The Diabetic Macular Edema Treated with Ozurdex (DMEO) Trial measured aqueous pro-permeability factors (PPFs) in diabetic macular edema (DME) patients before and after injection of dexamethasone implant or vascular endothelial growth factor (VEGF)-neutralizing protein and correlated changes in levels with changes in excess foveal thickness (EFT) to identify potential PPFs contributing to DME.
Prospective, randomized crossover clinical trial.
Twenty DME patients randomized to dexamethasone implant or VEGF-neutralizing protein had aqueous taps and spectral-domain optical coherence tomography (SDOCT) at baseline and every 4 weeks for 28 weeks. Aqueous levels of 55 vasoactive proteins were measured with protein array. Crossover at week 16 provided changes in protein levels after each intervention in all 20 patients.
After dexamethasone implant there was significant correlation between changes in levels of 13 vasoactive proteins with changes in EFT, including 3 known PPFs: angiopoietin-2 (r = 0.40, P = .001), hepatocyte growth factor (HGF; r = 0.31, P = .02), and endocrine gland-VEGF (EG-VEGF, r = 0.43, P < .001). Reduction of prolactin, insulin-like growth factor binding protein-3, and matrix metalloproteinase-9 correlated with edema reduction after injection of a VEGF-neutralizing protein as well as dexamethasone implant, suggesting their modulation is likely secondary to changes in edema rather than causative.
Correlation of edema reduction with reduction in the PPFs angiopoietin-2, HGF, and EG-VEGF provides potential insight into the multifactorial molecular mechanism by which dexamethasone implants reduce edema and suggest that additional study is needed to investigate the contributions of these 3 factors to chronic DME.
Diabetic macular edema (DME) is a common complication of diabetes, estimated to be present in 3.8% (or 746 000) of people aged ≥40 years in the United States in 2010. Owing to increasing obesity and demographic changes, the worldwide prevalence of diabetes is rapidly increasing, resulting in large increases in the prevalence of DME. Therefore, DME is a large public health problem that is getting larger.
Retinal hypoxia plays an important role in the pathogenesis of DME. In the hypoxic retina, stabilization of hypoxia-inducible factor-1α (HIF-1α) results in more binding to HIF-1β to form elevated levels of HIF-1 heterodimer and upregulation of vascular endothelial growth factor (VEGF) and other hypoxia-regulated gene products. A pilot trial demonstrated that suppression of VEGF caused remarkable reductions in DME, implicating VEGF as an important contributor to DME. Multiple large multicenter trials have confirmed that VEGF plays a key role in DME and determined that intraocular injections of a VEGF-neutralizing protein provide substantial visual benefit in most patients, but there are some patients in whom anti-VEGF injections are not sufficient to eliminate edema. This suggests that other pro-permeability factors in addition to VEGF contribute in some patients with DME.
Corticosteroids bind to cytoplasmic receptors that translocate to the nucleus and cause transcriptional repression of a large number of genes whose products promote inflammation, vascular leakage, and/or angiogenesis. The ability to reduce a large number of vasoactive factors provides a potential advantage, particularly when the identity of all of the contributors is unknown. The dexamethasone implant (Ozurdex; Allergan, Inc, Irvine, California, USA) causes significant reduction in DME and improvement in visual acuity, but its precise mechanism of action in DME is unknown. In this study, we investigated the mechanism of action of the dexamethasone implant in DME by measuring changes in aqueous vasoactive factors and correlating them with changes in edema. As a comparator, changes in aqueous vasoactive factors were correlated with changes in edema after intraocular injections of a VEGF-neutralizing protein.
The Diabetic Macular Edema Treated with Ozurdex (DMEO) Study was an investigator-initiated study sponsored by Allergan, Inc (Irvine, California, USA). The protocol was designed by the investigators, who controlled all aspects of the study with no influence from the sponsor. The protocol was approved by the Institutional Review Board of the Johns Hopkins Medical Institutions and was conducted in compliance with the Declaration of Helsinki, US Code 21 of Federal Regulations, and the Harmonized “Tripartite Guidelines for Good Clinical Practice” (1996). The study was registered on February 8, 2013, at www.clinicaltrials.gov ( NCT01790685 ). All patients provided informed consent. Twenty subjects with DME were randomized to Group 1 (dexamethasone implant/anti-VEGF) or Group 2 (anti-VEGF/dexamethasone implant) by the Reading Center and remained masked to group assignment. Randomization sequence was generated using Stata 9.0 (StataCorp, College Station, Texas, USA) statistical software and was stratified by the central subfield thickness (CST >450 μm or <450 μm) with a 1:1 allocation by the Reading Center.
Disease duration was determined from patient reporting and review of records, but only injections documented in records were used to determine the number of prior anti-VEGF injections. Response to prior anti-VEGF therapy was graded as good, moderate, or poor depending on whether all intraretinal fluid could be eliminated by monthly injections or how frequently anti-VEGF injections had to be given to maintain a dry macula. At baseline and at all subsequent visits, subjects had measurement of best-corrected visual acuity (BCVA) using the Early Treatment Diabetic Retinopathy Study (ETDRS) protocol with examiners masked with regard to treatment group, and ophthalmologic examination including measurement of intraocular pressure, spectral-domain optical coherence tomography (SDOCT) using the Spectralis machine (Heidelberg Engineering, Inc, Carlsbad, California, USA), and an anterior chamber tap. Aqueous samples were stored at −80 C. At baseline, Group 1 patients were given an intraocular injection of a dexamethasone implant in the study eye and Group 2 patients were given an anti-VEGF injection. For dexamethasone implant injections, povidone-iodine was used to clean the conjunctiva and 2% lidocaine was injected under the conjunctiva. The 22 gauge needle of the injector was inserted through the pars plana and the dexamethasone implant was injected into the vitreous cavity. The procedure was similar for anti-VEGF injections except that topical anesthesia and a 30 gauge needle were used. Patients in Group 1 were crossed over to pro re nata (prn) anti-VEGF injections and patients in Group 2 were crossed over to dexamethasone implant at the first visit after week 12 at which there was recurrent/persistent edema.
Anatomic and Functional Outcomes
The major anatomic outcome was excess foveal thickness (EFT), which provides an assessment of the amount of edema; it is calculated by subtracting edema-free CST from measured CST. Normal CST varies among patients, which is particularly true for patients with chronic macular edema who often experience some atrophy from the chronic edema resulting in a relatively thin edema-free CST. Using a single CST threshold such as 320 μm as an indicator of a dry retina could substantially underestimate edema reduction in many patients. Therefore, we used all information at our disposal to obtain the most accurate assessment of edema-free CST and, hence, change in EFT in each patient. For most patients, it was possible to identify 1 or more OCT scans obtained during the study or prior to enrollment on which there was no or minimal edema, but for the few subjects in whom such information was not available, 320 μm was used for normal CST. The functional outcome measure was change from baseline BCVA in ETDRS letter score at each visit. Mixed-effects regression models with a random intercept for eyes to account for the correlation among the repeated measures from the same eye were used to determine if BCVA or CST at each follow-up visit was different from baseline.
Vasoactive Protein Arrays
The levels of vasoactive proteins in aqueous at baseline and each subsequent visit were measured using the Human Angiogenesis Antibody Array Kit (catalog number ARY007; R&D Systems, Inc, Minneapolis, Minnesota, USA). Aqueous samples (115 μL) were blotted on the membrane, which was stored for 16 hours at 4°C and further processed according to the manufacturer’s instructions. Each array membrane was exposed to x-ray film for short-term and long-term exposures. The positive signals detected on the developed x-ray film were quantitated using TotalLab Quant software (Gentel Biosciences, Inc, Madison, Wisconsin, USA).
Correlation of Changes in Vasoactive Proteins With Changes in Edema
The diversity of protein levels and CST were calculated as fold change (log2) in their measurements at each visit relative to appropriate baseline:
FC Protein = log 2 ( Protein baseline / Protein visit )