ID NO:2 may fall within the definition
of galectin-7 provided herein. It will also be appreciated that as
defined herein, galectin-7 proteins may include regions represented
by the amino acid sequence of galectin-7 taken from other mammalian
species including but not limited to bovine, canine, feline,
caprine, ovine, porcine, murine, and equine species.
Preparation of Galectin-3 and Galectin-7
[0053] It will be appreciated by one of ordinary skill in the art,
that the galectins of this invention can be obtained from any
available source. These include but are not limited to proteins
isolated from natural sources, produced recombinantly or produced
synthetically, e.g., by solid phase procedures. In accordance with
the present invention, polynucleotide sequences which encode
galectin-3 or galectin-7 may be used in recombinant DNA molecules
that direct the expression of the galectins of this invention in
appropriate host cells. Cherayil et al., supra and Madsen et al.,
supra, describe in detail the cloning of human galectin-3 and -7
respectively. In order to express a biologically active galectin-3
or galectin-7, the nucleotide sequence encoding galectin-3,
galectin-7, or their functional equivalent, is inserted into an
appropriate expression vector, i.e., a vector which contains the
necessary elements for the transcription and translation of the
inserted coding sequence. Methods which are well known to those
skilled in the art can be used to construct expression vectors
containing a galectin-3-encoding or galectin-7-encoding sequence
and appropriate transcriptional or translational controls. These
methods include in vitro recombinant DNA techniques, synthetic
techniques and in vivo recombination or genetic recombination. The
introduction of deletions, additions, or substitutions can be
achieved using any known technique in the art e.g., using PCR based
mutagenisis. Such techniques are described in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,
Plainview, N.Y., 1989 and Ausubel et al., Current Protocols in
Molecular Biology, John Wiley & Sons, New York, N.Y., 1989. A
variety of expression vector/host systems may be utilized to
contain and express a galectin-3-encoding or galectin-7-encoding
sequence. These include but are not limited to microorganisms such
as bacteria transformed with recombinant bacteriophage, plasmid or
cosmid DNA expression vectors; yeast transformed with yeast
expression vectors; insect cell systems infected with virus
expression vectors (e.g., baculovirus); plant cell systems
transfected with virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or transformed with
bacterial expression vectors (e.g., Ti, pBR322, or pET25b plasmid);
or animal cell systems. Alternatively, the galectins of the present
invention could be produced using chemical methods to synthesize a
galectin-3 or galectin-7 amino acid sequence, whole or in part. For
example, peptide synthesis can be performed using various
solid-phase techniques (Roberge et al., Science 269:202, 1995) and
automated synthesis may be achieved, for example, using the 431A
peptide synthesizer (available from Applied Biosystems of Foster
City, Calif.) in accordance with the instructions provided by the
manufacturer.
Pharmaceutical Compositions
[0054] In one aspect of the present invention, pharmaceutical
compositions are provided, wherein these compositions comprise
galectin-3 and/or galectin-7, and optionally comprise a
pharmaceutically acceptable carrier. In certain embodiments, these
compositions optionally further comprise one or more additional
therapeutic agents. In certain embodiments, the additional
therapeutic agent or agents are selected from the group consisting
of growth factors, anti-inflammatory agents, vasopressor agents,
collagenase inhibitors, topical steroids, matrix metalloproteinase
inhibitors, ascorbates, angiotensin II, angiotensin III,
calreticulin, tetracyclines, fibronectin, collagen, thrombospondin,
transforming growth factors (TGF), keratinocyte growth factor
(KGF), fibroblast growth factor (FGF), insulin-like growth factors
(IGF), epidermal growth factor (EGF), platelet derived growth
factor (PDGF), neu differentiation factor (NDF), hepatocyte growth
factor (HGF), and hyaluronic acid.
[0055] As used herein, the term "pharmaceutically acceptable
carrier" includes any and all solvents, diluents, or other liquid
vehicle, dispersion or suspension aids, surface active agents,
isotonic agents, thickening or emulsifying agents, preservatives,
solid binders, lubricants and the like, as suited to the particular
dosage form desired. Remington's Pharmaceutical Sciences Ed. by
Gennaro, Mack Publishing, Easton, Pa., 1995 discloses various
carriers used in formulating pharmaceutical compositions and known
techniques for the preparation thereof. Some examples of materials
which can serve as pharmaceutically acceptable carriers include,
but are not limited to, sugars such as glucose, and sucrose;
starches such as corn starch and potato starch; cellulose and its
derivatives such as sodium carboxymethyl cellulose, ethyl
cellulose, and cellulose acetate; powdered tragacanth; malt;
gelatin; talc; excipients such as cocoa butter and suppository
waxes; oils such as peanut oil, cottonseed oil, safflower oil,
sesame oil, olive oil, corn oil, and soybean oil; glycols; such a
propylene glycol; esters such as ethyl oleate and ethyl laurate;
agar; buffering agents such as magnesium hydroxide and aluminum
hydroxide; alginic acid; pyrogen-free water; isotonic saline;
Ringer's solution; ethyl alcohol, and phosphate buffer solutions,
as well as other non-toxic compatible lubricants such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
releasing agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants can also be
present in the composition, according to the judgment of the
formulator.
Therapeutically Effective Dose
[0056] In yet another aspect, according to the methods of treatment
of the present invention, the re-epithelialization of wounds is
promoted by contacting the wounds with a pharmaceutical
composition, as described herein. Thus, the invention provides
methods for the treatment of wounds comprising administering a
therapeutically effective amount of a pharmaceutical composition
comprising active agents that include galectin-3 and/or galectin-7
to a subject in need thereof, in such amounts and for such time as
is necessary to achieve the desired result. It will be appreciated
that this encompasses administering an inventive pharmaceutical as
a therapeutic measure to promote the re-epithelialization of a
wound or as a prophylactic measure to minimize complications
associated with the slow re-epithelialization of wounds (e.g., as a
wound irrigation solution during and/or following surgery). In
certain embodiments of the present invention a "therapeutically
effective amount" of the pharmaceutical composition is that amount
effective for promoting the re-epithelialization of a wound. The
compositions, according to the method of the present invention, may
be administered using any amount and any route of administration
effective for healing a wound. Thus, the expression "amount
effective for promoting the re-epithelialization of a wound", as
used herein, refers to a sufficient amount of composition to heal a
wound. The exact dosage is chosen by the individual physician in
view of the patient to be treated. Dosage and administration are
adjusted to provide sufficient levels of the active agent(s) or to
maintain the desired effect. Additional factors which may be taken
into account include the severity of the disease state, e.g., wound
size and location; age, weight and gender of the patient; diet,
time and frequency of administration; drug combinations; reaction
sensitivities; and tolerance/response to therapy. Long acting
pharmaceutical compositions might be administered every 3 to 4
days, every week, or once every two weeks depending on half-life
and clearance rate of the particular composition.
[0057] The active agents of the invention are preferably formulated
in dosage unit form for ease of administration and uniformity of
dosage. The expression "dosage unit form" as used herein refers to
a physically discrete unit of active agent appropriate for the
patient to be treated. It will be understood, however, that the
total daily usage of the compositions of the present invention will
be decided by the attending physician within the scope of sound
medical judgment. For any active agent, the therapeutically
effective dose can be estimated initially either in cell culture
assays or in animal models, usually mice, rabbits, dogs, or pigs.
The animal model is also used to achieve a desirable concentration
range and route of administration. Such information can then be
used to determine useful doses and routes for administration in
humans. A therapeutically effective dose refers to that amount of
active agent which ameliorates the symptoms or condition.
Therapeutic efficacy and toxicity of active agents can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., ED50 (the dose is therapeutically
effective in 50% of the population) and LD50 (the dose is lethal to
50% of the population). The dose ratio of toxic to therapeutic
effects is the therapeutic index, and it can be expressed as the
ratio, LD50/ED50. Pharmaceutical compositions which exhibit large
therapeutic indices are preferred. The data obtained from cell
culture assays and animal studies is used in formulating a range of
dosage for human use.
Administration of Pharmaceutical Compositions
[0058] After formulation with an appropriate pharmaceutically
acceptable carrier in a desired dosage, the pharmaceutical
compositions of this invention can be administered to humans and
other mammals topically (as by powders, ointments, or drops),
orally, rectally, parenterally, intracisternally, intravaginally,
intraperitoneally, bucally, ocularly, or nasally, depending on the
severity and location of the wound being treated.
[0059] Liquid dosage forms for oral administration include, but are
not limited to, pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active agent(s), the liquid dosage forms may
contain inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, and perfuming agents.
[0060] Dosage forms for topical or transdermal administration of an
inventive pharmaceutical composition include ointments, pastes,
creams, lotions, gels, powders, solutions, sprays, inhalants, or
patches. The active agent is admixed under sterile conditions with
a pharmaceutically acceptable carrier and any needed preservatives
or buffers as may be required. For example, ocular or cutaneous
infections may be treated with aqueous drops, a mist, an emulsion,
or a cream. Administration may be therapeutic or it may be
prophylactic. Prophylactic formulations may be present or applied
to the site of potential wounds, or to sources of wounds, such as
contact lenses, contact lens cleaning and rinsing solutions,
containers for contact lens storage or transport, devices for
contact lens handling, eye drops, surgical irrigation solutions,
ear drops, eye patches, and cosmetics for the eye area, including
creams, lotions, mascara, eyeliner, and eyeshadow. The invention
includes opthalmological devices, surgical devices, audiological
devices or products which contain disclosed compositions (e.g.,
gauze bandages or strips), and methods of making or using such
devices or products. These devices may be coated with, impregnated
with, bonded to or otherwise treated with a disclosed
composition.
[0061] The ointments, pastes, creams, and gels may contain, in
addition to an active agent of this invention, excipients such as
animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc, zinc oxide, or mixtures
thereof.
[0062] Powders and sprays can contain, in addition to the agents of
this invention, excipients such as talc, silicic acid, aluminum
hydroxide, calcium silicates, polyamide powder, or mixtures of
these substances. Sprays can additionally contain customary
propellants such as chlorofluorohydrocarbons.
[0063] Transdermal patches have the added advantage of providing
controlled delivery of the active ingredients to the body. Such
dosage forms can be made by dissolving or dispensing the compound
in the proper medium. Absorption enhancers can also be used to
increase the flux of the compound across the skin. The rate can be
controlled by either providing a rate controlling membrane or by
dispersing the compound in a polymer matrix or gel.
[0064] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables. The
injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use. In order to
prolong the effect of an active agent, it is often desirable to
slow the absorption of the agent from subcutaneous or intramuscular
injection. Delayed absorption of a parenterally administered active
agent may be accomplished by dissolving or suspending the agent in
an oil vehicle. Injectable depot forms are made by forming
microencapsule matrices of the agent in biodegradable polymers such
as polylactide-polyglycolide. Depending upon the ratio of active
agent to polymer and the nature of the particular polymer employed,
the rate of active agent release can be controlled. Examples of
other biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the agent in liposomes or microemulsions which are
compatible with body tissues.
[0065] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the active
agent(s) of this invention with suitable non-irritating excipients
or carriers such as cocoa butter, polyethylene glycol or a
suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectum or vaginal
cavity and release the active agent(s).
[0066] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active agent is mixed with at least one inert, pharmaceutically
acceptable excipient or carrier such as sodium citrate or dicalcium
phosphate and/or a) fillers or extenders such as starches, sucrose,
glucose, mannitol, and silicic acid, b) binders such as, for
example, carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as
glycerol, d) disintegrating agents such as agar-agar, calcium
carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof.
[0067] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as milk sugar as well as high molecular weight
polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active agent(s) may be admixed with at least one inert diluent such
as sucrose or starch. Such dosage forms may also comprise, as is
normal practice, additional substances other than inert diluents,
e.g., tableting lubricants and other tableting aids such a
magnesium stearate and microcrystalline cellulose. In the case of
capsules, tablets and pills, the dosage forms may also comprise
buffering agents. They may optionally contain opacifying agents and
can also be of a composition that they release the active agent(s)
only, or preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions
which can be used include polymeric substances and waxes.
Uses of Pharmaceutical Compositions
[0068] As discussed above and described in greater detail in the
Examples, galectin-3 and galectin-7 are useful as promoters of the
re-epithelialization of wounds. In general, it is believed that
these galectins will be clinically useful in stimulating the
healing of wounds associated with any epithelial tissue including
but not limited to the skin epithelium; the corneal epithelium; the
lining of the gastrointestinal tract; the lung epithelium; and the
inner surface of kidney tubules, of blood vessels, of the uterus,
of the vagina, of the urethra, or of the respiratory tract. The
present invention encompasses the treatment of a variety of
epithelial wound types including but not limited to surgical
wounds, excisional wounds, blisters, ulcers, lesions, abrasions,
erosions, lacerations, boils, cuts, sores, and burns resulting from
heat exposure or chemicals. These wounds may be in normal
individuals or those subject to conditions which induce abnormal
wound healing such as diabetes, corneal dystrophies, uremia,
malnutrition, vitamin deficiencies, obesity, infection,
immunosuppression and complications associated with systemic
treatment with steroids, radiation therapy, non-steroidal
anti-inflammatory drugs (NSAID), anti-neoplastic drugs and
anti-metabolites.
[0069] Galectin-3 and/or galectin-7 could, for example, be used to
promote dermal re-establishment subsequent to dermal loss.
Alternatively, galectin-3 and/or galectin-7 could be used to
increase the adherence of skin grafts to a wound bed and to
stimulate re-epithelialization from the wound bed. Suitable skin
grafts include, but are not limited to, autografts, artificial
skin, allografts, autodermic grafts, autoepidermic grafts, avacular
grafts, Blair-Brown grafts, bone grafts, brephoplastic grafts,
cutis grafts, delayed grafts, dermic grafts, epidermic grafts,
fascia grafts, full thickness grafts, heterologous grafts,
xenografts, homologous grafts, hyperplastic grafts, lamellar
grafts, mesh grafts, mucosal grafts, Ollier-Thiersch grafts,
omenpal grafts, patch grafts, pedicle grafts, penetrating grafts,
split skin grafts, and thick split grafts.
[0070] Galectin-3 and/or galectin-7 could be used to treat
dermatitis herpetiformis in which blisters form at the
dermo-epidermal junction. Galectin-3 and/or galectin-7 could be
used to treat epidermolysis bullosa, a defect in adherence of the
epidermis to the underlying dermis which results in frequent, open
and painful blisters, by accelerating re-epithelialization of these
lesions. Galectin-3 and/or galectin-7 could further be used to
treat pemphigus diseases that involve loss of cell-cell adhesion
within the epidermis, or pemphigoid diseases that involve loss of
cell-cell adhesion at the dermo-epidermal junction. Galectin-3
and/or galectin-7 could be used to treat a variety of ulcers
including but not limited to diabetic ulcers, dermal ulcers,
decubitus ulcers, arterial ulcers, and venous stasis ulcers.
[0071] The present invention encompasses methods for the promotion
of corneal tissue healing. This includes treating corneal
epithelial defects caused by corneal ulcers, heat, radiation,
phlyctenulosis, corneal abrasions or lacerations, photorefractive
surgery for corrective myopia, foreign bodies and sterile corneal
infiltrates; chemical burns caused by exposure to acids or alkali
(e.g., hydrofluoric acid, formic acid, anhydrous ammonia, cement,
and phenol) or other chemical agents such as white phosphorus,
elemental metals, nitrates, hydrocarbons, and tar; keratopathies
such as neurotrophic keratopathy, diabetic keratopathy and
Thygeson's superficial punctate keratopathy; keratities such as
viral keratitis (e.g., metaherpetic or herpetic keratitis) and
bacterial keratitis; and corneal dystrophies such as lattice
dystrophy, epithelial basement membrane dystrophy (EBMD) and Fuch's
endothelial dystrophy.
[0072] Galectin-3 and/or galectin-7 could also be used to treat
gastrointestinal ulcers and help heal the mucosal lining and
regeneration of glandular mucosa and duodenal mucosal lining more
rapidly. Inflammatory bowel diseases, such as Crohn's disease and
ulcerative colitis, are diseases which result in destruction of the
mucosal surface of the small or large intestine, respectively.
Thus, galectin-3 and galectin-7 could be used to promote the
resurfacing of the mucosal surface to aid more rapid healing and to
prevent or attenuate progression of inflammatory bowel disease.
Galectin-3 and galectin-7 would be expected to bind mucin and
facilitate its adhesion to the apical surface of the epithelium and
could therefore be used to protect the gastrointestinal tract from
injurious substances that are ingested or following surgery.
Galectin-3 and/or galectin-7 could be used to reduce the side
effects of gut toxicity that result from the treatment of bacterial
infections, viral infections, radiation therapy, chemotherapy or
other treatments. Galectin-3 and/or galectin-7 may, for example, be
used prophylactically or therapeutically to prevent or attenuate
mucositis, esophagitis, or gastritis (e.g., to heal lesions
associated with oral, esophageal, intestinal, colonic, rectal, and
anal ulcers).
[0073] Galectin-3 and/or galectin-7 could be used to promote
urothelial healing. Tissue layers comprising urothelial cells may
be damaged by numerous mechanisms including catheterization,
surgery, or bacterial infection (e.g., infection by an agent which
causes a sexually transmitted disease, such as gonorrhea). The
present invention also encompasses methods for the promotion of
tissue healing in the female genital tract comprising the
administration of an effective amount of galectin-3 and/or
galectin-7. Tissue damage in the female genital tract may be caused
by a wide variety of conditions including Candida infections
trichomoniasis, Gardnerella, gonorrhea, chlamydia, mycoplasma
infections and other sexually transmitted diseases.
[0074] Galectin-3 and/or galectin-7 could be used to promote the
repair of renal epithelial cells and, thus, could be useful for
alleviating or treating renal diseases and pathologies such as
acute and chronic renal failure and end stage renal disease.
Galectin-3 and/or galectin-7 could be used to promote the repair of
breast tissue and therefore could be used to promote healing of
breast tissue injury due to surgery, trauma, or cancer. Galectin-3
and/or galectin-7 could further be used to promote healing and
alleviate damage of brain tissue due to injury from trauma, surgery
or chemicals.
[0075] Galectin-3 and/or galectin-7 could be administered
prophylactically to reduce or prevent damage to the lungs caused by
various pathological states. For example, galectin-3 and/or
galectin-7 could be used to promote the repair of alveoli and
bronchiolar epithelium to prevent, attenuate, or treat acute or
chronic lung damage. Emphysema, which results in the progressive
loss of alveoli, and inhalation injuries, i.e., resulting from
smoke inhalation and burns, that cause necrosis of the bronchiolar
epithelium and alveoli could be effectively treated using
galectin-3 and/or galectin-7 as could damage attributable to
chemotherapy, radiation treatment, lung cancer, asthma, black lung
and other lung damaging conditions.
[0076] It will be appreciated that the therapeutic methods
encompassed by the present invention are not limited to treating
wounds in humans, but may be used to treat wounds in any mammal
including but not limited to bovine, canine, feline, caprine,
ovine, porcine, murine, and equine species. When treating wounds in
a given species, it is preferred, but not required, that the
galectin-3 and/or galectin-7 used, have an amino acid sequence that
is substantially identical to the amino acid sequence of galectin-3
and/or galectin-7 as it occurs naturally in said species.
EXAMPLES
[0077] All animal treatments described in these examples conformed
to the Association for Research in Vision and Opthalmology
Resolution on the Use of Animals in Vision Research and the
recommendations of the NIH Guide for the Care and Use of Laboratory
Animals.
Example 1
Up-Regulation of Galectin-3 in Migrating Corneal Epithelium
Following Injury
[0078] To determine whether the expression level of galectin-3 is
altered in the epithelium of healing corneas following injury, mice
corneas with 2 mm excimer laser ablations and abrasion wounds, were
allowed to partially heal in vivo and were then processed for
immunostaining with rat anti-human galectin-3 mAb M3/38 (American
Type Culture Collection, Rockville, Md.). Corneal epithelium is a
prototype-stratified squamous epithelium. In mouse, it constitutes
20-25% of total corneal thickness and is composed of 5 to 6 layers
of cells. Posterior to the epithelial basement membrane is corneal
stroma, which in mouse represents 70-80% of the total corneal
thickness. Abrasion wounds remove epithelium leaving the corneal
stroma intact. In contrast, excimer laser treatment, which is
commonly used for correction of myopia, removes epithelium as well
as anterior corneal stroma.
[0079] Swiss Webster mice (Taconic Laboratory Animal Services,
Germantown, N.Y.) were anesthetized by an intramuscular injection
of 1.25% avertin (0.2 ml/10 kg body weight). Avertin was prepared
by mixing 2.5 g of 2,2,2 tribromoethanol, 5 ml 2-methyl-2-butanol
(Aldrich, Milwaukee, Wis.) and 195 ml distilled water. Proparacaine
eye drops (ALCAINE.TM. available from Alcon Labs, Fort Worth, Tex.)
were applied to the cornea as topical anesthetic. Transepithelial
excimer laser ablations were performed on the right eyes of a first
group of mice (2 mm optical zone; 42 to 44 .mu.m ablation depth,
PTK mode) using an APEX PLUS.TM. excimer laser (Summit Technology
of Waltham, Mass.). 2 mm abrasion wounds were produced on the right
eyes of a second group of mice using an Alger brush (Alger
Equipment Company of Lago Vista, Tex.).
[0080] Following surgery, all animals received an intramuscular
injection of buprenorphine (0.2 ml of 0.3 mg/ml, BUPRENEX.TM.
available from Reckitt & Colman Pharmaceuticals, Richmond, Va.)
as a pain killer. Antibiotic ointment (VETROPOLYCIN.TM. available
from Pharmaderm, Melville, N.Y.) was applied and the corneas were
allowed to partially heal in vivo for 16 to 18 hours. At the end of
the healing period the animals were anesthetized as described above
and were sacrificed by cervical dislocation. The eyes were then
fixed in formalin for two hours prior to embedding in paraffin wax.
Tissue sections (5 .mu.m thick) were cut in the place parallel to
the ocular axis. The sections were deparaffinized by treatment with
xyline and re-hydrated with graded ethanol solutions (100%, 70%,
and 30%). For immunostaining, tissue sections were incubated
sequentially with 3% H.sub.2O.sub.2 (37.degree. C., 10 min), and
2.5% normal goat serum to block endogenous peroxidase activity and
nonspecific binding, respectively. The sections were subsequently
incubated with mAb M3/38 (undiluted hybridoma fluid, 1 hour),
biotinylated anti-rat IgG for 1 hour (1:200, Vector Labs,
Burlingame, Calif.), a freshly prepared complex of avidin D and
biotinperoxidase for 20 hours (Vector Labs) and diaminobenzidine
(DAB)--H.sub.2O.sub.2 reagent (Kirkegaard & Perry Labs,
Gaithersburg, Md.). For negative controls, sections were treated
with an irrelevant mAb or media alone.
[0081] As shown in FIG. 9, immunohistochemical staining of paraffin
sections of normal (FIGS. 9 A and B) and healing (FIGS. 9 C and D)
corneas indicated that in both models of corneal wound healing, the
leading edge of the migrating epithelium of healing corneas stained
more intensely with mAb M3/38 compared to the normal epithelium,
especially in the basal and middle cell layers. In both healing as
well as normal corneal epithelium, immunostaining was more intense
at the site of cell-matrix attachment. While stromal cells of
normal corneas did not react with mAb M3/38, cells in the anterior
stroma under the healing corneas robustly expressed galectin-3,
especially in the region under the migrating epithelium.
[0082] The galectin-3 immunoreactivity in corneal epithelium was
similar when corneas were allowed to heal in serum-free Eagle's
minimum essential medium containing nonessential amino acids,
L-glutamine, antibiotics and 0.4% bovine serum albumin (BSA) in
organ culture for 16 to 18 hours. However, anterior stroma of
corneas that were allowed to heal in vitro lacked cells expressing
galectin-3, suggesting that the galectin-3 positive cells seen in
the stroma of corneas that were allowed to heal in vivo are most
likely leukocytes and not keratocytes.
[0083] To determine whether the carbohydrate recognition domain of
galectin-3 plays a role in corneal epithelial sheet migration
following injury, corneas with 2 mm excimer laser and abrasion
wounds were allowed to heal in organ culture in the presence and
absence of the disaccharides .beta.-lactose and sucrose. While
.beta.-lactose contains galactose and binds galectins, sucrose
lacks galactose and does not bind galectins. In these experiments,
the rate of re-epithelialization of corneal wounds was
significantly slower in the presence of .beta.-lactose, while
sucrose had no effect. As shown in FIG. 10, healing rates expressed
as mm.sup.2/h among the different groups (mean.+-.SEM of at least
two experiments) were: media alone, 0.088.+-.0.003 (N=29); media
plus .beta.-lactose, 0.063.+-.0.003 (N=19); media plus sucrose
0.084.+-.0.004 (N=10).
Example 2
Corneal Epithelial Wound Closure in Wild Type and Galectin-3
Deficient Mice
[0084] To determine whether the re-epithelialization of corneal
wounds is impaired in galectin-3 deficient mice, four different
models of corneal wound healing were used. Galectin-3 deficient
mice (gal-3.sup.-/-) were generated by targeted interruption of the
galectin-3 gene as described in Hsu et al., Am. J. Pathol.
156:1073, 2000. Specifically, the region coding for the CRD was
interrupted with a neomycin resistant gene. This involved
substituting a 0.5 kb intron 4-exon 5 segment with the antibiotic
resistant gene (neo). That the galectin-3 gene has been inactivated
was confirmed by Southern blot as well as Western blot
analysis.
[0085] Briefly, corneas with excimer laser ablations (as described
in Example 1) or alkali-burn wounds were allowed to partially heal
in vivo or in vitro (as described in Example 1). For alkali injury,
2 mm filter discs (Whatman 50, Whatman International, Maidstone,
UK) were prepared using a trephine, soaked in 0.5N NaOH, and placed
on the surface of the cornea of the right eyes of a second group of
mice for 30 seconds. The eyes were then rinsed with excess PBS. At
the end of the healing period, the wound areas were visualized by
staining with methylene blue. The stained wounds were then
photographed at a standard distance, and the outlines of the wound
areas were traced on paper from projected images of the stained
wounds. These outlines were digitized and quantified using
SIGMASCAN.TM. software (SPSS Science of Chicago, Ill.). Analysis of
the wound closure rate in gal-3.sup.+/+ mice in different models of
wound healing revealed that wound closure rate expressed as
mm.sup.2/h in gal-3.sup.+/+ mice was slower in corneas injured with
an excimer laser compared to those injured with an alkali-burn.
Also, regardless of the injury method used, the wound closure rate
was faster in corneas allowed to heal in vivo compared to those in
organ culture. As shown in FIG. 11, wound closure rates among
gal-3.sup.+/+ groups were: 0.076.+-.0.003 mm.sup.2/h for the
excimer laser/in vivo group, 0.050.+-.0.003 mm.sup.2/h for the
excimer laser/in vitro group, 0.182.+-.0.003 mm.sup.2/h for the
alkali-burn/in vivo group, and 0.106.+-.0.005 mm.sup.2/h for the
alkali-burn/in vitro group. Each group represents the mean.+-.SEM
of at least two experiments (N=9 or more in each group). Comparison
of the wound closure rate of gal-3.sup.+/+ groups with
gal-3.sup.-/- groups revealed that regardless of whether the
corneas were injured by excimer laser or by alkali treatment and
whether the corneas were allowed to heal in vivo or in vitro,
corneal epithelial wound closure rate expressed in mm.sup.2/h was
significantly slower in the gal-3.sup.-/- mice compared to that in
the gal-3.sup.+/+ mice. Wound closure rates among different
gal-3.sup.-/- groups were 0.060.+-.0.004 mm.sup.2/h for the excimer
laser/in vivo group, 0.036.+-.0.005 mm.sup.2/h for the excimer
laser/in vitro group, 0.150.+-.0.008 mm.sup.2 for the
alkali-burn/in vivo group, and 0.081.+-.0.004 mm.sup.2/h for the
alkali-burn/in vitro group. Again, all values are the mean.+-.SEM
of at least two experiments (N=8 or more in each group).
Example 3
Gene Expression Patterns in Migrating Corneal Epithelium of
Galectin-3 Deficient Mice Following Injury
[0086] In an attempt to understand why the re-epithelialization of
corneal epithelial wounds is perturbed in gal-3.sup.-/- mice, gene
expression patterns of healing gal-3.sup.+/+ and gal-3.sup.-/-
corneas were compared using cDNA microarrays and the results were
further confirmed by semiquantitative RT-PCR.
[0087] Transepithelial excimer laser ablations (2 mm diameter) were
produced on the right eye of 30 gal.sup.+/+ and 30 gal.sup.-/- mice
as described in Example 1. Corneas were allowed to partially heal
in vivo for 20 to 24 hours. At the end of the healing period,
animals were sacrificed and the corneas were excised and
immediately placed in liquid nitrogen and shipped to Clontech
Laboratories, Palo Alto, Calif. for analysis of gene expression
using SMART.TM. cDNA technology. Briefly, total RNA was isolated
using the reagents provided in the ATLAS.TM. Pure Total RNA
Labeling System. Yield of RNA from the 30 gal-3.sup.+/+ and 30
gal-3.sup.-/- corneas was 3.5 .mu.g and 2.6 .mu.g respectively. The
A260:A280 ratio of the RNA preparations of the corneas of
gal-3.sup.+/+ and gal-3.sup.-/- mice were 1.48 and 1.37
respectively. The ribosomal RNA 28S:18S ratio was 1.8 for both
preparations. This ensured that the quality of RNA preparation was
satisfactory. For probe preparation, first strand cDNA was
synthesized using 175 ng of RNA, a modified oligo(dT) primer (the
CDS primer), POWERSCRIPT.TM. reverse transcriptase, and SMART.TM.
II oligonucleotides. Controls involved incubation of samples
without reverse transcriptase. The cDNA was amplified by long
distance (LD)-PCR. To determine the optimal number of amplification
cycles, aliquots of reaction products were collected at 15, 18, 21
and 24 cycles and were analyzed by agarose gel electrophoresis. The
yield of amplified double stranded cDNA using an optimal number of
cycles, i.e., 23, was between 1 and 1.64 .mu.g. The amplified cDNAs
(500 ng) were radiolabeled using Klenow enzyme and
.sup.33P-.alpha.ATP as described in the instruction manual for
SMART.TM. cDNA probe synthesis for ATLAS.TM. microarrays
(Clontech). The labeled probes were purified by filtration on a
NUCLEOSPIN.TM. filter and were then hybridized to mouse 1.2k-I
ATLAS.TM. nylon cDNA microarrays (Clontech). This is a broad
spectrum array consisting of .about.1200 mouse genes. Following
hybridization, the membranes were exposed to a phosphorimager
screen and the results were analyzed by ATLAS IMAGE.TM. 2.0
software (Clontech). The data were verified by semiquantitative
RT-PCR.
[0088] For RT-PCR, total RNA and first strand cDNA were prepared
from healing gal-3.sup.+/+ and gal-3.sup.-/- corneas using the
procedures described earlier. PCR amplification was performed in 50
.mu.l volume using 14 ng of cDNA, gene-specific custom primers
purchased from Clontech and other reagents from the ADVANTAGE.TM. 2
PCR kit (Clontech). The annealing temperature used was 68.degree.
C. and reactions were subjected to varying number of cycles of PCR
amplification. For analysis of housekeeping genes, 5 .mu.l aliquots
of amplified product were collected at every 5.sup.th cycle,
beginning at the 18.sup.th cycle, whereas for analysis of
differentially expressed genes reaction amplified products were
collected at every other cycle, beginning at the 28.sup.th cycle.
Amplified products collected at various cycles were analyzed by
electrophoresis in 1.5% agarose/ethedium bromide gels (FIG.
12).
[0089] These experiments revealed that compared to healing corneas
of gal-3.sup.+/+ mice, healing corneas of gal-3.sup.-/- mice
contain markedly reduced levels of mRNA transcripts for galectin-7,
another galactose-binding protein, and tolloid-like protein (TLL),
a metalloproteinase. Overall, compared to healing gal-3.sup.+/+
corneas, healing gal-3.sup.-/- corneas contained about 12 times
less galectin-7 (FIG. 12) and 14 times less TLL gene transcripts
(data not shown). Expression levels of mRNA transcripts of various
housekeeping genes were similar in both healing gal-3.sup.+/+ and
gal-3.sup.-/- as detected by both microarray technology (FIG. 12),
and semi-quantitative RT-PCR (FIG. 12, GAPDH is
D-glyceraldehyde-3-phosphate dehydrogenase; RPS29 is ribosomal
protein S29; ODC is ornithine decarboxylase).
[0090] To determine whether the expression level of the galectin-7
protein is also reduced in healing corneas of gal-3.sup.-/- mice,
western blot analysis using detergent extracts of healing
gal-3.sup.+/+ and gal-3.sup.-/- corneas (FIG. 13A) and
immunohistochemical studies with an anti-galectin-7 polyclonal
antibody using paraffin sections derived from corneas of
gal-3.sup.+/+ and gal-3.sup.-/- mice (FIG. 13B) were performed. The
immunoreactivity was graded as intense (+++), moderate (++), weak
(+) or negative (-). Significantly less galectin-7 immunoreactivity
was detected in migrating epithelia of healing gal-3.sup.-/-
corneas compared to those of healing gal-3.sup.+/+ corneas:
gal-3.sup.+/+:+++36/42, ++5/42; + or less 1/42;
gal-3.sup.-/-:+++3/42., ++26/42, + or less 13/42. Also,
gal-3.sup.-/- mouse embryonic fibroblasts (MEF) grown in cell
culture expressed reduced levels of galectin-7 compared to
gal-3.sup.+/+ MEF cultures (FIG. 13C).
Example 4
Exogenous Galectin-3 Stimulates the Re-Epithelialization of Corneal
Wounds in Wild Type and Galectin-3 Deficient Mice
[0091] Having demonstrated that corneal epithelial wound closure
rate is perturbed in gal-3.sup.-/- mice (Example 2), it was of
interest to determine whether exogenous galectin-3 would stimulate
the re-epithelialization of healing corneas in organ culture. In
this study, corneas of gal-3.sup.+/+ and gal-3.sup.-/- mice with
alkali-burn wounds were incubated in serum free media in the
presence and absence of varying amounts of recombinant
galectin-3.
[0092] Recombinant full-length human galectin-3 was produced in
Eschericia coli and purified as described previously (Yang et al.,
Biochemistry 37:4086, 1998). Alkali-burn wounds (2 mm diameter)
were produced on both eyes of anesthetized mice using alkali-soaked
filter discs as described in Example 2. Following injury, the
animals were sacrificed and the eyes were excised and incubated in
the presence or absence of exogenous galectin-3 for 18 to 20 hours.
The left eyes of animals served as controls and were incubated in
serum free media alone. The right eyes were incubated in serum free
media containing various test reagents including: (i) galectin-3 (5
to 20 .mu.g/ml), (ii) galectin-3 (10 .mu.g/ml) plus 0.1 M
.beta.-lactose, (iii) galectin-3 (10 .mu.g/ml) plus 0.1 M sucrose,
(iv) 0.1 M .beta.-lactose, or (v) 0.1 M sucrose. At the end of the
healing period, the remaining wound areas were stained,
photographed and quantified as described in Example 2 using
SIGMASCAN.TM. software (SPSS Science of Chicago, Ill.). Each group
contained a minimum of three eyes and all experiments were
performed at least twice.
[0093] The exogenous galectin-3 had no influence on the rate of
re-epithelialization of corneal wounds in gal-3.sup.-/- mice (FIG.
14A), but it stimulated the rate of wound closure in a
concentration-dependent manner in gal-3.sup.+/+ mice (FIG. 14B) at
10 .mu.g/ml and 20 .mu.g/ml concentration (0 and 5 .mu.g/ml:
0.090.+-.0.010 mm.sup.2/h; 10 .mu.g/ml: 0.129.+-.0.010 mm.sup.2/h;
20 .mu.g/ml: 0.154.+-.0.004 mm.sup.2/h; mean.+-.SEM of at least two
experiments, N=7 or more). As shown in FIG. 15, the stimulatory
effect of galectin-3 on corneal epithelial wound closure in
gal-3.sup.+/+ mice was specifically inhibited by .beta.-lactose but
not sucrose (10 .mu.g/ml galectin-3: 0.127.+-.0.010 mm.sup.2/h; 10
.mu.g/ml galectin-3 plus 0.1 M .beta.-lactose: 0.103.+-.0.014
mm.sup.2/h; 10 .mu.g/ml galectin-3 plus 0.1 M sucrose:
0.130.+-.0.003 mm.sup.2/h. All values represent mean.+-.SEM of at
least two experiments, N=7 or more).
Example 5
Exogenous Galectin-7 Stimulates the Re-Epithelialization of Corneal
Wounds in Wild Type and Galectin-3 Deficient Mice
[0094] In a separate study, comparison of the gene expression
patterns of normal and healing corneas of gal-3.sup.+/+ mice using
cDNA microarrays (i.e., as in Example 3) revealed that in healing
corneas, expression of galectin-7 is markedly up-regulated. These
findings in conjunction with the studies described in Example 3
showing that galectin-7 expression is down-regulated in the healing
cornea of gal-3.sup.-/- mice, led to the design of experiments to
determine whether exogenous galectin-7 would stimulate the
re-epithelialization of healing corneas in organ culture. In this
study, corneas of gal-3.sup.-/- mice with alkali-burn wounds were
incubated in serum free media in the presence and absence of
varying amounts of recombinant galectin-7.
[0095] Recombinant full-length human galectin-7 was produced in
Eschericia coli by cloning the cDNA (available as an EST clone from
American Type Culture Collection of Manassas, Va.) into the pET25b
plasmid (available from Novagen, Madison, Wis.). Alkali-burn wounds
(2 mm diameter) were produced on both eyes of anesthetized animals
using alkali-soaked filter discs as described in Example 2.
Following injury, the animals were sacrificed and the eyes were
excised and incubated in the presence or absence of exogenous
galectin-7 for 18 to 20 hours. The left eyes of animals served as
controls and were incubated in serum free media alone. The right
eyes were incubated in serum free media containing various test
reagents including: (i) galectin-7 (20 .mu.g/ml), (ii) galectin-7
(20 .mu.g/ml) plus 0.1 M .beta.-lactose, or (iii) galectin-7 (20
.mu.g/ml) plus 0.1 M sucrose. At the end of the healing period, the
remaining wound areas were stained, photographed and quantified as
described in Example 2 using SIGMASCAN.TM. software (SPSS Science
of Chicago, Ill.). Each group contained a minimum of six eyes and
all experiments were performed at least twice.
[0096] As shown in FIG. 16, exogenous galectin-7 stimulated the
rate of wound closure (media alone: 0.036.+-.0.006 mm.sup.2/h; 20
.mu.g/ml galectin-7: 0.072.+-.0.004 mm.sup.2/h; mean.+-.SEM of at
least two experiments, N=10 or more). As shown in FIG. 16, the
stimulatory effect of galectin-7 on corneal epithelial wound
closure was specifically inhibited by .beta.-lactose but not by
sucrose (20 .mu.g/ml galectin-7: 0.072.+-.0.004 mm.sup.2/h; 20
.mu.g/ml galectin-7 plus 0.1 M .beta.-lactose: 0.050.+-.0.004
mm.sup.2/h; 20 .mu.g/ml galectin-7 plus 0.1 M sucrose:
0.079.+-.0.007 mm.sup.2/h. All values represent mean.+-.SEM of at
least two experiments, N=9 or more). As shown in FIG. 16, the rate
of wound closure was further enhanced (0.094.+-.0.003 gal-3.sup.+/+
mm.sup.2/h) when exogenous galectin-7 was added to the injured
corneas of gal-3.sup.+/+ mice instead of gal-3.sup.-/- mice.
Example 6
Skin Epithelial Wound Closure in Wild Type and Galectin-3 Deficient
Mice
[0097] Gal-3.sup.+/+ and gal-3.sup.-/- mice are anesthetized by an
intraperitoneal injection of 1.25% Avertin (0.2 ml/10 g body
weight). Prior to laser treatment, hair is shaved off from the
dorsal region using a razor blade. Six millimeter transepithelial
dorsal skin wounds are made using the excimer laser (Summit
Technology of Waltham, Mass.). After surgery, antibiotic ointment
is applied to the wound surface and buprenorphine (2 mg/kg body
weight) is given subcutaneously to minimize post-surgical pain. The
wounds are allowed to partially heal in vivo, and are examined 24,
48, and 72 hours after surgery. At the end of the healing period,
the mice are again anesthetized by an intraperitoneal injection of
1.25% Avertin (0.2 ml/10 g body weight), wound areas are
photographed and then quantitated using a Sigma scan software. The
wound closure rates between the two groups of animals (i.e.,
gal-3.sup.+/+ and gal-3.sup.-/- mice) are compared. The animals are
then sacrificed by carbon dioxide inhalation or an overdose of
pentobarbital.
Example 7
Effect of Exogenous Galectin-3 on the Re-Epithelialization of Skin
Wounds
[0098] Animals (Mice: 57BL/6 and 129 mixed genetic background; Age:
six to eight weeks old; Gender: mixed) are anesthetized by an
intraperitoneal injection of 1.25% Avertin (0.2 ml/10 g body
weight). Prior to laser treatment, hair is shaved off from the
dorsal region using a razor blade. Two 6-mm transepithelial dorsal
skin wounds (one on each side) are made using the excimer laser
(Summit Technology of Waltham, Mass.). After surgery, antibiotic
ointment is applied to the wound surfaces and buprenorphine (2
mg/kg body weight) is given subcutaneously to minimize
post-surgical pain. The wounds are then allowed to partially heal
in vivo. Every 4-6 hours, an ointment containing galectin-3 is
applied to the right wound and carrier only is applied to the left
wound which serves as a control. At the end of the healing period
(24 to 48 hours), the animals are anesthetized by an
intraperitoneal injection of 1.25% Avertin (0.2 ml/10 g body
weight), wound areas are photographed and quantitated using a Sigma
scan software. The wound closure rates between the two groups of
animals (galectin-3 treated and control) are compared. The animals
are then sacrificed by carbon dioxide inhalation or an overdose of
pentobarbital.
Example 8
Effect of Exogenous Galectin-7 on the Re-Epithelialization of Skin
Wounds
[0099] Animals (Mice: 57BL/6 and 129 mixed genetic background; Age:
six to eight weeks old; Gender: mixed) are anesthetized by an
intraperitoneal injection of 1.25% Avertin (0.2 ml/10 g body
weight). Prior to laser treatment, hair is shaved off from the
dorsal region using a razor blade. Two 6-mm transepithelial dorsal
skin wounds (one on each side) are made using the excimer laser
(Summit Technology of Waltham, Mass.). After surgery, antibiotic
ointment is applied to the wound surfaces and buprenorphine (2
mg/kg body weight) is given subcutaneously to minimize
post-surgical pain. The wounds are then allowed to partially heal
in vivo. Every 4-6 hours, an ointment containing galectin-7 is
applied to the right wound and carrier only is applied to the left
wound which serves as a control. At the end of the healing period
(24 to 48 hours), the animals are anesthetized by an
intraperitoneal injection of 1.25% Avertin (0.2 ml/10 g body
weight), wound areas are photographed and quantitated using a Sigma
scan software. The wound closure rates between the two groups of
animals (galectin-7 treated and control) are compared. The animals
are then sacrificed by carbon dioxide inhalation or an overdose of
pentobarbital.
CONCLUSION
[0100] It has been demonstrated that galectin-3 and galectin-7 play
a role in the re-epithelialization of corneal wounds. In Example 1
immunohistochemical studies revealed that following injury,
galectin-3 is located in high density at sites of corneal
epithelial cell-matrix adhesion, an ideal location for influencing
cell-matrix interactions and hence cell migration. In Example 2,
the re-epithelialization of corneal wounds was shown to be
significantly slower in the galectin-3 deficient mice compared to
that in wild-type mice. In Example 3, it was shown that following
injury, expression levels of galectin-7 are significantly reduced
in galectin-3 deficient mice compared to wild-type mice. In
Examples 4 and 5, exogenous recombinant galectin-3 and galectin-7
were shown to stimulate the re-epithelialization of corneal wounds
in gal3.sup.+/+ mice. It was further demonstrated in Example 1 that
the stimulatory effect of galectin-3 on the rate of corneal
epithelial wound closure can be almost completely-abrogated by a
competing disaccharide (.beta.-lactose), but not by an irrelevant
disaccharide (sucrose). This final result suggests that the
carbohydrate recognition domain (CRD) is directly involved in the
beneficial effect of the exogenous galectin-3 on wound closure.
[0101] Without wishing to be bound to any particular theory
regarding the mechanism by which galectin-3 and galectin-7 may
influence re-epithelialization of corneal wounds, the following
suggestions are presented.
[0102] As mentioned earlier, galectin-3 is thought to mediate
cell-cell and cell-matrix interactions by binding to complementary
glycoconjugates containing polylactosamine chains found in many ECM
and cell surface molecules such as certain isoforms of fibronectin,
laminin, and integrins (Liu, Clin. Immunol. 97:79, 2000 and
Perillo, supra). However, the finding presented herein that
exogenous galectin-3 does not accelerate the re-epithelialization
of wounds in gal3.sup.-/- mice (see Example 4) suggests that
intracellular galectin-3 contributes significantly to the process
of wound healing, most probably, by influencing the expression of
specific cell surface and/or ECM receptors, which in turn influence
cell-matrix interactions and cell migration. This idea is
consistent with published studies in which galectin-3 was stably
overexpressed in breast carcinoma cell lines, resulting in elevated
levels of .alpha.4.beta.7 and .alpha.6.beta.1 integrins and
enhanced adhesion to various ECM molecules including laminin,
fibronectin, and vitronectin as compared with parental cell lines
expressing little or no galectin-3 (Warfield, supra and Mattarese,
supra). In another study (Dudas et al., Gastroenterology 118:1553,
2000), colon cancer carcinoma cell lines transfected with
galectin-3 expressed elevated levels of a specific mucin, MUC2, a
major ligand of the lectin itself (Bresalier et al., Cancer
Research 56:4354, 1996). The fact that the stimulatory effect of
exogenous galectin-3 on the rate of re-epithelialization of wounds
in gal3.sup.+/+ mice is lactose inhibitable raises an intriguing
possibility that intracellular galectin-3 may in fact regulate
glycosylation of the proteins which serve as cell surface or ECM
receptors of the lectin itself. That intracellular galectin-3 has
the potential to act on the nuclear matrix to influence complex
biological processes is also suggested by findings that under
certain conditions the lectin can be found associated in the
nucleus with ribonucleoprotein complexes and can act as a pre-mRNA
splicing factor (Dagher et al., Proc. Natl. Acad. Sci. USA 92:1213,
1995). Also, Wang et al. have demonstrated that in prostate
adenocarcinoma cells, galectin-3 is associated with the nuclear
matrix and binds with both single-stranded DNA and RNA (Wang et
al., Biochem. Biophys. Res. Commun. 217:292, 1995).
[0103] Analysis of gene expression patterns of corneas of healing
gal3.sup.+/+ and gal3.sup.-/- mice using mouse cDNA microarrays
revealed that healing corneas of gal3.sup.-/- mice expressed
markedly reduced levels of galectin-7 compared to those of
wild-type mice (see Examples 3 and 5). Galectin-7 was first
reported in 1994 (Barondes, supra) and is not as well characterized
as galectin-3. Unlike galectin-3, galectin-7 exhibits a remarkable
degree of tissue specificity. In adult animals, its expression is
restricted to epithelia that are or are destined to become
stratified (Timmons et al., supra). The protein is thought to be
involved in cell-matrix and cell-cell interactions and in apoptosis
(Leonidas, Biochemistry 37:13930, 1998 and Bernerd et al., Proc.
Natl. Acad Sci. USA 96:11329, 1999). In general, an inverse
correlation exists between galectin-7 expression and keratinocyte
proliferation, and galectin-7 expression is abrogated in SV40
transformed keratinocytes as well as in cell lines derived from
epidermal tumors. The discovery described herein that exogenous
galectin-3 does not stimulate re-epithelialization of wounds in
gal3.sup.-/- corneas and that healing gal3.sup.-/- corneas contain
reduced levels of galectin-7 suggests that galectin-3 may influence
the re-epithelialization of wounds, at least in part, by modulating
galectin-7. Indeed, it has been found that unlike galectin-3,
galectin-7 accelerated re-epithelialization of wounds in
gal3.sup.-/- corneas in a lactose-inhibitable manner. Also, mouse
embryonic fibroblasts of gal3.sup.-/- mice expressed reduced level
of galectin-7.
[0104] Regardless of the mechanisms involved, the findings that
both galectin-3 and galectin-7 stimulate re-epithelialization of
corneal wounds have broad implications for the treatment of
epithelial wounds and non-healing epithelial wounds in particular.
At present, treatment of persistent epithelial defects of the
cornea is a major clinical problem. Moreover, the need continues
for effective treatment of post-transplantation wounds, chronic
wounds in the elderly, decubitus ulcers, and venous stasis ulcers
of the skin. A number of growth factors (e.g., EGF, TGF, FGF, KGF,
HGF) known to stimulate cell proliferation, have been tested for
usefulness in corneal as well as cutaneous epithelial wound healing
with overall disappointing results (Eaglstein, Surg. Clin. North
Am. 77:689, 1997; Singer and Clark, N. Engl. J. Med. 341:738, 1999;
Zieske and Gipson, pp. 364-372 in "Principle and Practice of
Opthalmology" Ed. by D. M. Albert and F. A. Jakobiec, W.B. Saunders
Company, Philadelphia, Pa., 2000; Schultz et al., Eye 8:184, 1994;
Kandarakis et al., Am. J. Opthalmol. 98:411, 1984; and Singh and
Foster, Am. J. Opthalmol. 103:802, 1987). The extent of
acceleration of re-epithelialization of wounds was far less in most
of these studies using growth factors than that observed with
galectins in the current study. Also, the epithelium of corneas
treated with growth factors such as EGF is hyperplastic (Singh and
Foster, Cornea 8:45, 1989), a clearly undesirable condition. In
this respect, the clinical potential of galectin-3 and galectin-7
may be more attractive than that of growth factors because the
lectins have not been shown to induce cell mitosis in epithelial
cells. Over the last decade, the potential of excimer laser
keratectomy to modify the corneal profile for correction of myopia
has been realized. Thousands of such procedures are performed each
week providing myopic individuals freedom from eye glasses and
contact lenses. In view of the fact that 25-30% of the adult
population worldwide is myopic, it has been estimated that nearly
half a million such procedures will be performed in the U.S. alone
in a given year. In some cases, following excimer laser surgery,
there is a delay in epithelial healing. Such a delay is highly
undesirable because it puts the cornea at risk of developing
postoperative haze, infectious keratitis and ulceration. Again,
galectin-based treatments may help promote re-epithelialization of
wounds in such cases.
OTHER EMBODIMENTS
[0105] Other embodiments of the invention will be apparent to those
skilled in the art from a consideration of the specification or
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with
the true scope of the invention being indicated by the following
claims.
Sequence CWU 1
1
281250PRTHuman 1Met Ala Asp Asn Phe Ser Leu His Asp Ala Leu Ser Gly
Ser Gly Asn1 5 10 15Pro Asn Pro Gln Gly Trp Pro Gly Ala Trp Gly Asn
Gln Pro Ala Gly 20 25 30Ala Gly Gly Tyr Pro Gly Ala Ser Tyr Pro Gly
Ala Tyr Pro Gly Gln 35 40 45Ala Pro Pro Gly Ala Tyr Pro Gly Gln Ala
Pro Pro Gly Ala Tyr Pro 50 55 60Gly Ala Pro Gly Ala Tyr Pro Gly Ala
Pro Ala Pro Gly Val Tyr Pro65 70 75 80Gly Pro Pro Ser Gly Pro Gly
Ala Tyr Pro Ser Ser Gly Gln Pro Ser 85 90 95Ala Thr Gly Ala Tyr Pro
Ala Thr Gly Pro Tyr Gly Ala Pro Ala Gly 100 105 110Pro Leu Ile Val
Pro Tyr Asn Leu Pro Leu Pro Gly Gly Val Val Pro 115 120 125Arg Met
Leu Ile Thr Ile Leu Gly Thr Val Lys Pro Asn Ala Asn Arg 130 135
140Ile Ala Leu Asp Phe Gln Arg Gly Asn Asp Val Ala Phe His Phe
Asn145 150 155 160Pro Arg Phe Asn Glu Asn Asn Arg Arg Val Ile Val
Cys Asn Thr Lys 165 170 175Leu Asp Asn Asn Trp Gly Arg Glu Glu Arg
Gln Ser Val Phe Pro Phe 180 185 190Glu Ser Gly Lys Pro Phe Lys Ile
Gln Val Leu Val Glu Pro Asp His 195 200 205Phe Lys Val Ala Val Asn
Asp Ala His Leu Leu Gln Tyr Asn His Arg 210 215 220Val Lys Lys Leu
Asn Glu Ile Ser Lys Leu Gly Ile Ser Gly Asp Ile225 230 235 240Asp
Leu Thr Ser Ala Ser Tyr Thr Met Ile 245 2502136PRTHuman 2Met Ser
Asn Val Pro His Lys Ser Ser Leu Pro Glu Gly Ile Arg Pro1 5 10 15Gly
Thr Val Leu Arg Ile Arg Gly Leu Val Pro Pro Asn Ala Ser Arg 20 25
30Phe His Val Asn Leu Leu Cys Gly Glu Glu Gln Gly Ser Asp Ala Ala
35 40 45Leu His Phe Asn Pro Arg Leu Asp Thr Ser Glu Val Val Phe Asn
Ser 50 55 60Lys Glu Gln Gly Ser Trp Gly Arg Glu Glu Arg Gly Pro Gly
Val Pro65 70 75 80Phe Gln Arg Gly Gln Pro Phe Glu Val Leu Ile Ile
Ala Ser Asp Asp 85 90 95Gly Phe Lys Ala Val Val Gly Asp Ala Gln Tyr
His His Phe Arg His 100 105 110Arg Leu Pro Leu Ala Arg Val Arg Leu
Val Glu Val Gly Gly Asp Val 115 120 125Gln Leu Asp Ser Val Arg Ile
Phe 130 1353139PRTArtificial sequenceThe protein was designed and
synthesized 3Pro Gly Leu Val Ala Leu Asn Leu Gly Leu Lys Pro Gly
Lys Thr Leu1 5 10 15Thr Val Lys Gly Thr Val Ala Pro Lys Asn Ala Lys
Arg Phe Ala Val 20 25 30Asn Leu Gly Lys Gly Ser Lys Glu Glu Asn Asp
Leu Val Leu His Phe 35 40 45Asn Pro Arg Phe Asn Glu Ala His Gly Asp
Gln Asn Thr Val Val Cys 50 55 60Asn Ser Lys Glu Asn Gly Asp Asn Glu
Trp Gly Thr Glu Gln Arg Glu65 70 75 80Ala Ala Phe Pro Phe Gln Ala
Gly Gln Pro Phe Glu Ile Ser Ile Ser 85 90 95Val Glu Glu Asp Lys Phe
Lys Val Lys Val Asn Asp Gly His Glu Phe 100 105 110Glu Phe Pro His
Arg Leu Lys Leu Glu Ala Val Gln Tyr Leu Gly Ile 115 120 125Lys Gly
Asp Ile Lys Leu Thr Ser Ile Lys Phe 130 1354242PRTRabbit 4Met Ala
Asp Gly Phe Ser Leu Asn Asp Ala Leu Ser Gly Ser Gly His1 5 10 15Pro
Pro Asn Gln Gly Trp Pro Gly Pro Trp Gly Asn Gln Pro Ala Gly 20 25
30Pro Gly Gly Tyr Pro Gly Ala Ala Tyr Pro Gly Ala Tyr Pro Gly His
35 40 45Ala Pro Gly Ala Tyr Pro Gly Gln Ala Pro Pro Gly Pro Tyr Pro
Gly 50 55 60Pro Gly Ala His Gly Ala Tyr Pro Gly Gln Pro Gly Gly Pro
Gly Ala65 70 75 80Tyr Pro Ser Pro Gly Gln Pro Ser Gly Ala Gly Ala
Tyr Pro Gly Ala 85 90 95Ser Pro Tyr Ser Ala Ser Ala Gly Pro Leu Pro
Val Pro Tyr Asp Leu 100 105 110Pro Leu Pro Gly Gly Val Met Pro Arg
Met Leu Ile Thr Ile Val Gly 115 120 125Thr Val Lys Pro Asn Ala Asn
Arg Leu Ala Leu Asp Phe Lys Arg Gly 130 135 140Asn Asp Val Ala Phe
His Phe Asn Pro Arg Phe Asn Glu Asn Asn Arg145 150 155 160Arg Val
Ile Val Cys Asn Thr Lys Val Asp Asn Asn Trp Gly Arg Glu 165 170
175Glu Arg Gln Thr Thr Phe Pro Phe Glu Ile Gly Lys Pro Phe Lys Ile
180 185 190Gln Val Leu Val Glu Pro Asp His Phe Lys Val Ala Val Asn
Asp Ala 195 200 205His Leu Leu Gln Tyr Asn His Arg Met Arg Asn Leu
Lys Glu Ile Asn 210 215 220Lys Leu Gly Ile Ser Gly Asp Ile Gln Leu
Thr Ser Ala Ser His Ala225 230 235 240Met Ile5262PRTChicken 5Met
Gln Ala Met Lys Ala Arg Cys Trp Gln Pro His Trp Met Leu Pro1 5 10
15Leu Leu Pro Leu Ser Ser Pro Leu His Pro Gln Leu Ser Asp Ala Leu
20 25 30Pro Ala His Asn Pro Gly Ala Pro Pro Pro Gln Gly Trp Asn Arg
Pro 35 40 45Pro Gly Pro Gly Ala Phe Pro Ala Tyr Pro Gly Tyr Pro Gly
Ala Tyr 50 55 60Pro Gly Ala Pro Gly Pro Tyr Pro Gly Ala Pro Gly Pro
His His Gly65 70 75 80Pro Pro Gly Pro Tyr Pro Gly Gly Pro Pro Gly
Pro Tyr Pro Gly Gly 85 90 95Pro Pro Gly Pro Tyr Pro Gly Gly Pro Pro
Gly Pro Tyr Pro Gly Gly 100 105 110Pro Thr Ala Pro Tyr Ser Glu Ala
Pro Ala Ala Pro Leu Lys Val Pro 115 120 125Tyr Asp Leu Pro Leu Pro
Ala Gly Leu Met Pro Arg Leu Leu Ile Thr 130 135 140Ile Thr Gly Thr
Val Asn Ser Asn Pro Asn Arg Phe Ser Leu Asp Phe145 150 155 160Lys
Arg Gly Gln Asp Ile Ala Phe His Phe Asn Pro Arg Phe Lys Glu 165 170
175Asp His Lys Arg Val Ile Val Cys Asn Ser Met Phe Gln Asn Asn Trp
180 185 190Gly Lys Glu Glu Arg Thr Ala Pro Arg Phe Pro Phe Glu Pro
Gly Thr 195 200 205Pro Phe Lys Leu Gln Val Leu Cys Glu Gly Asp His
Phe Lys Val Ala 210 215 220Val Asn Asp Ala His Leu Leu Gln Phe Asn
Phe Arg Glu Lys Lys Leu225 230 235 240Asn Gly Ile Thr Lys Leu Cys
Ile Ala Gly Asp Ile Thr Leu Thr Ser 245 250 255Val Leu Thr Ser Met
Ile 2606245PRTHamster 6Met Ala Asp Gly Phe Ser Leu Asn Asp Ala Leu
Ala Gly Ser Gly Asn1 5 10 15Pro Asn Pro Gln Gly Trp Pro Gly Ala Trp
Gly Asn Gln Pro Gly Ala 20 25 30Gly Gly Tyr Pro Gly Ala Ser Tyr Pro
Gly Ala Tyr Pro Gly Gln Ala 35 40 45Pro Pro Gly Ala Tyr Pro Gly Gln
Ala Pro Pro Gly Ala Tyr Pro Gly 50 55 60Pro Thr Ala Pro Gly Ala Tyr
Pro Gly Pro Ala Pro Gly Ala Tyr Pro65 70 75 80Gly Gln Pro Gly Ala
Ser Gly Ala Tyr Pro Ser Ala Pro Gly Ala Tyr 85 90 95Pro Ala Ala Gly
Pro Tyr Gly Ala Pro Thr Gly Ala Leu Thr Val Pro 100 105 110Tyr Lys
Leu Pro Leu Ala Gly Gly Val Met Pro Arg Met Leu Ile Thr 115 120
125Ile Met Gly Thr Val Lys Pro Asn Ala Asn Arg Ile Ile Leu Asn Phe
130 135 140Leu Arg Gly Asn Asp Ile Ala Phe His Phe Asn Pro Arg Phe
Asn Glu145 150 155 160Asn Asn Arg Arg Val Ile Val Cys Asn Thr Lys
Gln Asp Asn Asn Trp 165 170 175Gly Arg Glu Glu Arg Gln Ser Ala Phe
Pro Phe Glu Ser Gly Arg Pro 180 185 190Phe Lys Ile Gln Val Leu Val
Glu Ala Asp His Phe Lys Val Ala Val 195 200 205Asn Asp Ala His Leu
Leu Gln Tyr Asn His Arg Met Lys Asn Leu Arg 210 215 220Glu Ile Asn
Gln Met Glu Ile Ser Gly Asp Ile Thr Leu Thr Ser Ala225 230 235
240Ala Pro Thr Met Ile 2457136PRTRat 7Met Ser Ala Thr His His Lys
Thr Pro Leu Pro Gln Gly Val Arg Leu1 5 10 15Gly Thr Val Met Arg Ile
Arg Gly Val Val Pro Asp Gln Ala Gly Arg 20 25 30Phe His Val Asn Leu
Leu Cys Gly Glu Glu Gln Glu Ala Asp Ala Ala 35 40 45Leu His Phe Asn
Pro Arg Leu Asp Thr Ser Glu Val Val Phe Asn Thr 50 55 60Lys Gln Gln
Gly Lys Trp Gly Arg Glu Glu Arg Gly Thr Gly Ile Pro65 70 75 80Phe
Gln Arg Gly Gln Pro Phe Glu Val Leu Ile Ile Thr Thr Glu Glu 85 90
95Gly Phe Lys Thr Val Ile Gly Asp Asp Glu Tyr Leu His Phe His His
100 105 110Arg Met Pro Ser Ser Asn Val Arg Ser Val Glu Val Gly Gly
Asp Val 115 120 125Gln Leu His Ser Val Lys Ile Phe 130
1358136PRTMouse 8Met Ser Ala Thr His His Lys Thr Ser Leu Pro Gln
Gly Val Arg Val1 5 10 15Gly Thr Val Met Arg Ile Arg Gly Leu Val Pro
Asp Gln Ala Gly Arg 20 25 30Phe His Val Asn Leu Leu Cys Gly Glu Glu
Gln Gly Ala Asp Ala Ala 35 40 45Leu His Phe Asn Pro Arg Leu Asp Thr
Ser Glu Val Val Phe Asn Thr 50 55 60Lys Gln Gln Gly Lys Trp Gly Arg
Glu Glu Arg Gly Thr Gly Ile Pro65 70 75 80Phe Gln Arg Gly Gln Pro
Phe Glu Val Leu Leu Ile Ala Thr Glu Glu 85 90 95Gly Phe Lys Ala Val
Val Gly Asp Asp Glu Tyr Leu His Phe His His 100 105 110Arg Leu Pro
Pro Ala Arg Val Arg Leu Val Glu Val Gly Gly Asp Val 115 120 125Gln
Leu His Ser Leu Asn Ile Phe 130 13594PRTHuman 9Asn Phe Ser
Leu1103PRTHuman 10Thr Val Lys1113PRTHuman 11Ser Gly Lys1124PRTHuman
12Ser Leu His Asp1134PRTHuman 13Thr Lys Leu Asp1146PRTHuman 14Gly
Ala Trp Gly Asn Gln1 5156PRTHuman 15Gly Asn Gln Pro Ala Gly1
5166PRTHuman 16Gly Gly Tyr Pro Gly Ala1 5176PRTHuman 17Gly Ala Tyr
Pro Gly Gln1 5186PRTHuman 18Gly Ala Tyr Pro Gly Gln1 5196PRTHuman
19Gly Ala Tyr Pro Gly Ala1 5206PRTHuman 20Gly Ala Pro Gly Ala Tyr1
5216PRTHuman 21Gly Ala Tyr Pro Gly Ala1 52220PRTHuman 22Trp Gly Arg
Glu Glu Arg Gln Ser Val Phe Pro Phe Glu Ser Gly Lys1 5 10 15Pro Phe
Lys Ile 20234PRTHuman 23Asn Ala Ser Arg1243PRTHuman 24Ser Val
Arg1254PRTHuman 25Ser Leu Pro Glu1266PRTHuman 26Gly Ile Arg Pro Gly
Thr1 5276PRTHuman 27Gly Ser Asp Ala Ala Leu1 52820PRTHuman 28Trp
Gly Arg Glu Glu Arg Gly Pro Gly Val Pro Phe Gln Arg Gly Gln1 5 10
15Pro Phe Glu Val 20
* * * * *
References