Controlling IOP in glaucoma patients, whether or
not they have ocular hypertension, is important because
results from several clinical trials demonstrate that
significant, sustained intraocular pressure reduction
in people with glaucoma slows or halts vision loss
due to damage/death of retinal ganglion cells in the
retina. The finding of new and more effective way
to control IOP (and avoid laser treatment or surgery)
is an important step for treatment of many glaucoma
patients who no longer respond to available drugs.
Therefore, the patent application by Drs. Stamer and
Heimark represents a novel targeted therapy that has
not been identified in the ophthalmology literature
until their recent publication (Heimark, Kaocher &
Stamer 2002).
Interestingly, available pharmaceuticals that target
the diseased tissuethe conventional drainage pathway,
are not used to control IOP in the long term
due to unpleasant side effects. This is unfortunate
because the conventional pathway is the pressure-sensitive
pathway and responsible for 70-90% of total drainage
of aqueous humor. Instead, first-line treatments
utilize pharmaceutical agents that lower IOP by
targeting aqueous secretion and/or uveoscleral outflow;
ocular functions not directly responsible
for elevated IOP in glaucoma. In fact, treatment
with such current modalities decrease perfusion
of conventional outflow tissues (either by decreasing
pressure gradient that drives flow through the conventional
pathway or by shunting aqueous humor away) and are
thought to accelerate pathological processes. Thus,
developing a drug (such as the one proposed in our
current patent application) that targets the diseased
tissue in POAG, the conventional outflow pathway,
is critical for better long term outcomes.
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The conventional outflow tissue is unique
in its architecture and composed of the
trabecular meshwork and Schlemm’s
canal. Structurally, the trabecular meshwork
is a latticework of extracellular matrix
biopolymers that are covered by trabecular
endothelial-like cells and are situated
at the iridocorneal angle of the anterior
chamber of the eye (the angle formed by
the iris and cornea).
The cells of the trabecular meshwork maintain
the configuration of the trabecular lamellae,
the turnover of ground substances, and the
patency of the trabecular passages through
which aqueous humor travels (figure, arrows)
into Schlemm’s canal, a continuous
endothelial-lined channel that drains aqueous
humor from the eye interior into the general
venous circulation.
Thus, natural flow patterns will direct
therapeutics to Schlemm’s canal. The
walls of Schlemm’s canal provide the
only continuous cellular barrier for
intraocular fluid on its way out of the
eye. Cell-cell junctional complexes
between Schlemm’s canal cells maintain
the barrier and regulate the paracellular
movement of intraocular fluid. The patent
by Stamer and Heimark is the first to propose
the modulation of this cellular barrier
as a means to control IOP.
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Interestingly, while Schlemm’s canal endothelium
is morphologically similar to non-fenestrated tight
endothelium, the direction of fluid movement
across the endothelial cells that form Schlemm’s
canal is unique, in the basal to apical direction
(opposite of vascular endothelium). Thus, the control
of fluid flow across this unique endothelial cells
layer provides a novel drug target for therapeutic
development. In contrast, modulation of a similar
target in vascular endothelium is not physiologically
beneficial because it results in the leakage of
blood into the interstitial space. In these experiments
using intravenous administration of anti-cadherin-5
IgG, vasculature in the eye (including Schlemm’s
canal) was not affected because of ocular immune
privilege and because of the relationship of Schlemm’s
canal with venous drainage of the eye. A recent
review article by our group uncovers the unique
biology of Schlemm’s canal endothelia compared
to other endothelia (Ramos, Hoying, Witte and Stamer,
2007).
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New Preliminary Data in Supports VE-cadherin
as a Novel Drug Target for Glaucoma and
Addressed Concerns of Previous Patent Review
Human SC endothelial cells express VE-cadherin
as well as other endothelial markers. Since
intraocular pressure is related to the resistance
of the movement of aqueous humor across
Schlemm’s Canal, we began studies
to characterize the cell-cell junctions
of cells that form the inner wall of Schlemm’s
canal.
Previously we had shown in fresh frozen
sections of the human outflow pathway that
Schlemm’s canal endothelial cells
expressed PECAM1 and VE-cadherin, while
cells of the trabecular meshwork did not.
Further characterization here (figure 1)
shows that Human Schlemm’s Canal endothelial
cells isolated from non-glaucomatous cadaveric
eyes (SC42, SC44) express adherens junction
proteins: VE-cadherin, N-cadherin, β-catenin
and p120-catenin, similar to endothelial
cell controls (HMEC1). Messenger RNA corresponding
to the tight junction proteins: junctional
adhesion proteins 1, 2 and 3 (JAM1, JAM2,
JAM3), ZO-1, occludin and claudin 4 and
claudin 5 were examined.
Claudin 5 was limited to vascular endothelial
cells and SC cells, while claudin 4 was
expressed in all of the cell types. These
studies, shown in figure 1, confirm that
VE-cadherin is only expressed in Schelmm’s
canal endothelial cells and not in Trabecular
meshwork cells. Overall, the profile of
intercellular junction genes in Schlemm’s
canal is very similar to that of vascular
endothelial cells.
While Schlemm’s canal endothelial
(SCE) cells express many of the same proteins
as endothelial cells, the permeability properties
of Schlemm’s canal endothelial cells
compared to vascular endothelial cells are
quite different. In fact recent studies
by our laboratory show that SCE cells cultured
as monolayers on filters form a much less
permeable barrier than two different types
of endothelial cells (HUVECs and HMEC1)
treated in the identical fashion (figure
2).
While both vascular endothelial cell types
show a similar permeability, SCE cells were
60-90% less permeable. These data suggest
that SCE cells utilize the same protein
machinery to generate a tighter barrier
than other endothelial cells.
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The
intercellular junctions of Schlemm’s
canal endothelial cells have previously been
shown to be calcium sensitive by Dr Stamer’s
laboratory (Burke et al., 2004).
Thus, modulation of calcium dependent adhesion
molecules, such as the cadherins by neutralizing
antibodies or peptide antagonists constitutes
a novel approach to glaucoma therapy (the
focus of this patent).
Proof of concept for this idea was provided
when elevation in intraocular pressure were
shown to decrease the complexity of tight
junctional strands between cells of the inner
wall of Schlemm’s Canal (Ye et al, 1997)
and open spaces between the cells (Epstein
and Rohen, 1989).
The idea that complexity of cell-cell junctions
correlate with permeability has been shown
in ultrastructural analysis comparing arteries
and veins (Simonescu et al., 1976) and known
permeability of different vascular beds (Claude,
1973). |
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We conducted experiments to determine the
functionality of recombinant human VE-cadherin
utilizing the Biacore system to analyze
protein-protein interactions (screen for
potential interacting proteins). Recombinant
VE-Cadherin (extracellular “binding”
domain) cis-dimers were immobilized to the
surface of a CM5 sensor chip using a standard
amine chemistry protocol. Protein was diluted
to 10 mg/ml in 10mM Na-Acetate buffer and
100 ml was consumed in this process; creating
a surface density of 15,000 resonance units
(RU) of material. As a positive control
for the assay, we tested a monoclonal antibody
raised against the extracellular domain
of VE-cadherin (9H7) (Heimark and Hazelton,
1997). The antibody was prepared in the
two different buffers (PBS containing calcium
and PBS minus calcium) at a concentration
of 67 µM. These two buffer conditions
were used because anti-VE cadherin (9H7)
was previously characterized by its enhanced
binding activity in the absence of calcium.
Upon injection (arrow) into the Biacore
T100, anti-VE cadherin (9H7) was observed
to specifically interact with the cadherin
surface (Figure 3); showing a binding preference
in the absence of calcium.
Preliminary kinetic analysis of anti-VE
cadherin (9H7) binding was conducted. Typically
kinetic analysis is performed using a minimum
of 4 concentrations; however a single injection
can yield an estimate of the kinetics and
affinity of an interaction. Table 1 lists
the binding parameters for the 9H7 monoclonal
antibody to the extracellular domain of
VE-cadherin in calcium and calcium free
buffer.
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TABLE
1: anti-VE
cadherin 9H7
binding to Cadherin chimera surface
| Buffer
|
Kon
(1/mS) |
Koff
(1/S) |
KD
(nM) |
| Calcium |
4.32E3 |
2.684E-4 |
620 |
| Calcium
free |
4.61E3 |
3.365E-4 |
729 |
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To
develop peptides that could be used to compete
with VE-cadherin homotypic adhesion and increase
permeability across Schlemm’s canal
in vivo, we designed a series of peptides
that correspond to amino acids in extracellular
domains one and two of human VE-cadherin and
tested them in the Bicaore system with recombinant
VE-cadherin immobilized to the sensor (similar
to use with antibody). The binding of one
VE-cadherin peptide #4748 (Ac-HLTAVIVDKDTGEN-OH)
is shown in Figure 4. The peptide contains
amino acids 119 to 132 of human VE-cadherin.
There is a dose-related binding of 4748 in
the absence of calcium to recombinant extracellular
domain of VE-cadherin.
There is a significant alteration of the sensogram
at 25 and 50 mM. The negative deflection of
the sensogram in the presence of calcium suggests
that dissociation of VE-cadherin trans-dimers
had occurred.
Since VE-cadherin
peptide 4748 showed the ability to bind to
VE-cadherin, we tested its ability to increase
permeability of SC endothelial cells grown
in monolayer. VE-4748 was added to the luminal
surface of the monolayer at a concentration
of 100 mM and permeability was determined
by analysis of the rate of passage of horseradish
peroxidase from the upper luminal chamber
to the bottom chamber at 37oC (the same as
shown in figure 2).
Significantly, addition of 4748 to SCE monolayers
increased permeability to the level observed
for other endothelial monolayers. These effects
were interpreted as being specific because
other peptides failed to increase permeability
like 4748. Such results are consistent with
observations by Huang (Huang, 2000) stating
that “the prospects (of peptide regulation
of protein-protein interactions) appear bright
and that they represent new paradigms of drug
design and discovery”.
Taken together,
our new data emphasize the novelty and feasibility
of VE-cadherin as a target for the development
of therapeutics that will increase the permeability
of the conventional outflow pathway (decrease
intraocular pressure) in people with glaucoma.
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