Method of treating a nerve-related vision disorder in a non-diabetic
United States Patent: 7,829,532
Issued: November 9, 2010
Inventors: Gardner; Thomas
W. (Hummelstown, PA), Reiter; Chad E. (Hershey, PA)
Assignee: The Penn State
Research Foundation (University Park, PA)
Appl. No.: 11/755,232
Filed: May 30, 2007
Master of Science in Law
This invention provides reagents and
methods for delivering insulin, insulinomimetic agents, and the like to a
vertebrate eye via subconjunctival routes, sub-Tenon's routes, or
intravitreal routes for treatment of nerve-related vision disorders such
as diabetic retinopathy, and formulations useful in the practice of the
Description of the
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to a method for delivering insulin,
insulinomimetic agents, and the like to the eye for treatment of diabetic
retinopathy. More specifically, the method involves the periocular
administration of these drugs via subconjunctival routes, sub-Tenon's
routes, or intravitreal routes.
2. Background of the Related Art
Diabetes has reached epidemic proportions. Approximately 15 million people
in the United States are currently afflicted with the disease, and that
number is expected to rise to at least 21 million over the next 30 years.
In addition to (and as a consequence of) the metabolic disarray caused by
the disease, diabetes causes a variety of other, organ-specific
dysfunctions, including in particular diabetic retinopathy. Diabetic
retinopathy affects half of all Americans diagnosed with diabetes.
Diabetic retinopathy is an illness that occurs when diabetes damages tiny
blood vessels in the retina, affecting vision, and is a leading cause of
blindness. There are two clinical stages of retinopathy. The first stage
is known as nonproliferative retinopathy, in which the blood vessels
damaged by diabetes leak fluid and lipids onto the retina. When the fluid
accumulates in the center of the retina (i.e., the macula) it leads to
macular edema. The fluid makes the macula swell, which blurs vision. The
second stage is the proliferative stage, where new blood vessels grow
along the retina and in the clear, gel-like vitreous that fills the inside
of the eye. These new blood vessels can bleed, cloud vision, and destroy
the retina unless treated. There is also a preclinical phase in which
patients will generally have no symptoms, nor will there be any findings
on routine clinical examination. However, in the preclinical phase
sensitive tests reveal reduced contrast sensitivity, electrical responses
with an electroretinogram, or color vision.
There are several methods of treatment for diabetic retinopathy disclosed
in the art. However, none of these treatment approaches have proven
successful in addressing the primary metabolic disorder or in preventing
retinopathy. Conventional diabetic retinopathy treatments are limited to
controlling the diabetic state with systemic insulin administration or
oral hypoglycemic agents. The problem with these systemic approaches is
that they do not restore normal physiologic metabolic control or provide
overall effective levels of the drug to the eye. Secondary treatment
approaches include using diuretics to control blood pressure or
intravascular fluid overload. Attempts have also been recognized in the
arts for treating retinopathy with aldose reductase inhibitors, inhibitors
of nonenzymatic glycation (aminoguanidine), corticosteroids or
antihistamines. Methods of treatment for advanced retinopathy
complications include vitrectomy surgery and laser treatment, exposing an
intense beam of light to the small diseased areas of the retina. These
methods are palliative in nature, and none of these methods is
sufficiently effective to prevent or cure the disease.
Although diabetic retinopathy is extensively studied in the art, the
direct effects of insulin or insulinomimetics on diabetic retinopathy are
limited. It has been demonstrated that retinal neurons die in experimental
diabetes in rats and in humans. Moreover, insulin has been shown to be a
survival factor for retinal neurons in culture, and excess hexosamines
impair insulin's survival-promoting effects. In vivo, systemically and
intraocularly administered insulin activates the insulin receptor and
downstream signaling cascades that are involved in cell function and
survival. However, the ability to administer systemically sufficient
insulin or other insulinomimetic agents to be effective for prevention of
retinopathy is limited by the risk of hypoglycemia.
Accordingly, there is a great demand for safe and effective methods for
delivering agents effective in treating diabetic retinopathy. In
particular, there is a need in the art for treatment methods that maintain
retinal cell function and survival in the face of persistent
SUMMARY OF THE INVENTION
The invention describes methods and reagents for treating retinal
disorders, particularly retinal disorders having at least in part a
metabolic etiology. As provided herein, the inventive methods and reagents
permit compounds for treating ocular disorders, such as retinal
detachment, retinitis pigmentosa, central retinal artery occlusion,
central retinal vein occlusion, ischemic optic neuropathy, high tension
glaucoma, low tension glaucoma, and cataract, to be administered locally
in the eye. The invention specifically provides methods for preventing and
treating nerve-related vision disorders, including in particular diabetic
retinopathy. The inventive methods comprise periocular administration of a
sufficient amount of a drug by a subconjunctival, sub-Tenon's or
intravitreal route to be effective in treating such retinal disorders. In
certain embodiments, the drug is administered to an eye under its mucous
membrane or fascia.
Preferred drugs administered using the methods of the invention include
formulations of insulin, insulinomimetic agents, or peptides. Formulations
of insulin that may be used in the invention include, for example,
formulations of native insulin, naturally derived insulin, recombinant
insulin, any modification thereof containing buffers or modifying
proteins, or any other known formulations of insulin. The concentration of
the insulin formulation can range from 1 picomolar to 100 micromolar. If
the insulin formulation is a gel or liquid, the volume thereof can range
from 5 .mu.L to 1 mL. In the practice of the inventive methods, a
sufficient amount of any of these drugs is administered to the eye,
wherein a sufficient amount of insulin ranges from 5 to 100 .mu.L or 0.1
to 10 units of insulin. Formulations can also include augmenting drugs
from the thiazolidinediones (TZD) class, such as rosiglitazone,
pioglitazone, and troglitazone, as well as, non-peptide insulinomimetic
agents, such as TLK16998 (Telik), KRX-613, and L-783,281 (Merck).
The invention further comprises methods of administrating drugs to the
eye, where drugs are administered to more than one eye. These methods of
administration include via a solution, a polymeric base, or a pump.
Additionally, the method of administration may be by implanting a device,
where the device releases a formulation of a drug, preferably insulin, an
insulinomimetic agent or a peptide at a prescribed rate. One or more
devices may be administered to one eye.
The invention also provides formulations of insulin, insulinomimetic
agents or peptides adapted or prepared for use with the methods of the
invention. Preferably, the formulations of the invention comprise
pharmaceutical adjuvants, carriers, buffers or other components.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The invention provides reagents, pharmaceutical compositions and methods
for delivering drugs for treatment or prevention of nerve-related vision
disorders, particularly diabetic retinopathy. The method specifically
comprises periocular administration of insulin, insulinomimetic agents or
peptides via subconjunctival, sub-Tenon's or intravitreal routes.
More specifically, the method of the invention comprises periocularly
administering a sufficient amount of a formulation of insulin,
insulinomimetic agents or peptides to both eyes. Periocular delivery is
safer for the general health of an animal, preferably a human, undergoing
treatment for a nerve-related vision disorder since it involves less
ocular morbidity than laser or vitrectomy surgery.
The ability to systemically administer sufficient amounts or
concentrations of insulin or other insulinomimetic agents to prevent
retinopathy is largely limited by the risk of hypoglycemia. The invention
overcomes the consequences of systemic administration by achieving direct
local drug delivery of effective amounts of these agents by direct
administration to the subconjunctiva, sub-Tenon's capsule, or
intravitreous. Administering insulin or insulinomimetic agents via these
routes also provides effective intraocular drug penetration to maintain
retinal cell function and survival.
One aspect of this invention involves replacing a deficient insulin
receptor ligand and increasing activation of a down-regulated insulin
receptor, or its downstream signaling molecules. Specifically, the
inventive methods comprise direct insulin administration to the eye, and
specifically to cells of the retina. Any pharmaceutically acceptable
insulin formulation can be used with the methods of the invention.
Examples of useful insulin formulations include native insulin (preferably
human insulin, particularly recombinantly-produced human insulin such as
Humulin.RTM., or insulin isolated from any other mammalian species),
naturally derived or recombinant, and all of modifications thereof, such
as Regular to NPH, Ultralente (Eli Lilly & Co.), insulin glargin (Lantus.RTM.,
Aventis), Lispro.RTM., (Eli Lilly & Co.), Novolin.RTM. (Novo-Nordisk) and
formulations containing any modifying proteins (such as, for example,
prolamine) or buffers known or accepted in the art.
Moreover, the invention provides methods comprising administration of
insulinomimetic agents or nucleotides (aptamers) that mimic some or all of
insulin's actions. The invention further encompasses the administration of
drugs that augment insulin along with the insulin. These augmenting drugs
can be, inter alia, from the thiazolidinediones (TZD) class. They may also
be small non-peptide insulinomimetic agents such as TLK16998 (Telik),
KRX-613, and L-783,281 (Merck). Such compounds activate the proliferator-activator
gamma (PPAR-gamma) receptor to provide necessary actions of insulin in the
retina. Thus, their addition enhances the insulin effect on signaling to
retina cells. Examples of augmenting drugs include rosiglitazone,
pioglitazone, and troglitazone.
Concentrations of the drugs used in the invention can range from low
picomolar to micromolar concentrations. If the drug is a liquid or gel
insulin formulation, volumes can range from about 10 .mu.L to about 1 mL.
A sufficient dosage of the insulin will range from a few picomolars to
The inventive treatment provided herein permits a number of different
administration routes to be used to introduce an effective amount of a
drug to the eye. These include administering the drug via a pump, a
polymeric base, or a solution. The preferred method of administration is
by a polymeric base, including but not limited to polyester (PET),
polyethylene (PE), poly(L-lactic acid) (PLA), and polyurethane.
Additionally, drugs may be administered by implantation of a formulation
of the invention or a device that will release such a formulation at a
prescribed rate. The invention advantageously provides methods for
administering said formulation to both eyes simultaneously, although
embodiments having administration to one eye, as well as embodiments
having independent or non-contemporaneous administration to both eyes, are
also encompassed by the invention.
Drug formulations of the invention advantageously can be administered
under the mucous membrane of the eye or the Tenon's fascia of the eye.
More specifically, the drugs can be delivered to the subconjunctival
and/or sub-Tenon's space. As shown in FIG. 1 (see Original Patent), drug
formulations of the invention are injected, or otherwise administered,
under the eye's surface membrane so that the drugs are able to diffuse
through the sclera into the retina, vitreous, and the anterior chamber.
The inventive methods for treatment of nerve-related retinal disorders,
such as diabetic retinopathy, are suitable for prevention or treatment at
any stage of such retinopathic disorders. Specifically, the inventive
methods are equally effective for the preclinical, nonproliferative,
macular edema stages of such retinopathic disorders, as well as for the
proliferative stage of retinopathy. Other retinal disorders advantageously
treated using the methods of the invention, include retinal detachment,
glaucoma, retinitis pigmentosa, central retinal artery or central retinal
vein occlusion, ischemic optic neuropathy, high tension glaucoma, low
tension glaucoma, and cataract.
While the invention has been described with particular reference to
diabetic retinopathy treatment and other retinal disorders, it will be
understood by those skilled in the art that the invention has applications
in other medical fields, in particular whenever local insulin or
insulinomimetic agents are delivered to tissues at risk for complications.
For example, deliveries to kidneys and nerves since patients with diabetes
have impaired kidney function (nephropathy) and nerve function
(neuropathy). Thus, local application of insulin adjacent to these organs
and tissues may improve their function and prevent future deterioration.
The following Examples are intended to further illustrate certain aspects
of the above-described method and advantageous results. The following
examples are shown by way of illustration and not by way of limitation.
Intraportal insulin injection was performed to determine if a single bolus
insulin injection, and therefore an acute elevation in circulating
insulin, could activate retinal insulin receptors (IR) in vivo as it does
in other tissues. Intraportal insulin injection was conducted as follows.
Male Sprague-Dawley rats (Charles River, Mass.) weighing 200 350 g were
fasted 18 hours prior to being anesthetized with a 10:1 ketamine:xylazine
cocktail (53.5 mg/kg ketamine and 5.33 mg/kg xylazine) administered by
intramuscular injection. The fasted rats were administered a 500 .mu.g
bolus of insulin, nothing (sham), or vehicle (0.9% saline) via the portal
vein. At 15, 30, 45 and 60 minutes post injection (shown in FIG. 2A (see Original Patent)),
hindquarter skeletal muscle and retina were snap-frozen under liquid
nitrogen, and then stored at -80.degree. C. pending analysis by
immunoprecipitation and immunoblotting. There was no difference in insulin
receptor beta-subunit (IR.beta.) phosphorylation between vehicle (V) and
insulin (I) injection.
Tissue lysates were immunoprecipitated and immunoblotted as described by
Barber et al. (2001, J. Biol. Chem. 276: 32814 32831). Tissues were
homogenized in an immunoprecipitation (IP) lysis buffer (consisting of 50
mM HEPES, pH 7.3, 137 mM NaCl, 1 nM MgCl.sub.2, 2 mM NaVO.sub.4, 10 mM
Na.sub.2H.sub.2P.sub.2O.sub.7, 10 mM NaF, 2 mM EDTA, 2 mM PMSF, 10 mM
benzamidine, 10% glycerol, 1% NP-40, and 1 protease inhibitor tablet (Boehringer-Mannheim)
per 10 mL of buffer. Homogenates were rocked 15 minutes at 4.degree. C.
and then centrifuged at 14,000 rpm at 4.degree. C. Prior to
immunoprecipitation and immunoblotting, the resulting supernatant was
subjected to protein assay (Bio-Rad) and quantification.
Immunoprecipitations were performed as follows. Protein (250 .mu.g) was
diluted into 1 mL IP buffer containing one of the following antibodies: 5
.mu.L of anti-IR.beta. or anti-IGF-IR.beta. (Santa-Cruz, Santa Cruz), or 4
.mu.L of anti-IRS-1 or IRS-2 (Upstate Biotechnology, Lake Placid, N.Y.),
specific for these species of insulin responsive substrate, and 30 .mu.L
of a 50% protein A/Sepharose bead slurry (Amersham Pharmacia Biotech,
Piscataway, N.J.). The Sepharose bead complex was rocked overnight at
4.degree. C., washed twice with 200 .mu.L of IP buffer, and boiled with 30
.mu.L of 2.times. sample buffer (a solution of glycerol, SDS, TRIS buffer,
bromophenol blue and betamercapto-ethanol). Fifty .mu.g of protein per
sample were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Thereafter, gel-separated proteins were transferred to nitrocellulose
membranes (blocked with a solution of 0.05% Tween-20 and 5% non-fat milk
or 3% bovine serum albumin, dissolved in Tris-buffered saline) at room
temperature for 1 hour. The membranes were probed overnight in blocking
solution at 4.degree. C. at 1:1000 dilutions of the primary antibody. The
primary antibodies used in these assays included an anti-phosphotyrosine
antibody (Upstate Biotechnology), and anti-Act antibodies (Cell Signaling,
Beverly, Mass.). Secondary antibodies were diluted 1:4000 (for horseradish
peroxidase-conjugated anti-rabbit antibodies; obtained from Amersham
Pharmacia Biotech, Piscataway, N.J.), or 1:1000 (for biotin-conjugated
anti-mouse antibodies; obtained from Amersham Pharmacia Biotech). Tertiary
incubations with streptavidin-linked alkaline phosphatase were diluted
1:4000 (Gibco, Gaithersburg, Md.). Positive signals were detected with
electrochemiluminescence (ECL) kits (Cell Signaling) and
electrochemifluoresence (ECF) kits (Amersham) each performed according to
each manufacturer's protocol. Immunoblot quantitation was accomplished
using ImageQuant (Molecular Dynamics), NIH Image 1.6 (NIH, or GeneTools (SynGene)
software. Blots were re-incubated with different antibodies after being
stripped, at 50.degree. C. for 1 hour in a buffer containing 63 mM Tris
(pH 6.8), 2% SDS, and 0.07% 2-mercapthoethanol.
Western blots interrogated with anti-phosphotyrosine (PY) antibodies
showed a robust response in muscle, as expected. IR.beta. phosphorylation
was also increased in retinal tissue, but only after a 30 minute delay. In
addition, the response had a smaller magnitude than muscle. No difference
in IR.beta. phosphorylation was found in sham-operated rats. These results
indicated that retinal IR.beta. has greater levels of basal
phosphorylation than muscle in vehicle treated rats.
The results of experiments quantifying this Western blot analysis are
shown in FIG. 2B (see Original Patent), presented as a ratio of
phosphotyrosine (PY) levels to insulin receptor beta-subunit (IR.beta.)
amounts (PY/IR.beta.). In these experiments, Western blots interrogated
for muscle PY were stripped, reprobed for total IR.beta., quantified and
the data expressed in terms of PY/IR.beta. ratios. The zero time point was
set to a ratio of 1. The results in FIG. 2B (see Original Patent) show a
nearly 30-fold increase in phosphotyrosine content in muscle IR.beta. 15
minutes post-insulin injection, which increase peaked at 30 minutes and
declined by 60 minutes post-injection. There was a significant increase in
IR.beta. phosphorylation with insulin at all points examined. FIG. 2C (see Original Patent)
shows the result obtained in parallel experiments performed on retinal
tissue. Unlike the results shown in FIG. 2B (see Original Patent),
tyrosine phosphorylation did not significantly increase in retinal tissue
until 30 minutes post injection, was maximal after 45 min and remained
elevated 3 4 fold above vehicle-injected controls for 60 minutes. IR.beta.
phosphorylation did not change in vehicle injected animals. The
discrepancy in the fold increase in retinal tissue may be due to the
relatively higher basal IR.beta. phosphorylation in the retinas of vehicle
treated rats, or to the blood-retinal barrier limiting diffusion of
insulin into the retina.
In addition, 45 min after insulin administration retinal lysates were
analyzed for Akt activation by Western blotting specifically probed for
phosphorylation of Akt (serine 473). These Western blot results, shown in
FIG. 2D (see Original Patent) demonstrated that insulin induced a 48%
increase in phosphoserine 473 content of Akt over vehicle injected
controls when IR.beta. phosphorylation is maximal (45 minutes
post-injection). Quantification of Western blots, also shown in FIG. 2D
indicated there was no statistical difference between sham and vehicle
injected controls. These results indicated that systemic insulin injection
can effect changes in IR.beta. phosphorylation in retina, thereby
demonstrating that insulin crosses the blood-retinal barrier. Insulin
receptor phosphorylation and activation occurs in a different temporal
manner than muscle, however, and insulin increases Akt phosphorylation in
Thus, even though insulin penetrates the blood-retinal barrier, the
delayed kinetics and lower phosphorylation differential between activated
and inactive insulin receptor illustrates the limitations in systemic
delivery for affecting retinal diseases related to or dependent on insulin
levels, such as diabetic retinopathy.
Tyrosine phosphorylation in retinal IR.beta. and changes therein was
compared with other insulin responsive tissues under freely fed and
moderately fasted conditions as follows.
Six male Sprague-Dawley rats (Charles River) were fasted (18 hours) and
were compared to 6 rats that were freely fed overnight. All rats weighed
200 350 g and were anesthetized with a 10:1 ketamine:xylazine cocktail as
described in Example 1. Upon loss of motor reflex in the rats' retina,
tissue samples from liver and hindquarter skeletal muscle were obtained,
homogenized and immunoprecipitated as described in Example 1 and then
subjected to Western blot analysis, also as described in Example 1. The
blot in FIG. 3A (see Original Patent) shows that IR.beta. phosphorylation
was unaltered in retina, but increased in muscle and liver compared to
fasted rats. Phosphotyrosine-probed immunoblots were then reprobed for
total IR.beta. content with the immunoprecipitating antibody as described
in Example 1 to normalize the Western blot data.
The results of these experiments are shown in FIG. 3B(see Original Patent)
as the ratio of PY to total IR.beta.. Plasma insulin levels were
1.95.+-.0.36 ng/mL in fed rats versus 0.32.+-.0.049 ng/mL in fasted rats
(mean.+-.SEM, p=0.011). Similarly, blood glucose levels were 95.5.+-.2.6
mg/dL in fed rats versus 68.83.+-.2.4 mg/dL in fasted rats (mean.+-.SEM,
p=0.000008). Image analysis (software from Molecular Dynamics, Sunnyvale,
Calif.) of the Western blots from retinal tissue revealed no differences
in IR.beta. phosphorylation between freely fed and fasted rats. In
contrast, IR.beta. phosphorylation in liver and hindquarter skeletal
muscle was diminished 23% and 38%, respectively in the fasted rats. On the
other hand, IR.beta. phosphorylation in liver and muscle was significantly
greater than in retinal tissue for freely fed rats.
In summary, retinal IR remained relatively constant in response to the
physiological levels of insulin in circulation. Phosphotyrosine content of
the IR.beta. in retina did not change, unlike in liver and muscle, despite
changes in nutritional status and physiological increases in circulating
insulin and blood glucose. IR.beta. from muscle and liver displayed
increased tyrosine phosphorylation as expected in the freely fed state
compared to fasted rats. These results were likely due to the fluctuations
of circulating insulin in the freely fed condition.
Because tyrosine phosphorylation of the IR does not directly measure
enzymatic activity, an IR kinase assay was developed.
The IR.beta. from fasted and fed rats was immunoprecipitated from retina
and liver and analyzed by PY immunoblotting for autophosphorylation with
(+) and without (-) the addition of ATP to the kinase reaction. IR.beta.
immunoprecipitates obtained as described in Example 2 were subjected to IR
kinase assays as follows. The Sepharose bead complex, obtained as
described in Example 1, was washed three times with 200 .mu.L of kinase
buffer (50 mM HEPES, pH 7.3, 150 mM NaCl, 20 mM MgCl.sub.2, 2 mM
MnCl.sub.2, 0.05% bovine serum albumin, and 0.1% Triton X-100). Western
blotting was performed on these immunoprecipitates as described in Example
1; the results of these Western blot experiments (shown below) showed that
washing the Sepharose bead/immune complex in kinase buffer did not
diminish total bound IR.beta.. After the last (third) aspiration of kinase
buffer, 500 .mu.L of kinase buffer was added with or without 100 .mu.M ATP
(Sigma) to each aliquot of the washed Sepharose bead complex. These immune
complexes were then rocked at ambient temperature for 1 minute, and the
Sepharose beads then collected by brief centrifugation (approximately 3
seconds). The kinase buffer was aspirated, an equal volume of 2.times.
Laemmli sample buffer was added and the samples were boiled for 3 min. SDS-PAGE
and phosphotyrosine immunoblotting analysis was performed as described in
Results of these assays are shown in FIG. 4 (see Original Patent). The PY
immunoblot showed that retinal IR.beta. displays tonic tyrosine
phosphorylation (- lanes), and the rate of phosphate incorporated into the
IR.beta. is not different between fasted and fed rats (+lanes). Liver
IR.beta., however, displays less basal tyrosine phosphorylation, which
increases in the fed state (- lanes). As expected, the rate of IR
autophosphorylation is significantly greater in the freely fed rat liver
The results of these Western blot experiments shows that the kinase
reaction proceeded linearly though 5 minutes of incubation (R.sup.2>0.9)
and at a non-limiting dose of 100 .mu.M ATP. The time course of IR
autophosphorylation continued to proceed in a linear fashion over the
course of these experiments, and the concentration of ATP used did not
limit the rate of autophosphorylation.
These results further indicated that there was no change in retinal
IR.beta. autophosphorylation rate between freely fed and fasted rats.
These results also suggested a tonic level of tyrosine phosphorylation and
kinase activity despite changes in circulating insulin and glucose levels.
In contrast, in liver and skeletal muscle IR.beta. phosphorylation was
increased in the freely fed state, leading to a significant increase in
autophosphorylation activity. These results thus demonstrated the
difference in insulin signaling physiology within the whole animal.
Moreover, these results also indicated that the blood-retinal barrier may
discretely regulate insulin flux, unlike in tissues that undergo rapid
metabolic changes, the results having consequences for insulin
administration in diabetic retinopathy.
A tissue explant culture system was used to characterize insulin-signaling
transduction in retina without the hindrance of the blood retinal barrier
present in whole animal models. Such a tissue explant culture system was
developed as follows. Rats as described in Examples 1 and 2 were
anesthetized using sodium pentobarbital (7.5 mg/kg), and then decapitated
upon loss of motor reflexes. Rat retinas were removed by cutting across
the cornea, removing the lens, and squeezing the eyeball to rapidly
extract the retina. Retinas were pre-incubated in MEM (Sigma) supplemented
with 5 mM pyruvate and 10 mM HEPES for 15 minutes at 37.degree. C., 5%
CO.sub.2, and with gentle shaking. Ten nM insulin or vehicle was added
following pre-incubation. At 2, 5, 15, and 30 minutes after insulin
addition, retinas were snap-frozen in liquid nitrogen for future use.
To examine the time course of insulin signaling events in the tissue,
insulin-treated retinas were compared to retinas that received vehicle
(0.9% saline) and to untreated retinas. Tissues were analyzed by
immunoprecipitation and Western blotting as described in Example 1.
Western blot data were quantified, as shown in FIG. 5 (see Original Patent),
and revealed a significant increase in IR.beta. phosphorylation at all
time points examined when normalized to total IR.beta.. Within 2 minutes
of insulin stimulation, retinal IR.beta. exhibited a nearly 4-fold greater
phosphotyrosine immunoreactivity, which remained elevated at levels 3-fold
greater than baseline at the 30 minute time point. This analysis
demonstrated that retinal IR.beta. undergoes increased tyrosine
phosphorylation in response to a physiological dose of 10 nM insulin.
These results from the explant retina system were similar to the results
obtained using a cell culture model.
Insulin (10 nM) does not activate the IGF-IR.beta. in retinal explants.
Retinas were treated with either 1.3 nM IGF-1 or 0, 1, 10, 100, and 1000
nM insulin, and then retinal lysates were immunoprecipitated with an
insulin-like growth factor I receptor (IGF-1R.beta.)-specific antibody,
followed by PY Western blotting (shown in FIG. 6 (see Original Patent)).
The IGF1-R was immunoprecipitated and PY Western blotting performed as
described above. Insulin at 100 nM produced similar levels of IGF-IR.beta.
phosphorylation as were found when retinas were contacted with 1.3 nM
IGF-1. These results demonstrated that IGF-IR.beta. phosphorylation
remained unchanged in the presence of 10 nM insulin, but increased with
100 nM insulin. These data further indicated that 10 nM insulin does not
activate IGF-IR.beta. in retinal explants, but has specific effects via
the IR. Moreover, ATP levels over the time course experiments remained
constant, indicating that the energy status of the tissue was not
hindering the insulin signaling response.
These results suggested that isolated retinal tissue can respond robustly
to physiological insulin concentrations, this response including IR.beta.
tyrosine phosphorylation. The results also suggested that the
blood-retinal barrier plays a significant role in regulating circulating
insulin transport since the time course of the response is much faster
than observed in vivo. Moreover, intraportal insulin injection experiments
(Example 1) showed that insulin's access to the retina is limited by the
retinal vasculature, the blood-retinal-barrier (BRB). In explanted retina
tissue, in contrast, the BRB is essentially bypassed, and insulin
signaling characteristics can be analyzed in a shorter time frame since
insulin has direct access to the retina. A schematic comparison of
intraportal insulin injection and explanted retina tissue is shown in FIG.
7 (see Original Patent).
In clinical diabetes, hypoglycemia is the major factor that limits the
ability of patients to achieve the degree of intensive control of glycemia
needed to prevent or reduce retinopathy. Retinal cells die by apoptosis in
diabetes, and insulin is a survival factor for retinal neurons acting via
the PI3-kinase/Akt pathway, which is inhibited by hyperglycemia.
Systemically administered insulin activates the retinal insulin receptor,
and the activities of the retinal insulin receptor, PI3-kinase and Akt are
reduced after 4 weeks of streptozotocin-induced diabetes. Thus intensive
insulin therapy has direct effects on retinal cell survival and function.
Since intensive insulin therapy cannot be achieved by most patients due to
the hypoglycemic effects of systemic insulin administration, direct
application of insulin to the eye can provide the survival effects needed
to maintain retinal cell health in the presence of imperfectly controlled
To demonstrate the efficacy of the methods of this invention, insulin was
administered directly to the eye via the subconjunctival space of normal
Sprague-Dawley rats. This route of administration bypasses the BRB for a
more direct route of insulin action on retina. By injecting serial
dilutions of insulin in this manner, it was discovered that a dose of
0.0325 U/100 g insulin activated the IR and Akt kinase pathway without
lowering blood glucose values. As shown in FIG. 8 (see Original Patent),
the IR.beta. is phosphorylated in the eye receiving insulin (right
column), compared to the contralateral eye that received vehicle (left
column). The retina was processed for IR.beta. PY content as described in
Example 1. The results suggest it is feasible to administer insulin
directly to the retina in doses that will not lead to potentially harmful
hypoglycemic reactions and potently activate the insulin receptor.
These results demonstrated the in vivo efficacy of periocular insulin
administration as a treatment for diabetic retinopathy.
It should be understood that the foregoing disclosure emphasizes certain
specific embodiments of the invention and that alternatives equivalent
thereto are within the spirit and scope of the invention as set forth in
the appended claims.
Claim 1 of 4 Claims
1. A method of treating a nerve-related
vision disorder in a non-diabetic subject comprising the step of
administering a therapeutically effective amount of a formulation of
insulin, to an eye affected by the nerve-related vision disorder in in
said subject via a periocular route, wherein the therapeutically effective
amount is effective to achieve a local therapeutic effect without a
substantial systemic effect.
If you want to learn more
about this patent, please go directly to the U.S.
Patent and Trademark Office Web site to access the full