U.S. patent application number 14/922052 was filed with the patent office on 2016-04-21 for compositions and methods for macular degeneration.
The applicant listed for this patent is Case Western Reserve University, The Cleveland Clinic Foundation. Invention is credited to Bela Anand-Apte, John W. Crabb, Quteba Ebrahem, Kutralanathan Renganathan, Robert G. Salomon.
Application Number | 20160108114 14/922052 |
Document ID | / |
Family ID | 38792204 |
Filed Date | 2016-04-21 |
United States Patent
Application |
20160108114 |
Kind Code |
A1 |
Crabb; John W. ; et
al. |
April 21, 2016 |
Compositions And Methods For Macular Degeneration
Abstract
The present invention pertains to methods of inhibiting
angiogenesis (e.g., ocular angiogenesis choroidal
neovasculariztion) in an individual in need thereof comprising
administering to the individual an agent that inhibits one or more
CEP protein adducts wherein the angiogenesis is the result of 5
oxidative peptide modification of polyunsaturated fatty acids
(PUFA) in the individual, and administration of the agent inhibits
angiogenesis in the individual.
Inventors: |
Crabb; John W.; (Chagrin
Falls, OH) ; Salomon; Robert G.; (Mayfield Village,
OH) ; Anand-Apte; Bela; (Shaker Heights, OH) ;
Ebrahem; Quteba; (Shaker Heights, OH) ; Renganathan;
Kutralanathan; (Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Cleveland Clinic Foundation
Case Western Reserve University |
Cleveland
Cleveland |
OH
OH |
US
US |
|
|
Family ID: |
38792204 |
Appl. No.: |
14/922052 |
Filed: |
October 23, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13369964 |
Feb 9, 2012 |
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14922052 |
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12374747 |
Jan 22, 2009 |
8137991 |
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PCT/US2007/016619 |
Jul 23, 2007 |
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13369964 |
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60832898 |
Jul 24, 2006 |
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Current U.S.
Class: |
424/135.1 ;
424/145.1 |
Current CPC
Class: |
C07K 16/18 20130101;
C07K 16/44 20130101; A61P 27/02 20180101; A61K 2039/505 20130101;
C07K 2317/76 20130101; C07K 2317/622 20130101; A61P 9/00
20180101 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under GM
21249 (RGS) from The National Institutes of Health. The government
has certain rights in the invention.
Claims
1. A method of inhibiting angiogenesis in an individual in need
thereof, comprising administering to the individual an agent that
inhibits one or more carboxyethylpyrrole (CEP) protein adducts,
wherein the angiogenesis is the result of oxidative peptide
modification of polyunsaturated fatty acids (PUFA) in the
individual, and administration of the agent inhibits angiogenesis
in the individual.
2. The method of claim 1 wherein the PUFA is docosahexaenoate.
3. The method of claim 1 wherein the agent inhibits formation of
the CEP protein adducts, activity of the CEP protein adducts or a
combination thereof.
4. The method of claim 3 wherein the activity of the CEP protein
adducts comprises angiogenic activity.
5. The method of claim 4 wherein the agent binds to all or a
portion of the CEP protein adducts.
6. The method of claim 5 wherein the agent is an antibody or
antigen binding fragment thereof having binding specificity for the
one or more CEP protein adducts.
7. The method of claim 6 wherein the antibody is a monoclonal
antibody.
8. The method of claim 6 wherein the antibody is a single chain FV
(scFV) antibody.
9. The method of claim 1 wherein the one or more CEP protein
adducts is a CEP-albumin adduct.
10. The method of claim 1 wherein the individual is a primate.
11. The method of claim 10 wherein the primate is a human.
12. The method of claim 1, wherein the angiogenesis is ocular
angiogenesis.
13-21. (canceled)
22. The method of claim 12 wherein the ocular angiogenesis occurs
in the retina of the individual.
23. A method of inhibiting choroidal neovascularization in an
individual in need thereof, comprising administering to the
individual an agent that inhibits one or more carboxyethylpyrrole
(CEP) protein adducts wherein administration of the agent inhibits
choroidal neovascularization in the individual.
24. The method of claim 23 wherein the individual is at risk for
developing age-related macular degeneration.
25. The method of claim 23 wherein the individual is in an early
stage of age-related macular degeneration.
26-34. (canceled)
35. The method of claim 23 wherein the choroidal neovascularization
occurs in the retina of the individual.
36. A method of treating age-related macular degeneration (AMD) in
an individual in need thereof, comprising administering to the
individual an agent that inhibits one or more carboxyethylpyrrole
(CEP) protein adducts in the individual, thereby treating the
AMD.
37. The method of claim 36 wherein the AMD is at an advanced
stage.
38. The method of claim 37 wherein the advanced stage of the AMD is
characterized by choroidal neovascularization.
39-51. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/369,964, filed Feb. 9, 2012, which is a continuation of U.S.
application Ser. No. 12/374,747, filed on Jan. 22, 2009, which
issued as U.S. Pat. No. 8,137,991, which is the U.S. National Stage
of International Application No. PCT/US2007/016619, filed on Jul.
23, 2007, published in English, which claims the benefit of U.S.
Provisional Application No. 60/832,898, filed on Jul. 24, 2006. The
entire teachings of the above applications are incorporated herein
by reference.
BACKGROUND OF THE INVENTION
[0003] Age related macular degeneration (AMD) is the leading cause
of blindness in the elderly population in developed countries. Over
a third of those over 75 years currently have some form of this
disease. Slowing or preventing the progression of AMD is an urgent
public health goal. The clinical significance of ocular
angiogenesis is enormous, because choroidal neovascularization
(CNV) in late stage AMD is the chief cause of irreversible loss of
vision in elderly patients in the western hemisphere. CNV involves
abnormal vessel growth from the choriocapillaris through Bruch's
membrane resulting in hemorrhage, scarring, exudation and/or
retinal detachment with the ultimate consequence of severe loss of
high acuity central vision. The need for effective therapies to
prevent and treat CNV is rapidly growing with the increasing
population of people over the age of 65.
SUMMARY OF THE INVENTION
[0004] CNV, the advanced stage of AMD affects approximately 10% of
patients with AMD, yet accounts for more than 80% of all vision
loss in AMD. Carboxyethylpyrrole (CEP) protein modifications,
uniquely generated from oxidation of docosahexaenoate-containing
lipids are more abundant in ocular tissues from AMD than normal
donors and are concentrated in Bruch's membrane, the blood retinal
barrier. The investigation of whether CEP protein adducts stimulate
angiogenesis and contribute to CNV in AMD is described herein.
Human serum albumin (HSA) or dipeptide (acetyl-Gly-Lys-O-methyl
ester) were chemically modified to yield CEP-HSA or CEP-dipeptide.
The in vivo angiogenic properties of CEP-HSA and CEP dipeptide were
evaluated using the chick chorioallantoic membrane and rat corneal
micropocket assays. Low picomole amounts of CEP-HSA and CEP
dipeptide stimulated neovascularization. Monoclonal anti-CEP
antibody neutralized limbal vessel growth stimulated by CEP-HSA
while anti-vascular endothelial growth factor (anti-VEGF) antibody
only partially neutralized vessel growth. These results show that
anti-CEP modalities are useful as therapeutics in treating CNV in
AMD.
[0005] The studies described herein demonstrate the angiogenic
properties of CEP adducts and indicate that CEP plays a role in the
development of CNV in late stage AMD. Furthermore, the results show
that in vivo blood vessel growth stimulated by CEP can be
quantitatively neutralized by anti-CEP antibody but not by
anti-VEGF antibody, indicating CEP adducts stimulate angiogenesis
in part via a VEGF independent pathway.
[0006] Accordingly, the present invention is directed to a method
of inhibiting angiogenesis in an individual (e.g., primate such as
human) in need thereof, comprising administering to the individual
an agent that inhibits one or more carboxyethylpyrrole (CEP)
protein adducts, wherein the angiogenesis is the result of
oxidative peptide modification of polyunsaturated fatty acids
(PUFA), such as docosahexaenoate, in the individual, and
administration of the agent inhibits angiogenesis in the
individual. The agent can inhibit formation of the CEP protein
adducts, activity of the CEP protein adducts (e.g., angiogenic
activity) or a combination thereof. The agent can bind to all or a
portion of the CEP protein adducts, and includes antibodies or
antigen binding fragments thereof having binding specificity for
the one or more CEP protein adducts. In a particular embodiment,
the antibody is a monoclonal antibody or a single chain FV (scFV)
antibody.
[0007] The invention is also directed to a method of inhibiting
ocular angiogenesis in an individual in need thereof, comprising
administering to the individual an agent that inhibits one or more
CEP protein adducts wherein administration of the agent inhibits
ocular angiogenesis in the individual. In a particular embodiment,
the ocular angiogenesis occurs in the retina of the individual.
[0008] Also encompassed by the present invention is a method of
inhibiting choroidal neovascularization in an individual in need
thereof, comprising administering to the individual an agent that
inhibits one or more carboxyethylpyrrole (CEP) protein adducts
wherein administration of the agent inhibits choroidal
neovascularization in the individual. The individual can be at risk
for developing age-related macular degeneration or in an early
stage of age-related macular degeneration.
[0009] The invention is also directed to a method of treating AMD
in an individual in need thereof, comprising administering to the
individual an agent that inhibits one or more carboxyethylpyrrole
(CEP) protein adducts in the individual, thereby treating the AMD.
In a particular embodiment, the AMD is at an advanced stage. The
AMD can also be characterized by choroidal neovascularization.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIGS. 1a-1c are photographs showing that docosahexaenoate
lipid-derived oxidatively modified human serum albumin (CEP-HSA)
induces angiogenesis in the chorio allantoic membrane (CAM) assay.
Chicken CAMs were exposed to pellets containing (FIG. 1a) human
serum albumin (HAS) (0.5 .mu.g, .about.7.5 pmol), (FIG. 1b) CEP-HSA
(0.5 .mu.g, .about.7.5 pmol) or (FIG. 1c) vascular endothelial
growth factor (VEGF) (20 ng, 0.7 pmol) on day 6. Two days later the
vessels were injected with India ink and visualized using an
Olympus stereomicroscope.
[0011] FIG. 1d is a bar graph of the results.
[0012] FIGS. 2a-2f are photographs showing that CEP modified HSA as
well as dipeptide (Ac-Gly-Lys-OH) induces angiogenesis in a rat
corneal micropocket assay. Representative photographs of mouse
corneas at 7 days after implantation of pellets containing (FIG.
2a) phosphate buffered saline (PBS) (control), (FIG. 2b) VEGF (100
ng, .about.3.5 pmol), (FIG. 2c) HSA (1 .mu.g, .about.15 pmol),
(FIG. 2d) CEP-HSA (1 .mu.g, .about.15 pmol), (FIG. 2e) dipeptide
(41 ng, .about.112 pmol) or (FIG. 2f) CEP-dipeptide (37 ng, 101
pmol).
[0013] FIG. 2g is a bar graph of the results; peak vessel extension
was calculated as described in materials and methods. * P<0.005,
** p<0.001
[0014] FIGS. 3a-3d are photographs showing neutralization of
CEP-HSA induced angiogenesis by monoclonal anti-CEP antibody.
Representative photographs of mouse corneas at 7 days following
implantation of pellets containing (FIG. 3a) HSA (10 .mu.g,
.about.149 pmol), (FIG. 3b) CEP-HSA (1 .mu.g, .about.15 pmol),
(FIG. 3c) CEP-HSA (1 .mu.g) with non specific mouse IgM control
antibody and (FIG. 3d) CEP-has (1 .mu.g) with monoclonal anti CEP
antibody.
[0015] FIG. 3e is a bar graph of the results; peak vessel extension
was calculated as described in material and methods. *
P<0.05
[0016] FIGS. 4a-4d are photographs showing incomplete
neutralization of CEP-HSA induced angiogenesis by monoclonal
anti-VEGF antibody. Representative photographs of mouse corneas at
7 days following implantation of pellets containing (FIG. 4a)
CEP-HSA (1 .mu.g, .about.15 pmol), (FIG. 4b) CEP-HSA (1 .mu.g) with
anti-VEGF neutralizing antibody (FIG. 4c) VEGF (20 ng, .about.0.7
pmol) and (FIG. 4d) VEGF (20 ng) with anti-VEGF neutralizing
antibody.
[0017] FIG. 4e is a bar graph of the results; peak vessel extension
was calculated as described in material and methods. *P=0.06,
CEP-HSA+control IgG versus CEP-HSA+anti-VEGF.
[0018] FIG. 5 is a bar graph showing that CEP dipeptide does not
increase secretion of VEGF in RPE cells. Human ARPE-19 cells were
exposed to various concentrations of dipeptide or CEP-dipeptide.
VEGF concentrations were measured in the conditioned medium of
cells after 18 hours. VEGF is expressed in pg/mg of cellular
protein. All data are from n=4 independent experiments in which
each condition was assayed 6.times. per experiment; error bars
represent standard deviation.
[0019] FIG. 6 is an illustrates the generation of
2-(.omega.-carboxyethyl)pyrrole (CEP) adducts.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The clinical significance of ocular angiogenesis is
enormous, due largely to choroidal neovascularization (CNV) in
age-related macular degeneration (AMD) (Group, M. P. S. (1991) Arch
Ophthalmol 109, 1242-57; Group, M. P. S. (1991) Arch Ophthalmol
109, 1232-41; Campochiaro, P. A. (2000) J Cell Physiol 184, 301-10)
which is the chief cause of irreversible loss of vision in elderly
patients in the western hemisphere. CNV involves abnormal vessel
growth from the choriocapillaris through Bruch's membrane resulting
in hemorrhage, scarring, exudation and/or retinal detachment with
the ultimate consequence of a severe loss of high acuity central
vision. The molecular mechanisms involved in the development of CNV
are not well defined, but the need for effective therapies to
prevent and treat CNV is augmented with an increase in the
population of people over the age of 65 years.
[0021] CNV, the advanced stage of age-related macular degeneration
(AMD) accounts for more than 80% of vision loss in AMD.
Carboxyethylpyrrole (CEP) protein modifications, uniquely generated
from oxidation of docosahexaenoate-containing lipids are more
abundant in Bruch's membrane of AMD retinas. As described herein,
whether CEP protein adducts stimulate angiogenesis and contribute
to CNV in AMD was investigated. Human serum albumin (HSA) or
dipeptide (acetyl-Gly-Lys-O-methyl ester) were chemically modified
to yield CEP-HSA or CEP-dipeptide. The in vivo angiogenic
properties of CEP-HSA and CEP dipeptide were demonstrated using the
chick chorioallantoic membrane and rat corneal micropocket assays.
Low picomole amounts of CEP-HSA and CEP dipeptide stimulated
neovascularization. Monoclonal anti-CEP antibody neutralized limbal
vessel growth stimulated by CEP-HSA while anti-VEGF antibody was
found to only partially neutralize vessel growth. In vitro
treatments of human retinal pigment epithelial cells with
CEP-dipeptide or CEP-HSA did not induce increased VEGF secretion.
Overall, these results show that CEP induced angiogenesis utilizes
VEGF independent pathways and that anti-CEP therapeutic modalities
are likely of value in limiting CNV in AMD.
[0022] AMD is a progressive, multifactorial, polygenic disease with
poorly understood etiology. Early stages of the disease are
typically termed "dry" AMD and associated with the macular
accumulation of extracellular deposits (drusen) below the RPE on
Bruch's membrane. Geographic atrophy develops in the later stages
of dry AMD and is characterized by macular loss of RPE and
photoreceptor cells. Advanced stage disease or "wet" AMD is
characterized by CNV. Oxidative damage has long been suspected of
contributing to AMD (Beatty, S., et al. (2000) Sury Ophthalmol 45,
115-34; Winkler, B. S. et al. (1999) Mol Vis 5, 32), supported by
indirect evidence that smoking significantly increases the risk of
AMD (Seddon, J. M., et al. (1996) JAMA 276, 1141-6) and that
antioxidant vitamins and zinc can slow the progression of the
disease for select individuals (AREDS (2001) Arch Ophthalmol 119,
1417-36). Several laboratories have recently shown an association
between variants in the complement factor H gene and susceptibility
to AMD (Hageman, G. S., et al. (2005) Proc Natl Acad Sci USA 102,
7227-32; Klein, R. J., et al. (2005) Science 308, 385-9; Edwards,
A. O., et al. (2005) Science 308, 421-4; Haines, J. L., et al.
(2005) Science 308, 419-21) implicating inflammatory processes in
the pathophysiology of the disease (Bok, D. (2005) Proc Natl Acad
Sci USA 102, 7053-4). In addition, the recent observation that aged
mice exhibiting the apolipoprotein E4 genotype develop a full range
of AMD-like pathologies including CNV, when fed a high cholesterol
diet suggests that lipid oxidation in combination with genetic and
environmental factors might contribute to AMD (Malek, G., et al.
(2005) Proc Natl Acad Sci USA 102, 11900-5). A previous proteomic
study of drusen established a molecular link between oxidative
damage and AMD (Crabb, J. W., et al., Proc. Natl. Acad. Sci. USA,
99:14682-14887 (2002)) and demonstrated elevated oxidative protein
modifications in AMD tissues. Specifically, carboxyethylpyrrole
(CEP), a unique protein modification derived from the oxidation of
docosahexaenoate (DHA)-containing lipids, was found to be more
abundant in AMD compared to normal ocular tissues (Crabb, J. W., et
al. (2002) Proc Natl Acad Sci USA 99, 14682-7) and was localized in
Bruch's membrane between the blood-bearing choriocapillaris and
RPE. The outer segments of the photoreceptors contain high
concentrations of polyunsaturated fatty acids (PUFAs), especially
DHA in the membranes and are exposed to relatively high oxygen
tension, close to that found in arterial blood. The photooxidative
environment in the retina and the DHA rich photoreceptor outer
segments provide a ready source of reactive oxygen species for
generating oxidative modifications. PUFAs undergo oxidation in the
presence of oxygen or oxygen derived radical species, and elevated
levels of CEP-adducts and CEP autoantibodies are present in AMD
plasma (Gu, X. et al. (2003) J. Biol. Chem., 273:42027-42035; U.S.
Published Application No. 2004/0265924 A1). Described herein is the
investigation of whether these oxidative protein modifications are
a primary catalyst in drusen formation and play a role in the
development of choroidal neovascularization (Crabb, J. W., et al.
(2002) Proc Natl Acad Sci USA 99, 14682-7). The results of the
investigation show that CEP protein modifications induce
angiogenesis in vivo.
[0023] Levels of CEPs are significantly elevated in patients with
AMD versus unaffected individuals. Levels apparently rise strongly
in the earliest stage of the disease and then decline, although
they remain significantly elevated in all stages of the disease. It
is likely that the actual levels of CEPs are not detected because
these modified proteins are antigens that induce production of
autoantibodies that mask the antigens, especially in the later
stages of the disease. It is likely CEP-protein modifications as
well as consequent generation of autoantibodies play a role in the
etiology of AMD. For example, aggregation of CEP-modified proteins
by CEP autoantibodies may contribute to drusen accumulation and
promote retinal degeneration. It is likely that CEP levels appear
to be highest in the earliest stage of the disease because
autoantibodies are not yet present. In later stages of the disease,
levels of antigen continue to rise but are not detected because
they are present as circulating immune complexes. Plasma from AMD
patients exhibited more than a 2-fold higher average CEP
autoantibody titer than plasma from age-matched normal
controls.
[0024] Thus, the present invention pertains to methods of
inhibiting angiogenesis in an individual in need thereof comprising
administering to the individual an agent that inhibits one or more
CEP protein adducts wherein the angiogenesis is the result of
oxidative peptide modification of polyunsaturated fatty acids
(PUFA) in the individual, and administration of the agent inhibits
angiogenesis in the individual. In one embodiment, the invention is
directed to methods of inhibiting ocular angiogenesis in an
individual in need thereof comprising administering to the
individual an agent that inhibits the angiogenic activity of one or
more CEP protein adducts wherein administration of the agent
inhibits ocular angiogenesis in the individual. In another
embodiment, the invention is directed to methods of inhibiting
choroidal neovascularization in an individual in need thereof
comprising administering to the individual an agent that inhibits
the angiogenic activity of one or more CEP protein adducts wherein
administration of the agent inhibits choroidal neovascularization
in the individual.
[0025] The present invention is also directed to methods of
treating (prophylactic and/or therapeutic treatment) diseases
associated with the angiogenic activity of CEP protein adducts
using an agent that inhibits the angiogenic activity of CEP protein
adducts. In one embodiment, the invention is directed to a method
of treating AMD in an individual in need thereof comprising
administering to the individual an agent that inhibits one or more
CEP protein adducts.
[0026] The terms, "inhibiting" and "treatment" as used herein,
refer not only to ameliorating symptoms associated with the
condition or disease, but also preventing or delaying the onset of
the condition or disease, and/or lessening the severity or
frequency of symptoms of the condition or disease. The therapy is
designed to inhibit (partially, completely) activity and/or
formation of CEP protein adducts in an individual. For example, an
agent that inhibits CEP protein adducts can be administered in
order to decrease and/or prevent the activity and/or formation of
CEP protein adducts.
[0027] Carboxyethylpyrrole (CEP) protein adducts belong to a family
of 2-(.omega.-carboxyalkyl)pyrrole adducts generated from the
oxidation of polyunsaturated fatty acids (PUFA) (see Gu et al., J.
Biol. Chem., 278(43):42027-42035 (2003) and U.S. Published
Application No. 2004/0265924, both of which are incorporated herein
by reference). Docosahexaenoic acid (DHA) gives rise to
2-(.omega.-carboxyethyl)pyrrole adducts, by oxidative cleavage to
4-hydroxy-7-oxohept-5-enoic acid (HOHA) and reaction of the HOHA
with protein (FIG. 6). HOHA can form an adduct with a (one or more)
primary amine of a peptide (e.g., a dipeptide) or protein resulting
in a CEP epitope that is referred to as CEP-peptide or CEP-protein
adducts, respectively. For example, HOHA can form an adduct with,
or on, proteins such as albumin, and fragments thereof. CEP
epitopes can also be generated by the reaction of HOHA with the
primary amino group of ethanolamine phospholipids that are referred
to as ethanolamine phospholipid CEP adducts. Also phospholipids
containing an HOHA residue can form CEPs through reaction with
primary amino groups of biomolecules such as proteins followed by
phospholipolysis of the initially formed CEP phospholipid ester
derivative.
[0028] An agent that inhibits a (one or more) CEP protein adduct is
an agent or compound that inhibits the activity and/or formation
(expression) of a CEP protein adduct, as described herein (e.g., a
CEP protein adduct antagonist). An agent that inhibits a CEP
protein adduct can alter CEP protein adduct activity or CEP protein
adduct formation by a variety of means. The inhibition can be
partial or complete inhibition of CEP protein adduct activity
and/or formation. In addition, the agent can inhibit the CEP
protein adduct directly (specifically interact) or indirectly
(non-specifically interact).
[0029] For example, the agent for use in the methods of the present
invention can inhibit one or more biological activities of CEP
protein adducts. An example of a biological activity of a CEP
protein adduct is angiogenic activity. In one embodiment, the agent
binds to all or a portion (e.g., a portion of the CEP protein
adduct; the CEP portion of the CEP protein adduct; the protein or
peptide portion of the CEP protein adduct) of the CEP protein
adduct under conditions in which the angiogenic activity of the CEP
protein adduct is inhibited.
[0030] Alternatively, the agent for use in the methods of the
present invention can inhibit formation of the CEP protein adduct.
For example, the agent can prevent CEP protein adducts from
forming, and/or hydrolyze CEP protein adducts that have previously
formed, regenerating the primary amino group found in the
unmodified biomolecule. In one embodiment, the agent can interact
with HOHA or its esters, e.g., phospholipid derivatives containing
a HOHA acyl group esterified to the sn-2 position, and/or the
protein which forms an adduct with HOHA, prior to formation of the
CEP protein adduct, thereby preventing CEP protein adducts from
forming. In addition, the agent can interact with an upstream
product (e.g., DHA) of the reaction which leads to formation of CEP
protein adducts in order to prevent CEP protein adducts from
forming.
[0031] The agent can also interact with the CEP protein adduct or
portion thereof after CEP protein adducts have formed, for example,
under conditions in which the pyrrole moiety of the CEP and the
protein of the CEP protein adduct is disrupted. In particular
embodiments, the agent cleaves the CEP group from the protein.
[0032] Examples of agents which can inhibit receptor-mediated
effects of CEP protein adducts include the following: nucleic
acids, fragments or derivatives thereof and vectors comprising such
nucleic acids (e.g., a nucleic acid molecule, cDNA, and/or RNA);
polypeptides; peptidomimetics; fusion proteins or prodrugs thereof;
antibodies; ribozymes; aptamers; small molecules; and other
compounds that inhibit CEP protein adduct activity and/or
formation. One or more agents that inhibit CEP protein adducts can
be used concurrently (simultaneously) or sequentially in the
methods of the present invention, if desired.
[0033] In a particular embodiment, the agent or compound that
inhibits CEP protein adduct activity and/or formation is an
antibody (e.g., a polyclonal antibody; a monoclonal antibody). For
example, antibodies that bind all or a portion of one or more CEP
protein adducts and that inhibit CEP protein adduct activity can be
used in the methods described herein (Gu et al., J. Biol. Chem.,
278(43):42027-42035 (2003) and U.S. Application No. 2004/0265924,
both of which are incorporated herein by reference). In a
particular embodiment, the antibody is a purified antibody. The
term "purified antibody" as used herein refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site
that selectively binds all or a portion (e.g., a biologically
active portion) of a CEP protein adduct. A molecule that
selectively binds to a CEP protein adduct is a molecule that binds
to a CEP protein adduct or a fragment thereof, but does not
substantially bind other molecules in a sample (e.g., a biological
sample that naturally contains the CEP protein adduct). Preferably
the antibody is at least 60%, by weight, free from proteins and
naturally occurring organic molecules with which it naturally
associated. More preferably, the antibody preparation is at least
75% or 90%, and most preferably, 99%, by weight, antibody. Examples
of immunologically active portions of immunoglobulin molecules
include F(ab) and F(ab').sub.2 fragments that can be generated by
treating the antibody with enzymes such as pepsin or papsain, and
single chain FV (scFV) fragments.
[0034] The term "monoclonal antibody" or "monoclonal antibody
composition," as used herein, refers to a population of antibody
molecules that contain only one species of an antigen binding site
capable of immunoreacting with a particular epitope of a CEP
protein adduct of the invention. A monoclonal antibody composition
thus typically displays a single binding affinity for a particular
CEP protein adduct of the invention with which it immunoreacts.
[0035] Polyclonal antibodies can be prepared using known techniques
such as by immunizing a suitable subject with a desired immunogen,
e.g., a CEP protein adduct or fragment thereof. The antibody titer
in the immunized subject can be monitored over time by standard
techniques, such as with an enzyme linked immunosorbent assay
(ELISA) using immobilized polypeptide. If desired, the antibody
molecules directed against the CEP protein adduct can be isolated
from the mammal (e.g., from tissue, blood) and further purified by
well-known techniques, such as protein A chromatography to obtain
the IgG fraction.
[0036] At an appropriate time after immunization (e.g., when the
antibody titers are highest) antibody-producing cells can be
obtained from the subject and used to prepare monoclonal antibodies
by standard techniques, such as the hybridoma technique originally
described by Kohler and Milstein, Nature 256:495-497 (1975), the
human B cell hybridoma technique (Kozbor et al., Immunol. Today
4:72 (1983)), the EBV-hybridoma technique (Cole et al., Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96
(1985)) or trioma techniques. The technology for producing
hybridomas is well known (see generally Current Protocols in
Immunology, Coligan et al., (eds.) John Wiley & Sons, Inc., New
York, N.Y. (1994)). Briefly, an immortal cell line (typically a
myeloma) is fused to lymphocytes (typically splenocytes) from a
mammal immunized with an immunogen as described above, and the
culture supernatants of the resulting hybridoma cells are screened
to identify a hybridoma producing a monoclonal antibody that binds
a CEP protein adduct of the invention.
[0037] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating a monoclonal antibody to a CEP protein adduct
of the invention (see, e.g., Current Protocols in Immunology,
supra; Galfre et al., Nature, 266:55052 (1977); R. H. Kenneth, in
Monoclonal Antibodies: A New Dimension In Biological Analyses,
Plenum Publishing Corp., New York, N.Y. (1980); and Lerner, Yale J.
Biol. Med. 54:387-402 (1981)). Moreover, the ordinarily skilled
worker will appreciate that there are many variations of such
methods that also would be useful.
[0038] In one alternative to preparing monoclonal
antibody-secreting hybridomas, a monoclonal antibody to a CEP
protein adduct of the invention can be identified and isolated by
screening a recombinant combinatorial immunoglobulin library (e.g.,
an antibody phage display library) with the CEP protein adduct to
thereby isolate immunoglobulin library members that bind the
polypeptide. Kits for generating and screening phage display
libraries are commercially available (e.g., the Pharmacia
Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the
Stratagene SurfZAP.TM. Phage Display Kit, Catalog No. 240612).
Additionally, examples of methods and reagents particularly
amenable for use in generating and screening antibody display
library can be found in, for example, U.S. Pat. No. 5,223,409; PCT
Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT
Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT
Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT
Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs
et al., Bio/Technology 9:1370-1372 (1991); Hay et al., Hum.
Antibod. Hybridomas 3:81-85 (1992); Huse et al., Science
246:1275-1281 (1989); and Griffiths et al., EMBO J. 12:725-734
(1993).
[0039] Additionally, recombinant antibodies, such as chimeric and
humanized monoclonal antibodies, comprising both human and
non-human portions, which can be made using standard recombinant
DNA techniques, are within the scope of the invention. Such
chimeric and humanized monoclonal antibodies can be produced by
recombinant DNA techniques known in the art.
[0040] In a particular embodiment, the antibody is a scFV antibody
which binds CEP protein adducts. One of skill in the art can obtain
an scFV phage displayed combinatorial antibody library that can be
used to generate scFV antibodies which bind CEPs. Selection of
phages displaying scFV which bind CEPs can be accomplished using,
for example, CEP derivatives of biotinylated peptides anchored to
streptavidin-coated magnetic beads, a technology that facilitates
extensive washing that reduces non-specific interactions of the
phage (Sawyer, C., et al., J. Immunol. Methods, 204: 193-203
(1997)). To "pan" for scFV-CEP antibodies the efficacy of a CEP
derivative, such as btn-NH(CH.sub.2).sub.6--CEP prepared by a
general synthesis of biotinylated haptens can be determined. If
necessary, a biotinylated analogue, btn-GlyLys-CEP, of
Ac-GLyLys)OMe)-CEP (a biologically active "CEP-dipeptide"), can be
prepared. In addition, a longer flexible linker using a
CEP-modified analogue of the btn-GlySerGlyLys-isoLGE.sub.2-lactam
(SEQ ID NO:1) can be used.
[0041] The antibodies for use in the methods of the present
invention can also be capable of detection, for example, in order
to determine the efficacy of a given treatment regimen. Detection
can be facilitated by coupling the antibody to a detectable
substance. Examples of detectable substances include various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials, bioluminescent materials, and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, .beta.-galactosidase, and
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride and
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, green fluorescent protein, and aequorin, and examples of
suitable radioactive material include, for example, .sup.125I,
.sup.131I, .sup.35S, .sup.32P and .sup.3H.
[0042] The agents which inhibit CEP protein adducts are
administered in a therapeutically effective amount (i.e., an amount
that is sufficient to treat or inhibit the disease or condition,
such as by ameliorating symptoms associated with the disease or
condition, preventing or delaying the onset of the disease or
condition, and/or also lessening the severity or frequency of
symptoms of the disease or condition). The amount that will be
therapeutically effective in the treatment of a particular
individual's disorder or condition will depend on the symptoms and
severity of the disease, and can be determined by standard clinical
techniques. In addition, in vitro or in vivo assays may optionally
be employed to help identify optimal dosage ranges. The precise
dose to be employed in the formulation will also depend on the
route of administration, and the seriousness of the disease or
disorder, and should be decided according to the judgment of a
practitioner and each patient's circumstances. Effective doses may
be extrapolated from dose-response curves derived from in vitro or
animal model test systems.
[0043] The methods of the present invention can be used to treat
any suitable individual. In one embodiment, the individual is a
primate. In a particular embodiment, the individual is a human.
[0044] The agent (e.g., therapeutic compound) can be delivered in a
composition, as described above, or by themselves. They can be
administered systemically, or can be targeted to a particular
tissue. The therapeutic compounds can be produced by a variety of
means, including chemical synthesis; recombinant production; in
vivo production (e.g., a transgenic animal, such as U.S. Pat. No.
4,873,316 to Meade et al.), for example, and can be isolated using
standard means such as those described herein. A combination of any
of the above methods of treatment can also be used.
[0045] The compounds for use in the methods described herein can be
formulated with a physiologically acceptable carrier or excipient
to prepare a pharmaceutical composition. The carrier and
composition can be sterile. The formulation should suit the mode of
administration.
[0046] Suitable pharmaceutically acceptable carriers include but
are not limited to water, salt solutions (e.g., NaCl), saline,
buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable
oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates
such as lactose, amylose or starch, dextrose, magnesium stearate,
talc, silicic acid, viscous paraffin, perfume oil, fatty acid
esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well
as combinations thereof. The pharmaceutical preparations can, if
desired, be mixed with auxiliary agents, e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for
influencing osmotic pressure, buffers, coloring, flavoring and/or
aromatic substances and the like that do not deleteriously react
with the active compounds.
[0047] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. The
composition can be a liquid solution, suspension, emulsion, tablet,
pill, capsule, sustained release formulation, or powder. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, polyvinyl
pyrollidone, sodium saccharine, cellulose, magnesium carbonate,
etc.
[0048] Methods of introduction of these compositions include, but
are not limited to, intradermal, intramuscular, intraperitoneal,
intraocular, intravenous, subcutaneous, topical, oral and
intranasal. Other suitable methods of introduction can also include
gene therapy, rechargeable or biodegradable devices, particle
acceleration devises ("gene guns") and slow release polymeric
devices. The pharmaceutical compositions of this invention can also
be administered as part of a combinatorial therapy with other
compounds.
[0049] The composition can be formulated in accordance with the
routine procedures as a pharmaceutical composition adapted for
administration to human beings. For example, compositions for
intravenous administration typically are solutions in sterile
isotonic aqueous buffer. Where necessary, the composition may also
include a solubilizing agent and a local anesthetic to ease pain at
the site of the injection. Generally, the ingredients are supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampule or sachette
indicating the quantity of active compound. Where the composition
is to be administered by infusion, it can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water,
saline or dextrose/water. Where the composition is administered by
injection, an ampule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0050] For topical application, nonsprayable forms, viscous to
semi-solid or solid forms comprising a carrier compatible with
topical application and having a dynamic viscosity preferably
greater than water, can be employed. Suitable formulations include
but are not limited to solutions, suspensions, emulsions, creams,
ointments, powders, enemas, lotions, sols, liniments, salves,
aerosols, etc., that are, if desired, sterilized or mixed with
auxiliary agents, e.g., preservatives, stabilizers, wetting agents,
buffers or salts for influencing osmotic pressure, etc. The
compound may be incorporated into a cosmetic formulation. For
topical application, also suitable are sprayable aerosol
preparations wherein the active ingredient, preferably in
combination with a solid or liquid inert carrier material, is
packaged in a squeeze bottle or in admixture with a pressurized
volatile, normally gaseous propellant, e.g., pressurized air.
[0051] Compounds described herein can be formulated as neutral or
salt forms. Pharmaceutically acceptable salts include those formed
with free amino groups such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with free carboxyl groups such as those derived from sodium,
potassium, ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0052] In another embodiment, the invention is directed to agents
which inhibit CEP protein adducts for use as a medicament in
therapy. For example, the agents identified herein can be used in
the treatment of optic nerve damage. In addition, the agents
identified herein can be used in the manufacture of a medicament
for the treatment of AMD.
[0053] Use of the agents described herein to inhibit or treat
diseases or conditions associated with CEP protein adducts can be
used in conjunction with other known therapies for such diseases or
conditions. For example, anti-VEGF therapies with recombinant
humanized anti VEGF monoclonal antibody or aptamer are being
evaluated as treatments for CNV. However, the ability of VEGF
neutralizing antibody to only partially block CEP-HSA induced
angiogenesis indicates that additional therapeutics will be
required to effectively limit CNV. CEP neutralization modalities
can be effective independently or as a complement to anti-VEGF
therapies for the inhibition of CNV in AMD.
EXEMPLIFICATION
Methods
[0054] Synthesis of .omega.-Carboxyethylpyrrole-Modified Proteins
and Peptides
[0055] Unambiguous production of 2-(.omega.-carboxyethyl)pyrrole
(CEP) was established utilizing the .gamma.-ketoaldehydes,
4,7-dioxoheptanoic acid (DOHA) as described previously (Gu, X., et
al. (2003) J Org Chem 68, 3749-3761). Paal-Knorr condensation of
DOHA with human serum albumin (HSA) and with the dipeptide
acetyl-Gly-Lys-O-methyl ester were used to generate CEP-HSA and
CEP-dipeptide, respectively, which were characterized by mass
spectrometry and NMR as described previously (Gu, X., et al. (2003)
J Org Chem 68, 3749-3761). Pyrrole concentration was determined by
Ehlrich's assay with 4-(dimethyamino)benzaldehyde and absorbance at
570 nm. Protein was quantified by amino acid analysis (Crabb, J.
W., et al. (1997) Amino Acid Analysis (John Wiley and Sons, Inc.))
and the Bradford protein assay.
[0056] Chick Chorio Allantoic Membrane (CAM) Angiogenesis Assay
[0057] The CAM assay was performed as described previously (Nguyen,
M., et al. (1994) Microvasc Res 47, 31-40) with slight
modifications. Fertilized 3-day old white Leghorn eggs (CWRU,
Squire valley farms) were cracked, and embryos with the yolk intact
were placed in 100 mm.times.20 mm glass bottom Petri dishes.
Following incubation for 3 days at 37.degree. C. in 3% CO.sub.2, a
methylcellulose disc (Fisher Scientific, Fair Lawn, N.J.)
containing CEP-HSA or CEP-dipeptide, was placed on the CAM of
individual embryos. CAMs implanted with discs loaded with
unmodified dipeptide, HSA, control buffer or with vascular
endothelial growth factor (VEGF) were used as negative and positive
controls respectively. After 48 hours of incubation, India ink was
injected into the vascular system for better visualization of the
vessels by a stereomicroscope. Images were captured with a
Panasonic CCD camera. Samples were always compared on the same CAM
to avoid egg-to-egg variability. For quantitative analysis of
vessel density and leakage, CAM images were batch processed using
customized macros generated in Image-Pro Plus 5.0 (Media
Cybernetics, Silver Spring, Md.). Briefly, a region of interest
(ROI) was traced around grafted tissue in each image, each image
was then cropped to its ROI, converted to grayscale, and processed
using a large spectral filter to enhance the appearance of
vasculature whilst omitting presence of larger vessels (determined
by filter width). For skeletal density and vessel leakage
measurements images were skeletonized using morphological filters
(pixels representing branch points were excluded to divide
vasculature into distinct vessel segments). Lengths of skeletal
segments larger than 5 pixels (delineating vessels) were summed and
divided by total graft area for skeletal density. Conversely,
skeletal segments smaller than 5 pixels were summed for vessel
leakage measurement.
[0058] Rat Corneal Micropocket Assay
[0059] Hydron/sucralfate pellets containing unmodified or CEP
modified HSA or dipeptide with or without neutralizing antibodies
(monoclonal mouse anti-human VEGF, 1.5 .mu.g, R&D systems
(MAB293) or monoclonal anti-CEP antibody (Gu, X., et al. (2003) J.
Biol. Chem.), 4 .mu.g) were inserted into corneal micropockets (1
mm from the limbus) of Sprague-Dawley rats. Control mouse IgM, 4
.mu.g (eBioscience, cat. #14-4752) and mouse IgG, 1.5 .mu.g
(Southern Biotechnology Inc., cat. #0104-01) antibodies were used
in control pellets for comparison with anti-CEP and anti-VEGF
antibodies respectively. Corneas were examined daily with the aid
of a surgical microscope to monitor angiogenic responses to CEP
modified peptide or proteins. To photograph the angiogenic
response, animals were perfused with India ink to label the
vessels, and following enucleation and fixation, the corneas were
excised, flattened and photographed. A positive neovascularization
response was recorded only if sustained directional in-growth of
capillary sprouts and hairpin loops toward the pellet was observed.
A negative response was recorded when either no growth is observed
or only an occasional sprout or hairpin loop showing no evidence of
sustained growth was detected. All responses were compared to a
negative control (pellet and pellet containing buffer) and positive
control of VEGF. For neutralization studies, responses were
compared to a negative control of non-specific mouse immunoglobulin
(IgM) described above. Angiogenic response was analyzed for peak
vascular extension and total skeletal (vascular) length using
Image-Pro Plus 5.0 (Media Cybernetics, Silver Spring, Md.). Prior
to performing vessel measurements images were processed using
best-fit equalization filters, spectral filters, and large
pixel-width background removal filters to enhance vasculature and
eliminate image artifacts. For total skeletal length measurements,
processed images were skeletonized, summing pixel lengths of
resultant skeletal segments. To determine peak vessel extension,
processed images were thresholded for vasculature, filling in holes
between adjacent vessels using morphological filters. The resulting
image, a single segmented object representing the overall
dimensions of the vascular bed, was analyzed for maximum box-width,
i.e. extent of vessel penetration.
[0060] Cell Culture Conditions and VEGF Secretion Assay.
[0061] Human retinal pigment epithelium (ARPE-19) cells were
cultured in DMEM/F-12 medium with 10% fetal bovine serum, 100 U/ml
penicillin, and 100 .mu.g/ml streptomycin. The medium was changed
every four days and all studies were performed on confluent cells.
Endotoxin free media and serum were used. Confluent cultures in 24
well plates were starved for three days with serum free medium
before incubating with CEP-Dipeptide or CEP-HSA (0.1-10 .mu.M) and
unmodified dipeptide or HSA (as controls). The CEP-dipetide,
CEP-HSA and controls were quantified by amino acid analysis (Crabb,
J. W., et al. (1997) Amino Acid Analysis (John Wiley and Sons,
Inc.)). Supernatant media was collected to measure VEGF secretion
using an enzyme-linked immunosorbent assay (ELISA) according to the
manufacturer's protocol (Research Diagnostics, Flanders, N.J.).
Concanavalin (50 .mu.g/mL) was used as a positive control for VEGF
stimulation.
[0062] Statistical Analysis Data are presented as mean.+-.SD.
[0063] The statistical significance of differential findings
observed between experimental and control groups was determined
using one-way analysis of variance (ANOVA), and considered to be
significant if P values were <0.06.
[0064] Results
[0065] CEP-induced Angiogenesis in Chicken Embryo.
[0066] The potential consequence of CEP modified human serum
albumin (CEP-HSA) on angiogenesis was examined using the chick
chorioallantoic membrane (CAM) assay. The angiogenic response to
methylcellulose discs containing 0.5 .mu.g (n=4), 1 .mu.g (n=3),
and 10 .mu.g (n=4) of CEP-HSA or unmodified HSA (n=8) was analyzed.
The protein preparations were analyzed for endotoxin and determined
to be free of contamination. Representative results from these CAM
assays are depicted in FIGS. 1a-1d. CEP-HSA (FIG. 1b) induced
sprouting of new blood vessels that appeared to be tortuous and
leaky when perfused with India ink. The average skeletal density of
CAM vessels with 0.5 .mu.g of CEP-HSA was .about.3.1. Unmodified
HSA (0.5 .mu.g) did not show this effect (FIG. 1a) with
quantitation revealing a lower background skeletal density of
.about.2.5. Vascular endothelial growth factor (VEGF 20 ng) was
used as a positive control (FIG. 1c) and showed an average skeletal
density of .about.3.1 in the CAM assay. CEP-HSA (161 ng) induced a
maximal response compared with minimal or absent response with HSA
at doses up to 0.5 .mu.g. The angiogenic response of 161 ng CEP-HSA
(2.4 pmol) was similar to the half maximal response of VEGF at a
dose of 20 ng (0.7 pmol).
[0067] CEP-Induced Angiogenesis in Rat Cornea.
[0068] The results from the CAM assay were confirmed and extended
in rats using an additional in vivo angiogenesis assay, the corneal
micropocket assay. Pellets containing CEP-HSA, (1 .mu.g, FIG. 2d)
or CEP modified dipeptide (CEP-dipeptide, 37 ng, FIG. 2f) when
implanted 1 mm from the limbus of rat cornea stimulated the growth
of limbal blood vessels towards the pellet. The newly formed
capillaries reached the pellet by day 7 in all the animals
implanted with 1 .mu.g or more of CEP-HSA (n=5), or 37 ng or more
of CEP-dipeptide (n=7). Notably, unmodified HSA (1 .mu.g n=3, FIG.
2c) or dipeptide (41 ng, n=6, FIG. 2e) did not induce this effect.
Discs containing no protein or peptide were used as a negative
control (FIG. 2a) and VEGF discs (100 ng, FIG. 2b) generated the
positive control. A statistically significant increase in peak
vessel extensions were observed in response to CEP-HSA (.about.2.7
fold) or CEP-dipepetide (.about.3.1 fold) when compared with
unmodified parent molecules (FIG. 2g).
[0069] Neutralization of CEP Induced Angiogenesis with Anti-CEP but
not Anti-VEGF Antibodies.
[0070] To confirm that the angiogenesis was induced by CEP
modification of HSA, pellets were prepared by premixing anti-CEP
antibody (see U.S. Published Application No. 2004/0265924) or
anti-VEGF antibody (R&D Systems, cat. #MAB293) and CEP-HSA. The
monoclonal anti-CEP antibody almost completely inhibited the
formation of new blood vessels from CEP-HSA implants (FIG. 3d) in
the corneal micropocket assay. Neutralizing VEGF antibody only
partially inhibited the CEP-HSA induced neovascularization response
(FIG. 4a, 4b, 4e) while completely inhibiting VEGF induced response
(FIG. 4c, 4d). Control mouse IgM or IgG antibodies did not show
inhibition of CEP-HSA mediated corneal neovascularization (FIG. 3c,
4e). Quantitation of peak vessel extensions indicates that the
observed neutralization of CEP-HSA induced angiogenesis by anti-CEP
was of greater statistical significance than by anti-VEGF
antibodies (FIG. 3e, 4e).
[0071] CEP Adducts do not Stimulate VEGF Secretion In Vitro.
[0072] Another type of oxidized protein modification, namely
advanced glycation end products (AGEs), stimulate angiogenesis in
vivo (Okamoto, T., et al. (2002) Microvasc Res 63, 186-95) and
induce VEGF secretion in vitro (Hirata, C., et al. (1997) Biochem
Biophys Res Commun 236, 712-5; Hoffmann, S., et al. (2000) Invest
Ophthalmol Vis Sci 41, 2389-93). To explore whether CEP adducts
influence VEGF secretion in vitro, human retinal pigment epithelial
(RPE) cells were treated with CEP-dipeptide (0.1-10 .mu.M) and VEGF
protein quantified in the growth media by ELISA. CEP-dipetide
treated ARPE 19 cells did not exhibit increased VEGF in the growth
media relative to the unmodified dipeptide or media alone (FIG. 5).
ARPE19 cells treated with CEP-HSA (0.1-10 .mu.M) also exhibited no
increase in VEGF secretion (data not shown).
[0073] Discussion
[0074] CEP protein adducts belong to a family of
2-(.omega.-carboxyalkyl)pyrrole adducts generated from the
oxidation of polyunsaturated fatty acids (PUFAs) (Kaur, K., et al.
(1997) Chem Res Toxicol 10, 1387-96). For example, oxidative
fragmentation of linoleic acid or arachidonic acid can generate
2-(.omega.-carboxyheptyl)pyrrole (CHP) or
2-(w-carbosypropyl)pyrrole (CPP) adducts, respectively. The
phosphatidylcholine (PC) esters of the oxidatively truncated PUFA
progenitors of these adducts are biologically active and present in
atherosclerotic plaques (Podrez, E. A., et al. (2002) J Biol Chem
277, 38517-23; Podrez, E. A., et al. (2002) J Biol Chem 277,
38503-16; Subbanagounder, G., et al. (2002) Vascul Pharmacol 38,
201-9; Sun, M., et al. (2002) J Org Chem 67, 3575-84). However,
while CPP or CHP protein adducts can also arise from oxidation of
other common PUFAs, CEP protein adducts uniquely are generated from
oxidation of DHA (Gu, X., et al. (2003) J Org Chem 68, 3749-3761).
Although rare in most human tissues, DHA accounts for approximately
80 mol % of the polyunsaturated lipids in photoreceptor outer
segments (Fliesler, S. J. & Anderson, R. E. (1983) Prog Lipid
Res 22, 79-131). The abundance of DHA in photoreceptors, the high
photooxidative stress in the retina as well as the fact that DHA is
the most oxidizable fatty acid in humans, all contribute to the
higher levels of CEP-adducts in AMD. Interestingly, CEP
immunoreactivity and CEP autoantibody titer are also significantly
elevated in plasma from AMD donors (Gu, X., et al. (2003) J. Biol.
Chem.), and are likely of diagnostic utility as biomarkers for
predicting AMD susceptibility. Other oxidative modifications such
as advanced glycation end products (AGEs), generated from oxidized
carbohydrate products also accumulate during aging (Handa, J. T.,
et al. (1999) Invest Ophthalmol Vis Sci 40, 775-9; Wu, J. T. (1993)
J Clin Lab Anal 7, 252-5), especially in the choriocapillaris,
Bruch's membrane (Handa, J. T., et al. (1999) Invest Ophthalmol Vis
Sci 40, 775-9) and CNV membranes (Ishibashi, T., et al. (1998) Arch
Ophthalmol 116, 1629-32). Several studies have shown that AGEs can
stimulate the proliferation of choroid endothelial cells, the
expression of MMP-2 and growth factors such as VEGF (Hoffmann, S.,
et al. (2002) Graefes Arch Clin Exp Ophthalmol 240, 996-1002) and
angiogenesis in vivo (Okamoto, T., et al. (2002) Microvasc Res 63,
186-95).
[0075] The present study demonstrates the angiogenic properties of
CEP adducts and suggests the possibility of CEP playing a role in
the development of the wet (exudative) form of AMD. However, the
molecular mechanism by which CEP induces angiogenesis has not yet
been determined. A likely indirect mechanism for in vivo CEP
stimulation of angiogenesis is that CEP induces the release of
angiogenic factors such as VEGF or basic fibroblast growth factor
(bFGF) by epithelial cells, or inhibits the secretion of
angiogenesis inhibitors that might contribute to the induction of
angiogenesis. The ability of VEGF neutralizing antibody to only
partially block CEP-HSA induced angiogenesis in vivo and the lack
of increase in VEGF secretion in RPE cells exposed to CEP-modified
dipeptide or CEP-HSA, indicates the utilization of additional VEGF
independent pathways.
[0076] All publications and patent documents cited in this
disclosure are incorporated by reference in their entirety. The
citation of any references herein is not an admission that such
references are prior art to the present invention.
[0077] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *