Bull Vet Inst Pulawy 54, 251-258, 2010
CLINICAL EVALUATION OF TACROLIMUS EYE DROPS FOR
CHRONIC SUPERFICIAL KERATITIS TREATMENT IN DOGS
IRENEUSZ BALICKI AND ALEXANDRA TRBOLOVA1
Department and Clinic of Animal Surgery, Faculty of Veterinary Medicine,
University of Life Sciences in Lublin, 20-612 Lublin, Poland
1
Department and Clinic of Animal Surgery,
University of Veterinary Medicine, 041- 81 Košice, Slovak Republic
[email protected]
Received for publication November 27, 2009
Abstract
The aim of this study was to assess the efficacy of 0.02% tacrolimus ophthalmic drops application for chronic superficial
keratitis (CSK) treatment in dogs. The studies included 14 German Shepherd dogs – eight males and six females, aged 2-10 years
affected with CSK. The drops were administered to the ocular surface three times a day. Prior to the treatment onset, and after the 5
week medical therapy, an estimation of a conjunctiva redness, ocular discharge, depigmentation of the third eyelid, and blood vessel
ingrowth in each corneal quadrant and corneal pigmentation was conducted. The photo images with calibrated grid enabled to
calculate the percentage of corneal area surface affected by inflammatory process. Tacrolimus did not exert any irritant effects
throughout the treatment. The therapy has led to the decrease in corneal inflammatory infiltrate and blood vessel ingrowth in all the
patients. Median corneal area surface affected by the condition showed a statistically significant decrease from 46% to 27% (P<0.01)
in case of the left corneas and from 58% to 33% for the right ones. Out of 27 corneas affected by pigmentation, 13 corneas in eight
patients exhibited decreased pigmentation. The increased pigmentation was observed on eight corneas in five patients. The studies
proved that 0.02% ophthalmic drops of tacrolimus have been effective topically in CSK therapy. A treatment response was observed
by reduced granulation tissue and corneal neovascularisation, still in some cases tacrolimus failed to inhibit the pigmentation
formation.
Key words: dog, chronic superficial keratitis, tacrolimus, therapy.
Chronic superficial keratitis (CSK) is a
progressive condition with a main clinical manifestation
of a fibrovascular infiltration commencing at the lower
temporal corneal quadrants, combined with corneal
neovascularisation and eventually followed by
pigmentation. The initial clinical appearance of the
disease includes a slight inflammatory infiltrate in the
region of the limbus of lower temporal corneal quadrant.
The abnormal ingrowth of blood vessels into the cornea
and an inflammatory infiltration develops in the form of
a pink fibrovascular tissue (12, 17, 23). Initially, it is
thin but as the disease progresses, the infiltrate gets
thicker and occasionally, the corneal surface becomes
uneven. In addition, centrally located corneal focal
macular opacities of grey colour are detected. Most dogs
manifest the typical CSK lesions in the cornea as well as
oedema and depigmentation of the third eyelid margin.
The inflammatory processes are bilateral but the corneal
changes are usually asymmetrical. Some dogs
experience loss of the corneal transparency in short time,
while others manifest slow progression of the condition
with maintained good vision acuity (8). CSK is thought
to be immune mediated corneal disease (26). The
disease may develop for a few months or even some
years and in severe cases may cause blindness a year
after the symptom onset (9).
The main therapy for the CSK condition
includes administration of topical corticosteroids in the
form of ophthalmic drops or ointment directly to the
conjunctival sack (3, 5, 10, 17, 25). They may be also
applied as subconjunctival injections. Cyclosporine used
alone or in conjunction with topical corticosteroids is
effective in cases of CSK (3, 7, 25). Williams et al. (25)
compared the medical outcomes of the CSK therapeutic
processes with 0.2% cyclosporine and 0.1%
dexamethasone. They found that cyclosporine
application implicated the disappearance of blood
vessels and corneal infiltration in a similar manner to
dexamethasone action (25). There were also some
clinical studies on the use of ophthalmic drop of
dimethyl sulfoxide (DMSO) for CSK condition therapy
(3, 4). Its combined application with dexamethasone
proved to be more efficient than dexamethasone alone
(40). Recently, the use of pimecrolimus in CSK therapy
became an object of researches. They revealed that in
some clinical cases pimacrolimus caused the regression
of both, blood vessel ingrowing and corneal
pigmentation (19). However, with the current therapies,
252
chronic superficial keratitis can usually be controlled by
medical or surgical therapy, by it cannot be cured (12).
Tacrolimus is a macrolide antibiotic with
immunosuppressant and anti-inflammatory properties
isolated from Streptomyces tsukubaensis (14). The
medication acts on early activation of T lymphocytes,
most likely preventing the transcription of T lymphocyte
stimulation genes (IL-2, IL-3, IL-4, IFN-y, TNF-α, GMCSF, c-myc) (20, 21). Tacrolimus is commonly
employed in human and animal therapy, predominantly
in transplantology and dermatology but its use in
ophthalmology is still limited (11, 13, 15, 18, 24).
Ophthalmic drops of 0.02% tacrolimus were used to
treat canine keratoconjunctivitis sicca (6). Topical
0.03% tacrolimus ointment appeared to be a well
tolerated and potent drug for human allergic
conjunctivitis treatment (2). The positive effects of
0.03% tacrolimus employment in the therapeutic process
of blepharokeratoconjunctivitis, keratoconjunctivitis,
and chronic follicular conjunctivitis were reported (13).
Conjunctival cytology, as well as clinical examination of
the patients, who underwent the 0.03% tacrolimus
therapy
towards
blepharoconjunctivitis
or
keratoconjunctivitis were also performed. The studies
have revealed the reduced infiltration of inflammatory
cells in the conjunctiva and improvement in
conjunctivitis (24).
The objective of the presented research has
been to assess the efficacy of 0.02% tacrolimus
ophthalmic drops application for chronic superficial
keratitis treatment in dogs.
formation or regression, increased or decreased
transparency rate, and calculation of a number of
corneas affected by pigmentation prior to, and 5 weeks
post the treatment in all the patients. Calculation of the
blood vessel ingrowth into each corneal quadrant –
graded from 1 to 4 grade points was also conducted.
The research results were analysed statistically.
The Kolmogorov-Smirnov test showed a non-normal
distribution of data, therefore the Mann-Whitney test
was used to compare the parameters before and after the
5-week treatment. The χ-square test was used to
compare totals of corneal quadrants involved in
neovascularisation.
Results
Tacrolimus application reduced the conjunctiva
redness. None of the patients displayed irritated
conjunctivas as the effect of the medication action,
manifested by increasingly red and itching eyes. Before
and onset of the therapy, three patients were diagnosed
as having mucopurulent exudate visible in the medial
angle of the lid. The exudate gradually disappeared on
10 to 14 d of the gentamicin therapy.
The overall treatment outcomes obtained for
each patient are summarised in Table 1. Tacrolimus
therapy was associated with reduced neovascularisation
of the cornea (Figs 1 and 2).
Material and Methods
The studies included 14 German Shepherd dogs
– eight males and six females aged 2-10 years, affected
with keratitis superficialis chronica. The patients
underwent the detailed ophthalmic examinations using a
slit lamp, as well as the indirect and direct
ophthalmoscopy. The patients were treated with eye
drops of 0.02% tacrolimus in 0.9% sodium chloride
formulated by a specialist in the ophthalmic pharmacy
field. The drug was administered three times a day to the
ocular surface. Prior to the therapy, and 5 weeks after it,
the following features were determined: conjunctiva
redness - lack (-), present (+); occurrence of ocular
discharge; depigmentation of the third eyelid margin present (+), absent (-); corneal area surface affected by
inflammatory process, as well as the occurrence of
pigmentation and corneal neovascularisation. When
mucopurulent exudate was observed, the tacrolimus
therapy was used in conjunction with 0.3% gentamicin
ophthalmic drops administered three times a day for 14
d. The repeated photographic documentation of all
patients was taken. The calibrated grid was placed on
photographs to calculate a percentage of the corneal area
involved
in
inflammatory
process,
i.e.
neovascularisation, granulation tissue, superficial
macular opacities, and pigmentation (Fig. 1). The
corneal pigmentation was evaluated on the basis of its
Fig. 1. Case 13. Before tacrolimus treatment – superficial
macular opacities, pigmentation, and neovascularisation of the
temporal cornea quadrants (cornea with calibrated grid).
Fig. 2. Case 13. After a 5 week treatment with tacrolimus –
regression of corneal neovascularisation.
Age
Sex
Eye
Before
treatment
After
treatment
Depigmentation of the
third eyelid margin
Before
treatment
After
treatment
1
3
♂
OD
D
D
4
4
OS
D
D
4
3
OD
D
D
4
2
2
10
♀
OS
D
D
4
2
OD
D
R
4
2
3
9
♂
OS
D
R
3
1
OD
D
R
1
1
4
4
♀
OS
D
R
1
1
OD
D
R
1
1
5
3
♀
OS
D
R
1
1
OD
D
R
1
1
6
3
♂
OS
D
R
1
0
OD
D
R
3
1
7
9
♀
OS
D
R
2
0
OD
D
R
4
3
8
6
♂
OS
D
R
3
2
OD
D
D
2
1
9
7
♀
OS
D
D
1
0
OD
D
R
2
2
10
4
♂
OS
D
R
0
0
OD
D
R
4
1
11
6
♂
OS
D
R
3
1
OD
D
R
4
2
12
4
♂
OS
D
R
3
2
OD
D
R
4
3
13
7
♀
OS
D
R
2
0
OD
D
R
3
1
14
8
♂
OS
D
R
2
1
Explanation: D- depigmentation, R- repigmentation, (?) difficult to evaluated.
Case
Table 1
68
52
85
63
100
82
20
17
24
43
17
23
47
46
78
65
38
24
38
0
71
67
78
55
76
33
48
44
Before
treatment
52
42
50
27
82
46
14
10
20
24
18
10
30
36
62
55
7
8
33
0
32
14
60
36
48
22
32
30
After
treatment
Responses to tacrolimus therapy
Corneal
Corneal area affected
neovascularisation
(%)
(number of quadrant)
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0
+
+
+
+
+
+
+
+
Before
treatment
+
0
+
+
+
+
+
+(?)
+
+
+
+
+
+
+
Decrease
+
+
+
+
+(?)
+
+(?)
0
+(?)
+
+
+(?)
+(?)
+(?)
+(?)
+
Development
After treatment
Corneal pigmentation
Decreased
density
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-(?)
0
+
+
+
+
253
253
254
Table 2
Area of affected cornea and median number of quadrants
with neovascularisation before and after a 5-week treatment
Before treatment
After treatment
Right cornea Left cornea Right cornea
Left
cornea
Median of corneal area affected
(%)
(min-max)
58
(100-17)
46
(82-17)
33
(82-7)
27**
(55-8)
Median of quadrants
with neovascularisation
(min-max)
3.5
(4-1)
2
(4-1)
1.5*
(4-1)
1**
(3-0)
Number of all quadrants
with neovascularisation
41
30
25**
14**
(min-max) – minimal and maximal values,
* P<0.05; ** P<0.01.
Table 3
Corneal pigmentation
Before treatment
After treatment
Number of corneas affected Decrease Development Decreased density
Corneal pigmentation
27
Fig. 3. Case 10. The ventral quadrants covered
by thick granulation tissue.
13; 1(?)
8; 7(?)
20
Fig. 4. Case 10. The complete disappearance of granulation
tissue, remaining of corneal neovascularisation.
It was hard to estimate the progression or regression
of pigmentation because of thick granulation tissue
before treatment.
255
Fig. 5. Case 3. The whole cornea covered
by superficial macular opacities, neovascularisation
and pigmentation.
Fig. 7. Case 7: the pigmentation and corneal
neovascularisation with grayish granulation tissue.
Fig. 9. Case 6. Neovascularisation of the ventrotemporal
cornea quadrant and depigmentation of the free
margin of nictitans membrane.
Fig. 6. Case 3. A decrease in superficial
macular opacities and pigmentation.
Fig. 8. Case 7: disappearance of neovascularisation
and granulation tissue, the increase and decrease
of pigmentation on the same cornea after a 5 week
treatment with tacrolimus.
Fig. 10. Case 6. The disappearance of
corneal neovascularisation. The repigmentation
of the third eyelid margin and progression
of corneal pigmentation.
256
Before the therapy, a total of 71 corneal
quadrants were involved in neovascularisation, whereas
their number after the treatment was 39. A median right
corneal quadrants with neovascularisation was reported
to show a statistically significant decrease from 3.5 to
1.5 while for the left corneas from 2 to 1.
It was found that tacrolimus therapy provided
benefits to all the patients by reducing of corneal area
surface affected by inflammatory process (Table 2). In
addition, this medication contributed to the decrease in
granulation tissue thickness (Figs 3 and 4). Median
corneal area surface affected by the condition showed a
statistically significant decrease from 46% to 27%
(P<0.01) in case of the left corneas and from 58% to
33% for the right ones. The occurrence of pigmentation
was recognised in all of the patients before the therapy
but in one of them, the pigmentation involved only one
cornea. Out of 27 corneas affected by pigmentation, 13
corneas in eight patients exhibited decreased
pigmentation (Table 3; Figs 5 and 6), whereas increased
pigmentation was observed on eight corneas in five
patients. The increase and decrease in pigmentation on
the same cornea was noted in patient No. 7 (Figs 7 and
8). It was not possible to accurately assess a decrease or
increase in pigmentation on eight corneas because prior
to the treatment, and at the beginning, a thick
inflammatory infiltration was found in the sites where
later pigmentation appeared (Figs 3 and 4). The
tacrolimus therapy has decreased the density of
pigmentation in 20 corneas.
Before the therapy, the depigmentation of the
third eyelid margin was diagnosed in 14 patients. The
outcome of the treatment was repigmentation of the
third eyelid margin in 11 patients and persistent
depigmentation in three dogs (Table 1; Figs 9 and 10).
Discussion
Corticosteroids have been the mainstay in CSK
therapy. The authors indicate that the drugs are to be
used at high frequency in the first period of therapeutic
process (12, 22). It is believed that 1% prednisolone
should be applied every 4 h for the first 7-10 d (17). The
presented study has revealed that the therapeutic effects
were obtained when tacrolimus was administered only
three times a day for 5 weeks.
Tacrolimus as an immunosuppressive agent has
proven to be a useful tool for the treatment of numerous
immune-mediated diseases, but its therapeutic range for
dogs has not been defined. Therefore, tacrolimus therapy
requires special precautions due to potential adverse
events that might develop (16). Having that in mind, this
medication was used three times a day for only 5 weeks.
The studies on its level in blood serum after 0.03%
tacrolimus topical application twice a day did not show
any significant systemic absorption after the 2-week
application period (1).
Topical cyclosporine therapy led to side effects
in some patients, including conjunctival irritation
reactions (7, 25). However, the treatment of human giant
papillary conjunctivitis did not produce any adverse
events associated with topical 0.03% tacrolimus
ointment applied for 3 months (15). Similarly, no
common treatment-related side effects associated with
0.03% ointment of tacrolimus applied for human allergic
conjunctivitis therapy were reported (2). The studies
have confirmed good tolerability of the medication. The
treated dogs did not display any signs of irritated eyes,
i.e. conjunctiva redness or itching eyes as a response to
0.02% ophthalmic ointment of tacrolimus.
As for the CSK cases, it remains challenging to
define the corneal surface area involved in granulation
tissue or pigmentation. The pigmentation may cover the
granulation tissue located underneath, or in some cases it
is impossible to accurately localise pigmentation as it
may develop under a thick granulation tissue. The
examinations indicated the occurrence of the
pigmentation after regression of granulation tissue. It is
difficult to state if it became developed or regressed as it
was not visible before the therapy commencement.
Therefore, the authors estimate the percentage of corneal
surface area affected by the disease, i.e.
neovascularisation, granulation tissue, superficial
macular opacities, and pigmentation. Additionally, the
evaluation of the development or regression, and
increase or decrease in the transparency of visible
pigmentation was performed.
Regarding the CSK progression, the blood
vessel ingrowing into the cornea is preceded by the
development of granulation tissue. Owing to this fact,
corneal neovascularisation regression appears to be a
promising sign of the disease remission. The conducted
studies have revealed a statistically significant reduction
of the corneal neovascularisation. A number of
quadrants under corneal neovascularisation declined by
45% as a therapeutic outcome of the 5-week therapy.
Nell et al. (19) found that in some cases the corneal
neovascularisation regressed as early as 2 weeks after
pimecrolimus therapy and completely disappeared after
11 weeks. Pimecrolimus application caused a decrease
in corneal opacity, vascularisation, and pigmentation.
The presented study has shown that as for the right
corneas, median corneal surface area involved in the
condition decreased by 44.8%, and by 34.7% for the left
corneas. Balicki (3) carried out the studies in the same
manner and found that 0.5% prednisole applied for the
CSK treatment has induced a decrease in the affected
corneal surface area by 35% for the right corneas, and
41% for the left corneas. Importantly, in this study,
tacrolimus treatment resulted not only in a considerable
disease regression but additionally, in the cases where a
thick granulation tissue was formed, it effectively
reduced its thickness.
The most challenging element of the CSK
treatment is to suppress the pigmentation development
and cause its regression. Progressing remission of
corneal pigmentation was observed as a therapeutic
effect of the pimecrolimus application (19). The study
showed a reduced corneal pigmentation on 13 corneas in
eight patients but pigmentation development was
observed on eight cornea surface areas in five patients. It
was found that pigmentation progressed despite a
257
decrease in the granulation tissue. Interestingly, in many
clinical cases tacrolimus failed to suppress pigmentation
production but increased its transparency. These
findings are consistent with other results reported by
Williams et al. (25). The authors found that when the
dogs affected by CSK were treated with both
cyclosporine and dexamethasone, the density of
pigmentation was reduced even when a decreased
response rate of the pigmentation-affected area of the
cornea was not observed. In the severe cases, when the
cornea has undergone extensive pigmentation localised
in its superficial layers of the stroma, its regression is
frequently impossible. Then, the inhibition of
pigmentation formation and decrease in its density may
be considered as a positive therapeutic outcome.
Besides, the evaluation of the corneal pigmentation in
some CSK clinical cases is not easy. In the presented
investigations, the pigmentation occurred in the site
where a thick granulation tissue regressed. It was hard to
confirm its presence and extensiveness before and at the
beginning of the treatment, as on the other cornea of
these patients the pigmentation progressed.
The studies revealed repigmentation of the third
eyelid margin in 11 patients. It is difficult to estimate the
complete repigmentation of the third eyelid margin.
Bigelbach (7) observed complete repigmentation of
nicitating membrane after long-term treatment. In the
presented study, the repigmentation of the third eyelid
margin seemed complete in many cases but a detailed
examination with the use of computer photos enabled to
notice very little depigmentation spots. Therefore, the
decreased depigmentation was determined as
repigmentation.
In conclusion, the studies proved that 0.02%
ophthalmic drops of tacrolimus have been effective
topically in canine chronic superficial keratitis
treatment. A treatment response was visualised by
reduced
granulation
tissue
and
corneal
neovascularisation. Still in some cases tacrolimus failed
to inhibit the pigmentation formation.
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