Purpose
To determine intra-individual long-term stability of vascular endothelial growth factor (VEGF) suppression time in eyes with neovascular age-related macular degeneration (AMD) treated with ranibizumab.
Design
Nonrandomized, prospective clinical study.
Methods
Eighty-three eyes of 83 patients with neovascular AMD undergoing intravitreal ranibizumab injections were included in the study. A total of 859 aqueous humor specimens were taken before each intravitreal ranibizumab injection. Vascular endothelial growth factor A was measured by multiplex bead analysis.
Results
Ranibizumab resulted in complete VEGF suppression within a mean period of 36.4 days (standard deviation ±6.7 days; range, 26-69 days). Intra-individual suppression time was stable within a period of up to 3 years. Among 859 VEGF measurements, only 5 (0.58%) deviated from this pattern. Nonsuppressed VEGF levels did not differ significantly between baseline and recurrence (68.0 pg/mL vs 69.3 pg/mL) and did not correlate with choroidal neovascularization size and lesion type.
Conclusions
Both the long-term stability and the broad range of individual suppression times after ranibizumab injections would allow and justify adjustment of continuous injections individually in order to achieve permanent VEGF suppression in patients.
Age-related macular degeneration (AMD) is a common cause of vision loss. Neovascular AMD is characterized by choroidal neovascularization (CNV), an outgrowth of blood vessels that invade the subretinal and intraretinal spaces, causing exudation, hemorrhage, and, subsequently, rapid visual decline. Targeting vascular endothelial growth factor (VEGF) is an effective treatment for the preservation and improvement of visual acuity. The best outcome is achieved by intravitreal injection of anti-VEGF drugs at monthly intervals or using an optical coherence tomography (OCT)-guided pro re nata regimen. Recently, we determined that ranibizumab resulted in complete VEGF suppression over a mean period of 37.8 days. Aqueous humor samples can be used, as aqueous humor levels correlate with vitreous levels. However, it remained unclear whether suppression periods maintain long-term stability. In this study, we therefore measured VEGF suppression in aqueous humor specimens of 83 patients over a period of up to 36 months. If it were true that VEGF suppression periods after ranibizumab injections show intra-individual long-term stability, this would potentially allow tailoring of individual regimens instead of using fixed monthly injections. Our data suggested that suppression periods remained intra-individually stable.
Subjects and Methods
Study Population
This prospective, observational study comprised 83 eyes of 83 patients 60 years of age or older with active subfoveal CNV secondary to AMD. All eyes were examined and treated at the Department of Ophthalmology, University of Cologne, Cologne, Germany. The study was performed in accordance with the tenets of the Declaration of Helsinki. Approval was obtained from the Ethics Committee of the University of Cologne (reference number 11-027), and written informed consent was provided by all participants. The study was registered at ClinicalTrials.gov (Identifier NCT01213667 ).
Inclusion and Exclusion Criteria
All patients had active subfoveal or juxtafoveal CNV attributable to AMD confirmed by fluorescein angiography (FA) with indocyanine green and spectral-domain optical coherence tomography (SDOCT). Further criteria in the study eye were a best-corrected visual acuity ≥20 Early Treatment of Diabetic Retinopathy Study (ETDRS) letters and no previous treatment for neovascular AMD, such as photodynamic therapy or intravitreal injections in the study eye. Exclusion criteria included any previous ophthalmic surgery, except for cataract removal.
Diagnostics and Treatment
Patients initially received 3 consecutive, monthly intravitreal injections of 0.5 mg ranibizumab. After this first series of treatments, patients were monitored monthly. Evaluations included SDOCT, standardized ETDRS visual acuity, and fundus examination. FA and indocyanine green were used in unclear cases only. Recurrence or persistence of CNV activity was defined as fluid seen by OCT or leakage seen on FA, loss of ≥5 letters in ETDRS visual acuity, or new macular intraretinal or subretinal hemorrhage. Recurrences were again treated with a series of 3 consecutive, monthly ranibizumab injections. As in Germany ranibizumab is only re-funded after a written request, those delays lead to variable reinjection intervals over time, allowing us to determine VEGF suppression periods without experimental assignment of injection intervals. The CNV size (mm 2 ) was measured on fluorescein angiograms with the HRA-2 software (Heidelberg Engineering, Heidelberg, Germany).
Measurement of Vascular Endothelial Growth Factor
Prior to the intravitreal injection of ranibizumab, about 0.1 mL of aqueous humor was collected via a limbal paracentesis with a 30-gauge needle connected to an insulin syringe and immediately stored at −80 C. The samples were analyzed with the Luminex xMAP microbead multiplex technology (Luminex 200; Luminex Inc, Austin, Texas, USA) according to the manufacturer’s instructions (Angiogenesis Panel; R&D Systems, Wiesbaden, Germany). Standard curves for VEGF were generated using the reference standard supplied with the kit and showed a detection threshold of 4 pg/mL for VEGF.
Statistical Analysis
Study data were tested for statistically significant changes of VEGF levels or correlations using Spearman correlations or nonparametric tests as specified. Statistical evaluation was performed at a significance level of α = 0.05. Statistical analysis was conducted using SPSS software (v. 21.0; IBM, Chicago, Illinois, USA).
Results
Eighty-three patients were prospectively recruited within an interventional case series. The clinical characteristics of the study population are depicted in the Table . Mean follow-up was 18.8 ± 10.3 months (range 1.8-35.9), with 56 patients reaching a follow-up time of at least 12 months, and 24 months follow-up for 38 patients. While a total of 942 intravitreal injections were administered, we were able to acquire 859 anterior chamber tap specimens. VEGF levels were determined an average of 10.3 times (SD ±5.1, range 3-21) per patient. VEGF levels at baseline and at re-treatment at the beginning of a new injection series are shown in the Table . There was no statistical difference between these 2 time points ( P = .65, Wilcoxon). Baseline VEGF levels did not correlate with CNV type and lesion size (r s = −0.015/−0.031, P = .894/.793, Spearman).
| Study participants | 83 patients |
| Sex, n (%) | 35 (42%) men |
| 48 (58%) women | |
| Age (y) at first ranibizumab treatment, mean ± SD (range) | 76.8 ± 7.2 (60-95) |
| Eyes | 83 eyes; 51% right, 49% left |
| Type of CNV, n (%) | 46 (55%) occult |
| 4 (5%) minimally classic | |
| 20 (24%) predominantly classic | |
| 13 (16%) retinal angiomatous proliferation | |
| Number of intravitreal injections per patient, mean ± SD (range) | 11.4 ± 5.9 (3-24) |
| VEGF suppression time (d), mean ± SD (range) | 36.4 ± 6.7 (26-69) |
| Aqueous VEGF concentration (baseline) (pg/mL), mean ± SD (range) | 68.0 ± 32.0 (17.8-179.6) |
| Aqueous VEGF concentration (new treatment series) (pg/mL), mean ± SD (range) | 69.3 ± 31.6 (9.0-179.0) |
| CNV size (mm 2 ), mean ± SD (range) | 5.0 ± 3.7 (0.25-20.7) |
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