Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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OCULAR GROWTH AND NICOTINIC ANTAGONISTS
Field of the Invention
The present invention relates to the control of eye growth by nicotinic
receptor
antagonists, more particularly to the inhibition of postnatal ocular growth
and the
prevention of myopia in a host animal by ocular administration of nicotinic
receptor
antagonists.
Background of the Invention
Visual input dominates the regulation of post-natal eye growth and the
development
of refractive errors. Eye growth appears largely controlled locally in the
eye, likely through
the retina; specific roles for other components of the nervous system, such as
the brain or
peripheral nervous system, remain unclear (Stone, 1997, Myopia Updates:
Proceedings of
the 6th International Conference on Myopia, pp. 241-254; Wallman, 1993,
Progress in
Retinal Research, 12:133-153). As complex qualities of the visual image such
as blur
influence eye growth, it seems reasonable that neurons in the proximal retina
might
comprise the elements of a local regulatory mechanism. Indeed, much current
evidence
implicates several classes of retinal amacrine cells in the pathway linking
visual input and
eye growth control. The data most strongly supports a role for dopaminergic
amacrine cells
(Stone, 1997, Myopia Updates: Proceedings of the 6th International Conference
on
Myopia, pp. 241-254; Stone et al., 1989, Proc. Natl. Acad. Sci. U.S.A., 86:704-
6). While
evidence implicating other retinal neurons is either less fully developed or
controversial,
other subtypes of retinal amacrine cells hypothesized to influence refractive
development
include those containing vasoactive intestinal peptide (Pickett Seltner and
Stell, 1995,
Vision Res., 35:1265-1270; Stone et al., 1988, Proc. Natl. Acad. Sci. U.S.A.,
85:257-60),
glucagon (Fischer et al., 1999a, Nature Neuroscience, 2:706-12), nitric oxide
(Fujikado et
al., 1997, Curr. Eye Res., 16:992-6), enkephalin (Pickett Seltner et al.,
1997, Vis.
Neurosci., 14:801-809) and acetylcholine (Stone et al., 1991, Exp. Eye Res.,
52:755-8).
Cholinergic mechanisms, acting through muscarinic receptors, seem involved in
eye
growth control because the muscarinic antagonist atropine retards the
development of
myopia in chick (Stone et al., 1991, Exp. Eye Res., 52:755-8), tree shrew
(McKanna and
Casagrande, 1981, Documenta Ophthalmologica Proceedings Series, 28:187-192),
monkey
(Raviola and Wiesel, 1985, N. Engl. J. Med., 312:1609-15; Tigges et al., 1999,
Optometry
& Vision Science, 76:397-407) and human (Brodstein et al., 1984, Ophthalmol.,
91:1373-
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1379). Identifying the specific cholinergic neurons responsible for the
regulation of eye
growth, however, has proved difficult.
Because of the clinical association of myopia development with near work, it
has
long been hypothesized that accommodation underlies the mechanism causing
myopia and
that cycloplegia explains the anti-myopia activity of atropine, thus
suggesting a role for the
cholinergic neurons of the ciliary ganglion and the muscarinic receptors of
the ciliary
muscle. However, little experimental work supports a role for accommodation in
the
development of myopia despite many attractive features of the hypothesis.
Ciliary
ganglionectomy (Lin et al., 1996, Curr. Eye Res., 15:453-60; Raviola and
Wiesel, 1985, N.
Engl. J. Med., 312:1609-15) sectioning of the ciliary nerves (Shih et al.,
1994, Invest.
Ophthalmol. Yis. Sci., 35:3691-701) or lesioning the pre-ganglionic input to
the ciliary
ganglion at the Edinger-Westphal nucleus (Troilo, 1990, Ciba Foundation
Symposium,
155:89-102; discussion 102-14) each fail to have a major impact on the
development of
experimental myopia. Pharmacologic evidence also argues against a role for
accommodation in myopia development. An M1-selective muscarinic antagonist
with
minimal cycloplegic activity is at least as effective as atropine against
experimental myopia
in the rhesus monkey (Tigges et al., 1999, Optometry & Vision Science, 76:397-
407).
Further, it has been observed that the chick ciliary body contains striated
rather than smooth
muscle, that avian accommodation is controlled by nicotinic rather than
muscarinic
mechanisms, and that atropine fails to paralyze accommodation in the chick
(Stone et al.,
1991, Exp. Eye Res., 52:755-8). That atropine blocks myopia in the chick
further argues
against an accommodation mechanism (Stone, 1997, Myopia Updates: Proceedings
of the
6th International Conference on Myopia, pp. 241-254).
Alternative cholinergic mechanisms have been sought to explain refractive
development. Retinal cholinergic neurons and muscarinic receptors have been
proposed,
based on the activity of an M1-selective, but not M3-selective, muscarinic
subtype receptor
antagonists in chick myopia (Stone et al., 1991, Exp. Eye Res., 52:755-8).
This notion is
also supported by the anti-myopia activity of an M1-selective muscarinic
antagonist in tree
shrew (Cottriall and McBrien, 1996, Invest. Ophthalmol. Vis. Sci., 37:1368-79)
and rhesus
monkey (Tigges et al., 1999, Optometry & Vision Science, 76:397-407). However,
in
studies carned out to identify the cholinergic neurons involved, it was found
that the
activity of the biosynthetic enzyme for acetylcholine (choline
acetyltransferase) was
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unaltered in the retina of myopic chick eyes but depressed in the ciliary
ganglion (Pendrak
et al., 1995, Exp. Eye Res., 60:237-43). Based upon responses of cultured
chick scleral
cells (Lind et al., 1998, Invest. Ophthalmol. Vis. Sci., 39:2217-2231) and the
responses of
the chick eye to retinal toxins that lesion cholinergic amacrine cells
(Fischer et al., 1998b,
Brain Res., 794:48-60), it has been suggested that muscarinic antagonists
might inhibit
myopia development by acting at extra-retinal sites such as sclera or choroid.
Further limiting our understanding the role of cholinergic neurons in eye
growth
control is the paucity of studies addressing cholinergic nicotinic mechanisms.
Both
intravitreal and subconjunctival nicotine induce accommodation in chicks
(Refiner et al.,
1995, Vision Res., 35:1227-1245). Twice daily intravitreal injections of
nicotine for two
weeks induce in chicks about a 2 diopter myopic shift in refraction compared
to
contralateral non-injected eyes; however, intravitreal saline injection had
the same effect
(Refiner et al., 1995, Vision Res., 35:1227-1245). Daily subconjunctival
nicotine injections
in chicks did cause a slight myopic refractive shift of 0.75 diopters compared
to non-treated
eyes, a response not seen for subconjunctival saline (Refiner et al., 1995,
Vision Res.,
35:1227-1245); but this degree of refractive shift in chicks may be of little
biological
significance because it approximates the focal depth of the chick eye (Schmid
and
Wildsoet, 1997, Ophthal. Physiol. Opt., 17:61-7). Nicotine's high
lipophilicity would
permit rapid diffusion from the eye, such that potential action at extra-
ocular sites further
limits mechanistic interpretation of these results. When vecuronium bromide, a
neuromuscular blocking agent and nicotinic antagonist, was applied to chick
corneas, it
paralyzed accommodation but failed to influence the ocular elongation
following spectacle-
induced hyperopic defocus, again arguing against an accommodative mechanism
for
myopia (Schwahn and Schaeffel, 1994, Invest. Ophthalmol. Vis. Sci., 35:3516-
24).
Charged antagonists at the neuromuscular junction, of which d-tubocurarine is
a prototype,
typically penetrate poorly into the central nervous system and bind to all
nicotinic receptor
subtypes with low affinity (Gotti et al., 1997, Progress in Neurobiology,
53:199-237).
Vecuronium bromide is also highly charged; thus, while it diffuses readily to
block the
neuromuscular junctions of intraocular muscles, it may not have access to
receptor sites in
lipophilic tissues potentially involved in eye growth control, such as the
neural retina.
Besides the nicotinic acetylcholine receptors at the neuromuscular junctions
of the
intraocular muscles, the chick eye also has well-characterized nicotinic
receptor subtypes in
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both retina (Hamassaki-Britto et al., 1994a, Vis. Neurosci., 11:63-70;
Hamassaki-Britto et
al., 1994b, J. Comp. Neurol., 347:161-170; Keyser et al., 1993, J. Neurosci.,
13:442-454;
Vailate et al., 1999, Mol. Pharmacol., 56:11-19) and ciliary ganglion (Berg et
al., 1998,
Neuronal Nicotinic Receptors: Pharmacology and Therapeutic Opportunities, pp.
187-
196; Conroy and Berg, 1995, J. Biol. Chem., 270:4424-4431; Halvorsen and Berg,
1990, J.
Neurosci., 10:1711-1718; Horch and Sargent, 1995, J. Neurosci., 15:7778-7795;
Pugh et
al., 1995, Mol. Pharmacol., 47:717-725).
Summary of the Invention
The invention concerns the use of nicotinic antagonists of suitable solubility
to penetrate to the relevant targets that regulate postnatal eye growth and
that inhibit
postnatal ocular growth and the development of myopia.
The invention provides a method of controlling postnatal ocular growth by
ocular
administration of therapeutically effective amounts of a nicotinic antagonist
to control
postnatal ocular growth. Further provided is a method of inhibiting the
abnormal postnatal
axial growth of the eye of a host animal by administering therapeutically
effective amounts
of a nicotinic antagonist during postnatal development. The invention also
provides a
method of inhibiting abnormal equatorial expansion of the eye of a host animal
by
administering therapeutically effective amounts of a nicotinic antagonist
during postnatal
development. The invention further provides a method of inhibiting the
abnormal vitreous
cavity expansion of the eye of a host animal by administering therapeutically
effective
amounts of a nicotinic antagonist during postnatal development. Another aspect
of the
invention provides a method of preventing or inhibiting development of myopia
by ocular
administration of therapeutically effective amounts of a nicotinic antagonist.
The invention provides for the use of a nicotinic antagonist for the
preparation of a
medicament adapted for ocular administration for the control of postnatal
ocular growth.
The invention further provides for the use of a nicotinic antagonist for the
preparation of a
medicament for uses such as inhibiting the abnormal axial growth of the eye of
a host
animal during postnatal development, inhibiting the abnormal equatorial
expansion of the
eye of a host animal during postnatal development. inhibiting the abnormal
vitreous cavity
expansion of the eye of a host animal during postnatal development. The
invention also
provides for the use of a nicotinic antagonist for the preparation of a
medicament adapted
for ocular administration for the prevention or treatment of myopia.
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In one embodiment of the invention, the nicotinic antagonist may be a
competitive
nicotinic antagonist such as methyllcaconitine or dihydro-(3-erythroidine. In
another
embodiment, the nicotinic antagonist may be a channel-blocking nicotinic
antagonist such
as chlorisondamine or mecamylamine. In a further embodiment of the invention,
the
nicotinic antagonist may be a noncompetitive nicotinic antagonist such as
sertraline,
paroxetine, nefaxodone, venlafaxine, fluoxetine, buproprion, phencyclidine,
and ibogaine.
In another embodiment of the invention, the nicotinic antagonist may be an
antibody
inhibiting nicotinic receptor function. In yet another embodiment of the
invention, the
nicotinic antagonist may be an agonist that acts like a nicotinic antagonist.
The invention provides method of detecting the ability of a nicotinic
antagonist to
control postnatal ocular growth of the eye of a host animal by contacting an
animal eye
with a therapeutically effective amount of a nicotinic antagonist, detecting
the change in
growth of the eye exposed to a therapeutically effective amount of a nicotinic
antagonist,
then applying a known control agent in a second eye, observing the results of
the control
agent on the change in growth of the second eye, and comparing the change in
growth of
the first eye exposed to a therapeutically effective amount of a nicotinic
antagonist with the
change in growth of the second eye exposed to a known control agent, thereby
identifying
the nicotinic antagonist as having the ability to control postnatal ocular
growth. Further
provided is a method of making a pharmaceutical including the steps of
identifying a
nicotinic antagonist as an active agent having the ability to control
postnatal ocular growth
and combining the active agent in admixture with a pharmaceutical excipient.
The invention provides a method of identifying compounds that can be used to
modulate myopia including the steps of incubating a cell that expresses a
nicotinic receptor
in the presence and absence of a test compound, determining whether the test
compound
binds to at least one nicotinic receptor, selecting a test compound that binds
to at least one
nicotinic receptor, administering the selected test compound to a test animal,
determining
whether the test compound alters the development of myopia of the test animal,
and
selecting a compound that alters the development of myopia of a test animal.
Brief Description of the Drawings
Figure 1 shows drug effects on refractions of goggled eyes. Chlorisondamine
(CHL; P < 0.001 ), mecamylamine (MEC; P < 0.001 ) and methyllycaconitine (MLA;
P =
0.04) each influenced the myopic refraction occurring beneath a goggle, as
assessed by
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ANOVA; but dihydro-(3-erythroidine (DHBE) had no effect on the refraction of
visually
deprived eyes. For the results of pairwise comparisons by the Tukey test, see
Table 1. The
number of chicks in each experimental group is indicated here. Data are shown
as the
difference between the goggled and contralateral open eye (mean t S.E.M.). To
facilitate
comparisons, the bar for each control group is cross-hatched.
Figure 2, A-D, shows drug effects on ocular dimensions of goggled eyes. Figure
2A shows drug effects on axial length measured by ultrasound. Figure 2B shows
drug
effects on vitreous~cavity length measured by ultrasound. Figure 2C shows drug
effects on
axial length measured by digital calipers. Figure 2D shows drug effects on
equatorial
diameter measured by calipers. Chlorisondamine (CHL), mecamylamine (MEC) and
methyllycaconitine (MLA) each reduced the excessive ocular growth occurring
beneath a
goggle; the statistically significant effects (P < 0.05) and suggestive trends
are identified by
the ANOVA results directly on each data set. The effects of dihydro-(3-
erythroidine
(DHBE) were weaker as only the axial measurements by calipers showed a
statistically
significant drug effect. For the results of pairwise comparisons by the Tukey
test, see Table
1. The number of chicks in each experimental group is provided in Figure 1.
Data are
shown as the difference between the goggled and contralateral open eye (mean ~
S.E.M.).
To facilitate comparisons, the bar for each control group is cross-hatched.
Figure 3 shows effects of chlorisondamine on non-goggled eyes. Unilateral
administration of chlorisondamine to eyes of never-goggled chicks shifted
overall
refraction towards hyperopia (ANOVA on ranks: P = 0.004), reduced axial length
(ANOVA on ranks: ultrasound, P = 0.03; calipers, P = 0.03) and inhibited the
axial
expansion of the vitreous cavity (ANOVA on ranks: P = 0.01). For the results
of pairwise
comparisons by the Tukey test, see Table 1. N = 9-20 chicks per group. The
data are
illustrated as the difference between the drug treated and contralateral
vehicle treated eye
(mean ~ S.E.M.).
Table A presents a summary of nicotinic receptor subtypes.
Table 1 shows post hoc pairwise comparisons of drug effects using the Tukey
test
Table 2 shows the longer term effects of a single-dose of chlorisondamine
(200p.g)
on form deprivation myopia.
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Detailed Description of the Invention
The nervous system, in large part through the retina, controls eye growth
postnatally, and the development of refractive errors (the need for glasses)
appears to be
chiefly dependent on neural mechanisms. The most common refractive error
clinically is
myopia. The present disclosure includes a pharmaceutical drug class, nicotinic
antagonists,
with activity against an experimental model of myopia, but which in addition
also inhibits
"normal" eye growth.
In the present invention, the inventors tested the effects on eye chick growth
of
several nicotinic antagonists with favorable pharmacologic properties, using
the efficient
delivery route of intravitreal injection. Four nicotinic antagonists selected
were
chlorisondamine (CHL), mecamylamine (MEC), methyllcaconitine (MLA) and dihydro-
(3-
erythroidine (DHBE). Animal models using chicks offer advantages of good
optics as a
model for the human eye. Using antagonists with established profiles against
neuronal
nicotinic receptors and having lipophilic properties compatible with diffusion
into neural
tissue, we found evidence for an actual role, perhaps a central role, of
nicotinic receptors in
control of ocular growth.
The invention is directed to the use of nicotinic antagonists having suitable
solubility properties to penetrate to the relevant target sites that regulate
postnatal eye
growth and that inhibit postnatal ocular growth or the development of myopia.
All drug
classes previously identified for controlling eye growth show activity only
against the form
deprivation myopia model (eye remains covered by a goggle beginning at
approximately 1
week of age). The present invention identifies a drug class that is also
active against open
eyes (vision has not been deprived by goggles during maturation); and it is
the first class of
agents identified to show this activity. This activity against open eyes is
advantageous in
application to human myopia where form deprivation is not the usual
circumstance. While
chicks utilize nicotinic receptors in the control of pupil size and
accommodation,
mammalian eyes use a subclass of muscarinic receptors to control pupil size
and
accommodation; therefore, nicotinic antagonists are expected to be well
tolerated following
local application in the human eye, without inducing pupil dilation and
paralysis of
accommodation in children.
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Section 1. Nicotinic receptor subtypes
Nicotinic acetylcholine receptors subtypes are composed of five homologous
subunits that form an acetylcholine-gated cation channel (Lindstrom, 1997,
Mol.
Neurobiology, 15:193-222). There are 17 known nicotinic receptor subunits (al-
10, (31-4,
y, b, and s). Each subunit possesses four transmembrane domains. The
acetylcholine
binding site is formed by at least three peptide loops on the a-subunit
(principal
component), and two on the adjacent subunit (complementary component). All a-
subunits
possess two tandem cysteine residues near to the site involved in
acetylcholine binding, and
subunits not named a lack those tandem cysteines. Alternatively spliced forms
of the a4
subunit (a4-1 and a4-2; rat) and al subunit (human) have been identified.
(Mandelzys, A.
et al. (1995) J. Neurophysiol. 74, 1212-1221; Papke, R.L. et al. (1996)
Neurosci. Lett. 213,
201-204; Albuquerque, E.X. et al. (1997) J. Pharmacol. Exp. Ther. 280, 1117-
1136;
deFiebre, C.M. et al. (1995) Mol. Pharmacol. 47, 164-171)
The receptors fall into three general classes: a muscle class and two neural
classes.
The muscle types exist in only two forms - a fetal and an adult form, each
with a 1 subunits
and other subunits specific for muscle receptors. One class of neuronal
receptors binds a-
bungarotoxin and is composed of a7, a8, a9 or a10 subunits, often as homomeric
receptors.
The other class of neural receptors does not bind a-bungarotoxin and is formed
from
combining a2, a3, a4 or a6 subunits with (32 or (34 subunits. Rapid
desensitization and
limited availability of selective drugs suited for in vivo studies have
impaired defining
physiologic functions for these biochemically defined receptor subtypes. A
summary of
nicotinic receptor subtypes is presented in Table A.
The nicotinic receptor subcommittee of NC-IUPHAR has recommended a
nomemclature and classification scheme for nicotinic acetylcholine (nACh)
receptors based
on the subunit composition of known, naturally-expressed nACh receptor
subtypes and/or
on subtypes formed by heterologous expression (Lukas et al. (1999) IIJPHAR XX.
Current
status of the nomenclature for nicotinic acetylcholine receptors and their
subunits. Pharm.
Rev. 51, 397-401). Headings for Table A reflect abbreviations designating nACh
receptor
subtypes based on the predominant a subunit contained in that receptor
subtype. An
asterisk following the indicated a subunit means that other subunits are known
to or might
assemble with the indicated a subunit to form the designated nACh receptor
subtype(s).
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The absence of an asterisk indicates that the indicated subunit is known to
assemble into a
homomeric nACh receptor subtype. Where subunit stoichiometries are known,
numbers of
a particular subunit in a specific nACh receptor subtype are indicated by a
subscript
following the subunit in brackets. All subunits are of mammalian origin with
the exception
of a8 (avian).
Section 1.1. Nicotinic receptor subtypes and the eye.
The chick retina contains several classes of cholinergic neurons (Miller et
al., 1987,
Neurosci., 21:725-743). Besides several subtypes of muscarinic acetylcholine
receptors
(Fischer et al., 1998a, J. Comp. Neurol., 392:273-84), the chick retina
expresses a
multiplicity of nicotinic acetylcholine receptor subunits, including a3, a6,
a7, a8 and ~2,
(33, (34. The cellular patterns of neural localization of nicotinic receptors
in chick retina are
complex (Hamassaki-Britto et al., 1994a, Vis. Neurosci., 11:63-70; Hamassaki-
Britto et
al., 1994b, J. Comp. Neurol., 347:161-170; Keyser et al., 1993, J. Neurosci.,
13:442-454;
Vailate et al., 1999, Mol. Pharmacol., 56:11-19). The chick ciliary ganglion
is similarly
enriched with a diversity of nicotinic receptor subtypes that include the a3
subunits that
typifies autonomic ganglia, a5, a7, (32 or (34 subunits, with both synaptic
and extrasynaptic
localizations (Berg et al., 1998, Neuronal Nicotinic Receptors: Pharmacology
and
Therapeutic Opportunities, pp. 187-196; Conroy and Berg, 1995, J. Biol. Chem.,
270:4424
4431; Horch and Sargent, 1995, J. Neurosci., 15:7778-7795; Pugh et al., 1995,
Mol.
Pharmacol., 47:717-725).
Section 2. Nicotinic anta og nists.
The function of nicotinic acetycholine receptors (nicotinic receptors, or NR)
can be
antagonized by various compounds. The action of these compounds against NR may
be
complex, may involve more than one mechanism of antagonism, and may not yet
have been
fully characterized in all details. Specific drugs for discussion purposes are
classified in
terms of their currently best understood mechanism of action. Nicotinic
antagonists are
defined as compounds that inhibit, block, compete, prevent, or otherwise
interfere with any
effect of a nicotinic agonist on a target. We claim use of competitive
nicotinic antagonists
in this invention. Competitive nicotinic antagonists are defined as compounds
that appear
to compete for the agonist binding sites on nicotinic receptors, where
competitive
antagonists appear to inhibit receptor function by preventing activation of
the receptor by
agonists. Examples of competitive antagonists may include, but are not limited
to, dihydro-
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(3-erythroidine, bungarotoxins, tubocurarine, methyllcaconitine, the peptide
conotoxins
derived from snails, including MI, EI, GI, SI, SIA, SII, as well as other
naturally occurring
peptide antagonists and synthetic peptide antagonists derived from expression
libraries
(Lindstrom, 1997, Mol. Neurobiology, 15:193-222).
We claim use of channel-blocking nicotinic antagonists in this invention.
Channel-
blocking nicotinic antagonists are defined as compounds that appear to block
the ion
channel of nicotinic receptors (NR), thereby preventing the transmembrane ion
flux
required for nicotinic receptor function. Examples of channel-blocking
nicotinic
antagonists may include, but are not limited to, chlorisondamine,
mecamylamine,
hexamethonium, amantadine, memantine, dizocilpine [(+)-MK-801], 8
(dethylamino)octyl-
3,4,5-trimethoxybenzoate (TMB-8), and zinc. (Bencherif et al., 1995, J.
Pharmacol. and
Exper. Therapeutics, 275: 1418-1426; Buisson and Bertrand, 1998, Mol.
Pharmacol., 53:
555-563). While these compounds appear to act preferentially on open channels
(Peng et
al., 1997, Mol. Pharmacol., 51: 776-784; Buisson and Bertrand, 1998, Mol.
Pharmacol.,
53: 555-563), compounds that block closed channels are also suitable for use
in this
invention.
We claim use of noncompetitive nicotinic antagonists in this invention.
Noncompetitive nicotinic antagonists are defined as compounds that antagonize
the
functions of nicotinic receptors (NR), but do not appear to block the ligand
binding site or
directly block the ion channel. Functional blockade by a noncompetitive
nicotinic
antagonist of ion flux through the ion channel of nicotinic receptors is
insurmountable by
increasing agonist concentration (Fryer and Lukas, 1999a, J. Pharmacol. and
Exper.
Therapeutics, 288: 88-92; Fryer and Lukas, 1999b, J. Neurochem., 72: 1117-
1124).
Ethanol and volatile anesthetics including tetracaine and procaine are
noncompetitive
functional nicotinic antagonists for diverse nicotinic receptor subtypes
(Bencherif et al.,
1995, J. Pharmacol. and Exper. Therapeutics, 275: 1418-1426; Lindstrom, 1997,
Mol.
Neurobiology, 15:193-222). Unexpected noncompetitive nicotinic antagonists
include
psychoactive compounds such as buproprion, phencyclidine, ibogaine,
sertraline,
paroxetine, nefaxodone, venlafaxine, and fluoxetine (Fryer and Lukas, 1999a,
J.
Pharmacol. and Exper. Therapeutics, 288: 88-92; Fryer and Lukas, 1999b, J.
Neurochem.,
72: 1117-1124). Other noncompetitive nicotinic antagonists may act as negative
allosteric
effectors, acting via allosteric sites used by known postive allosteric
effectors such as
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ivermectin, or acting on distinct sites on the receptor (Krause et al., 1998,
Mol. Pharmacol.,
53: 283-294). Certain voltage-dependent mechanisms can also function as
noncompetitive
antagonists, including voltage-sensitive MgZ+ block of nicotinic receptor ion
channels,
voltage-dependent channel blockage by intracellular spermine, and nicotinic
receptor
inactivation triggered by membrane depolarization.
We claim use of antibodies that act as nicotinic antagonists in this
invention.
Antibodies can also act as nicotinic antagonists; for example, the monoclonal
antibody
mAb 319 blocks the function of nicotinic receptors (NR) when injected into
cells (Cuevas
and Berg, 1998, J Neurosci. 18: 10335-10344). Antibodies include polyclonal
antibodies,
monoclonal antibodies, humanized or chimerized antibodies, single chain
antibodies, FAb
fragments F(Ab)'2 fragments, fragments produced by a FAb expression library,
anti-
idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the
above.
We claim use of agonists that function as nicotinic antagonists in this
invention.
Agonists can also function as nicotinic antagonists under certain
circumstances, for
example, based on their time-averaged antagonist effects. Reversible
desensitization is
observed following stimulation by all agonists including, but not limited to,
acetylcholine,
nicotine, epibatidine, cytisine, methylcarbamylcholine, and DMPP. Some
compounds have
a bifunctional effect, acting as an agonist for some nicotinic receptor
subtypes and as an
antagonist for others. The heterocyclic substituted pyridine derivative (+/-)-
2-(-3-
pyridinyl)-1-azabicyclo [2.2.2]octane, also known as RJR-2429, selectively
activates
human muscle nicotinic receptors and a putative x,3(34-containing receptor,
but inhibits
nicotinic receptors in preparations of rat thalamus. This compound is a
partial agonist on
nicotinic receptors mediating dopamine release from rat synaptosomal
preparations.
(Bencherif et al., 1998, J. Pharmacol. and Exper. Therapeutics, 284: 886-894).
At
sufficiently high concentrations, any agonist can cause reversible and
irreversible
desensitization and/or inactivation of nicotinic receptors. Exposure to high
agonist
concentrations thus give rise to time-averaged antagonist effects on the
exposed cell. By
way of example, this phenomenon is observed following chronic nicotine
exposure at
concentrations comparable to circulating nicotine levels found in human
tobacco smokers,
and can lead to inactivation of certain nicotinic receptor subtypes
(Lindstrom, 1997, Mol.
Neurobiology, 15:193-222).
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Section 3. Screenin assays for compounds that modulate nicotinic receptor
activity
The following assays are designed to identify compounds that interact with
nicotinic
receptors (NR) that control postnatal eye growth, compounds that interfere
with NR, and
compounds which modulate the activity of NR genes or modulate the levels of
NR. Assays
may additionally be utilized which identify compounds which bind to NR and
which may
modulate NR levels.
The compounds which may be screened in accordance with the invention include,
but are not limited to peptides, antibodies and fragments thereof, and other
organic
compounds (e.g., peptidomimetics) that bind to NR and either mimic the
activity triggered
by the natural ligand (i.e., agonists) or inhibit the activity triggered by
the natural ligand
(i.e., antagonists), as well as peptides, antibodies or fragments thereof, and
other organic
compounds that mimic a domain of the NR (or a portion thereof) and bind to and
"neutralize" natural ligand.
Such compounds may include, but are not limited to, naturally occurring
peptides
such as conotoxins, synthetic peptides such as, for example, soluble peptides,
including but
not limited to members of random peptide libraries, (see, e.g., Lam et al.,
1991, Nature,
354:82-84; Houghten et al., 1991, Nature, 354:84-86), and combinatorial
chemistry-
derived molecular library made of D- and/or L- configuration amino acids,
phosphopeptides (including, but not limited to, members of random or partially
degenerate,
directed phosphopeptide libraries; (see, e.g., Songyang et al., 1993, Cell,
72:767-778),
antibodies (including, but not limited to, polyclonal, monoclonal, humanized,
anti
idiotypic, chimeric or single chain antibodies, and FAb, F(Ab')Z and FAb
expression library
fragments, and epitope-binding fragments thereof), and small organic or
inorganic
molecules.
Other compounds which can be screened in accordance with the invention include
but are not limited to small organic molecules that are able to cross the
blood-retinal or
blood-aqueous humor barrier, gain entry into an appropriate cell and affect
the expression
of the NR gene or some other gene involved in the NR signal transduction
pathway (e.g.,
by interacting with the regulatory region or transcription factors involved in
gene
expression); or such compounds that affect the activity of the NR or the
activity of some
other intracellular factor involved in the NR signal transduction pathway.
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Computer modelling and searching technologies permit identification of
compounds, or the improvement of already identified compounds, that can
modulate NR
activity. Having identified such a compound or composition, the active sites
or regions are
identified. Such active sites might typically be ligand binding sites, such as
the interaction
domains of ligands with NR itself. The active site can be identified using
methods known
in the art including, for example, from the amino acid sequences of peptides,
from the
nucleotide sequences of nucleic acids, or from study of complexes of the
relevant
compound or composition with its natural ligand. In the latter case, chemical
or X-ray
crystallographic methods can be used to find the active site by finding where
on the factor
the complexed ligand is found. Next, the three dimensional geometric structure
of the
active site is determined. This can be done by known methods, including X-ray
crystallography, which can determine a complete molecular structure. On the
other hand,
solid or liquid phase NMR can be used to determine certain intra-molecular
distances. Any
other experimental method of structure determination can be used to obtain
partial or
complete geometric structures. The geometric structures may be measured with a
complexed ligand, natural or artificial, which may increase the accuracy of
the active site
structure determined.
If an incomplete or insufficiently accurate structure is determined, the
methods of
computer based numerical modelling can be used to complete the structure or
improve its
accuracy. Any recognized modelling method may be used, including parameterized
models
specific to particular biopolymers such as proteins or nucleic acids,
molecular dynamics
models based on computing molecular motions, statistical mechanics models
based on
thermal ensembles, or combined models. For most types of models, standard
molecular
force fields, representing the forces between constituent atoms and groups,
are necessary,
and can be selected from force fields known in physical chemistry. (Bencherif
et al., 1998,
.l. Pharmacol. and Exper. Therapeutics, 284: 886-894) The incomplete or less
accurate
experimental structures can serve as constraints on the complete and more
accurate
structures computed by these modeling methods.
Finally, having determined the structure of the active site, either
experimentally, by
modeling, or by a combination, candidate modulating compounds can be
identified by
searching databases containing compounds along with information on their
molecular
structure. Such a search seeks compounds having structures that match the
determined
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active site structure and that interact with the groups defining the active
site. Such a search
can be manual, but is preferably computer assisted. These compounds found from
this
search are potential NR modulating compounds.
Alternatively, these methods can be used to identify improved modulating
compounds from an already known modulating compound or ligand. The composition
of
the known compound can be modified and the structural effects of modification
can be
determined using experimental and computer modelling methods such as those
described
above applied to the new composition. The altered structure is then compared
to the active
site structure of the compound to determine if an improved fit or interaction
results. In this
manner systematic variations in composition, such as by varying side groups,
can be
quickly evaluated to obtain modified modulating compounds or ligands of
improved
specificity or activity.
Further experimental and computer modeling methods useful to identify
modulating
compounds based upon identification of the active sites of NR, and related
transduction and
transcription factors will be apparent to those of skill in the art.
Examples of molecular modelling systems are the CHARMM and QUANTA
programs (Polygen Corporation, Waltham, Mass.). CHARMM performs the energy
minimization and molecular dynamics functions. QUANTA performs the
construction,
graphic modelling and analysis of molecular structure. QUANTA allows
interactive
construction, modification, visualization, and analysis of the behavior of
molecules with
each other.
A number of articles review computer modelling of drug interactions with
specific
proteins, such as Rotivinen et al., 1988, Acta Pharmaceutical Fennica 97:159-
166; Ripka,
New Scientist 54-57 (Jun. 16, 1988); McKinaly and Rossmann, 1989, Annu. Rev.
Pharmacol. Toxiciol. 29:111-122; Perry and Davies, OSAR: Quantitative
Structure-Activity
Relationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and
Dean,
1989, Proc. R. Soc. Lond. 236:125-140 and 141-162; and, with respect to a
model receptor
for nucleic acid components, Askew et al., 1989, J. Am. Chem. Soc. 111:1082-
1090. Other
computer programs that screen and graphically depict chemicals are available
from
companies such as BioDesign, Inc. (Pasadena, Calif.), Allelix, Inc.
(Mississauga, Ontario,
Canada), and Hypercube, Inc. (Cambridge, Ontario). Although these are
primarily
designed for application to drugs specific to particular proteins, they can be
adapted to
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design of drugs specific to regions of DNA or RNA encoding NR, once that
region is
identified.
Although described above with reference to design and generation of compounds
which could alter binding, one could also screen libraries of known compounds,
including
natural products or synthetic chemicals, and biologically active materials,
including
proteins, for compounds which are inhibitors or activators.
Compounds identified via assays such as those described herein may be useful,
for
example, in elaborating the biological function NR, and for ameliorating
myopia. Assays
for testing the effectiveness of compounds, identified by, for example,
techniques such as
those described in Section 3.1 through 3.3, are discussed, below, in Section
3.4.
Section 3.1. In Vitro Screening Assays for Compounds that Bind to NR
In vitro systems may be designed to identify compounds capable of interacting
with
(e.g., binding to) NR. Compounds identified may be useful, for example, in
modulating the
activity of wild type and/or mutant NR gene products; may be useful in
elaborating the
biological function of NR; may be utilized in screens for identifying
compounds that
disrupt normal NR interactions; or may in themselves disrupt such
interactions.
The principle of the assays used to identify compounds that bind to NR
involves
preparing a reaction mixture of the NR and the test compound under conditions
and for a
time sufficient to allow the two components to interact and bind, thus forming
a complex
which can be removed and/or detected in the reaction mixture. The NR species
used can
vary depending upon the goal of the screening assay. For example, where
agonists of the
natural ligand are sought, the full length NR, or a truncated NR, a peptide
corresponding to
the extracellular domain or a fusion protein containing the NR ligand binding
site fused to a
protein or polypeptide that affords advantages in the assay system (e.g.,
labeling, isolation
of the resulting complex, etc.) can be utilized. Where compounds that interact
with the
cytoplasmic domain (CD) are sought to be identified, peptides corresponding to
the NR CD
and fusion proteins containing the NR CD can be used.
The screening assays can be conducted in a variety of ways. For example, one
method to conduct such an assay would involve anchoring the NR protein,
polypeptide,
peptide or fusion protein or the test substance onto a solid phase and
detecting NR/test
compound complexes anchored on the solid phase at the end of the reaction. In
one
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embodiment of such a method, the NR reactant may be anchored onto a solid
surface, and
the test compound, which is not anchored, may be labeled, either directly or
indirectly.
In practice, microtiter plates may conveniently be utilized as the solid
phase. The
anchored component may be immobilized by non-covalent or covalent attachments.
Non
covalent attachment may be accomplished by simply coating the solid surface
with a
solution of the protein and drying. Alternatively, an immobilized antibody,
preferably a
monoclonal antibody, specific for the protein to be immobilized may be used to
anchor the
protein to the solid surface. The surfaces may be prepared in advance and
stored.
In order to conduct the assay, the nonimmobilized component is added to the
coated
surface containing the anchored component. After the reaction is complete,
unreacted
components are removed (e.g., by washing) under conditions such that any
complexes
formed will remain immobilized on the solid surface. The detection of
complexes anchored
on the solid surface can be accomplished in a number of ways. Where the
previously
nonimmobilized component is pre-labeled, the detection of label immobilized on
the
surface indicates that complexes were formed. Where the previously
nonimmobilized
component is not pre-labeled, an indirect label can be used to detect
complexes anchored on
the surface; e.g., using a labeled antibody specific for the previously
nonimmobilized
component (the antibody, in turn, may be directly labeled or indirectly
labeled with a
labeled anti-Ig antibody).
Alternatively, a reaction can be conducted in a liquid phase, the reaction
products
separated from unreacted components, and complexes detected; e.g., using an
immobilized
antibody specific for NR protein, polypeptide, peptide or fusion protein or
the test
compound to anchor any complexes formed in solution, and a labeled antibody
specific for
the other component of the possible complex to detect anchored complexes.
Alternatively, cell-based assays can be used to identify compounds that
interact with
NR. To this end, cell lines that express NR, or cell lines that have been
genetically
engineered to express NR (e.g., by transfection or transduction of NR DNA) can
be used.
Interaction of the test compound with, for example, the heterologous NR
expressed by the
host cell can be determined by comparison or competition with native ligands.
Section 3.2. Assavs for Intracellular Proteins that Interact with the NR
Any method suitable for detecting protein-protein interactions may be employed
for
identifying transmembrane proteins or intracellular proteins that interact
with NR. Among
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the traditional methods which may be employed are co-immunoprecipitation,
crosslinking
and co-purification through gradients or chromatographic columns of cell
lysates or
proteins obtained from cell lysates and the NR to identify proteins in the
lysate that interact.
with the NR. For these assays, the NR component used can be a full length NR,
a soluble
derivative lacking the membrane-anchoring region (e.g., a truncated NR in
which the
transmembrane region is deleted resulting in a truncated molecule containing
the
extracellular domain fused to the cellular domain), a peptide corresponding to
the cellular
domain or a fusion protein containing the cellular domain of NR. Once
isolated, such an
intracellular protein can be identified and can, in turn, be used, in
conjunction with standard
techniques, to identify proteins with which it interacts. For example, at
least a portion of
the amino acid sequence of an intracellular protein which interacts with the
NR can be
ascertained using techniques well known to those of skill in the art, such as
via the Edman
degradation technique. (See, e.g., Creighton, 1983, Proteins: Structures and
Molecular
Principles, pp.34-49). The amino acid sequence obtained may be used as a guide
for the
generation of oligonucleotide mixtures that can be used to screen for gene
sequences
encoding such intracellular proteins. Screening may be accomplished, for
example, by
standard hybridization or PCR techniques. Techniques for the generation of
oligonucleotide
mixtures and the screening are well-known. (See, e.g., PCR Protocols: A Guide
to Methods
and Applications, 1990).
Additionally, methods may be employed which result in the simultaneous
identification of genes which encode the transmembrane or intracellular
proteins interacting
with NR. These methods include, for example, probing expression libraries, in
a manner
similar to the well known technique of antibody probing of ~,gtl l libraries,
using labeled
NR protein, or an NR polypeptide, peptide~or fusion protein, e.g., an NR
polypeptide or NR
domain fused to a marker (e.g., an enzyme, fluor, luminescent protein, or
dye), or an IgG-
Fc domain.
One method which detects protein interactions in vivo, the two-hybrid system,
is
described in detail for illustration only and not by way of limitation. One
version of this
system has been described (Chien et al., 1991, Proc. Natl. Acad. Sci. USA,
88:9578-9582)
and is commercially available from Clontech (Palo Alto, Calif.).
Briefly, utilizing such a system, plasmids are constructed that encode two
hybrid
proteins: one plasmid consists of nucleotides encoding the DNA-binding domain
of a
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transcription activator protein fused to a nucleotide sequence encoding NR, an
NR
polypeptide, peptide or fusion protein, and the other plasmid consists of
nucleotides
encoding the transcription activator protein's activation domain fused to a
cDNA encoding
an unknown protein which has been recombined into this plasmid as part of a
cDNA
library. The DNA-binding domain fusion plasmid and the cDNA library are
transformed
into a strain of the yeast Saccharomyces cerevisiae that contains a reporter
gene (e.g., HBS
or lacZ) whose regulatory region contains the transcription activator's
binding site. Either
hybrid protein alone cannot activate transcription of the reporter gene: the
hybrid
containing the DNA-binding domain cannot activate transcription because it
does not
provide activation function, and the hybrid containing the activation domain
cannot because
it cannot localize to the activator's binding sites. Interaction of the two
hybrid proteins
reconstitutes the functional activator protein and results in expression of
the reporter gene,
which is detected by an assay for the reporter gene product.
The two-hybrid system or related methodology may be used to screen activation
domain libraries for proteins that interact with the "bait" gene product. By
way of example,
and not by way of limitation, NR may be used as the bait gene product. Total
genomic or
cDNA sequences are fused to the DNA encoding an activation domain. This
library and a
plasmid encoding a hybrid of a bait NR gene product fused to the DNA-binding
domain are
cotransformed into a yeast reporter strain, and the resulting transformants
are screened for
those that express the reporter gene. For example, and not by way of
limitation, a bait NR
gene sequence, such as the open reading frame can be cloned into a vector such
that it is
translationally fused to the DNA encoding the DNA-binding domain of the GAL4
protein.
These colonies are purified and the library plasmids responsible for reporter
gene
expression are isolated. DNA sequencing is then used to identify the proteins
encoded by
the library plasmids.
A cDNA library of the cell line used to detect proteins that interact with
bait NR
gene product, can be made using methods routinely practiced in the art.
According to the
particular system described herein, for example, the cDNA fragments can be
inserted into a
vector such that they are translationally fused to the transcriptional
activation domain of
GAL4. This library can be co-transformed along with the bait NR gene-GAL4
fusion
plasmid into a yeast strain which contains a lacZ gene driven by a promoter
which contains
GAL4 activation sequence. A cDNA encoded protein, fused to GAL4
transcriptional
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activation domain, that interacts with bait NR gene product will reconstitute
an active
GAL4 protein and thereby drive expression of the HIS3 gene. Colonies which
express
HIS3 can be detected by growth on petri dishes containing semi-solid agar
based media
lacking histidine. The cDNA can then be purified from these strains, and used
to produce
and isolate the protein that interacts with the bait NR gene product using
techniques
routinely practiced in the art.
Section 3.3. Assays for Compounds that Interfere with NR/Intracellular or
NR/Transmembrane Macromolecule Interaction
The macromolecules that interact with the NR are referred to, for purposes of
this
discussion, as "binding partners". These binding partners are likely to be
involved in the
NR signal transduction pathway, and therefore, in the role of NR in
controlling postnatal
ocular growth. Therefore, it is desirable to identify compounds that interfere
with or
disrupt the interaction of such binding partners with NR which may be useful
in regulating
the activity of NR and control of postnatal ocular growth associated with NR
activity.
The basic principle of the assay systems used to identify compounds that
interfere
with the interaction between the NR and its binding partner or partners
involves preparing a
reaction mixture containing NR protein, polypeptide, peptide or fusion protein
as described
in Sections 3.1 and 3.2 above, and the binding partner under conditions and
for a time
sufficient to allow the two to interact and bind, thus forming a complex. In
order to test a
compound for inhibitory activity, the reaction mixture is prepared in the
presence and
absence of the test compound. The test compound may be initially included in
the reaction
mixture, or may be added at a time subsequent to the addition of the NR moiety
and its
binding partner. Control reaction mixtures are incubated without the test
compound, or
with a placebo. The formation of any complexes between the NR moiety and the
binding
partner is then detected. The formation of a complex in the control reaction,
but not in the
reaction mixture containing the test compound, indicates that the compound
interferes with
the interaction of the NR and the interactive binding partner. Additionally,
complex
formation within reaction mixtures containing the test compound and normal NR
protein
may also be compared to complex formation within reaction mixtures containing
the test
compound and a mutant NR. This comparison may be important in those cases
wherein it
is desirable.to identify compounds that disrupt interactions of mutant but not
normal NRs.
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The assay for compounds that interfere with the interaction of the NR and
binding
partners can be conducted in a heterogeneous or homogeneous format.
Heterogeneous
assays involve anchoring either the NR moiety product or the binding partner
onto a solid
phase and detecting complexes anchored on the solid phase at the end of the
reaction. In
homogeneous assays, the entire reaction is carried out in a liquid phase. In
either approach,
the order of addition of reactants can be varied to obtain different
information about the
compounds being tested. For example, test compounds that interfere with the
interaction
by competition can be identified by conducting the reaction in the presence of
the test
substance; i.e., by adding the test substance to the reaction mixture prior to
or
simultaneously with the NR moiety and interactive binding partner.
Alternatively, test
compounds that disrupt preformed complexes, e.g. compounds with higher binding
constants that displace one of the components from the complex, can be tested
by adding
the test compound to the reaction mixture after complexes have been formed.
The various
formats are described briefly below.
In a heterogeneous assay system, either the NR moiety or the interactive
binding
partner is anchored onto a solid surface, while the non-anchored species is
labeled, either
directly or indirectly. In practice, microtiter plates are conveniently
utilized. The anchored
species may be immobilized by non-covalent or covalent attachments. Non-
covalent
attachment may be accomplished simply by coating the solid surface with a
solution of the
NR or binding partner and drying. Alternatively, an immobilized antibody
specific for the
species to be anchored may be used to anchor the species to the solid surface.
The surfaces
may be prepared in advance and stored.
In order to conduct the assay, the partner of the immobilized species is
exposed to
the coated surface with or without the test compound. After the reaction is
complete,
unreacted components are removed (e.g., by washing) and any complexes formed
will
remain immobilized on the solid surface. The detection of complexes anchored
on the solid
surface can be accomplished in a number of ways. Where the non-immobilized
species is
pre-labeled, the detection of label immobilized on the surface indicates that
complexes were
formed. Where the non-immobilized species is not pre-labeled, an indirect
label can be
used to detect complexes anchored on the surface; e.g., using a labeled
antibody specific for
the initially non-immobilized species (the antibody, in turn, may be directly
labeled or
indirectly labeled with a labeled anti-Ig antibody). Depending upon the order
of addition of
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reaction components, test compounds which inhibit complex formation or which
disrupt
preformed complexes can be detected.
Alternatively, the reaction can be conducted in a liquid phase in the presence
or
absence of the test compound, the reaction products separated from unreacted
components,
and complexes detected; e.g., using an immobilized antibody specific for one
of the binding
components to anchor any complexes formed in solution, and a labeled antibody
specific
for the other partner to detect anchored complexes. Again, depending upon the
order of
addition of reactants to the liquid phase, test compounds which inhibit
complex or which
disrupt preformed complexes can be identified.
In an alternate embodiment of the invention, a homogeneous assay can be used.
In
this approach, a preformed complex of the NR moiety and the interactive
binding partner is
prepared in which either NR or its binding partners is labeled, but the signal
generated by
the label is quenched due to formation of the complex (see, e.g., U.S. Pat.
No. 4,109,496 by
Rubenstein which utilizes this approach for immunoassays). The addition of a
test
substance that competes with and displaces one of the species from the
preformed complex
will result in the generation of a signal above background. In this way, test
substances
which disrupt the interaction between NR and an intracellular binding partner
can be
identified.
In a particular embodiment, an NR fusion can be prepared for immobilization.
For
example, NR or a peptide fragment can be fused to a glutathione-S-transferase
(GST) gene
using a fusion vector, such as pGEX-SX-1, in such a manner that its binding
activity is
maintained in the resulting fusion protein. The interactive binding partner
can be purified
and used to raise a monoclonal antibody, using methods routinely practiced in
the art. This
antibody can be labeled with the radioactive isotope'zSI, for example, by
methods routinely
practiced in the art. In a heterogeneous assay, e.g., the GST-NR fusion
protein can be
anchored to glutathione-agarose beads. The interactive binding partner can
then be added
in the presence or absence of the test compound in a manner that allows
interaction and
binding to occur. At the end of the reaction period, unbound material can be
washed away,
and the labeled monoclonal antibody can be added to the system and allowed to
bind to the
complexed components. The interaction between NR (as a gene product) and the
interactive binding partner can be detected by measuring the amount of
radioactivity that
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remains associated with the glutathione-agarose beads. A successful inhibition
of the
interaction by the test compound will result in a decrease in measured
radioactivity.
Alternatively, the GST-NR fusion protein and the interactive binding partner
can be
mixed together in liquid in the absence of the solid glutathione-agarose
beads. The test
compound can be added either during or after the species are allowed to
interact. This
mixture can then be added to the glutathione-agarose beads and unbound
material is
washed away. Again the extent of inhibition of the NR/binding partner
interaction can be
detected by adding the labeled antibody and measuring the radioactivity
associated with the
beads.
In another embodiment of the invention, these same techniques can be employed
using peptide fragments that correspond to the binding domains of the NR
and/or the
interactive or binding partner (in cases where the binding partner is a
protein), in place of
one or both of the full length proteins. Any number of methods routinely
practiced in the
art can be used to identify and isolate the binding sites. These methods
include, but are not
limited to, mutagenesis of the gene encoding one of the proteins and screening
for
disruption of binding in a co-immunoprecipitation assay. Compensating
mutations in the
gene encoding the second species in the complex can then be selected. Sequence
analysis
of the genes encoding the respective proteins will reveal the mutations that
correspond to
the region of the protein involved in interactive binding. Alternatively, one
protein can be
anchored to a solid surface using methods described above, and allowed to
interact with
and bind to its labeled binding partner, which has been treated with a
proteolytic enzyme,
such as trypsin. After washing, a short, labeled peptide comprising the
binding domain
may remain associated with the solid material, which can be isolated and
identified by
amino acid sequencing. Also, once the gene coding for the intracellular
binding partner is
obtained, short gene segments can be engineered to express peptide fragments
of the
protein, which can then be tested for binding activity and purified or
synthesized.
For example, and not by way of limitation, a GST-NR fusion protein can be
prepared and anchored to a solid material as described, for example by
allowing it to bind
to glutathione agarose beads. The interactive binding partner can be labeled
with a
radioactive isotope, such as 355, and cleaved with a proteolytic enzyme such
as trypsin.
Cleavage products can then be added to the anchored GST-NR fusion protein and
allowed
to bind. After washing away unbound peptides, labeled bound material,
representing the
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intracellular binding partner binding domain, can be eluted, purified, and
analyzed for
amino acid sequence by well-known methods. Peptides so identified can be
produced
synthetically or fused to appropriate facilitative proteins using recombinant
DNA
technology.
Section 3.4. Assays for Identification of Compounds that Ameliorate Abnormal
Postnatal Ocular Growth
Compounds, including but not limited to binding compounds identified via assay
techniques such as those described, above, in Sections 3.1 through 3.3, can be
tested for the
ability to ameliorate abnormal postnatal ocular growth, including myopia. The
assays
described above can identify compounds which affect NR activity (e.g.,
compounds that
bind to the NR, inhibit binding of the natural ligand, and either activate
signal transduction
(agonists) or block activation (antagonists), and compounds that bind to the
natural ligand
of the NR and neutralize ligand activity); or compounds that affect NR gene
activity (by
affecting NR gene expression, including molecules, e.g., proteins or small
organic
molecules, that affect or interfere with splicing events so that expression of
the full length
or the truncated form of the NR can be modulated). However, it should be noted
that the
assays described can also identify compounds that modulate NR signal
transduction (e.g.,
compounds which affect downstream signalling events, such as inhibitors or
enhancers of
tyrosine kinase or phosphatase activities which participate in transducing the
signal
activated by ligand binding to the NR). The identification and use of such
compounds
which affect another step in the NR signal transduction pathway in which the
NR gene
and/or NR gene product is involved, and by affecting this same pathway may
modulate the
effect of NR on the development of abnormal postnatal ocular growth, are
within the scope
of the invention. Such compounds can be used as part of a therapeutic method
for the
treatment of myopia and other conditions resulting from abnormal postnatal
ocular growth.
The invention encompasses cell-based and animal model-based assays for the
identification of compounds exhibiting such an ability to ameliorate myopia
symptoms,
signs or characteristics. Such cell-based assay systems can also be used as
the "gold
standard" to assay for purity and potency of natural ligands, including
recombinantly or
synthetically produced ligands.
Cell-based systems can be used to identify compounds which may act to
ameliorate
myopia symptoms, signs or characteristics. Such cell systems can include, for
example,
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recombinant or non-recombinant cells, such as cell lines, which produce NR.
For example,
retinal cells or cell lines derived from retina can be used. In addition,
expression host cells
(e.g., COS cells, CHO cells, fibroblasts) genetically engineered to express a
functional NR
and to respond to activation by the natural ligand, e.g., as measured by a
chemical or
phenotypic change, induction of another host cell gene, change in ion flux
(e.g., Na+, K+),
tyrosine phosphorylation of host cell proteins, etc., can be used as an end
point in the assay.
In utilizing such cell systems, cells may be exposed to a compound suspected
of
exhibiting an ability to ameliorate myopia symptoms in intact eyes, at a
sufficient
concentration and for a time sufficient to elicit amelioration of myopia-
related cellular
phenotypes or cell functions in the exposed cells. After exposure, the cells
can be assayed
to measure alterations in gene expression, e.g., by assaying cell lysates for
mRNA
transcripts (e.g., by Northern analysis) or for NR protein expressed in the
cell; compounds
which regulate or modulate expression of the NR gene are good candidates as
therapeutics.
Alternatively, the cells are examined to determine whether one or more myopia-
related
cellular phenotypes or cell functions has been altered to resemble a more
normal or more
wild type, non-myopic phenotype, or a phenotype more likely to produce a lower
incidence
or severity of myopia symptoms. Still further, the expression and/or activity
of components
of the signal transduction pathway of which NR is a part, or the activity of
the NR signal
transduction pathway itself can be assayed.
For example, after exposure, the cell lysates can be assayed for the presence
of
tyrosine phosphorylation of host cell proteins, as compared to lysates derived
from
unexposed control cells. The ability of a test compound to inhibit tyrosine
phosphorylation
of host cell proteins in these assay systems indicates that the test compound
inhibits signal
transduction initiated by NR activation. The cell lysates can be readily
assayed using a
Western blot format; i.e., the host cell proteins are resolved by gel
electrophoresis,
transferred and probed using a anti-phosphotyrosine detection antibody (e.g.,
an anti-
phosphotyrosine antibody labeled with a signal generating compound, such as
radiolabel,
fluor, enzyme, etc.) (See, e.g., Glenney et al., 1988, J. Immunol. Methods,
109:277-285;
Frackelton et al., 1983, Mol. Cell. Biol., 3:1343-1352). Alternatively, an
ELISA format
could be used in which a particular host cell protein involved in the NR
signal transduction
pathway is immobilized using an anchoring antibody specific for the target
host cell
protein, and the presence or absence of phosphotyrosine on the immobilized
host cell
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protein is detected using a labeled anti-phosphotyrosine antibody. (See, King
et al., 1993,
Life Sciences, 53:1465-1472). In yet another approach, ion flux, such as
sodium or
potassium ion flux, can be measured as an end point for NR stimulated signal
transduction.
Membrane depolarization can also be measured as an end point for NR stimulated
effects.
In addition, animal-based myopia models may be used to identify compounds
capable of ameliorating myopia-like symptoms. Such animal models may be used
as test
substrates for the identification of drugs, pharmaceuticals, therapies and
interventions
which may be effective in treating such disorders. For example, animal models
may be
exposed to a compound suspected of exhibiting an ability to ameliorate myopia
symptoms,
at a sufficient concentration and for a time sufficient to elicit such an
amelioration of
myopia symptoms in the exposed animals. The response of the animals to the
exposure
may be monitored by assessing the reversal of characteristics, signs, or
symptoms
associated with myopia. With regard to intervention, any treatments which
reverse any
aspect of myopia-like characteristics, signs or symptoms should be considered
as
candidates for human myopia therapeutic intervention. Dosages of test agents
may be
determined by deriving dose-response curves.
Section 4. Pharmaceutical compositions
The nicotinic antagonists of this invention have been found to possess
valuable
pharmacological properties. Nicotinic antagonists regulate postnatal growth of
the eye,
with the particularly desirable effect of inhibiting postnatal ocular growth
and preventing
the development of myopia. This effect can be demonstrated, for example, using
the
methods described in the Examples below.
Thus, these compounds can be used to control postnatal growth of the eye,
inhibit
postnatal ocular growth, prevent myopia, control abnormal postnatal ocular
growth, inhibit
abnormal postnatal axial growth of the eye, inhibit abnormal equatorial
expansion of the
eye, inhibit vitreous cavity expansion, inhibit the progression of myopia,
inhibit the onset
of myopia, and reverse myopia. These compounds are particularly useful to
inhibit the
development of myopia.
The compounds of this invention are generally administered to animals,
including
but not limited to mammals including humans, as well as to birds, monotremes,
reptiles, or
fish.
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The pharmacologically active compounds of this invention can be processed in
accordance with conventional methods of galenic pharmacy to produce medicinal
agents
for administration to patients, e.g., mammals including humans. The compounds
of this
invention can be employed in admixture with conventional excipients, i.e.,
pharmaceutically acceptable organic or inorganic Garner substances suitable
for parenteral,
enteral (oral) or topical ocular application which do not deleteriously react
with the active
compounds. Suitable pharmaceutically acceptable Garners include but are not
limited to
water, salt solutions, alcohols, vegetable oils, benzyl alcohols, polyethylene
glycols,
gelatine, carbohydrates such as lactose, amylose or starch, magnesium
stearate, talc, silicic
acid, viscous paraffin, fatty acid monoglycerides and diglycerides,
pentaerythritol fatty acid
esters, hydroxymethylcellulose, polyvinyl pyrrolidone, or any other suitable
Garner. The
pharmaceutical preparations can be sterilized and if desired, mixed with
auxiliary agents,
e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers,
salts for influencing
osmotic pressure, buffers, and the like which do not deleteriously react with
the active
compounds. They can also be combined where desired with other active agents,
e.g.
vitamins.
For parenteral application, particularly suitable are injectable, sterile
solutions,
perferably aqueous or oily solutions, as well as suspensions, emulsions, or
implants.
For enteral application, particularly suitable are tablets, dragees, liquids,
drops,
suppositories, or capsules. A syrup, elixir, or the like can be used wherein a
sweetened
vehicle is employed.
For topical application, there are employed liquid to viscous to semi-solid or
solid
forms comprising a carrier compatible with topical application and having a
dynamic
viscosity that might be preferably greater than water. Suitable formulations
include but are
not limited to, solutions, suspensions, emulsions, creams, ointments, gels,
powders,
liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed
with auxiliary
agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for
influencing
osmotic pressure, etc. For topical application, also suitable are sprayable
aerosol
preparations wherein the active ingredient, prefereably in combination with a
solid or liquid
inert carrier material, is packaged in a squeeze bottle or otherwise propelled
through a
vehicle capable of aerosolizing the preparation.
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The compounds of the instant invention are useful in treating or preventing
the
development of myopia. Therapy to inhibit axial elongation or equatorial
expansion,
control postnatal growth of the eye, inhibit postnatal ocular growth, prevent
myopia,
control abnormal postnatal ocular growth, inhibit abnormal postnatal axial
growth of the
eye, inhibit abnormal equatorial expansion of the eye, inhibit vitreous cavity
expansion,
inhibit the progression of myopia, inhibit the onset of myopia, and reverse
myopia, can be
administered by the use of the agent in eye drops. Eye drops are typically
made up at a
concentration of active agent between about 0.005% and 10% in the ophthalmic
medium,
advantageously between about 0.01 % and 5%, and preferably between about 0..1
% and 2%.
A pH of about 3.5 to 8.5, advantageously about 4.0 to 8.0, and preferably
about 4.5 to 7.5,
may be expected to be acceptable as an ophthalmic drop. Phosphate buffering is
also
common for eye drops, but other buffers can be used. A common regimen for
application
of eye drops is one to four times a day spaced evenly throughout waking hours.
More
effective agents may require fewer applications or enable the use of more
dilute solutions.
It will be appreciated that the actual preferred amounts of active compounds
in a
specific case will vary according to the specific compound being utilized, the
particular
compositions formulated, the mode of application, and the particular situs and
organism
being treated. Dosages for a given host can be determined using conventional
considerations, e.g., by customary comparison of the differential activities
of the subject
compounds and of a known agents, e.g., by means of an appropriate,
conventional
pharmacological protocol.
Examples
Methods. One-day-old white leghorn chicks (Truslow Farms, Chestertown, MD)
were reared in brooders on a 12 hour light-dark cycle with General Electric
chroma 50
fluorescent lighting with irradiance of approximately 50pW/cmz at chick eye
level. The
chicks received Purina Chick Chow~ food and water ad libitum.
Experiments started at one week of age. For some chicks, a unilateral
translucent
white plastic goggle was glued to the periorbital feathers with cyanoacrylate
glue to induce
form-deprivation myopia, a commonly studied experimental model that induces
ipsilateral
myopia in newly hatched chicks and in those nearing maturity (Papastergiou et
al., 1998,
Vision Res., 38:1883-8). Under aseptic conditions, the goggled eye received a
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intravitreal injection of either drug or saline vehicle at that time. Other
chicks were non-
goggled but similarly received intravitreal injections of either drug or
vehicle to one eye. In
most experimental groups, drug and vehicle were administered by intraocular
injections
daily or every other day at approximately four hours into the light phase. In
each series, the
experimental eye was alternated between left and right, and all contralateral
eyes received
injections of saline vehicle at the same time as injections to the
experimental eye. Chicks
were anesthetized with inhalation ether for all goggle applications and drug
injections.
After one week of treatment and at two weeks of age, the chicks were
anesthetized
with an intramuscular mixture of ketamine (20mg/kg) and xylazine (Smg/kg), and
ocular
refractometry and A-scan ultrasonography were performed as described (Stone et
al., 1995,
Vision Res., 35:1195-202). No intraocular injections were administered on the
day of
examination. While still under general anesthesia, the chicks were decapitated
and the axial
and equatorial dimensions of enucleated eyes were measured with digital
calipers. The
coronal profile of the chick eye is elliptical, and the equatorial diameter is
reported as the
mean of the shortest and longest equatorial dimensions of the eye.
Data are provided as mean ~ S.E.M. and were analyzed with SigmaStat (SPSS,
Inc.
Chicago, IL). Neither visual deprivation or drug treatments to these eyes
affected lens
thickness, and these data are not reported for goggled chicks. A one-way
analysis of
variance (ANOVA), using the differences between visually deprived and
contralateral eyes
on goggled chicks, was performed to ascertain drug efficacy against
experimental myopia.
Because the ultrasound data on axial length following mecamylamine treatment
to goggled
eyes did not meet conditions of normality, these data were assessed with a
Kruskal-Wallis
one-way ANOVA on ranks on the differences between experimental and
contralateral eyes.
Data from different cohorts of chicks tested with the same drug, along with
the respective.
vehicle-treated controls, were pooled for analysis (Figure 1). Because the
drug effects in the
never-goggled chicks also were not normally distributed, drug treated non-
goggled eyes
and vehicle treated contralateral eyes were compared with a Friedman repeated
measures
ANOVA on ranks. In series when the ANOVA identified a treatment effect, post
hoc
multiple pairwise comparisons of the treatment groups were made with the Tukey
test,
using a value of P < 0.05 for statistical significance. In assessing acute
drug effects on
ocular refractions and ultrasounds, the measurements before and after drug
injection were
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compared with a Student's paired t-test. The experiments conformed with the
ARVO
Resolution on the Use of Animals in Ophthalmic and Vision Research.
Example 1
The following drugs were administered daily: dihydro-(3-erythroidine
hydrobromide
(RBI/Sigma; Natick, MA), mecamylamine (RBI/Sigma) and methyllcaconitine
citrate
(RBI/Sigma). Because it is a long-acting nicotinic antagonist in mammalian
brain (El-Bizri
and Clarke, 1994, Br. J. Pharmacol., 113:917-925), chlorisondamine diiodide
(Tocris
Cookson; Ballwin, MO) was generally administered every other day by
intravitreal
injection in most experiments.
Goggled chicks As expected from previous studies, the cohorts of vehicle
treated
control chicks wearing a unilateral goggle developed ipsilateral myopia of
about -7 to -12
diopters compared to the contralateral non-goggled eyes. The axial lengths in
the goggled
eyes were increased by some 0.4-0.6 mm compared to the contralateral eyes. In
general,
the axial length difference between goggled and open eyes was greater as
measured by
ultrasound which records to the inner limiting membrane than as measured by
calipers
which records to the outer scleral surface. Besides the greater variability of
the caliper
measurements, this disparity may at least partly be physiologic as both the
choroid and
retina of young chicks thins during goggle wear. The vitreous cavity of
goggled eyes was
enlarged in both the axial and equatorial dimensions, with the vitreous cavity
elongation
largely accounting for the increase in overall axial length of the eye. Goggle
wearing alone
induced no significant effect on anterior chamber depth in most cohorts of
vehicle treated
chicks.
Two relatively non-selective nicotinic antagonists were tested,
chlorisondamine and
mecamylamine. Chlorisondamine reduced the myopic refractive error (Figure 1;
ANOVA:
P < 0.001), inhibited the excessive axial elongation developing beneath a
goggle (Figures
2A, 2B, and 2C ANOVA: ultrasound, P < 0.001; calipers, P = 0.008) and reduced
the
vitreous cavity expansion in both axial (ANOVA: P < 0.001) and equatorial
(ANOVA: P =
0.001) dimensions. Chlorisondamine had no statistically significant effect on
anterior
chamber depth. Post hoc pairwise comparisons by the Tukey test (Table 1)
showed
significant drug effects compared to the vehicle-treated controls for
refraction, axial length
and vitreous cavity depth measurements and for several other intragroup
comparisons.
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The effects of mecamylamine on goggled eyes were more complex, with a
multiphasic response that differed between the low and high drug doses. It had
a maximal
anti-myopia effect at the intermediate dose and tended toward stimulating the
growth and
myopic refractive shift of goggled eyes at the lowest doses. Overall,
mecamylamine altered
refraction of goggled eyes (Figure 1; ANOVA: P c 0.001). Although all three
higher drug
doses reduced the induced myopia, only the SOpg dose differed significantly
from the
controls by post hoc pairwise comparison testing (Table 1) and virtually
eliminated the
induced myopia. While the refractions at the 1 and lOpg doses were not
individually
different from the controls by post hoc pairwise comparisons, significant
differences
occurred between the 1 ~g dose and each of the 50, 100 and 200pg doses as well
as between
the 10 and SOpg doses (Table 1). The anatomical effects of mecamylamine on
goggled eyes
tended to follow the refractive effects: larger eyes developing with doses
that did not reduce
the myopia and smaller eyes with doses that did (Figure 2). For axial length
(Figuress 2A
and 2C), there was only a trend towards a drug effect by ultrasound (Figure 2A
ANOVA: P
- 0.07); no statistical effect on axial length by caliper measurements was
apparent.
Mecamylamine influenced the ultrasound measurements of vitreous cavity length
(Figure
2B ANOVA: P = 0.007); post hoc pairwise comparison testing identified the 1pg
dose as
different from the SOpg dose but not from the controls (Table 1). Similarly,
overall
equatorial diameter of goggled eyes was influenced by mecamylamine (Figure 2D
ANOVA: P = 0.003); post hoc pairwise comparison testing did not identify any
individual
doses that differed from the controls but showed that the 1 and SOpg doses
differed from
each other and that the 10~g dose differed from both the 50 and 200pg doses
(Table 1).
There also may have been an anterior chamber affect from mecamylamine
(ANOVA: P = 0.009), but post hoc pairwise comparison testing did not identify
any
individual differences. In reviewing the data on anterior chamber depth, the
vehicle treated
goggled eyes in the mecamylamine experiments had an anterior chamber depth
slightly
shallower than the contralateral non-goggled eyes (1.22~0.04mm in goggled eyes
versus
1.34~0.04mm in non-goggled eyes). The differences in anterior chamber depth
between
goggled and contralateral eyes were similar for the low mecamylamine doses;
but for the
100 and 200pg doses, the anterior chamber depths in the drug treated goggled
eyes relative
to the contralateral controls were no longer reduced but instead were equal
(data not
shown).
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Of the antagonists with some subtype selectivity, methyllycaconitine showed
the
greater efficacy of the two drugs and was similar to mecamylamine in that the
strongest
effects seemed to occur at the intermediate drug doses. Methyllycaconitine
affected the
myopic refraction (ANOVA: P = 0.04), axial length (ANOVA: ultrasound, P = 0.05
(Figure 2A); calipers, P = 0.002 (Figure 2C)), and equatorial expansion of the
vitreous
cavity (ANOVA: P = 0.02 (Figure 2D) in goggled eyes (Figures 1, 2). A trend
towards an
influence on vitreous cavity length did not reach significance with this drug
(Figure 2B
ANOVA: P = 0.09). With post hoc pairwise multiple comparisons by the Tukey
test, a
significant difference from controls was only identified for the inhibition of
equatorial
expansion beneath a goggle at the Spg dose; additionally, the Spg dose reduced
axial length
by calipers compared to the 0.05, 0.5 and SOpg doses (Table 1). There was no
effect from
methyllycaconitine on the anterior chamber depth of goggled eyes.
Dihydro-(3-erythroidine exhibited only a weak effect against experimental
myopia
(Figures 1, 2). The drug induced a significant reduction only in axial length
as measured by
calipers (ANOVA: P = 0.02 (Figure 2C), but no individual drug dose was
identified by the
post hoc pairwise multiple comparison testing. Otherwise, none of the
differences in
refraction, ultrasound measurements or caliper measurements of the equatorial
diameter
reached statistical significance by ANOVA.
Non-goggled chicks. Chicks were reared and treated as described in the Methods
section above, but without goggles. Under aseptic conditions, one non-goggled,
or "open"
eye received a lOp,l intravitreal injection of either drug or saline vehicle.
All contralateral
eyes received injections of saline vehicle at the same time as injections to
the experimental
eye. Drug and vehicle were administered by intraocular injections daily or
every other day
at approximately four hours into the light phase.
Unilateral intravitreal administration of chlorisondamine reduced the axial
growth
of drug-treated eyes in never-goggled chicks (Figure 3; ANOVA on ranks:
ultrasound, P =
0.03; calipers, P = 0.03). The growth reduction was confined to the vitreous
cavity
(ANOVA on ranks: P = 0.01) and was reflected in a hyperopic shift in
refraction (ANOVA
on ranks: P = 0.004). The effect on equatorial expansion of the vitreous
cavity did not reach
statistical significance. Pairwise comparisons with the Tukey test identified
the refractions
of the eyes treated with 200pg and lOpg and the vitreous cavity depths of the
eyes treated
with 100pg and SO~,g as different from each other (Table 1). An effect on lens
thickness
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also was noted (ANOVA on ranks: P < 0.001), comprising an increase of about
0.1 mm in
both eyes in the 10~g group compared to chicks receiving the 50, 100 or 200pg
doses as
well as other chicks who received saline injections to both eyes; no pairwise
comparisons
of the lenses were identified as significant by the Tukey test, however.
In contrast to the chlorisondamine effects on open eyes, daily intravitreal
injections
of mecamylamine had no influence on the growth or refraction of. non-goggled
eyes after
one week at doses of SO~g (the dose with the strongest effect against form-
deprivation
myopia) or of either 10 or 1 ~g (the doses that tended to stimulate the myopic
response to a
goggle).
Example 2
Acute drug effects. To assess acute drug effects, other two-week old chicks
(n=5/group) received a single unilateral intravitreal injection of one of the
nicotinic
antagonists at doses chosen based on drug effects on the growth of goggled
eyes: 200~g
chlorisondamine, SOpg and leg mecamylamine, Spg methyllycaconitine or SOpg
dihydro-
(3-erythroidine. Just before and at two hours and 24 hours after injection,
both eyes were
examined by refractometry and ultrasonography by the above methods. Because
chicks in
the eye growth studies did not receive drug on the day of measurements, the 24
hour
examination point was selected specifically to identify a potential residual
drug effect on
the intraocular muscles at a time relevant to the eye growth measurements.
Mean baseline pupil diameter measured 2.4~O.Smm. Two hours after injection,
each
of the drugs induced some pupillary dilation (change from baseline: ZOOpg
chlorisondamine, 0.8~O.lmm, P < 0.01; SOpg mecamylamine, 0.3t0.lmm, not
significantly changed; lpg mecamylamine, 0.8t0.2mm, P < 0.05; Spg
methyllycaconitine
0.4~0.2mm, not significantly changed; SOpg dihydro-(3-erythroidine 1.O~O.lmm,
P < 0.01).
Although dilated, the pupils in each group still constricted in response to
light but were
sluggish. By 24 hours, the pupil had returned to normal in all but two groups
(change from
baseline: chlorisondamine, 0.7~0.2mm, P < 0.05; mecamylamine SOpg, 0.4~O.lmm,
P <
0.05). None of the drug applications had a significant effect on refraction at
either 2 or 24
hours. By ultrasonography, chlorisondamine induced a 0.16~0.04mm (P < 0.05)
reduction
in axial length and a 0.20t0.07mm (P < 0.05) reduction in posterior chamber
depth at 2
hours, each of which returned to baseline at 24 hours; chlorisondamine also
reduced lens
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thickness by 0.12~0.04mm (P < 0.05) at 2 hours and by 0.16~O.OSmm (P < 0.05)
at 24
hours. None of the other drugs influenced the ultrasound measurements.
In these experiments, the eyes were examined 2 and 24 hours after
administration of
selected doses of nicotinic antagonists to learn if any acutely influenced the
tone of the
intraocular muscles. Each drug induced some mydriasis (pupil dilation),
reversible to light;
presumably any cycloplegic (inhibition of accommodation) effect also was
partial. Based
on the results, these nicotinic antagonist drugs neither shifted refraction
acutely nor
uncovered any basal accommodative tone under the conditions of the
examinations. Only
chlorisondamine acutely altered ocular dimensions by ultrasound, transiently
reducing axial
and vitreous cavity lengths at the 2 hour but not at the 24 hour reading.
Further, only
chlorisondamine influenced lens thickness, reducing it and likely increasing
the focal
length; if any increase in lens focal length had modulated development of open
eyes
receiving this drug daily, it would have stimulated eye growth (Schaeffel F,
Glasser A,
Howland HC, 1988, Vision Res., 28:639-657) and not inhibited it (Figure 3).
Because all
measurements of drug influences on eye growth were made 24 hours after the
last dosing,
none of the observations on growth or refractive development shown in Examples
1 and 3
can be explained by an acute drug effect, muscular or otherwise, on refraction
or eye
component measurements; that is, the effects of nicotinic antagonists on
ocular refraction
and eye growth (Examples 1 and 3) are effects on the development of the eye
and constitute
developmental modification of ocular refraction and/or dimensions.
Example 3
Longer term effects of single-dose chlorisondamine. In a separate experiment,
another group of goggled chicks received a single intravitreal dose of 200pg
of
chlorisondamine to the form deprived eye and saline to the contralateral eye
only at the
time of goggle application. They were compared to a group of unilaterally
goggled chicks
receiving a single saline injection to both eyes only at the time of goggle
application.
Neither group received subsequent intraocular injections. These chicks were
evaluated by
refractometry and ultrasound 4 days later, using ketamine/xylazine anesthesia.
At one
week after goggle application and single drug injection, the same chicks were
evaluated
again with refractometry, ultrasonography and caliper measurements of
enucleated eyes as
described above.
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A single dose of 200~g of chlorisondamine given at the time of goggle
application
significantly blunted the response for form deprivation over the next week
(Table 2). The
effect is manifest at four days by significant attenuations in refraction
(p<0.05) and axial
length (p<0.01) by ultrasonography of goggled eyes. While the effectiveness
against the
myopic shift in refraction is diminished at one week, statistically
significant attenuations
are evident at one week in the ultrasound measurements of axial length
(p<0.05) and
vitreous cavity depth (p=0.05) and in the caliper measurements of axial length
(p<0.05) and
equatorial diameter (p<0.005). The action of a single dose of chlorisodamine
against
myopic eye growth seems equivalent at four days and one week because the
ratios of axial
and vitreous cavity lengths of the drug treated to vehicle treated eyes are
equivalent at both
times (Table 2).
Example 4
Histopathological effects. To identify potential histopathologic effects in
other
groups of monocularly deprived or never-goggled one-week old chicks,
chlorisondamine
(200 or 100~g every other day; n = 5-6/group), mecamylamine (200 or SO~g
daily; n = 5-
8/group) or saline vehicle (n = 3-9/group) was administered by intravitreal
injection to the
goggled eyes or to one of the open eyes of never-goggled chicks with vehicle
to the
coritralateral eye, using the identical protocol as above. After one week of
treatment, the
above protocols provided refraction, ultrasound and caliper measurements. The
eyes were
then immersion fixed in 3% glutaraldehyde/0.5% paraformaldehyde in O.1M
phosphate
buffer, pH 7.4. The posterior segments were either embedded in paraffin, cut
at S~m
thickness and stained with hematoxylin and eosin, or embedded in historesin,
cut at 3~m or
S~m thickness and stained with 0.5% azure II/0.5% methylene blue in 1% borate.
With chlorisondamine 200~g every other day, gross inspection of the eye cup of
most of the goggled eyes (4/5) and all of the never-goggled eyes (n=5) showed
mild to
marked mottling and depigmentation of the mid-peripheral fundus; a variably
sized
geographic area appeared relatively spared or normal in the central fundus
region. The
tissue sections revealed marked disruption and clumping of cells of the
retinal pigment
epithelium (RPE) in regions corresponding to the peripheral depigmented areas.
Pigment
containing cells, presumably macrophages, were occasionally noted in the outer
retina, and
outer segments were sometimes disrupted overlying the disrupted epithelium.
The retina
otherwise appeared intact. Presumed inflammatory cells infiltrated the
peripheral
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choriocapillaris beneath the most involved areas of the RPE, but the choroid
was otherwise
unaffected. The central regions of these eyes showed either normal histology
or less
marked changes. The goggled eye treated with 200pg chlorisondamine that had a
normal
gross examination also exhibited normal histology. Of the goggled or non-
goggled eyes
treated with chlorisondamine 100pg every other day, gross inspection of the
eye cups
showed either a normal fundus or only mild peripheral pigmentary changes. Some
eyes
had normal histology, some showed a single large, smooth hyper-pigmented
inclusion
within a rare RPE cell as the sole detectable histologic change, and some had
a small
isolated peripheral patch of the marked RPE/choriocapillaris pathology as
described above.
Importantly, the growth and refractive responses of goggled or open eyes to
chlorisondamine was not clearly related to the degree of retinal
histopathology.
The retinas of goggled and non-goggled eyes treated daily with either 200 or
SO~g
of mecamylamine were indistinguishable grossly or histologically from vehicle
treated
control eyes.
All of the references cited herein, whether research articles, patent
documents, or
other cited references, are hereby incorporated by reference in their entirety
as though
individually incorporated by reference. It can be understood by those of skill
in the art that
numerous and various modifications can be made without departing from the
spirit of the
present invention. Therefore, it should be clearly understood that the forms
of the present
invention are illustrative only and are not intended to limit the scope of the
present
invention.
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TABLE A Nicotinic Receptors
Acetylcholine Receptors (Nicotinic)
Nomenclature* al* a2* a3* a4*
Natural locations) muscle CNS ANS, CNS CNS, sensory
ganglia
Agonistsfi iso; sub; ACh; ABT418b; cytisinea, cytisinea, ABT418b,
nicotine;
epibatidine; BAC; epibatidined;epibatidined;nicotine, epibatidined;
DMPP; DMPP;
DMAP; DMPP; anatoxin nic; AUh; anatoxin; DMPP;
; cytisine; ABT-418; ACh
carbamylcholine; ACh; sub anatoxin;
DMPP;
sue; cytisine; nicotine sub; iso;
BAC;
carbamylcholine;
DMAP; sue
Selective competitive a-bungarotoxin, dihydro-a- dihydro-(3- dihydro-(3-
erythroidine
antagonists dTC [high (al/y) and erythroidine erythroidine
low (a1/8) affinity
sites] '
Other competitive a-conotoxins GI & dTC a-conotoxin MII; methyllycaconitine;
antagonists MI; alcuronium; a-conotoxin AuIB; alcuronium; dTC; eser;
nstx; trim; ben; decamethonium decamethonium
mecamylamine;
lobeline; eser;
decamethonium;
atropine; gallamine;
hemicholinium'
strychnine;
neostigmine;
dihydro-(3-
erythroidine;
hexamethonium;
choline
Noncompetitivegallamine hexamethonium,hexamethonium,hexamethonium,
channel blocker mecamylamine mecamylamine mecamylamine
antagonists chlorisondamine;
Radioligands ['H] or ['ZSI]-['H]-acetylcholine['H]-acetylcholine['H]-
acetylcholine
bungarotoxin(ACh) (ACh) (ACh)
['H]-cytisine['H]-cytisine['H]-cytisine
['H]-epibatidine['H]-epibatidine['H]-epibatidine
['H]- ['H]- ['H]-methylcarbamyl-
methylcarbamyl-methylcarbamyl-choline
choline choline ['H]-nicotine
['H]-nicotine['H]-nicotine
Effector Int.cat. Int. cat. Int. cat. Int. cat. (PCa/PNa
(PCa/PNa (Na', CaZ'; (Na', Caz';
0.2 - 1) PCa~Na) PCa/PNa) 0.5-G)
Structural
Information
Receptor subtypes(al)2(ily8 a2[32b, a2[34a3*, a3[34*,a3a5(34,a4*, (a4)2((32)3
-
identified, embryonic a3a5/32[i4, a4a5(32
in vivo
(a 1 )2[31 a3 (323 [33
e8 - adult (34
Functional (al)2(31y8, a2/[32, a2/(34a3(32d a3(34a,a4(32a,b,c,d
receptor a4a4a,c
subtypes created(al)2[31e8, a3a5[i2, a3a5[34,a4a5(32, a4(32(34
by
heterologous al(32y8,a1(34y8 a3[33[34 a4[32~33(34
expression
Binding site ally or al/e a3/(32d, a3/(34a
interfaces and a4/[32, a4/(34
a 1 /8
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Table A, continuedAcetylcholine
Receptors
(Nicotinic)
Nomenclature* a6* a7, a7* a8 (avian), a9, a9*
a8*
(avian)
Natural locations)CNS ANS, CNS retina, CNS pituitary, cochlea
Agonistst epibatidined;anatoxin, cytisine; ACh
cholinel-3 nicotine;
cytisine; DMAC4; ACh; DMPP;
DMPP, TMA
nicotine, epibatidine;
ACh OH-
GTS-21; DMPP;
cytisine;
nicotine;
GTS-21; ABT-418;
ACh, TMA,
carbamylcholine
Selective antagonistsdihydro-a- a-bungarotoxin,a-bungarotoxina-bungarotoxin,
erythroidinemethyllycaconitine nicotine,
Other antagonistsdTC a-conotoxin atropine; strychnine,
IMI; a- dTC; atropine;
bungarotoxin;strychnine dTC; muscarine
dTC;
atropine;
dihydro-(3-
erythroidine
Channel blockerhexamethonium,- - -
mecamylamine
Radioligands - [3H]/[I125I]_[3H]/[1251]__
bungarotoxin bungarotoxin
Effector Int.cat. Int. cat. Int. cat. Int. cat. (Na',
(Na', CaZ';(PCa/PNa (PCa/PNa Caz')
)
PCa~Na) 6 - 20)
Structural
Information
Receptor subtypesa6a3[32, (a7)5, a7a8, (a8)5, a7a8 a9*
a6a3(i4, a7*
identified, a6~32[34,
in vivo
a6/[l4d
Functional a6a3[I2, (a7)5, a7/a8 (a8)5, a7a8 (a9)5, a9a10
receptor a6a4[32
subtypes createda6(33[34,
by a6(34
heterologous
expression
Binding site a6/(32, a7/a7, a7/a8 a8/a8, a8/a7a9/a9 (not a105)
interfaces a6(34 (avian)
tSuperscripts (a,b,c and d) indicate ligands showing some selectivity for the
binding site interfaces, similarly
labelled
Chemical names:
ABT418, (,S~-3-methyl-5-(1-methyl-2-pyrrolidinyl) isoxazole; ACh,
acetylcholine; ben,
benzoquinonium; BAC, bromoacetylcholine; DMAB, 3-(4)-dimethylaminobenzylidine
anabaseine; DMAC,
3-(4)-dimethylaminocinnamylidine; DMAP, N-N-dimethyl-4-acetylpiperzinium;
DMPP, 1,1-dimethyl-4-
phenylpiperazinium; dTC, d-tubocurarine, or (+)-tubocurarine; eser,
eser(physostigmine); GTS-21, 3-(2,4)-
diemthoxybenzylidine anabaseine (DMXB); iso, isoarecolone methiodide; nstx,
neosurugatoxin; OH-GTS-
21, 3-(4-hydroxy, 2-methoxy)benzylidine anabaseine; sub, suberyldicholine;
sue, succinyl(di)choline; TMA,
tetramethylammonium; trim, trimethaphan.
_3~_ .
CA 02400803 2002-08-26
WO 01/52832 PCT/USO1/01692
Table 1. Post Hoc Pairwise Comparisons of Drug Effects Using Tukey Test
Ultrasound Caliper
measurements measurements
refractionaxial lengthvitreous axial lengthequatorial
cavity
depth diameter
Condition
and
dru
GoQQled chicks
200~g vs. 200~g vs. 200~g vs. 200~g vs. 200~g
vs.
chlorisondaminecontrol, control, control, control, 1 ~g
50, 10 50, 10 50, 10 50 &
&
& 1~g & 1~g 1~g 10~g
100~g
vs.
100~g vs. 100~g vs. 100~g vs. 1 ~g
50 & 50~g
control, 1 ~g
50, 10
& 1 ~g
200~g vs. 50~g vs. 200~g
1 ~g 1 ~g vs.
mecamylamine --- --- 10~g
100~g vs.
1 ~g
50~g vs.
10
50~g vs. & 1 ~g
control,
10 &
leg
methyll- --- --- --- 5~g vs. 5~g vs.
50, 0.5
caconitine & 0.05~g control
dihydro-(3- --- --- --- --- --
erythroidine
Non- og g~led
chicks
chlorisondamine200~g vs. --- 100~g vs. --- --
10~g 50~g
treated eyes
The statistically significant (defined as P < 0.05) post hoc pairwise
comparisons by the
Tukey test are shown for each condition and drug for which the ANOVA
identified a
treatment effect (see text and Figures 1-3).
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Table 2. Longer Term Effects of Single-dose Chlorisondamine (200p,g) on
Form Deprivation Myopia.
Time After Drug
Administration
4 Days 1 Week
Refraction Difference
vehicle n = 7 - 8.82 + 1.07 diopters-11.72 + 1.19
diopters
chlorisondamine n = 10 - 2.98 + 1.88 diopters- 8.58 + 2.06
** diopters
Ultrasound Measurements
axial length difference
vehicle 0.43 + 0.04 mm 0.61 + 0.06 mm
chlorisondamine 0.20 + 0.07 mm *** 0.33 + 0.11 mm
**
ratio - chlorisondamine/vehicle0.47 0.54
vitreous cavity length difference
vehicle 0.40 + 0.04 mm 0.69 + 0.06 mm
chlorisondamine 0.28 + 0.06 mm 0.49 + 0.08 mm
ratio - chlorisondamine/vehicle0.70 0.71
Caliper Measurements
axial length difference
vehicle ------ 0.51 + 0.06 mm
chlorisondamine ------ 0.22 + 0.10 mm
**
equatorial diameter difference
vehicle ------ 0.51 + 0.06 mm
chlorisondamine ------ 0.17 + 0.07 mm****
* p = 0.05; ** p < 0.05; *** p < 0.01; **** p < 0.005, comparing the drug
treated to vehicle
treated groups, using one-way analysis of variance on the difference of
goggled and
contralateral eyes. The number of chicks in each group are shown with the
refractions.
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