Aims To determine the level of vascular endothelial growth factor (VEGF) in the plasma of patients with diabetic macular edema (DME) and of patients with exudative age-related macular degeneration (ARMD) before and after intravitreal injection of bevacizumab, ranibizumab or pegaptanib.
Methods 30 patients with DME and 30 patients with ARMD were included in this randomized controlled study. Patients were randomized to treatment with ranibizumab (0.5 mg), bevacizumab (1.25 mg) or pegaptanib (0.3 mg). 10 patients with DME received bevacizumab, 10 ranibizumab and 10 pegaptanib. The same randomized treatment allocation applied to the 30 patients with ARMD. The concentrations of VEGF were measured by ELISA just before the injection, after 7 days and 1 month.
Results Plasma VEGF in patients with exudative ARMD before the injection of bevacizumab was 89.7 pg/ml. It was significantly reduced to 25.1 pg/ml after 7 days (p=0.01), and to 22.8 pg/ml after 1 month (p=0.008). In patients with DME the same systemic reduction by bevacizumab was observed with a significant decrease of baseline VEGF level from 72.2 pg/ml to 13.7 pg/ml after 7 days (p=0.008) and 17.1 pg/ml at 4 weeks with (p=0.012). No significant reductions of plasma VEGF levels were observed in patients receiving ranibizumab or pegaptanib during follow-up.
Conclusions Bevacizumab significantly reduces the level of VEGF in the blood plasma for up to one month in patients with DME as well as in those with ARMD. No significant systemic effects of intravitreal ranibizumab or pegaptanib on plasma VEGF could be observed.
- Treatment Medical
- Clinical Trial
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Intravitreal injections for exudative age-related macular degeneration (ARMD), diabetic macular oedema (DME) and retinal vein occlusions are becoming the most frequent procedures in ophthalmology. Due to the availability in early days and high costs of ranibizumab (Lucentis; Novartis Pharma AG, Basel, Switzerland and Genentech Inc, South San Francisco, California, USA), the use of bevacizumab (Avastin; Genentech, Inc, San Francisco, California, USA) is expanding worldwide. The role of pegaptanib (Macugen; Pfizer/Eyetech Pharmaceuticals, New York, New York, USA) is more or less marginal, but approved for the treatment of wet ARMD.
Bevacizumab is a recombinant human monoclonal IgG1 antibody that inhibits human vascular endothelial growth factor (VEGF). It has been approved by the US Food and Drug Administration for intravenous use for metastatic colorectal cancer. Several prospective studies have been published on the off-label use of intravitreal bevacizumab in VEGF-mediated diseases.1–4
Intravitreal bevacizumab has been shown to be associated with a substantial decrease in the aqueous VEGF levels and plasma levels in patients with diabetic retinopathy and ARMD.5–9 For ranibizumab, only data of patients with wet ARMD are available, and no significant reduction in systemic VEGF plasma levels was found.9
VEGF is a multifunctional cytokine that regulates antiapoptotic pathways of endothelial cells in adult vasculature. Plasma VEGF acts as a vascular protective factor and is essential for maintaining the integrity and the antithrombogenic as well as anti-inflammatory properties of the endothelium.10 ,11 Prolonged suppression of plasma VEGF levels resulting from serial intravitreal anti-VEGF injections might, therefore, raise concern for unwanted systemic effects.
To the best of our knowledge, data have not been published of a prospective series determining the blood levels of VEGF after an intravitreal injection of bevacizumab, ranibizumab and pegaptanib in patients with exudative ARMD as well as patients with DME.
Thus, the purpose of this study was to determine the blood levels of VEGF after a single intravitreal injection of bevacizumab, ranibizumab or pegaptanib in patients with diabetic retinopathy, and in patients with exudative ARMD
Material and methods
This prospective study was elaborated according to the Declaration of Helsinki, and was performed after approval from the institutional review committee of the Medical University Innsbruck. Informed consent was obtained from all included patients. All patients did not receive intravitreal injections for at least 3 months prior to inclusion. Patients with anti-inflammatory treatment, like steroids, were excluded. Patients with proliferative diabetic retinopathy were excluded if they had neovascular glaucoma, rubeosis of the iris or progressive proliferative diabetic retinopathy. Eyes that had undergone vitrectomy were excluded.
Controls were established from 11 participants without any history of systemic and other ocular pathologies; subjects with ARMD, chorioretinal abnormalities, diabetes, hypertension and vasoproliferative disorders were excluded.
Ninety-seven patients with ARMD und DME were identified, 60 patients reached the inclusion criteria and were randomised by permuted block randomisation to treatment with bevacizumab, ranibizumab or pegaptanib. Of the 30 patients with decreased visual acuity and DME, 10 received bevacizumab, 10 were treated with ranibizumab and 10 with pegaptanib. The 30 patients with exudative ARMD were randomised to 10 patients treated with bevacizumab, 10 with ranibizumab and 10 with pegaptanib. Loses to follow-up are due to failure to attend medical appointments (no show) in all cases.
The intravitreal dose of bevacizumab was 1.25 mg/0.05 ml and for ranibizumab 0.5 mg/0.05 ml. A compounding pharmacy using aseptic methods placed the medication in a 0.5 ml syringe with an integrated 30-gauge needle by. For pegaptanib, the prefilled syringe of the manufacturer was used resulting in a dosage of 0.3 mg/0.09 ml. All intravitreal injections were performed under sterile conditions in the operating room. The medication was administered by injection 3.5–4.0 mm posterior to the limbus. After removal of the needle, a sterile cotton tip applicator was used to prevent reflux. Postoperatively, gentamicin eye drops were given four times per day.
Collecting blood samples
Blood samples were obtained 1–3 h prior to intravitreal injections, 1 week and 4 weeks after intravitreal injection.
Blood samples were drawn from all patients by venous puncture with minimal stasis. For the VEGF assay, blood samples were collected in tubes containing EDTA. Centrifugation was done at 3000 rpm for 20 min within 1 h after sampling. Plasma was stored at −20°C until the assay, which was done within 4 weeks after sampling.
VEGF plasma levels were determined by ELISA (Quantikine VEGF ELISA Kit, R&D Systems Europe, Abingdon, OX14 3NB, UK, #DVE00) as described by the manufacturer. Briefly, 100 µl of assay diluents RD1W was added to each well of 96-well polystyrene microplates, then 100 µl of standard or samples (EDTA-plasma) was added to each well, mixed by gently tapping the plate frame for 1 min, and incubated for 2 h at room temperature. Afterwards, washing with wash buffer (400 µl) was performed three times followed by addition of 200 µl of human VEGF conjugate to each well, incubation for 2 h at room temperature and washing again with wash buffer three times. Subsequently, 200 µl of substrate solution was added to each well, incubated for 25 min at room temperature and finally, 25 µl of stop solution was added to each well and the concentration was determined by an ELISA reader at 450 nm.
For statistical analyses, the baseline characteristics of the study groups were evaluated with the Kruskal–Wallis Test. The pairwise comparisons of baseline characteristics were evaluated with the Mann–Whitney Test, 2-tailed. The same tests were applied for the interpretation of follow-up data at 1 week and 4 weeks. Analyses within one group were performed with the Friedman test and the Wilcoxon signed rank test. p Values of less than 0.05 were considered to indicate statistical significance. IBM SPSS software V.19 was used for statistical analysis.
The mean plasma VEGF concentration of controls was 50.0±49.9 pg/ml. There were no significant differences comparing VEGF plasma levels of the control group and the baseline plasma concentrations of patients with ARMD or DME.
The mean (±SD) VEGF concentration in the plasma of patients with exudative ARMD before the injection of bevacizumab was 89.7±106.4 (range 31–382) pg/ml. It was significantly reduced to 25.1±10.5 (range 11–41) pg/ml after 7 days (p=0.01), and to 22.8±12.4 (range 4–38) pg/ml even after 1 month (p=0.008), respectively. The VEGF levels in patients treated with ranibizumab did not change significantly from baseline 91.3±59.9 (range 15–217) pg/ml to 135.5±133.3 (range 10–473) pg/ml at 7 days and 77.1±46.6 (range 43–178) pg/ml at 4 weeks of follow-up. Also in the pegaptanib group, no significant plasma VEGF variations could be observed with a baseline value of 105.5±131.7 (range 13–393) pg/ml, 139.7±125.1 (range 18–449) pg/ml at 7 days and 151.7±209.0 (range 9–585) pg/ml after 4 weeks (figure 1).
The same systemic reduction of plasma VEGF was observed in patients with DME treated with bevacizumab. The mean baseline plasma VEGF level significantly decreased from 72.2±64.4 (range 15–217) pg/ml to 13.7±11.9 (range 4–42) pg/ml after 7 days (p=0.008). This reduction persisted throughout 4 weeks with a plasma measurement of 17.1±10.3 (range 3–32) pg/ml (p=0.012). No significant reductions of plasma VEGF levels were observed in patients with DME receiving ranibizumab or pegaptanib at any point of follow-up time; in the ranibizumab group, the baseline VEGF level was 43.5±52.4 (range 15–191) pg/ml, 52.9±72.4 (range 12–238) pg/ml after 7 days, and 41.7±28.0 (range 17–99) pg/ml after 4 weeks. In the pegaptanib group, the baseline VEGF level was 57.1±49.5 (range 19–177) pg/ml, 57.1±43.4 (range 22–158) pg/ml after 7 days, and 76.9±56.4 (range 20–164) pg/ml after 4 weeks (figure 2).
Comparing the amounts of reduction of plasma VEGF after intravitreal bevacizumab injection in the ARMD and DME groups, no significant differences could be observed. The after-treatment reduction of plasma VEGF induced by bevacizumab in the ARMD group was mean −71.1±107.8 pg/ml (range −347–3) after 7 days, and −73.44±105.6 pg/ml (range −345 to −12) compared with baseline levels after 4 weeks.
In the DME group, the mean plasma VEGF reduction after intravitreal bevacizumab was −69.1±66.4 pg/ml (range −213 to −11) at 7 days, and −61.9±66.2 pg/ml (range −185 to −2) at 4 weeks.
Since the publication of the comparison of AMD treatment trials (CATT) and Inhibit VEGF in Age-related choroidal Neovascularisation (IVAN) study results, the off-label use of bevacizumab has been justified in ARMD, but concerns remain especially in diabetic patients.1 ,4 The paper ‘Effects of intravitreally injected bevacizumab on VEGF in fellow eyes’ by Matsuyama, and the follow-up paper about VEGF plasma levels in diabetic patients after a single injection of bevacizumab was not really appealing.7 ,8 In these studies, a significant decrease of plasma VEGF after the intravitreal injection of bevacizumab at 1 day, 7 days and even at 1 month in patients with diabetic retinopathy was observed.
Intravitreal anti-VEGF therapy is able to maintain vision in the majority of patients, but does not cure the underlying disease entity. Patients with neovascular ARMD, as well as patients with DME, need frequent retreatment for more than 2 years, the longest period of time currently documented by level I evidence. In our study, we found a significant reduction of VEGF in the plasma that persisted throughout 1 month in those patients treated with bevacizumab for ARMD. The same systemic effect was seen in patients with DME. We could not observe a reduction of plasma VEGF in those patient cohorts receiving ranibizumab or pegaptanib.
Patients with ARMD together with diabetics, represent a patient population with multiorgan comorbidities resulting in high cardiovascular risk and increased risk of thromboembolic events in which VEGF may play an important factor.9 ,12–16
VEGF is a multifunctional cytokine that regulates biological function in endothelial cells in adult vasculature. Systemic VEGF acts as a vascular protective factor by stimulating antiapoptotic signalling pathways to enhance endothelial integrity and maintain the antithrombogenic and anti-inflammatory properties of the endothelium.10 ,11
Intravitreal bevacizumab is a full-length monoclonal immunoglobulin G (IgG; 149 kD) that neutralises all isoforms of VEGF-A. Being a full IgG molecule, there has been some controversy about its ability to penetrate the retina, but immunohistochemistry of primate eyes clearly found immunoreactivity in the choroid and the inner layers of the retina as early as 1 day after the injection and spread to the outer layers and the choroid within the following days, in particular accumulating in vessel walls. Bevacizumab labelled with 125I showed radioactivity in blood serum 1 day after the intravitreal injection and remained relatively stable until day 7.17 ,18
Ranibizumab is an engineered Fab (48 kD) of humanised anti-VEGF with the similar pan-VEGF-A affinity.19 The Fabs are more diffusible than the IgGs with more rapid penetration and consecutive clearance of the retina.20
Different pharmacodynamics of anti-VEGF therapeutics are a possible cause for the observed significant suppression of systemic plasma VEGF in patients with ARMD as well as DME by bevacizumab. There are two main differences in pharmacodynamics of bevacizumab and ranibizumab. On the one hand, bevacizumab is the larger full-length IgG molecule with slower retinal clearance and, therefore, prolonged diffusion into systemic circulation.17 On the other hand, the systemic half-life of a Fab molecule, like ranibizumab, is a few hours, while that of a full-length IgG is up to 3 weeks in general circulation.21 Therefore, systemic exposure to an anti-VEGF agent is strikingly minimised by using a Fab. The degree of exposure is an important consideration in the clinical setting, as side effects such as hypertension, arterial thrombotic events, bleeding and proteinuria were associated with systemic anti-VEGF therapy.19 ,22–24
Regarding pegaptanib, we were not able to observe an effect on the measured VEGF plasma concentrations. Pegaptanib is a small 28-base RNA aptamer that specifically binds and blocks the 165-amino-acid isoform (VEGF165) and, therefore, has no pan-VEGF activity.25 ,26 The available data for systemic pharmacokinetics of pegaptanib refer to measurements after intravenous injection in rhesus monkeys. The measured elimination half-live was short with 9.3 h.27 ,28 The lack of pan-VEGF activity is suspected to be the cause for its inferior therapeutic effectivity in ARMD treatment compared with data available for ranibizumab or bevacizumab.3 ,4 ,29 This could also be responsible for the unaffected systemic VEGF measurements in our study.
In summary, our findings indicate that intravitreally injected bevacizumab does penetrate through all layers of the retina into choroidal blood vessels and rapidly enters systemic blood circulation. By contrast with ranibizumab and pegaptanib, bevacizumab then significantly reduces the level of VEGF in the blood plasma for up to 1 month in patients with diabetic retinopathy, as well as in those with ARMD.
Assuming a greater risk in diabetic retinopathy, where the blood-retinal barrier is broken at a very early stage of the disease, observing a similar reduction of plasma VEGF by intravitreal bevacizumab in ARMD patients is disturbing.15 ,30
Consequences of prolonged suppression of systemic VEGF levels resulting from serial intravitreal bevacizumab injections required for the treatment of retinal disease might become apparent after longer follow-up.3 ,4 The limitations of the present study are the small sample size in the individual subgroups, and losses to follow-up. The strengths are a prospective study design, and the randomised treatment allocation directly evaluating the systemic effects of the three currently used anti-VEGF therapeutics for DME as well as ARMD.
As our ophthalmic patients with ARMD, and especially diabetic patients, represent a high-risk group for cardiovascular events, we should be careful about the potential risks for adverse advents resulting from suppression of vasculoprotective effects of systemic VEGF by bevacizumab.
Digesting the findings of the recent CATT/IVAN studies, and considering the demand of national insurers to approve bevacizumab, our findings shed a different light on the widespread use of bevacizumab as an equivalent alternative to ranibizumab in our daily clinical practice.
Contributors Each author certifies that they have made substantial contribution to the work reported in this manuscript by participating in at least the following three areas: (1) substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; (2) drafting the article or revising it critically for important intellectual content; and (3) final approval of the version to be published.
Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None.
Patient consent Obtained.
Ethics approval Provided by the local ethics committee of the Medical University Innsbruck.
Provenance and peer review Not commissioned; externally peer reviewed.