Purpose
To determine the duration of suppression of aqueous humor concentrations of vascular endothelial growth factor (VEGF) in eyes with neovascular age-related macular degeneration (AMD) treated with aflibercept.
Design
Nonrandomized prospective clinical study.
Methods
Twenty-seven eyes of 27 neovascular AMD patients receiving intravitreal aflibercept injections on a pro re nata regimen driven by spectral-domain optical coherence tomography (SD OCT) were included in this study. A total of 132 aqueous humor specimens were collected before intravitreal aflibercept injections and their VEGF-A concentrations assayed by multiplex bead analysis.
Results
Mean aqueous humor VEGF concentrations before treatment initiation were 90.6 ± 37.1 pg/mL (range 23.4–190.3 pg/mL). Intravitreal injection of aflibercept suppressed the aqueous VEGF concentrations to below the lower limit of quantification (<4 pg/mL) in all patients. The mean duration of VEGF suppression below the lower limit of quantification was >71 ± 18 days. The earliest time after injection at which the VEGF concentration recovered to above the lower limit of quantification was 55 days in 1 patient and >56 days, the recommended aflibercept treatment interval, in 20 patients. The aqueous VEGF recovery status of 6 patients was uncertain after 56 days.
Conclusions
On average, VEGF concentrations in the aqueous humor were suppressed below the lower limit of quantification after intravitreal aflibercept injections for about 10 weeks. This aqueous suppression time suggests durable VEGF inhibition for most patients dosed with aflibercept every 8 weeks.
Age-related macular degeneration (AMD) is a major cause of vision loss. The neovascular variant is characterized by choroidal neovascularization (CNV), in which formation of blood vessels leads to sub- and intraretinal macular edema, hemorrhage, fibrosis, and visual decay. Effective treatments have been developed recently, focusing on neutralizing vascular endothelial growth factor (VEGF) with antibodies (bevacizumab), antibody fragments (ranibizumab), or fusion proteins (aflibercept). Major clinical trials found 4-weekly injections of ranibizumab to result in best visual outcome for ranibizumab, and found that injections of aflibercept every 8 weeks (following a loading phase) provided similar functional benefits. As an alternative to fixed dosing intervals, pro re nata (PRN) treatments based on optical coherence tomography (OCT)-determined activity achieve similar functional results.
Clinical trials with aflibercept suggest a longer duration of VEGF suppression than with bevacizumab or ranibizumab, which is also supported by pharmacokinetic models. We have recently determined the average time for which aqueous humor VEGF concentrations are suppressed below the lower limit of quanitification of 4 pg/mL following intravitreal ranibizumab injections to be 37 days on average, with individual VEGF suppression times ranging from 26 to 69 days. Aqueous humor concentrations appear suitable for assessing ocular VEGF levels as they correlate well with vitreous VEGF concentrations, extrapolated from retinal vascular occlusive disease and diabetic retinopathy.
This study aimed to determine intraocular VEGF suppression duration following aflibercept treatment for neovascular AMD by sampling aqueous humor VEGF levels.
Materials and Methods
Study Population
This prospective, observational study enrolled 27 eyes of 27 patients who were 60 years of age or older and had active CNV secondary to AMD. All eyes were examined and treated at the Department of Ophthalmology, University of Cologne, Germany. The study was performed in accordance with the tenets of the Declaration of Helsinki. The protocol was approved by the Ethics Committee of the University of Cologne (reference number 11–027), and all participants gave written informed consent. The study was registered at ClinicalTrials.gov (Identifier NCT01213667).
Inclusion and Exclusion Criteria
All included patients were suffering from an active sub- or juxtafoveal CNV attributable to neovascular AMD. This was confirmed by fluorescein angiography and indocyanine green angiography as well as spectral-domain optical coherence tomography (SD OCT) (HRA-2 and Spectralis OCT; Heidelberg Engineering, Heidelberg, Germany). CNV size (mm 2 ) was determined from fluorescein angiograms using the HRA-2 software (Heidelberg Engineering). An additional inclusion criterion in the study eye was a best-corrected visual acuity ≥20 Early Treatment of Diabetic Retinopathy Study (ETDRS) letters. Exclusion criteria were any previous intraocular surgery (apart from cataract removal) or photodynamic therapy; any treatment with intraocular steroids; any ranibizumab/bevacizumab/pegaptanib treatment within the previous 90 days; and any previous aflibercept treatment.
Diagnostics and Treatment
Patients initially received 3 2-mg loading dose injections of aflibercept at intervals ranging from 4 to 6 weeks. After this first treatment phase, patients were monitored monthly by SD OCT, ETDRS best-corrected visual acuity tests, and fundus examinations. Fluorescein angiography was repeated only in unclear cases. CNV persistences or recurrences were treated by additional aflibercept injections on a PRN regimen mainly driven by morphologic findings in SD OCT. Recurrent or persistent CNV activity was detected as sub- or intraretinal fluid by SD OCT, leakage in fluorescein angiography, a loss of ETDRS letters if attributable to CNV activity, or new sub- or intraretinal macular hemorrhages. Because of variable treatment approval times of health insurances, individual disease activities, and patient appointment preferences, variable reinjection intervals occurred without any experimental study design.
Aqueous Humor Vascular Endothelial Growth Factor Measurements
Samples were acquired only upon necessary treatment. Prior to each aflibercept injection, approximately 0.1 mL of aqueous humor was collected via a sterile limbal puncture with a 30 gauge needle connected to an insulin syringe. The procedure of sample collection immediately followed by aflibercept injection was randomly performed by 3 surgeons. No surgeon was assigned to specific patients, cancelling out possible dosing variabilities. Samples were immediately stored at −80 C in polypropylene tubes until they were analyzed on a Luminex xMAP microbead multiplex platform (Luminex 200; Luminex Inc, Austin, Texas, USA) following the manufacturer’s assay instructions (Human Angiogenesis Panel; R&D Systems, Wiesbaden, Germany). Standard curves for VEGF were generated using the reference standard supplied with the kit. The lower limit of quantification for VEGF was 4 pg/mL.
Results
Intraocular VEGF concentrations were assayed in samples of aqueous humor from 27 patients undergoing PRN aflibercept treatment for neovascular AMD. The clinical characteristics of the study population are listed in the Table . We analyzed 132 aqueous humor samples of 149 intravitreal aflibercept injections administered during the study. VEGF levels (y-axis) were plotted in relation to the interval from the previous aflibercept injection (x-axis) for each patient; the very first aflibercept injection was defined as day 0. Representative examples are depicted in Figure 1 . Complete aqueous humor VEGF suppression was assumed when VEGF levels were below the lower limit of quantification of the analytical method (4 pg/mL).
| Study participants | 27 patients |
|---|---|
| Sex, n (%) | 13 (48%) male |
| 14 (52%) female | |
| Age at first aflibercept treatment (y), mean ± SD (range) | 77.5 ± 6.4 (64–90) |
| Eyes, n (%) | 27 eyes: 9 (33%) right, 18 (67%) left |
| Follow-up time per patient (mo), mean ± SD (range) | 7.7 ± 2.1 (4.1–12.0) |
| Number of intravitreal injections per patient, mean ± SD (range) | 5.5 ± 1.3 (3–8) |
| Aqueous VEGF concentration on day 0 (pg/mL), mean ± SD (range) | 90.6 ± 37.1 (23.4–190.3) |
| VEGF suppression time (days), mean ± SD (range) | 70.5 ± 18.0 (41–109) |
| Type of choroidal neovascularization, n | 18 occult |
| 4 mixed | |
| 2 classic | |
| 3 RAP | |
| Size of choroidal neovascularization (mm 2 ), mean ± SD (range) | 4.5 ± 3.4 (0.3–12.7) |
| Best-corrected visual acuity on day 0 (ETDRS letters), mean ± SD (range) | 59.5 ± 17.0 (20–85) |
| Central retinal thickness on day 0 (μm), mean ± SD (range) | 392 ± 132 (210–658) |
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