2~ ~~~~s
WO 95/17178 PCT/US94/14068
USE OF NON-STEROIDAL CYCLOOXYGENASE INHIBITORS TO TREAT ELEVATED
INTRAOCULAR PRESSURE
Field of the Inaention
" This invention relates to methods for the
prevention and treatment of elevated intraocular
pressure which is a factor involved in developing
optic nerve damage and loss of vision. More
particularly, this invention relates to new methods
for treating the eye with non-steroidal
cyclooxygenase inhibitors (or non-steroidal
. anti-inflammatory, NSAI, agents) to prevent or treat
elevated intraocular pressure due to administered or
endogenous steroids (glucocorticoids).
Acknowledgement of Government SuoDOrt
This invention was made with Government
support under Grant Nos. EY-02477 and EY-08973,
awarded by the National Institute of Health. The
Government has certain rights in this invention.
Hackaround of the Invention
Glucocorticoids (also known as anti-
inflammatory corticosteroids, corticosteroids, or
simply as "steroids" in the ophthalmic literature)
have been known since the early 1950's as effective
therapeutic agents~for reducing ocular inflammation.
The introduction of anti-inflammatory steroid
therapy, using a variety of local and systemic routes
of delivery, continues to provide a means to decrease
the inflammation which otherwise would damage and
" impair the vision of the patient. ophthalmic steroid
therapy proved useful to a wide variety of
inflammatory and irritating conditions in the eye
where other therapies either did not work as well or
were ineffective. Steroids were not useful for many
other conditions, especially chronic ones in which
WO 95/17178 217 ~ 5 7 8 PCT/US94/14068
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inflammatory mechanisms did not~play a clear ,,
pathogenic role (including cataract, most forms of
glaucoma, and macular degeneration).
As ophthalmic steroid therapy became more
widespread, and more potent and stable steroids
became available, it became clear that their use
involved a number of potential side-effects, one of
the first of which noticed was elevation of pressure
within the eye (intraocular pressure [IOP]). Concern
over this side-effect has become a limitation on the
long-term ophthalmic use of both potent and less
active steroids, especially because a number of cases
were reported in which irreversible blindness has
occurred due to unrecognized increased IOP.
15 When detailed evaluations of the potent
ophthalmic steroid formulations was conducted in
large populations using standardized testing
protocols (four times a day administration of
ophthalmic 0.1% dexamethasone eyedrops), it was
20 determined that elevated IOP occurred relatively
frequently in the general population within three to
six weeks. Potentially serious elevations in IOP
occurred in some patients within the first week of
ophthalmic steroid administration. The longer the
25 duration and the more frequent the steroid
administration, the more likely a given patient was
shown to develop elevated IOP. Approximately 5
percent of the general population show an accentuated
IOP elevation to topical ophthalmic steroids (in
30 which IOP can increase from its normal 10 to 20 mm Hg
to over 35 mm Hg) occurring in the one-to-six week
time frame of steroid administration. Some patients
who did not show a high response to the normal
WO 95/17178 PCT/US94/14068
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steroid eyedrop testing protocol did show a large IOP
rise with longer treatments and/or different routes
(e. g. injectable steroids) of steroid administration.
If undetected, such high elevations of IOP las weir
as elevations in the range of 20 to 30 mm Hg) were
known to put the patient at substantial risk of
glaucomatous damage as evidenced by loss of vision in
visual field testing and optic nerve damage. Even
minor increases in IOP can be detrimental in some
patients such as those with low-tension glaucoma.
Elevated IOP is currently recognized as an
important risk factor for the development of optic
nerve damage and visual field loss in otherwise
normal individuals. The concern is even greater for
patients who are already demonstrating such damage
due to glaucoma. Individuals with primary open-angle
glaucoma (POAG), also,called "chronic glaucoma",
which is the most prevalent type of glaucoma, have
additional problems with ophthalmic steroid therapy
in that these patients (and their genetically related
family members) appear predisposed to high IOP
elevations due to steroids. Patients with pigmentary
glaucoma also appear to have an increased sensitivity
to steroid administration, while other forms of
glaucoma do not appear to have this increased
sensitivity.
In addition to direct ophthalmic steroid
use, dermatologic steroids used on the face and
eyelids are sufficient to produce elevated IOP and
visual loss in some individuals. Elevated IOP occurs
also with systemic steroid use, increased endogenous
'steroids as occurs with Cushing's Syndrome, and POAG
WO 95/17178 2 ~ 7 ~ ~ ~ ~ PCT/US94/14068
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itself may involve an increased activity or response
to excessive endogenous glucocorticoid activity.
Efforts have been made to substitute
non-steroidal anti-inflammatory (NSAI) agents, from
the group of drugs known as cyclooxygenase inhibitors
(a number of which were known useful in treating
systemic inflammatory medical conditions), for the
use of steroids in the treatment of ocular
inflammation and ocular pain syndromes. These agents
10 have not shown the same propensity to produce side-
effects in ocular tissues as do ophthalmic steroids
(in addition to the IOP rise, steroid side effects
include steroid cataract, delayed wound healing, and
the masking or spreading of infections), although a
15 concern has remained that NSAI agents might elevate
IOP or produce other steroid-related complications in
the eye since their accepted mechanism of action is
to inhibit the production of prostaglandins and other
eicosanoids similar to steroids. Because
20 prostaglandin administration does appear to decrease
IOP and to increase outflow, blockage of endogenous
prostagladins by steroid (or NSAI agents) in
trabecular meshwork cells from the aqueous outflow
pathway could contribute to a rise in IOP. See,
25 e.g., Weinreb et al., Arachidonic Acid Metabolism in
HTM Cells, Investigative Ophthalmology & Visual
Science, Vol. 29, No. 11 (1988) and Weinreb et al.,
Prostaglandin Production by HTM Cells: In Vitro
Inhibition by Dexamethasone,~Investigative
30 Ophthalmology & Visual Science, Vol. 24, No. 12 ,
(1983).
Doulakas (U. S. Patent No. 4,829,088)
discloses the use of an ophthalmic medicament
WO 95/17178 PCT/US94/14068
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containing diclofenac-sodium in aqueous solution for
the treatment of inflammations of the eye.
Diclofenac-sodium is a non-steroidal
anti-inflammatory agent which is said to be a
suitable alternative for the treatment of severe
acute or chronically recurrent inflammatory symptoms
in the eye. The aqueous solution is made suitable
for the local treatment of inflammations of the eye
due to its stability against chemical decomposition
of the diclofenac-sodium and preservation properties
and toleration by the eye.
Nagy (U. S. Patent No. 4,960,799) also
discloses aqueous ophthalmic solutions containing
diclofenac-sodium. The solutions, having a pH of
about 7.0 to about 7.8, comprise per milliliter of
solution about 0.1-to about 5.0 milligrams of
(a) pharmaceutically acceptable salt of
ortho-(2,6-dichlophenyl-)aminophenyl acetic acid;
(b) about 0.1 to about 10 milligrams of a
pharmaceutically acceptable salt of ethylene diamine
tetraacetic acid, (c) about 0.5 to about 200
milligrams of a pharmaceutically acceptable
solubilizer, (d) about 0.01 to about 5.0 milligrams
of a pharmaceutically acceptable bacteriostat and
(e) the remainder water. The ophthalmic solutions
are used for topical administration to the eye for
the control or treatment of ocular inflammation.
Cherng-Chyi et al. (U. S. Patent
No. 5,110,493) relates to ophthalmic non-steroidal
~ 3o anti-inflammatory drug formulations containing a
quaternary ammonium preservative and a non-ionic
surfactant. The formulations are useful for treating
WO 95/17178 217 7 ~ 7 8 PCT/US94/14068
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diseases that are either caused by, associated with
or accompanied by inflammatory processes.
The NSAI agents used in the eye have been
tested empirically for different ocular inflammatory
conditions, using drugs that had been selected
previously in systemic studies for their ability to
suppress prostaglandin production and to decrease
inflammatory responses in animals and humans. In the
eye, these NSAI agents appear to provide at least
1o some benefit to prevent particular side-effects of
surgical trauma, fluid accumulating in the back of
the eye, appearance of inflammatory cells and vessel
leakage in the anterior chamber, and the presence of
pain. Although potentially useful to these conditions
and ocular disease states, and some ocular conditions
such as post surgical macular edema appear to respond
better to some NSAIs than steroid administration,
NSAI agents do not appear as fully-effective
alternatives for steroid treatment of ocular
20 inflammation in many other conditions, and in a
variety of individual clinical settings ophthalmic
steroid therapy is still preferred.
As mentioned earlier, steroids
(glucocorticoids) are believed to alleviate
25 inflammation at least in part by inhibiting the
production of prostaglandins and other eicosanoids at
early stage involving the utilization of arachidonic
acid (by interactions with lipomodulin-like
molecules), although it is clear that other steroid
30 actions also contribute to their antiinflammatory ,
effects. NSAIs also are believed to inhibit the
formation of prostaglandins and other eicosanoids at
a later step (by interaction with the enzyme
WO 95/17178 PCT/US94/14068
. cyclooxygenase) as the major mechanism for their
antiinflammatory effects. However, since steroids
and NSAI agents both effectively inhibit
prostaglandin and other eicosanoid pathways, a
concern has been that both classes of drugs might
elevate IOP.
8ummarv and Obiects of the Invention
It is a primary objective of the present
invention to provide novel~methods and compositions
for decreasing the propensity of steroids
(glucocorticoids) to produce IOP elevation.
Yet another object of the present invention
is to provide novel methods and compositions capable
of preventing or ameliorating particular eye diseases
such as primary open-angle glaucoma and pigmentary
glaucoma whose conditions are associated with an
increased propensity for steroid-induced IOP
elevation (and whose pathogeneses may involve
endogenous glucocorticoids).
Still other objects of the present
invention are to provide novel methods which employ
NSAI (cyclooxygenase inhibitors) to prevent or treat
elevated IOP induced by glucocorticoids also termed
steroids.
With regard to the above, certain NSAI
agents show quite unexpected activities to block the
appearance of major protein markers for steroid
effects on IOP using human trabecular meshwork
(termed HTM) cells exposed to the potent
glucocorticoid dexamethasone. Such agents for this
reason represent useful therapeutic agents for
treating certain chronic, non-inflammatory forms of
WO 95/17178 PCT/US94/14068
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glaucoma such as POAG and pigmentary glaucoma. Since .
certain NSAI agents were shown to help alleviate
glucocorticoid inductions in cultured HTM cells, both
in the cell layer and in the media surrounding the
5 cells (based on their mobilities on SDS-gel
electrophoresis these major glucocorticoid inductions
have been termed herein "55 kDa" and "66 kDa"
protein/glycoprotein marker inductions, although they
appear in a range of about 52-56 kDa and about 64-70
10 kDa, in the cell and in the media, respectfully).
These proteins have dose response and time course
characteristics that set them apart from most of the
other glucocorticoid regulated proteins in HTM cells,
and these characteristics correspond to those
15 observed for steroid induced IOP changes in human
subjects. For this reason, these proteins have been
identified as "markers" for the glucocorticoid
induced response of IiTM cells related to IOP changes.
Since the 55 kDa protein in the HTM cell layer is
20 rapidly secreted into the media in a glycosylated
form (i.e., the 66 kDa protein), and since the 66 kDa
protein is readily separated from other background
proteins detected using labeled amino-acid protein
precursors, this marker was preferred to evaluate the
25 ability of drugs to block glucocorticoid induced
effects on the cells related to IOP changes. The
NSAI agents may also help the HTM cells to function
more normally and preserve themselves, i.e., maintain
their nonaal extracellular environment, as well as
30 their shape and cell number, when exposed to
glucocorticoids. '
The above findings were quite unexpected
since inhibition of prostaglandins by NSAI agents
R'O 95117178 2 ~ ~ 7 ~ ~ ~ PCT/U894/14068
-g-
would add to the inhibition of prostaglandins by
glucocorticoids in IiTM cells. Such inhibition by
cyclooxygenase inhibitors of these pathways would
not
be expected to produce beneficial effects. In this
regard, the NSAIs as non-steroidal eicosanoid
inhibiting agents of the present invention were
believed to further inhibit pathways needed to
maintain normal outflow pathways and IOP. Thus, the
effective use of such agents to ameliorate elevated
IOP induced by steroid treatment is not expected.
In one aspect, the present invention
. involves methods of arresting processes causing
damage to the eye of a human or other animal that
is
subject to intraocular damage and in need of
.
maintaining visual function or
prevention of its loss
' from such damage, wherein certain non-steroidal
cyclooxygenase inhibitors or NSAI agents which
function as therapeutic agents are administered in
an
inert vehicle to eye tissue by intraocular injection
or topically. The term "inert vehicle" is broadly
used herein to optionally include adjuvants,
preservatives, buffers, demulcents and anything else
that is essentially inert relative to the therapeutic
function of the non-steroidal cyclooxygenase
inhibitors or NSAI agents.
In another aspect, the present invention
involves methods of preventing or treating ophthalmic
diseases or disorders in a human or other animal
that
is subject to intraocular damage and in need of
improved visual function or prevention of its loss
from such damage, wherein an ophthalmologically
effective amount of certain non-steroidal
cyclooxygenase inhibitors or NSAI agents which
WO 95/17178 PCT/US94/14068
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function as a therapeutic agent are administered, in '
an inert vehicle, to arrest processes. and
particularly, those processes induced by steroids
which are damaging to the eye. As used herein,
5 "ophthalmically effective amount" is that amount
which, in the composition administered and by the
technique administered, provides an amount of
therapeutic agent to the involved eye tissues
sufficient to reduce intraocular pressure or prevent
its rise associated with steroid treatments.
The present invention also involves methods
of arresting processes causing damage to the eye,
wherein certain non-steroidal cyclooxygenase
inhibitors or NSAI agents which function as
15 therapeutic agents are administered topically. When
the administration is topical (either dermal or
topically ophthalmic), a topical formulation
containing between 0.001 and 10%, preferably
between 0.05 and 1%, and more preferably between 0.05
and 0.6% by weight; and preferably it is administered
as a topical ophthalmic formulation in an aqueous
polymeric solution, aqueous suspension, ointment, or
gel vehicle. In other preferred topical
formulations, between 0.001 and 0.009% by weight of
25 the agent is employed. Except for ointments, these
vehicles may contain liposomes for creating a
reservoir of dissolved agent for contact with the
tear film.
The present invention also involves methods
30 of arresting processes causing damage to the eye,
wherein certain NSAI agents or non-steroidal
cyclooxygenase inhibitors which function as
therapeutic agents are administered by intraocular
WO 95/1?178 PC"T/US94/14068
i
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injection. When the intraocular injection is
r
subconjunctival, a formulation containing
between 0.001 and 5%, preferably between 0.01
and 1.0%, by weight of the therapeutic agent is
administered; and preferably it is administered in a
polymeric carrier such as methylcellulose
polycarbophil, hydroxymethylcellulose or dextran,
with the formulations containing additives such as
disodium edetate, sodium sulfite, and/or sodium
chloride, and sodium hydroxide or hydrogen chloride
for pH adjustment. When the intraocular injection is
intracameral or intravitreal, a formulation
containing between 0.001 and 1%, preferably
between 0.01 and 1.0% especially when in solution, by
weight of the therapeutic agent is administered; and
preferably it is administered in a vehicle containing
phosphate buffered saline, citrate buffered saline,
or chondroitin sulfate, or in a polymeric vehicle
such as sodium hyaluronate, or hyaluronic acid,
purified polyacrylamide or polysorbate 80, with the
formulation containing sodium hydroxide or hydrogen
chloride for pH adjustment.
The present invention also involves methods
of arresting processes causing damage to the eye,
wherein certain non-steroidal cyclooxygenase
inhibitors or NSAI agents which function as
therapeutic agents are administered systemically.
One such systemic administration is by intramuscular
injection. Such formulations may contain
between 0.01 and 10%, preferably between 0.5 and 5%,
by weight of the therapeutic agent, preferably in a
polysorbate 80, methyl cellulose, or other polymeric
vehicle.
*rB
WO 95/17178 21 l l ~ ~ ~ pCT~S94/14068
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Another such systemic administration is by
oral dosage. When administered orally in an aqueous
solution, aqueous suspension, elixir or other liquid,
formulations containing between 0.1 and 50%,
preferably between 1.0 and 10%, by weight of the
therapeutic agent may be employed. When administered
orally as a solid, tablets, caplets or capsules
containing between 1 and 1000 mg of the therapeutic
agent may be employed.
Another such systemic administration is by
intravenous injection. When administered
intravenously, formulations containing between 0.05
and 5%, preferably between 0.1 and 1% by weight, of
the therapeutic agent is employed, preferably in a
citrate buffer or borate buffer carrier or in a lipid
emulsion, unilamellar liposome or multilamellar
liposome formulation.
In a further aspect, the present invention
involves a composition for preventing or treating
ophthalmic diseases or disorders, comprising: (a) a
selected non-steroidal cyclooxygenase inhibiting
therapeutic agent or NSAI agent, and (b) an inert
ophthalmic vehicle, suitable for a topical,
preferably topically ophthalmic, systemic or
intraocular application, selected from polymeric
solutions, suspensions, ointments or gels. The
aqueous carriers may contain liposomes for creating a
reservoir of dissolved agents for contact with the
tear film. Gels for topical ophthalmic applications
are the most preferred carriers. The composition ,
contains an ophthalmically effective amount of an
cyclooxygenase inhibiting agent or NSAI agent to
arrest processes damaging to the eye resulting from
WO 95/17178 ; ; PCT/US94/14068
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increased intraocular pressure associated with
steroid treatment.
In another aspect of the invention, there
is a novel ointment, gel or drop ophthalmic
composition containing an amount of NSAI less than or
equal to that typically required to reduce
inflammation. Such composition includes a
cyclooxygenase inhibiting agent and or NSAI agent and
an inert carrier suitable for application to the eye.
Preferably, the composition contains 0.001 to 0.009,
preferably 0.005 to 0.008 percent by weight
cyclooxygenase inhibiting agent.
Advantageously, the cyclooxygenase
inhibiting agents or NSAI agents may be administered
chronically or acutely, while preventing tissue
damage and minimizing any acute injury and preventing
the onset of the diseased state.
With the foregoing as well as other
objects, advantages, features and aspects of the
invention that will become hereinafter apparent, the
nature of the invention may be more clearly
understood by reference to the detailed description
of the invention and to the appended claims.
Detailed Description of Preferred Embodiments
The present invention encompasses methods
and compositions for preventing or treating an
- ophthalmic disease or disorder, wherein an
ophthalmically effective amount of a non-steroidal
cyclooxygenase inhibiting, or non-steroidal
anti-inflammatory (NSAI) therapeutic agent, and an
'inert ophthalmic vehicle, are administered to prevent
onset of or reduce abnormally high IOP associated
*rB
WO 95/17178 PCT/US94/14068
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with treatment with steroids, corticosteroids or
glucocorticoids. The steroids, corticosteroids or
glucocorticoids may be used as a treatment of an
ocular or other condition such as inflammation or
5 naturally produced by the organism treated, i.e.,
Coshing syndrome.
Non-steroidal anti-inflammatory agents are
widely prescribed to reduce pain and inflammation in
a wide number of tissues. This includes their
10 application as topical agents in the eye, in which
their ability to suppress inflammatory responses and
to prevent particular side-effects of surgical trauma
(on the pupil preventing surgical meiosis), fluid
accumulating in the back of the eye after cataract
15 surgery (post-surgical macular edema) and the
appearance of inflammatory cells and vessel leakage
in the anterior chamber. Topical application of NSAI
agents in the eye also appear to relieve some of the
itching due to allergic conjunctivitis. These
20 conditions fit in the normal and expected effects of
NSAI agents in inflammation and pain.
The present invention, however, relates to
new methods of treatment and compositions for
relieving or arresting processes which induce
25 elevated IOP associated with steroid use. While not
wishing to be bound by theory, steroid elevations of
IOP appear to be due to an increased resistance in
the trabecular meshwork pathway, the major pathway
through which aqueous humor fluid produced by the
30 ciliary body must flow.
The invention also includes treatment of
types of glaucoma such as steroidal glaucoma and POAG
and pigmentary glaucoma which are not thought to
WO 95/17178 PCTIUS94/14068
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~ involve inflammation as a pathogenic mechanism.
Thus, the present invention relates to prevention and
treatment of elevated IOP associated with steroid or
glucocorticoid treatment.
Prior to discussing examples of the
invention, a brief discussion is provided concerning
non-steroidal cyclooxygenase inhibiting agents. As
used herein, °cyclooxygenase inhibiting agents"
include those compounds which inhibit prostaglandin
and other eicosanoid or cyclooxygenase pathways which
are believed to affect IOP. Compounds considered
within the classification of cyclooxygenase
inhibitors include certain NSAI agents.
NSAI agents have been documented by
J. Lombardino in "Nonsteroidal Antiinflammatory
Drugs", Wiley-Interscience, New York, 1985. Examples
of compounds of this class of antiinflammatory drugs
include but are not limited to the following:
aspirin, benoxaprofen, benzofenac, bucloxic acid,
butibufen, carprofen, cicloprofen, cinmetacin,
clidanac, clopirac, diclofenac, etodolac, fenbufen,
fenclofenac, fenclorac, fenoprofen, fentiazac,
flunoxaprofen, furaprofen, flurbiprofen, furobufen,
furofenac, ibuprofen, ibufenac, indomethacin,
indoprofen, isoxepac, ketoprofen, lactorolac,
lonazolac, metiazinic, miroprofen, naproxen,
oxaprozin, oxepinac, phenacetin, pirprofen,
pirazolac, protizinic acid, sulindac, suprofen,
tiaprofenic acid, tolmetin, and zomepirac.
. 30 Non-steroidal cyclooxygenase inhibiting
compounds can be prepared in the form of
pharmaceutically acceptable salts, esters and other
prodrugs. Derivative salts~include relatively
WO 95/17178 PCT/US94/14068
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non-toxic inorganic or organic acid addition salts or
alkaline earth metal salts of the therapeutic
compounds, which can be prepared in situ during the
final isolation and purification of the compounds or
by separately reacting the free base with a suitable
organic or inorganic acid. Where the compounds
include a basic functionality such as amine or
alkylamine, representative salts include
hydrochloride, sulfate, acetate, maleate, lauryl
sulphate, and the like. Where an acidic
functionality is present, salts such as sodium,
calcium, potassium and magnesium salts may be formed.
The phrase "NSAI agent" as used herein is
intended to mean any non-narcotic analgesic/non-
steroidal anti-inflammatory compound useful as a
cyclooxygenase inhibitor, including but not limited
to the derivatives of (1) propionic acid, (2) acetic
acid derivatives, (3) fenamic acid,
(4) biphenylcarboxylic acid and (5) oxicams.
While some of these agents are primarily
used at the present time as anti-inflammatory agents
and others are primarily used as analgesics, in fact
it is believed that all of the contemplated compounds
have both analgesic and anti-inflammatory activity
and can be used at appropriate dosage levels for
either purpose in various compositions.
The compounds in groups (1) through (4)
typically contain a carboxylic acid function;
however, those acids are sometimes administered in
the form of their pharmaceutically acceptable acid
addition or alkali metal salts, e.g., sodium salts.
The propionic acid derivatives include, but
are not limited to, ibuprofen, naproxen,
2 i 71578
WO 95/17178 PCT/US94/14068
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benoxaprofen, flurbiprofen, fenoprofen, fenbufen,
ketoprofen, indoprofen, pirprofen, carprofen,
oxaprozin, pranoprofen, miroprofen, tioxaprofen,
suprofen, alimoprofen, tiaprofenic acid, fluprofen
and bucloxic acid. Structurally related propionic
acid derivatives having similar analgesic and
anti-inflammatory properties are also intended to be
encompassed by this group.
Thus, "propionic acid derivatives" as
defined herein are non-narcotic analgesics/non
steroidal anti-inflammatory drugs having a free
-CH (CH3) COOH or -CHzCH2COOH group (which optionally can
be in the form of a pharmaceutically acceptable salt
group, e.g., -CH(CH3)COO-Na+), typically attached
directly or via a carbonyl function to a ring system,
preferably to an aromatic ring system.
The acetic acid derivatives as defined
herein include, but are not limited to, indomethacin,
sulindac, tolmetin, zomepirac, diclofenac,
fenclofenac, alclofenac, ibufenac, isoxepac,
furofenac, tiopinac, zidometacin, acemetacin,
fentiazac, clidanac and oxpinac. Structurally
related acetic acid derivatives having similar
analgesic and anti-inflammatory properties are also
intended to be encompassed by this group.
Thus, "acetic acid derivatives" as defined
herein are non-narcotic analgesics/non-steroidal
. anti-inflammatory drugs having a free -CHZCOOH group
(which optionally can be in the form of a
pharmaceutically acceptable salt group, e.g.,
-CHZCOO'Na+), typically attached directly to a ring
system, preferably to an aromatic or heteroaromatic
ring system.
WO 95/17178
PCT/US94/14068
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The fenamic acid derivatives as defined
herein include, but are not limited to, mefenamic
acid, meclofenamic acid, flufenamic acid, niflumic
acid and tolfenamic acid. Structurally related
fenamic acid derivatives having similar analgesic and
anti-inflammatory properties are also intended to be
encompassed by this group.
Thus, "fenamic acid derivative" as defined
herein are non-narcotic analgesics/non-steroidal
anti-inflammatory drugs which contain the basic
structure
COOH
which can bear a variety of substituents and in which
the free -COOH group can be in the form of a
pharmaceutically acceptable salt group, e.g.,
--c00'NA+ .
The biphenylcarboxylic acid derivatives as
defined herein include, but are not limited to,
diflunisal and flufenisal. Structurally related
biphenylcarboxylic acid derivatives having similar
analgesic and anti-inflammatory properties are also
intended to be encompassed by this group. '
Thus, "biphenylcarboxylic acid derivative"
as defined herein are non-narcotic analgesics/non- '
steroidal anti-inflammatory drugs which contain the
basic structure
WU 95/17178 PCT/US94/14068
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COON
which can bear a variety of substituents and in which
the free -COOH group can be in the form of a
pharmaceutically acceptable salt group, e.g.,
-COO~TA* .
The oxicams as defined herein include, but
are not limited to, piroxicam, sudoxicam, isoxicam,
and CP-14,304. Structurally related oxicams having
l0 similar analgesic and anti-inflammatory properties
are also intended to be encompassed by this group. A
preferred member of this group is piroxicam.
Thus, "oxicams" as defined herein are
non-narcotic analgesics/non-steroidal
anti-inflammatory drugs which have the general
formula
OH
O
2 0 ~ ~ . \ C-NH-R
~3
~Z
wherein R is an aryl or heteroaryl ring system.
2i 1~~78
WO 95/17178 PCT/L1S94/14068
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Also included within the non-steroidal
cyclooxygenase inhibitors or NSAI agents of the
present invention are inhibitors as described by
Flach, "Cyclooxygenase Inhibitors in Ophthalmology,"
Survey of Ophthalmology; Vol. 36, No. 4,
(Jan.-Feb. 1992). Cyclooxygenase inhibitors are also
non-steroidal antiinflammatory drugs that have become
available as ophthalmic eyedrops for treatment of
inflammation. These inhibitors may be grouped'into
six different classes: salicylates, fenamates,
indoles, phenylalkanoic acids and pyrazolones.
Specific drugs within the respective groups are
summarized below.
Cyclooxygenase Inhibitors
Chemical Class Generic Name
Salicylates Aspirin, Salicylic Acid,
Diflunisol
Indoles Indomethacin, Sulinda,
Tolmetin
Phenylalkanoic acids Fenoprofen, Flurbiprofen,
_ Ibuprofen, Ketoprofen,
Ketorolac, Naproxen,
Piroxicam, Suprofen
Phenylacetic acids Diclofenac
Pyrazolons Oxyphenbutazone,
Phenylbutazone, Antipyrine,
Aminopyrine, Azapropazone
The precise type of and amount of NSAI
agent or non-steroidal cyclooxygenase or other
eicosanoid inhibitor for use in the present
compositions will vary depending, for example, on the
specific drug chosen, the dosage form thereof, i.e.,
WO 95/17178 PCT/US94/14068
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standard versus sustained release, the condition for
which the drug is administered and the size and kind
of the organism treated.
It is within the ability of those skilled
in the art to determine, upon reading this
disclosure, which of the foregoing cyclooxygenase
inhibiting agents will function to prevent an
increase of IOP or actually decrease IOP associated
with steroid, corticosteroid or glucocorticoid
10 treatment. Preferably, those compounds of the
present invention include all non-steroidal
cyclooxygenase inhibitors which provide a reduction
in or prevention of enhanced IOP induced by
glucocorticoid treatment when used in an amount
15 sufficient to provide a concentration of 1 x 10-s M or
less, preferably an amount of about 1 x 10'~ M to
about 1 x 10-s M, more preferably about 1 x 10'~ M to
about 1 x 10'~ M in the aqueous or treated tissue of
the eye.
20 By formulating the above-described
therapeutic agents into an appropriate inert vehicle
or carrier, it is possible to reduce or treat
elevated intraocular pressure associated with
steroid, corticosteroid or glucocorticoid treatment.
25 In order to maintain an adequate therapeutic level of
drug in the eye, the present invention also
contemplates the treatment of an ophthalmic disease
by administration of an opthalmically effective
amount of the non-steroidal cyclooxygenase inhibiting
30 agents of the present invention (including salts,
hydrates, or solvates), in a suitable carrier, by
oral, intramuscular and intravenous routes, in
WO 95/1?178 PCTlUS94/14068
-22-
addition to the convenient topical route or by
intraocular injection.
In general, ophthalmic formulations
suitable for topical and intraocular administration
5 may be formulated and administered in accordance with
techniques known to persons skilled in the art. The
formulations are preferably prepared in an anaerobic
environment by making all formulations under an inert
gas. The finished formulations are preferably stored
10 in opaque or brown containers to protect them from
light exposure, and under an inert atmosphere.
Aqueous polymeric solutions, aqueous
suspensions, ointments, and gels are preferably used
for topical formu7.ations. The aqueous formulations
15 may also contain liposomes for creating a reservoir
of dissolved therapeutic agent. Particularly
preferred among topical formulations are gels, which
enhance pre-corneal retention without the
inconvenience and impairment of vision associated
20 with ointments.
Topical ophthalmic or other topical
formulations should generally include between 0.001
and 10% by weight, preferably between 0.05 and 1% by
weight and most preferably 0.05 and 0.6% by weight,
25 of the therapeutic agent in a suitable polymeric
carrier. Other preferred formulations contain
between 0.001 to 0.009% by weight of the therapeutic
agent. As will be appreciated by those skilled in
the art, the amounts of non-steroidal agent needed to
30 reduce IOP associated with steroid treatments include
those amounts which will not effectively reduce
inflammation, i.e., amounts lower than currently used
in topical antiinflammatory formulations.
WO 95/17178 PCT/US94/14068
-23-
Suitable polymeric carriers include lightly
crosslinked carboxy-containing polymers (such as
polycarbophil), dextran, cellulose derivatives,
polyethyleneglycol 40o and other polymeric
demulcents.
A preferred system includes lightly
crosslinked polymers of acrylic acid or the like,
which are well known in the art. In a preferred
embodiment, such polymers are ones prepared from at
10 least about 90%, and preferably from about 95% to
about 99.9% by weight, based on the total weight of
monomers present, of one or more carboxyl-containing
monoethylenically unsaturated monomers. Acrylic acid
is the preferred carboxyl-containing
15 monoethylenically unsaturated monomer, but other
unsaturated, polymerizable carboxyl-containing
monomers, such as methacrylic acid, ethacrylic acid,
~B-methylacrylic acid (crotonic acid),
cis-a-methylcrotonic acid (angelic acid),
20 trans-a-methylcrotonic acid (tiglic acid),
a-butylcrofionic acid, a-phenylacrylic acid,
a-benzylacrylic acid, a-cyclohexylacrylic acid,
~-phenylacrylic acid (cinnamic acid), coumaric acid
(o-hydroxycinnamic acid), umbellic acid
25 (p-hydroxycoumaric acid), and the like can be used in
addition to or instead of acrylic acid.
Such polymers are crosslinked by using a
small percentage, i.e., from about 0.01% to about 5%,
and preferably from about 0.1% to about 2%, based on
30 the total weight of monomers present, of a
polyfunctional crosslinking agent. Included among
such crosslinking agents are non-polyalkenyl
polyether difunctional crosslinking monomers such as
WO 95/17178
217 7 ~ ~ ~ pCT~S94/14068
-24-
divinyl glycol; 2,3-dihydroxyhexa-1,5-diene;
2,5-dimethyl-1,5-hexadeone; divinylbenzene;
N,N-diallylacrylamide; N,N-diallylmethacrylamide and
the like. Also included are polyalkenyl polyether
crosslinking agents containing two or more alkenyl
ether groupings per molecule, preferably alkenyl
ether groupings containing terminal HZC=C< groups,
prepared by etherifying a polyhydric alcohol
containing at least four carbon atoms and at least
three hydroxyl groups with an alkenyl halide such as
allyl bromide or the like, e.g., polyallyl sucrose,
polyallyl pentaerythritol, or the like; see, e.g.,
Brown, U.S. Patent No. 2,798,053. Diolefinic
non-hydrophilic macromeric crnsslinking agents having
molecular weights of from about 400 to about 8,000,
such as insoluble di-and polyacrylates and
methacrylates of diols and polyols, diisocyanate-
hydroxyalkyl acrylate or methacrylate reaction
products, and reaction products of isocyanate
terminated prepolymers derived from polyester diols,
polyether diols or polysiloxane diols with
hydroxyalkylmethacrylates, and the like, can also be
used as the crosslinking agents; see, e.g., Mueller
et al., U.S. Patents Nos. 4,192,827 and 4,136,250.
The lightly crosslinked polymers can of
course be made from a carboxyl-containing monomer or
monomers as the sole monoethylenically unsaturated
monomer present, together with a crosslinking agent -
or agents. They can also be'polymers in which up to
about 40%, and preferably from about 0% to about 20% -
by weight, of the carboxyl containing
monoethylenically unsaturated monomer or monomers has
been replaced by one or more non-carboxyl-containing
WO 95/17178 ~ PCT/US94/14068
-25-
monoethylenically unsaturated monomers containing
only physiologically and ophthalmologically innocuous
substituents, including acrylic and methacrylic acid
esters such as methyl methacrylate, ethyl acrylate,
butyl acrylate, 2-ethyl-hexylacrylate, octyl
methacrylate, 2-hydroxyethyl-methacrylate, 3-
-hydroxypropylacrylate, and the like, vinyl acetate,
N-vinylpyrrolidone, and the like; see Mueller et al.,
U.S. Patent No. 4,548,990 for a more extensive'
listing of such additional monoethylenically
unsaturated monomers. Particularly preferred
polymers are lightly crosslinked acrylic acid
polymers wherein the crosshinking monomer is
2,3-dihydroxyhexa-1,5-diene or
2,3-dimethylhexa-1,5-diene.
The lightly crosslinked polymers used in
practicing this invention are preferably prepared by
suspension or emulsion polymerizing the monomers,
using conventional free radical polymerization
catalysts, to a dry particle size of not more than
about 50 ;cm in equivalent spherical diameter; e.g.,
to provide dry polymer particles ranging in size from
about 1 to about 30 ;cm, and preferably from about 3
to about 20 um, in equivalent spherical diameter. In
general, such polymers will range in molecular weight
estimated to be greater than 2,000,000.
Aqueous suspensions formulated in
accordance with this invention containing polymer
particles prepared by suspension or emulsion
polymerization whose dry particle size is appreciably
larger than about 50 ;Cm in equivalent spherical
diameter are less comfortable when administered to
the eye than suspensions otherwise identical in
WO 95/17178 217 7 5 7 8 p~~s94/14068
-26-
composition containing polymer particles whose
. equivalent spherical diameters are, on the average,
below about 50 Vim. Lightly crosslinked polymers of
acrylic acid or the like prepared to a dry particle
size appreciably larger than about 50 ~cm in
equivalent spherical diameter and then reduced in
size, e.g., by mechanically milling or grinding, to a
dry particle size of not more than about 50 ~cm in
equivalent spherical diameter do not work as well as
polymers made from aqueous suspensions. One possible
explanation for the difference of such mechanically
milled or ground polymer particles as the sole
particulate polymer present is that grinding disrupts
the spatial geometry or configuration of t:~e larger
than 50 um lightly crosslinked polymer particles,
perhaps by removing uncrosslinked branches from
polymer chains, by producing particles having sharp
edges or protrusions, or by producing ordinarily too
broad a range of particle sizes to afford
satisfactory delivery system performance. A broad
distribution of particle sizes will impair the
viscosity-gelation.relationship. In any event, such
mechanically reduced particles are less easily
hydratable in aqueous suspension than particles
prepared to the appropriate size by suspension or
emulsion polymerization, and also are less able to
gel in the eye under the influence of tear fluid to a
sufficient extent and are less comfortable once
gelled than gels produced in the eye using the
aqueous suspensions of this invention. However, up
to about 40% by weight, e.g., from about 0% to
over 20% by weight,'based on the total weight of
lightly crosslinked particles present, of such milled
WO 95/17178 ~ PCT/US94/14068
-27-
or ground polymer particles can be admixed with
solution or emulsion polymerized polymer particles
having dry particle diameters of not more than
about 50 um when practicing this invention. Such
mixtures will also provide satisfactory viscosity
levels in the ophthalmic medicament delivery systems
with ease and comfort of administration and
satisfactory sustained release of the medicament to
the eye, particularly when such milled or ground
polymer particles, in dry form, average from
about 0.01 to about 30 ~tm, and preferably from
about 1 to about 10 Vim, in equivalent spherical
diameter.
In the most preferred embodiment of the
invention, the particles have a narrow particle size
distribution. The use of a monodisperse particle
will give maximum viscosity and an increased eye
residence time of the ophthalmic medicament delivery
systems for a given particle size. Monodisperse
particles having a particle size of 30 ~m and below
are most preferred. Good particle packing is aided
by a narrow particle size distribution.
The particles are not only subject to the
upper size limits described above, but also to a
narrow particle size distribution. Such use of a
monodispersion of particles, which aids in good
particle packing, yields a maximum increased
viscosity upon contact of the suspension with the
tears and increases eye residence time. At least
about 80%, more preferably at least about 90% and
most preferably at least about 95%, of the particles
should be within a no more than about 10 ~Cm band of
major particle size distribution, and overall (i.e.,
WO 95/17178 PCT/US94/14068
~ ~ ~'~~~~8
-28-
considering particles both within and outside such
band) there should be no more than about 20%,
preferably no more than about 10% and most preferably
no more than about 5% fines (i.e., particles of a
5 size below 1 ~Cm). It is also preferred that as the
average particle size is lowered from the upper limit
of 50 ~Cm, more preferably 30 ~Cm, to lower sizes such
as 6 ~cm, that the band of major particle distribution
be also narrowed, for example to 5 ~Cm. Preferred
10 sizes for particles within the band of major particle
distribution are less than about 30 ~cm, more
preferably less than about 20 hem, most preferably
from about 1 ~m to about 5 ~Cm.
The aqueous suspensions of this invention
15 may preferably contain amounts of lightly crosslinked
polymer particles ranging from about 0.1% to
about 6.5% by weight, and preferably from about 0.5%
to about 4.5% by weight, based on the total weight of
the aqueous suspension. They will preferably be
20 prepared using pure, sterile water, preferably
deionized or distilled, having no physiologically or
ophthalmologically harmful constituents, and will be
adjusted to a neutral pH of about 7.0 to about 7.4
using any physiologically and ophthalmologically
25 acceptable pH adjusting acids, bases or buffers,
e.g., acids such as acetic, boric, citric, lactic,
phosphoric, hydrochloric, or the like, bases such as
sodium hydroxide, sodium phosphate, sodium borate,
sodium citrate, sodium acetate, sodium lactate, THAM
30 (trishydroxymethylamino-methane), or the like and .
salts and buffers such as citrate/dextrose, sodium
' bicarbonate, ammonium chloride and mixtures of the
aforementioned acids and bases.
WO 95/17178 PCT/US94/14068
21 X1518
-29-
When formulating the aqueous suspensions of
this invention, their osmotic pressure ( rr ) will be
adjusted to from about 10 milliosmolar (mOsM) to
about 400 mOsM, and preferably from about 100 to
about 250 mOsM, using appropriate amounts of
physiologically and ophthalmologically acceptable
salts. Sodium chloride is preferred to approximate
physiologic fluid, and amounts of sodium chloride
ranging from about 0.01% to about 1% by weight and
preferably from about 0.05% to about 0.45% by weight,
based on the total weight of the aqueous suspension,
will give osmolalities within the above-stated
ranges. Equivalent amounts of one or more salts made
up of cations such as potassium, ammonium and the
like and anions such as chloride, citrate, ascorbate,
borate, phosphate, bicarbonate, sulfate, thiosulfate,
bisulfite and the like, e.g., potassium chloride,
sodium thiosulfate, sodium bisulfite, ammonium
sulfate, and the like can also be used in addition to
or instead of sodium chloride to achieve osmolalities
within the above-stated ranges.
The amounts of lightly crosslinked polymer
particles, the pH,~and the osmotic pressure chosen
from within the above-stated ranges will be
correlated to give aqueous suspensions preferably
having viscosities ranging from about 1,000 to
about 30,000 centipoise, and preferably from
about 5,000 to about 30,000 centipoise, as measured
at room temperature (about 25°C) using a Brookfield
Digital LVT Viscometer equipped with a number 25
spindle and a 13R small sample adapter at 12 rpm.
Higher viscosities may also be employed, and
WO 95/17178 PCT/US94/14068
2~~1~%8
-30-
formulations of less than 100,000 centipoise can be
administered as a ribbon.
The viscous gels that result from fluid
eyedrops delivered by means of the aqueous
suspensions of this invention have residence times in
the eye ranging from about 2 to about 12 hours, e.g.,
from about 3 to about 6 hours. The medicaments
contained in these drug delivery systems will be
released from the gels at rates that depend on .such
factors as the drug itself and its physical form, the
extent of drug loading and the pH of the system, as
well as on any drug delivery adjuvants, such as ion
exchange resins compatible with the ocular surface,
which may also be present. Preferably, the aqueous
suspensions provide a sustained concentration of
cyclooxygenase inhibitor of between 10'° and 10'~ M, and
more preferably between 10~ and 10's M, in the aqueous
or treated tissue of the eye for at least two hours,
preferably at least three hours.
The aqueous suspension topical ophthalmic
medicament delivery systems of this invention can be
formulated in any of several preserved or
nonpreserved ways. For example the drug, the lightly
crosslinked polymer particles, and the osmolality-
adjusting salt can be pre-blended in dry form, added
to all or part of the water, and stirred vigorously
until apparent polymer dispersion is complete, as
evidenced by the absence of visible polymer
aggregates. Sufficient pH adjusting agent is then
added incrementally to reach the desired pH, and more _
water to reach 100 percent formula weight can be
added at this time, if necessary. Another convenient
method involves adding the drug to about 95 percent
WO 95/17178
PCT/US94/14068
-31-
of the final water volume and stirring for a
sufficient time to saturate the solution. Solution
saturation can be determined in known manner, e.g.,
using a spectrophotometer. The lightly crosslinked
polymer particles and the osmolality-adjusting salt
are first blended in dry form and then added to the
drug-saturated suspension and stirred until apparent
polymer hydration is complete. Following the
incremental addition of sufficient pH adjusting agent
to reach the desired pH, the remainder of the water
is added, with stirring, to bring the suspension
to 100 percent formula weight.
These aqueous suspensions can be packaged
in preservative-free, single-dose non-reclosable
containers. This permits a single dose of the
medicament to be delivered to the eye one drop at a
time, with the container then being discarded after
use. Such containers eliminate the potential for
preservative-related irritation and sensitization of
the corneal epithelium, as has been observed to occur
particularly from ophthalmic medicaments containing
mercurial preservatives. Multiple-dose containers
can also be used, ~if desired, particularly since the
relatively low viscosities of the aqueous suspensions
of this invention permit constant, accurate dosages
to be administered dropwise to the eye as many times
each day as necessary. In those suspensions where
preservatives are to be included, suitable
preservatives are chiorobutanol, Polyquat,
benzalkonium chloride, cetyl bromide, and the like.
Other additives which are desirably
' ' ~ included in the topical formulations include sodium
chloride, EDTA (disodium edetate), surfactants, and
*rB
WO 95/17178 PCT/US94/14068
-32-
preservatives like BAK (benzalkonium chloride).
Administration of the formulation to the eye will
typically be carried out between one and four times a
day, depending on the particular problem being
treated.
Formulations suitable for ocular injection
fall into two categories. For subconjunctival
injection, the formulations should generally include
between 0.001 and 5% by weight, preferably
between 0.01 and 1% by weight of therapeutic agent.
Any suitable carriers may be employed, preferably
polymeric carriers such as dextran or polysorbate 80.
Other additives which desirably may be included in
the formulations are disodium edetate and sodium
sulfite. To administer the formulations to the eye,
the drug formulations will be slowly injected into
the bulbar conjunctiva of the eye.
For intracameral or intravitreal
injections, the suitable formulation should include
phosphate buffered saline, citrate buffered saline,
chondroitin sulfate, or a polymeric carrier such as
sodium hyaluronate.(or hyaluronic acid), purified
polyacrylamide or polysorbate 80. other additives
which are desirably included in the ocularly
injectable formulations are sodium chloride, sodium
hydroxide and hydrogen chloride, where sodium
hydroxide and hydrogen chloride are used for
adjustment of pH. Typically, the formulations
contain between 0.001 and 1%, preferably between 0.01
and 1.0% especially when in solution, by weight of
the agent.
When the agent is substantially in
solution, it is rapidly available to exert its
WO 95/17178 ~ PCT/US94/14068
-33-
therapeutic function and lower concentrations may
therefore be administered to achieve effective levels
without causing tissue intolerance. When the agent
is substantially in suspension, higher concentrations
may be administered to achieve a sustained effective
level, again without causing tissue intolerance.
Hence, with solutions, lower concentrations are
employed to avoid local tissue damage. With a
suspension, higher concentrations are employed
because a smaller dissolved amount is introduced for
immediate activity.
To administer the formulations
intravitreally to the eye, the drug formulation will
be injected through the sclera layer of the eye into
the vitreous cavity. To administer the formulations
intracamerally, the drug formulations will be
injected through the cornea into the anterior chamber
of the eye.
Formulations for intravenous,
intramuscular, and oral administration are likewise
prepared in accordance with techniques well known to
persons skilled in the art. Intravenous formulations
for ophthalmic use~in methods of the present
invention may be prior art formulations used for
other purposes and will typically include
between 0.01 and 50.0% by weight and preferably
between 1.0 and 10.0% by weight of the therapeutic
agent. Suitable carriers for such NSAI agents are
those well known to persons skilled in the art such
. 30 as citrate buffer, borate buffer and others. Other
additives which may be desirably added to intravenous
formulations include sodium chloride, sodium sulfite,
disodium edetate and benzyl alcohol. Alternative
WO 95/17178 ~ PCT/US94/14068
-34-
formulations suitable for intravenous administration -
include carriers such as lipid emulsions containing
the therapeutic agent. To administer the intravenous
formulations for treatment of the eye, the drug
formulations are preferably dose injected or infused
into a major vein (e.g.,-in the arm area), or
introduced by continuous intravenous drip.
Intramuscular formulations will typically
include between 0.01 and 10.0% by weight and
preferably between 0.5 and 5.0% by weight of the
therapeutic agent. Suitable adjuvants in aqueous
solution or suspension for intramuscular lazaroid
formulations are those well known to persons skilled
in the art such as polysorbate.80, methyl cellulose,
and other demulcents. Other additives desirably
added to intramuscular formulations include sodium
chloride and sodium bisulfite. To administer the
intramuscular formulations for treatment of the eye,
the drug formulations will be injected for example
into the upper outer quadrant of the gluteal muscle.
Formulations suitable for oral
administration will include both liquid formulations
(aqueous solutions, aqueous suspension, elixirs, and
the like) and solid dosage forms, both containing
i5 additives and adjuvants well known to persons skilled
in the art. Aqueous solutions and suspensions for
liquid oral administration will typically contain
between 0.05 and 50% by weight and preferably
between 1.0 and 10.0% by weight of the NSAI agent.
Suitable adjuvants may be used as carriers to provide
wetability and stability such as propylene glycol,
lightly crosslinked carboxy-containing polymers such
as polycarbophil, ethyl cellulose, hydroxypropyl
WO 95/17178 ~ PCT/US94/14068
-35-
cellulose and methyl cellulose. Other additives,
including sodium edetate, methyl and propyl parabens,
flavoring agents and colorants may be employed if
desirable. Solid dosage forms for oral
administration may also be prepared as capsules,
caplets or tablets with the aid of fillers,
lubricants and stabilizers. To administer oral
formulations for treatment of the eye, the drug is
swallowed in solid dosage form or as a solution or
suspension.
Studies of human trabecular meshwork (HTM)
cells grown in tissue culture in which biochemical
and morphological responses have been evaluated have
provided a model system to evaluate the mechanisms
for the development of steroid effects to raise IOP,
and provide a means to investigate new therapeutic
approaches. Under appropriate cell culture
conditions, HTM cells may be propagated using
sufficiently high split ratios to obtain populations
of these cells at early passages for reproducible
experimental evaluations. ,~ vitro studies of
confluent, stable monolayers of HTM cells reveal a
variety of structural and functional properties of
the trabecular meshwork cell type which appear
important for normal maintenance of the aqueous humor
outflow pathway. Using these cells, it is possible
to consider alterations produced by steroids and
other drugs that may be related to effects on
intraocular pressure (IOP).
Investigations of HTM cells with topical
glucocorticoid treatment proved that steroids such as
dexamethasone produced major new protein inductions
in HTM cells which became progressively more
WO 95/17178 ~ ~ 7 7 5 7 8 pCT~s94/14068
-36-
noticeable between 1 and 3 weeks of 100 nM
dexamethasone exposure. The correlation between
dexamethasone effects on these protein inductions and
the clinically observed rise in IOP suggested that
prolonged glucocorticoid treatments on HTM cells
provide a model system to study steroid effects on
outflow facility, as described in Polansky et al.,
"Glucocorticoid regulation of cultured human
trabecular cells: a model system to study effects of
steroids on IOP," Invest Ophthalmol Vis
Sci 26:5 (1985). Studies of the HTM model system
have reported that inductions of
protein/glycoproteins in the molecular weight range
of about 54-56 kDa (glucocorticoid-induced protein at
55 kDa, termed GIP-55 in the above-cited publication)
and about 65-67 kDa (glucocorticoid-induced protein
at 66 kDa, termed GIP-66 in the above-cited
publication) were found in cytosol and media
fractions, respectively, of dexamethasone treated HTM
cultures. Thus, these proteins/glycoproteins provide
a suitable marker for steroid induced elevated IOP
since these inductions were not observed in the non-
treated controls evaluated in this study. As
mentioned above, these markers are referred to herein
as 55 kDa and 66 kDa protein/glycoprotein marker
inductions but, of course, as will be appreciated by
those skilled in the art, the actual molecular weight
of the protein according to the methods described
herein, i.e., gel electrophoresis, is within a range
of the recited values and when referred to herein
such marker induction should include the major
induction within the range.
WO 95/17178
PCT/US94114068
-37-
Using the above models, it has now
unexpectedly been found that the non-steroidal
cyclooxygenase inhibiting agents or NSAI agents of
the present invention do not induce, or induce to a
minimal extent, the protein markers for elevated IOP
in the model system. In fact, it has unexpectedly
been found that conjoint treatment of steroids and
NSAI agents provides protein/glycoprotein marker
reduction and, thus, would be expected to help
minimize or prevent the elevated IOP found with
steroid treatment.
According to preferred embodiments of the
invention, the cyclooxygenase inhibiting agents or
NSAI agents of the present invention provide a
protein/glycoprotein marker reduction of steroid
induced glycoprotein markers, i.e., either 55 kDA
or 66 kDa proteins, where the marker reduction equals
induction of glycoprotein induction of glycoprotein
with steroid treatment alone - with combination of steroid
2 0 and non-steroid treatment
% 100$
induction of glycoprotein
with steroid treatment alone
Of course, steroid induced elevated IOP may also be
subsequently treated using the non-steroidal
cyclooxygenase agents of the present invention. In
such case, the agent may be applied to reduce
WO 95/17178 1 ~ PCT/ITS94/14068
-38-
elevated IOP. In this case, the marker reduction
equals
induction of glycoprotein induction of glycoprotein
with steroid treatment alone - after treatment with non-
steroidal agent
X 100%
induction of glycoprotein
with steroid treatment alone
Most preferably, the non-steroidal
10 inhibiting agents or NSAI agents used in the methods
and compositions of the present invention are used in
an amount sufficient to provide a protein marker
reduction of at /east about 5%, more preferably at
least about 10%, even more preferably at least
15 about 20% and most preferably at least about 40%. Of
course, the amount of protein marker reduction
depends on the type end amount of non-steroidal
cyclooxygenase inhibiting agent or NSAI agent used.
In view of the above, the non-steroidal
20 cyclooxygenase inhibiting agents or NSAI agents used
in the methods and compositions of the present
invention are used. in an amount sufficient to provide
reduced IOP by at least about 5%, more preferably at
least about 10%, even more preferably at least
25 about 20%, and even more preferably at least
about 40%. Again, however, the amount of IOP
reduction depends on the amount and type of
non-steroidal cyclooxygenase inhibiting agent or NSAI
agent used.
30 In order to evaluate the effects of
glucocorticoids and non-steroidal cyclooxygenase
inhibitors on the eye, cell cultures of human
trabecular meshwork cells are studied. A discussion
CA 02177578 2005-07-18
-39-
of how such cell cultures are prepared and evaluated
is set forth below.
Cell culture and cell crrowth experiments
Human trabecular meshwork (HTM) cells are
used for the experiments described below. The HTM
cells are prepared as described in Polansky et al.,
Trabecular meshwork cell culture in glaucoma
research, Ophthalmology, 1984; 91: 580-595; Polansky
et al., Human trabecular cells I: Establishment in
tissue culture and growth characteristics, Invest
Ophthalmol Vis Sci, 1979; 18:1043-1049; Alvarado et
al., Humban trabecular cells II: Ultrastructural
c':aracteristics of cultured trabecular cells, Invest
Ophthalmol Vis Sci, 1982; 23:464-478; and Polansky et
al., Studies on human trabecular cells propagated ~
vitro, Vision Res, 1981; 21:155.
To perform the experiments described below,
third to fifth passage HTM cultures are removed from
cryopreserved stocks, plated at approximately 10,000
cells/cm2, and grown seven to ten days post-confluency
in Dulbecco's modified Eagle's (DME) medium with 10%
fetal calf serum (FCS) to obtain stable
endothelial-like monolayers. The protein synthesis
studies presented below are performed on these stable
HTM monolayer cultures.
The glucocorticoid effects on HTM cell
division are evaluated on growing cultures using DME
medium with 10% FCS. Glucocorticoid and
non-steroidal cyclooxygenase inhibiting agent
treatments begun the day after HTM cells are plated
at 2,500 cells/cm2. Effects are measured during log
2 ~ 77578
WO 95/17178 PCT/US94/14068
-4 0-
phase of growth (7 days) and after the control
cultures reaches confluency (which varies between 3
to 6 weeks, depending on the HTM cell line and the
serum in the culture medium).
The ability of non-steroidal cyclooxygenase
inhibiting agents to influence glucocorticoid effects
are evaluated by addition of the non-steroidal
cyclooxygenase inhibiting agents in the desired
concentration when treating the HTM cell cultures
with the glucocorticoid, i.e., dexamethasone.
Various agents are compared at various concentrations
as described below.
Evaluation of Non-steroidal Cyclooxygenase
Synthesis
Glucocorticoid and non-steroidal
cyclooxygenase inhibiting agent effects on specific
protein/glycoprotein synthesis are evaluated by
addition of [35S] methionine to label newly
synthesized proteins in the HTM cells. Confluent
cultures which have been exposed to the
glucocorticoid (dexamethasone) and non-steroidal
cyclooxygenase inhibiting agent for varying times
(media changes 24 hours prior to labelling) are
placed into methionine-free medium with 100-500 ;CCi
[3sS]-methionine per ml media (New England Nuclear;
specific activity 1100 ci/mmol, l0% FCS and the
appropriate concentration of dexamethasone and/or
inhibiting agent. Dexamethasone inductions in the
cytosol are evaluated 15 to 60 minutes after addition
of [35S] methionine (short labeling times providing an
estimate of protein synthetic rates).
*rB
CA 02177578 2005-07-18
-41-
Proteins/glycoproteins present in the HTM tissue
culture medium are from 2 hour serum-free DME
collection after a 3 hour initial cell labeling as
described above. Pulse-chase experiments are also
performed which helps in the choice of these
conditions.
Immediately after collection, each sample
of cytosol proteins is exposed to lysis buffer (20 mM
Tris HCI, pH 7.6; lOmM MgS04; 0.1% TX100;
chymostatin, 2 ~g/ml; leupeptin, 4 ~g/ml; bacitracin,
25 ~/ml; PMSF, 1 mM) at 4°C. The trichloroacetic
acid precipitable counts of the cell lysates are used
to normalize the amounts of lysate or media added to
the gel electrophoresis lanes. The samples are
diluted according to TCA assay results and gel buffer
is added to a ffinal concentration of 10% glycerol, 2%
SDS, 5% beta-Mercaptoethanol, and 62.5 mM Tris, pH
6.8. Samples are then boiled for 2 minutes, cooled,
spun and loaded onto gels.
Neuraminidase diqestions and tunicamycin treatments
To evaluate the possibility of sialic acid
residues on the major glycoprotein induction in the
dexamethasone treated HTM cultures, two hour media
collections of secreted proteins are performed as
described above. Enzyme digestions using
Neuraminidase Type X N2133 (affinity purified from
Clostridium perfringes, Sigma?"", St. Louis) are
conducted according to Schwartz et al.,
Characterization of the sialoglycoprotein receptor in
_ 30 a continuous hepatoma line, J. Biol Clem, 1981; 256:
8878-888. Samples are prepared using 0.1 M
sodium acetate
CA 02177578 2005-07-18
-42-
buffer containing i mg/ml human serum albumin, 0.2%
NaN" pH5 and incubated with neuraminidase (0.05 U/ul)
for 5 hours at 37°C. The reactions are stopped by
boiling the samples, with all further preparation for
electrophoresis performed on ice. Tunicamycin
effects on the secreted HTM proteins/glycoproteins
are evaluated by placing dexamethasone treated (300
nM) cultures into methionine-free medium with 10% FCS
containing tunicamycin (1-6 ~g/ml). The labeled
lysate and media proteins are collected and the
TCA-precipitable counts are determined as described
above.
Phosphorimager 3uantitation and gel electrophores~~.s
Previously published methods involving
computerized spot matching (PDQuest) programs on
three different autoradiogram exposures of large
format 2-D gels according to Polansky et al.,
Eicosanoid production and glucocorticoid regulator
mechanisms in cultured human trabecular meshwork
2~ cells, Prog. Clin Biol Res, 1989; 312:113-138,
are used to
quantitate dexamethasone effects on specific protein
synthesis in the HTM cultures. Phosphorimager
quantitations, greatly reduces the time and cost of
obtaining dose-response and time-course data in
different HTM lines. This method is primarily useful
in evaluating glucocorticoid effects on secreted
proteins/glycoproteins in HTM cells because of the
relatively few secreted proteins and the large size
of the dexamethasone inductions of interest. For the
studies shown here, a discontinuous buffer system is
il% acrylamide, pH s.8. Gels are run for
CA 02177578 2005-07-18
-43-
approximately five hours at a constant current
(30mA/gel) in a running buffer consisting of 25 mM
Tris, 192mM Glycine, and 0.1% SDS. Gels are stained
in a solution consisting of 0.5% Coomassie R-250, 50%
ethanol, and 10% acetic acid for 30 minutes and then
destained, first in 40% ethanol/10% acetic acid, then
in 10% ethanol/5% acetic acid for one hour per bath.
Destained gels are then soaked in deionized water for
30 minutes and dried on WhatmanT"" 3 mm under vacuum at
60°C. Dried gels are then pressed flat overnight
before exposure to a Phosphorimager'"' screen (Phosphor
Screen, Molecular Dynamics, Sunnyvale, Ca) at room
temperature, or standard x-ray film at -70°C to
visualize the labeled protein bands. The phosphor
screens require approximately 10% of the time
necessary for an autoradiogram exposure using x-ray
film. For quantitation, the screens are scanned
using a Phosphorimager model 400E, with gel files
stored on a magneto-optical disk. Analyses are
performed using the Image Quant v.3.22 software
package, which allow the quantitation of radiolabled
protein bands over a four log-unit range of
intensity. This method overcomes the substantial
problems involved in obtaining quantitative
information from densitometry of standard
autoradiograms, which must have internal standards,
at least three x-ray exposures, and difficult
computer software to overcome the non-linearity of
the film.
Results
Table 1 below provides the area integration in units
of Phosphorimager~" counts (i.e. pixel values, which is
WO 95/17178 PCT/US94/14068
-44-
directly related to 35S-methionine incorporation) in
the samples for the 66 kDa protein/glycoprotein
marker induction used as a marker in the media and
for the 55 kDa protein/glycoprotein marker induction
used as a marker in the cell.
WO 95/17178 PCT/US94/14068
-45-
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WO 95/17178
21 l 7 5 7 8 PCT/US94/14068
-47-
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WO 95/17178 PCT/US94/14068
-48-
As indicated above, the use of certain NSAI
agents in proper concentrations reduces the
protein/glycoprotein induction (marker)
significantly, whereas others do not. Of course, in
view of the above, those skilled in the art will be
able to readily determine the appropriate NSAI agent
and concentration needed.
Preuaration of Preferred Tanical Formulations
A hydrated polymeric dispersion is prepared
by slowly dispersing 1.0 part of Noveon"' AA-1 type
acrylic polymer, available from e.F Goodrich, into a
beaker fitted with an overhead stirrer containing
two-thirds of the final deionized water content and
stirring for one hour. Then, 0.10 parts of edetate
disodium is added to the dispersion followed by .
stirring for 10 minutes. The resulting dispersion
possessing a pH of about 3.0-3.5 is sterilized by
autoclaving at 121°C for 20 minutes. Diclofenac
sodium frequently used in the treatment of ocular
inflammation is dissolved separately in approximately
one-fifth of the final weight of water, added to the
polymer mixture by sterile filtration (0.22 ~m
filter) and stirred for 10 minutes. The mixture is
adjusted to pH 7.2 with lON sodium hydroxide, brought
to final weight with Water by sterile filtration and
aseptically filled into unit-dose containers.
Table 2 sets forth the amounts of each
component in the sample formulations.
WO 95/17178 ~ ~ ~ ~ ~ ~ ~ PCT/US94/14068
-49-
Table 2
Amount of Each Component
In Sample Formulations
Sample 1 Sample Sample 3
2
INGREDIENTS
Weight Weight Weight
Percent Percent Percent
C%WIW~ ~$WIW~ ~$WIW~
Diclofenac Sodium 0.01 0.05 0.1
Noveon~'-AA-1 1. 0 1. 0 1. 0
Edetate Disodium 0.1 0.1 0.1
Sodium Chloride 0.7 0.7 0.7
Sodium Hydroxide g.s. to g.s. to g.s. to
ION pH 7.2 pH 7.2 pH 7.2
Purified Water 100.0 100.0 100.0 I
The above discussion of this invention is
directed primarily to preferred embodiments and
practices thereof. It Will be readily apparent to
those skilled in the art that further changes and
modifications in actual implementation of the
concepts described herein can easily be made without
departing from the spirit and scope of the invention
as defined by the following claims.
