Purpose
To evaluate serum and plasma vascular endothelial growth factor (VEGF) concentrations in neovascular age-related macular degeneration patients treated bimonthly with an intravitreal injection of aflibercept or ranibizumab.
Design
Prospective, interventional case series.
Methods
This study includes 17 eyes of 17 patients treated with 2 mg aflibercept (the aflibercept group), 15 eyes of 15 patients treated with 0.5 mg ranibizumab (the ranibizumab group), and 12 patients with cataract (the control group). Serum and plasma VEGF concentrations were quantified using the enzyme-linked immunosorbent assay.
Results
At baseline, mean serum VEGF concentration (in picograms per milliliter) did not differ significantly among the 3 groups ( P = .99). In the aflibercept group, it was 28.3 pg/mL at baseline, decreased to below the detectable limit at 1 week ( P < .0001), increased to 11.7 pg/mL at 1 month, which was still significantly less than the baseline level ( P < .001), and returned to 23.9 pg/mL ( P = .35) at 2 months. In the ranibizumab group, there were no significant differences. At baseline, mean plasma VEGF concentration did not differ significantly among the 3 groups ( P = .64). In the aflibercept group, it was 16.2 at baseline, decreased to less than the detectable limit at 1 week ( P < .01) and at 1 month ( P < .05), and returned to 13.6 pg/mL at 2 months ( P = .73). In the ranibizumab group, there were no significant differences.
Conclusions
Aflibercept significantly decreased serum and plasma VEGF concentrations 1 month after injection; however, ranibizumab had no significant effect on either serum or plasma VEGF level.
Neovascular age-related macular degeneration (AMD) characterized by choroidal neovascularization is a primary cause of irreversible central vision loss in ageing populations of developed countries. With the identification of vascular endothelial growth factor (VEGF) as a key mediator of angiogenesis and the discovery of an elevated amount in eyes with AMD, intravitreal injection of VEGF inhibitor became an effective therapy for preserving and improving visual acuity in AMD patients.
Ranibizumab (Lucentis; Genentech, Inc, South San Francisco, California, USA) is a recombinant, humanized monoclonal antibody Fab fragment that blocks all active degradation products of VEGF-A. As the first anti-VEGF agent approved by the United States Food and Drug Administration, it has been shown to promote anatomic as well as functional recovery in patients with AMD.
Aflibercept (Eylea; Regeneron Pharmaceuticals, Tarrytown, New York, USA; Bayer AG, Leverkusen, Germany), the latest VEGF antagonist, is a fusion protein consisting of the binding domains of VEGF receptor-1 and VEGF receptor-2 with the ability to bind VEGF-A, VEGF-B, and placental growth factor. Clinical evidence from the VIEW 1 and VIEW 2 (VEGF Trap: Investigation of Efficacy and safety in Wet age-related macular degeneration) clinical trials suggests that aflibercept intravitreally injected bimonthly after 3 loading doses is noninferior to ranibizumab intravitreally injected monthly.
Although VEGF plays a prominent role as a pathologic factor in the development of neovascular AMD, it also participates in processes necessary for normal accommodation in vivo, including promoting endothelial cell growth and survival, regulating blood pressure, and maintenance of the microvasculature in many organs. Therefore, the systemic effect of intravitreal use of anti-VEGF agents has been a concern since they became first-line drugs for the treatment of AMD.
Previous reports have proved that intravitreal injection of ranibizumab can reduce aqueous VEGF level in AMD patients significantly, without affecting systemic VEGF level. So far, there are few published data on serum and plasma VEGF levels after intravitreal injection of aflibercept in patients with AMD. The present study aimed to evaluate the change in serum and plasma VEGF concentrations over a 2-month period after 1 intravitreal injection of aflibercept or ranibizumab in patients with AMD.
Methods
This prospective study was registered at http://www.umin.ac.jp (No. UMIN000010312). Its protocol was approved by the Institutional Review Board of Shiga University of Medical Science Hospital. All patients provided written informed consent before the study started.
Patients
This prospective, interventional case series investigated the levels of VEGF in serum and plasma from patients with active choroidal neovascularization secondary to AMD. Forty-four participants were enrolled, including 32 patients with choroidal neovascularization resulting from AMD (18 with neovascular AMD, 12 with polypoidal choroidal vasculopathy, and 2 with retinal angiomatous proliferation), and 12 age-matched patients scheduled for cataract surgeries served as a control group. Patients with any other ocular disease that had systemic pathologic features were excluded from the study.
Treatment
Treatment-naïve patients with AMD underwent bimonthly intravitreal injection of aflibercept or ranibizumab without a loading phase of monthly injection. Seventeen eyes of 17 patients received 1 intravitreal aflibercept injection (the aflibercept group, 2.0 mg/0.05 mL; Eylea; Regeneron Pharmaceuticals, Inc, Tarrytown, New York, USA; and Bayer HealthCare Pharmaceuticals, Berlin, Germany). Fifteen eyes of another 15 AMD patients received 1 intravitreal ranibizumab injection (the ranibizumab group, 0.5 mg/0.05 mL; Lucentis; Novartis, Basel, Switzerland).
The bimonthly intravitreal aflibercept or ranibizumab injection were administered as described in a previous report. In brief, eyes were anesthetized with topical 4% lidocaine using a sterile lid speculum. After the ocular surface was sterilized using a povidone–iodine solution, aflibercept or ranibizumab was injected. A topical ophthalmic antibiotic was applied 4 times daily starting 3 days before the day of injection and continuing for 3 days after the injection.
Sample Collecting
Venous blood samples were collected from patients just before the first injection, and then 1 week, 1 month, and 2 months after the injection. For serum preparation, blood samples from 17 patients in the aflibercept group, 15 patients in the ranibizumab group, and 12 patients in the control group were obtained (allowed to clot at room temperature for 30 minutes) and then centrifuged as mentioned in a previous article. For plasma preparation, blood samples from 7 patients in the ranibizumab group, 7 patients in the aflibercept group, and 10 patients in the control group were collected in tubes with sodium citrate, theophylline, adenosine, dipyridamole (catalog no. 367599; Becton Dickinson, Rutherford, New Jersey, USA) and centrifuged for 10 minutes at 1700 g within 30 minutes after sampling. Harvested serum and plasma samples were frozen immediately and stored at −80 C until assay.
Measurement of Vascular Endothelial Growth Factor Concentrations
Serum and plasma VEGF concentrations were measured by enzyme-linked immunosorbent assay of human VEGF (SVE00; R&D Systems, Quantikine, Minneapolis, Minnesota, USA) according to the instructions given by the manufacturer and mentioned previously. The lower limit of VEGF detection was 9.0 pg/mL. All samples were analyzed together in duplicate.
Statistical Analysis
SigmaStat statistical software version 3.1 (Systat Software, Inc, Richmond, California, USA) was used for data analysis. Baseline characteristic of the 3 groups were evaluated with a 1-way analysis of variance. The Friedman repeated-measures analysis of variance on ranks was used to compare baseline and follow-up data in each group. A P value of less than .05 indicated statistical significance. For analytic purposes, a VEGF concentration less than the lower limit of detection was considered to be 0.
Results
The baseline characteristics of the participants are presented in the Table . There was no difference among the 3 groups. No eye or systemic adverse events were observed during the 2-month observation period.
| Characteristics | Control Group | Aflibercept Group | Ranibizumab Group | P Value |
|---|---|---|---|---|
| Patients (n) | ||||
| Serum | 12 | 17 | 15 | — |
| Plasma | 10 | 7 | 7 | — |
| Age (y) | ||||
| Serum | 76.2 ± 8.6 | 75.6 ± 6.9 | 76.5 ± 7.4 | .95 |
| Plasma | 73.0 ± 8.2 | 74.6 ± 9.4 | 77.4 ± 10.6 | .63 |
| Gender (male/female) | ||||
| Serum | 4/8 | 13/4 | 11/4 | — |
| Plasma | 2/8 | 4/3 | 6/1 | — |
At baseline, the mean ± standard deviation serum VEGF concentration was 28.3 ± 9.8 pg/mL (range, 12.1 to 40.2 pg/mL) in the control group, 28.1 ± 10.6 pg/mL (range, 14.0 to 50.2 pg/mL) in the aflibercept group, and 27.5 ± 13.7 pg/mL (range, 9.3 to 53.7 pg/mL) in the ranibizumab group. There were no significant differences among the 3 groups ( P = .99; Figure 1 ).
In the aflibercept group, the mean serum VEGF concentration was significantly less than the detectable limit at 1 week after the injection in all the patients ( P < .0001) and increased to 11.7 ± 12.6 pg/mL (range, 0 to 49.7 pg/mL) at 1 month after the injection. The VEGF level in 6 (35.3%) of 17 patients was still less than the lower limit of detection, meaning that it was still significantly less than the baseline level ( P < .001) and returned to near-baseline levels (to 23.9 ± 18.7 pg/mL; range, 9.7 to 75.3 pg/mL) at 2 months after the injection ( P = .35; Figure 2 ).
In the ranibizumab group, the mean serum VEGF concentration was 36.2 ± 20.8 pg/mL (range, 0 to 82.1 pg/mL) at 1 week, 31.2 ± 19.4 pg/mL (range, 0 to 74.2 pg/mL) at 1 month, and 32.8 ± 31.6 pg/mL (range, 0 to 139.9 pg/mL) at 2 months ( Figure 3 ). There was no significant difference among the time points ( P = .36).
At baseline, the plasma VEGF concentrations were 29.5 ± 44.3 pg/mL (range, 0 to 153.8 pg/mL) in the control group, 16.2 ± 9.4 pg/mL (range, 10.1 to 29.6 pg/mL) in the aflibercept group, and 19.7 ± 10.8 pg/mL (range, 0 to 30.8 pg/mL) in the ranibizumab group. No significant difference was found among the 3 groups ( P = .64; Figure 4 ).
In the aflibercept group, plasma VEGF concentration was less than the limit of detection in all cases at 1 week ( P < .01). It remained less than the limit of detection and significantly less than the baseline level: in 5 (71.4%) of 7 patients, the VEGF concentrations were still less than the lower limit of detection at 1 month ( P < .05). Plasma VEGF concentration was at near-baseline levels (13.6 ± 5.1 pg/mL; range, 0 to 32.8 pg/mL) at 2 months ( P = .73; Figure 5 ).
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