RETINAL DISORDERS

Aqueous vascular endothelial growth factor and ranibizumabconcentrations after monthly and bimonthly intravitrealinjections of ranibizumab for age-relatedmacular degeneration

Xiying Wang & Tomoko Sawada & Masashi Kakinoki & Taichiro MiyakeHajime Kawamura & Yoshitsugu Saishin & Ping Liu & Masahito Ohji

Received: 29 July 2013 /Revised: 7 October 2013 /Accepted: 16 October 2013# Springer-Verlag Berlin Heidelberg 2013

AbstractPurpose To evaluate vascular endothelial growth factor(VEGF) and ranibizumab concentrations in eyes with age-related macular degeneration (AMD) after monthly and bi-monthly intravitreal ranibizumab (IVR) injections.Methods Aqueous humor samples were obtained from 26eyes with AMD before and after IVR injections. Nine eyesreceived three monthly injections and 17 eyes received twobimonthly injections. The VEGF and ranibizumab concentra-tions were measured by enzyme-linked immunosorbentassay.Results The aqueous VEGF concentrations in the monthlyinjection group decreased below the lowest detectable limitin eight of nine eyes 1 month after the first injection and sevenof nine eyes 1 month after the second injection (P <0.001,mean baseline value, 94.7 pg/ml); the aqueous VEGF concen-trations in the bimonthly injection group decreased below thelowest detectable limit in two of 17 eyes 2 months after thefirst injection (P <0.001, mean baseline value, 152.4 pg/ml).The mean aqueous ranibizumab concentrations with monthlyinjections were 71.2 ng/ml 1 month after the first injection,and 96.3 ng/ml 1 month after the second injection. The mean

aqueous ranibizumab concentrations in the bimonthly injec-tion group were 2.5 ng/ml in 15 of 17 eyes, and below thelowest detectable limit in two of 17 eyes 2 months after thefirst injection.Conclusions In this pilot study with limited follow-up, intra-vitreal injection of ranibizumab can suppress aqueous VEGFcompletely for 1 month in most cases. Its effect does not lastfor 2 months enough to suppress VEGF completely in mostcases, although aqueous VEGF at 2 months after intravitrealinjection of ranibizumab is less than that before injection inmost cases.

Keywords Age-related macular degeneration . Aqueoushumor . Ranibizumab . Bimonthly intravitreal injection .

Vascular endothelial growth factor

Introduction

Vascular endothelial growth factor (VEGF) plays a key role inthe development of neovascularization in exudative age-related macular degeneration (AMD) [1–7], the leading causeof irreversible blindness among aging populations in devel-oped countries [8–11]. The aqueous VEGF concentration ineyes with AMD and polypoidal choroidal vasculopathy(PCV) increases compared with normal eyes [12–16].

Ranibizumab (Lucentis, Genentech Inc., South SanFrancisco, CA, USA) is a recombinant, humanizedmonoclonalantibody Fab fragment that neutralizes all biologically activeforms of VEGF-A. Anti-VEGF therapy with ranibizumab sig-nificantly decreased the VEGF concentrations in the aqueoushumor of eyes with AMD [14–16]. Monthly injections of0.5 mg of ranibizumab prevent visual loss, and the mean visualacuity (VA) increased after 2 years in patients with all types of

The Institutional Review Board of Shiga University of Medical ScienceHospital approved this study, which is registered at http://www.umin.ac.jp (No. UMIN000005691). All patients providedinformed written consent before participation.

X. Wang : T. Sawada (*) :M. Kakinoki : T. Miyake :H. Kawamura :Y. Saishin :M. OhjiDepartment of Ophthalmology, Shiga University ofMedical Science,Seta Tukinowacho, Otsu, Shiga 520-2192, Japane-mail: tsawada@belle.shiga-med.ac.jp

X. Wang : P. LiuKey Laboratory of Harbin Medical University Eye Center, EyeHospital, First Affiliated Hospital, Harbin Medical University,Harbin, People’s Republic of China

Graefes Arch Clin Exp OphthalmolDOI 10.1007/s00417-013-2505-2

choroidal neovascularization (CNV) secondary to AMD[17–19]. It is unclear if monthly intravitreal ranibizumab(IVR) injections are needed. Monthly injections may increasethe risk of intraocular infection and impose a heavy economicburden on patients. Some investigators administered quarterlyIVR injections after three monthly loading doses to treat AMD,but this regimen did not achieve noninferiority to monthlyinjections, which means that a regimen with more frequentdrug administration is needed [20–22].

Increasing interest also has focused on aflibercept (VEGFTrap-Eye, Regeneron Pharmaceuticals, Tarrytown, NY, USA).Unlike ranibizumab and bevacizumab (Avastin, GenentechInc.), aflibercept is a recombinant fusion protein comprised ofportions of human VEGFR1 and VEGFR2 extracellular do-mains fused to the Fc portion of human immunoglobulin G1[23, 24]. With an extraordinarily high VEGF binding affinity,aflibercept binds all VEGF-A, VEGF-B, and placental growthfactor. The VIEW I andVIEW2 studies, two parallel, phase III,double-masked, randomized, multicenter trials, evaluated theefficacy and safety of bimonthly injections of aflibercept afterthree monthly injections of the drug, compared with monthlyinjections of ranibizumab. The results indicated that bimonthlyinjections of aflibercept were not inferior to monthly injectionsof ranibizumab over a 1-year period [25]. However, little dataare available on bimonthly injections of ranibizumab. Thecurrent study investigated the aqueous VEGF and ranibizumabconcentrations in patients with AMD after monthly and bi-monthly IVR injections.

Material and methods

Patients

Twenty-six eyes of 26 patients were enrolled between August2009 and August 2012. The monthly injection group includednine eyes, and the bimonthly injection group included 17 eyes.Inclusion criteria included age over 50 years, subfoveal lesionswith CNV secondary to neovascular AMD or PCV cases diag-nosed according to fluorescein angiography (FA) depending onthe classification of previous paper [26] and indocyanine greenangiography (IA). Patients who received previous AMD treat-ments and any retina surgery or vitrectomy history wereexcluded.

The Institutional Review Board of Shiga University of Med-ical Science Hospital approved this study, which is registered athttp://www.umin.ac.jp (No. UMIN000005691). All patientsprovided informed written consent before participation.

Treatment and aqueous humor preparation

Anesthesia was induced using topical 4 % xylocaine beforethe intravitreal injections were administered. A sterile lid

speculum was used in all cases. The ocular surface wasprepared using a povidone–iodine solution. Undiluted aque-ous humor samples were obtained from the eyes using a 29-gauge needle, followed by an IVR injection (0.5 mg/ 0.05ml).The samples were stored at −80 °C until analysis. Patientswere treated with a topical ophthalmic antibiotic four timesdaily starting 3 days before the day of injection and continuingfor 3 days after the injection.

Measurement of VEGF concentrations

Aqueous VEGF concentrations were measured using anenzyme-linked immunosorbent assay (ELISA) (QuantikineHuman VEGF Immunoassay, R&D Systems, Minneapolis,MN, USA), according to the manufacturer’s instructions[16]. The lowest detectable limit of the VEGF concentrationwas 9.0 pg/ml.

Measurement of ranibizumab concentrations

Aqueous ranibizumab concentrations were measured byELISA. Ninety-six-well plates were coated with recombinanthuman VEGF165 (R&D Systems) at a concentration of1.0 μg/ml (100μl/well) overnight at 4 °C. After washing threetimes with phosphate-buffered saline (PBS) containing0.05 % Tween-20, the wells were blocked with 3 % bovineserum albumin (BSA)/PBS overnight at 4 °C (200 μl/well).The wells then were washed five times with PBS containing0.05%Tween-20 and stored dry at 4 °C for later use. Aqueoushumor samples diluted in 0.1 % BSA/PBS were added to theplates overnight at 4 °C (50 μl/well). Ranibizumab was de-tected by horseradish peroxidase-goat anti-human IgG/F(ab’)2 (Pierce Biotechnology Inc., Rockford, IL, USA) at aconcentration of 1 μg/ml after a 2-hour incubation at roomtemperature. After five washes, color development was per-formed with a 100 μl 3, 3’, 5, 5”-tetramethylbenzidine sub-strate, and the reaction was stopped by adding 1 M hydrogenchloride (100μl). The optical density wasmeasured at 450 nmwith correction at 570 nm. The minimal quantifiable concen-tration of ranibizumab in the aqueous humors was 15.6 pg/ml.Because the sample volumes were small, we diluted them to10 times and carried out the measurement. Therefore, thelowest detectable limit of the ranibizumab concentration was156 pg/ml (0.156 ng/ml).

Examinations

The best-corrected VA (BCVA) and the central retinal subfieldthickness (CRST) were measured before and monthly after theinitial intravitreal injection. The BCVAwas measured using aLandolt C chart and converted to the logarithm of the mini-mum angle of resolution (logMAR) VA and analyzed. TheCRSTwas measured using spectral-domain optical coherence

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tomography (SD-OCT) (Cirrus HD-OCT, Carl Zeiss Meditec,Dublin, CA, USA). The average retinal thickness in the cen-tral 1-mm area obtained from the macular thickness analysiswas analyzed. In all eyes, FA and IA were performed beforethe first injection.

Statistical analysis

Statistical analysis was performed using the Sigma Stat statis-tical software version 3.1 (Systat Software Inc., Richmond,CA, USA). One-way repeated measures analysis of variance,Friedman repeated-measures analysis of variance by ranks, orthe paired t -test was used to compare the differences in theaqueous VEGF concentrations, BCVA, and CRST before andafter the IVR injections. P <0.05 was considered significant.AVEGF concentration below the lowest detectable limit wascalculated as 0 pg/ml when the data were calculated.

Results

The baseline characteristics of the study patients are summa-rized in Table 1. No major differences were found between thetwo study groups except for the baseline aqueous concentra-tion. According to the classification as published previously[26], in the monthly injection group, seven eyes of ninepatients were diagnosed with neovascular AMD, includingtwo eyes with a minimally classic lesion and five eyes with anoccult lesion with no classic lesions identified by fluoresceinangiography (FA); the other two eyes were diagnosed withPCV, which was identified by indocyanine green angiography.In the bimonthly injection group, 13 eyes of 17 patients werediagnosed with typical AMD (one eye with a predominantlyclassic lesion, four eyes with minimally classic lesions, andeight eyes had occult and no classic lesions identified by FA).The other four eyes were diagnosed with PCV.

In the monthly injection group, the aqueous VEGF con-centrations ranged from 47.2 to 155.1 pg/ml (mean±standarddeviation [SD], 94.7±32.0 pg/ml) at baseline, and decreasedto below the lowest detectable limit in eight of nine eyes and64.5 pg/ml in one of nine eyes at month 1 (1 month after thefirst injection) and decreased to below the lowest detectablelimit in seven of nine eyes, 17.7 pg/ml and 58.9 pg/ml in theother two eyes respectively at month 2 (1 month after thesecond injection). The aqueous VEGF concentration in themonthly injection group decreased significantly (P <0.001,for both comparisons) at months 1 and 2 (Fig. 1).

In the bimonthly injection group, the aqueous VEGF con-centrations ranged from 41.9 to 380.7 pg/ml (mean±SD,152.4±80.1 pg/ml) at baseline and decreased to below thelowest detectable limit in two eyes at month 2 (2 months afterthe first injection). The aqueous VEGF concentrations rangedfrom 10.3 to 216.1 pg/ml (mean±SD, 69.4±60.9 pg/ml) in 15

of 17 eyes at month 2, and decreased significantly at month 2(P <0.001, Fig. 2).

The ranibizumab concentrations in the monthly injectiongroup ranged from 9.0 to 163.0 ng/ml (mean±SD, 71.2±48.6 ng/ml) at month 1 (1 month after the first injection) andfrom 16.3 to 222.7 ng/ml (mean±SD, 96.3±65.4 ng/ml) atmonth 2 (1 month after the second injection). Theranibizumab concentrations in the bimonthly injection groupranged from 0.2 to 6.9 ng/ml (mean±SD, 2.5±2.2 ng/ml) infifteen of 17 eyes, and were below the lowest detectable limitin the other two of 17 eyes at month 2 (2 months after the firstinjection) (Fig. 3). When the ranibizumab and VEGF concen-trations were evaluated, the aqueous VEGF concentrationdecreased and the aqueous ranibizumab concentration in-creased, with the exception of a few cases. An aqueousranibizumab concentration of about 5 ng/ml or greater seemedto suppress the aqueous VEGF concentrations to below thelowest detectable limit (Fig. 4).

The mean logMAR BCVAvalues in the monthly injectiongroup were 0.66±0.32 at baseline, 0.69±0.35 at month 1, and0.61±0.33 at month 2 (95 % confidence interval: −0.15 to

Table 1 Baseline characteristics of the study patient

Monthly injectiongroup (n=9)

Bimonthly injectiongroup (n =17)

P value

Age, years 67.9±8.5 73.0±9.3 0.18

Sex, male/female 8/1 12/5 0.38

VA 0.66±0.32 0.44±0.42 0.09

CRST, μm 398.4±207.3 363.1±84.1 0.66

Aqueous VEGFconcentration,pg/ml

94.7±32.0 152.4±80.1 0.02

Prior therapy forAMD

No No –

PCV patients 2 4 –

Fig. 1 The aqueous VEGF concentrations in eyes treated with monthlyIVR injections. The VEGF concentrations decrease significantly (P<0.001for both comparisons) after IVR injections at months 1 and 2

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0.10 and −0.07 to 0.18, baseline versus month 1 and baselineversus month 2 respectively). No significant differences werefound in the logMARBCVA in the monthly injection group atbaseline and after treatment. The mean logMARVAvalues inthe bimonthly injection group were 0.44±0.42 at baseline,0.40±0.41 at month 1, and 0.36±0.40 at month 2 (95 %confidence interval, −0.03 to 0.10 and −0.12 to 0.16; baselineversus month 1 and baseline versus month 2 respectively).The VA tended to improve, although there were no significantdifferences between baseline and months 1 or 2 in the twotreatment groups (Fig. 5a).

The mean CRST values in the monthly injection groupwere 398±207 μm at baseline, 262±77 μm at month 1, and221±60 μm at month 2 (95 % confidence interval, −33.78 to305.78 and 29.59 to 325.96, baseline versus month 1 andbaseline versus month 2 respectively). The CRST value inthe monthly injection group at month 1 decreased significant-ly (P <0.05) compared with baseline. In the bimonthly injec-tion group, the mean CRST values were 363±84 μm atbaseline, 263±72 μm at month 1, and 297±103 μm at month

2 (95 % confidence interval: 70.25 to 130.93 and 17.45 to114.31, baseline versus month 1 and baseline versus month 2respectively). The CRST decreased significantly (P <0.05) atmonth 1 compared with baseline. The differences in the CRSTvalues between baseline and months 1 and 2 in the bimonthlyinjection group were significant (P <0.001 for both compari-sons) (Fig. 5b).

Discussion

To the best of our knowledge, the current study was the first toanalyze the levels of aqueous VEGF and ranibizumab in eyeswith AMD treated with bimonthly IVR injections.Ranibizumab was detected for at least 2 months, and aqueousVEGF concentrations decreased significantly 2 months afterthe first bimonthly IVR injection in most eyes. We measuredthe VEGF concentration in the aqueous humor rather than thevitreous humor because of the high correlation between aque-ous and vitreous levels of VEGF [27–29] and ease and lowerrisk of obtaining aqueous humor samples compared withvitreous samples.

In the current study, the baseline VEGF level of two groupshas a significant difference (P=0.02) which may result fromthe small and uneven number of patients in this study andfrom coincidence.

The aqueous VEGF was completely suppressed (decreasedto the lower limit of detection) with monthly IVR injections,similar to previous reports [14–16]. Meanwhile, in eyes thatreceived bimonthly IVR injections, a significant decrease inthe VEGF level also was observed in 14 (82.3 %) patients atmonth 2, although the aqueous VEGF concentrations in onlytwo eyes (11.8 %) were suppressed completely. The

Fig. 2 The aqueous VEGF concentrations in eyes treated with bimonthlyIVR injections. The VEGF concentrations decrease significantly (P<0.001)after IVR injections

Fig. 3 The ranibizumab concentrations in eyes treated with monthly andbimonthly injections of the drug. The lowest detectable limit is 0.156 ng/ml.Ranibizumab is detectable in eight eyes treated with bimonthly injections

Fig. 4 Correlation between aqueous VEGF and ranibizumab concentra-tions in eyes treated with monthly and bimonthly injections. With a fewexceptions, the aqueous VEGF level decreases and the aqueousranibizumab concentration increases. Since the ranibizumab concentra-tion increases to 5 ng/ml or higher, most aqueous VEGF is suppressedbelow the lowest detectable limit

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significant decrease of VEGF level seen at month 2 is inter-esting, because in rabbits and monkeys treated with one IVRinjection the half-life of intraocular ranibizumab was about3 days. From these studies, the period for ranibizumab toinhibit intraocular VEGF activity in patients with AMD wasexpected to last up to 30 days [28, 29]. However, Krohne andcolleagues confirmed that the aqueous half-life of 0.5 mg IVRinjections in human nonvitrectomized eyes with AMD, dia-betic retinopathy, and retinal vein occlusion was a mean of7.19 days [30], much longer than in animals. That finding wasconsistent with the present results in vivo.

Incomplete suppression of VEGF at month 2 in bimonthlygroup may indicate that the ranibizumab dose may be insuf-ficient to suppress aqueous VEGF for 2 whole months, andthe aqueous VEGF level may begin to increase at a certaintime point between 1 month and 2 months. Our result showedthat one intrealvireal injection of ranibizumab may suppress

VEGF for an extended time, which was more than 1 monthbut less than 2 months in human eyes with AMD.

In the bimonthly injection group, there were three eyes ofthree patients whose aqueous VEGF level didn’t change sig-nificantly 2 months after the first injection (P=0.23). Onepatient’s VEGF concentration increased from 101.01 pg/mlto 161.95 pg/ml 2 months after first injection. This may resultfrom the fact that this patient seemed to be a non-responder toranibizumab treatment, because the CRST of the patient didnot decrease (217 μm at baseline, 217 μm 1 month after thefirst injection and 216 μm 2 months after the first injection)and the logMAR VA did not improve after rainibizumabinjection either (0.82 at baseline, 1.0 one month after the firstinjection and 1.0 two months after the first injection). Theother two patients’ aqueous VEGF levels were slightly in-creased after the first injection. This may be due to variabilityof VEGF concentration in aqueous humor; their VEGF

Fig. 5 Clinical outcomes of twogroups. The solid circles indicatethe monthly injection group; theopen triangles indicate thebimonthly injection group. a ThelogMAR BCVA levels in the twogroups. There are no significantdifferences between baseline andmonths 1 or 2. b The CRSTmeasurements in the two groups.In the bimonthly injection group,there are significant (P<0.001)differences between baseline andmonths 1 and 2. In the monthlyinjection group, the CRST issignificantly (P<0.05) different atmonth 1 compared with baseline

Fig. 6 Findings on OCT in eyestreated with bimonthly injectionsat baseline and months 1 and 2. aThe OCT images obtained from apatient whose aqueous VEGFconcentration at month 2 hasdecreased to less than the lowestdetectable limit. b The OCTimages obtained from a patientwhose aqueous VEGFconcentration at month 2 is higherthan the lowest detectable limit.LV=left visual acuity

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baseline levels (41.85 pg/ml and 42.73 pg/ml) were muchlower than in other patients, which may be the reason for thesmall change in VEGF level after ranibizumab injection.

The aqueous ranibizumab level in the monthly injectiongroup was higher than that at month 2 in the bimonthlyinjection group after the initial injection. This is justifiablebecause of drug metabolism and excretion. The present studywas the first to describe aqueous ranibizumab concentrationsin human eyes with AMD 2 months after IVR injections.Surprisingly, in the current study the ranibizumab was detect-able at month 2 in eyes treated with bimonthly injections.According to our observations, the VEGF concentration de-creased with increases in the aqueous ranibizumab concentra-tion. The current measurements indicated that an aqueousconcentration of about 5 ng/ml or greater of ranibizumabsuppressed the aqueous VEGF level under the lowest thresh-old. We deduced that 5 ng/ml or greater is the lowest effectivedose of ranibizumab that can suppress the VEGF level belowthreshold. In a primate model, the ranibizumab concentrationin the vitreous was 2- to 3.3-fold higher than in the aqueous[29]. Furthermore, the half maximal inhibitory concentrationof ranibizumab for reducing VEGF was estimated to be 11 to27 ng/ml [31]. Therefore, the lowest ranibizumab concentra-tion of 5 ng/ml that can completely suppress the VEGFactivity in the aqueous in the current study can be estimatedto be 10 to 16.5 ng/ml in the vitreous, which was consistentwith a previous report [31].

Interestingly, when the aqueous VEGF level was below thelowest detectable limit in most eyes, the CRST was main-tained through the second month (Fig. 6a). In contrast, whenthe aqueous VEGF level was higher than the lowest detectablelimit, the CRST decreased at month 1 but recurrent thickeningwas seen at month 2 (Fig. 6b). Therefore, the VEGF level mayindicate the changes in the CRST after IVR treatment in eyeswith AMD. Although in Fig. 6b the CRST at month 2 in-creased compared with month 1, it was still lower than base-line, which also indicates the benefit of bimonthly injections.

In the current study, although there was no significantdifference in the VA results at months 1 and 2 compared withbaseline in the two groups, the VA improvement was clinical-ly relevant. In the bimonthly injection group, the mean VAimproved by 5.5 letters, and in the monthly injection group,the mean VA improved by 2.5 letters at month 2, while asignificant improvement in the CRST occurred in the eyes inthe monthly injection group at month 1 and the eyes in thebimonthly injection group at months 1 and 2. This may haveoccurred because the changes in the CRST preceded thechanges in the BCVA [32, 33]. The 2-month follow-up inthe current study may have been too short for significantBCVA improvements in a small number of eyes.

The current study should be considered a pilot study with asmall number of eyes and a short observation period. Fromour study, although bimonthly IVR injections would not

ensure complete VEGF inhibition in all patients, it not onlyprovides morphologic but also functional benefits to patientswith AMD. In eyes with AMD, morphologic recurrences(increased central retinal thickness) occurred at an averageof 93.7 days and functional recurrence (decreased VA) oc-curred at an average of 114.3 days [33], both of which aremuch longer than 2 months. Based on this, the injectioninterval of 60 days is enough to prevent significantrecurrences.

Compared with monthly injections, the number of bi-monthly intravitreal injections administered during 1 yearwas six, fewer than 12 monthly injections, and the bimonthlyinjection regimen eases the patients’ treatment burden eco-nomically and psychologically. Based on the positive resultsof the CLEAR-IT 2 study, the VIEW I and the VIEW 2 studycompared the efficacy and safety of monthly and bimonthlyinjections of aflibercept with ranibizumab injections, the goldstandard, and both studies reported noninferiority in visualoutcomes and safety at 1 year [25, 34]. Although the outcomesof the VIEW 1 and VIEW 2 studies showed that afliberceptwith 8-week dosing was not inferior to monthly IVR injec-tions of ranibizumab over a 1-year period, according to thecurrent findings, another study with more patients and alonger follow-up period is warranted to evaluate the efficacyof bimonthly injections of ranibizumab for treating AMD.

In conclusion, in this pilot study with limited follow-up,intravitreal injection of ranibizumab can suppress aqueousVEGF completely for 1 month in most cases. Its effect doesnot last sufficiently for 2 months to suppress VEGF complete-ly in most cases, although aqueous VEGF at 2 months afterintravitreal injection of ranibizumab is less than that beforeinjection in most cases.

Acknowledgments Supported in part by a grant from the Ministry ofEducation, Culture, Sports, Science and Technology of Japan (#24592668)and a grant from the Ministry of Health, Labour and Welfare.

The authors have no proprietary interest in any aspect of this study. Thefunding organizations had no role in the design or conduct of this study.

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