Nan Su, Fu-Guang Li, Ulrich Schraermeyer

1Basic Medical College, Zhengzhou University, Zhengzhou 450001, Henan Province, China

2Department of Ophthalmology, the Fourth Affiliated Hospital of Zhengzhou University, Zhengzhou 450044，Henan Province, China

3Section of Experimental Vitreoretinal Surgery, Centre for Ophthalmology, Tuebingen 72076, Germany

Correspondence to:Fu-Guang Li. Basic Medical College, Zhengzhou University, Zhengzhou 450001, Henan Province, China. lifuguang@zzu.edu.cn

Received： 2015-06-15　　Accepted： 2016-04-05

?

The effects of anti-VEGF drugs on the retinal pigment epithelium and inner segment after intravitreal injection in the monkeys

Nan Su1,2, Fu-Guang Li1, Ulrich Schraermeyer3

1Basic Medical College, Zhengzhou University, Zhengzhou 450001, Henan Province, China

2Department of Ophthalmology, the Fourth Affiliated Hospital of Zhengzhou University, Zhengzhou 450044，Henan Province, China

3Section of Experimental Vitreoretinal Surgery, Centre for Ophthalmology, Tuebingen 72076, Germany

Correspondence to:Fu-Guang Li. Basic Medical College, Zhengzhou University, Zhengzhou 450001, Henan Province, China. lifuguang@zzu.edu.cn

Received： 2015-06-15Accepted： 2016-04-05

猴眼玻璃體腔內注射抗VEGF藥物對視網(wǎng)膜色素上皮層和光感受器內節(jié)VEGF含量的影響

蘇楠1,2, 李付廣1, Ulrich Schraermeyer3

(作者單位：1450001中國河南省鄭州市，鄭州大學基礎醫(yī)學院；2450044中國河南省鄭州市，鄭州大學第四附屬醫(yī)院眼科；3德國，圖賓根 72076，圖賓根眼科中心實驗室)

目的：研究比較抗血管內皮生長因子(VEGF)藥物(貝伐單抗、雷珠單抗、阿柏西晉)行猴眼玻璃體腔內注射后對視網(wǎng)膜色素上皮層和眼內節(jié)VEGF的作用效果。

方法：選取14只健康獼猴，4只雙眼注射貝伐單抗，4只雙眼注射雷珠單抗，4只雙眼注射阿柏西晉，每種注射藥物的4只猴雙眼分別于注射藥物后第一天和第七天分別摘除2只猴的眼球，剩余2只未注射任何藥物猴的雙眼作為對照。所有摘除后的眼球福爾馬林固定，石蠟包埋，切片后給予抗VEGF抗體，用光學顯微鏡觀察，經(jīng)Image-Pro Plus軟件處理圖片，用JMP10.0進行統(tǒng)計學分析。

結果：這三種藥物均能降低視網(wǎng)膜色素上皮(RPE)和光感受器內節(jié)(inner segment)的VEGF水平，貝伐單抗作用在三種藥物中作用最強，雷珠單抗在注射后第一天與阿柏西晉注射后第一天相比，雷珠單抗作用較強，但二者在注射后第七天，作用基本相似。

結論：這三種藥物均能降低RPE和inner segment的VEGF水平。

引用：蘇楠，李付廣，Schraermeyer U. 猴眼玻璃體腔內注射抗VEGF藥物對視網(wǎng)膜色素上皮層和光感受器內節(jié)VEGF含量的影響.國際眼科雜志2016;16(6):1014-1018

Abstract

?AIM: To compare the effects on the retina inner segment and retinal pigment epithelium (RPE) of intravitreally injecting bevacizumab, ranibizumab and aflibercept into monkey eyes.

?METHODS: Fourteen healthy cynomolgus monkeys (Macaca fascicularis, aged 3-8y,10 males,4 females) were raised at the Covance Laboratories under standard conditions. The 14 monkeys were grouped into 4 groups. Three of the groups with 4 monkeys each were injected intravitreally with one of the drugs, either bevacizumab, ranibizumab or aflibercept, while the 4thgroup with 2 monkeys served as a negative control. On 1d and 7d of injection, 2 monkeys from each drug treatment group were sacrificed under general anaesthesia and the 4 eyes were enucleated. All the enucleated eyes were fixed in formalin, embedded in paraffin wax, cut into 4.0 μm sections and deparaffinized according to standard procedures. Image-Pro Plus was used for all the photos to measure the content of vascular endothelial growth factor (VEGF)in the inner segment and RPE. The ANOVA test from JMP10.0 statistical program was used to evaluate the results.

?RESULTS: Retinal sections were checked for their anti-VEGF immune reactivity. The untreated control samples had the highest level of VEGF in the RPE and inner segment. All of these three drugs can reduce the level of VEGF in the RPE and inner segment, but Avastin seems to be more effective than Eylea in this regard. Lucentis treatment at 1d seems to be more effective than Eylea at VEGF 1d. But at 7d, both Lucentis and Eylea have the same effect on reducing VEGF expression level in the RPE and inner segment.

?CONCLUSION: All of these three drugs can reduce the level of VEGF in the RPE and inner segment.

KEYWORDS:?anti-vascular endothelial growth factor drugs; bevacizumab; ranibizumab; aflibercept; retinal pigment epithelium; inner segment; vascular endothelial growth factor

Citation:Su N, Li FG, Schraermeyer U. The effects of anti-VEGF drugs on the retinal pigment epithelium and inner segment after intravitreal injection in the monkeys.GuojiYankeZazhi(IntEyeSci) 2016;16(6):1014-1018

INTRODUCTION

Ocular neovascularization forms one of the most common pathological changes and clinical manifestations of a variety of eye diseases, such as age-related macular degeneration (AMD), diabetic retinopathy in adults, and retinopathy of prematurity in infants. The blinding complications of the disease make it a major cause of global visual morbidity in many countries[1-2].Among these diseases, AMD is the leading cause of blindness in patients over the age of 65y in developed countries, with choroidal neovascularization (CNV) being the main factor responsible for vision loss in AMD. It accounts for approximately 90% of AMD-related blindness[3]. Although the pathogenesis of AMD is not completely clear, several growth factors have been implicated in the disease process as they induce the development of blood vessels [i.e. basic fibroblast growth factor, transforming growth factor β, insulin-like growth factor-1, epidermal growth factor, interleukins and vascular endothelial growth factor (VEGF)]. However, only VEGF appears to be sufficient and essential for both physiological and pathological angiogenesis[4]. Furthermore, VEGF has been considered an important stimulus for CNV formation and persistence[5]. Thus VEGF has been found to be an important therapeutic target in neovascular AMD[6].

Since 2004, anti-VEGF therapy has become the standard treatment for wet AMD and has revolutionized the management of this disease. Between 2004 and 2006, three anti-VEGF drugs were introduced to ophthalmology after receiving regulatory approval for the treatment of AMD , ranibizumab (Lucentis, Genentech/Novartis),aflibercept (VEGF Trap-Eye/Eylea, Regeneron/Bayer) and bevacizumab (Avastin,Genentech/Roche). Among the most well-known medications injected are the anti-VEGF agents. Bevacizumab is a humanized full-length monoclonal antibody that binds to and inhibits VEGF. It is widely used in an off-label manner to inhibit VEGF in the eye[7]. Ranibizumab is a recombinantly produced humanized antibody (Fab) fragment that binds all active forms of VEGF-A, and is Food and Drug Administration (FDA) approved for ophthalmic use[8]. Aflibercept is a new anti-VEGF agent recently approved by the Food and Drug Administration. It is a fully human, recombinant fusion protein composed of the second immunoglobulin (Ig) binding domain of VEGFR1 and the third Ig-binding domain of VEGFR2, fused to the fragment crystallizable (Fc) region of human IgG1[9].

The aim of this study was to compare the effects of bevacizumab, ranibizumab and aflibercept on the monkey retina inner segment and retinal pigment epithelium (RPE). The monkey is the ideal model for thisinvivostudy, in contrast to rodents, the interactions between the Fc domain and the Fc receptors mimic those present in humans[10].

MATERIALS AND METHODS

Animals and Study ProtocolFourteen healthy cynomolgus monkeys (Macaca fascicularis, aged 3-8y, 10 males, 4 females) were raised at the Covance Laboratories (Muenster, Germany) under standard conditions. The 14 monkeys were grouped into 4 groups. Three of the groups with 4 monkeys each were injected intravitreally with one of the drugs, either bevacizumab, ranibizumab or aflibercept, while the 4thgroup with 2 monkeys served as a negative control. At 1d and 7d of injection, 2 monkeys from each drug treatment group were sacrificed under general anaesthesia and the 4 eyes were enucleated.

All animals were housed and handled in strict accordance with good animal practice under supervision of veterinarians and were monitored for evidence of disease and changes in attitude, appetite or behaviour suggestive of illness. Handling and housing of the animals at Covance Laboratories GmbH was done in accordance with the German Animal Welfare Act. Before the experiments were started, all monkeys were approved as experimental animals by a veterinarian of the site veterinary service and local authorities. Moreover, all animals underwent a range of ophthalmic examinations (described in ‘Ophthalmic examinations’) during the predose phase in order to detect possible ocular disorders that would have not been related to the injection of these three drugs. For each monkey, one eye was fixed for light and electron microscopy and the other eye for immunohistochemistry. For the notice of approval by the appropriate institutional animal care and use committee, please see Covance Studies 8260977 and 8274007.

Intravitreal Injection of Bevacizumab, Ranibizumab and AfliberceptThe animals were sedated by an intramuscular injection of medetomidine (Domitor) and ketamine hydrochloride, the eyes having previously been examined for any signs of inflammation. Pupils were dilated (Mydriasis with 1% tropicamide) and anaesthetised (proxymetacaine; Proparakain-POS 0.5%; Ursapharm). The conjunctival and corneal surface was disinfected (povidone iodine 10%). After sterile coating and insertion of a lid speculum, 1.25mg (25mg/ml) bevacizumab ,0.5 mg (10mg/ml) ranibizumab or 2 mg (40mg/ml) aflibercept were injected into the vitreous cavity using a 27-gauge canula. When removing the canula, the injection site was compressed with forceps to prevent reflux and a topical antibiotic (gentamicin) was administered. Animals were monitored for signs of inflammation until sacrificed.

Ophthalmic ExaminationsFor all ophthalmic examinations, a mydriatic agent (tropicamide) and a local ophthalmic anaesthetic (proxymetacaine) were instilled in the eyes of the sedated monkeys before examinations.

EnucleationOn 1d and 7d after the intravitreal injection, the animals were sacrificed under general anaesthesia, that is,intramuscular injection of ketamine hydrochloride followed by an intravenous sodium pentobarbitone (Lethabarb, Virbac, Australia) overdose. The eyes were enucleated 5min postmortem, cleaned of orbital tissue and were slit carefully at the limbus without damaging the ora serrata. Then 25 μL of the fixative were carefully injected into the vitreous before the eyes were fixed at 4℃ by immersion into 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4, Sigma, St. Louis, Missouri, USA) overnight for electron microscopy or into

Figure 1Light micrographs of untreated control eye (A), eye treated with Eylea on 1d (B), eye treated with Lucentis on 1d (C), eye treated with Avastin on 1d (D), eye treated with Eylea on 7d (E), eye treated with Lucentis on 7d (F), eye treated with Avastin on 7d (G)This can be seen by the reduction in the red staining after treatment with Avastin， Lucentis and Eylea. The red staining in Avastin 1d and Avastin 7d are more reduced than the staining in others. Compare Eylea 1d and Lucentis 1d, the red staining in Lucentis 1d is more reduced than the staining in Eylea 1d. IS: Inner segment; RPE: Retinal pigment epithelium.

formalin (Carl Roth, Karlsruhe, Germany) for immunohistochemistry. The eyes of two healthy monkeys without treatment were handled in the same manner.

ImmunohistochemistryAll the enucleated eyes were fixed in formalin, embedded in paraffin wax, cut into 4.0 μm sections and deparaffinized according to standard procedures.

Bevacizumab, ranibizumab and aflibercept were detected respectively.

Using the first antibody Monoclonal Mouse Anti-Human Vascular Growth Factor, Clone VG1,the antibody labels the VEGF-165,and VEGF-121,and VEGF-189 isoforms of VEGF. (Dako Cytomation Denmark, Code No.M7273)dilution 1:140; and REALTMDetection System, Alkaline Phosphatase/RED, Rabbit/Mouse(Code k5005,Dako) is the second antibody, dilution 1:25. and inspected with a light microscope (Axioskop; Carl Zeiss, Oberkochen, Germany).

Semi-quantification of VEGF Staining in Inner Segment of the RetinaWe obtained images from each section of the specimens, both from untreated eyes and treated eyes at 1d and 7d, at a magnification of 630-fold using Zeiss Axioskop with AxioVision. For each sample there were two eyes coming from different monkeys (except for one of the samples treated with Avastin on 1d, because the monkey had endophthalmitis after the injection, so we did not take a photo of that sample). Almost every sample came from the same place (i.e. the layer which included the optic nerve) for each eye. One section was selected for photography, and we then took 6 photos in different areas of the inner segment and RPE in each eye. All images were made using Image-Pro Plus, in order to measure the content of VEGF in the inner segment. The first step in these photos was to define all the inner segment and RPE as the area of interest (AOI). We took 5 AOIs for each image, and each one we selected was isolated using Image-Pro Plus software. The next step was to stain each AOI area using a color cube base which absorbed the red color in the AOI.

The intensity of the red staining from the control was set as a standard value, and then every other section needing color was set against this standard to ensure that each film used the same standard and parameters.

Statistical AnalysisThe ANOVA test from JMP10.0 statistical program (SAS, Heidelberg, Germany) was used to evaluate the results.

RESULTS

ImmunohistochemistryRetinal sections were checked for their anti-VEGF immune reactivity. The red color depicting VEGF in the RPE and inner segment was most intense in the untreated controls. On 1d after the injection, it seemed that all the drugs could reduce VEGF in the RPE and inner segment, if depending only on the basis of color. This was most obvious with bevacizumab on 1d compared to the other drugs and other days. The second most effective on 1d was ranibizumab.

Light MicroscopyAll sections of the untreated eyes, and the eyes treated with bevacizumab, ranibizumab and aflibercept were examined under identical conditions using 630 magnification, the focus being on the RPE and inner segment.

On 1d after the injection, it seemed that all the drugs could reduce VEGF in the RPE and inner segment, if depending only on the basis of color. This was most obvious with bevacizumab on 1d compared to the other drugs and other days. The second most effective on 1d was ranibizumab (Figure 1).

We used the application Image-Pro Plus to measure integral optical density (IOD) in all sections, and the area of VEGF in the inner segment and RPE of the retina. After calculation, we obtained the optical density of the inner segment and RPE. The ANOVA test from JMP10.0 statistical program was used to evaluate the results.

A difference ofP<0.001 between the controls and all the treated monkey eyes was considered statistically significant. In the comparison of all treated monkeys on 1d and 7d, we compared all the 6 databases with each other, Eylea 1d and Lucentis 1d, Eylea 1d and Avastin 1d, Eylea 1d and Avastin 7d, and whenPvalues were <0.05, the difference was considered statistically significant. But otherPvalues of them>0.05 when considering the difference between the treated

Table 1Pvalue of untreated and after injection

Figure 2Box-plot of semi-quantification of VEGF in the inner segment and RPE of the retina.

monkey eyes on other days were not taken to be statistically significant (Table 1, Figure 2).

DISCUSSION

Our results show that in the comparison between the controls and the treated monkeys, the difference was considered statistically significant whenP<0.001. This demonstrates that the VEGF-A inhibitors bevacizumab, ranibizumab and aflibercept can all reduce the level of VEGF in the RPE and inner segment.

The differences between Eylea 1d and Lucentis 1d,P=0.0080; Eylea 1d and Avastin 1d,P=0.0099; Eylea 1d and Avastin 7d,P=0.0172,P<0.05 were considered statistically significant. When these results are combined with the images (Figure 1), Avastin appears to be more effective than Eylea in reducing VEGF in the RPE and inner segment. Moreover, if we only compare Lucentis 1d and Eylea 1d, Lucentis 1d also appears more effective than Eylea 1d. The otherP>0.05 values indicate that between Eylea 7d and Lucentis 7d; Eylea 1d and Lucentis 7d; Lucentis 7d and Lucentis 1d; Lucentis 7d and Avastin 1d; Eylea 1d and Eylea 7d; Lucentis 7d and Avastin 7d; Avastin 7d and Lucentis 1d; Avastin 7d and Avastin 1d; Avastin 1d and Lucentis 1d, there are not any obviously different effects in reducing VEGF in the RPE and inner segment. Thus according to these results, Avastin appears to be the strongest of the three drugs, Lucentis is stronger than Eylea on 1d, but on 7d these two drugs have the same effect in reducing VEGF in the RPE and inner segment.

Medications injected into the vitreous cavity reach the systemic circulation and can even exert an effect on the contralateral eye[11]. So this is the reason why we chose monkey eyes which had not been injected in either eye as the control.

Monoclonal antibodies (anti-VEGF agents) have been used to target VEGF[12]. VEGF was originally identified as an endothelial cell-specific growth factor that can stimulate angiogenesis and enhance vascular permeability. VEGF-A is the most well-studied member of the VEGF family and is a key target for antiangiogenic therapy[13].VEGF was also shown to be a survival factor for photoreceptor cells, Müller cells and ganglion cells[14-15].

All of these three drugs can reduce VEGF in the RPE and inner segment of retina. But the current study reports a continuous increase in sub-retinal fibrosis during the first year of bevacizumab treatment. At the beginning of the study, it was present in approximately 33% of analyzed scans and after 12mo of treatment in 52% of scans in 3D imaging mode with visible sub-retinal fibrosis increased, mean central retinal thickness decreased[16].

Furthermore, other studies confirm that even if there is an incredible short-term gain in visual acuity after anti-VEGF drugs, this effect diminishes after at least 24mo of treatment[17]. Moreover, RPE is also responsible for the passage of nutrients and oxygen from the choroid towards the retina, but if damaged, the RPE may not be able to fulfil this task, which would lead to disturbance in photoreceptor renewal. The choriocapillaris is dependent on RPE-derived VEGF. Mouse studies showed that aggressively destroying VEGF could unintentionally destroy the choriocapillaris and furthermore produce geographic atrophy[14,18-19]. In addition, evidence exists for long-term retinal complications including atrophy with anti-VEGF use in AMD, and the drugs are not freely available in a sterile form in many parts of the world[20].

Until very recently the efficacy of ranibizumab seemed greatest, and remains accompanied by a large body of evidence, and a good ocular safety profile. Very recent evidence has emerged from a large randomized controlled trial (RCT) that aflibercept may be more efficacious in patients with poor vision at baseline[21].

Invitro, the same constellation with ranibizumab instead of bevacizumab did not induce formation of protein complexes[22]. Strong haemolysis in the choriocapillaris and in deeper choroidal vessels as well as the presence of extracellular haemoglobin known to be toxic were other important aspects in which aflibercept differs from ranibizumab and the controls[23]. Ranibizumab-treated eyes showed weaker haemolysis[24].

Aflibercept, like bevacizumab, is produced in Chinese hamster ovary (CHO) cells and therefore contains sugar residues in contrast to ranibizumab, which is produced in Escherichia coli. Thus, it is possible that the Fc domain and sugar residues[25-26]of aflibercept (and on bevacizumab)[27]facilitate interference of the VEGF trap with the physiological metabolism or functioning of cells and can result in complement-mediated red blood cell death[24].

In this study, one case had endophthalmitis after injection of Avastin. Further research needs to be done in the future as to whether this is the natural course of an injection complication or a side-effect of Avastin.

To summarize, Avastin might be more effective than Lucentis and Eylea for treating wet AMD and ocular neovascular diseases, but excessively reducing VEGF might induce geographic atrophy, sub-retinal fibrosis haemolysisetc. In this study there seems to be no obvious difference in the effectiveness of Lucentis and Eylea, so this must be further investigated.

It should be noted that this study has examined only limited samples, and antibodies were used to see the effects of the anti-VEGF drugs. We have to point out that we do not think this is sufficient. Unfortunately, we cannot determine the effect from this data alone. But without standing its limitation, this study does suggest that all these three drugs can reduce VEGF in the inner segment and RPE. Furthermore, they should have some different effects, because they have different structures. In the future, these problems could be solved if we also studied the level of platelet-derived growth factor (PDGF), the antilog of VEGF; and analyzed the agents in the inner segment and RPE etc. Apart from this, we would like to obtain more information on how the anti-VEGF drugs influence the cellular structures through electron microscopy studies.

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摘要

關鍵詞：抗血管內皮生長因子藥物；貝伐單抗；雷珠單抗；阿柏西晉；視網(wǎng)膜色素上皮；光感受器內節(jié)；血管內皮生長因子

作者簡介：蘇楠，畢業(yè)于河南醫(yī)科大學，學士，主任醫(yī)師，研究方向：眼底病。

通訊作者：李付廣，畢業(yè)于鄭州大學，博士，教授，免疫學系副主任，碩士生導師，研究方向：腫瘤免疫. lifuguang@zzu.edu.cn

DOI:10.3980/j.issn.1672-5123.2016.6.03

·Original article·