METHOD FOR PREVENTING INCREASED IRIDIAL PIGMENTATION DURING PROSTAGLANDIN TREATMENT

METHODE EMPECHANT L'ACCROISSEMENT DE LA PIGMENTATION IRIDIENNE PENDANT UN TRAITEMENT A LA PROSTAGLANDINE

English Abstract

Anti-inflammatory agents are used to prevent iridial pigmentation during
prostaglandin treatment and to manufacture pharmaceuticals for this purpose. A
method for preventing iridial pigmentation as well as a method for treating
glaucoma is based on the concomitant or simultaneous administration of a
prostaglandin or prostaglandin analogue and an anti-inflammatory agent. Both
non-steroid and steroid anti-inflammatory agents can be employed according to
the invention.

French Abstract

L'invention concerne des agents anti-inflammatoires qu'on utilise pour prévenir la pigmentation iridienne au cours d'un traitement à la prostaglandine, et pour fabriquer des médicaments à cette fin. Une méthode de prévention de la pigmentation iridienne et de traitement du glaucome est fondée sur l'administration concomitante ou simultanée d'une prostaglandine ou son analogue et d'un agent anti-inflammatoire. Selon l'invention, des agents anti-inflammatoires tant stéroïdiens que non stéroïdiens peuvent être employés.

Claims

14


Claims



1. Use of an anti-inflammatory agent for the manufacture of a pharmaceutical
composition for the prevention of increased iridial pigmentation during
prostaglandin
treatment of the eye.
2. The use according to claim 1, characterized in that the anti-inflammatory
agent is of steroid type.
3. The use according to claim 1, characterized in that the anti-inflammatory
agent is of non-steroid type.
4. The use according to claim 3, characterized in that the non-steroid type
anti-
inflammatory agent is a cyclo-oxygenase inhibitor.
5. The use according to claim 3, characterized in that the non-steroid type
anti-
inflammatory agent is a cyclo-oxygenase-2 inhibitor.
6. The use according to claim 3, characterized in that the non-steroid type
anti-
inflammatory agent is a cyclo-oxygenase inhibitor chosen from the following
group:
indomethacin, sulindac, etodolac, diclofenac, ketorolac, aceclophenac,
piroxicam, tenoxicam,
lornoxicam, meloxicam, fenoprofen, ibuprofen, naproxen, ketoprofen,
flurbiprofen,
nabumeton, azapropazon, mefenamic acid, oxaprosin, tolmetin, acetylsalicylic
acid, salicylic
acid, salsalate, valeryl salicylate, bismuth subsalicylate, acetoaminophen, 6-
MNA, ninesulide,
DuP 697, L 745,337, NS-398, celecoxib, rofecoxib and a combination thereof.
7. The use according to claim 3, characterized in that the non-steroid type
anti-
inflammatory agent is a cyclo-oxygenase inhibitor chosen from the following
group:
meloxicam, nabumeton, NS-398, DuP 697, L 745,337, celecoxib, rofecoxib and a
combination thereof.
8. The use according to claim 2, characterized in that the steroid type anti-
inflammatory agent is chosen from a group of steroid anti-inflammatory agents
comprising
dexamethasone, prednisolone, methylprednisolone, prednisone, cortisone,
hydrocortisone,
fluorometholone, triamcinolol, betametasone, fludrocortisone, deflazacort and
a combination
thereof.
9. Use of an anti-inflammatory agent in combination with a prostaglandin for
the
manufacture of a pharmaceutical composition for the treatment of glaucoma.
10. The use according to claim 9, characterized in that the anti-inflammatory
agent is of steroid type.
11. The use according to claim 9, characterized in that the anti-inflammatory
agent is of non-steroid type.




15



12. The use according to claim 11, characterized in that the non-steroid type
anti-inflammatory agent is a cyclo-oxygenase inhibitor.
13. The use according to claim 11, characterized in that the non-steroid type
anti-inflammatory agent is a cyclo-oxygenase-2 inhibitor.
14. The use according to claim 11, characterized in that the non-steroid type
anti-inflammatory agent is a cyclo-oxygenase inhibitor chosen from the
following group:
indomethacin, sulindac, etodolac, diclofenac, ketorolac, aceclophenac,
piroxicam, tenoxicam,
lomoxicam, meloxicam, fenoprofen, ibuprofen, naproxen, ketoprofen,
flurbiprofen,
nabumeton, azapropazon, mefenamic acid, oxaprosin, tolmetin, acetylsalicylic
acid, salicylic
acid, salsalate, valeryl salicylate, bismuth subsalicylate, acetoaminophen, 6-
MNA, ninesulide,
DuP 697, L 745,337, NS-398, celecoxib, rofecoxib and a combination thereof.
15. The use according to claim 11, characterized in that the non-steroid type
anti-inflammatory agent is a cyclo-oxygenase inhibitor chosen from the
following group:
meloxicam, nabumeton, NS-398, DuP 697, L 745,337, celecoxib, rofecoxib and a
combination thereof.
16. The use according to claim 10, characterized in that the steroid type anti-

inflammatory agent is chosen from a group of steroid anti-inflammatory agents
comprising
dexamethasone, prednisolone, methylprednisolone, prednisone, cortisone,
hydrocortisone,
fluorometholone, triamcinolol, betametasone, fludrocortisone, deflazacort and
a combination
thereof.
17. Method for prevention of increased iridial pigmentation during
prostaglandin treatment of the eye, characterized in that an anti-inflammatory
agent is
administered to the eye during the prostaglandin treatment.
18. The method according to claim 17, characterized in that the anti-
inflammatory agent is of steroid type.
19. The method according to claim 17, characterized in that the anti-
inflammatory agent is of non-steroid type.
20. The method according to claim 19, characterized in that the non-steroid
type anti-inflammatory agent is a cyclo-oxygenase inhibitor.
21. The method according to claim 19, characterized in that the non-steroid
type anti-inflammatory agent is a cyclo-oxygenase-2 inhibitor.
22. The method according to claim 19, characterized in that the non-steroid
type anti-inflammatory agent is a cyclo-oxygenase inhibitor chosen from the
following group:
indomethacin, sulindac, etodolac, diclofenac, ketorolac, aceclophenac,
piroxicam, tenoxicam,




16



lornoxicam, meloxicam, fenoprofen, ibuprofen, naproxen, ketoprofen,
flurbiprofen,
nabumeton, azapropazon, mefenamic acid, oxaprosin, tolmetin, acetylsalicylic
acid, salicylic
acid, salsalate, valeryl salicylate, bismuth subsalicylate, acetoaminophen, 6-
MNA, ninesulide,
DuP 697, L 745,337, NS-398, celecoxib, rofecoxib and a combination thereof.
23. The method according to claim 19, characterized in that the non-steroid
type anti-inflammatory agent is a cyclo-oxygenase inhibitor chosen from the
following group:
meloxicam, nabumeton, NS-398, DuP 697, L 745,337, celecoxib, rofecoxib and a
combination thereof.
24. The method according to claim 18, characterized in that the steroid type
anti-inflammatory agent is chosen from a group of steroid anti-inflammatory
agents
comprising dexamethasone, prednisolone, methylprednisolone, prednisone,
cortisone,
hydrocortisone, fluorometholone, triamcinolol, betametasone, fludrocortisone,
deflazacort and
a combination thereof.
25. The method according to any one of claims 17 - 24, characterized in that
the prostaglandin is a prostaglandin analogue.
26. The method according to any one of claims 17 - 24, characterized in that
the prostaglandin is latanoprost.
27. The method according to any one of claims 17 - 24, characterized in that
the prostaglandin is isopropyl unoprostone.
28. The method according to any one of claims 17 - 24, characterized in that
the prostaglandin is travaprost.
29. The method according to any one of claims 17 - 24, characterized in that
the prostaglandin is any one of AGN 190910, AGN 191129 or AGN 192024.
30. Method for the treatment of glaucoma, characterized in that that an anti-
inflammatory agent is administered to the eye together with a prostaglandin.
31. The method according to claim 30, characterized in that the anti-
inflammatory agent is of steroid type.
32. The method according to claim 30, characterized in that the anti-
inflammatory agent is of non-steroid type.
33. The method according to claim 32, characterized in that the non-steroid
type anti-inflammatory agent is a cyclo-oxygenase inhibitor.
34. The method according to claim 32, characterized in that the non-steroid
type anti-inflammatory agent is a cyclo-oxygenase-2 inhibitor.




17

35. The method according to claim 32, characterized in that the non-steroid
type anti-inflammatory agent is a cyclo-oxygenase inhibitor chosen from the
following group:
indomethacin, sulindac, etodolac, diclofenac, ketorolac, aceclophenac,
piroxicam, tenoxicam,
lornoxicam, meloxicam, fenoprofen, ibuprofen, naproxen, ketoprofen,
flurbiprofen,
nabumeton, azapropazon, mefenamic acid, oxaprosin, tolmetin, acetylsalicylic
acid, salicylic
acid, salsalate, valeryl salicylate, bismuth subsalicylate, acetoaminophen, 6-
MNA, ninesulide,
DuP 697, L 745,337, NS-398, celecoxib, rofecoxib and a combination thereof.

36. The method according to claim 32, characterized in that the non-steroid
type anti-inflammatory agent is a cyclo-oxygenase inhibitor chosen from the
following group:
meloxicam, nabumeton, NS-398, DuP 697, L 745,337, celecoxib, rofecoxib and a
combination thereof.

37. The method according to claim 31, characterized in that the steroid type
anti-inflammatory agent is chosen from a group of steroid anti-inflammatory
agents
comprising dexamethasone, prednisolone, methylprednisolone, prednisone,
cortisone,
hydrocortisone, fluorometholone, triamcinolol, betametasone, fludrocortisone,
deflazacort and
a combination thereof.

38. The method according to any one of claims 30 - 37, characterized in that
the prostaglandin is a prostaglandin analogue.

39. The method according to any one of claims 30 - 37, characterized in that
the prostaglandin is latanoprost.

40. The method according to any one of claims 30 - 37, characterized in that
the prostaglandin is isopropyl unoprostone.

41. The method according to any one of claims 30 - 37, characterized in that
the prostaglandin is travaprost.

42. The method according to any one of claims 30 - 37, characterized in that
the prostaglandin is any one of AGN 190910, AGN 191129 and AGN 192024.

Description

CA 02349427 2001-05-03
WO 00/25771 PCT/SE99/01993
METHOD FOR PREVENTL~IG INCREASED I1ZIDIAL PIGMENTATION
DURING PROSTAGLANDIN TREATMENT
The present invention is related to a method whereby increased iridial
pigmentation which
occurs during topical prostaglandin treatment can be prevented, avoided or at
least largely
reduced. Prostaglandin analogues are commonly used for the treatment of
glaucoma, and one
of the local side-effects of prostaglandin treatment in the eye is increased
iridial pigmentation.
The invention also relates to ophthalmic compositions containing medicaments
that prevent
the development of increased iridial pigmentation during prostaglandin
treatment.
Background
Glaucoma is an eye disorder characterised by increased intraocular pressure,
excavation of the
optic nerve head, and gradual loss of the visual field. An abnormally high
intraocular pressure
is known to be the most important risk factor for the development of glaucoma.
The exact
pathophysiological mechanism of open angle glaucoma, however, is still
unknown. Unless
treated glaucoma may lead to blindness, the course of the disease typically
being slow with
progressive loss of vision. The intraocular pressure in humans is normally in
the range of
12-21 mmHg. At higher pressures e.g. above 21 mmHg there is an increased risk
that the eye
may be damaged. In one particular form of glaucoma, normal tension glaucoma,
damage may,
however, occur at intraocuiar pressure levels that are within the normal
physiological range.
The opposite situation is also known when the intraocular pressure exceeds the
normal range
without causing damage to the eye. These cases are referred to as ocular
hypertensives. In all
these cases it is generally regarded that a reduction of the intraocular
pressure is beneficial for
the eye.
Glaucoma can be treated by drugs, laser or surgery. Recently, prostaglandin
analogues have
been introduced for the treatment of glaucoma and such analogues are now being
commonly
used in many parts of the world. The prostaglandin analogues effectively
reduce the pressure
in the eye by increasing the drainage of aqueous humor from the eye. Two
analogues are
currently on the market, namely latanoprost (Xalatan~, Pharrnacia & Upjohn
Co., USA) and
isopropyl unoprostone (Rescula~, Ueno Fine Chemicals, Ltd., Japan), and
information on
both drugs can be found in the literature (e.g. Stjernschantz and Alm, Curr.
Opin. Ophtalmol.,
7; 11-17, 1996; and Yamamoto et al., Surv. Opthalmol. 41, Suppl. 2, S99-5103,
1997).
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Prostaglandins are endogenous fatty acids usually derived from the precursors
eicosatrienoic,
eicosatetraenoic and eicosapentaneoic acid through metabolic steps involving
oxygenation.
The precursors are released from the phospholipids of the cell membrane by
lipases, in
particular phospholipase A2. The oxygenation of the precursors to the
endoperoxide
intermediates is catalysed by the cyclo-oxygenase enzyme. There are two cyclo-
oxygenase
isoenzymes, cyclo-oxygenase-1 (COX-1), and cyclo-oxygenase-2 (COX-2). COX-1 is
a
constitutive enzyme that continuously generates small amounts of
prostaglandins for
physiologic purpose, while COX-2 is an inducible enzyme which is typically
expressed
during inflammation and certain other pathological conditions.
The prostaglandins carry a cyclopentane ring to which two carbon chains
attach, the upper
usually being called the alpha chain and the lower the omega chain. The
prostaglandins are
classified in subgroups A, B, C, D, E, F, G, H, I, and J depending on the
structure and
substituents in the cyclopentane ring as demonstrated in Fig. 1.
The alpha chain is a 7 carbon carboxy-terminated aliphatic chain while the
omega chain
contains 8 carbons including a terminal methyl group. Subscripts of 1-3 are
given depending
on th:e number of double bonds. In prostaglandins with subscript 1 the double
bond is situated
between carbons 13 and 14 in the omega chain. In prostaglandins with subscript
2 an
additional double bond is situated between carbons 5 and 6 in the alpha chain,
and
prostaglandins with subscript 3 contain a third double bond between carbons 17
and 18 in the
omega chain. The molecular structures of latanoprost, isopropyl unoprostone
and travaprost
(Alcon Inc., USA) are shown in Fig. 2. While currently only two prostaglandin
analogues
have been introduced on the market for glaucoma treatment it is anticipated
that several
different prostaglandins analogues will reach the market in the future, and
that many if not all
of these will cause increased iridial pigmentation as a side-effect. The
present invention thus
also applies to future prostaglandin analogues that cause increased iridial
pigmentation.
Furthermore, recently prostaglandin derivatives called "Novel ocular
hypotensive lipidsTM"
(Allergan Inc., USA) in which the carboxylic acid moiety has been substituted
with an alcohol
or ether group have been presented and may be marketed in the future. Such
derivatives are
also considered as prostaglandins according to the present invention. Examples
of such
specific analogues include AGN 190910, AGN 191129 and 192024 (Allergan Inc.,
USA).
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As mentioned above a novel side-effect that is sometimes encountered during
topical
prostaglandin treatment is increased pigmentation of the iris (Selen et al.,
Surv. Opthalmol.
41, Suppl.,2; 5125-S128, 1997; Wistrand et al., Surv. Opthalmol. 41, Suppl. 2;
S129-138,
1997). Both latanoprost (Wistrand et al., J. Glaucoma, 6; 430-432, 1997) and
isopropyl
unoprostone (Yamamoto and Kitazawa, 1997) have been reported to cause this
side-effect
during chronic therapy in man. Consequently in some patients the eye colour
may turn darker
in the direction of brown. While this side-effect apparently has no harmful
medical
consequences it nevertheless is a disadvantage particularly from a cosmetic
point of view in
some patients. It would thus be desirable to identify a method whereby the
increase of iridial
pigmentation during prostaglandin therapy could be avoided.
Summary of the invention
The above problem is solved in an unexpected way, that is through the use of
anti-
inflammatory drugs and the methods of treatment disclosed in the attached
claims. Further
embodiments and advantages of the present invention will be evident from the
description and
examples.
Description
The present inventors have unexpectedly found that latanoprost, used as the
free acid,
stimulates the production of endogenous prostaglandins, at least PGE2 and
PGF2a, in iridial
melanocytes, and that this production of endogenous prostaglandins by
latanoprost acid can
be prevented or markedly reduced by non-steroid anti-inflammatory agents such
as
indomethacin and NS-398 as well as with the steroid (corticosteroid) anti-
inflammatory agent
dexamethasone. Since it is known from previous studies that both PGEZ and
PGFZQ can elicit
increased pigmentation of the iris of monkeys in a similar way as latanoprost
(Selen et al.,
1997) it is now apparent that the latanoprost effect is mediated by endogenous
prostaglandins
such as PGF2a and PGEZa since our experiments demonstrate that latanoprost
stimulated the
formation of these prostaglandins in the iridial melanocytes. Thus it may be
possible to
prevent the formation of pigment in the iridial meianocytes during chronic
latanoprost
treatment by concomitant treatment with an anti-inflammatory agent,
preferentially a COX-I
or COX-2 inhibitor.
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Accordingly, patients that are being treated with either latanoprost or
isopropyl unoprostone,
and in the future possibly with other prostaglandin derivatives or analogues
who are at risk of
developing increased iridial pigmentation may be treated simultaneously with
an
anti-inflammatory agent to prevent increase in iridial pigmentation. Such anti-
inflammatory
agents include but are not limited to: indomethacin, ibuprofen, diclofenac,
etodolac,
flurbiprofen, ketorolac, acetosalicylic acid, salicylic acid, salsalate,
valeryl salicyiate, bismuth
subsalicylate, aceto-aminophen, sulindac, aceclofenac, piroxicam, tenoxicam,
lomoxicazn,
meloxicam, naproxen, nabumeton, ketoprofen, azapropazon, fenoprofen, mefenamic
acid,
oxaprosin, tolinetin, 6-MNA, NS-398, nimesulide, DuP 697, L 745,337,
celecoxib, rofecoxib
and steroid anti-inflammatory agents such as dexamethasone, prednisolone,
methylprednisolone, prednisone, cortisone, hydrocortisone, fluorometholone,
triamcinolol,
betamethasone, fludrocortisone and deflazacort, and combinations thereof.
The anti-inflammatory agent can be administered locally, e.g. as eye drops,
ointments or
inserts, or it can be given systemically e.g. orally. Prodrugs, e.g. for
enhancing stability or
bioavailability of the anti-inflammatory agents are also covered by the
present invention. Such
prodrugs comprise e.g. sulindac and nabumetone.
According to one embodiment, the present invention defines a method of using
per se known
anti-inflammatory drugs, preferentially so called non-steroid anti-
inflammatory agents, for the
prevention of increased iridial pigmentation during topical prostaglandin
treatment. The
method of preventing increased iridial pigmentation during prostaglandin
treatment comprises
administering the anti-inflammatory drug that prevents endogenous
prostaglandin synthesis
either directly on the eye or systemically, i.e. orally, once or several times
daily, or possibly
even with longer intervals.
According to another embodiment, the present invention defines ophthalmologic
compositions for administering the anti-inflammatory agents topically on the
eye whenever
such compositions of the specified compounds have not been disclosed
previously. In
particular compositions containing both the prostaglandin analogue and the
anti-
inflammatory agent are novel and highly useful for the patients. The anti-
inflammatory agent
(and whenever possible together with the prostaglandin analogue) is mixed with
an ophthal-
mologically compatible vehicle known per se. The vehicle which may be employed
for
preparing compositions of this invention comprises aqueous solutions, such as
physiological
MH/42401
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saline, oil solutions, creams and ointments. The vehicle may furthermore
contain
ophthalmologically compatible preservatives such as benzalkonium chloride,
surfactants e.g.
polysorbate 80, liposomes or polymers, e.g. methyl cellulose, polyvinyl
alcohol, polyvinyl
pyrrolidone and hyaluronic acid; these polymers may be employed to increase
the viscosity
of the solutions. Inclusion complexes such as cyclodextrins can be used to
enhance stability
and delivery. Incorporation of the active drugs into soluble and insoluble
drug inserts is also
possible.
According to a third embodiment, the present invention relates to the
preparation of
ophthalmologic compositions containing anti-inflammatory agents to be used
concomitantly
with prostaglandins in the treatment of glaucoma, and in particular
compositions that include
both the prostaglandin analogue and the anti-inflammatory agent.
The invention is illustrated by means of the following non-limiting examples:
Methods:
Iridial melanocytes were isolated from bovine eyes obtained from a local
slaughter house. The
melanocytes were cultured in a DMEM, Glutamax/Ham's f 12 culture medium
containing
10% fetal calf serum, 1 ng/ml basic human recombinant fibroblast growth
factor, 10 ng/rnl
cholera toxin, 0.1 rnM isobutylmethylxanthine (IBMX), 50 pg/ml gentamicin, and
0.25 p.g/ml
Fungizone. The cells, usually used at passages 2-4, were plated into 48 or 96
well microtiter
plates, and grown to confluence. Each well usually contained around 50.000
cells. The test
substances, latanoprost, used as the free acid, and the various blocking
agents were
administered to the cells by adding them to the culture medium. The cells were
incubated for
24 hours in the presence of latanoprost and the blocking agents at 37°C
in an atmosphere of
95% oxygen and 5% carbon dioxide. For investigating the blocking effect of
dexamethasone
the cells were preincubated with dexamethasone for 24 hours before the
exposure to
latanoprost acid and dexamethasone. After the incubation 0.2 ml of the culture
medium was
aspirated and centrifuged in an Eppendorff centrifuge at 8000 rpm for 5 min.
The
concentration of PGE2 and PGF2« in the culture medium was then measured using
commercial enzyme immunoassay kits for PGEZ (Amersham Life Science), and PGFZ«
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(Cayman Chemicals) following appropriate dilutions. The assays were performed
according
to the manual of the respective commercial enzyme immunoassay kit.
Test substances:
Latanoprost acid was obtained by synthesising according to the method
previously described
(Resul et al., J. Med. Chem. 36; 243-248, 1993). The acid of latanoprost was
used instead of
the isopropyl ester because it is not known whether the melanocyte cultures
express esterases
that can hydrolyse latanoprost to the free acid. In the human eye latanoprost
( 13,1
4-dihydro- 1 7-phenyl- 18,1 9,20-trinor-PGFZa isopropyl ester)is hydrolysed to
the free acid.
Indomethacin was obtained from Sigma Chemicals, and was freshly prepared by
dissolving
in water solution using Na2C03. NS-398 (N-(2Cyclohexyloxy-4-nitrophenyl)
methane-
sulphonamide) was obtained from Calbiochem-Novabiochem and was dissolved in
dimethyl-
sulphoxide (DMSO). Dexamethasone was obtained from Sigma Chemical Company and
was
dissolved in ethanol. The final concentrations of the test substances in the
experiments were
the following; latanoprost acid 10'$, 10'7, 10'6, and 10'5 M, indomethacin
10'5 M, NS 398
10'$, 1 and 10'6 M, and dexamethasone 10'~ M. The concentrations of the
various test
substances are generally known to be effective.
Results:
Latanoprost acid caused a release of PGE2 and PGFZa into the culture medium
from the
meianocytes in a dose dependent manner (Table I). Maximum effect was seen with
a
concentration of latanoprost of 10'5 M. A near maximum effect was seen with
the ten times
lower concentration of 10'~ M. As can be seen in Table I the melanocytes
produced much
more PGEZ than PGFza when exposed to latanoprost,
Table I.
Effect of latanoprost acid on the production and release of PGE~ and PGF~a
from bovine
iridial melanocytes in culture, and blocking effect of indomethacin ( 10'5 M).
(Mean t SEM;
ng = nanogram)
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Test compound ConcentrationPGEZ PGFz~,


(Moles/1) (ng/well) (ng/well)


Control (vehicle) 0 9.7 t 2.0 9.4 t 0.1


S Latanoprost acid 10-g 13.5 t 0 ---
3


Latanoprost acid 10-7 --- 11.6 t 1.1


Latanoprost acid 10'~ 22.6 t 1 12.1 t 0 8
9


Latanoprost acid 10-5 23.7 t 0.4 12,8 t 0.8


Latanoprost acid + Indom.10'5 3.1 t 0.1 7.9 t 0.1


Indomethacin, a non-selective inhibitor of the COX- 1 and COX-2 enzymes
completely
blocked the latanoprost acid induced release of both PGEz and PGFZa from the
iridial
melanocytes (Table I). Likewise the relatively selective COX-2 blocking agent
NS-398 at
high concentrations (10'~ M) markedly blocked the formation of PGE2, but even
at lower
concentrations the formation of PGE~ was partly blocked (Table II). Since the
ICSO value of
NS-398 for blocking the COX-2 enzyme has been reported to be around 10-7 M,
while the
corresponding value for blocking the COX- 1 enzyme was reported to about 10'SM
(Patrono
et al., Improved non-steroid anti-inflammatory drugs; COX-2 enzyme inhibitors
(Eds.
Vane, J., et al.) Kluvrier Acad. Publ., 121-131, 1996), the results suggest
that a large part if
not all of the endogenous PGEZ was produced through the COX-2 enzyme pathway
when
the cells were exposed to latanoprost acid. As can be seen in Table III
dexamethasone, a
steroid anti-inflammatory drug that inhibits the phospholipase A2 enzyme
through the
induction of lipocortin partly blocked the formation of PGEZ by the
melanocytes during
exposure to latanoprost acid. Since the anti-inflammatory agents used, either
deprive the
cells of free arachidonic acid or block its conversion into cyclic
endoperoxides the results of
the blocking experiments also apply to PGFza, and any other endogenous
prostaglandin and
thromboxane.
Table II.
Blocking effect of NS-398, a selective cyclo-oxygenase II inhibitor, on
latanoprost
acid-induced production of endogenous PGEZ in bovine iridial meianocytes.
Latanoprost
acid was used at a concentration of 10-5 M (Mean ~ SEM; ng = nanogram).
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Test compounds Concentration PGE2
of blocking agent (ng/well)
{Moles/1)
Vehicle 0 24.5 ~ 0.5
Latanoprost acid 0 104.1 ~ 0.3
Latanoprost acid + NS-398 10'9 65.5 ~ 17 0
Latanoprost acid + 10-8 32.6 1.2
NS-398


Latanoprost acid + 10'' 11.8 1 4
NS-398


Latanoprost acid + 10'~ 6.8 0.4
NS-398


(The reason for the larger amount of PGEz as compared to Table I was a larger
number
of cells/well)
Table III. Blocking effect of dexamethasone on latanoprost acid-induced
production of PGEZ
in bovine iridial melanocytes. Latanoprost acid was used at a concentration of
10'5 M. (Mean
t SEM; ng = nanogram)
Test compounds Concentration PGE2
of blocking agent (ng/well)
(moles/1)
Vehicle 0 - 20.5 ~ 2.0
Latanoprost acid 0 43.1 t 1.2
Latanoprost acid + Dexamethasone 10'~ 23.9 t 1.4
From these experiments it is obvious that latanoprost, a synthetic
prostaglandin analogue,
causes endogenous formation of PGE2 and PGFz~, in iridial melanocytes, and
that the effect
can be blocked by the inhibition of the necessary enzymes in the formation of
the
endogenous prostaglandins. From previous experiments it is clear that both
PGFZa and PGEz
have the capacity to induce increased pigmentation in primates under in vivo
conditions. It is
thus likely that endogenous prostaglandins mediate the melanogenic effect of
latanoprost a
synthetic prostaglandin analogue.
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Accordingly, medicaments that interfere with the prostaglandin synthesis can
be predicted to
prevent the formation of melanin in the meianocytes. Such medicaments may
inhibit either
the cyclo-oxygenase 1 or 2 enzymes or the phospholipase A2 or phospholipase C
enzymes or
any other enzyme necessary in the production of endogenous prostaglandins.
Various non steroid anti-inflammatory agents as well as steroid anti-
inflammatory agents
have the ability to interfere with the enzymes necessary for prostaglandin
synthesis and may
therefore be employed according to the present invention. Thus, patients at
risk may be
treated with anti-inflammatory agents in parallel with prostaglandin treatment
used for the
reduction of the intraocular pressure in glaucoma therapy.
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Experimental hart added during the priority vear~
Additional experiments have been performed to demonstrate the general validity
of the
inventive concept, i.e. that anti-inflammatory agents that block the
prostaglandin synthesis in
5 melanocytes prevent melanogenesis and thus increased iridiaI pigmentation
during topical
prostaglandin treatment of the eye. Topical prostaglandin preparations are
used as
medicaments for the treatment of glaucoma. Two series of experiments have been
performed;
1) to show that not only latanoprost causes increased production of endogenous
prostaglandins (e.g. PGE2) but also other prostaglandins, including analogues
that are used or
10 being developed for the treatment of glaucoma (PGFZa, fluprostenol and
unoprostone acid),
and 2) to show that simultaneous treatment of pigment producing cells with
indomethacin and
latanoprost results in a reduced production of melanin in the cells as
compared to latanoprost
treatment only demonstrating a true blockade of the melanogenic effect of
prostaglandins by
anti-inflammatory agents such as indomethacin.
The prostaglandins used in the additional experiments were the following:
PGFZQ (prostaglandin FZa)
Latanoprost ( 13,14-dihydro-17-phenyl-18,19,20-trinor-PGF2a)
Fluprostenol ( 16-phenoxy-3-trifluormethyl-18,19,20-trinor-PGFZn)
Unoprostone acid ( 13,14-dihydro-15-keto-PGFZQ-20-ethyl)
All prostaglandin analogues were used as acids instead of esters which are
used clinically for
pharmacokinetic reasons.
1. Increase of the production of endogenous PGE2 in iridial melanocytes
exposed to
PGFZa, fluprostenol and unoprostone acid.
These experiments were performed using bovine iridial melanocytes essentially
as described
in the Methods section above. PGF2a (obtained from Chinoin Chemical and
Pharmaceutical
Works, Budapest, Hungary), fluprostenol (obtained from Cayman Chemicals, Ann
Arbor,
Michigan) and unoprostone acid (obtained from Pharrnacia & Upjohn, Uppsala,
Sweden)
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11
were used at concentrations ranging from 10'8 M to 10'~ M, and PGEZ was
measured with a
radioimmunoassay kit as previously described. The following results were
obtained:
Experiment 1.
Prostaglandin Concentration PGEz


(Molesll) (ng/well


PGFza 0 19


10'5 37


10'~ 24


10'7 22


10-8 12


PGF~~, + indomethacin 10'5 17


Fluprostenol 0 6


10'5 28


10'~ 26


10'7 24


10'8 7


Fluprostenol + indomethacin10'5 0


Unoprostone acid 0 10


10'5 17


10'~ 12


10'7 12


10'g 11


Unoprostone acid + indomethacin 10'5 0


This single experiment clearly demonstrates that the endogenous prostaglandin
production in
meianocytes is induced by several exogenous prostaglandins and that this thus
is a general
effect of exogenous prostaglandins. It should also be pointed out that we have
utilised PGEZ
as an indicator of endogenous prostaglandin synthesis, but other endogenous
prostaglandins
such as PGFza, PGD2, PGIZ and TxA2 may also be produced and be of importance
in the
induction of melanogenesis.
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2. Inhibition of prostaglandin-induced melanogenesis by indomethacin
The purpose of this experiment was to demonstrate that anti-inflammatory drugs
of the
NSAID type inhibiting endogenous prostaglandin production have the ability to
prevent or
reduce melanin formation (melanogenesis) in pigment cells exposed to exogenous
prostaglandins. The experiment was performed in the following way:
Cloudman S91 mouse melanoma cells (CCL-53.1 ) at passages 25-31 were used for
the
experiment since they produce melanin within a sufficiently short term period
to allow
measurement of melanogenesis which usually ordinary melanocytes do not. The
cells were
cultured in a medium containing HAM's F-10 (82.5%), fetal calf serum (2.5%)
and horse
serum (15%) in a routine way. The cells were grown to confluence in 80 cm2
flasks and
incubated with the test substances in the flasks. To one flask only control
medium was added,
to one latanoprost acid at a final concentration of 10'6 M and to the third
latanoprost acid at a
final concentration of 10~ M and freshly prepared indomethacin at a final
concentration of
10'6 M. The medium including the drugs was replaced after 2 days, and the
total incubation
period in the presence of the drugs was 4 days. Thereafter the cells were
trypsinized and
lysated with 1 M NaOH and the melanin content was measured
spectrophotometrically at 475
nm with appropriate standards. The melanin content of the cultures were found
to be the
following:
Experiment 2.
Treatment Total melanin content Increase compared Reduction
to control of melanin
formation*
Control 163.43 ug 0 0
Latanoprost 10'6 M 182.39 p.g 18.96 p.g 0
Latanoprost 10'6 M + 175.80 p.g 12.37 pg 34.8
Indomethacin 10'6 M
* compared with latanoprost group
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This experiment demonstrates that latanoprost increases formation of melanin
in melanoma
cells that normally have a high rate of melanin production. When indomethacin
is used
concomitantly with latanoprost the increase in melanin production was reduced
by about 35
%. Although the stimulatory effect of latanoprost of the melanin production is
relatively
modest it should be kept in mind that the Cloudman S91 melanoma cells have a
high rate of
spontaneous melanin production and an additional stimulation of the melanin
production
therefore by the prostaglandin cannot be anticipated to be very marked.
Nevertheless the
experiment shows that the cells were stimulated to produce more melanin by
latanoprost and a
considerable part of the stimulation could be blocked by indomethacin, a
specific blocker of
the cyclo-oxygenase enzyme and thereby endogenous prostaglandin production in
the cells.
Thus it is obvious that exogenous prostaglandins e.g. PGFza, latanoprost acid,
fluprostenol
and unoprostone acid stimulate the formation of endogenous prostaglandins in
melanocytes
which in turn somehow trigger melanin formation. This prostaglandin induced
melanin
formation can be blocked by anti-inflammatory agents of the NSAID type.
Finally, the present inventors have also obtained in vivo data supporting the
mechanism
suggested by the in vitro data in an ongoing study. In this study 15
cynomolgus monkeys are
being treated twice daily with commercially available latanoprost (0.005 %)
eye drops
(control group), while another 15 cynomolgus monkeys are being treated with
commercially
available flurbiprofen (0.03 %) eye drops twice daily and the same dose of
latanoprost
(experimental group). Flurbiprofen is an anti-inflammatory agent which
inhibits the cyclo-
oxygenase enzyme and thus endogenous prostaglandin formation. After three
months
treatment 2 of the animals in the control group (2/15), but none of the
animals in the
experimental group (0/15), had developed clear-cut increase of iridial
pigmentation. Thus it
appears that blockade of endogenous formation of prostaglandins in the iris
prevents
increased iridial pigmentation induced by exogenous prostaglandins such as
latanoprost. It
should be noted that study is scheduled for a longer duration of 12 months.
Although the invention has been described with regard to its preferred
embodiments, which
constitute the best mode presently known to the inventors, it should be
understood that
various changes and modifications as would be obvious to one having the
ordinary skill in this
art may be made without departing from the scope of the invention which is set
forth in the
claims appended hereto.
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