July 2010
Volume 51, Issue 7
Free
Retina  |   July 2010
Long-Term Tolerability and Serum Concentration of Bevacizumab (Avastin) when Injected in Newborn Rabbit Eyes
Author Affiliations & Notes
  • Wei-Chi Wu
    From the Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan;
    the College of Medicine, Chang Gung University, Taoyuan, Taiwan; and
  • Chi-Chun Lai
    From the Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan;
    the College of Medicine, Chang Gung University, Taoyuan, Taiwan; and
  • Kuan-Jen Chen
    From the Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan;
    the College of Medicine, Chang Gung University, Taoyuan, Taiwan; and
  • Tun-Lu Chen
    From the Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan;
    the College of Medicine, Chang Gung University, Taoyuan, Taiwan; and
  • Nan-Kai Wang
    From the Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan;
    the College of Medicine, Chang Gung University, Taoyuan, Taiwan; and
  • Yih-Shiou Hwang
    From the Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan;
    the College of Medicine, Chang Gung University, Taoyuan, Taiwan; and
  • Ling Yeung
    the College of Medicine, Chang Gung University, Taoyuan, Taiwan; and
    the Department of Ophthalmology, Chang Gung Memorial Hospital, Keelung, Taiwan.
  • Lien-Min Li
    From the Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan;
  • Corresponding author: Wei-Chi Wu, Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan; weichi666@gmail.com
Investigative Ophthalmology & Visual Science July 2010, Vol.51, 3701-3708. doi:10.1167/iovs.09-4425
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      Wei-Chi Wu, Chi-Chun Lai, Kuan-Jen Chen, Tun-Lu Chen, Nan-Kai Wang, Yih-Shiou Hwang, Ling Yeung, Lien-Min Li; Long-Term Tolerability and Serum Concentration of Bevacizumab (Avastin) when Injected in Newborn Rabbit Eyes. Invest. Ophthalmol. Vis. Sci. 2010;51(7):3701-3708. doi: 10.1167/iovs.09-4425.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: To test the long-term effects and systemic exposure level after single or multiple bevacizumab (Avastin) intravitreal injections in newborn rabbit eyes.

Methods.: Four groups of newborn New Zealand rabbits received a single intravitreal bevacizumab injection at a concentration of 1.25 mg/0.05 mL at the ages of 2 (group 1), 4 (group 2), 6 (group 3), and 12 (group 5) weeks. The other group of rabbits (group 4) received three consecutive injections of bevacizumab at a concentration of 1.25 mg/0.05 mL at weeks 2, 6, and 10. Eight days after injection, the serum concentration of bevacizumab was determined in groups 1, 2, 3, and 5. Morphologic and functional changes were evaluated 12 months after bevacizumab injection.

Results.: Twelve months after either single or multiple intravitreal injections of bevacizumab, newborn rabbit eyes showed no significant differences compared with control eyes on examination with funduscopy, histopathology, or electroretinogram. The serum concentrations when the injections were performed at the ages of 2 (19.4 ± 8.1 μg/mL) and 4 (10.2 ± 2.3 μg/mL) weeks were significantly higher than the serum level detected when the injection was performed at 12 weeks of age (2.8 ± 1.2 μg/mL, P = 0.02 and P = 0.024, respectively).

Conclusions.: After 1 year, single and three consecutive intravitreal injections of 1.25 mg bevacizumab in newborn rabbit eyes are well tolerated. Systemic exposure is higher when the injection is performed at an early age.

Retinopathy of prematurity (ROP) is one of the leading causes of childhood blindness. There are two phases in ROP. 1 In phase I, cessation of vessel growth and loss of vessels are noted at the time of premature birth. Oxygen-regulated growth factors are suppressed by higher than normal levels of oxygen and loss of factors normally provided by the mother in utero. As the retina matures after birth, it becomes more metabolically active, and the avascular retina becomes hypoxic, leading to phase II of ROP. The hypoxia of phase II induces a rapid expression of vascular endothelial growth factor (VEGF), leading to neovascularization of the retina. 1,2 Neovascularization leads to retinal traction, retinal detachment, and retinal funnel configuration, eventually affecting vision. Bevacizumab (Avastin; Genentech Inc., South San Francisco, CA) is a humanized anti-VEGF monoclonal antibody, 3 and it is the first antiangiogenic agent for the treatment of metastatic colorectal cancer. 3 Bevacizumab has two binding sites, binds directly to VEGF, and has been shown to inhibit more than one of the nine VEGF isoforms. It has shown promising results in treating many retinopathies associated with VEGF elevation, including age-related macular degeneration (ARMD), 46 diabetic retinopathy, 79 vitreous hemorrhage, 1012 neovascular glaucoma, 13 and retinal vascular occlusion. 1417 These encouraging results show the potential of bevacizumab in the treatment of ROP. 
So far, there have been only a few case reports or small case series of bevacizumab used in ROP. 1826 Mintz-Hittner and Kuffel 22 showed that a single injection of bevacizumab prevented progression to retinal detachment in 22 eyes of 11 patients with stage 3 ROP in zone I or posterior zone II, without any noticeable ocular or systemic complications. However, long-term safety data for bevacizumab used in newborn patients' eyes are not yet available. Results of studies have suggested that VEGF is an important growth factor during the development of the retina. 2,27,28 The visual system in newborn infants is different from that in adults, in that it is still rapidly developing. Whether anti-VEGF antibody usage affects the development of the visual system, especially the retina, is unclear. In addition, whether there is systemic exposure after the injection of bevacizumab in newborns remains unknown. To answer these questions, this study was designed to evaluate the morphologic and functional changes in the eyes 12 months after either a single or three consecutive bevacizumab injections in newborn rabbit eyes. Serum levels of bevacizumab were also checked to test for systemic exposure when the injections were performed in these newborn animals. 
Materials and Methods
Animals and Grouping
Newborn New Zealand rabbits were used in the study. The rabbits were housed in a 12/12 hour light–dark cycle. This study was approved by the animal committee at Chang Gung Memorial Hospital, Taoyuan, Taiwan. All the experimental procedures adhered to the ARVO Statement for the Treatment of Animals in Ophthalmic and Vision Research and to institutional guidelines. Before intravitreal injection and electrophysiological recordings, the rabbits were anesthetized with intramuscular injections of 1.5 mL/kg of an equal volume mixture of 2-(2.6-xylidino)-5.6-dihydro-4H-1.3-thiazine-hydrochloride, methylparaben (Rompun; Bayer AG, Leverkusen, Germany) and 50 mg/mL ketamine (Ketomin; Nang Kuang Pharmaceutical Co., Tainan, Taiwan). Topical anesthesia (Alcaine; Alcon-Couvreur, Puurs, Belgium) was administered to reduce the animals' discomfort. The pupils were fully dilated with 1% cyclopentolate hydrochloride. The rabbits underwent clinical inspection by slit lamp, indirect ophthalmoscopy, and electroretinograms (ERGs). After all the experiments were finished, the rabbits were killed by an intravenous injection of an overdose of pentobarbital sodium (80 mg/kg body weight). 
Four groups of newborn rabbits received single intravitreal bevacizumab injection at a concentration of 1.25 mg/0.05 mL at the ages of 2 (group 1), 4 (group 2), 6 (group 3), and 12 (group 5) weeks. The other group of rabbits (group 4) received three consecutive injections of bevacizumab at the same concentration at weeks 2, 6, and 10. In clinical use of bevacizumab for the treatment of ROP, the doses range from 1.25 to 0.5 mg. 1826 Because the main purpose of this study was to explore the safety profile of bevacizumab in newborn eyes, we used the higher dose (1.25 mg) to test the tolerance of the eye. Two weeks after birth was the earliest time we could perform the experiment, as young rabbits open their eyes at around 10 days of age. The time points of 4, 6, 10, and 12 weeks were chosen to mimic the time at which ROP starts to develop after birth and treatment is needed. The grouping of animals, number of animal used, and examinations performed are shown in Table 1. In all, 27 newborn albino rabbits were included in the study. 
Table 1.
 
Treatment Groups
Table 1.
 
Treatment Groups
Group IVI Mode Injection Time Evaluation
1 (single injection at 2 weeks) Single injection 2 wk after birth Clinical examinations, histology, IHC, ERG, ELISA
2 (single injection at 4 weeks) Single injection 4 wk after birth Clinical examinations, histology, IHC, ERG, ELISA
3 (single injection at 6 weeks) Single injection 6 wk after birth Clinical examinations, histology, IHC, ERG, ELISA
4 (three injections at 2, 6, and 10 weeks) Three consecutive injections 2, 6, 10 wk after birth Clinical examinations, histology, IHC, ERG
5 (single injection at 12 weeks) Single injection 12 wk after birth ELISA
Surgical Technique for Intravitreal Bevacizumab Injection
The right eye of each rabbit was injected intravitreally, and the left eye of each rabbit was left untreated and served as a control according to a published technique. 29 After povidone iodine (5%) was placed on the conjunctiva, a 30-gauge needle attached to a 1.0-mL tuberculin syringe was inserted into the vitreous approximately 1 mm posterior to the limbus. The syringe was directed under visual control with a surgical microscope (M691; Wild Heerbrugg, Heerbrugg, Switzerland) toward the center of the vitreous above the optic disc. A volume of 0.05 mL was then slowly injected. 
Clinical Observations
One year after the intravitreal injection, the rabbits underwent clinical examination for the detection of any abnormalities in the cornea, anterior chamber, lens, vitreous, or retina. Cells and flares in the anterior chamber were checked by a portable slit lamp (SL-15; Kowa, Tokyo, Japan). Vitreous opacity and the presence of any retinal abnormalities were determined via indirect ophthalmoscopy (Omega 500; Heine, Herrsching, Germany). External photos and color fundus photos were then taken to document the findings. 
Electroretinograms
At baseline and 1 year after the intravitreal bevacizumab injection, flash ERGs were recorded to assess the retinal function of bevacizumab-injected and control eyes. After 1 hour of dark adaptation, ERGs were recorded from both eyes separately with an ERG recording system (RETIport ERG; Roland Consult, Brandenburg, Germany). ERGs were recorded with a contact lens electrode carrying light-emitting diodes as a stimulator and referenced to an electrode on the forehead. The ground electrode was attached to the ear. The luminance of the stimulus was 3 cd/m2, with a duration of 10 ms. Scotopic 0-dB ERGs were recorded with a standard white flash and a dark background. Twenty responses elicited by identical flashes applied at 10-second intervals were averaged in the dark-adapted state. The amplitudes and the implicit times of the a- and b-waves were measured and averaged. To minimize the effect of individual and daily variation on the ERG, the ratio of the study eye b-wave amplitude to the control eye b-wave amplitude was calculated. The ratio was also calculated for the a-wave amplitude. 
Immunohistochemistry
Immunohistochemistry was used to visualize cells in different retinal layers 1 year after bevacizumab injection. The integrity of intermediate filament proteins of Müller cells, neurofilaments in ganglion and horizontal cells, synaptic vesicles in plexiform layers, and photoreceptors were checked. After the cornea, lens, and vitreous were removed, the eye cup was cut into two halves along the optic nerve and medullary ray. The retinas were fixed in 4% paraformaldehyde overnight. Then, they were incubated in 30% sucrose (USB Corp., Cleveland, OH) overnight at 4°C, embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA), and sectioned with a microtome cryostat (CM3050S; Leica, Wetzlar, Germany). The sections were placed on slides that had been coated with silane (Muto Pure Chemicals, Tokyo, Japan) to promote adhesion of the sections to the glass surface. Samples were blocked with 1% bovine serum albumin (in PBS) for 60 minutes after washing in PBS. After removal of the blocking serum, the following primary antibodies were added: anti-glial fibrillary acidic protein (GFAP; 1:50; Santa Cruz Biotechnology, Santa Cruz, CA); anti-vimentin (ready to use; Dako, Glostrup, Denmark); anti-synaptophysin (1:20; Dako); anti-neurofilament (ready to use; Dako); anti-red/green opsin (1:100; Santa Cruz Biotechnology); anti-rhodopsin (1:50; Santa Cruz Biotechnology); and anti-blue opsin (1:100; Santa Cruz Biotechnology). For the selection and specificity of the antibodies used, see Table 2
Table 2.
 
Selection and Specificity of Antibodies Used
Table 2.
 
Selection and Specificity of Antibodies Used
Antibody Specificity in the Retina
Anti-GFAP; anti-vimentin Intermediate filament proteins of Müller cells
Anti-neurofilament Neurofilaments in ganglion cells and in horizontal cells
Anti-synaptophysin Synaptic vesicles in plexiform layers
Anti-red/green opsin; anti-blue opsin Cones
Anti-rhodopsin Rods
Anti-goat or mouse IgG-fluorescein isothiocyanate (FITC) was used as a secondary antibody, depending on the origin of the primary antibody. The resulting sections were then viewed on a fluorescence microscope (BX50; Olympus, Tokyo, Japan). 
Morphology and Electron Microscopy Study
After the cornea, lens, and vitreous were removed, the eye cup was cut into halves transiting the optic nerve head. The retinas were fixed in 2.5% glutaraldehyde in sodium phosphate buffer for 2 hours. The tissue was then fixed in phosphate-buffered osmium tetroxide (1%) for 1 hour and embedded in Spurr's resin. The central and peripheral retina were sectioned at 0.5 μm, counterstained with toluidine blue, examined by light microscopy (Eclipse E800; Nikon, Osaka, Japan), sectioned at 90 nm, and examined by electron microscopy (JEM-2000 EX; JEOL, Tokyo, Japan). The observers were masked when they interpreted the morphology data. 
Enzyme-Linked Immunosorbent Assay
A bevacizumab pharmacokinetic study has shown that the serum level of bevacizumab reaches its highest point 8 days after bevacizumab injection into the vitreous cavity. 30 Therefore, 8 days after intravitreal injection, an ELISA was used to measure the amount of systemic bevacizumab in these newborn rabbits. The measurement was performed in groups 1, 2, and 3 (n = 6 each) as well as in group 5 (n = 3) that received intravitreal injections at the age of 12 weeks. A venous blood sample 0.5 to 1 mL was taken from each rabbit. Serum was obtained after centrifugation at 3000 rpm and frozen at −80°C until tested. A 96-well ELISA plate was coated with VEGF (0.1 μg/mL in PBS buffer; R&D, Minneapolis, MN) at 4°C overnight. After the plate was washed with PBST (1× PBS buffer with 0.5% Tween-20) three times, 300 μL of antibody diluent (bovine serum albumin; Sigma-Aldrich, St. Louis, MO) was added for 1 hour and used for blocking. Diluted serum samples (100 μL) were then added and incubated for 1 hour. After the plate was washed with PBST, goat anti-human IgG (Abcam, Cambridge, UK) conjugated with biotin was added for 1 hour. After another wash of the plate with PBST, streptavidin-horseradish peroxidase (Streptavidin-HRP; R&D) and the HRP:tetramethylbenzidine substrate (Clinical Science Products, Inc., Mansfield, MA) was added for color developing. The reaction was stopped by adding 2 N H2SO4 (J. T. Baker, Phillipsburg, NJ). The results were read by a microplate reader with a wavelength of 450 nm. The background was read at the wavelength of 570 nm. For each assay, a standard curve was graphed based on known concentrations of bevacizumab (range, 4–0.0625 ng/mL). 
Statistical Analysis
The Wilcoxon signed-ranks test was used, and a two-sided probability was computed to detect statistically significant differences in the ERG results between bevacizumab-injected and noninjected eyes, as well as for the baseline data and the data obtained 1 year after bevacizumab injection. The Mann-Whitney test was used, and a two-sided probability was computed to detect statistically significant differences in the peak serum concentrations after intravitreal bevacizumab injection at different time points (SPSS version 13.0; SPSS Inc., Chicago, IL). Data are expressed as the mean ± SD, with P < 0.05 considered to be significant. 
Results
Clinical Observation
One year after the intravitreal injection, no cell or flare was noted in any group of eyes (Fig. 1A–1E). The lens remained clear up to 1 year after either a single or three consecutive intravitreal bevacizumab injections. Dilation of the fundus revealed no signs of vitreous opacity, retinal detachment, vessel occlusion, or retinal necrosis (Figs. 1F–1J). 
Figure 1.
 
External photos and color fundus photos of rabbit eyes 1 year after intravitreal injection of 1.25 mg bevacizumab. External photos (A–E) reveal a clear cornea and crystalline lens. No cell or flare was noted in the anterior chamber. Color fundus photos (F–J) show no signs of vitreous opacity, retinal detachment, vessel occlusion, or retinal necrosis. Group 1, single injection at 2 weeks; group 2, single injection at 4 weeks; group 3, single injection at 6 weeks; and group 4, three consecutive injections at 2, 6, and 10 weeks.
Figure 1.
 
External photos and color fundus photos of rabbit eyes 1 year after intravitreal injection of 1.25 mg bevacizumab. External photos (A–E) reveal a clear cornea and crystalline lens. No cell or flare was noted in the anterior chamber. Color fundus photos (F–J) show no signs of vitreous opacity, retinal detachment, vessel occlusion, or retinal necrosis. Group 1, single injection at 2 weeks; group 2, single injection at 4 weeks; group 3, single injection at 6 weeks; and group 4, three consecutive injections at 2, 6, and 10 weeks.
Retinal Histology and Electron Microscope Results
One year after bevacizumab injection into the vitreous, retinal morphology remained intact and showed no differences compared with that of the control eyes. No sign of retinal degeneration, thinness, dissolution, or loss in the layers of either the central or peripheral retina was detected (Fig. 2). Ultrastructural morphology did not show any difference in the retinal layers of the ultrathin sections between bevacizumab-injected eyes and control eyes, even in eyes with multiple bevacizumab injections (Fig. 3). In the inner segments of the photoreceptor layers, swelling, and disruption of mitochondria with a loss of cristae were noted in some areas (Fig. 4). Localized, minor mitochondrial changes were found in all the samples studied, both study and control eyes. However, in 12 samples treated with bevacizumab, 3 had more extensive morphologic changes in mitochondria. None of the 12 samples from the control eyes had extensive morphologic changes in mitochondria. 
Figure 2.
 
Histology of the retina in rabbit eyes 1 year after bevacizumab injection. No sign of retinal degeneration, thinness, dissolution, or loss in the layers of either the central or peripheral retina was detected. No noticeable differences were observed between any treatment group and control eyes. Groups are as stated in Figure 1. RGC, retinal ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments of the photoreceptor layers; OS, outer segments of the photoreceptor layers; RPE, retinal pigment epithelium. Magnification, ×400.
Figure 2.
 
Histology of the retina in rabbit eyes 1 year after bevacizumab injection. No sign of retinal degeneration, thinness, dissolution, or loss in the layers of either the central or peripheral retina was detected. No noticeable differences were observed between any treatment group and control eyes. Groups are as stated in Figure 1. RGC, retinal ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments of the photoreceptor layers; OS, outer segments of the photoreceptor layers; RPE, retinal pigment epithelium. Magnification, ×400.
Figure 3.
 
Electron microscopy results in rabbit eyes 1 year after three consecutive intravitreal injections of 1.25 mg/0.05 mL bevacizumab (group 4). Normal morphology was noted in the inner nuclear layer (A), the outer segment in the photoreceptor layer (B), and the junction of the retinal pigment epithelium and outer segment in the photoreceptor layer (C) after multiple bevacizumab injections. Magnification: ×5000.
Figure 3.
 
Electron microscopy results in rabbit eyes 1 year after three consecutive intravitreal injections of 1.25 mg/0.05 mL bevacizumab (group 4). Normal morphology was noted in the inner nuclear layer (A), the outer segment in the photoreceptor layer (B), and the junction of the retinal pigment epithelium and outer segment in the photoreceptor layer (C) after multiple bevacizumab injections. Magnification: ×5000.
Figure 4.
 
Changes in the inner segments of the photoreceptor layers in rabbit eyes 1 year after intravitreal injection of 1.25 mg/0.05 mL bevacizumab, as revealed by electron microscopy. Swelling and disruption of mitochondria (arrows) with a loss of cristae were noted in some areas in the bevacizumab-injected (A) and control (B) eyes. Magnification: ×20,000.
Figure 4.
 
Changes in the inner segments of the photoreceptor layers in rabbit eyes 1 year after intravitreal injection of 1.25 mg/0.05 mL bevacizumab, as revealed by electron microscopy. Swelling and disruption of mitochondria (arrows) with a loss of cristae were noted in some areas in the bevacizumab-injected (A) and control (B) eyes. Magnification: ×20,000.
Immunohistochemical Analysis of Specific Retinal Cells
To probe whether bevacizumab injection causes long-term toxicity in the retina, we used specific antibodies to identify different cell components in the retina 1 year after intravitreal injection. These retinal cell layers included intermediate filament proteins of Müller cells; neurofilaments in ganglion cells and horizontal cells; synaptic vesicles in the plexiform layers; and the photoreceptors. Losses of cell layers, structure, or lack of expression of these markers were considered abnormal. We found that there were no significant differences between bevacizumab-injected eyes and control eyes in the retinal cell components in any group of animals (Fig. 5). 
Figure 5.
 
Representative images from immunohistochemistry 1 year after single 1.25-mg/0.05 mL (group 1) or three consecutive 1.25 mg/0.05 mL bevacizumab (group 4) injections in rabbit eyes. There was no statistically significant difference in the retinal cell components between eyes receiving single or multiple bevacizumab injections and control eyes. Magnification, ×200.
Figure 5.
 
Representative images from immunohistochemistry 1 year after single 1.25-mg/0.05 mL (group 1) or three consecutive 1.25 mg/0.05 mL bevacizumab (group 4) injections in rabbit eyes. There was no statistically significant difference in the retinal cell components between eyes receiving single or multiple bevacizumab injections and control eyes. Magnification, ×200.
ERG Results
Visual function results were evaluated by ERG 1 year after single or three consecutive bevacizumab injections. The results are shown in Table 3. The ratios of the a- and b-waves of treated eyes and control eyes were close to one, showing no statistically significant difference between bevacizumab-injected eyes and naïve eyes in any groups of animals. There was also no statistically significant difference from baseline to 1 year after intravitreal injection of bevacizumab in any groups of animals. The results of the ERG implicit time also showed no difference between bevacizumab-injected eyes and naïve eyes in any groups of animals (data not shown). These results indicate that there was no abnormal visual response in the retina for 1 year after bevacizumab injection. 
Table 3.
 
ERG Results
Table 3.
 
ERG Results
a-Wave (μV) b-Wave (μV) a-Wave Ratio (Treated Eyes versus Nontreated Eyes) b-Wave Ratio (Treated Eyes versus Nontreated Eyes)
Group 1 (single injection at 2 weeks)
    Baseline 65 ± 19 143 ± 45 0.96 0.96
    One year 51 ± 27 139 ± 25 1.09 0.92
    P 0.207 0.752 1.112 0.753
Group 2 (single injection at 4 weeks)
    Baseline 58 ± 23 127 ± 29 1.04 1.02
    One year 46 ± 15 122 ± 33 1.12 1.03
    P 0.463 0.917 0.249 0.248
Group 3 (single injection at 6 weeks)
    Baseline 44 ± 24 111 ± 28 1.02 0.86
    One year 49 ± 26 108 ± 16 1.05 0.90
    P 0.463 0.917 1.10 0.600
Group 4 (three injections at 2, 6, and 10 weeks)
    Baseline 53 ± 16 135 ± 36 1.10 1.02
    One year 49 ± 17 132 ± 13 1.32 1.03
    P 0.593 0.593 0.144 0.593
Serum Bevacizumab Concentration 8 Days after Intravitreal Bevacizumab Injection
Serum bevacizumab concentrations were highest when the intravitreal injection of bevacizumab was performed 2 weeks (group 1; 19.3 ± 8.1 μg/mL) after birth. Serum bevacizumab levels were lower when the injection occurred 4 (group 2; 10.2 ± 2.3 μg/mL) or 6 (group 3; 4.4 ± 1.3 μg/mL) weeks after birth. The serum concentrations when the injections were performed at the ages of 2 or 4 weeks were significantly higher than the serum levels detected when the injection was performed at 12 weeks of age (2.9 ± 1.6 μg/mL, P = 0.02 and P = 0.024 respectively). The serum concentration when the injection was performed 6 weeks after birth (4.4 ± 1.3 μg/mL) was close to that when the injection was performed at the age of 12 weeks (P = 0.184) (Fig. 6). 
Figure 6.
 
Serum concentrations of bevacizumab after intravitreal injection of 1.25 mg bevacizumab in rabbit eyes at different times after birth. The serum concentrations were significantly higher when the injection was performed at an early age. When the injection was performed 6 weeks after birth, the serum concentration of bevacizumab was close to that observed when the injection was performed at the age of 12 weeks. *P < 0.05 compared with the data from injection at 12 weeks.
Figure 6.
 
Serum concentrations of bevacizumab after intravitreal injection of 1.25 mg bevacizumab in rabbit eyes at different times after birth. The serum concentrations were significantly higher when the injection was performed at an early age. When the injection was performed 6 weeks after birth, the serum concentration of bevacizumab was close to that observed when the injection was performed at the age of 12 weeks. *P < 0.05 compared with the data from injection at 12 weeks.
Discussion
VEGF has been shown to be an important neurotrophic factor for central nervous system 3133 and retinal 2,27,28 development. Because neurons in newborns are still rapidly developing, it is important to know whether bevacizumab, an anti-VEGF antibody, causes any toxicity in the eye. In this study, we did not find any morphologic or functional changes in the retina 1 year after either single or three consecutive bevacizumab injections of 1.25 mg in the newborn rabbit vitreous cavity. These results have clinical implications, suggesting that bevacizumab is well tolerated in the newborn eye and does not cause apparent long-term ocular toxicity. 
Previous studies have shown an excellent safety profile for bevacizumab in adult eyes with ARMD, 46 diabetic retinopathy, 79 vitreous hemorrhage, 1012 neovascular glaucoma, 13 and retinal vascular occlusion. 1417 A few case reports and small case series have shown bevacizumab to be helpful in treating ROP without apparent ocular toxicity. 1826 Further, the disease diminishes and vasculosa lentis disappears after bevacizumab injection. One study showed that a single injection of bevacizumab prevented progression to retinal detachment in all eyes with posterior zone-I ROP, even without the need for laser ablation. 22 The results are encouraging, because roughly 27% to 47% of posterior zone-I cases progress to retinal detachment, even with the application of peripheral retinal ablation. 3436 However, there are no current animal studies in which intravitreal bevacizumab injection in newborn animals was examined. Our study has extended the data of other studies conducted in adult animals, suggesting that the tolerability of 1.25 mg bevacizumab is relatively good in the eyes of newborn animals. 
We found in newborn rabbits that serum bevacizumab concentrations were significantly higher when the intravitreal injection of bevacizumab was performed 2 weeks after birth (19.4 ± 8.1 μg/mL). The serum concentration when the injection was performed 6 weeks after birth (4.4 ± 1.3 μg/mL) was close to that when the injection was performed at 12 weeks (P = 0.184). The serum concentrations of bevacizumab were higher when the injections were performed on younger animals. This phenomenon could be explained by the following reasoning. First, the blood–retinal barrier may be less well developed in the newborn rabbit eye than in the adult rabbit eye, 37 permitting more bevacizumab to leak into the systemic circulation. Second, newborn rabbits have a smaller vitreous cavity and smaller serum compartment than do adult rabbits. These factors may result in altered pharmacokinetics in comparison to that in adult rabbits. More studies are needed to investigate the effects of systemic bevacizumab on neuronal development after intravitreal injection of bevacizumab in the newborn. 
In the inner segments of the photoreceptor layers, swelling and disruption of mitochondria with loss of cristae were noted in some areas in both bevacizumab-treated and control eyes. This finding suggests that the phenomenon is related to a sample-processing artifact. Although this disruption was seen in both control eyes and bevacizumab-injected eyes, it was more common in bevacizumab-injected eyes. Although morphologic changes have been observed after intravitreal injection of bevacizumab, functional study of the retina using ERGs did not show any toxicity associated with the injection of single or three consecutive 1.25-mg doses. Using both ERG and light microscopy, Inan et al. 38 found no evidence of retinal toxicity with intravitreal bevacizumab at doses of 1.25 and 3 mg. However, electron microscopic assessment revealed mitochondrial damage in the inner segments of the photoreceptors. Extensive apoptotic protein expression in study eyes and minimal expression in control eyes were noted. Their study differs from ours in that they used adult rabbit tissue from animals killed 28 days after the intravitreal injection. In addition, they used a higher dose volume in half of the animals in their study. A definitive judgment regarding the relationship between this phenomenon and the injection of bevacizumab awaits further investigation. 
Currently, the recommended treatment for type-1 ROP is peripheral ablation. Although cryotherapy has been used in the past, laser therapy has significantly replaced that procedure. In addition, the timing of treatment has been moved to an earlier stage of the disease, as documented in the Early Treatment for Retinopathy of Prematurity Study (ETROP). 39 Although laser or cryotherapy effectively halts the progression of stage 3 ROP to stage 4 in 90% of patients, these treatments actually destroy almost two thirds of the retina. Some patients progress to retinal detachment despite laser or cryotherapy. The functional outcomes are still not satisfying in stage 4B or 5 ROP, even after vitrectomy or scleral buckling. 4042 A new treatment that could either decrease the need for laser treatment or vitreoretinal surgery or facilitate the success rate of vitreoretinal surgery would be worth pursuing. Currently, there are ongoing, multiple center, randomized clinical trials of bevacizumab for use in ROP. The animal data presented in this article may provide further references for these clinical trials. 
This study is limited by somewhat subjective outcome measures (e.g., ophthalmoscopy, slit lamp, and retinal morphology). The fellow untreated eye was used as the control, yet there is literature suggesting that fellow eyes may take up a small fraction of bevacizumab. 30 Furthermore, a newborn animal model instead of an ROP model was used in the present study. However, we tried to be as objective as possible in our data interpretation. The ERG results after intravitreal injection were also compared with those before injection (baseline), to correct the possible effect of drug circulation in the contralateral eye. In addition, the data were interpreted when the observers were masked to treatment. The specimens were also checked repeatedly by multiple researchers. An oxygen-induced retinopathy (OIR) model using newborn mice to mimic ROP was not used in this study, because rodent VEGF does not interact with the humanized VEGF antibody bevacizumab. 43,44  
In conclusion, single and three consecutive intravitreal injections of 1.25 mg bevacizumab in newborn rabbit eyes did not cause morphologic or functional changes for up to 1 year. However, systemic exposure was higher when the injection was performed at an early age. Although bevacizumab seems to be well tolerated in these newborn rabbits' eyes, the systemic effect remains unknown after intravitreal injections in newborns. Further study of the systemic effects of bevacizumab after intraocular injection is recommended. 
Footnotes
 Supported by Grant CMRPG 370281 from Chang Gung Memorial Hospital Research Grant, Taoyuan, Taiwan.
Footnotes
 Disclosure: W.-C. Wu, None; C.-C. Lai, None; K.-J. Chen, None; T.-L. Chen, None; N.-K. Wang, None; Y.-S. Hwang, None; L. Yeung, None; L.-M. Li, None
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Figure 1.
 
External photos and color fundus photos of rabbit eyes 1 year after intravitreal injection of 1.25 mg bevacizumab. External photos (A–E) reveal a clear cornea and crystalline lens. No cell or flare was noted in the anterior chamber. Color fundus photos (F–J) show no signs of vitreous opacity, retinal detachment, vessel occlusion, or retinal necrosis. Group 1, single injection at 2 weeks; group 2, single injection at 4 weeks; group 3, single injection at 6 weeks; and group 4, three consecutive injections at 2, 6, and 10 weeks.
Figure 1.
 
External photos and color fundus photos of rabbit eyes 1 year after intravitreal injection of 1.25 mg bevacizumab. External photos (A–E) reveal a clear cornea and crystalline lens. No cell or flare was noted in the anterior chamber. Color fundus photos (F–J) show no signs of vitreous opacity, retinal detachment, vessel occlusion, or retinal necrosis. Group 1, single injection at 2 weeks; group 2, single injection at 4 weeks; group 3, single injection at 6 weeks; and group 4, three consecutive injections at 2, 6, and 10 weeks.
Figure 2.
 
Histology of the retina in rabbit eyes 1 year after bevacizumab injection. No sign of retinal degeneration, thinness, dissolution, or loss in the layers of either the central or peripheral retina was detected. No noticeable differences were observed between any treatment group and control eyes. Groups are as stated in Figure 1. RGC, retinal ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments of the photoreceptor layers; OS, outer segments of the photoreceptor layers; RPE, retinal pigment epithelium. Magnification, ×400.
Figure 2.
 
Histology of the retina in rabbit eyes 1 year after bevacizumab injection. No sign of retinal degeneration, thinness, dissolution, or loss in the layers of either the central or peripheral retina was detected. No noticeable differences were observed between any treatment group and control eyes. Groups are as stated in Figure 1. RGC, retinal ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments of the photoreceptor layers; OS, outer segments of the photoreceptor layers; RPE, retinal pigment epithelium. Magnification, ×400.
Figure 3.
 
Electron microscopy results in rabbit eyes 1 year after three consecutive intravitreal injections of 1.25 mg/0.05 mL bevacizumab (group 4). Normal morphology was noted in the inner nuclear layer (A), the outer segment in the photoreceptor layer (B), and the junction of the retinal pigment epithelium and outer segment in the photoreceptor layer (C) after multiple bevacizumab injections. Magnification: ×5000.
Figure 3.
 
Electron microscopy results in rabbit eyes 1 year after three consecutive intravitreal injections of 1.25 mg/0.05 mL bevacizumab (group 4). Normal morphology was noted in the inner nuclear layer (A), the outer segment in the photoreceptor layer (B), and the junction of the retinal pigment epithelium and outer segment in the photoreceptor layer (C) after multiple bevacizumab injections. Magnification: ×5000.
Figure 4.
 
Changes in the inner segments of the photoreceptor layers in rabbit eyes 1 year after intravitreal injection of 1.25 mg/0.05 mL bevacizumab, as revealed by electron microscopy. Swelling and disruption of mitochondria (arrows) with a loss of cristae were noted in some areas in the bevacizumab-injected (A) and control (B) eyes. Magnification: ×20,000.
Figure 4.
 
Changes in the inner segments of the photoreceptor layers in rabbit eyes 1 year after intravitreal injection of 1.25 mg/0.05 mL bevacizumab, as revealed by electron microscopy. Swelling and disruption of mitochondria (arrows) with a loss of cristae were noted in some areas in the bevacizumab-injected (A) and control (B) eyes. Magnification: ×20,000.
Figure 5.
 
Representative images from immunohistochemistry 1 year after single 1.25-mg/0.05 mL (group 1) or three consecutive 1.25 mg/0.05 mL bevacizumab (group 4) injections in rabbit eyes. There was no statistically significant difference in the retinal cell components between eyes receiving single or multiple bevacizumab injections and control eyes. Magnification, ×200.
Figure 5.
 
Representative images from immunohistochemistry 1 year after single 1.25-mg/0.05 mL (group 1) or three consecutive 1.25 mg/0.05 mL bevacizumab (group 4) injections in rabbit eyes. There was no statistically significant difference in the retinal cell components between eyes receiving single or multiple bevacizumab injections and control eyes. Magnification, ×200.
Figure 6.
 
Serum concentrations of bevacizumab after intravitreal injection of 1.25 mg bevacizumab in rabbit eyes at different times after birth. The serum concentrations were significantly higher when the injection was performed at an early age. When the injection was performed 6 weeks after birth, the serum concentration of bevacizumab was close to that observed when the injection was performed at the age of 12 weeks. *P < 0.05 compared with the data from injection at 12 weeks.
Figure 6.
 
Serum concentrations of bevacizumab after intravitreal injection of 1.25 mg bevacizumab in rabbit eyes at different times after birth. The serum concentrations were significantly higher when the injection was performed at an early age. When the injection was performed 6 weeks after birth, the serum concentration of bevacizumab was close to that observed when the injection was performed at the age of 12 weeks. *P < 0.05 compared with the data from injection at 12 weeks.
Table 1.
 
Treatment Groups
Table 1.
 
Treatment Groups
Group IVI Mode Injection Time Evaluation
1 (single injection at 2 weeks) Single injection 2 wk after birth Clinical examinations, histology, IHC, ERG, ELISA
2 (single injection at 4 weeks) Single injection 4 wk after birth Clinical examinations, histology, IHC, ERG, ELISA
3 (single injection at 6 weeks) Single injection 6 wk after birth Clinical examinations, histology, IHC, ERG, ELISA
4 (three injections at 2, 6, and 10 weeks) Three consecutive injections 2, 6, 10 wk after birth Clinical examinations, histology, IHC, ERG
5 (single injection at 12 weeks) Single injection 12 wk after birth ELISA
Table 2.
 
Selection and Specificity of Antibodies Used
Table 2.
 
Selection and Specificity of Antibodies Used
Antibody Specificity in the Retina
Anti-GFAP; anti-vimentin Intermediate filament proteins of Müller cells
Anti-neurofilament Neurofilaments in ganglion cells and in horizontal cells
Anti-synaptophysin Synaptic vesicles in plexiform layers
Anti-red/green opsin; anti-blue opsin Cones
Anti-rhodopsin Rods
Table 3.
 
ERG Results
Table 3.
 
ERG Results
a-Wave (μV) b-Wave (μV) a-Wave Ratio (Treated Eyes versus Nontreated Eyes) b-Wave Ratio (Treated Eyes versus Nontreated Eyes)
Group 1 (single injection at 2 weeks)
    Baseline 65 ± 19 143 ± 45 0.96 0.96
    One year 51 ± 27 139 ± 25 1.09 0.92
    P 0.207 0.752 1.112 0.753
Group 2 (single injection at 4 weeks)
    Baseline 58 ± 23 127 ± 29 1.04 1.02
    One year 46 ± 15 122 ± 33 1.12 1.03
    P 0.463 0.917 0.249 0.248
Group 3 (single injection at 6 weeks)
    Baseline 44 ± 24 111 ± 28 1.02 0.86
    One year 49 ± 26 108 ± 16 1.05 0.90
    P 0.463 0.917 1.10 0.600
Group 4 (three injections at 2, 6, and 10 weeks)
    Baseline 53 ± 16 135 ± 36 1.10 1.02
    One year 49 ± 17 132 ± 13 1.32 1.03
    P 0.593 0.593 0.144 0.593
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