Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF PREVENTING PROLIFERATION OF RETINAL
PIGMENT EPITHELIUM BY RETINOIC ACID RECEPTOR AGONISTS
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to pharmacological uses of
retinoids. More particularly, this invention relates to
use of retinoids in treatment of ocular disorders.
2. Description of Related Art
The retinal pigment epithelium (RPE) forms a monolayer of
cells beneath the sensory retina that is normally
mitotically inactive except when it is participating in
retinal wound repair, in which it plays a central role.
When wound repair is complete, the RPE usually stops
proliferating; failure to do so can result in blinding
disorders such as proliferative vitreoretinopathy (PVR)
and disciform scarring. For instance, after detachment
of the sensory retina, the. RPE changes in morphology and
begins to proliferate. Multilayered' colonies of
dedifferentiated RPE cells are formed. Cells then begin
to migrate into the subretinal space where they engulf
rod outer segments. In some instances cells migrate onto
the surface of the retina and form epiretinal membranes.
These events have been implicated in the pathogenesis of
proliferative vitreoretinopathy, severe scarring
occurring in association with macular degeneration, and
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poor or delayed recovery of vision after retinal
reattachment.
Age-related macular degeneration (AND) is the major cause
of blindness in patients over the age of 60 in the United
States. Severe loss of vision in patients with AND is
usually due to the development of choroidal
neovascularization (CNV). Laser treatment can ablate CNV
and help to preserve vision in selected cases not
involving the center of the retina; however, the
treatment benefit is often transient due to the high rate
of recurrent CNV (50% over 3 years and approximately 60%
at 5 years) (Macular Photocoagulation Study Group, Arch.
Ophthalmol. 204: 694-701, 1986). In addition, many
patients who develop CNV are not good candidates for
laser therapy because the CNV is too large for laser
treatment, or the location cannot be determined so that
the physician cannot accurately aim the laser.
Despite these important consequences, little is known
about the stimuli involved in RPE dedifferentiation and
loss of density-dependent growth control. However, it is
known that cultured human RPE rapidly become depleted of
retinoids when maintained in media supplemented with
fetal bovine serum (FBS) (S.R. Das, et al., Biochem. J.,
250:459, 1988). Retinoids have been implicated in
cellular differentiation (S. Strickland, et al., Cell,
15:393-403, 1978; T.R. Brietman, et al., PNAS, 77:2936-
2940, 1980; and are normally present in high levels in
RPE in vivo. Retinoids play a prominent role in visual
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transduction and therefore their recycling is needed for
normal visual function. This recycling occurs through an
intimate relationship between the photoreceptors and the
RPE. Disruption of this intimate relationship during
retinal detachment prevents recycling of retinoids and
may be one reason for outer segment degeneration and
dedifferentiation of the RPE (P.A. Campochiaro, et al.,
Invest. Opthaimol. Vie. Sci., 32:65-72, 1991).
Incubation of cultured RPE cells with all-trans retinoic
acid (RA) inhibits cell proliferation and promotes a
morphologic appearance like RPE in situ (P.A.
Campochiaro, et al., supra; J. W. Doyle, et al., Curr.
Eye Res. 11:753-765, 1992). All-trans RA and other
derivatives of vitamin A (generally referred to as
retinoids) affect the growth and differentiation of many
cell types (S. Strickland, et al., supra; T.R. Breitman,
et al., Proc. Natl. Acad. Sci. USA, 7:2936-2940, 1980).
Therefore, retinoic acid or a related molecule may be one
of the signals that helps to maintain or re-establish
quiescence of RPE and other cells that participate in
PVR.
The biological effects of retinoids are mediated through
nuclear receptors which are ligand-induced trans-acting
factors that bind to DNA response elements, modulating
the transcription of genes containing those response
elements.
These receptors are divided into two major families,
retinoic acid receptors (RARs) and retinoid X receptors
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(RXRs). For each family there are three separate gene
products constituting three subtypes designated a, a, and
y (H.G. Stunnenberg, Bio' Essays, X5:309-315, 1993).
Alternative splicing of mRNA for these subtypes results
in different isoforms and even greater diversity. The
level of expression of RAR and RXR subtypes differs from
tissue to tissue, and differences in activity of subtypes
may provide some tissue specificity of retinoid effects
(P. Dolle, et al., Nature, 342:702-705, 1989; J.L. Rees,
et al., Biochem. J., 259:917-919', 1989). Retinoid
receptors up-regulate gene expression by binding to the
promoter regions of RA-responsive genes as
transcriptionally active RAR-RXR heterodimers (S. Nagpal,
et al., EMBO J., 12:2349-2360, 1993) or RXR homodimers
(X. Zhang, et al., Nature, 358:587-591, 1992); whereas
they down-regulate expression of other genes by
antagonizing the effect of transcription factors such as
AP-1 (M. Pfahl, Endocrine Review, 14:651-658, 1993;
Nagpal, et al . , J. Biol. Chem 270:923-927, 1995). AP-1
components c-Jun and c-Fos are products of immediate
early genes which are produced in response to the
mitogenic signals (e.g., growth factors and tumor
promoters) arriving at the cell membrane. The c-Jun/c-Fos
complex, in turn, activates the expression of AP-1-
responsive genes involved in cell division and cell
proliferation (T. Curran and B.R. Franzo, Jr., cell,
55:395-397, 1988; I.R. Hart, et al., Biochim. Biophys.
Acta. 989:65-84, 1989; P.K. Vogt and T.J. Bos, Trends
Biochem. Sal., 14:172-175, 1989). On the other hand,
retinoids inhibit cell proliferation and induce
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differentiation (L.J. Guoas, et al., in The Retinoids:
Biology, Chemistry and medicine, eds. M. B . Sporn, et al.,
Raven Press Ltd., New York, pp 443-520, 1994).
Therefore, in the light of the above mentioned
observations, retinoid mediated antagonism of AP-1-
dependent gene expression provides a basis of their anti-
proliferative effects. Another level of regulation is
provided by differences in ligand-receptor affinities.
All trans-RA is the endogenous ligand for RARs, while
that for RXRs is believed to be 9-cis-RA (R.A. Heyman, et
al., Cell, 68:397-406, 1992; A.A. Levin, et al., Nature,
355:359-361, 1992); however, 9-cis-RA can bind to and
activate the RARs as well. 9-cis RA is a stereoisomer
of all-trans RA and is generated from all-trans RA in
vivo during metabolism (R.A. Heyman, et al., supra).
Proliferative vitreoretinopathy (PVR) is the most common
cause of failure following rhegmatogenous retinal
detachment surgery. Despite meticulous surgical membrane
removal and the use of tamponades such as silicone oil
(SiO), failure occurs in a large number of cases due to
difficulty with complete removal and continuous growth of
the membranes. To date, the goals of surgery for PVR are
to relieve traction by removal of epiretinal membranes
and portions of foreshortened retina when necessary,
surround all retinal breaks with retinopexy, and inject
gas or silicone oil into the vitreous cavity to provide
retinal tamponade for a sufficiently long period of time
that all retinal breaks are sealed. Using these
techniques, retinal reattachment is achieved in 35-45% of
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eyes with PVR with one operation, and in up to 71% of
eyes with multiple operations. However, with each
operation the visual prognosis worsens (Silicone Study
Group, Silicone Study Report No. 2., Arch Ophthalmol,.
110:780-792, 1992). The major cause of failure is
reproliferation with regrowth of epiretinal membranes
resulting in traction and recurrent detachment.
Therefore, prevention of reproliferation is a major goal
in the treatment of PVR.
A number of experiments have been reported using
different antiproliferative agents, such as daunomycin,
alone or in combination with vitreoretinal surgery.
Retinoids are lipid soluble, as most antiproliferative
agents are not.. All-trans RA dissolved in SiO was tested
in a rabbit model of PVR, and produced a significant and
lasting reduction in cellular proliferation. At doses of
2 to 20 ug/ml SiO in 3 kilogram rabbits no histological
evidence of retinal toxicity was found, and the rate of
retinal detachment was decreased from 100% in untreated
controls to 44.5% in treated rabbit eyes at 8 weeks (J.J.
Araiz, et al., Invest. Ophthalmol. Vis. Sci., 34:522-30,
1993). This mode of retinoid delivery is suitable for
patients with advanced PVR in whom silicone oil is often
used, but is not applicable for use in patients with
early or less severe PVR or those patients at high risk
for PVR after retinal reattachment.
The rabbit model has also been used to test sustained
delivery of RA from microspheres of biodegradable polymer
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in treatment of PVR (G.G. Giordano, et al., Invest.
Ophthalmol. Vis. Sci., 344:2743-2751, 1993). Filling the
vitreous cavity with a suspension of biodegradable
microspheres into which a total of about 100pg of all-
trans RA was incorporated significantly decreased
tractional retinal detachment (TRD) in eyes treated with
gas compression vitrectomy and fibroblast injection.
Toxicity was limited to localized areas of inflammation
felt to represent a foreign body reaction.
Retinoic acid, its geometric isomer 13-cis-retinoic acid,
and synthetic derivatives have numerous biological
effects in several tissues. Retinoids are currently
used as the first line treatment for acute myelogenous
leukemia (R.P. Warrell, Jr., et al., N. Engl. J. Med.,
324:1385-1393, 1991), as adjuvant therapy for several
types of metastatic carcinomas (K. Dhingra, et al.,
Invest. New Drugs, 11:39-43, 1993; S.M. Lipman, et al.,
J. Natl. Can. Inst., 85:499-500, 1993), and for
treatment of psoriasis and other skin disorders. While
these varied effects of retinoids provide multiple
clinical applications, they are also the basis for
undesired effects and toxicity that can impede the
treatment of any one particular disorder. For instance,
13-cis-retinoic acid is associated with teratogenic
effects when administered to pregnant females of child-
bearing age. Thus, the need exists for additional
synthetic retinoid analogs that avoid these toxic effects
for use in treatment of PVR. Also, understanding the
mechanism by which retinoids exert their
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antiproliferative effect in RPE cells will enable the
development of new and adjunctive therapeutic agents for
PVR and related diseases.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a
method of preventing proliferation of retinal pigment
epithelium by retinoic acid receptor agonists. In
accordance with an aspect of the invention there is
provided a method for amelioration of disease associated
with proliferation of retinal pigment epithelium (RPE)
comprising contacting the retinal pigmented epithelial
cells of a subject in need thereof with a therapeutic
amount of a retinoic acid receptor (RAR) agonist, except
for retinoic acid.
In accordance with an embodiment of the invention there is
provided a method for amelior
anon of disease associated
with proliferation of retinal pigment epithelium (RPE)
comprising contacting the retinal pigmented epithelial
cells of a subject in need thereof with a therapeutic
amount of a retinoic acid receptor (RAR) agonist, except
for retinoic acid, wherein the therapeutic amount is in
the range from about 50 to 150 g, wherein the contacting
is by slow release, and wherein the RAR agonist is
formulated for compaction into microparticulates.
In accordance with an embodiment of the invention there is
provided a method for amelioration of disease associated
with proliferation of retinal pigment epithelium (RPE)
comprising contacting the retinal pigmented epithelial
cells of a subject in need thereof with a therapeutic
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amount of a retinoic acid receptor (RAR) agonist, except
for retinoic acid, wherein the therapeutic amount is in
the range from about 50 to 150 g, wherein the contacting
is by slow release, wherein the RAR agonist is formulated
for compaction into microparticulates, and wherein the
microparticulates are administered to the scleral pocket
or subconjunctival space.
In accordance with another aspect of the invention there
is provided a method for amelioration of disease
associated with proliferation of retinal pigment
epithelium (RPE) comprising contacting the retinal
pigmented epithelial cells of a subject in need thereof
with a therapeutic amount of an APi antagonist.
In accordance with another aspect of the invention there
is provided a method for amelioration of traction retinal
detachment comprising contacting the retinal pigmented
epithelial cells of a subject in need thereof with a
therapeutic amount of an AP-1 antagonist.
In accordance with another aspect of the invention there
is provided a method for amelioration of traction retinal
detachment comprising contacting the retinal pigmented
epithelial cells of a subject in need thereof with a
therapeutic amount of a retinoic acid receptor (RAR)
agonist.
In accordance with another aspect of the invention there
is provided a composition for treatment of proliferation
of retinal pigment epithelium comprising a therapeutic
amount of a retinoic acid receptor (RAR) agonist except
for retinoic acid.
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In accordance with another aspect of the invention there
is provided a method for amelioration of an ocular disease
associated with choroidal neovascularization comprising
contacting the retinal pigmented epithelial cells of a
subject in need thereof with a therapeutic amount of an
AP-1 antagonist.
In accordance with another aspect of the invention there
is provided a method for amelioration of an ocular disease
associated with choroidal neovascularization comprising
contacting the retinal pigmented epithelial cells of a
subject in need thereof with a therapeutic amount of a
retinoic acid receptor (RAR) agonist.
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Proliferation of retinal' pigment epithelium following
surgery or trauma or in ocular diseases associated with
choroidal neovascularization, such as age related macular
degeneration and histoplasmosis syndrome, is treated by
contacting retinal pigment epithelium cells with a
therapeutic amount of a retinoic acid receptor (RAR)
agonist, preferably one with specific activity, for
retinoic acid receptors and with potent ability in
inhibiting APi-dependant gene expression. This
proliferation is also treated with therapeutic amounts of
other agents that inhibit APi-dependent activity,. used
singly or in combination with RAR agonists. The
contacting can be accomplished by bolus injection into
the vitreous cavity or by providing the RAR agonist in a
slow release format, such as encapsulated into liposomes
or dissolved in a liquid tamponade injected into the
vitreous cavity or periocular space. Formulations for
preventing proliferation of retinal pigment epithelium
are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 is a graph showing molar concentration-dependent
potency of retinoid agonists for inhibition of serum
stimulated DNA synthesis in human RPE cells after
incubation for 7 days. 745 = Compound 745; 183 =
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Compound 183; RA = retinoic acid; 9-cis-RA = 9-cis-
retinoic acid; 659 = Compound 659; 701 = Compound 701.
FIGURE 2 is a series of micrographs at magnification x
150 using a phase contrast microscope showing the effect
of retinoid agonists on RPE cell morphology.
Magnification x 150.
Figures 2 A-D show cells (4 x 104) from a 60 year-old
donor grown in four different media. Figure 2A = serum
alone; 2B = 5% serum supplemented with 1pM all-trans
retinoic acid; 2C = 5% serum supplemented with fpm of
Compound 701; 2D = 5% serum supplemented with fpm of
Compound 183.
Figures 2 E-H show cells (4 x 104) from a 76 year-old
donor grown in four different media. Figure 2A = serum
alone; 2B = 5% serum supplemented with 1}1M all-trans
retinoic acid; 2C = 5% serum supplemented with ipm of
Compound 701; 2D = 5% serum supplemented with 1pm of
Compound 183.
FIGURE 3 is a graph showing the effect of several nuclear
receptor agonists alone or in combination with retinoic
acid in RPE proliferation. RA = retinoic acid; Dex and
Dexa = dexamethasone; T3 = thyroid hormone; D3 = 1,25-
dihydroxyvitamin D3.
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A DETAILED DESCRIPTION OF THE INVENTION
Provided herein is a method for treating proliferative
vitreoretinopathy (PVR) and other disorders of retinal
wound repair by contacting retinal pigmented epithelial
(RPE) cells with a therapeutic amount of one or more
compounds having activity as a retinoic acid receptor
(RAR) agonist. It has been discovered that RAR agonists
prevent the proliferation of RPE cells in vitro by
promoting density arrest and a differentiated morphology
in cultured RPE mediated through an RAR-activated
pathway, and are also clinically effective for inhibiting
traction retinal detachment in animals, such as humans,
as is commonly suffered following eye surgery and other
wounds to the retina that cause detachment of the RPE
from the photoreceptors. These results indicate that
inhibition of traction retinal detachment is mediated by
a RAR-activated pathway, but not by a RXR-mediated
pathway. Thus, RAR agonists are generally useful in the
treatment of conditions in which there is excessive
proliferation of RPE cells leading to retinal scarring or
retinal detachment.
On the other hand, RXR agonists have no such useful
clinical effects. Further, the primary mechanism by
which RAR agonists inhibit RPE proliferation is by
antagonism of API activity. Thus, RAR agonists with
specific anti-API activity, other agents that block AP1
activity, or combinations of these other agents and RAR
agonists with anti-API activity can be used to treat PVR.
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it is shown in the Examples herein that the above-
described retinoid compounds of this invention produce a
differentiated phenotype in retinal epithelial cells. One
of the differentiated functions of retinal pigmented
epithelial cells is to prevent growth of new blood
vessels from the choroid through Bruch's membrane into the
space beneath the RPE, or into the subretinal space.
This process is called choroidal neovascularization. It
occurs in several disease processes, the most common of
which is age-related macular degeneration. Consequently,
treatment with the retinoid compounds of this invention
helps to maintain RPE in the differentiated state and,
therefore, helps to prevent choroidal neovascularization
in patients.
The patients with age-related macular degeneration
represent the largest group in which choroidal
neovascularization is a major problem. In addition,
younger patients who suffer choroidal neovascularization,
such as those with ocular histoplasmosis syndrome, are
also benefited by treatment with the retinoid compounds
of this invention.
In the method of this invention, a therapeutically
effective amount of an RAR. agonist having potent activity
as an antagonist of API or another anti-API agent or a
combination of these agents is introduced into the
vitreous cavity or periocular space of a patient who has
PVR or choroidal neovascularization, or is at risk of
developing these conditions due to disease, surgery,
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trauma or aging. In the case of surgery, it is
particularly preferred that the RAR agonist or other
anti-API agent or combination be given after surgery for
PVR to prevent reproliferation of RPE cells and
redetachment of the retina.
As used herein, the term "a therapeutically effective
amount" of an RAR agonist is an amount calculated to
achieve and maintain a therapeutic level in the vitreous
cavity, if introduced directly into the vitreous cavity
or periocular space, or in the bloodstream, if
administered peripherally. over the period of time
desired in a human or animal such as to substantially
inhibit proliferation and thus restore differentiation of
RPE. It is preferred that the therapeutic amount be an
amount sufficient to inhibit proliferation of at least 50
percent, more preferably 80 percent of the RPE cells in
an eye under treatment. The therapeutic amount will vary
with the potency of each RAR agonist, the amount required
for the desired therapeutic or other effect, the rate of
elimination or breakdown of the substance by the body
once it has entered the vitreous cavity or bloodstream,
and the amount of the RAR agonist in the formulation. In
accordance with conventional prudent formulating
practices, a dosage near the lower end of the useful
range of a particular agent is usually employed
initially, and the dosage is increased or decreased as
indicated from the observed response, as in the routine
procedure of the physician.
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For administration directly into the vitreous cavity of
the eye, an amount in the range between about 50 and 150
pg administered within about 24 hours following surgery
or trauma generally provides protection against
development of PVR. In an alternative embodiment, a
second dosage of the RAR agonist after an interval of
several hours, usually between about 8 and 36 hours,
preferably about 24 hours, is injected intravitreally or
subconjunctivally. Alternatively, a combination of
intravitreal and subconjunctival injection of the
retinoid, either simultaneously or at the above described
spaced interval, can be used to administer the retinoid.
For intravitreal injection, it is preferred that the RAR
agonist be injected into the anterior vitreous cavity
using topical or retrobulbar anesthesia. In an
alternative embodiment, the RAR agonist is introduced
intravitreally using a drug delivery vehicle. For
instance, the RAR agonist can be dissolved in a
biologically inert fluid that is also useful as a
mechanical tamponade to help keep the retina in place,
preferably an oil such as silicone oil in which the
retinoid is soluble. However, for RAR agonists having
partial miscibility, a liquid other than an oil can be
used.
However, intravitreous injection of the retinoids as a
bolus injection can result in focal areas of retinal
damage. In addition, it has been discovered that the
therapeutic effects of the retinoids of this invention
are delayed in onset and reversible. Therefore, it is
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advantageous to administer the retinoids utilizing a
method of a slow release, for instance by intravitreal
injection of the dose of retinoid encapsulated in a
microvesicle, such as a liposome, from which the dose is
released over the course of several days, preferably
between about 3 to 20 days. Alternatively, the drug can
be formulated for slow release, such as incorporation
into a slow release polymer from which the dosage of drug
is slowly released over the course of several days, for
example from 2 to 30 days.
As is known in the art, liposomes are generally derived
from phospholipids or other lipid substances. Liposomes
are formed by mono- or multilamellar hydrated liquid
crystals that are dispersed in an aqueous medium. Any
non-toxic, physiologically acceptable and metabolizable
lipid capable of forming liposomes can be used. The
present compositions in liposome form can contain
stabilizers, preservatives, excipients, and the like in
addition to the agent. The preferred lipids are the
phospholipids and the phosphatidyl cholines (lecithins),
both natural and synthetic.
Methods to form liposomes are known in the art. See, for
example, Prescott, Ed., Methods in Cell Biology, Volume
XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq.
Regardless of the mode of administration, the RAR agonist
can be either naturally occurring or a synthetic
retinoid, preferably having selective activity as an
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agonist for RARs and high potency.in antagonism of AP-1-
dependent gene expression. Examples of naturally
occurring retinoids with activity as RAR agonists are
all-trans retinoic acid (all-trans RA) and 9-cis retinoic
acid (9-cis RA), which are stereoisomers, all-trans RA
being naturally converted into 9-cis RA during metabolism
(J.G. Allen, et al., Pharmac. Ther., 4Q:1-27, 1989).
However, 9-cis RA has activity as agonists for both RARs
and RXRs, and all-trans-RA, because of its metabolism or
chemical isomerization to 9-cis-RA, can also effectively
activate both RARs and RXRs; whereas the preferred
retinoid compounds for use in the practice of this
invention have specific activity as RAR agonists.
Synthetically prepared retinoids are well known in the
art. For example, U.S. Patent No. 5,234,926 discloses
methods of synthesizing disubstituted acetylenes
bearing heteroaromatic and heterobicyclic groups with
selective activity as RAR agonists. U.S. Patent No.
4,326,055 discloses methods for synthesizing 5,6,7,8-
tetrahydro naphthyl and indanyl stilbene derivatives
with retinoid-like activity. Since it is known that
proliferation of cultured RPE cells is inhibited in
vitro by retinoid compounds having activity as RAR
agonists and not by compounds having activity as RXR
agonists, anti-proliferative retinoid compounds can
readily be selected by determining whether they have
RAR activity, for instance by utilizing well known in
vitro
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transacivation assay techniques such as that disclosed by
M. Pfahl, et al., Methods in Enzymology, L__:256-270,
1990, and as illustrated. in Example 1 of this
invention. A RAR selective agonist will prevent cell
overgrowth, resulting in a cell morphology
indistinguishable from that caused by all-trans RA.
Examples of synthetic RAR agonists suitable for use in
the practice of this invention are ethyl 6-[2-(4,4-
dimethylthiochroman-6-yl)ethynyl]nicotinate (Compound
168) and 6-[2-(4,4-dimethylchroman-6-yl)ethynyl] nicotinic
acid (Compound 299), whose synthesis is disclosed in U.S.
Patent No. 5,234,926 as Examples 6 and 24, respectively;
and p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-
naphthyl)propenyl]-benzoic acid (Compound 183), whose
synthesis is disclosed in U.S. Patent Number 4,326,055.
By contrast, an example of an RXR selective agonist is 2-
[ (E) - 2- (5 , 6, 7, 8- t e t r a h y d r o- 3, 5, 5, 8, 8-
pentamethylnaphthaleen-2-yl)propen-1-yl]thiophene-4-
carboxylic acid (Compound 701), whose synthesis is
disclosed in U.S. Patent Number 5,324,840, Example 11.
Preferably the RAR agonist is selected to be
metabolically stable and to remain completely specific
for the RAR pathway. Thus, all-trans RA, which
isomerized to 9-cis RA during metabolism and results in
activation of both the RAR and RXR pathways, is not
preferred for use in the practice of this invention.
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The cross-talk between the retinioid and AP-1 signal
transduction pathways can be manipulated for therapeutic
benefit in hyperproliferative diseases. This has been
demonstrated in this invention since the retinoids that
are STRONG antagonists of AP-1-dependent gene expression,
are also STRONG INHIBITORS of RPE cell proliferation.
Potent anti-proliferative RAR-agonists can be screened by
determining their ability to inhibit AP-1-dependent gene
expression in a transient transfection CAT assay as
illustrated in Example 2 of this invention.
The following examples illustrate the manner in which the
invention can be practiced. It is understood, however,
that the examples are for the purpose of illustration and
the invention is not to be regarded as limited to any of
the specific materials or conditions therein.
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EXAMPLE 1
The transactivation properties of retinoids were
determined by measuring their ability to induce
transcription in the HeLa cells by transiently
cotransfecting them with a receptor gene construct and a
reporter gene. Since retinoid receptors are members of
the steroid receptor family of nuclear receptors that are
characterized by homologous functional domains, hybrid
receptors were used that contain the amino terminus and
DNA-binding domain of the estrogen receptor and the
hormone-binding domain of the retinoid receptors, either
RAR-a, 8, y, or RXR-a. These ER/R.AR receptors activate
transcription by binding to promoter sequences recognized
by the estrogen receptor (estrogen response element), but
do so in response to a retinoid (D. Benbrook, et al.,
Nature, 333:669-672, 1988). Previous studies have shown
that the activation characteristics of hybrid receptors
are determined by their ligand binding domain. To
determine activation of the hybrid constructs by the
retinoids, an estrogen receptor-responsive reporter gene
was used that cannot be activated by endogenous retinoid
receptors, which are present in most, if not all,
mammalian cells.
The Cationic Liposome Mediated Transfection Assay by
which the activity of a test compound as a potential
agonist of the RXR and RAR receptor sites is determined,
is performed substantially as reported by P . L. Feigner,
et al., Focus, 11:2, 1989,
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and is described below first in principle and
thereafter in the form of specific instructions how to
perform the assay.
In connection with this assay it is known that retinoic
acid receptors are a member of the steroid/thyroid
receptor super family and that they contain domains which
are interchangeable within individual receptors. Thus,
plasmids for chimeric retinoid receptors containing
estrogen DNA binding domain and estrogen response element
chloramphenicol acetyl-transferase (CAT) enzyme are
constructed and are grown in specific cultured bacteria.
These plasmids respectively code for chimeric RARO, RARe,
RAR,,, or RXRa receptor proteins, and for the chloramphen-
icol acetyl A transferase (CAT) enzyme protein. The
bacteria with these plasmids are obtainable in accordance
with the procedure set forth in the article titled
"Nuclear Retinoic Acid Receptors: Cloning, Analysis, and
Function", M. Pfahl, et al., Methods in Enzymology, 189:
256-270, 1990). The detailed procedure for isolating
the DNA plasmids from the respective bacteria is also
set forth below in detail, in the form of specific
instructions under the title "Supercoiled Plasmid
Isolation".
Thus, in accordance with the test procedure, a DNA
plasmid that codes for one of the chimeric RARa, RARE,
RARY, or RXRa receptor proteins is transfected into
cultures of HeLa cells. It is for this purpose that HeLa
cells are grown in a medium during the first day of the
CA 02558199 1996-01-31
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assay detailed below as the "Cationic Liposome Mediated
Transfection Assay". In the transfection procedure,
which is performed during the second day of the
transfection assay, the DNA plasmid coding for the CAT
enzyme is also added to each cell culture, in addition to
the respective chimeric RARa, RAR8, RARY, or RXRU coding
plasmid.
As is known and will be readily understood by those
skilled in the art, especially in view of the above-cited
M. Pfahl, et al, article, chimeric retinoid receptors
involved in this assay include a ligand binding domain
that recognizes and binds specific agonist molecules,
such as retinoic acid and analogs. These chimeric
protein receptors (which were constructed in accordance
with the teachings of the M. Pfahl, et al. article) also
contain a DNA binding domain, which is capable of binding
to the "estrogen response element" (a DNA fragment)
attached to the DNA plasmid coding for the CAT enzyme.
The nature of the interaction is such that only when an
agonist (such as retinoic acid or analog) is bound to the
ligand binding domain of the respective RARa, RAR., RAR ,
or RXRQ receptor, is the receptor bound through its
DNA-binding domain to the estrogen response element of
the estrogen-response-element-chloramphenicol-acetyl
transf erase-construct (ERE-CAT) and capable of initiating
transcription of messenger RNA for the CAT enzyme. In
other words, through multiple interactions CAT enzyme is
manufactured by the HeLa cell in this assay only if an
CA 02558199 1996-01-31
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appropriate agonist ligand binds to the ligand binding
site of the respective retinoid receptor.
The estrogen response-element-chioramphenicol
acetyl-transferase construct (ERE-CAT) is itself
obtained in accordance with the procedure described in
G.U. Ryssel, et al. (Cell, 46:1053-1061, 1986). This
procedure per se is well known in the art. The specific
detailed procedure for isolating and obtaining the
estrogen-response-element chloramphenicol-acetyl-
transferase-construct (ERE-CAT) from bacteria is
described in the procedure titled "Supercoiled Plasmid
Isolation".
in addition to the foregoing, lipofectin (LF) is also
added to each cell culture. The purpose of the
lipofectin is to facilitate transport of plasmids through
the cell membrane. The lipofectin used in the procedure
is available commercially.
As will be well understood by those skilled in the art,
as a result of transfection with the 25 respective DNA
plasmids coding for RARa, RARE, RAR,, or RXRd chimeric
receptors and as a result of transfection with the
ERA-CAT (which codes for the CAT enzyme as described
above), the aforementioned plasmids are incorporated into
the HeLa cells cultured in the assay. The retinoid
receptor plasmids undergo transcription (into m-RNA) and
subsequent translation into the corresponding chimeric
CA 02558199 1996-01-31
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receptor protein. Therefore, the HeLa cells cultures
obtained in this manner manufacture the respective RARU,
RAP., RAH, or RXRa chimeric receptor protein. As a result
of transfection with the ERA-CAT the cell cultures of
this assay also contain the genetic information for
manufacturing the CAT enzyme. However, as is noted
above, the latter genetic information is not transcribed,
and the CAT enzyme is not manufactured by the respective
cell cultures of this assay, unless an appropriate
agonist compound binds to and activates the respective
RARa, RAR,, RAR., or RXRa, chimeric receptor protein in
the cell and this activated agonist-receptor complex
binds to the estrogen response element of the ERE-CAT
construct.
The assay procedure is continued by adding, on the third
day of the assay, an appropriate reference compound and
the test compound (agonist or prospective agonist) to the
respective HeLa cell culture, preferably in varying
concentrations. As a result of this addition, if the test
compound is an agonist, it binds to the respective RARa,
RARE, RAR., or RXRa chimeric receptor protein, and
consequently the genetic information which codes for the
CAT enzyme is transcribed in the cell, whereby CAT enzyme
is made by the cell.
After lysis of the cell, which is performed on the fourth
day of the below-detailed assay procedure, the activity
of CAT enzyme in aliquot portions of the lysate is
measured. This is done by incubating the lysate with
CA 02558199 1996-01-31
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chioramphenicol and tritium labeled acetyl coenzyme A.
As a final measurement, the amount of tritium labeled
acetyl chioramphenicol, which is formed in the enzymatic
reaction involving the CAT enzyme, is measured in a
scintillation counter.
The reference compound is retinoic acid (all trans) for
the assays involving the RAR2, RARE, and RAR , receptors,
and 4-(E)-2-(5,6,7,8-tetrahydro 3,5,5,8,8-pentamethyl-
naphthalen-2-yl)-propen-1-yl benzoic acid (also
designated Compound 440 in this application) for the RXRa
chimeric receptor. The data obtained in the assay are
evaluated and expressed as follows. For each test
compound and for each subspecies of the RAR receptors, a
graph (or the mathematical equivalent of a graph) is
prepared where the "counts per minute" (cpm) obtained in
the scintillation counter measurements are plotted (on
the y axis) against the concentration of the test
compound. A similar graph (or mathematical equivalent) is
prepared for retinoic acid. EC50 of the test compound is
defined as that concentration of the test compound which
provides one-half (50%) of the maximum cpm number
(maximum CAT enzyme activity) obtained in the same
receptor in the same assay with the reference compound
retinoic acid.
To evaluate and express the data obtained in the assay
for the RXRU the same type of graph (or
mathematical-equivalent) is prepared for the test
compound, and also for the reference compound, Compound
CA 02558199 1996-01-31
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440. 'This reference compound is a known agonist of the
RXRa receptor site. ECG is that concentration of the test
compound which gives one half (500) of the counts per
minute (CAT enzyme activity) of the maximum cpm obtained
with Compound 440 on the same receptor in the same assay.
SUPERCOILED PLASMID ISOLATION
Large Scale 1L Prep
DNA isolation
1. Place cells on ice for 15 minutes. Harvest the
bacterial cells (E. coli) by spinning down in 250 ml
nalgene tubes at 7k rpm, 10 minutes at 4 C using JA14
rotor, Beckman J2-21 M centrifuge. Discard the
supernatant.
2. To each cell pellet add 1.0 ml Solution I, vortex to
resuspend the pellet. Transfer the 1.0 ml of cells from
one bottle to another. Transfer this to a 50 ml Oakridge
tube. Use 4ml of Solution I and wash the bottles again
transferring from one bottle to the next. Transfer this
also into the Oakridge tube. Using a pipet bring up the
total volume to 16m1 with Solution I and mix the
solution. Transfer 8 ml to a second Oak ridge tube. Store
at room temperature for 5 minutes.
CA 02558199 1996-01-31
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Solution I
50 mM glucose, 25 mM Tris-Ci pH 8, 10 mM EDNA pH 8
3. Add to each tube ml of freshly prepared Solution II.
Mix contents gently by inverting the tube several times.
Store on ice for 10 minutes. After this time the liquid
should be clear with no aggregates. (If there are
clumps, then the cells were not resuspended well enough
previously.)
Solution II
1o sodium dodecylsulfate (SDS), 0.2N NaOH (4 ml 10%
SDS, 0.8 ml ION NaOH, 35.2 ml water)
4. Add 12 ml, (or as much as will fit) of ice-cold
Solution III. Mix the contents of tube by inverting it
sharply several times. A white flocculent precipitate
should appear. Store on ice for 10 minutes.
Solution III
Prepare as follows: to 60 ml 5M potassium acetate,
add 11.5 ml of glacial acetic acid and 28.5 ml water
5. Centrifuge at 4'C in a Beckman J2-21M centrifuge
JA20 rotor, (Beckman Instruments, Carlsbad, CA) at 17k
rpm for 30 minutes.
6. Pipet approximately 12 ml of supernatant from the
Oakridge tubes into 6 baked Corexo tubes. Add 0.6 volumes
of isopropanol (7.2 ml) mix by inversion and store at
room temperature for 15 minutes to precipitate DNA.
CA 02558199 1996-01-31
= -27-
7. Warm Beckman centrifuge by spinning JA20 rotor at
14k rpm for 15 minutes at 20'C.
8. Pellet DNA at 20'C in the J2-21M centrifuge, JA20
rotor at 10.5k rpm for 30 minutes (use adapters for corex
tubes).
9. Pour off supernatant, dry inside of tube with
pasteur pipet on a vacuum flask.
10. Dry in vacuum dessicator for 10 minutes (longer
drying time will make it hard to dissolve pellet).
Purify plasmid DNA by centrifugation to equilibrium in
CsC1 density gradients.
11. Dissolve pellet by adding 1 ml TE (10 mM Tris-Ci pH
8, 1 mM EDNA pH8) to each Corex tube. Place tubes in
37'C water bath to help pellets to dissolve faster (15-30
minutes).
12. Transfer liquid from like batch into one tube.
Bring volume to 8.5 ml with TE.
13. Add 1001.il RNase, DNase free (2U/}11, source
Boehringer Mannheim Biochemical (BMB) (Indianapolis, IN).
14. Add 400p1 of 10mg/ml Ethidium Bromide.
15. Add 9.0 g CsC1 and mix using a pasteur pipet.
CA 02558199 1996-01-31
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16. Add solution to two 13x51 mm Beckman polyallomer
quick-seal centrifuge tubes.
17. Spin at 50k rpm for 12 hours in Beckman
ultracentrifuge, VTi65.2 rotor, 200C.
18. After ultracentrifugation, two bands of DNA should
be visible. The upper band consists of linear bacterial
DNA and nicked circular plasmid DNA. The lower band
consists of closed circular plasmid DNA. Only the lower
CsCl-banded DNA is removed from the tube with a 3-ml
syringe fitted to an 21-gauge needle (Needle is inserted
into the side of the tube and 1.5-2 ml is removed).
19. Preparation for Second CsCl centrifugation:
(9 ml - vol 1st CsCl band) - number g CsCl
(9 ml - vol 1st band - 100u1 10mg/ml Ethidium
Bromide - 50u1 RNase) - ml TE pH 8.0
Combine 1st band, TE, CsCl, RNase and EtBr.
20. Add solution to 2 quick-seal tubes.
21. Spin at S0k for 12 hours or 60k rpm for 4 hours in
ultracentrifuge, VTi65.2 rotor, 20'C.
22. Remove twice CsCl-banded DNA (lower band only) to a
ml Falcon snap tube (as.in step 18).
CA 02558199 1996-01-31
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Extraction of ethidium bromide
23. Under fumehood add an equal volume isoamyl alcohol,
vortex, spin at room temperature at 1500 rpm in Beckman
TJ-6 centrifuge for 3 minutes.
24. Transfer bottom aqueous layer to fresh tube. Repeat
3-4 times or until aqueous layer is clear (no pink
color).
25. Transfer clear aqueous layer to Spectra/Por 3
dialysis tubing, mwco 3500. (Tie a knot in the bottom of
tubing before clamping dialysis tubing.) Add liquid
using a pasteur pipet. Clamp top or dialysis tubing.
Using a rubber band suspend tubing in 2.8 L TE (28 ml 1M
Tris-C1, pH8, 5.6 ml 0.500M EDNA, pH8). Always handle
dialysis tubing carefully, with gloves.
26. Dialyze aqueous phase against several changes of
2.8 L TE pH8 (lx 2-4 hours, overnight and lx 2-4 hours
the next day).
27. In the tissue culture hood transfer the dialyzed DNA
into sterile microcentrifuge tubes. Label tubes and
store at -20 C.
CATIONIC LIPOSOME-MEDIATED TRANSFECTION
Reference: P.L. Feigner, et al., Focus, 11: 2, 1989.
CA 02558199 1996-01-31
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USE STERILE TECHNIQUE TEROUGHOUT.
Grow up HeLa or CV-1 cells in T-125 culture flask. Cells
are passed twice a week, usually on Monday and Friday
(0.5 ml cells into 15 ml medium).
DAY 1: Plating cells
1. Trypsinize and collect cells from T-162 cm2 culture
flask. Count cells using a hemocytometer. Usually, this
amount of cells is enough for sixteen 12-well plates.
2. Based on the cell number, dilute cells in medium
(D-MEM low glucose, 10% fetal bovine serum (FBS), 2 mM
Glu) to a concentration of 60,000 cells per well.
Example cell calculation:
want 40,000 cells/well and 200 wells
have (X) cells/ml
15 therefore, 40,000 cells/well x 200 wells - total # ml
cells
(X) cells/ml
needed
Using a 200m1 filter unit receiver add total #ml cells to
20 medium and bring final volume to 200 ml. Mix well by
pipetting.
3. Add 1.0 ml of cells per well using a sterile 12.5 ml
dropper. Shake plates back and forth (do not swirl).
Incubate at 37 C in a humidified 5% CO2 environment
overnight. Cells are about 40% confluent prior to
transfection.
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Transfection: DAY 2 PREPARATION DNA/LIPOFECTIN COMPLEX
1. Using 50 ml polystyrene tubes prepare Lipofectin
(LF) and DNA separately. Determine vol of LF and DNA
needed for 2 jig LF/well, 500 ng ERE-CAT DNA /well, 100 ng
ER/RAR DNA per well. Determine total volume needed for
experiment. (DNA concentration will vary between each
plasmid prep). Separately dilute LF and DNA in Opti-Mem
media (Gibco-BRL; Gaithersburg, MD) a volume of 25 ul x
#wells: Vol Opti-Mem
1 = (25 ul x #wells) - total vol. DNA or LF.
2. Add the diluted LF to the diluted DNA and swirl tube
gently. Let sit at room temperature for 10 min.
3. Aspirate off the medium from the wells and wash 2X
using 0.5 ml Opti-Mem I (sterile 12.5 ml combitip,
setting 2).
4. Add the DNA/LF complex to vol of Opti-Mem: (450111 x
# wells). Invert tube to mix. Using a sterile 12.5 ml
dropper add 500 111 to each well. Shake plates back and
forth to mix, do not swirl.
5. Incubate the cells for 6 hours at 37'C in a
humidified 5% CO2 incubator.
6. After 6 hours add 0.5 ml medium to each well
(Dulbecco's Modified Eagle's Media(D-MEM) low glucose,
20% FBS charcoal treated, 2 mM Glu) Use 12.5 dropper and
place back in the incubator.
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DAY 3: Drug addition
1. 18 hours after the start of transfection add
retinoids in triplicate (1'0 pi) using a sterile 0.5 ml
dropper and incubate for 20-24 hours at 37 C in a
5 humidified 5o CO2environment.
DRUG DILUTIONS
weight (a) X 1 X 100 ml = ml
ACETONE
mol. wt (g/mol) .005 mol/L L
10 Example: Retinoids are dissolved in acetone to a
concentration of 5 mM and further diluted to 1mM in EtOH.
If retinoids do not go into solution, place tube in hot
water for 5 seconds followed by vigorous vortexing. Each
experiment may have a different dilution scheme. For 2
concentrations per order of magnitude use a 3.16-fold
dilution as follows: To labeled sterile 12x75 mm tubes
add 1080 ul of 100% EtOH. Using the 1 mM solution,
transfer 500 ul to the next tube (316pM) . Vortex and
repeat the transfer to the next tube down the line. Some
retinoids are light sensitive, especially RA and 13-cis
RA, and should be used with a red or very dim light.
CA 02558199 1996-01-31
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Example
Drug Dilution Vol add to well Final: -log (conc.]
mM (initial)
1 mm 10 5.0
5 316p1 10 5.5
OpM 10 6.0
31.6}1M 10 6.5
10ptM 10 7.0
3.16pM 10 7.5
10 1pM 10 8.0
316nM 10 8.5
100nM 10 9.0
31.6nM 10 9.5
lOnM 10 10.0
3.16nM 10 10.5
1.OnM 10 11.0
Day 4 MIXED PHASE CAT ASSAY
1. Wash cells in 12 mm wells once with 0.50 ml 1X PBS
(no Ca/mg).
2. Using a 5 ml pipet add 100 pl of a ice cold 10i
Triton, 1 mM Tris-C1 pH7.8, 2mM EDTA pH8, DNase I.
Prepared as follows:
CA 02558199 1996-01-31
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LYSIS BUFFER (hypotonic buffer)
2.0 mg DNase I (Sigma, St. Louis, MO)
4.925 ml water
50.0 p1 100o Triton X-100 (BMB)
5.0 p1 1M Tris-C1 pH 7.8
20.0 p1 0 .5M EDTA pH 8
5.0 ml
3. Place on ice for 60 minutes. Agitate occasionally.
4. Transfer 50 ul of lysate from 3 wells at a time
using titertrek multichannel pipet with tips attached to
channels #1, #3, #6 to 96 U-bottom well (Costar,
Cambridge, MA). Place (unused lysate) plates at -20'C.
5.' Using a 1.25 ml combipipet (setting 1) add 50 ul
premix per well, gently shake plates and incubate 37'C
for 2 hours.
CA 02558199 1996-01-31
-35-
Vol. per Vol. per
Blank reaction X (#assays) = total
vol.
47.0 27.0 p1 buffer I (250 mM Tris-C1 pH 7.8,
5mM EDTA
1.5 1.5 ill 1 mM HCl
*** 20.0 ul 5 mM Chloramphenicol (make fresh
in buffer 1)
0.75 0.75 ul 4 mM Acetyl CoA in water (make
fresh)
0.80 0.80 p1 3H-Acetyl CoA (New England
Nuclear, Boston, MA #NET-290L, 200mCi/mmol
6. Using a titertrek multichannel pipet, add 100 41 of
7M Urea (Mallincrokt, Chesterfield, MD) into each
reaction well to quench the reaction. Do six at a time.
7. Using a titertrek multichannel pipet, transfer 200
41 reaction mixture into a 5 ml plastic scintillation
vial (Research Products International #125514, Mount
Prospect, IL). Do three reactions at a time.
8. Add 1 ml 0.8% 2,5 Diphenyloxazole(PPO) /toluene (3.2
g PPO (Mall inckrodt -RPI) /4L Toluene (Mallinckrodt
ScintillAR'). Vortex vigorously for 5 seconds and allow
the phases to separate for 15 minutes. Count cpm for 2.0
min-Beckman LS 3801.
The retinoid activities were determined at receptor
subtypes RAR-a, RAR-0, RAR-y and RXR-a.
CA 02558199 1996-01-31
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Table 1 below shows the EC50 for activation of RAR-a, 8,
y, and RXR-a for each of the retinoids used in this
study. Four of the agents showed good selectivity for
activation of RAR receptors compared to RXR receptors.
Two agents, 9-cis-RA and synthetic agonist Compound 659
activate both RAR and RXR receptors.
TABLE 1
POTENCY OF RETINOID AGONISTS AT INDUCING
TRANCRIPITIONAL ACTIVATION THROUGH SPECIFIC RECEPTORS
Compound No. RARa RARE RAR Rim-32
183 30 2 1 15,000
745 45 235 590 N/A
All trans RA 5 1.5 0.5 )10'000
9-cis RA 100 3 5 20
659 N/A 20 100 15
701 N/A 3000 1000 100
EXAMPLE 2
Primary cultures of human retinal pigment epithelium
(RPE) cells were established from eyes obtained from the
Old Dominion Eye Bank (Richmond, VA) or the Eye Bank of
Maryland (Baltimore, MD) using the technique described in
P.A. Campochiaro, et al., Invest. Ophthalmol. Vis. Sci. ,
27:1615-1621, 1986.
CA 02558199 1996-01-31
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The RPE cell lines used in this study were from two
donors aged 60 and 76 years, respectively, and each was
stained uniformly for cytokeratins using a known
technique (K.H. Leschey, et al., Invest. Ophthalmol. Vis.
S Sci., 31:839-846, 1990). All-trans RA was obtained from
Sigma (St. Louis, MO) and 9-cis retinoic acid and
synthetic retinoid agonists were obtained from Allergan,
Inc. (Irvine, CA).
For [3H]thymidine incorporation, RPE cells at passage 3
or 4 were lightly trypsinized and plated in 16-mm wells
of 24-well plates. The transfected cells were allowed to
attach overnight and then the media containing 5o fetal
bovine serum (FBS) were supplemented with retinoids or
vehicle alone. Stock solutions (10"3M) of retinoids were
prepared in dimethylsulfoxide (DMSO) and stored as frozen
aliquots until used; the highest final concentration of
DMSO was 0.11; and was also used for control cultures.
The media containing freshly prepared retinoids were
changed every three days. At 7 or 10 days 2pCi/ml of
[3H]thymidine (specific activity, 6.7 Ci/mM; New England
Nuclear, Boston, MA) was added to the cultures and
incorporation was measured as previously described
(Leschey, supra).
The potency of retinoid agonists for inhibition of serum
stimulated DNA synthesis in human RPE cells was tested.
RPE cells (4x104 cells) were plated in 16-mm wells of 24-
well plates and grown for 6 days in DMEM containing 10a
fetal bovine serum (Life Technologies, Inc.) supplemented
CA 02558199 1996-01-31
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with various concentrations of one of the following
compounds: Compound #745, 183, 659, or 701, all-trans
retinoic acid (RA) or 9-cis retinoic acid (9-cis-RA). The
cells were shifted to serum-free media containing the
same concentration of retinoid and then pulsed with 10%
serum for 18 hours, after which [3H]thymidine
incorporation was measured. Thymidine incorporation for
cells grown in control medium was used to calculate the
percent inhibition of thymidine incorporation induced by
four concentrations of each retinoid (each concentration
tested in triplicate) which were then used to generate
each line.
As shown by the results summarized in Figure 1,
incubation of RPE cells for 7 days in all-trans-RA or 9-
cis-RA results in dose-dependent inhibition of
[3H] thymidine incorporation.
As shown by the results summarized in Table 2, a 10 day
incubation results in greater inhibition and the
potencies of all-trans RA and 9-cis-RA are very similar.
Incubation of RPE cells for 7 or 10 days in each of four
synthetic retinoids that selectively activate RAR
receptors results in strong inhibition of [3H]thymidine
incorporation.
CA 02558199 1996-01-31
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TABLE 2
EFFECT OF RETINOID TREATMENT FOR TEN DAYS
ON [3H1 THYMIDINE INCORPORATION IN RPE CELLS
Percent Inhibition
Compound No. 5x10-8M 10-7M 10-6M
183 76.7 98.6 98.8
745 79.2 98.0 98.6
All trans RA 52.6 78.3 99.4
9-cis RA 57.8 79.8 98.2
659 20.3 73.2 98.7
701 12.0 48.8 74.6
The RXR receptor-selective agonist (Compound 701) was a
much less effective inhibitor of RPE [3H]thymidine
incorporation than the RAR-selective agonists,
and agonists that activate both RXR and RAR receptors (9-
cis RA and Compound 659) were not better inhibitors than
those that activate only RAR receptors.
EXAMPLE 3
The anti-AP-1 properties of retinoids are determined by
measuring their ability to inhibit AP-1-dependent gene
expression in HeLa cells by transiently cotransfecting
them with a reporter gene and a receptor expression
vector. Since the DNA binding domain of the RARs is
involved in the inhibition of AP-1-dependent gene
expression (R. Schule, et al., Proc. Natl. Acad. Sci.
USA, 88:6092-6096, 1991), holoreceptors of RARs (a, J3,
CA 02558199 1996-01-31
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USA, 88:6092-6096, 1991), holoreceptors of RARs (a, R,
and y) are used in transfection assays to quantitate the
relative potency of retinoids in antagonism of AP-1-
dependent gene expression.
Recombinant plasmids
The expression vectors for RARs (a, R and y) have been
described (E.A. Allegretto, et al., J. Biol. Chem.,
268:26625-26633, 1993). AP-1-reporter plasmid construct
Str-AP-1-CAT was prepared by cloning -84 to +1 base pairs
of rat stromelysin-1 promoter (L.M. Matrisian, et al.,
6:1679-1686, 1986) in Hind III-Bam HI sites of pBLCAT3
(B. Luckow and G. Schutz, Nucl. Acids Res., 15:5490,
1987). This sequence of stromelysin-1 promoter contains
an AP-1 motif as its sole enhancer element (R.C.
Nicholson, et al., EMBO J., 9:4443-4454, 1990). The
promoter sequence was prepared by annealing two synthetic
oligonucleotides 5'-AGAAGCTT ATG GAA GCA ATT ATG AGT CAG
TTT GCG GGT GAC TCT GCA AAT ACT GCC ACT CTA TAA AAG TTG
GGC TCA GAA AGG TGG ACC TCG A GGATCCAG-3' (SEQ ID NO:1)
AND 5'-CT GGATCC TCG AGG TCC ACC TTT CTG AGC CCA ACT TTT
ATA GAG TGG CAG TAT TTG CAG AGT CAC CCG CAA ACT GAC TCA
TAA TTG CTT CCA T AAGCTT CT-3' (SEQ ID NO: 2) containing
Hind III and Bam HI restriction sites at their ends. The
specific details for obtaining the supercoiled plasmid
expression and reporter vectors have been described in
detail in Example 1.
CA 02558199 1996-01-31
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Transfection of cells an CAT assays
For retinoid-mediated AP-1-antagonism assay, HeLa cells
grown in Dulbecco's modified Eagle's medium (DMEM),
containing 10% fetal bovine serum (FBS, Life
Technologies, Inc.) are transfected using the cationic
liposome-mediated transfection procedure (P.L. Feigner,
et al., Focus, 111:2, 1989). Cells are plated 18h before
transfection at about 40% confluence (40,000-50,000
cells/well) in a 12-well tissue culture plate (Costar,
Cambrigde, MA). Cells are transfected with lug of
reporter construct Str-AP-1-CAT and 0.2 jig of human RAR
a, (i, or y expression vectors, along with 2ug of
Lipofectamine (Life Technologies, Inc.) for each well in
a total volume of 500ul. The details of plating of
recombinant HeLa cells have been described in Example 1.
DNA/Lipofectamine complexes obtained by mixing 2ug
Lipofectamine/well, lug Str-AP-1-CAT/well, and 0.2ug RAR
expression vector in a 50 ml polystyrene tube are treated
and incubated with HeLa cells in exactly the same manner
as described for DNA/Lipofectin complexes in Example 1 of
this invention. DNA is precipitated with Lipofectamine
for 30 min at room temperature before transfer to cells.
Five hours post-transfection, 500 ul of DMEM containing
20% charcoal treated FBS (Gemini Bioproducts, Inc., CA)
is added. All the transfections are performed in
triplicate. Test retinoids (at 10-10 to 10-7 M
concentrations) are added 18h post-transfection and 6h
later the cells are treated with 12-0-tetradecanoyl
phorbyl-14-acetate (TPA) to induce AP-1 activity.
CA 02558199 1996-01-31
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Retinoids are dissolved in acetone to a concentration of
5mM and further diluted from this stock solution using
ethanol. The next day after washing with phosphate
buffered saline without calcium and magnesium (Life
Technologies, Inc.), the cells are harvested and lysed
for 60 min with occasional agitation using a hypotonic
buffer (100 }xl/well) containing Dnase I, Triton X-100,
Tris-HC1 and EDTA as described in the mixed phase CAT
assay section of Example 1 of this invention. CAT
activity is assayed in 50 pl of the lysed cell extract
using [3H] acetyl CoA (DuPont NEN) in a 96-well U-bottom
plate (Costar). The CAT activity is quantified by
counting the amount of 3H-acetylated forms of
chloramphenicol using a liquid scintillation counter.
The detailed procedures of CAT assay and scintillation
counting of labeled acetylated forms of chloramphenical
are described in the Example 1 of this invention
CA 02558199 1996-01-31
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TABLE 3
API INHIBITION AND RPE CELL GROWTH INHIBITION
AP 1 Inhibition
Compound RPE 148 EC., (nM)b
Number Structure ICs- ( RARa RAR(3 RARy
All-trans RA <10 0.07 0.23 <0.01
13-cis RA 30 0.08 10 1
521 10 0.01 0.4 0.01
121 30 0.6 5 1.9
509 30 22 NA 0.01
183 <10 0.17 <0.01 0.53
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TABLE 3 Cont'd. -44-
Page 2.
AP 1 Inhibition
Compound RPE 148 EC50 (nM)b
Number Structure -MSJnM RARa RARI3 RARy
659 10 >102 14 22
967 >103 >102 >102 >102
870 >103 >102 >102 >102
NA = Not Available
' Retinoid (nM) required for 50% inhibition of RLPE cell growth relative to
mock
treated RPE cells.
b Retinoid (nM) required for 50% inhibition of Str-API-CAT activity in
transient
transfection assays.
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The effects of retinoid agonists in inhibiting APi
activity as described in Example 3 are shown in Table 3.
Also shown in Table 3 are the effects of the same
retinoids in inhibiting RPE cell proliferation measured
as described in Example 2. Compounds that are potent
inhibitors of API activity (e.g., compounds 521, 183 and
659) are all effective inhibitors of RPE cell
proliferation. In contrast, compounds that are
ineffective inhibitors of API activity (e.g., compounds
867 and 810) are also ineffective in inhibiting RPE cell
proliferation.
EXMPLE 4
Other nuclear receptors, including the thyroid hormone
receptor, which is activated by thyroid hormone (T3), the
glucocorticoid receptor, which is activated by
dexamethasone (Dex), and the vitamin D receptor, which is
activated by 1,25-dihydroxyvitamin D3 (D3), can mediate
anti-AP1 activity. The effect of dexamethasone, T3 and
D3 on RPE cell proliferation (measured as described in
Example 2) was examined alone and in combination with
retinoic acid (RA). The results are shown in Figure 3.
Dexamethasone inhibited RPE cell proliferation, but T3
and D3 did not. However, each of these agents had a
synergistic effect when used in combination with retinoic
acid. This provides supportive evidence that the
mechanism by which RAR agonists inhibit RPE cell
proliferation is by antagonism of API activity and
suggest that other effective inhibitors of API activity,
CA 02558199 1996-01-31
-46-
when used alone or in combination with RAR agonists, will
be useful for the treatment of PVR.
EXAMPLE 5
To determine the effect of various retinoids on RPE cell
morphology, RPE cells from the two donors described in
Example 1 were plated in 35-mm wells and grown on plastic
for 10 days in media containing 52k serum with or without
one of three retinoids (1pm); all-trans RA; a RAR
selective agonist (Compound 183); or a RXR selective
agonist (Compound 701).
As shown in Figures 2A and 2B, the RPE cells grown in 5%
serum containing media without other additions showed
extensive overgrowth with numerous processes from one
cell extending over neighboring cells. Cells treated
with all-trans RA did not exhibit cell overgrowth,
resulting in a morphology more like RPE in situ as shown
in Figures 2C and 2D. The RAR selective agonist also
prevented cell overgrowth, resulting in a morphology
indistinguishable from that caused by all-trans RA
(Figures 2E and 2F); whereas the RXR-selective agonist
failed to prevent cell overgrowth, resulting in a
morphology indistinguishable from the controls, as shown
in Figures 2G and 2H. Trypan blue exclusion showed
staining of less than 10% of cells in all cultures
supplemented with each of the retinoids and was not
statistically different from controls.
CA 02558199 1996-01-31
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EXAMPLE 6
The effect of intravitreous injections of retinoids was
investigated in the rabbit cell injection model of PVR
first described by H.A. Sen, et al. (Arch. Ophthalmol.,
106:1291-1294, 1988). Briefly, pigmented rabbits were
anesthetized with a subcutaneous injection of 5 mg/kg
of xylazine and 25 mg/kg of ketamine, and the pupil of
one eye was dilated with 2%% phenylephrine. Under
direct observation 5 x 105 RPE cells were injected into
the vitreous cavity just anterior to the optic nerve.
In initial experiments intravitreous injections of
100 g of all-trans-RA, a selective RXR agonist
(Compound 701), a selective RAR agonist (Compound 183),
or vehicle were administered to the rabbits.
CA 02558199 1996-01-31
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CA 02558199 1996-01-31
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Rabbits were examined by indirect ophthalmoscopy on days
7, 14 and 28 and traction retinal detachments were
graded. After injection of all trans-RA, a localized
yellow cloud of precipitate appeared in the vitreous and
remained for several weeks, and injection of the other
agonists resulted in a while cloud. The localized clouds
decreased in size and cleared over 2 to 3 weeks. There
were no visible changes in the retina. As shown by the
data summarized in Table 4, eyes injected with all-trans
RA had fewer (p(0.05) and less severe traction retinal
detachments than eyes injected with vehicle alone, while
eyes injected with the RXR-selective agonist did not
differ from controls.
In the initial group of rabbits, five received two
intravitreous injections of the RAR-selective agonist.
None of these rabbits developed retinal detachments, but
all experienced hair loss and loss of appetite, and two
rabbits died before completion of the 28 day observation
period. It was felt that the hair loss and possibly the
loss of appetite and death were due to systemic toxicity
from drug that had gotten out of the eye. Another group
of rabbits was tested in which a single intravitreous
injection of 50pg of the RAR agonist, 50 pg and 100 jig of
RA, or 100 pg of RXR agonist (701), or vehicle alone was
given one day after RPE cell injection. The eyes
injected with the RAR agonist or with RA had fewer
(p(0.05) and less severe retinal detachments than the
control eyes, but the eyes injected with the RXR agonist
were not statistically different from controls. None of
CA 02558199 1996-01-31
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the rabbits experienced hair loss and none died; their
appetites appeared to be normal.
EXAMPLE 7
To examine for ocular toxicity, rabbits were given a
single intravitreous injection of 100pg of all-trans-RA,
the RAR-selective agonist (compound 183), the RXR-
selective agonist, (compound 701), an agonsit that
activates both RAR and RXR (compound 659), or vehicle
alone. The rabbits were killed two weeks after
injection. The results of histopathologic examination of
the retinas of the rabbits are summarized in Table 5. In
general, the retinas were well-preserved and showed only
mild changes believed to represent no more than artifact.
As shown by the data in Table 5 below, all of the eyes,
including those injected with vehicle, showed mild
vitritis. One of the eyes injected with the RXR agonist
and one injected with the agonist showing activity with
both RXR and RAR receptors exhibited a few focal areas of
retinal necrosis. The same RXR-injected eye also showed
a few focal areas of inner retinal edema, and similar
focal areas of inner edema were seen in one eye injected
with the RAR agonist. Occasional focal areas of
photoreceptor degeneration were seen in one eye injected
with the RAR agonist and in one eye injected with the
agonist having both RXR and RAR activity.
CA 02558199 1996-01-31
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TABLE 6
EFFECT OF INTRAVITREAL INJECTION OF THE RAR AGONISTS 168
AND 299 ON THE DEVELOPMENT OF TRACTION RETINAL DETACHMENT
IN RABBITS AFTER INJECTION OF HUMAN RPE CELLS
Compound RD at 7 Days RD at 14 Days RD at 28 Days % Total % Any
No. N Partial Total Partial Total Partial Total RD RD
Control 12 0 0 0 0 6 0 0 50
168 12 1 0 1 0 7 1 8 80
299 10 0 0 1 1 4 4 40 80
RA 2 0 0 0 0 2 0 0 100
Areas of retinal whitening were noted in several of the
drug-injected eyes, and were associated with drug
precipitates, suggesting that localized areas of high
concentration of Compound 168 or 299 might cause retinal
damage that could be responsible for the high rate of
retinal detachment. It was concluded that the poor
solubility of retinoids in the vitreous resulted in
precipitates and localized areas of high concentration
that resulted in toxicity.
CA 02558199 1996-01-31
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EXAMPLE 9
Due to the toxicity of certain RAR agonists and because
the effects of retinoids are delayed in onset and
reversible, methods for sustained release of the
retinoids were investigated. To explore alternative
methods of sustained release delivery, rabbits were given
subconjunctival injections of 0.3 mg of Compound 168,
299, or RA once a day for five days after intravitreous
injection of 5 x 105 human RPE cells. The results as
shown in Table 7 demonstrate that, as compared with the
control, subconjunctival injections of all three
retinoids administered over a course of five days
decreased TRD. The therapeutic effect of the RAR
specific agonists was greater than that of RA, which is
an agonist for both RXRs and RARs. There were no signs
of any toxicity to the retina.
CA 02558199 1996-01-31
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TABLE 7
EFFECT OF SUBCONJUNCTIVAL INJECTION OF THE RAR AGONISTS 168
AND 299 ON THE DEVELOPMENT OF TRACTION RETINAL DETACHMENT
IN RABBITS AFTER INJECTION OF HUMAN RPE CELLS
Compound RD at 7 Days RD at 14 Days RD at 28 Days % Total % Any
No. N Partial Total Partial Total Partial Total RD RD
Control 12 0 0 0 0 6 0 0 50
168 6 0 0 0 0 1 0 16 16
299 4 0 0 0 0 0 0 0 0
RA 4 0 0 0 0 0 0 0 0
EXAMPLE 10
The effect of subconjunctival injection of RAR agonists
was tested in a rabbit model of PVR as described in
Example 7, except that human dermal fibroblasts were
injected into the vitreous cavity in the place of RPE
cells. Generally, this model tends to have a higher rate
of retinal detachment and more severe detachments than
does the model utilizing RPE cells. However, as shown by
the data in Table 8, subconjunctival injections of
Compounds 168, 299, and all-trans RA all decreased the
number of retinal detachments and slowed the rate at
which they developed as compared with the results of the
tests described in Example 9, although the improvement
over the control was not as great as in Example 9.
CA 02558199 1996-01-31
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TABLE B
EFFECT OF SUBCONJUNCTIVAL INJECTION OF THE RAR AGONISTS 168
AND 299 ON THE DEVELOPMENT OF TRACTION RETINAL DETACHMENT
IN RABBITS AFTER INJECTION OF HUMAN DERMAL FIBROBLASTS
Compound RD at 7 Days RD at 14 Days RD at 28 Days % Total % Any
No. N Partial Total Partial Total Partial Total RD RD
Control 10 2 0 5 0 7 1 10 80
168 10 0 0 0 0 4 2 20 60
299 10 0 0 0 0 4 0 0 40
RA 10 0 0 0 0 5 0 0 50
EXAMPLE 11
In this experiment the retinoid was incorporated into
lipid microvesicles utilizing standard techniques.
Microvesicles encapsulating all-trans RA, Compounds 168
and 299, and vehicle were injected into the vitreous
cavity of rabbits on the day after intravitreous
injection of 5x105 human dermal fibroblasts. As shown
by the data in Table 9 below, microvesicles containing
Compound 168 were very effective in preventing traction
retinal detachment, while those containing Compound 299
CA 02558199 1996-01-31
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or all-trans RA were not effective as compared with the
control group.
TABLE 9
EFFECT OF INTRAVITREOUS INJECTION OF LIPID MICROVESICLES CONTAINING
168 OR 299 ON THE DEVELOPMENT OF TRACTION RETINAL DETACHMENT
IN RABBITS AFTER INJECTION OF HUMAN DERMAL FIBROBLASTS
Compound RD at 7 Days RD at 14 Days RD at 28 Days % Total % Any
No. N Partial Total Partial Total Partial Total RD RD
Control 11 2 0 5 1 7 2 18 82
168 7 0 0 0 0 0 2 29 29
299 3 0 0 0 0 0 3 100 100
RA 6 1 0 1 1 0 4 67 67
Retinoids were incorporated into lipid microvesicles and
injected subconjunctivally into the eyes of rabbits on
the day after intravitreous injection of 5 x 105 human
dermal fibroblasts. As shown by the data in Table 10
below, Compound 168 was substantially more effective at
inhibiting tractional retinal detachment than were the
other retinoids or vehicle.
CA 02558199 1996-01-31
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ABLE 10
EFFECT OF SUBCONJUNCTIVAL INJECTION OF LIPID MICROVESICLES
CONTAINING 168 OR 299 ON THE DEVELOPMENT OF TRACTION RETINAL
DETACHMENT IN RABBITS AFTER INJECTION OF HUMAN DERMAL FIBROBLASTS
Compound RD at 7 Days RD at 14 Days RD at 28 Days % Total % Any
No. N Partial Total Partial Total Partial Total RD RD
Control 2 0 0 1 0 0 2 100 100
168 8 0 0 1 0 0 2 33 33
299 8 0 0 0 1 1 4 50 63
RA 8 0 0 0 1 4 1 13 63
The foregoing description of the invention is exemplary
for purposes of illustration and explanation. It should
be understood that various modifications can be made
without departing from the spirit and scope of the
invention. Accordingly, the following claims are
intended to be interpreted to embrace all such
modifications.
CA 02558199 1996-01-31
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SUMARY OF SEQUENCES
SEQ ID NO: 1 is an oligonucleotide primer for the
stromelysin-1 promoter.
SEQ ID NO: 2 is an oligonucleotide primer for the
stromelysin-1 promoter.
CA 02558199 1996-01-31
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SEQUENCE LISTING
(3.) GENERAL U FOMOLTICN:
(i) APPLICANT: THE JOHNS HOPKINS tNIVERSITY
SCHOOL OF ) DIC33 E
(ii) TITLE OF nIVENTION: NETHOD OF PBEVENT3NG PROLTRERATICN OF
BETnQL PIGMENT EPITHELIt3M BY RETMOIC ACID RECEPTOR
AGONISTS
(iii) NUER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Fish 6 Richardson P.C.
(B) STE=T: 4225 Executive Square, Suite 1400
(C) CITY: La Jolla
(D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 92037
(v) CCHFUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) CCHPUTER: IBL( PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTFIlsiBE: Patentln Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NU ER: PCT/US95/
(B) FILING DATE:
(C) CLASSIFICATION:
CA 02558199 1996-01-31
-59-
(viii) ATTORNEY/AGENT ZtTF'OBMATICN:
(A) NAm: Learn, June M.
(B) REGISTRATION NT1R: 31,238
(C) REFEREN'CE/DOCKET NCR: 07265/023WC)1
(ix) TELECC ICATION INFOR aLTIOiN:
(A) TELEPHCTTE : (619) 678-5070
(B) TELEFAX: (619) 678-5099
(2) IITICN FOR SEQ ZD NO:1:
(i) SEQUENZ CHARACTERISTICS:
(A) LENGTH: 101 base pairs
(B) TYPE: nucleic acid
(C) STRMMWEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/BEY: CDS
(B) LOCATION: 1..101
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AGAAGCTTAT GGAAGCAATT ATGAGTCAGT TTGCGGGTGA CTCTGCAAAT ACTGCCACTC 60
TATAAAAGTT GGGCTCAGAA AGGTGGACCT CGAGGATCCA G 101
(2) INFORATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 101 base pairs
(B) TYPE: nucleic acid
(C) ST MDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1. .3.01
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CTGGATCCTC GAGGTCCACC TTTCTGAGCC CAACTTTTAT AGAGTGGCAG TATTTGCAGA 60
GR4'AC000CA AACTGACTCA TAATTGCTTC CATAAGCTTC T 101
