Download The effect of subconjunctival ranibizumab on corneal and anterior

DOI: 10.5301/ejo.5000391
Eur J Ophthalmol 2014; 24 ( 3 ): 299-308
Original Article
free on line
The effect of subconjunctival ranibizumab on corneal
and anterior segment neovascularization: study on an
animal model
Vasilios S. Liarakos1-3, Dimitrios Papaconstantinou4, Ioannis Vergados2, Maria Douvali2,
Panagiotis G. Theodossiadis2
Naval Hospital, Athens - Greece
2nd Department of Ophthalmology, “Attikon” Hospital, University of Athens - Greece
3
Netherlands Institute for Innovative Ocular Surgery, Rotterdam - the Netherlands
4 st
1 Department of Ophthalmology, University of Athens - Greece
1
2
Purpose: To evaluate the effect of subconjunctival anti–vascular endothelial growth factor (VEGF)
ranibizumab on corneal and anterior segment neovascularization.
Methods: In this experimental study and laboratory investigation, chemical cauterization was utilized
to induce corneal neovascularization in 16 rabbits randomly divided in 2 equal groups. Cauterized
eyes were either treated with 0.1 mL (1 mg) of subconjunctival ranibizumab or administered a sham
injection. A third group of 4 rabbits served as control for side effects after ranibizumab administration.
All animals were monitored daily for 14 days and the extent of corneal scarring and neovascularization
was measured on days 1, 7, and 14. After enucleation, ocular tissues were separated under a surgical
microscope and VEGF levels were measured with ELISA. Statistical analysis was performed to compare the extent of corneal neovascularization and VEGF levels between treated and untreated eyes.
Results: Subconjunctival ranibizumab inhibited corneal neovascularization significantly both in the
first and the second week compared to untreated controls (p = 0.006 and p = 0.001, respectively).
The VEGF levels were significantly lower in all anterior segment tissues like the cornea, iris, aqueous
humor, and conjunctiva of the treated eyes (p<0.01). The reduction of VEGF levels ranged from 19% to
73% in different ocular tissues. Corneal scarring was not significantly affected by anti-VEGF treatment
(p = 0.7). No side effects were noticed.
Conclusions: Early subconjunctival administration of ranibizumab may successfully inhibit alkali-induced
corneal neovascularization in an animal model. Subconjunctival ranibizumab reduces VEGF levels significantly not only in the cornea and the bulbar conjunctiva but also in the aqueous humor and the iris.
Keywords: Cornea, Iris, Neovascularization, Ranibizumab, Vascular endothelial growth factor
Accepted: October 22, 2013
INTRODUCTION
Corneal clarity and avascularity are important for the
proper optical performance of the cornea. Corneal neovascularization is a sight-threatening condition usually associated with inflammatory or infectious disorders of the
ocular surface. There is a balance between angiogenic factors and anti-angiogenic molecules in the cornea, which
tilts towards angiogenesis in case of various inflammatory, infectious, degenerative, or traumatic disorders (1).
Corneal neovascularization is a common clinical problem
with serious consequences in vision; it can compromise
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Subconjunctival ranibizumab for anterior segment neovascularization
corneal transparency and plays a major role in corneal
graft rejection (2) and the prognosis of penetrating keratoplasty (3-5) by breaching corneal immune privilege (6). Diseases associated with corneal neovascularization include
inflammatory disorders, corneal graft rejection, infectious
keratitis, contact lens–related hypoxia, alkali burns, stromal ulceration, aniridia, and limbal stem cell deficiency.
In these conditions, the balance between angiogenic and
antiangiogenic factors may be tilted in favor of neovascularization due to the upregulation of angiogenic factors
and/or the downregulation of antiangiogenic factors (7).
Twenty percent of corneal specimens obtained during
corneal transplantation show histopathologic evidence of
neovascularization (4, 5, 8). Subsequently, topical antiangiogenic therapy could reduce the need for corneal transplantation and retransplantation by improving long-term
graft survival.
Vascular endothelial growth factor (VEGF)–A is upregulated in inflamed and vascularized corneas in both human
and animal models (9-11). Enhanced VEGF production
has been shown in hypoxia and inflammatory response.
The importance of VEGF in corneal angiogenesis has
been demonstrated, among others, also by the inhibition
of neovascularization with the administration of anti-VEGF
agents in rabbit cornea models (12, 13). Additional VEGF
members include VEGF-B, VEGF-C, and VEGF-D, which
bind differently to VEGF receptors and regulate angiogenesis and lymphangiogenesis (14). Ranibizumab is an anti-VEGF agent already approved for age-related macular
degeneration (15), diabetic retinopathy (16), and retinal
vein occlusions (17, 18).
The primary purpose of our study was to evaluate the effect of subconjunctival ranibizumab on corneal neovascularization. Additionally, we aimed to evaluate the potential
effect of subconjunctival ranibizumab on VEGF levels in
various ocular tissues.
MATERIALS AND METHODS
New Zealand albino rabbits were used in this experimental study. Twenty male 4-month-old animals weighing 2.83.5 kg were included in the protocol. The animals were
housed under a 12 hour:12 hour light-dark cycle with free
access to standard chow and water ad libitum. All rabbits were examined prior to the experimental study and
confirmed to have no apparent corneal abnormality. All
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procedures were performed with animals under general
and topical anesthesia. General anesthesia was induced
by intramuscular injection of 5 mg/kg ketamine hydrochloride (Ketalar®; Pfizer, Hellas) and 2 mg/kg xylazine
hydrochloride (Rompun®; Bayer Schering Pharma AG,
Leverkusen, Germany), as previously described (13). The
eyes were additionally anesthetized topically with 0.5%
proparacaine hydrochloride (Alcaine®; Alcon Hellas) before
manipulation. Every procedure was conducted according
to the ARVO statement for the use of animals in ophthalmic and vision research. Every procedure was conducted
in accordance with the Declaration of Helsinki, to Council Directive 86/609/EEC of 24-11-1986 of the European
Union, as well as Greek laws and regulations (Presidential
Decree 160/1991, Act No 2015/2001) regarding the protection of animals used for experimental and other scientific
purposes. The experimental protocol was approved by
the Ethics Committee of Attikon University Hospital. No
human material or human data were used in this study.
Induction of corneal neovascularization
The rabbit model of corneal neovascularization subsequent to corneal alkali injury has been utilized effectively in
the past (13, 19). Therefore, an alkali cauterization model
was generated for this study and checked for reproducibility. This is a model of corneal angiogenesis characterized
by epithelial defect and stromal scarring. An alkali burn
injury was induced in only 1 eye of 16 rabbits. Whatman filter
papers (5 x 5mm) were soaked in 5% NaOH solution for
10 seconds and then applied on the surface of the upper
half of the cornea for 60 seconds. The eyelids were held
open for another 30 seconds after application and were
then rinsed thoroughly with 20 mL of balanced salt solution (BSS). To increase the reproducibility of the alkali burn
model, the whole process was carried out on all animals in
the same manner by the same investigator. Only the one
eye was cauterized so that the animals were not blinded.
Group formation and follow-up
Immediately after alkali cauterization, rabbits were randomly and equally divided in 2 groups. One hour after
the chemical cauterization, 0.1 mL (1 mg) of ranibizumab
(Lucentis®; Novartis, Hellas, Greece) was administered
subconjunctivally in the cauterized eye of the one group
(treated group) with a 30-G needle, 2 mm from the superior
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Liarakos et al
limbus. A sham injection of 0.1 mL of BSS was administered also with a 30-G needle, 2 mm from the superior
limbus, in the cauterized eyes of the second group (untreated eyes). A third group of 4 rabbits was only injected
subconjunctivally with 0.1 mL (1 mg) of ranibizumab in the
one eye in order to evaluate possible side effects.
A topical antibiotic (Tobramycin; Tobrex®, Alcon, Hellas)
was administered every 4 hours for the first 24 hours to
avoid bacterial infection. In case of infection or corneal
perforation during follow-up, eyes had to be excluded from
the study.
All 20 animals were monitored daily. To minimize observer
bias, all observations were performed by an investigator
who was masked to the allocation of the animals in each
group. Digital pictures (Sony 8.1 megapixels digital camera, Carl Zeiss lens, Oberkochen, Germany) were taken
on days 1, 7, and 14. Image analysis was performed on
corneal digital photographs using an image processing and analysis software program (IMAGEnet 2000®
v2.5x for Windows). The area of corneal scarring and neovascularization was measured in pixels and expressed as
the percentage of corneal surface, as described previously
(13). In order to achieve a more detailed evaluation of the
neovascularization, a qualitative evaluation of the density
and diameter of the vessels was performed. The precise
measurement of the diameter of the vessels has been
suggested for human patients in the literature (20); however, it was difficult to apply in laboratory conditions.
Laboratory investigation
All animals were euthanased on day 14 and eyes were
enucleated. First, the aqueous humor was aspirated immediately after enucleation with a 27-G needle inserted
from the limbus parallel to the iris plane. Then, the vitreous
humor was aspirated with a 16-G needle inserted 2 mm
behind the limbus. Ocular tissues were surgically separated under a surgical microscope. The cornea was excised
at the corneal limbus (including the limbus) with a curved
cornea scissors. The iris was separated from the ciliary
body manually with a forceps and a blade. A 7 x 7-mm
piece of the superior bulbar conjunctiva was also cut with
a Westcott tenotomy scissors and excised. All samples
were placed in preweighed laboratory microcentrifuge
tubes. Then, they were weighed with an electronic precision
laboratory balance (Kertus, Hellas, Greece) and stored in
a −80oC freezer.
Cornea, iris, and conjunctiva samples were homogenized
after adding phosphate buffered saline (PBS), and then centrifuged at 1000 g, at 4oC for 10 minutes. The supernatant
was used to measure the concentration of VEGF. Aqueous
and vitreous samples were also diluted with PBS. VEGF levels were determined by a commercially available ELISA kit
(R&D Systems; Minneapolis, Minnesota, USA), as described
before (21-23). Previous studies have shown that the antibody against human VEGF crossreacts with rabbit VEGF
(24) and comparison of the nucleotide sequences of rabbit
and human VEGF revealed a homology of 91%. The assay
was performed according to the manufacturer’s instructions.
The tissue samples’ VEGF concentrations were calculated
from the standard curve, corrected for total protein, and expressed in picograms of protein per gram of tissue (pg/g).
Statistical analysis
Statistical analysis was performed using SPSS® v15.0 for
Windows (SPSS Inc., Chicago, Illinois, USA). Nonparametric Mann-Whitney U test was used to compare corneal neovascularization, scarring, and VEGF levels between treated
and untreated eyes. One-way analysis of variance (ANOVA)
was used to evaluate the differences in corneal neovascularization and scarring over time as well as the differences
in the VEGF levels of different tissues in the same eyes.
Statistical significance was defined as a p<0.05. Values
were expressed as mean ± SD. The standard error of the
mean (SEM) regarding VEGF levels is mentioned to indicate 95% confidence interval (95% CI).
RESULTS
The effect of subconjunctival ranibizumab on
corneal scarring and neovascularization
Profound corneal scarring was evident in all cauterized eyes
within 24 hours after the alkali cauterization. Corneal opacity
extended slightly beyond the primary alkali injury and was
similar between the untreated and the treated eyes (36.75 ±
4.98% and 34.88 ± 3.00%, respectively; p = 0.385, MannWhitney U test). Adjacent bulbar conjunctiva appeared
hyperemic in both groups during the first 5 days following
cauterization; however, these signs regressed during followup. During the 14-day follow-up, no significant change was
observed in the area of corneal scarring in both groups
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Subconjunctival ranibizumab for anterior segment neovascularization
TABLE I - C
ORNEAL SCAR AND NEOVASCULARIZATION IN UNTREATED EYES AND EYES TREATED WITH SUBCONJUNCTIVAL RANIBIZUMAB
Corneal scar, %
Corneal neovascularization, %
Untreated,
mean ± SD
Treated,
mean ± SD
Mann-Whitney
U, p value
Untreated,
mean ± SD
Treated,
mean ± SD
Mann-Whitney
U, p value
Day 1
37.63 ± 5.24
34.88 ± 3.00
0.197
0
0
NA
Day 7
42.63 ± 8.55
40.75 ± 7.34
0.632
9.25 ± 3.20
3.63 ± 3.02
0.006a
Day 14
37.75 ± 6.14
36.38 ± 4.66
0.709
22.63 ± 4.03
6.13 ± 4.22
0.001a
ANOVA
p = 0.266
p = 0.095
p<0.001a
p = 0.002a
ANOVA = analysis of variance; NA = not applicable.
The area of corneal neovascularization changed significantly over time in both groups (ANOVA) but it was significantly smaller in the treated eyes compared to the
untreated eyes during the 14-day follow-up (Mann-Whitney U). The area with corneal scar did not change significantly over time (ANOVA) and was similar between
the 2 groups (Mann-Whitney U).
a
Statistically significant difference.
Fig. 1 - Evolution of corneal neovascularization and scar in eyes treated
with subconjunctival ranibizumab vs
untreated eyes. Corneal scar and neovascularization (NV) are expressed as
a percentage of the corneal surface
in eyes treated with subconjunctival
ranibizumab (dark gray) and untreated
eyes (light gray). Median values and
95% confidence intervals are shown.
*The difference is statistically significant
at the 0.01 level (Mann-Whitney U test).
#The difference is not statistically significant (p>0.05; Mann-Whitney U test).
(p = 0.404 and p = 0.095, respectively, one-way ANOVA), as
shown in Table I and Figure 1.
Mild peripheral neovascularization consisting of multiple,
short (<2 mm long), fine and dense vessels emerging from
the limbus appeared during the first week in all cauterized
corneas in the untreated group (Fig. 2). Fourteen days after
cauterization, there was clear evidence of neovascularization
covering almost one quarter of the cornea, with major centripetal vessels and multiple smaller branch vessels (Fig. 2).
Neovascular tissue progressed significantly from 8.38% ±
3.29% of the corneal surface on day 7 to 21.88% ± 5.03%
on day 14 (p<0.001, one-way ANOVA), as shown in Table I.
Very restricted scattered areas of faint neovascularization were observed in eyes treated with ranibizumab
302
7 days after cauterization (Fig. 2). Although neovascularization progressed during the second week (p = 0.002;
one-way ANOVA), neovascular area did not exceed
10% of the cornea in most of the treated eyes (Fig. 2).
The area of neovascularization was significantly smaller
in the corneas treated with subconjunctival ranibizumab, compared to untreated corneas, on both days 7 and
14 (p = 0.01 and p<0.001, respectively; Mann-Whitney
U test), as shown in Table I and Figure 1. No recurrence
or significant progression of corneal neovascularization
was recorded after day 10.
No significant inflammation, corneal melt, or other major
complication that would be a reason for exclusion were
noted during follow-up.
© 2013 Wichtig Editore - ISSN 1120-6721
Liarakos et al
Control corneas in the third group presented neither scarring nor neovascularization. Moreover, no apparent clinical
ophthalmic or systemic side effects were noted.
The effect of subconjunctival ranibizumab on
VEGF levels in different ocular tissues
Fig. 2 - Pictures of corneal neovascularization in untreated eyes
and after subconjunctival ranibizumab. Corneal scar and neovascularization in untreated control eyes 7 days (A) and 14 days (B) after alkali burn injury. Mild peripheral neovascularization appeared
by the end of the first week (A) and progressed significantly during
the second week, covering almost one-quarter of the cornea with
major centripetal and multiple smaller branch vessels (B). Corneal
scar and neovascularization in eyes treated with subconjunctival
ranibizumab 7 days (C) and 14 days (D) after alkali burn injury followed by a single injection of 1 mg of ranibizumab. Only restricted
scattered areas of faint neovascularization were observed 7 days
after cauterization (C). Neovascular area did not exceed 10% of
the cornea in most of the treated eyes 14 days after chemical injury (D).
All tissue samples were excised from the enucleated
globes as described above and were weighed before preparing them for the ELISA. Differences between treated
and untreated eyes regarding the average weight of the
tissue samples were not statistically significant (p>0.1,
Mann-Whitney U test), as shown in Table II. The VEGF
levels (per unit of tissue mass) were found to be lower in
the vitreous humor and, by ascending order of concentration, were higher in the aqueous humor, even higher in the
cornea, and much higher in the iris and the conjunctiva
(Tab. II).
Corneal VEGF levels were found to be significantly lower
in treated eyes compared to untreated ones by more than
45% (p = 0.009; Mann-Whitney U test). The VEGF levels
were measured to be lower in the conjunctiva, the iris, and
the aqueous humor of treated eyes compared to untreated
ones by 44%-71% (p = 0.001; Mann-Whitney U test), as
shown in Table II and Figure 3. Although vitreous VEGF
levels were also lower in the treated group, the difference
was not statistically significant (p = 0.093).
TABLE II - VEGF LEVELS AND TISSUE WEIGHT IN UNTREATED EYES AND EYES TREATED WITH SUBCONJUNCTIVAL
RANIBIZUMAB
Tissue weight
VEGF
Untreated, mg
Treated, mg
MannWhitney
U, p value
Untreated, pg/g,
mean ± SEM
Treated, pg/g,
mean ± SEM
Difference, %
MannWhitney
U, p value
Cornea
80.92 ± 3.16
71.36 ± 12.41
0.150
2365.46 ± 213.32
1166.88 ± 150.09
-50.7
0.005a
Iris
46.90 ± 9.08
34.29 ± 7.24
0.078
23186.80 ± 2064.31
6343.35 ± 251.33
-72.6
0.001a
Conjunctiva
107.05 ± 76.48
126.38 ± 54.23
1.000
28411.14 ± 1517.47
16432.25 ± 929.24
-42.2
0.001a
Aqueous humor
217.52 ± 33.74
195.54 ± 44.28
0.873
424.73 ± 31.83
211.02 ± 25.46
-50.2
0.001a
1264.86 ± 92.95
1542.27 ± 382.58
0.140
112.75 ± 4.69
91.89 ± 8.19
-18.8
0.059
p<0.001a
p<0.001a
Vitreous
ANOVA
ANOVA = analysis of variance; VEGF = vascular endothelial growth factor.
The VEGF levels were found to be significantly lower in the eyes treated with subconjunctival ranibizumab in almost all ocular tissues. No significant differences were
noted between the 2 groups regarding the weight of the tissue samples.
a
Statistically significant difference.
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Subconjunctival ranibizumab for anterior segment neovascularization
are not aware of any other experimental or clinical study
addressing the effect of subconjunctival ranibizumab on
iris neovascularization.
Advantages of early subconjunctival administration
Fig. 3 - Significant decrease of vascular endothelial growth factor
(VEGF) levels in all anterior segment tissues after subconjunctival
administration of ranibizumab. The VEGF levels in different tissue
samples of eyes treated with subconjunctival ranibizumab (dark
gray) and untreated eyes (light gray). Mean values and 95% confidence intervals are shown. *The difference is statistically significant
at the 0.01 level (Mann-Whitney U test). #The difference is not statistically significant (p>0.05; Mann-Whitney U test).
DISCUSSION
Ranibizumab is an anti-VEGF agent already approved for
ophthalmic use. Our initial results regarding subconjunctival administration of ranibizumab in the early posttraumatic period were encouraging (Liarakos VS, et al. IOVS 2008;
49: ARVO E-Abstract 3743) and triggered further experimental research. Recently, the effectiveness of subconjunctival ranibizumab for corneal neovascularization was
well-documented by an experimental study on rats (25).
In our study, we used a dose twice as high as in the study
of Sener et al (25) and extended the follow-up to 15 days
instead of 8, because, according to Dastjerdi et al (26), the
concentration of the anti-VEGF agent in the stroma begins
to fade after that time. During this period, ranibizumab
proved to be well-tolerated when administered subconjunctivally even at a dose twice as high as the intravitreal
dose and twice as high as used by Sener et al (25).
Moreover, we tried to provide additional evidence based
on the effect of ranibizumab on the actual levels of VEGF in
the cornea as well as the iris and other ocular tissues. We
304
We chose to administer subconjunctival ranibizumab in
the early posttraumatic period, soon after cauterization,
because we have already documented the favorable effect
of early administration of anti-VEGF treatment in a corneal
neovascularization model (13). Early administration of antiVEGF treatment achieves a desirable concentration in the
early phases of corneal wound healing, as described by
Azar (7). This is a period when keratocytes proliferate and
transform to fibroblasts and then migrate to populate the
wound. This procedure is accompanied by the production of high levels of epithelial and stromal growth factors
(tumor growth factor [TGF]–α, TGF-β, basic fibroblast
growth factor, platelet-derived growth factor) as well as
cytokines (tumor necrosis factor–α, interleukin [IL]–1) (3)
and apparently also VEGF (27).
We chose subconjunctival administration instead of eyedrops because it has been shown that subconjunctival
bevacizumab may achieve favorable results when compared to eyedrops (28). Although bevacizumab eyedrops
are known to penetrate the cornea and enter the anterior
chamber (29), prolonged topical application may cause
epitheliopathy and even corneal thinning (30). In a recent
experimental study, it has been shown that subconjunctival administration of bevacizumab is superior to topical
application in the form of drops with regard to stromal penetration, especially in intact corneas (26).
Targeting VEGF to treat corneal neovascularization
Topical steroids remain a first-choice treatment for corneal
neovascularization. However, steroids are not always ideally indicated, as they can either delay corneal wound healing, both in humans (31) and in experimental animal models
(32), or trigger the onset or recurrence of herpetic or fungal
inflammation (33). It is also known that chronic treatment
may lead to glaucoma and cataract formation. In terms of
the pathophysiology of corneal neovascularization, inflammation is not necessarily the only reason. Corneal hypoxia,
limbal stem cell deficiency, persistent or recurrent epithelial
defects, and keratocyte activation should also be considered (7, 34). Moreover, polymorphonuclear chemoattractants
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Liarakos et al
IL-8 and cytokines like IL-6 and IL-1β are upregulated by
closed-eye conditions or contact lens wear (35). The VEGF
expression is localized to corneal epithelial cells, corneal
endothelial cells, vascular endothelial cells of limbal vessels and of newly formed stromal vessels, and weakly by
keratocytes (36). In addition, VEGF expression has markedly
increased in epithelial cells of inflamed corneas, vascular
endothelial cells, macrophage infiltrates, and fibroblasts in
corneal scar tissue (7). The VEGF concentrations are significantly higher in vascularized corneas than in normal control
corneas (27, 36). Additionally, VEGF promotes several steps
of angiogenesis, including proteolytic activities (dissolution
of the membrane of the original vessel), vascular endothelial cell proliferation, migration, and capillary tube formation.
Subsequently, adjuvant or alternative treatments for corneal
neovascularization also targeting VEGF should be evaluated.
Pharmacokinetics of ranibizumab
Although both ranibizumab and bevacizumab neutralize
all isoforms of VEGF-A, thus having a similar clinical effect at least on choroidal neovascularization (Comparison
of AMD Treatments Trials study [37]), their small structural
differences do not ensure identical behavior in all ocular
tissues. In vitro studies showed that the binding affinity of
ranibizumab is much more specific than bevacizumab, but
bevacizumab has a longer half-life than ranibizumab (38).
When administered intravitreally, ranibizumab’s concentration is described to decline in a monoexponential fashion
with a similar half-life in both the vitreous and the aqueous
humor (2.88 and 2.84 days, respectively) (38). Subsequently, pharmacokinetics may differ not only in the retina (39)
but also in other tissues.
In our study, VEGF levels were significantly lower in the
treated eyes 14 days after a single subconjunctival ranibizumab injection. A time-dependent diffusion of subconjunctival bevacizumab in the rabbit cornea has been
described (19). Although diffusion extends during the first
days after administration, it declines significantly after the
first week. However, its clinical effect may last longer (13).
Possible effect of subconjunctival ranibizumab
on iris neovascularization
The VEGF levels in the cornea of untreated cauterized
eyes were almost 5 times higher than VEGF levels in the
aqueous humor and almost 20 times higher than VEGF
levels in the vitreous of the same eyes. On the contrary,
VEGF levels in the cornea of the same eyes were
10-14 times lower than VEGF levels in highly vasculated
tissues, like the iris and the conjunctiva. This shows that,
in general, VEGF levels are related to the vasculature of
the ocular tissues, either normally preexisting or newly
established.
Based on the significant reduction of VEGF levels in the
various ocular tissue samples of the treated cauterized
eyes, it seems that ranibizumab not only diffuses into the
conjunctiva and the adjacent cornea. It probably penetrates the ocular surface and enters into the anterior
chamber, where it reduces VEGF levels in the iris and the
aqueous humor significantly. Ranibizumab could have
been distributed into the ocular tissues via the systemic
circulation and reached the anterior chamber through the
ciliary vessels or through direct diffusion into the tissues.
A similar pattern of systemic distribution has been described for subconjunctival bevacizumab (40). However,
this has not been confirmed for ranibizumab. The VEGF
levels were reduced in the iris of treated eyes by more
than 70%. Based on the reduction of VEGF levels, the effect on the iris was larger than the effect on the conjunctiva, which happened to be the primary site of ranibizumab
injection. This indicates that subconjunctival ranibizumab
may also be a therapeutic alternative for secondary iris
neovascularization. So far, mostly intravitreal injections of
bevacizumab have been suggested for treating iris neovascularization secondary to diabetic retinopathy (41) or
other ischemic retinal disorders (42). Intravitreal ranibizumab may also inhibit iris neovascularization secondary
to central retinal vein occlusion (43). However, subconjunctival administration is safer, more convenient for the
patient, and easier for the doctor to perform.
Although the minimum therapeutic concentration of ranibizumab within the cornea is not known, our study
demonstrated that ranibizumab was clinically effective
and reduced VEGF levels significantly in almost all ocular
tissues, at least in the first 14 days after administration.
Its potency in animal models should be further evaluated
with regard to the dosage regimen before interpretation
of the experimental results.
Clinical application
We have already depicted the effect of subconjunctival
bevacizumab on experimental corneal neovascularization
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Subconjunctival ranibizumab for anterior segment neovascularization
and we have documented the supremacy of early administration (13). Other experimental studies agree with
these results (12, 19, 21). Experimental results encouraged
clinical application with several reports of subconjunctival
bevacizumab for corneal neovascularization associated
with various corneal or ocular surface pathologies (4446). It is shown that a single subconjunctival injection of
bevacizumab in its usual concentration of 25 mg/mL may
achieve better results than topical instillation in the form
of eyedrops at least in the first week of treatment (28). In a
clinical study, it is shown that subconjunctival bevacizumab repeated every 4-7 days is superior to intense topical
administration of bevacizumab eyedrops (47). There are no
such reports with ranibizumab. The frequency of administration also could be related to the severity of the corneal
neovascularization as well as its etiology. Further clinical
studies are needed to determine this. In an ongoing clinical study regarding administration of a single subconjunctival injection of 0.5 mg ranibizumab in primary pterygium
preoperatively or intraoperatively, ranibizumab is well-tolerated and does not affect the survival of the conjunctival
autograft (48). However, the possible effect of ranibizumab
on VEGF levels or the vessels themselves was not evaluated. Prompt regression of conjunctival microvessels was
also reported in a single patient with pterygium (49). Subconjunctival ranibizumab was well-tolerated by 5 patients
who were injected 3 days to 2 months prior to pterygium
excision (50).
CONCLUSIONS
REFERENCES
6.
1.
2.
3.
4.
5.
306
Chang JH, Gabison EE, Kato T, Azar DT. Corneal neovascularization. Curr Opin Ophthalmol 2001;12:242-9.
Garcia DD, Shtein RM, Musch DC, Elner VM. Herpes simplex
virus keratitis: histopathologic neovascularization and corneal allograft failure. Cornea 2009;28:963-5.
Cursiefen C, Cao J, Chen L, et al. Inhibition of hemangiogenesis and lymphangiogenesis after normal-risk corneal transplantation by neutralizing VEGF promotes graft survival.
Invest Ophthalmol Vis Sci 2004;45:2666-73.
Cursiefen C, Küchle M, Naumann GO. Angiogenesis in corneal diseases: histopathologic evaluation of 254 human corneal
buttons with neovascularization. Cornea 1998;17:611-3.
Dana MR, Schaumberg DA, Kowal VO, et al. Corneal neovascularization after penetrating keratoplasty. Cornea
1995;14:604-9.
This experimental and laboratory study indicates that early
subconjunctival administration of ranibizumab may successfully inhibit alkali-induced corneal neovascularization
in an animal model. Subconjunctival ranibizumab reduces
VEGF levels significantly not only in the cornea and the
bulbar conjunctiva but also in the aqueous humor and the
iris, suggesting a possible treatment for secondary neovascularization of the anterior segment of the eye.
ACKNOWLEDGEMENT
Dr. Liarakos received the 2011 Annual Scholarship from the
Hellenic Society of Intraocular Implants and Refractive Surgery.
Financial Support: No financial support was received for this submission.
Conflict of Interest Statement: None of the authors has conflict of
interest with this submission.
Meeting Presentation: Primary results were presented at the 2008 ARVO
meeting (Liarakos VS, et al. IOVS 2008; 49: ARVO E-Abstract 3743).
Address for correspondence:
Vasilios S. Liarakos
Adrianoupoleos 5
Papagou 15669
Athens
Greece
[email protected]
Cursiefen C, Colin J, Dana R, et al. Consensus statement on
indications for anti-angiogenic therapy in the management
of corneal diseases associated with neovascularisation: outcome of an expert roundtable. Br J Ophthalmol 2012;96:3-9.
7. Azar DT. Corneal angiogenic privilege: angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis,
and wound healing (an American Ophthalmological Society
thesis). Trans Am Ophthalmol Soc 2006;104:264-302.
8. Lee P, Wang CC, Adamis AP. Ocular neovascularization: an
epidemiologic review. Surv Ophthalmol 1998;43:245-69.
9. Amano S, Rohan R, Kuroki M, Tolentino M, Adamis AP. Requirement for vascular endothelial growth factor in woundand inflammation-related corneal neovascularization. Invest
Ophthalmol Vis Sci 1998;39:18-22.
10. Kvanta A, Sarman S, Fagerholm P, Seregard S, Steen B. Expression of matrix metalloproteinase-2 (MMP-2) and vascular
endothelial growth factor (VEGF) in inflammation-associated
corneal neovascularization. Exp Eye Res 2000;70:419-28.
© 2013 Wichtig Editore - ISSN 1120-6721
Liarakos et al
11. Mastyugin V, Mosaed S, Bonazzi A, Dunn MW, Schwartzman
ML. Corneal epithelial VEGF and cytochrome P450 4B1 expression in a rabbit model of closed eye contact lens wear.
Curr Eye Res 2001;23:1-10.
12. Kim TI, Kim SW, Kim S, Kim T, Kim EK. Inhibition of experimental corneal neovascularization by using subconjunctival injection of bevacizumab (Avastin). Cornea 2008;27:
349-52.
13. Papathanassiou M, Theodossiadis PG, Liarakos VS, Rouvas
A, Giamarellos-Bourboulis EJ, Vergados IA. Inhibition of corneal neovascularization by subconjunctival bevacizumab in
an animal model. Am J Ophthalmol 2008;145:424-31.
14. Li X, Eriksson U. Novel VEGF family members: VEGF-B,
VEGF-C and VEGF-D. Int J Biochem Cell Biol 2001;33:421-6.
15. Rosenfeld PJ, Brown DM, Heier JS, et al; MARINA Study
Group. Ranibizumab for neovascular age-related macular
degeneration. N Engl J Med 2006;355:1419-31.
16. Mitchell P, Bandello F, Schmidt-Erfurth U, et al; RESTORE
study group. The RESTORE study: ranibizumab monotherapy or combined with laser versus laser monotherapy for
diabetic macular edema. Ophthalmology 2011;118:615-25.
17. Campochiaro PA, Brown DM, Awh CC, et al. Sustained benefits from ranibizumab for macular edema following central
retinal vein occlusion: twelve-month outcomes of a phase III
study. Ophthalmology 2011;118:2041-9.
18. Brown DM, Campochiaro PA, Bhisitkul RB, et al. Sustained
benefits from ranibizumab for macular edema following
branch retinal vein occlusion: 12-month outcomes of a
phase III study. Ophthalmology 2011;118:1594-602.
19. Chen WL, Lin CT, Lin NT, et al. Subconjunctival injection of
bevacizumab (Avastin) on corneal neovascularization in different rabbit models of corneal angiogenesis. Invest Ophthalmol Vis Sci 2009;50:1659-65.
20. Dastjerdi MH, Al-Arfaj KM, Nallasamy N, et al. Topical bevacizumab in the treatment of corneal neovascularization: results of a prospective, open-label, noncomparative study.
Arch Ophthalmol 2009;127:381-9.
21. Kim WJ, Jeong HO, Chung SK. The effect of bevacizumab
on corneal neovascularization in rabbits. Korean J Ophthalmol 2010;24:230-6.
22. Li Z, Van Bergen T, Van de Veire S, et al. Inhibition of vascular endothelial growth factor reduces scar formation after glaucoma filtration surgery. Invest Ophthalmol Vis Sci
2009;50:5217-25.
23. Ambati BK, Anand A, Joussen AM, Kuziel WA, Adamis AP,
Ambati J. Sustained inhibition of corneal neovascularization by genetic ablation of CCR5. Invest Ophthalmol Vis Sci
2003;44:590-3.
24. Kinnunen K, Korpisalo P, Rissanen TT, et al. Overexpression
of VEGF-A induces neovascularization and increased vascular leakage in rabbit eye after intravitreal adenoviral gene
transfer. Acta Physiol 2006;187:447-57.
25. Sener E, Yuksel N, Yildiz DK, et al. The impact of subconjuctivally injected EGF and VEGF inhibitors on experimental
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
corneal neovascularization in rat model. Curr Eye Res
2011;36:1005-13.
Dastjerdi MH, Sadrai Z, Saban DR, Zhang Q, Dana R. Corneal penetration of topical and subconjunctival bevacizumab.
Invest Ophthalmol Vis Sci 2011;52:8718-23.
Zheng M, Deshpande S, Lee S, Ferrara N, Rouse BT. Contribution of vascular endothelial growth factor in the neovascularization process during the pathogenesis of herpetic
stromal keratitis. J Virol 2001;75:9828-35.
Hashemian MN, Z-Mehrjardi H, Moghimi S, Tahvildari M,
Mojazi-Amiri H. Prevention of corneal neovascularization:
comparison of different doses of subconjunctival bevacizumab with its topical form in experimental rats. Ophthalmic
Res 2011;46:50-4.
Yoeruek E, Ziemssen F, Henke-Fahle S, et al. Safety, penetration and efficacy of topically applied bevacizumab: evaluation of eyedrops in corneal neovascularization after chemical
burn. Acta Ophthalmol 2008;86:322-8.
Kim SW, Ha BJ, Kim EK, Tchah H, Kim TI. The effect of topical bevacizumab on corneal neovascularization. Ophthalmology 2008;115:e33-8.
Borderie VM, Touzeau O, Bourcier T, Allouch C, Laroche L.
Graft reepithelialization after penetrating keratoplasty using organ-cultured donor tissue. Ophthalmology 2006;113:
2181-6.
Sarchahi AA, Maimandi A, Tafti AK, Amani M. Effects of acetylcysteine and dexamethasone on experimental corneal
wounds in rabbits. Ophthalmic Res 2008;40:41-8.
Wilhelmus KR, Dawson CR, Barron BA, et al; Herpetic Eye
Disease Study Group. Risk factors for herpes simplex virus
epithelial keratitis recurring during treatment of stromal keratitis or iridocyclitis. Br J Ophthalmol 1996;80:969-72.
Ellenberg D, Azar DT, Hallak JA, et al. Novel aspects of corneal angiogenic and lymphangiogenic privilege. Prog Retin
Eye Res 2010;29:208-48.
Thakur A, Willcox MD. Contact lens wear alters the production of certain inflammatory mediators in tears. Exp Eye Res
2000;70:255-9.
Philipp W, Speicher L, Humpel C. Expression of vascular
endothelial growth factor and its receptors in inflamed and
vascularized human corneas. Invest Ophthalmol Vis Sci
2000;41:2514-22.
Martin DF, Maguire MG, Ying GS, Grunwald JE, Fine SL,
Jaffe GJ; CATT Research Group. Ranibizumab and bevacizumab for neovascular age-related macular degeneration. N
Engl J Med 2011;364:1897-908.
Bakri SJ, Snyder MR, Reid JM, Pulido JS, Ezzat MK, Singh
RJ. Pharmacokinetics of intravitreal ranibizumab (Lucentis).
Ophthalmology 2007;114:2179-82.
Veurink M, Stella C, Tabatabay C, Pournaras CJ, Gurny
R. Association of ranibizumab (Lucentis®) or bevacizumab
(Avastin®) with dexamethasone and triamcinolone acetonide: an in vitro stability assessment. Eur J Pharm Biopharm
2011;78:271-7.
© 2013 Wichtig Editore - ISSN 1120-6721
307
Subconjunctival ranibizumab for anterior segment neovascularization
40. Nomoto H, Shiraga F, Kuno N, et al. Pharmacokinetics of
bevacizumab after topical, subconjunctival, and intravitreal administration in rabbits. Invest Ophthalmol Vis Sci
2009;50:4807-13.
41. Oshima Y, Sakaguchi H, Gomi F, Tano Y. Regression of iris
neovascularization after intravitreal injection of bevacizumab
in patients with proliferative diabetic retinopathy. Am J Ophthalmol 2006;142:155-8.
42. Wakabayashi T, Oshima Y, Sakaguchi H et al. Intravitreal
bevacizumab to treat iris neovascularization and neovascular glaucoma secondary to ischemic retinal diseases in
41 consecutive cases. Ophthalmology 2008;115:1571-80.
43. Risard SM, Pieramici DJ, Rabena MD, et al. Intravitreal ranibizumab for macular edema secondary to central retinal
vein occlusion. Retina 2011;31:1060-7.
44. Bahar I, Kaiserman I, McAllum P, Rootman D, Slomovic A.
Subconjunctival bevacizumab injection for corneal neovascularization. Cornea 2008;27:142-7.
45. Georgakopoulos CD, Vasilakis P, Makri OE, Beredima E,
Pharmakakis N. Subconjunctival bevacizumab for corneal
308
46.
47.
48.
49.
50.
neovascularization secondary to topical anesthetic abuse.
Cutan Ocul Toxicol 2011;30:320-2.
Yeung SN, Lichtinger A, Kim P, Amiran MD, Slomovic AR.
Combined use of subconjunctival and intracorneal bevacizumab injection for corneal neovascularization. Cornea
2011;30:1110-4.
Dastjerdi MH, Saban DR, Okanobo A, et al. Effects of topical and subconjunctival bevacizumab in high-risk corneal
transplant survival. Invest Ophthalmol Vis Sci 2010;51:
2411-7.
Galor A, Yoo SH, Piccoli FV, Schmitt AJ, Chang V, Perez VL.
Phase I study of subconjunctival ranibizumab in patients
with primary pterygium undergoing pterygium surgery. Am J
Ophthalmol 2010;149:926-31.
Mansour AM. Treatment of inflamed pterygia or residual
pterygial bed. Br J Ophthalmol 2009;93:864-5.
Mandalos A, Tsakpinis D, Karayannopoulou G, et al. The
effect of subconjunctival ranibizumab on primary pterygium:
a pilot study. Cornea 2010;29:1373-9.
© 2013 Wichtig Editore - ISSN 1120-6721