Retinal Toxicity of Intravitreal Trastuzumab in a Rabbit Model: Preliminary Results of an Experimental Study

Abstract

Purpose:

To evaluate the retinal toxicity of intravitreal trastuzumab in a rabbit model.

Methods:

Results:

The clinical examination was unremarkable on either sham or intravitreal injection of trastuzumab. Conversely, the ERG was greatly affected and in 2 cases extinguished 14 days after trastuzumab injection. Consistent with electrophysiological abnormalities of the retina, signs of retinal edema in experimented eyes, suggesting morphologic retinal damage, were observed. In contrast, in the sham injected eyes, the ERG was normal without histopathologic retinal changes.

Conclusions:

Intravitreal trastuzumab seems to be toxic to the retina in albino rabbits even at a concentration of 5 mg/mL. Further studies are needed to evaluate the safety of intravitreal trastuzumab in models of choroidal neovascularization, as well as to obtain experience concerning the intravitreal toxicity of trastuzumab in primates too and not only in rabbits.

Introduction

The epidermal growth factor (EGF) is a growth factor that stimulates cell growth, proliferation, and differentiation by binding to its receptor (EGFR).¹ The EGF can be found in human platelets, macrophages, urine, saliva, milk, and plasma.¹ The EGFR belongs to the ErbB membrane receptors of the tyrosine kinase receptor family.¹ There is evidence indicating that EGFR signaling is involved in a variety of cellular processes, such as growth, differentiation, and survival of retinal pigment epithelial (RPE) cells in vitro.^2–5 Interestingly enough, Sugino et al. supported that the EGF and its signaling pathways are significant factors associated with the survival, proliferation, adhesion, and migration of RPE in age-related macular degeneration models.⁶ In addition, the EGF is known to be an angiogenic factor, as well as an upregulator of the expression of other angiogenic factors, such as the vascular endothelial growth factor (VEGF) and interleukin-8.^7–10

Trastuzumab, a humanized monoclonal antibody against the EGFR2 (HER2), was approved by the Food and Drug Administration for the treatment of ErbB2/HER2 overpresenting breast cancers in 1998.¹¹ It reduces signaling from EGFR-related pathways, and therefore promotes cell cycle arrest and apoptosis.^11,12 Furthermore, it is considered to inhibit angiogenesis and expression of multiple proangiogenic factors in vitro and in vivo.^9,13,14 Recently, Güler et al. demonstrated that systemic administration of trastuzumab reduced the extent of experimentally induced corneal neovascularization in a rat model.¹⁵ In consideration of the above, trastuzumab could be also a potential therapeutic approach in the prevention of choroidal or retinal neovascularization.

Intravitreal injections are widely performed in ophthalmology to treat retinal and choroidal diseases and were associated with low incidence of complications.¹⁶ Retinal toxicity is a primary concern when using intravitreal drugs.^17,18 To the best of our knowledge, the retinal toxicity of trastuzumab had not been yet analyzed. Therefore, the purpose of our study was to evaluate the retinal toxicity of trastuzumab in a rabbit model, using electroretinography (ERG) and light microscopy.

Methods

Fourteen New Zealand albino rabbits, weighing between 2.5 and 3 kg were used for this study. In the first group (n=6), 0.1 mL trastuzumab 10 mg/mL was injected into the vitreous. In the second group (n=6), the same volume of sterile balanced saline solution was injected intravitreally (sham injection). Additionally, 0.1 mL of 2 other concentrations of trastuzumab (7.5 and 5 mg/mL, respectively), was injected into the vitreous of 2 rabbits. The animals were treated according to the Association for Research in Vision and Ophthalmology guidelines for the Use of Animals in Ophthalmic and Vision Research. The study was approved by the Institutional Animal Care and Use Committee, as well as the Institutional Review Board of our department. Slit-lamp examination and indirect funduscopic examination were performed to all animal eyes at baseline. Animals that showed corneal/lens opacities, retinal damage or inter-eye difference in b-wave amplitude more than 10% before the study were excluded. The animals were anesthetized with intramuscular mixture of ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (5 mg/kg). Topical anesthesia was applied using proparacaine 0.5%. The eyes were dilated by topical application of tropicamide 0.5%.

Intravitreal injections

All procedures were performed under sterile conditions. Tobramycin antibiotic was used topically before and after all surgical procedures. A paracentesis of the anterior chamber was executed with a 27-gauge needle, withdrawing 0.1 mL of aqueous fluid to ensure adequate hypotony and the safer introduction of trastuzumab in to the vitreous cavity without loss of vitreous or drug reflux after injection. Intravitreal injection of trastuzumab was performed using a 30-gauge needle attached to a tuberculin syringe inserted ∼1.5 mm posterior to the limbus and guided into the vitreous cavity under ophthalmoscopic control. Only the right eye (OD) was injected, while left eye (OS) served as control.

Standard commercial solution of trastuzumab (Herceptin, Roche) was diluted appropriately with sterile normal saline solution to obtain a concentration of 10, 7.5 and 5 mg/mL. Intravitreal injections of 0.1 mL of trastuzumab in different concentrations as well as sterile balanced saline solution were performed, as described above. Slit-lamp examinations and indirect ophthalmoscopy were performed on all eyes immediately after the injections and 14 days thereafter, all animals were observed for signs of infection, inflammation, and toxicity.

Electrophysiologic test

The ERG using the Retimax (CSO Strumenti Oftalmici) system was performed before the intravitreal injection and 14 days after injection. Once the rabbits were well anesthetized, they were placed in a restrained box that supported the head of the animals. This facilitated a uniform positioning of the eyes relative to the light stimulus. The ERG responses were recorded simultaneously from the injected and normal control eye. Standard ERG was performed after full adaptation for 5 min after papillary dilation. Unipolar contact lenses were placed on both corneas, the negative electrode was placed in the subcutaneous space of the forehead, and the ground electrode was clipped to the earlobe with some electric gel. Light stimuli were obtained from the Ganzfeld light source with a maximum of energy of 5.76–5/m². ERG signals were amplified (×10.000) and filtered (0.3–300 Hz). At each scheduled examination, before and 14 days after the intravitreal injection, ERGs were repeated 3 times. The ERG estimation was based on the measurement of the b-wave amplitude from the trough of a-wave to the peak of the b-wave. ERG changes were considered significant if the differences in the b-wave amplitude were <30% from the preinjection values.

Histologic examination

Following ERG tests, animals were euthanized with a lethal overdose of intraperitoneal pentobarbital (100 mg/kg). The eyes were enucleated, preserving the globe integrity, and fixed immediately in 10% buffered formalin. After 72 h, the lens was removed and the eye was cut along the cornea-optic nerve axis into halves. Tissues were processed, embedded in paraffin, sectioned at a thickness of 5 μm, and stained with hematoxylin–eosin. The light microscope was used for histologic examinations. Specifically, the histological slides were stained with hematoxylin–eosin and photos of the slights were taken by light microscopy (Nikon Eclipse 80i; Nikon Corp.) connected with digital camera (DS-2 MW; Nikon Corp.). Magnification was 4× for all figures. Digital photos were then analyzed (Image-Pro Plus v 5.1; Media Cybernetics) and the thickness of the retina was measured. In addition, the immunocytochemistry study using anti-CD15 and anti-EGFR was performed.

Results

The clinical examination of sham-injected eyes as well as of eyes injected with trastuzumab was unremarkable during follow-up. No signs of inflammation were observed after the intravitreal injection. The cornea, lens, and vitreous remained clear, while the retina and optic nerve appeared unaltered without any lesion from the injection. As a result, there was no clinical evidence of ocular changes in any rabbit.

ERG responses showed a significant decrease of the b-wave amplitude 2 weeks after the intravitreal injection of trastuzumab in all concentrations. Conversely, no changes of ERG were observed in sham-injected eyes (Fig. 1). Table 1 shows that in 2 experimented eyes of group 1, ERG was almost extinguished and in the other 4 eyes, the decrease of the b-wave amplitude was statistically significant (P<0.001 in all cases), amounting to 71.24%–90.0% from the normal. On the other hand, there was no statistically significant difference between the b-wave amplitude in ERG at baseline and 14 days after the intravitreal injection in control eyes (50.45±10.17 μV vs. 50.21±11.02 μV, P=0.970, Mann–Whitney–Wilcoxon test). In the other 2 rabbits, where 0.1 mL trastuzumab of 7.5 and 5 mg/mL was injected, there was a statistically significant decrease of the b-wave amplitude (P<0.001 and P<0.01, respectively), amounting to 58.72%–76.3% from the normal (Fig. 2).

FIG. 1.

(A) Electroretinogram (ERG) recorded from both eyes in a control rabbit. In the right eye (OD), the b-wave amplitude is 50.88 μV and in the left eye (OS), 48.26 μV, (B) ERG recorded from both eyes in an experimental rabbit, 14 days after intravitreal injection of 0.1 mL trastuzumab 10 mg/mL in the right eye (OD). B-wave amplitude of ERG in the injected eye is greatly decreased (9.13 μV). B-wave amplitude in the left eye (OS) is normal (52.37 μV).

FIG. 2.

(A, B) ERG recorded from the right eye of an experimental rabbit, before (A) and after (B) intravitreal injection of 0.1 mL trastuzumab 7.5 mg/mL, (C, D) ERG recorded from the right eye of an experimental rabbit, before (C) and after (D) intravitreal injection of 0.1 mL trastuzumab 5 mg/mL.

Table 1.

b-Wave Amplitude Before and 14 Days After the Intravitreal Injection of trastuzumab 10 mg/mL in Control and Experimental Eyes

S. No.	Before injection b-wave amplitude	14 days after injection b-wave amplitude
Control eyes
1.	61.63 μV	61.09 μV
2.	50.88 μV	52.41 μV
3.	38.24 μV	36.82 μV
4.	48.26 μV	47.23 μV
5.	62.65 μV	61.99 μV
6.	41.02 μV	39.73 μV
Experimental eyes
1.	65.75 μV	17.4 μV (73.24% decrease)
2.	53.27 μV	9.13 μV (80.15% decrease)
3.	37.0 μV	10.56 μV (71.5% decrease)
4.	46.5 μV	13.38% (71.3% decrease)
5.	56.3 μV	Extinguished
6.	37.56 μV	Extinguished

There were differences in the histological appearance between the eyes injected with trastuzumab and those injected with a sterile saline solution. We found extensive signs of edema in experimental eyes, suggesting morphologic retinal damage of the ganglion cell layer and the inner layers (Fig. 3). The immunocytochemistry study showed infiltrating leukocytes (Fig. 4). In addition, binding of EGF receptors was observed in some ganglion cells (Fig. 5).

FIG. 3.

Photomicrographs of hematoxylin–eosin stained sections of experimental and control retinas from 3 rabbits 2 weeks after intravitreal injection of trastuzumab. (A) A normal retina of the control eye, (B) diffuse signs of edema of the ganglion cell layer of the experimental eye (arrow), (C) signs of edema of inner layers (arrow). PRL, photoreceptor layer; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

FIG. 4.

Immunostaining with anti-CD15 (PAP×250), showing infiltrating leukocytes (arrow).

FIG. 5.

Immunostaining with anti-EGFR (PAP×250), showing the positive ganglion cell (arrow).

Discussion

Angiogenesis is a dynamic process regulated by a great deal of pro- and antiangiogenic molecules.^14,17,19 The VEGF is considered to be the major factor concerning intraocular neovascularization in ocular diseases, such as age-related macular degeneration, proliferative diabetic retinopathy, retinal vascular occlusion, retinopathy or prematurity, and other ischemic vascular diseases.^17–20 Additionally, the EGF, another angiogenic factor, upregulates the expression of VEGF, taking an active part in pathologic angiogenesis.^7,8 Anti-VEGFs are currently considered to be a treatment alternative in certain ocular diseases involving neovascularization.^{17–19,21–24} Therefore, there is a need of discovering new anti-VEGF agents, which will be less expensive, less toxic, and effective in less frequent dosing.

Trastuzumab is a molecule, acting against EGF2 and is used for the treatment mainly of breast or other types of cancer, as it inhibits pathologic angiogenesis.^25–27 Systemically administered trastuzumab is not reported to be toxic.²⁸ There is only a case report published, describing bilateral macular ischemia and severe visual loss after trastuzumab therapy.²⁹ Intravitreal injection has become a common practice in ophthalmology for the treatment of retinal and choroidal diseases with a relatively low incidence of complications in comparison with systemic administration.¹⁶ To our knowledge, the safety of intravitreal injection of trastuzumab has never been reported. We used the doses of 10, 7.5, and 5 mg/mL to evaluate the retinal safety profile of intravitreal trastuzumab.

The principal message of our study is that intravitreal trastuzumab shows a clearly toxic effect on the retina of a rabbit model even at a dose of 5 mg/mL 2 weeks after its delivery. ERG findings confirmed the fact that intravitreal trastuzumab was toxic for the rabbit retina, as the b-wave was extinguished or greatly affected in experimental eyes and remained normal in control ones. As far as light microscopy findings, the presence of edema and degeneration not only in the ganglion cell layer, but also in the inner layers was another sign of toxicity, as those morphologic retinal changes were probably induced by the intravitreal drug.

In conclusion, our preliminary study suggests that intravitreally injected trastuzumab is toxic for the rabbit eye. It should be mentioned that toxicity in the rabbit retina may not necessarily correlate with primate or human retinal toxicity, as the retinal anatomy of the rabbit model is not the same as that of human eyes. Further studies are needed to evaluate the safety of intravitreal trastuzumab in models of choroidal neovascularization, as well as to obtain experience concerning the intravitreal toxicity of trastuzumab in primates too and not only in rabbits. Moreover, observational studies investigating the potential retinal toxicity of systemically administered trastuzumab in patients with cancer will be worthwhile, as our results may raise a flag on the potential safety issue in these patients.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

References

Huang

T.S.

, Rauth

, Das Gupta

T.K.

Overexpression of EGF receptor is associated with spontaneous metastases of a human melanoma cell line in nude mice. Anticancer Res., 16:3557–3563. 1996.

Yan

, Hui

Y.N.

, Li

Y.J.

, Guo

C.M.

, Meng

Epidermal growth factor receptor in cultured human retinal pigment epithelial cells. Ophthalmologica., 221:244–250. 2007.

Defoe

D.M.

, Grindstaff

R.D.

Epidermal growth factor stimulation of RPE cell survival: contribution of phosphatidylinositol 3- kinase and mitogen-activated protein kinase pathways. Exp. Eye Res., 79:51–59. 2004.

Steindl-Kuscher

, Krugluger

, Boulton

M.E.

et al. Activation of the beta-catenin signaling pathway and its impact on RPE cell cycle. Invest. Ophthalmol. Vis. Sci., 50:4471–4476. 2009.

Khaliq

, Jarvis-Evans

, McLeod

, Boulton

Oxygen modulates the response of the retinal pigment epithelium to basic fibroblast growth factor and epidermal growth factor by receptor regulation. Invest. Ophthalmol. Vis. Sci., 37:436–443. 1996.

Sugino

I.K.

, Wang

, Zarbin

M.A.

Age-related macular degeneration and retinal pigment epithelium wound healing. Mol. Neurobiol., 28:177–194. 2003.

Gasparini

, Longo

, Torino

, Morabito

Therapy of breast cancer with molecular targeting agents. Ann. Oncol., 16:28–36. 2005.

Russell

K.S.

, Stern

D.F.

, Polverini

P.J.

, Bender

J.R.

Neuregulin activation of ErbB receptors in vascular endothelium leads to angiogenesis. Am. J. Physiol., 277:2205–2211. 1999.

Sener

, Yuksel

, Yildiz

D.K.

, Yilmaz

, Ozdemir

, Caglar

, Degirmenci

The impact of subconjuctivally injected EGF and VEGF inhibitors on experimental corneal neovascularization in rat model. Curr. Eye Res., 36:1005–1013. 2011.

10.

Jonas

J.B.

, Tao

, Neumaier

, Findeisen

Cytokine concentration in aqueous humour of eyes with exudative age-related macular degeneration. Acta. Ophthalmol., 90:e381–e388. 2012.

11.

Roskoski

Jr.

The ErbB/HER receptor protein-tyrosine kinases and cancer. Biochem. Biophys. Res. Commun., 319:1–11. 2004.

12.

Press

M.F.

, Lenz

H.J.

EGFR, HER2 and VEGF pathways: validated targets for cancer treatment. Drugs., 67:2045–2075. 2007.

13.

Izumi

, Xu

, di Tomaso

, Fukumura

, Jain

R.K.

Tumour biology: herceptin acts as an anti-angiogenic cocktail. Nature., 416:279–280. 2002.

14.

Petit

A.M.

, Rak

, Hung

M.C.

et al. Neutralizing antibodies against epidermal growth factor and ErbB-2/neu receptor tyrosine kinases down-regulate vascular endothelial growth factor production by tumor cells in vitro and in vivo: angiogenic implications for signal transduction therapy of solid tumors. Am. J. Pathol., 151:1523–1530. 1997.

15.

Güler

, Yilmaz

, Ozercan

, Elkiran

The inhibitory effects of trastuzumab on corneal neovascularization. Am. J. Ophthalmol., 147:703–708. 2009.

16.

Jager

R.D.

, Aiello

L.P.

, Patel

S.C.

, Cunningham

E.T.

Risks of intravitreous injection: a comprehensive review. Retina., 24:676–698. 2004.

17.

Fiore

, Iaccheri

, Pietrolucci

et al. Retinal toxicity of intravitreal genistein in a rabbit model. Retina., 30:1536–1541. 2010.

18.

Manzano

R.P.

, Peyman

G.A.

, Khan

, Kivilcim

Testing intravitreal toxicity of bevacizumab (Avastin)

Retina., 26:257–261. 2006.

19.

Aiello

L.P.

, Avery

R.L.

, Arrigg

P.G.

et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N. Engl. J. Med., 331:1480–1487. 1994.

20.

Abouammoh

, Sharma

Ranibizumab versus bevacizumab for the treatment of neovascular age-related macular degeneration. Curr. Opin. Ophthalmol., 22:152–158. 2011.

21.

Sultan

M.B.

, Zhou

, Loftus

, Dombi

, Ice

K.S.

A phase 2/3, multicenter, randomized, double-masked, 2-year trial of pegaptanib sodium for the treatment of diabetic macular edema. Ophthalmology., 118:1107–1118. 2011.

22.

Salam

, Mathew

, Sivaprasad

Treatment of proliferative diabetic retinopathy with anti-VEGF agents. Acta. Ophthalmol., 85:405–411. 2011.

23.

Ciulla

T.A.

, Rosenfeld

P.J.

Anti-vascular endothelial growth factor therapy for neovascular ocular diseases other than age-related macular degeneration. Curr. Opin. Ophthalmol., 20:166–174. 2009.

24.

Sacu

, Pemp

, Weigert

et al. Response of retinal vessels and retrobulbar hemodynamics to intravitreal anti-VEGF treatment in eyes with branch retinal vein occlusion. Invest. Ophthalmol. Vis. Sci., 52:3046–3050. 2011.

25.

Nahta

, Esteva

F.J.

Herceptin: mechanisms of action and resistance. Cancer Lett., 232:123–138. 2006.

26.

Valabrega

, Montemurro

, Aglietta

Trastuzumab: mechanism of action, resistance and future perspectives in HER2-overexpressing breast cancer. Ann. Oncol., 18:977–984. 2007.

27.

De Vita

, Giuliani

, Silvestris

et al. Human epidermal growth factor receptor 2 (HER2) in gastric cancer: a new therapeutic target. Cancer Treat. Rev., 36,suppl 3:S11–S15. 2010.

28.

Hall

P.S.

, Hulme

, McCabe

et al. Updated cost-effectiveness analysis of trastuzumab for early breast cancer: a UK perspective considering duration of benefit, long-term toxicity and pattern of recurrence. Pharmacoeconomics., 29:415–432. 2011.

29.

Saleh

, Bourcier

, Noel

, Speeg-Schatz

, Gaucher

Bilateral macular ischemia and severe visual loss following trastuzumab therapy. Acta. Oncol., 50:477–478. 2011.