Graefes Arch Clin Exp Ophthalmol (2009) 247:273–281
DOI 10.1007/s00417-008-0967-4
MEDICAL OPHTHALMOLOGY
Intravitreal administration of the anti-TNF monoclonal
antibody Infliximab in the rabbit
Panagiotis G. Theodossiadis & Vasilios S. Liarakos &
Petros P. Sfikakis & Alexander Charonis &
Georgios Agrogiannis & Nikolaos Kavantzas &
Ioannis A. Vergados
Received: 10 August 2008 / Revised: 4 October 2008 / Accepted: 6 October 2008 / Published online: 4 November 2008
# Springer-Verlag 2008
Abstract
Background Tumor necrosis factor (TNF) is known to play
an important role in various immune-mediated ocular
diseases; intravenous administration of the anti-TNF mono-
clonal antibody infliximab has proved beneficial in such
cases. Since intravitreal injection (when available) is a
substitute for systemic administration of various drugs
targeting the eye, we aimed to evaluate the safety of
intravitreal injection of infliximab in the rabbit eye.
The authors have no financial disclosure regarding this manuscript.
All the authors have full control of all primary data, and agree to allow
Graefe’s Archive for Clinical and Experimental Ophthalmology to
review all data if requested. Every procedure involving animals was
conducted in accordance with the Declaration of Helsinki and the
ARVO guidelines, and 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.
P. G. Theodossiadis : V. S. Liarakos : A. Charonis : I. A. Vergados
2nd Department of Ophthalmology,
“Attikon” University Hospital, University of Athens,
Athens, Greece
P. P. Sfikakis
1st Department of Propedeutic Medicine, “Laikon” Hospital,
University of Athens,
Athens, Greece
G. Agrogiannis : N. Kavantzas
Pathology Department, Medical School, University of Athens,
Athens, Greece
P. G. Theodossiadis (*)
13 Lykiou Street,
10675 Athens, Greece
e-mail: patheo@med.uoa.gr
Methods Seven groups of New Zealand white rabbits
(four animals in each group) received a single unilateral
intravitreal injection (0.1 ml) of increasing doses of
infliximab (namely 1, 2, 5, 8, 10 or 20 mg infliximab
[Remicade]) or a sham injection respectively. Slit-lamp
biomicroscopy, fundoscopy and electrophysiology
recordings, i.e. scotopic, photopic and flicker responses,
were performed at baseline and after 1, 5, 10, 15, 30 and
45 days. Infliximab-injected eyes were compared with
sham-injected and with uninjected fellow eyes (n=28).
Animals were euthanized on day 45 for histopathological
examination of the retinas.
Results Clinical examination and electrophysiological test-
ing were consistently unremarkable after either sham or
1 mg or 2 mg infliximab injections. In contrast, electro-
physiological recordings were significantly reduced in a
dose-dependent manner from day 1 through day 45, after 5,
8, 10 and 20 mg infliximab injections. Flicker responses
were the most sensitive in detecting the lower toxic dose of
5 mg. Histopathological findings were similar in uninjected
and sham-injected eyes, as well as after 1 mg or 2 mg
infliximab injections. Consistent with the functional abnor-
malities, retinal deformities and diffuse edema were
observed after injection of 5 mg or higher doses of
infliximab.
Conclusions Intravitreal infliximab may be safely adminis-
tered up to a dose of 2 mg in the rabbit eye. Such doses can
be used in the design of future clinical trials assessing the
effects of infliximab for selected patients with immune-
mediated ocular conditions.
Keywords Infliximab . anti-TNF . Intravitreal injection .
Remicade . Uveitis . Age-related macular degeneration .
AMD . Rabbit . Electroretinogram . ERG . Histology .
Toxicity . Safety . TNF
274
Introduction
A growing body of evidence supports a role of the
pleiotropic cytokine tumor necrosis factor (TNF) in various
immune-mediated ocular diseases [1]. Experimental studies
in rabbit and murine models of retinal neovascularization
suggest that TNF is a mediator of undesirable angiogenesis
in the eye through its proinflammatory properties [2–4].
TNF may have important pathogenetic role(s) in neo-
vascular age-related macular degeneration and proliferative
vitreoretinopathy [5–8], as well as in non-infectious uveitis
[9]. Consequently, there is increasing interest in ophthal-
mology in the exploration of the therapeutic potential of
drugs that target TNF action, and a growing optimism
regarding their possible efficacy [10, 11].
Infliximab (Remicade) is a human-mouse chimeric IgG
anti-TNF antibody, which has been and/or is currently used
in the treatment of Crohn’s disease and ulcerative colitis,
rheumatoid arthritis, ankylosing spondylitis, psoriasis and
psoriatic arthritis in over 1,000,000 patients worldwide.
Infliximab is administered intravenously to adults with
systemic disease, at doses ranging from 3 to 7.5 mg/kg,
usually every 8 weeks. Ocular inflammation associated with
these chronic diseases responds to intravenous infliximab,
with symptomatic improvement and a rapid decrease in
anterior chamber reaction [12, 13]. Doses as high as 20 mg/
kg have been administered to children with uveitis
secondary to systemic disease, with good results and no
adverse effects [14]. Moreover, patients with Adamantiades-
Behcet’s disease and sight-threatening panuveitis may re-
spond to a single infliximab infusion [15]. Repeated infusions
may also prevent ocular relapses and maintain visual acuity
[16–18]. Beneficial preliminary results following systemic
administration of infliximab have been reported in patients
with refractory ocular sarcoidosis [19, 20], neurosarcoidosis
with optic disk swelling [21], chronic ocular inflammation-
associated refractory cystoid macular edema [22] and
diabetic macular edema [23]. Regression of experimentally-
induced choroidal neovascularization after treatment with
anti-TNF agents has been demonstrated in animal models
[24, 25], >as well as in patients with choroidal neovascula-
rization secondary to age-related macular regeneration [26].
These data not only further support a pathogenetic role of
TNF in ocular immune-mediated disease, but also point to
infliximab as a plausible treatment approach.
With regard to ocular disease, infliximab has always
been administered intravenously at conventional doses
used for treating patients with the approved indications
[1]. The amount of drug reaching the eye, which is the
primary target for the ophthalmologist, is not known.
Moreover, similar amounts reach both eyes, even when
ocular disease is unilateral. In addition, blocking TNF
actions by long-term systemic administration of infliximab
Graefes Arch Clin Exp Ophthalmol (2009) 247:273–281
has been associated with tuberculosis, opportunistic
infections, and autoimmune disease, including exacerba-
tions of pre-existing multiple sclerosis [27–29]. Although
controversial, history of cancer or congestive heart failure
is currently included among the contraindications for
systemic anti-TNF treatment [30].
Intravitreal administration of various drugs has recently
become common practice. Triamcinolone and anti-vascular
endothelial growth factor (anti-VEGF) agents (ranibizumab,
pegaptanib, bevacizumab) are administered intravitreally
with minimal side effects [31]. It is thus likely that
therapeutic intraocular concentrations of infliximab can be
achieved following intravitreal administration with minimal
systemic absorption, minimizing, if not eliminating, adverse
effects. The present study was conducted to evaluate the
ocular safety of infliximab administered intravitreally at
increasing doses in a rabbit model. Clearly, determination
of toxicity levels is a major prerequisite for the design of
future clinical trials to assess the use of intravitreal
infliximab in selected patients with immune-mediated
ocular conditions.
Methods
Experimental protocol
Male New Zealand white rabbits weighing between 2.3 and
3.1 kg (mean 2.7 kg) were studied. Animals were treated
according to the Association for Research in Vision and
Ophthalmology (ARVO) guidelines, fed standard laborato-
ry food, and allowed free access to water in an air-
conditioned room with a 12 h light-dark cycle. Animals
that demonstrated corneal or lens opacities or retinal
damage, or asymmetrical electroretinogram (ERG)
responses between their eyes, were excluded. All selected
animals presented initial inter-eye differences in b-wave
amplitude and implicit time lower than 11% and 8%
respectively. We used ketamine for anesthesia, because,
unlike other anesthetics [32], ketamine has not been
reported to induce any notable effect on ERG amplitudes.
Twenty-eight animals were randomly and equally allo-
cated to seven groups. Group 1 was administered a sham
injection of 0.1 ml sterile water, whereas groups 2–7 were
administered 0.1 ml of infliximab solution containing 1 mg,
2 mg, 5 mg, 8 mg, 10 mg and 20 mg of the drug
respectively. Infliximab (Remicade®, 100 mg vial, Scher-
ing-Plough, Greece) was reconstituted with 10 ml of sterile
water to form a 10 mg/ml solution; 5 ml, 2 ml, 1.25 ml,
1 ml and 0.5 ml of sterile water were added to different
vials to form solutions of 20 mg/ml, 50 mg/ml, 80 mg/ml,
100 mg/ml and 200 mg/ml respectively. After 20 minutes,
the crystals initially formed had dissolved completely,
Graefes Arch Clin Exp Ophthalmol (2009) 247:273–281
resulting in clear and homogenous solutions. All solutions
were nearly neutral with a pH of 7.40, 7.25, 7.03, 6.92,
6.88 and 6.73 corresponding to 10 mg/ml, 20 mg/ml,
50 mg/ml, 80 mg/ml, 100 mg/ml and 200 mg/ml respec-
tively. Additionally, osmolarity was directly proportional to
the concentration of infliximab in each solution, namely
116 mosm/kg, 306 mosm/kg, 770 mosm/kg, 1324 mosm/
kg, 1610 mosm/kg and 3185 mosm/kg respectively.
Infliximab was administered simultaneously to the four
rabbits of each group. The drug was injected intravitreally
1.5 mm from the limbus with a 30-gauge needle under
general anesthesia with ketamine (50 mg/kg) and xylazine
(5 mg/kg) together with topical anesthesia with alcaine
drops and under sterile conditions. Only the right eye was
injected, while the left eye served as control. Injected eyes
were instilled with topical antibiotics (ofloxacin 0.3%)
immediately after the injection, and were observed thereaf-
ter for signs of infection, inflammation or toxicity.
Slit-lamp biomicroscopy and fundus examinations, as
well as electrophysiology recordings, as described below,
were performed in all eyes at baseline and 1, 5, 10, 15, 30
and 45 days after infliximab or sham injection. Animals
were euthanized on day 45 with intravenous injection of
100 mg/ml sodium pentobarbital under deep general
anesthesia. Enucleated eyes were fixed in 4% formaldehyde
for 48 hours and then left to dry before paraffin embedding
and sectioning. Four-μm thick sections were obtained from
the paraffin blocks and routinely processed for standard
hematoxylin-eosin staining.
Electrophysiology recordings
Full-field light-evoked electroretinography (ERG) was
performed using a computerized Ganzfeld ERG system
TOMEY EP-1000 (Tomey GmbH, Erlangen, Germany). A
2-channel converter box with amplifier was used. All
rabbits were dark adapted for 40 minutes after pupil
dilation before scotopic ERG recording. Unipolar gold-
plated ERG jet electrode lenses were applied on rabbit
corneas. The negative skin Ag-electrodes were applied onto
depilated bare skin near the lateral canthus of each eye
using conductive paste. The ground electrode was clipped
to the earlobe with conductive paste. A notch filter for 50/
100 Hz was applied to all measurements. Impedance was
kept ideally below 6 kOhm or at least below 10 kOhm. For
scotopic ERG responses, a dim white flash 2.5 log units
below the recommended standard white flash intensity of
1.7 cd/m2 was used on dark background to collect at least
three dark-adapted rod responses, according to the stand-
ards of the International Society for Clinical Electrophys-
iology of Vision (ISCEV) [33]. Consequently, white flash
stimulus with the standard intensity of 1.7 cd/m2 was used
to collect at least three maximal responses, the mean of
275
which was used for the respective comparisons. After light
adaptation for 10 minutes with 23.1 cd/m2 background
light, the same standard white flash stimulus (1.7 cd/m2)
was used to record at least three photopic responses.
Subsequently, a flicker white stimulus (30 Hz base
frequency) with an intensity of 1.7 cd/m2 was used on
16.8 cd/m2 background light to record at least ten flicker-
evoked light-adapted responses. Photopic responses to
white flash stimulus of various intensities (1.0, 1.7 and
3.0 cd/m2) were also recorded (a mean of 20 reliable
readings). Implicit time and amplitude of b-wave were
always recorded. Decreases in b-wave amplitude equal to or
greater than 30%, compared with the fellow untreated eye,
were considered subnormal.
Statistical analysis
Statistical analysis was performed using SPSS® v13.0 for
Windows. Non-parametric Mann-Whitney U test and
Kruskal-Wallis tests were used for independent samples.
Wilcoxon test and analysis of variance (one-way ANOVA)
were used for related samples. Statistical significance was
defined as p<0.05.
Results
Slit-lamp and fundus examination
Clinical examination of uninjected eyes (n=28), sham-
injected eyes (n=4), as well as of eyes injected with 1 mg
or 2 mg of infliximab (n=8) was unremarkable from day 1
through day 45, with the exception of two eyes with
subconjunctival hemorrhage observed immediately after the
injection. In total, abnormal ocular findings included
perilimbal hyperemia (n=6, observed between days 1 and
5 after the injection of 8 mg or higher doses), vitreous flare
(n=9, observed on day 5 and persisting up to day 45 after
the injection of 8 mg or higher doses) and lens opacification
(n=1, recorded after 15 days, probably caused by the
injection procedure).
ERG responses
Significant differences in b-wave amplitude were observed
between the seven groups 1 day after intravitreal injection
of infliximab (p<0.001; Kruskal-Wallis test). As shown in
Table 1, eyes injected with 1 mg or 2 mg of infliximab
presented similar b-wave amplitude when compared with
sham-injected eyes, as well as with the fellow uninjected
eye. In contrast, significant dose-dependent reductions in b-
wave amplitude in different ERG responses were observed
as early as 24 hours after the injection of infliximab in
276 Graefes Arch Clin Exp Ophthalmol (2009) 247:273–281

Table 1 ERG b-wave amplitude (μV; mean ± SD) recorded 24 hours after increasing doses of intravitreal infliximab administered to groups of
rabbits (four animals each) compared with a sham-injected group and with uninjected eyes

Sham Infliximab Infliximab Infliximab Infliximab Infliximab Infliximab Uninjected eyes

injection 1 mg 2 mg 5 mg 8 mg 10 mg 20 mg (n=28)

Scotopic 95.83± 100.88± 105.92± 110.54± 89.20± 53.99± 50.06± 113.33±18.08

(log2.5 filter) 8,8 a 9.26 a 9.72 a 5.60 a 10.12 d 10.32 d 10.52 d
Scotopic 187.84± 189.43± 200.25± 186.23± 120.76± 108.16± 82.93± 182.30±37.56
maximal 39.18 a 48.40 a 39.76 a 36.98 b 25.93 d 37.11 d 26.99 d
response e
Photopic 126.11± 132.75± 121.25± 121.13± 79.65± 53.08± 47.06± 118.85±24.93
13.53 a 14.24 a 7.59 a 15.55 a 14.46 c 13.45 d 12.51 d
Flash 1.0 cd/m2 155.28± 115.90± 132.08± 129.85± 79.95± 59.56± 49.48± 132.24±30.97
44.55 a 15.75 a 32.87 a 29.60 a 28.71 c 27.85 d 21.18 d
Flash 1.7 cd/m2 178.74± 174.75± 172.25± 155.52± 89.12± 62.44± 45.44± 166.38±40.20
69.79 a 60.53 a 37.56 a 17.73 b 27.30 c 26.48 d 26.48 d
Flash 3.0 cd/m2 167.69± 202.50± 163.50± 152.04± 86.58± 97.59± 76.59± 179.24±38.55
17.60 a 61.07 a 25.20 a 33.47 a 34.13 d 14.31 d 14.31 d
Flicker (30Hz 16.51± 17.38± 19.05± 14.86± 13.10± 12.33± 10.52± 19.60±2.70
Basefrequency) 0.66 a 0.69 a 1.46 a 1.14 d 1.58 d 1.64 d 1.71 d
a
p>0.05; b p<0.05; c p<0.01; d p<0.001 (Mann-Whitney U test).
e
dark-adapted eyes; mean of three responses; 1.7 cd/m2 white flash on dark background

groups of rabbits that received 5 mg of infliximab and Histological findings

higher doses (Table 1).
There was no evidence of abnormal scotopic, photopic
or flicker responses in animals injected with either 1 mg
(not shown) or 2 mg of infliximab (Fig. 1 and Table 2)
from day 1 through day 45, which confirmed the ocular
safety of these particular doses. In contrast, ERG abnor-
malities were detected at 5 mg and higher doses on day 1
(Table 1 and Fig. 1), and persisted through day 45 (not
shown). High doses of infliximab did not affect the
contralateral eye, since ERG responses of the uninjected
eyes were similar in all seven groups, and remained
unaltered during follow up.
Overall, detectable ERG responses were often subnormal
(defined as a reduction of greater than 30%) from day 1 after
intravitreal injection of 5 mg infliximab or higher doses. As
shown in Table 3, subnormal responses were recorded in all
injected eyes in groups 6 and 7 (10 mg and 20 mg
respectively), but in none in groups 2 and 3 (1 mg and
2 mg respectively). As shown in Fig. 1, ERG responses to
30 Hz flickering stimulus were more sensitive for detecting
retinal toxicity of 5 mg infliximab dose. In general, results
obtained on day 1 were sustained through day 45.
With regard to b-wave implicit time, no significant
changes were noticed in any group between different time
points (p>0.1; one-way ANOVA). Moreover, no significant
differences were found between groups or between injected
and fellow eyes, confirming that implicit time was not
affected by intravitreal administration of infliximab.
Retinal histology was examined by light microscopy
using ×40, ×100, ×200 and ×400 magnification. All
samples were taken from the mid-periphery. Photo-
micrographs of hematoxylin-eosin (H-E) sections were
captured and compared with control retinal images. As
shown in Fig. 2, eyes that had received sham injection or
1 mg or 2 mg of infliximab presented a similar histological
appearance, without any signs of inflammation or dis-
turbed retinal anatomy. Similarly, normal retinal appear-
ance was noted in the fellow uninjected eyes in all groups
(not shown). In contrast, abnormal retinal appearance was
evident in all eyes injected with 5 mg or higher doses of
infliximab (Fig. 2). Such dose-dependent infliximab-
induced abnormalities consisted of excessive diffuse
edema and various anatomical deformities of the ganglion
cells, nerve fibers layer and the inner plexiform layer with
multiple microcysts formation. In some areas, thickening
of the outer nuclear layer was also observed. Only a few
deformities were noticed in the inner nuclear layer.
Although distal retina appeared less affected, at the
specific time-point examined, subnormal ERG responses
suggest that functional damage had indeed occurred.
Additionally, infiltrating plasmatocytes and lymphocytes
were noted in the nerve fiber layer and the ganglion cell
layer as well as in the posterior vitreous of eyes injected
with 5 mg or higher doses of infliximab. Moreover, at
infliximab doses of 8 mg or higher, the choroid appeared
Graefes Arch Clin Exp Ophthalmol (2009) 247:273–281 277

Fig. 1 Representative ERG responses (scotopic, top; photopic, middle; flicker, bottom) recorded 24 hours after intravitreal injection of 2 mg (left
column), 5 mg (middle column), or 8 mg (right column) of infliximab in the right eye

relatively edematous, with vessel dilation and blood
congestion (not shown).
Discussion
Using increasing doses of intravitreal infliximab in an
established experimental model, we aimed to determine
the highest safe dose, as assessed by clinical examination
and ERG performed 1, 5, 10, 15, 30 and 45 days after
injection. Regarding intravitreal injection technique, the
volume of 0.1 ml was well tolerated by the rabbit eye, and
did not require withdrawal of aqueous fluid from the
anterior chamber [34]. Perilimbal hyperemia and vitreous
flare observed after the injection were attributable to
infliximab toxicity, because they were only observed in
eyes injected with high concentrations (≥8 mg). In
contrast, subconjunctival hemorrhage and lens opacifica-
tion (n=2 and n=1 respectively) were possibly caused by
the injection procedure per se.
By comparing clinical and ERG findings after infliximab
administration with baseline findings and with sham-
injected eyes, we found that intravitreal infliximab doses
of 1 mg and 2 mg were not associated either with clinical
abnormalities or with early or late abnormalities in ERG.
The ocular safety of these doses of infliximab was further
established by comparing every injected eye with the fellow
“control” eye. This comparison is also meaningful, because
the difference between simultaneous recordings from the
two eyes in the sham-injected group (inter-eye difference)
278 Graefes Arch Clin Exp Ophthalmol (2009) 247:273–281

Table 2 Unaltered ERG responses (μV; mean ± SD; n=4) from baseline through day 45 after intravitreal injection of 2 mg of infliximab in the
rabbit eye

Baseline Day 1 Day 5 Day 10 Day 15 Day 30 Day 45

Scotopic 119.12±17.07 105.92±9.72 109.86±17.51 121.86±15.33 117.03±8.17 131.99±21.51 128.07±19.44

(log2.5 filter)
Scotopic 195.35±36.20 200.25±39.76 198.03±43.28 180.58±33.94 200.33±46.49 202.68±48.73 190.28±20.76
a
maximal response
Photopic 130.08±15.59 121.25±7.59 137.75±18.64 132.50±10.03 116.10±4.00 142.35±21.57 130.50±15.01
Flash 1.0 cd/m2 139.98±33.07 132.08±32.87 144.70±43.55 138.05±37.76 151.05±42.66 134.23±24.68 139.75±35.01
Flash 1.7 cd/m2 168.82±34.60 172.25±37.56 182.25±37.72 186.75±32.08 154.75±26.55 151.08±41.27 170.83±29.92
Flash 3.0 cd/m2 185.74±35.26 163.50±25.20 190.75±47.09 209.00±42.21 191.58±45.93 174.88±19.90 184.75±27.83
Flicker (30 Hz 19.45±2.37 19.05±1.46 21.05±4.61 18.78±1.58 20.40±3.03 18.85±0.49 18.58±0.98
Basefrequency)
a
dark-adapted eyes; mean of three responses; 1.7 cd/m2 white flash on dark background

did not exceed 11%. Histological evaluation of the retina
confirmed the absence of toxicity 45 days after the injection
of 1 mg or 2 mg of infliximab. It is of note that Manzano et
al. reported recently that intravitreal injection of 0.5 mg of
adalimumab is not associated with retinal toxicity 14 days
after the injection in the same rabbit model [35]. Adalimu-
mab, a humanized anti-TNF monoclonal antibody sharing
the same indications with infliximab, is routinely adminis-
tered subcutaneously at doses of 0.5–1 mg/kg, whereas
infliximab is administered intravenously at doses up to
10 mg/kg. Similarly, intravitreal etanercept, a soluble TNF
receptor inhibiting TNF actions, has also been shown to be
safe up to a dose of 0.1 mg (almost 200 times lower than
usual systemic dosage) in a rabbit model [36].
In doses higher than 5 mg, infliximab suppressed
scotopic and photopic ERG responses to flash stimuli in a
dose-dependent manner, as early as the first post-injection
day. On the other hand, only responses to a 30 Hz flickering
stimulus (but not scotopic and photopic responses) were
significantly reduced at the 5 mg dose, indicating a higher
sensitivity of flicker ERG for detecting retinal toxicity of
infliximab at this particular dose. The toxic effect of doses
equal to or higher than 5 mg was subsequently confirmed
with retinal histology. A possible explanation could be that,
although both a and b waves in the ERG originate mainly in
the outer retinal layers, some components of flicker ERG
originate also in the proximal inner retina [37], which was
indeed affected by the 5 mg dose of infliximab (Fig. 2,
bottom left). Additional studies utilizing visual evoked
potentials (VEP) could further address this matter, because
VEP could verify damage to the inner retina, with particular
attention to the ganglion cells and the nerve fibre layer.
All eyes with retinal toxicity presented subnormal ERG
responses as early as 1 day after infliximab injection,
without subsequent significant improvement. This is
compatible with an acute infliximab-induced effect on
retinal function. No self-repair reaction during the 45 days
of follow up was evident, indicating a permanent retinal
damage induced by high doses of infliximab. Moreover, in
immunohistochemical experiments with an anti-idiotype
antibody recognizing infliximab (Centocor Inc., Horsham,
PA, USA), the successful distribution of infliximab, at

Table 3 Subnormal ERG responses, defined as at least 30% reduction in b-wave amplitude compared with fellow uninjected eyes, recorded 24
hours after escalating doses of intravitreal infliximab administered to groups of rabbits (four animals each)

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7

(sham) (1 mg) (2 mg) (5 mg) (8 mg) (10 mg) (20 mg)

Scotopic (log2.5 filter) 0/4 0/4 0/4 0/4 2/4 4/4 4/4
Scotopic maximal 0/4 0/4 0/4 0/4 2/4 4/4 4/4
response a
Photopic 0/4 0/4 0/4 0/4 1/4 4/4 4/4
Flash 1.0 cd/m2 0/4 0/4 0/4 0/4 2/4 4/4 4/4
Flash 1.7 cd/m2 0/4 0/4 0/4 0/4 3/4 4/4 4/4
Flash 3.0 cd/m2 0/4 0/4 0/4 0/4 3/4 4/4 4/4
Flicker (30 Hz 0/4 0/4 0/4 2/4 4/4 4/4 4/4
Basefrequency)
a
dark-adapted eyes; mean of three responses; 1.7 cd/m2 white flash on dark background
Graefes Arch Clin Exp Ophthalmol (2009) 247:273–281
Fig. 2 Normal appearance of retinal anatomy (samples obtained from
the mid-periphery) in sham-injected eyes and eyes injected with 1 mg
or 2 mg of infliximab (a, b and c respectively). Dose-dependent
infliximab-induced abnormalities after injection of 5 mg, 10 mg or
20 mg (d, e and f respectively) consisting of excessive diffuse edema
least in the proximal retinal layers, was observed 5 days
after intravitreal injection (Liarakos VS et al., unpub-
lished). These findings are compatible with the retinal
penetration pattern of bevacizumab [38], a drug which has
a similar molecular weight to infliximab (149 and
144.1 kDaltons respectively).
Our study’s main result, that 1 mg and 2 mg doses of
intravitreally injected infliximab are safe for the rabbit eye,
confirms recent observations by Giansanti et al. [39]. In
their pilot study on the same animal model, the ocular
toxicity of infliximab at 1 mg, 1.7 mg and 3.3 mg was
assessed. The 1.7 mg dose was found to be safe, whereas
the 3.3 mg dose-induced retinal damage was detected on
histology, which was similar to our findings. Giansanti et
al. found discordant functional and structural effects of
intravitreal infliximab at 3.3 mg, i.e. normal scotopic ERG
responses with disturbed retinal anatomy. Such a discrep-
ancy was not found in our study because, in addition to
flash stimuli (which gave normal responses at a dose of
5 mg), we used 30 Hz flicker ERG responses, which were
indeed affected.
Taken together, these results indicate that a dose of
infliximab between 1 mg and 2 mg reconstituted in 0.1 ml
solution is safe for the rabbit eye. Given that the volume of
the rabbit vitreous cavity is smaller compared with the
human, a 2 mg dose could be considered safe for the human
eye. However, differences in vitreous and retinal anatomy
279
and various anatomic deformities of the ganglion cells, the nerve fiber
layer and the inner plexiform layer; infiltrating plasmatocytes and
lymphocytes are seen in the proximal retina and the posterior vitreous
(hematoxylin-eosin staining; x200)
may lead to different patterns of pharmacokinetics and
subsequent clinical effects, especially in a state of ocular
inflammation. Whether an intravitreal dose of 1 mg or 2 mg
of infliximab exerts a pharmacologic effect was not
determined in this study. Notably, the therapeutic dose of
intravitreal bevacizumab (1.25 mg) is almost 500-fold
lower than the average intravenous dose; whereas the safe
dose of 2 mg of infliximab in the rabbit is only 200-fold
lower than the average intravenous dose in humans,
implying that an intravitreal injection of 1–2 mg may be
effective. Our preliminary results of intravitreal infliximab
injection in three human patients with age-related macular
degeneration suggest that these doses indeed interfere with
TNF intraocular actions (Theodossiadis P et al., manuscript
submitted for publication).
To conclude, given that intravitreal injections assure
delivery of a drug at the desired site, intravitreal injection
of infliximab at a dose of 2 mg or lower may be used
for the design of future clinical trials. These trials may
include selected patients with immune-mediated ocular
diseases, in whom systemic administration of anti-TNF
agents is beneficial, as suggested by preliminary results
[1]. Patients likely to benefit are mainly those with
refractory non-infectious uveitis, as well as those with
cystoid macular edema, including patients with diabetes
mellitus, and patients with neovascular age-related macular
degeneration.
280
References
1. Theodossiadis PG, Markomichelakis NN, Sfikakis PP (2007)
Tumor necrosis factor antagonists: preliminary evidence for an
emerging approach in the treatment of ocular inflammation.
Retina 27:399–413, doi:10.1097/MAJ.0b013e3180318fbc
2. Majka S, McGuire PG, Das A (2002) Regulation of matrix
metalloproteinase expression by tumor necrosis factor in a murine
model of retinal neovascularization. Invest Ophthalmol Vis Sci
43:260–266
3. Spranger J, Meyer-Schwickerath R, Klein M, Schatz H, Pfeiffer A
(1995) TNF-alpha level in the vitreous body. Increase in neo-
vascular eye diseases and proliferative diabetic retinopathy. Med
Klin (Munich) 90:134–137
4. Ueda T, Ueda T, Fukuda S, Browne R, Jenis E, Spengler R,
Chou R, Buch P, Aljada A, Dandona P, Sasisekharan R,
Dorey CK, Armstrong D (1998) Lipid hydroperoxide-induced
tumor necrosis factor (TNF)-alpha, vascular endothelial
growth factor and neovascularization in the rabbit cornea:
effect of TNF inhibition. Angiogenesis 1:174–184, doi:10.
1023/A:1018377621102
5. Armstrong D, Augustin AJ, Spengler R, Al-Jada A, Nickola T,
Grus F, Koch F (1998) Detection of vascular endothelial growth
factor and tumor necrosis factor alpha in epiretinal membranes of
proliferative diabetic retinopathy, proliferative vitreoretinopathy
and macular pucker. Ophthalmologica 212:410–414, doi:10.1159/
000027378
6. Oh H, Takagi H, Takagi C, Suzuma K, Otani A, Ishida K,
Matsumura M, Ogura Y, Honda Y (1999) The potential angiogenic
role of macrophages in the formation of choroidal neovascular
membranes. Invest Ophthalmol Vis Sci 40:1891–1898
7. Yang P, McKay BS, Allen JB, Jaffe GJ (2004) Effect of NF-
kappa B inhibition on TNF-alpha-induced apoptosis in human
RPE cells. Invest Ophthalmol Vis Sci 45:2438–2446, doi:10.
1167/iovs.03–0805
8. Yang P, Wiser JL, Peairs JJ, Ebright JN, Zavodni ZJ, Bowes
Rickman C, Jaffe GJ (2005) Human RPE expression of cell
survival factors. Invest Ophthalmol Vis Sci 46:1755–1764,
doi:10.1167/iovs.04–1039
9. Santos Lacomba M, Marcos Martin C, Gallardo Galera JM,
Gomez Vidal MA, Collantes Estevez E, Ramirez Chamond R,
Omar M (2001) Aqueous humor and serum tumor necrosis factor-
alpha in clinical uveitis. Ophthalmic Res 33:251–255, doi:10.
1159/000055677
10. Murray PI, Sivaraj RR (2005) Anti-TNF-alpha therapy for uveitis:
Behcet and beyond. Eye 19:831–833 doi:10.1038/sj.eye.6701792
11. Nussenblatt R (2005) Treating intraocular inflammatory disease in
the 21st century. Arch Ophthalmol 123:1000–1001, doi:10.1001/
archopht.123.7.1000
12. El-Shabrawi Y, Hermann J (2002) Anti-tumor necrosis factor-
alpha therapy with infliximab as an alternative to corticosteroids
in the treatment of human leukocyte antigen B27-associated acute
anterior uveitis. Ophthalmology 109:2342–2346, doi:10.1016/
S0161–6420(02)01292–7
13. Fries W, Giofre MR, Catanoso M, Lo Gullo R (2002) Treatment
of acute uveitis associated with Crohn’s disease and sacroileitis
with infliximab. Am J Gastroenterol 97:499–500, doi:10.1111/
j.1572–0241.2002.05514.x
14. Kahn P, Weiss M, Imundo LF, Levy DM (2006) Favorable
response to high-dose infliximab for refractory childhood uveitis.
Ophthalmology 113:860–864, e862
15. Sfikakis PP, Theodossiadis PG, Katsiari CG, Kaklamanis P,
Markomichelakis NN (2001) Effect of infliximab on sight-
threatening panuveitis in Behcet’s disease. Lancet 358:295–296,
doi:10.1016/S0140–6736(01)05497–6
Graefes Arch Clin Exp Ophthalmol (2009) 247:273–281
16. Ohno S, Nakamura S, Hori S, Shimakawa M, Kawashima H,
Mochizuki M, Sugita S, Ueno S, Yoshizaki K, Inaba G (2004)
Efficacy, safety, and pharmacokinetics of multiple administration
of infliximab in Behcet’s disease with refractory uveoretinitis. J
Rheumatol 31:1362–1368
17. Sfikakis PP, Kaklamanis PH, Elezoglou A, Katsilambros N,
Theodossiadis PG, Papaefthimiou S, Markomichelakis N (2004)
Infliximab for recurrent, sight-threatening ocular inflammation in
Adamantiades-Behcet disease. Ann Intern Med 140:404–406
18. Tugal-Tutkun I, Mudun A, Urgancioglu M, Kamali S, Kasapoglu E,
Inanc M, Gul A (2005) Efficacy of infliximab in the treatment of
uveitis that is resistant to treatment with the combination of
azathioprine, cyclosporine, and corticosteroids in Behcet’s disease:
an open-label trial. Arthritis Rheum 52:2478–2484, doi:10.1002/
art.21231
19. Baughman RP, Bradley DA, Lower EE (2005) Infliximab in
chronic ocular inflammation. Int J Clin Pharmacol Ther 43:7–11
20. Roberts SD, Wilkes DS, Burgett RA, Knox KS (2003) Refractory
sarcoidosis responding to infliximab. Chest 124:2028–2031,
doi:10.1378/chest.124.5.2028
21. Katz JM, Bruno MK, Winterkorn JM, Nealon N (2003) The
pathogenesis and treatment of optic disc swelling in neuro-
sarcoidosis: a unique therapeutic response to infliximab. Arch
Neurol 60:426–430, doi:10.1001/archneur.60.3.426
22. Markomichelakis NN, Theodossiadis PG, Pantelia E, Papaefthimiou
S, Theodossiadis GP, Sfikakis PP (2004) Infliximab for chronic
cystoid macular edema associated with uveitis. Am J Ophthalmol
138:648–650, doi:10.1016/j.ajo.2004.04.066
23. Sfikakis PP, Markomichelakis N, Theodossiadis GP, Grigoropoulos V,
Katsilambros N, Theodossiadis PG (2005) Regression of sight-
threatening macular edema in type 2 diabetes following
treatment with the anti-tumor necrosis factor monoclonal
antibody infliximab. Diabetes Care 28:445–447, doi:10.2337/
diacare.28.2.445
24. Olson JL, Courtney RJ, Mandava N (2007) Intravitreal infliximab
and choroidal neovascularization in an animal model. Arch
Ophthalmol 125:1221–1224, doi:10.1001/archopht.125.9.1221
25. Shi X, Semkova I, Muther PS, Dell S, Kociok N, Joussen AM
(2006) Inhibition of TNF-alpha reduces laser-induced choroidal
neovascularization. Exp Eye Res 83:1325–1334, doi:10.1016/j.
exer.2006.07.007
26. Markomichelakis NN, Theodossiadis PG, Sfikakis PP (2005)
Regression of neovascular age-related macular degeneration
following infliximab therapy. Am J Ophthalmol 139:537–540,
doi:10.1016/j.ajo.2004.09.058
27. Sfikakis PP, Iliopoulos A, Elezoglou A, Kittas C, Stratigos A
(2005) Psoriasis induced by anti-tumor necrosis factor therapy: a
paradoxical adverse reaction. Arthritis Rheum 52:2513–2518,
doi:10.1002/art.21233
28. Suhler EB, Smith JR, Wertheim MS, Lauer AK, Kurz DE, Pickard TD,
Rosenbaum JT (2005) A prospective trial of infliximab therapy for
refractory uveitis: preliminary safety and efficacy outcomes. Arch
Ophthalmol 123:903–912, doi:10.1001/archopht.123.7.903
29. Wegscheider BJ, El-Shabrawi L, Weger M, Ardjomand N,
Hermann J, Aberer E, El-Shabrawi Y (2007) Adverse skin
reactions to infliximab in the treatment of intraocular inflamma-
tion. Eye 21:547–549
30. Keystone EC (2005) Safety of biologic therapies—an update. J
Rheumatol Suppl 74:8–12
31. Jager RD, Aiello LP, Patel SC, Cunningham ET Jr (2004) Risks of
intravitreous injection: a comprehensive review. Retina 24:676–
698, doi:10.1097/00006982–200410000–00002
32. van Norren D, Padmos P (1975) Cone dark adaptation: the
influence of halothane anesthesia. Invest Ophthalmol 14:212–227
33. Marmor MF, Holder GE, Seeliger MW, Yamamoto S (2004)
Standard for clinical electroretinography (2004 update). Doc
Graefes Arch Clin Exp Ophthalmol (2009) 247:273–281
Ophthalmol 108:107–114, doi:10.1023/B:DOOP.0000036793.
44912.45
34. Kivilcim M, Peyman GA, Kazi AA, Dellacroce J, Ghobrial RN,
Monzano R (2007) Intravitreal toxicity of high-dose etanercept.
J Ocul Pharmacol Ther 23:57–62, doi:10.1089/jop.2006.0083
35. Manzano RP, Peyman GA, Carvounis PE, Kivilcim M, Khan P,
Chevez-Barrios P, Takahashi W (2008) Ocular toxicity of
intravitreous adalimumab (Humira) in the rabbit. Graefes Arch
Clin Exp Ophthalmol 246:907–911, doi:10.1007/s00417–008–
0765-z
36. Fauser S, Kalbacher H, Alteheld N, Koizumi K, Krohne TU,
Joussen AM (2004) Pharmacokinetics and safety of intravitreally
delivered etanercept. Graefes Arch Clin Exp Ophthalmol
242:582–586, doi:10.1007/s00417–004–0895-x
281
37. Porciatti V, Moretti G, Ciavarella P, Falsini B (1993) The second
harmonic of the electroretinogram to sinusoidal flicker: spatio-
temporal properties and clinical application. Doc Ophthalmol
84:39–46, doi:10.1007/BF01203281
38. Shahar J, Avery RL, Heilweil G, Barak A, Zemel E, Lewis GP,
Johnson PT, Fisher SK, Perlman I, Loewenstein A (2006)
Electrophysiologic and retinal penetration studies following intra-
vitreal injection of bevacizumab (Avastin). Retina 26:262–269,
doi:10.1097/00006982–200603000–00002
39. Giansanti F, Ramazzotti M, Vannozzi L, Rapizzi E, Fiore T,
Iaccheri B, Degl’ Innocenti D, Moncini D, Menchini U (2008) A
pilot study on ocular safety of intravitreal infliximab in a rabbit
model. Invest Ophthalmol Vis Sci 49:1151–1156, doi:10.1167/
iovs.07–0932