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Vision Research journal homepage: www.elsevier.com/locate/visres
Novel therapeutic targets in diabetic macular edema: Beyond VEGF Elizabeth A. Uriasa, George A. Uriasa, Finny Monickaraja,b,c, Paul McGuireb, Arup Dasa,b,c, a b c
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Department of Surgery, University of New Mexico School of Medicine, Albuquerque, NM, United States Department of Cell Biology & Physiology, University of New Mexico School of Medicine, Albuquerque, NM, United States New Mexico VA Health Care System, Albuquerque, NM, United States
A R T I C L E I N F O
A B S T R A C T
No of reviewers = 2
The leading cause of major vision loss in diabetic persons is diabetic macular edema (DME). The hallmark feature of diabetic retinopathy is the alteration of the blood–retinal barrier (BRB). Inflammation plays a crucial role in DME with involvement of several chemokines and cytokines including vascular endothelial growth factor (VEGF). VEGF is a potent cytokine and vaso-permeability factor that has been targeted in multiple, large clinical trials. Multiple anti-VEGF drugs are widely used in the treatment of diabetic macular edema (DME) as the first line of treatment, and have been shown to be effective in vision improvement and prevention of vision loss. However, many DME patients do not show complete response to anti-VEGF drugs despite multiple intravitreal injections with these drugs. Also, the effect seems to be transient in those responders, and many patients do not show complete resolution of fluid. This article summarizes the mechanisms other than VEGF, and how these novel factors can be targeted as promising therapies of DME.
Keywords: Blood retinal barrier Diabetes mellitus Diabetic retinopathy Inflammation VEGF
1. Introduction Diabetes mellitus (DM) is a rapidly growing epidemic that affects 415 million people world-wide, and the projection is that this number will reach 642 million by the year 2040 (IDF, 2017). Diabetic retinopathy (DR) is a microvascular complication of diabetes and is present in approximately 35% of persons with diabetes (Yau, Rogers, Kawasaki, Meta-analysis doe eye disease (META-EYE) study group, et al., 2012). Diabetic macular edema (DME) has a prevalence of 6.8% amongst people with DM and it is the leading cause of blindness in diabetic persons. Macular edema can occur in any stage of diabetic retinopathy, either non-proliferative or proliferative retinopathy. DME is caused by excess fluid and lipid accumulation in the macula due to a breakdown in the blood retinal barrier. When the edema extends into the fovea, the patient becomes symptomatic with metamorphopsia and vision loss (Das, McGuire, & Rangasamy, 2015). The advent of intraocular anti-vascular endothelial growth factor (anti-VEGF) drugs has revolutionized the treatment of DME because it has largely decreased the use of laser therapy. However, the response to anti-VEGF drugs in DME is not as robust as in proliferative diabetic retinopathy, and many patients with DME do not show complete resolution of fluid despite multiple injections (DRCR, 2010; Sang & D’Amore, 2008). Recent animal and clinical studies suggest that diabetic retinopathy is an inflammatory disease with many cytokines and chemokines involved in the process (Adamis & Berman, 2008; Kern,
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2007). Thus, the molecular mechanisms beyond VEGF are being explored, and several clinical trials are in progress to examine the efficacy of these new therapies. A brief overview of the pathogenesis of DME and current pharmacotherapy available for treatment of DME will be briefly discussed here. Additionally, we will comprehensively review the results of various clinical trials that have evaluated therapeutic agents other than anti-VEGF drugs for the treatment of DME. 2. Overview of pathophysiology of DME 2.1. Blood-retinal barrier The blood-retinal barrier (BRB) is a tight, physiologic barrier that tightly regulates the balance of electrolytes, protein, and water in the retina, and it is composed of both an outer and an inner portion. The inner BRB is formed by tight junctions between retinal capillary endothelial cells, basement membrane surrounding it and pericytes outside (Das et al., 2015). The outer BRB consists of the retinal pigment epithelium (RPE) cells, located between the fenestrated choriocapillaris and the outer retina. An intact BRB is essential in maintaining normal visual function through these processes. 2.2. Alteration of blood retinal barrier Three important changes in the BRB occur in diabetes: 1)
Corresponding author at: Department of Surgery, Ophthalmology, MSC10-5610, 1 University of New Mexico, Albuquerque, NM 87131, United States. E-mail address: [email protected] (A. Das).
http://dx.doi.org/10.1016/j.visres.2017.06.015 Received 13 March 2017; Received in revised form 5 June 2017; Accepted 9 June 2017 0042-6989/ © 2017 Published by Elsevier Ltd.
Please cite this article as: Urias, E.A., Vision Research (2017), http://dx.doi.org/10.1016/j.visres.2017.06.015
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Fig. 1. Alteration of the blood retinal barrier in diabetes mellitus. Chronic inflammation in diabetes leads to production of chemokines (including Chemokine ligand-2, CCL2) that result in leukostasis, diapedesis and influx of monocytes into the retina and extravascular space. Monocytes are differentiated into macrophages which along with activated microglia produce an array of cytokines and chemokines including VEGF, IL-6, TNF-α and Ang-2. These mediators then break down the cell-cell junction molecules resulting in alteration of the blood- retinal barrier. (Reproduced from Das et al., Indian J Ophthalmol. 2016; 64:4–13).
leads to increased monocyte adhesion to endothelial cell layer and trafficking into the retina (Rangasamy et al., 2014). In the lumen of retinal blood vessels, leukocytes continuously roll along the endothelium. The integrin-ligand interactions lead to stable adhesion, or leukostasis. These cells then migrate through the endothelium into the extravascular space where they differentiate into activated macrophages and secrete various cytokines (Fig. 1). The disruption of the BRB in diabetes involves numerous factors and cascade of events within the vascular lumen and in the retinal parenchyma. A prominent factor contributing to the breakdown of the BRB is VEGF (vascular endothelial growth factor). But there are other factors involved including TNF-α and interleukin-1β, growth factors such as hepatocyte growth factor, FGF, and insulin-like growth factor-1 (IGF-1), cytokines such as ICAM-1, IL-6, MCP-1, and histamine (Adamis & Berman, 2008).
breakdown of cell-cell junctions between endothelial cells, 2) pericyte loss, and 3) basement membrane thickening (Fig. 1). Loss of cell-to-cell junctions in the endothelium results in the leakage of red blood cells, plasma and lipid (Frank, 2004). This leakage can be seen clinically on funduscopic exam as intraretinal hemorrhages, edema and hard exudates respectively. Pericytes are contractile smooth muscle –like cells that regulate blood flow in the microcirculation, and their loss leads to focal endothelial cell proliferation and microaneurysms, the earliest clinical lesions present in diabetic retinopathy. Hyperglycemia is the major risk factor contributing towards DME. It induces four major biochemical pathways (polyol pathway, advanced glycation end-product pathway, protein kinase C pathway, and hexosamine pathway) that all lead to increased oxidative stress and inflammation (Brownlee, 2005). Although the DCCT study has shown that tight glucose control prevents development and slows down progression of diabetic retinopathy, further analysis of the DCCT cohort has revealed that the total glycemic exposure (A1C and duration of diabetes) explains only ∼11% of the variation in retinopathy risk in the complete cohort, so that other factors may presumably explain the remaining 89% of the variation in risk among subjects independent of A1C (Lachin et al., 2008). The oxidative stress in diabetes upregulates multiple cytokines and chemokines, such as VEGF, angiopoietins, tumor necrosis factor (TNF), interleukins (ILs) and matric metalloproteinases (MMPs) that leads to breakdown of the BRB. Also, retinal capillary obstruction (leukostasis) or dropout (apoptosis of vascular cells) in diabetes leads to tissue ischemia and hypoxia causing increased retinal VEGF expression through transcriptional regulation by hypoxia-inducible factor 1 alpha (HIF-1α) (Arjamaa & Nikinmaa, 2006). Genetic factors that alter gene expression predispose to or protect against the progression of diabetic retinopathy, or its vision-threatening consequences.
3. Anti-VEGF drugs 3.1. Current therapy VEGF is a potent vasopermeability factor that has been targeted extensively in DME and PDR. Out of all the isoforms, VEGF 165 is the major pro-inflammatory cytokine. The VEGF levels are significantly elevated in vitreous of DME patients when compared with nondiabetic eyes (Funatsu, Noma, Mimura, Eguchi, & Hori, 2009). VEGF induces the phosphorylation of cell-cell junctional molecules such as VE-cadherin, occludin, and ZO-1 and thus causes a breakdown of the BRB. Drugs that directly inhibit the VEGF molecule are anti-VEGF aptamer, pegaptanib (Macugen, OSI), monoclonal antibody fragment ranibizumab (RBZ) (Lucentis, Genentech), full-length antibody bevacizumab (BVZ) (Avastin, Genentech) and soluble VEGF receptor analogs, aflibercept (VEGF-Trap (Regeneron). Other anti-VEGF molecules include small interfering RNAs, bevasiranib (Opko Health), and rapamycin (Sirolimus, MacuSight) (Das et al., 2015). Anti-VEGF drugs are injected intravitreally every 4 weeks under topical anesthesia as office procedures. All the larger clinical trials RIDE/RISE, DRCR, and VIVID/VISTA have shown significant vision improvement and reduction of central retinal thickness in DME patients. Currently, anti-VEGF agents are considered the first line of treatment in center-involving DME, where
2.3. Inflammation Leukostasis occurs early in the DME disease process. The increased leukostasis leads to upregulation of intercellular adhesion molecule (ICAM-1) and retinal vascular leakage (Adamis & Berman, 2008). Use of antibodies to CD-18 or ICAM-1, or genetic knockout of these animals, inhibits retinal leukostasis and breakdown of the BRB (Joussen et al., 2004). Also, increased expression of MCP-1 gene expression in diabetes 2
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Fig. 2. Novel therapeutic strategies in diabetic macular edema. Hyperglycemia in diabetes activates different bio chemical pathways that lead to increased hypoxia, inflammation and oxidative stress with production of several cytokines. This mediators then cause break down of the inner and outer blood retinal barrier through endothelial/ pericyte loss, thickening of basement membrane and photoreceptor/ glial cell dysfunction. The current anti-VEGF therapies have limitations because they target only VEGF rather than other inflammatory molecules. Many novel therapies targeting the other molecules beyond VEGF are now in clinical trials. As shown above.
endogenous VEGF is critical for trophic support for retinal function. A recent study has suggested that chronic suppression of locally synthesized VEGF in the eye may damage the choriocapillaris and cone function (Kurihara, Westenskow, Bravo, Aguilar, & Friedlander, 2012). Knocking out VEGF-A in adult mice results in damage to retinal pigment epithelial (RPE) cells, ablation of the choriocapillaris, and rapid dysfunction of cone photoreceptors. However, there is no strong evidence of such neuronal damage documented in human clinical studies.
the indication of focal/grid laser is limited to non-center-involving DME. However, all the large clinical trials have shown that only 33–45% of DME patients on anti-VEGF agents show 3 lines or more of visual improvement. The rest of DME patients show intermediate response (5–9 letters of improvement) or poor response (< 5 letters of improvement or worse). Eyes with suboptimal early vision response showed poorer long-term visual outcomes than eyes with pronounced early response (mean improvement 3.0 vs 13.8 letters at 3 years) (Gonzalez et al., 2016). The poor response to anti-VEGF agents could be explained by the presence of other factors beyond VEGF as a result of inflammation that are not targeted by the anti-VEGF drugs. Thus, many anti-VEGF-treated eyes with DME do not achieve visual acuity of 20/20 or complete resolution of retinal edema, thus, there is a need for supplemental treatments that might improve visual acuity in eyes with persistent edema despite anti-VEGF therapy (Figs. 2, 3). Although systemic administration of bevacizumab has been involved with side effects such as hypertension, thromboembolic events, similar incidence in patients receiving intravitreal anti-VEGF drugs is not significantly high. Thus, small doses of anti-VEGF drugs for intravitreal injections (almost 1/400th dose of systemic dose) have so far been found to be safe. VEGF has a neuroprotective role, and
3.2. High-dose anti-VEGF therapy As there is a wide range of VEGF levels in vitreous of DME patients, it is possible that those with high levels of VEGF may need higher doses of anti-VEGF drugs. Based on this rationale, the READ-3 study has compared the efficacy of two different doses of anti-VEGF drugs (0.5 and 2.0 mg of ranibizumab) in DME patients. The higher dose of ranibizumab (2.0 mg) did not result in better visual acuity (Sepah et al., 2016). A very similar result was obtained in the RIDE/RISE trials that showed equal efficacy with both the 0.3 and 0.5 mg doses of ranibizumab. As there is less chance of potential systemic risks with the lowest dose (0.3 mg) of ranibizumab, the FDA approved the dose of 3
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Fig. 3. List of novel drugs based on mechanisms of action in diabetic macular edema: 1. Anti-VEGF, 2. Anti-inflammatory, 3. Non-Steroidal anti-inflammatory drugs (NSAIDs), 4. Inhibitors of multiple growth factors, 5. Integrin inhibitors, 6. Cytokine and chemokine inhibitors, 7. Others.
compared to the standard of care. A phase II trial using the VEGF DARPin (abicipar pegol) (Allergan) in wet ARMD patients showed improvement in vision and decreases in central retinal thickness in a way very similar to that of monthly ranibizumab injections (Campochiaro et al., 2013). The data indicated that abicipar can be injected every 12 weeks following loading doses. A phase III clinical trial for abicipar for wet AMD is in progress now. A Phase 2 clinical trial, PALM, has evaluated abicipar pegol in DME patients, and the drug met its study endpoints and the efficacy of abicipar was shown in all three treatment groups (molecularpartners.com).
0.3 mg ranibizumab in DME patients (Brown et al., 2013). 3.3. Extended delivery of anti-VEGF therapy Monthly anti-VEGF injections are difficult for patients to comply with. Different Long – acting delivery systems such as biodegradable implants, biodegradable microspheres, encapsulated cells, and gene therapy are currently under investigation. A phase 1 study testing refillable, non-biodegradable, long-term delivery implant (Genetech) of ranibizumab has been completed on 20 patients with wet age-related macular degeneration (ARMD). The implant is placed under the conjunctiva in the pars plana, and requires a 3.2 mm surgical incision without sutures. The implant is refilled when needed intervals. The results demonstrated improvement in visual acuity of 10 letters throughout 1 year (Rubio, 2014). A phase II clinical trial (Study of the Efficacy, 2017) is now being done in wet ARMD patients to study the efficacy, safety and pharmacokinetics of three different formulations of ranibizumab delivered via the ranibizumab port delivery system compared with the standard of care of intravitreal injections of ranibizumab (ClinicalTrials.gov NCT02510794). As there is a more consistent pharmacokinetic profile with fewer peaks and troughs, sustained delivery of anti-VEGF drugs offers better long-term visual outcomes and disease modification, and thus the treatment burden may be decreased.
4. Steroids 4.1. Current steroid therapy Steroid injections suppress proinflammatory chemokines, and inhibit leukostasis and phosphorylation of cell-junction proteins. The DRCR protocol I trial has shown the efficacy of intravitreal triamcinolone in DME (DRCR Network, 2010). There has been visual improvement and reduction in central retinal thickness with triamcinolone in a similar way to that of ranibizumab up to 24 weeks, but after that period the effect of triamcinolone drops due to high rates of cataract formation and elevation of intraocular pressure (IOP). In a subgroup analysis of pseudophakic patients, the triamcinolone plus laser group was found to be superior to the laser treatment alone group in terms of vision improvement, and equivalent to the ranibizumab group. Sustained release corticosteroids were developed in effort to decrease the number of injections a patient with DME requires. The dexamethasone implant (Ozurdex; Allergan, Inc.) is a biodegradable copolymer that releases the dexamethasone over a 6 month-period and
3.4. VEGF designed ankyrin repeat protein (DARPin) Designed ankyrin repeat proteins (DARPins) are genetically engineered antibody mimetic protein that show highly specific and highaffinity target protein binding. Its small size, high potency, and long intra-vitreal half-life offers the potential for less frequent injections 4
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danazol in these patients.
the implant can be placed in the office, using a 23-gauge needle. The safety and efficacy of the implant has been evaluated in multiple phase II studies, which found that the implant is effective in improving BCVA and in decreasing vascular leakage. In the MEAD Study, dexamethasone intravitreal implant resulted in visual improvement of > 15 letters in 22% patients (0.7 mg) and 18% (0.35 mg) compared with 12% in the sham group (Boyer et al., 2014). Cataract formation was seen in up to 68% of treated patients, and increases in IOP could be managed with medication, or no therapy. The implant is currently FDA approved for use of treatment of DME. The newer generation of flucinolone acetonide implant (Iluvien, Alimera Sciences) is a non-biodegradable microimplant containing 190 μg of drug, with release of 0.2 μg of drug per day. In the randomized Fluocinolone Acetonide for macular Edema (FAME) study, there was significant vision improvement in DME patients (Campochiaro et al., 2011). This implant is now FDA approved for treatment of DME in patients who have not previously demonstrated IOP spikes when using steroids.
5. Cytokine and chemokine Inhibitors 5.1. Angiopoietin-2 inhibitor Angiopoietins are a family of growth factors that bind to endothelial receptor tyrosine kinases, also known as Tie receptors (Rangasamy et al., 2011). When activated, Tie receptors play a role in vascular development and function. Angiopoietin (Ang)-1 and -2 share 60% amino acid identity and bind with similar affinity to Tie-2. The activity of Tie2 is differentially regulated by the two ligands. Ang-1 is a strong agonist of the Tie-2 receptor, and Ang-2 acts as an agonist or antagonist in a context-dependent manner. Ang-2 is an endogenous weak, partial agonist that competes with Ang-1, thereby suppressing Tie2 phosphorylation. Ang-2 is predominantly expressed in endothelial cells, stored in vesicles known as Weibel-Palade bodies, and is rapidly released in response to specific stimuli. Emerging evidence indicates that Ang-2 is upregulated in response to hyperglycemia and plays an important role in the alteration of the BRB in diabetes. Increased Ang-2 leads to decreased phosphorylation of Tie-2 which results in increased retinal vascular permeability (Rangasamy et al., 2011). We previously showed that the up-regulation of proteinases in microvascular endothelial cells by Ang-2 may be an important early response during the development of retinal neovascularization (Das et al., 2003). Many systemic conditions (sepsis, acute lung injury, systemic capillary leak syndrome, disseminated intravascular coagulation and acute kidney injury) with systemic vascular leakage have been reported to be associated with high serum Ang-2 levels. In summary, the benefits of angiopoietin inhibition in diabetic macular edema include decrease in vaso-permeability as well as decrease in inflammation in the retina by decreasing leukocyte adhesion to retinal vessel wall, decreasing cell adhesion molecule, ICAM-1, decreasing chemokines, CCL2 and CCL5 in retinas, and decreasing monocyte trafficking and microglia activation in retinas (Nitta et al., 2015). There are three ongoing clinical trials ongoing targeting angiopoietins in DME patients. A small molecule, AKB-9778 (Aerpio Therapeutics) is an inhibitor of an enzyme, vascular endothelial protein tyrosine phosphatase (VE-PTP) that stimulates phosphorylation of Tie2 in vascular endothelial cells and suppresses VEGF-induced leakage. Levels of both angiopoietin 2 and VE-PTP are increased by hypoxia, accentuating VEGF-mediated retinal neovascularization and vascular leakage. Inhibitors of VE-PTP (AKB-9778) could activate Tie-2 receptors and thus provide a strategy of inhibiting retinal vascular leakage (Campochiaro et al., 2015). The TIME-2 study found that subcutaneous injections of AKB-9778 combined with VEGF inhibitor (ranibizumab) causes a significantly greater reduction in DME than that seen with suppression of VEGF alone (Campochiaro et al., 2016). One advantage of this drug is its subcutaneous administration route that can be easily done by diabetic patients themselves. A phase II trial, BOULEVARD (Roche, Basel, Switzerland) is using an approach of targeting both VEGF and Ang-2 by using a bispecific IgG1 monoclonal antibody (RO6867461) that has one arm binding to VEGF and the other arm binding to Ang-2. Participants are randomized into each arm group (1:1:1): ranibizumab group 0.3 mg, intravitreal injection of R06867461 1.5 mg, and R06867461 6 mg (Clinicaltrials.govBOULEVARD). The study will have duration of 28 weeks. A similar approach is being followed in a phase II trial, RUBY (Regeneron) using a co-formulation of nesvacumab (REGN910) and aflibercept (Clinicaltrials.gov-RUBY). The nesvacumab is a fully human, monoclonal antibody that binds to Ang-2, and aflibercept is a VEGF trap molecule, that binds to VEGF. The advantage of using an inhibitor of Ang-2 is that it also blocks the inflammatory cascade in addition to its beneficial effect of inhibiting the retinal vascular permeability.
4.2. Combination of steroid + anti-VEGF therapy A Phase II DRCR randomized clinical trial is investigating whether the addition of steroid (dexamethasone) to an anti-VEGF treatment regimen (ranibizumab) in eyes with persistent center-involved DME despite anti-VEGF treatment increases visual acuity and decreases DME in the short term, compared with continued anti-VEGF treatment alone (ranibizumab) (DRCR.net). Furthermore, this phase II study is being conducted to provide information for a Phase III trial. 4.3. Triamcinolone acetonide with aflibercept A phase II trial, TANZANITE study recently evaluated the safety and efficacy of a single suprachoroidal injection of, triamcinolone acetonide (Zuprata; Clearside Biomedical, Alpharetta, GA) injectable suspension, given along with an intravitreal injection of aflibercept compared to aflibercept alone in subjects with retinal vein occlusion (RVO). Results demonstrated that 78% of patients in the combination arm required no additional treatments over 3 months compared with 30% in the control arm (clearsidebio.com). Based on the results of this trial, an open-labeled multi-center Phase I/II trial (HULK) has been started using the same combination therapy in DME patients. 4.4. Betamethasone microspheres Betamethasone is a steroid used for a number of diseases including rheumatoid arthritis and systemic lupus erythematosus, dermatitis and psoriasis, allergic conditions such as asthma and angioedema. A phase II/III clinical trial evaluated the efficacy and safety of sub-tenon injection of biodegradable, sustained release betamethasone microspheres (DE-102; Santen Pharmaceutical Co.) in DME patients (clinicaltrials.gov). The study was completed but results are not yet published. 4.5. Danazol Danazol is an orally administered steroid that is already being used to treat other conditions, mainly endometriosis. It is a synthetic steroid analog of a 17-a-ethinyl testosterone, that when given at extremely low doses, is able to strengthen cell-cell junctions and reduce capillary permeability. A safety and efficacy study of oral danazol for the treatment of DME was recently completed (Ampio pharmaceuticals) (clinicaltrials.gov). The study evaluated the efficacy of ultra-low dose danazol (Optina) vs placebo in DME patients. In addition, there was a significant reduction in central retinal thickness compared to those in the placebo group (Singer, Orlando, & Baro-Or, 2016). There was no side effect related to insulin resistance and hyperglycemia in using 5
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activates PPK into catalytically active PKal. PKal further activates bradykinin (BK), which in turn activates the BK B2 receptor (B2R). Both the B2R and B1R receptors are highly expressed in the retina. Specifically, retinal expression of B1R is increased in diabetes. It is speculated that hemorrhage, activated platelets, and unfolded proteins activate the KKS in diabetic retinopathy (Liu & Feener, 2013). Vitreous proteomics analysis in advanced stages of diabetic retinopathy have shown increased levels of plasma KK system components including plasma kallikrein, coagulation factor XII, and high molecularweight kininogen (Kita et al., 2015). A Phase I study (Kalvista Pharmaceutical, Cambridge, MA) studied the safety and tolerability of a novel plasma kallikrein inhibitor, intravitreal KVD001, in patients with DME (clinicaltrials.gov-KVD001). Promising results with increases in VA and decreases in CRT lasting up to 12 weeks have been reported, and based on the outcome, the company is planning a phase II trial in DME in 2017.
5.2. Interleukin Inhibitors IL-6 is an important cytokine that has been consistently elevated in vitreous of DME patients. Intravitreal injection of an antibody against interleukin-6 potentially inhibits choroidal neovascularization in an animal model (Schmidt, Tisdale, Lowden, Kovalchin, & Furfine, 2014). There are two clinical trials investigating the blockage of IL-6 in DME patients. The first trial involves an IL-6 antibody (EBI-031; Eleven Biotherapeutics, Roche), a humanized monoclonal antibody that potentially binds IL-6 and inhibits all known forms of IL-6 cytokine signaling in the eye (ir.elevenbio.com). The second study, READ-4 trial will investigate the safety, tolerability and efficacy of ranibizumab and intravenous tocilizumab alone and in combination in DME patients (Clinicaltrials.gov-READ-4). Tocilizumab, a humanized monoclonal antibody against IL-6 receptor, is an immunosuppressant drug that is used in treatment of rheumatoid arthritis and systemic juvenile idiopathic arthritis. 6. Inhibitors of adhesion molecules
8. Inhibitors of multiple growth factors
6.1. Integrin
Squalamine is a small molecule anti-angiogenic drug that counteracts multiple growth factors in the cascade of angiogenesis including VEGF, platelet- derived growth factor, (PDGF) and basic fibroblast growth factor (Sills et al., 1998). It does not block all VEGF mediated inflammatory pathways, but it inhibits mitogen-activated protein kinase, p38 inflammatory and vascular endothelial-cadherin and other signaling pathways. This compound was discovered in tissues of dogfish sharks that have anti-angiogenic and antiviral properties. A phase II study (IMPACT) in wet ARMD patients demonstrated that more than twice the proportion of patients on combination of squalamine lactate eye drops (0.2%, OHR-102; Ohr Pharmaceutical, New York, NY) and ranibizumab achieved ≥3 line gains in visual acuity at 9 months compared with the ranibizumab alone (clinicaltrials.govsqualamine). A phase II trial using squalamine eye drops is currently ongoing in DME patients.
Integrins are cell-surface receptors that regulate attachments among cells and between cells. In a broad sense, they control the way in which cells communicate with each other in the extracellular matrix. In the retina, integrins are located on endothelial cells of blood vessels and serve to interact with growth factors and regulate their functions. The pathologic process of retinal angiogenesis is largely dependent on specific integrins like αvβ3, αvβ5, and α5β1. An integrin antagonist, Luminate (ALG-1000) (Allegro Ophthalmics, San Juan Capistrano, CA) is an oligopeptide that binds to multiple integrin receptor sites (αvβ3, αvβ5, and α5β1) and can inhibit integrins in multiple pathways. This antagonist also binds to α3β1 that mediates attachment of vitreous to the retinal interface, and induces vitreolysis. Liquefying the vitreous and inducing a posterior vitreous detachment lowers the VEGF levels and prevent them from continuing to accumulate over the macula, and thus helps in reducing the retinal vascular leakage in DME. Recently, the DEL MAR phase IIb clinical trial demonstrated that Luminate (1.0 mg) was non-inferior to intravitreal bevacizumab injections in terms of visual acuity gains and decreases in central retinal thickness and patients needed half the number of injections (allegroeye.com-DEL MAR). A larger, phase II study is currently evaluating the safety and exploring the efficacy of Luminate compared to that of bevacizumab injection and focal laser photocoagulation.
9. Conclusions Currently, anti-VEGF drugs are the first line of treatment in centerinvolving diabetic macular edema. Once the focal/grid laser therapy as the standard treatment for DME, is now being reserved only for noncenter-involving macular edema. Over the last ten years, we have seen the rapid, wide use of anti-VEGF drugs in treatment of many retinal diseases including diabetic macular edema. No doubt, anti-VEGF therapy is an effective therapy in DME. But compared to other angiogenic diseases like proliferative diabetic retinopathy and wet macular degeneration, the effect of anti-VEGF drugs is much less robust in DME patients. Multiple, frequent monthly anti-VEGF injections are needed in DME patients, and a significant number of patients poorly respond to these anti-VEGF drugs. DME appears to be a heterogeneous disease with good responders (33–45%) to anti-VEGF drugs on one end of the spectrum, poor responders on the other end of the spectrum, and many intermediate responders in between. In the latter two groups, probably factors other than VEGF are responsible for alteration of the BRB. The cytokine/chemokine profile of DME patients varies from one to another. No biomarkers are currently available to predict good, poor, or intermediate responders. Probably, genetic factors play an important role in this anti-VEGF responsiveness. Anti-VEGF therapy will still be the first line of treatment in DME patients in the coming years. However, we will see combination therapies of novel inhibitors targeting the molecules beyond VEGF in the coming years in management of diabetic retinopathy. Also, novel drug delivery systems using nanotechnology, sustained-release delivery implants, and stem cell therapy are on the horizon.
6.2. Vascular adhesion protein (VAP-1) Vascular adhesion protein-1 is an endothelial adhesion molecule that is expressed on the surface of endothelial cells. It is involved in leukocyte transmigration during inflammation. In a diabetic rat model, orally given VAP-1 inhibitor prevented increased retinal vascular leakage (Inoue et al., 2013). ASP8232 (Astellas; Northbrook, IL) is an orally administered VAP-1 inhibitor that is currently being studied in DME patients in the phase II VIDI trial (clinicaltrials.gov-VIDI trial). The study involves 3 intervention arms: ASP8232 + sham injections, ASP8232 + ranibizumab injections, placebo + ranibizumab injections. 7. Inhibitors of kallikrein-kinin (KK) system The kallikrein-kinin system (KKS) contributes to the vascular inflammatory response associated with hereditary angioedema (Liu & Feener, 2013). The KKS also mediates vascular hyperpermeability, leukostasis, cytokine production, and retinal thickening in rodent models of diabetic retinopathy. The proinflammatory effects of the KKS are mediated by plasma kallikrein (PKal), a protease derived from a circulating zymogen named plasma prekallikrein (PPK). Factor XII 6
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protein-1 (VAP-1) inhibitors for diabetic macular edema treatment. Bioorganic & Medicinal Chemistry, 21, 3873–3881. Joussen, A. M., Poulaki, V., Le, M. L., Koizumi, K., Esser, C., Janicki, H., et al. (2004). A central role for inflammation in the pathogenesis of diabetic retinopathy. FASEB Journal, 18, 1450–1452. Kern, T. S. (2007). Contributions of inflammatory processes to the development of the early stages of diabetic retinopathy. Experimental Diabetes Research, 2007, 95103. Kita, T., Clermont, A. C., Murugesan, N., Zhou, Q., Fukisawa, K., Ishibashi, T., et al. (2015). Plasma kallikrein-kinin system as a VEGF-independent mediator of diabetic macular edema. Diabetes, 64, 3588–3599. Kurihara, T., Westenskow, P. D., Bravo, S., Aguilar, E., & Friedlander, M. (2012). Targeted deletion of VEGFA in adult mice induces vision loss. Journal of Clinical Investigation, 122, 4213–4217. Lachin, J. M., Genuth, S., Nathan, D. M., Zinman, B., Rutledge, B. N., & DCCT/EDIC Research Group (2008). Effect of glycemic exposure on the risk of microvascular complications in the diabetes control and complications trial–revisited. Diabetes, 57, 995–1001. Study of the Efficacy and Safety of the Ranibizumab Port Delivery System for Sustained Delivery of Ranibizumab in Participants With Subfoveal Neovascular Age-Related Macular Degeneration (LADDER). https://clinicaltrials.gov/ct2/show/ NCT02510794. Accessed March 1, 2017. Liu, J., & Feener, E. P. (2013). Plasma kallikrein-kinin system and diabetic retinopathy. Biological Chemistry, 394, 319–328. Nitta, C. F., Monickaraj, F., McGuire, P. M., & Das, A. (2015). Angiopoeitin-2 and chemokine ligand-2 plays independent and complimentary roles in the retinal inflammatory cascade. Abstract. Poster presentation at ARVO-NIH meeting on Diabetic Retinopathy, Bethesda, MD. Rangasamy, S., McGuire, P. G., Franco Nitta, C., Monickaraj, F., Oruganti, S. R., & Das, A. (2014). Chemokine mediated monocyte trafficking into the retina: role of inflammation in alteration of the blood-retinal barrier in diabetic retinopathy. PLoS One, 20, e108508. Rangasamy, S., Srinivasan, R., Maestas, J., McGuire, P. G., & Das, A. (2011). A potential role of angiopoietin-2 in the regulation of the blood-retinal barrier in diabetic retinopathy. Investigative Ophthalmology & Visual Science, 52, 3784–3791. Rubio, R. (2014). Phase I clinical trial using a refillable, non-biodegradable long-term drug delivery implant of ranibizumab. World Congress of Ophthalmology, Tokyo, Japan. Sang, D. N., & D’Amore, P. A. (2008). Is blockade of vascular endothelial growth factor beneficial for all types of diabetic retinopathy? Diabetologia, 51, 1570–1573. Schmidt, M., Tisdale, A., Lowden, P., Kovalchin, J., & Furfine, E. S. (2014). Optimized IL-6 blockade for the treatment of diabetic macular edema. Abstract. Association for Research in Vision & Ophthalmology (ARVO) Meeting. Orlando, Florida. Sepah, Y. J., Sadiq, M. A., Boyer, D., Callanan, D., Gallemore, R., Bennett, M., et al. (2016). Twenty-four-month outcomes of the ranibizumab for edema of the macula in diabetes - protocol 3 with high dose (READ-3) study. Ophthalmology, 123, 2581–2587. Sills, A. K., Jr., Williams, J., Tyler, B. M., Epstein, D. S., Sipos, E. P., Davis, J. D., et al. (1998). Squalamine inhibits angiogenesis and solid tumor growth in vivo and perturbs embryonic vasculature. Cancer Research, 58, 2784–2792. Singer, M., Orlando, A., & Baro-Or, D. (2016). Potential beneficial effect of low dose danazol in combination with renin angiotensin inhibitors in diabetic macular edema: A 12-week double blind randomized controlled trial. Poster presentation at the Association for Research in Vision and Ophthalmology, Seattle, WA. Yau, J. W., Rogers, S. L., Kawasaki, R., Meta-analysis doe eye disease (META-EYE) study group, et al. (2012). Global prevalence and risk factors of diabetic retinopathy. Diabetes Care, 35, 556–564.
Funding This work was supported by the National Institutes of Health EY022327 and Veterans Affairs Merit Review Grant BX001801. References Adamis, A. P., & Berman, A. J. (2008). Immunological mechanisms in the pathogenesis of diabetic retinopathy. Seminars in Immunopathology, 30, 65–84. Arjamaa, O., & Nikinmaa, M. (2006). Oxygen-dependent diseases in the retina: role of hypoxia-inducible factors. Experimental Eye Research, 83, 473–483. Boyer, D. S., Yoon, Y. H., Belfort, R., Jr., Bandello, F., Maturi, R. K., Augustin, A. J., et al. (2014). Three-year, randomized, sham-controlled trial of dexamethasone intravitreal implant in patients with diabetic macular edema. Ophthalmology, 121, 1904–1914. Brown, D. M., Nguyen, Q. D., Marcus, D. M., Boyer, D. S., Patel, S., Feiner, L., et al. (2013). Long-term outcomes of ranibizumab therapy for diabetic macular edema: the 36-month results from two phase III trials: RISE and RIDE. Ophthalmology, 120, 22. Brownlee, M. (2005). The pathobiology of diabetic complications: a unifying mechanism. Diabetes, 54, 1615–1625. Campochiaro, P. A., Brown, D. M., Pearson, A., Ciulla, T., Boyer, D., Holz, F. G., et al. (2011). Long-term benefit of sustained-delivery fluocinolone acetonide vitreous inserts for diabetic macular edema. Ophthalmology, 118, 626–635. Campochiaro, P. A., Channa, R., Berger, B. B., Heier, J. S., Brown, D. M., Fiedler, U., et al. (2013). Treatment of diabetic macular edema with a designed ankyrin repeat protein that binds vascular endothelial growth factor: a phase I/II study. American Journal of Ophthalmology, 155, 697–704. Campochiaro, P. A., Khanani, A., Singer, M., Patel, S., Boyer, D., Dugel, P., et al. (2016). Enhanced benefit in diabetic macular edema from AKB-9778 Tie2 activation combined with vascular endothelial growth factor suppression. Ophthalmology, 123, 1722–1730. Campochiaro, P. A., Sophie, R., Tolentino, M., Miller, D. M., Browning, D., Boyer, D. S., et al. (2015). Treatment of diabetic macular edema with an inhibitor of vascular endothelial-protein tyrosine phosphatase that activates Tie2. Ophthalmology, 122, 545–554. Das, A., Fanslow, W., Cerretti, D., Warren, E., Talarico, N., & McGuire, P. (2003). Angiopoietin/Tek interactions regulate mmp-9 expression and retinal neovascularization. Laboratory Investigation, 83, 1637–1645. Das, A., McGuire, P. G., & Rangasamy, S. (2015). Diabetic macular edema: Pathophysiology and novel therapeutic targets. Ophthalmology, 122, 1375–1394. Diabetic Retinopathy Clinical Research Network, Elman, M. J., Aiello, L. P., Beck, R. W., Bressler, N. M., Bressler, S. B., et al. (2010). Randomized trial evaluating ranibizumab plus prompt or deferred laser or triamcinolone plus prompt laser for diabetic macular edema. Ophthalmology, 117(6), 1064–1077 e35. Frank, R. N. (2004). Diabetic retinopathy. The New England Journal of Medicine, 1, 48–58. Funatsu, H., Noma, H., Mimura, T., Eguchi, S., & Hori, S. (2009). Association of vitreous inflammatory factors with diabetic macular edema. Ophthalmology, 116, 73–79. Gonzalez, V. H., Campbell, J., Holekamp, N. M., Kiss, S., Loewenstein, A., Augustin, A. J., et al. (2016). Early and long-term responses to anti-vascular endothelial growth factor therapy in diabetic macular edema: Analysis of protocol I data. American Journal of Ophthalmology, 172, 72–79. IDF Diabetes Atlas. Brussels, Belgium: International Diabetes Federation, Facts and figures, accessed www.idf.org on March 1, 2017. Inoue, T., Morita, M., Tojo, T., Nagashima, A., Moritomo, A., & Miyake, H. (2013). Novel 1H-imidazol-2-amine derivatives as potent and orally active vascular adhesion
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Contents lists available at ScienceDirect
Vision Research journal homepage: www.elsevier.com/locate/visres
Novel therapeutic targets in diabetic macular edema: Beyond VEGF Elizabeth A. Uriasa, George A. Uriasa, Finny Monickaraja,b,c, Paul McGuireb, Arup Dasa,b,c, a b c
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Department of Surgery, University of New Mexico School of Medicine, Albuquerque, NM, United States Department of Cell Biology & Physiology, University of New Mexico School of Medicine, Albuquerque, NM, United States New Mexico VA Health Care System, Albuquerque, NM, United States
A R T I C L E I N F O
A B S T R A C T
No of reviewers = 2
The leading cause of major vision loss in diabetic persons is diabetic macular edema (DME). The hallmark feature of diabetic retinopathy is the alteration of the blood–retinal barrier (BRB). Inflammation plays a crucial role in DME with involvement of several chemokines and cytokines including vascular endothelial growth factor (VEGF). VEGF is a potent cytokine and vaso-permeability factor that has been targeted in multiple, large clinical trials. Multiple anti-VEGF drugs are widely used in the treatment of diabetic macular edema (DME) as the first line of treatment, and have been shown to be effective in vision improvement and prevention of vision loss. However, many DME patients do not show complete response to anti-VEGF drugs despite multiple intravitreal injections with these drugs. Also, the effect seems to be transient in those responders, and many patients do not show complete resolution of fluid. This article summarizes the mechanisms other than VEGF, and how these novel factors can be targeted as promising therapies of DME.
Keywords: Blood retinal barrier Diabetes mellitus Diabetic retinopathy Inflammation VEGF
1. Introduction Diabetes mellitus (DM) is a rapidly growing epidemic that affects 415 million people world-wide, and the projection is that this number will reach 642 million by the year 2040 (IDF, 2017). Diabetic retinopathy (DR) is a microvascular complication of diabetes and is present in approximately 35% of persons with diabetes (Yau, Rogers, Kawasaki, Meta-analysis doe eye disease (META-EYE) study group, et al., 2012). Diabetic macular edema (DME) has a prevalence of 6.8% amongst people with DM and it is the leading cause of blindness in diabetic persons. Macular edema can occur in any stage of diabetic retinopathy, either non-proliferative or proliferative retinopathy. DME is caused by excess fluid and lipid accumulation in the macula due to a breakdown in the blood retinal barrier. When the edema extends into the fovea, the patient becomes symptomatic with metamorphopsia and vision loss (Das, McGuire, & Rangasamy, 2015). The advent of intraocular anti-vascular endothelial growth factor (anti-VEGF) drugs has revolutionized the treatment of DME because it has largely decreased the use of laser therapy. However, the response to anti-VEGF drugs in DME is not as robust as in proliferative diabetic retinopathy, and many patients with DME do not show complete resolution of fluid despite multiple injections (DRCR, 2010; Sang & D’Amore, 2008). Recent animal and clinical studies suggest that diabetic retinopathy is an inflammatory disease with many cytokines and chemokines involved in the process (Adamis & Berman, 2008; Kern,
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2007). Thus, the molecular mechanisms beyond VEGF are being explored, and several clinical trials are in progress to examine the efficacy of these new therapies. A brief overview of the pathogenesis of DME and current pharmacotherapy available for treatment of DME will be briefly discussed here. Additionally, we will comprehensively review the results of various clinical trials that have evaluated therapeutic agents other than anti-VEGF drugs for the treatment of DME. 2. Overview of pathophysiology of DME 2.1. Blood-retinal barrier The blood-retinal barrier (BRB) is a tight, physiologic barrier that tightly regulates the balance of electrolytes, protein, and water in the retina, and it is composed of both an outer and an inner portion. The inner BRB is formed by tight junctions between retinal capillary endothelial cells, basement membrane surrounding it and pericytes outside (Das et al., 2015). The outer BRB consists of the retinal pigment epithelium (RPE) cells, located between the fenestrated choriocapillaris and the outer retina. An intact BRB is essential in maintaining normal visual function through these processes. 2.2. Alteration of blood retinal barrier Three important changes in the BRB occur in diabetes: 1)
Corresponding author at: Department of Surgery, Ophthalmology, MSC10-5610, 1 University of New Mexico, Albuquerque, NM 87131, United States. E-mail address: [email protected] (A. Das).
http://dx.doi.org/10.1016/j.visres.2017.06.015 Received 13 March 2017; Received in revised form 5 June 2017; Accepted 9 June 2017 0042-6989/ © 2017 Published by Elsevier Ltd.
Please cite this article as: Urias, E.A., Vision Research (2017), http://dx.doi.org/10.1016/j.visres.2017.06.015
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Fig. 1. Alteration of the blood retinal barrier in diabetes mellitus. Chronic inflammation in diabetes leads to production of chemokines (including Chemokine ligand-2, CCL2) that result in leukostasis, diapedesis and influx of monocytes into the retina and extravascular space. Monocytes are differentiated into macrophages which along with activated microglia produce an array of cytokines and chemokines including VEGF, IL-6, TNF-α and Ang-2. These mediators then break down the cell-cell junction molecules resulting in alteration of the blood- retinal barrier. (Reproduced from Das et al., Indian J Ophthalmol. 2016; 64:4–13).
leads to increased monocyte adhesion to endothelial cell layer and trafficking into the retina (Rangasamy et al., 2014). In the lumen of retinal blood vessels, leukocytes continuously roll along the endothelium. The integrin-ligand interactions lead to stable adhesion, or leukostasis. These cells then migrate through the endothelium into the extravascular space where they differentiate into activated macrophages and secrete various cytokines (Fig. 1). The disruption of the BRB in diabetes involves numerous factors and cascade of events within the vascular lumen and in the retinal parenchyma. A prominent factor contributing to the breakdown of the BRB is VEGF (vascular endothelial growth factor). But there are other factors involved including TNF-α and interleukin-1β, growth factors such as hepatocyte growth factor, FGF, and insulin-like growth factor-1 (IGF-1), cytokines such as ICAM-1, IL-6, MCP-1, and histamine (Adamis & Berman, 2008).
breakdown of cell-cell junctions between endothelial cells, 2) pericyte loss, and 3) basement membrane thickening (Fig. 1). Loss of cell-to-cell junctions in the endothelium results in the leakage of red blood cells, plasma and lipid (Frank, 2004). This leakage can be seen clinically on funduscopic exam as intraretinal hemorrhages, edema and hard exudates respectively. Pericytes are contractile smooth muscle –like cells that regulate blood flow in the microcirculation, and their loss leads to focal endothelial cell proliferation and microaneurysms, the earliest clinical lesions present in diabetic retinopathy. Hyperglycemia is the major risk factor contributing towards DME. It induces four major biochemical pathways (polyol pathway, advanced glycation end-product pathway, protein kinase C pathway, and hexosamine pathway) that all lead to increased oxidative stress and inflammation (Brownlee, 2005). Although the DCCT study has shown that tight glucose control prevents development and slows down progression of diabetic retinopathy, further analysis of the DCCT cohort has revealed that the total glycemic exposure (A1C and duration of diabetes) explains only ∼11% of the variation in retinopathy risk in the complete cohort, so that other factors may presumably explain the remaining 89% of the variation in risk among subjects independent of A1C (Lachin et al., 2008). The oxidative stress in diabetes upregulates multiple cytokines and chemokines, such as VEGF, angiopoietins, tumor necrosis factor (TNF), interleukins (ILs) and matric metalloproteinases (MMPs) that leads to breakdown of the BRB. Also, retinal capillary obstruction (leukostasis) or dropout (apoptosis of vascular cells) in diabetes leads to tissue ischemia and hypoxia causing increased retinal VEGF expression through transcriptional regulation by hypoxia-inducible factor 1 alpha (HIF-1α) (Arjamaa & Nikinmaa, 2006). Genetic factors that alter gene expression predispose to or protect against the progression of diabetic retinopathy, or its vision-threatening consequences.
3. Anti-VEGF drugs 3.1. Current therapy VEGF is a potent vasopermeability factor that has been targeted extensively in DME and PDR. Out of all the isoforms, VEGF 165 is the major pro-inflammatory cytokine. The VEGF levels are significantly elevated in vitreous of DME patients when compared with nondiabetic eyes (Funatsu, Noma, Mimura, Eguchi, & Hori, 2009). VEGF induces the phosphorylation of cell-cell junctional molecules such as VE-cadherin, occludin, and ZO-1 and thus causes a breakdown of the BRB. Drugs that directly inhibit the VEGF molecule are anti-VEGF aptamer, pegaptanib (Macugen, OSI), monoclonal antibody fragment ranibizumab (RBZ) (Lucentis, Genentech), full-length antibody bevacizumab (BVZ) (Avastin, Genentech) and soluble VEGF receptor analogs, aflibercept (VEGF-Trap (Regeneron). Other anti-VEGF molecules include small interfering RNAs, bevasiranib (Opko Health), and rapamycin (Sirolimus, MacuSight) (Das et al., 2015). Anti-VEGF drugs are injected intravitreally every 4 weeks under topical anesthesia as office procedures. All the larger clinical trials RIDE/RISE, DRCR, and VIVID/VISTA have shown significant vision improvement and reduction of central retinal thickness in DME patients. Currently, anti-VEGF agents are considered the first line of treatment in center-involving DME, where
2.3. Inflammation Leukostasis occurs early in the DME disease process. The increased leukostasis leads to upregulation of intercellular adhesion molecule (ICAM-1) and retinal vascular leakage (Adamis & Berman, 2008). Use of antibodies to CD-18 or ICAM-1, or genetic knockout of these animals, inhibits retinal leukostasis and breakdown of the BRB (Joussen et al., 2004). Also, increased expression of MCP-1 gene expression in diabetes 2
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Fig. 2. Novel therapeutic strategies in diabetic macular edema. Hyperglycemia in diabetes activates different bio chemical pathways that lead to increased hypoxia, inflammation and oxidative stress with production of several cytokines. This mediators then cause break down of the inner and outer blood retinal barrier through endothelial/ pericyte loss, thickening of basement membrane and photoreceptor/ glial cell dysfunction. The current anti-VEGF therapies have limitations because they target only VEGF rather than other inflammatory molecules. Many novel therapies targeting the other molecules beyond VEGF are now in clinical trials. As shown above.
endogenous VEGF is critical for trophic support for retinal function. A recent study has suggested that chronic suppression of locally synthesized VEGF in the eye may damage the choriocapillaris and cone function (Kurihara, Westenskow, Bravo, Aguilar, & Friedlander, 2012). Knocking out VEGF-A in adult mice results in damage to retinal pigment epithelial (RPE) cells, ablation of the choriocapillaris, and rapid dysfunction of cone photoreceptors. However, there is no strong evidence of such neuronal damage documented in human clinical studies.
the indication of focal/grid laser is limited to non-center-involving DME. However, all the large clinical trials have shown that only 33–45% of DME patients on anti-VEGF agents show 3 lines or more of visual improvement. The rest of DME patients show intermediate response (5–9 letters of improvement) or poor response (< 5 letters of improvement or worse). Eyes with suboptimal early vision response showed poorer long-term visual outcomes than eyes with pronounced early response (mean improvement 3.0 vs 13.8 letters at 3 years) (Gonzalez et al., 2016). The poor response to anti-VEGF agents could be explained by the presence of other factors beyond VEGF as a result of inflammation that are not targeted by the anti-VEGF drugs. Thus, many anti-VEGF-treated eyes with DME do not achieve visual acuity of 20/20 or complete resolution of retinal edema, thus, there is a need for supplemental treatments that might improve visual acuity in eyes with persistent edema despite anti-VEGF therapy (Figs. 2, 3). Although systemic administration of bevacizumab has been involved with side effects such as hypertension, thromboembolic events, similar incidence in patients receiving intravitreal anti-VEGF drugs is not significantly high. Thus, small doses of anti-VEGF drugs for intravitreal injections (almost 1/400th dose of systemic dose) have so far been found to be safe. VEGF has a neuroprotective role, and
3.2. High-dose anti-VEGF therapy As there is a wide range of VEGF levels in vitreous of DME patients, it is possible that those with high levels of VEGF may need higher doses of anti-VEGF drugs. Based on this rationale, the READ-3 study has compared the efficacy of two different doses of anti-VEGF drugs (0.5 and 2.0 mg of ranibizumab) in DME patients. The higher dose of ranibizumab (2.0 mg) did not result in better visual acuity (Sepah et al., 2016). A very similar result was obtained in the RIDE/RISE trials that showed equal efficacy with both the 0.3 and 0.5 mg doses of ranibizumab. As there is less chance of potential systemic risks with the lowest dose (0.3 mg) of ranibizumab, the FDA approved the dose of 3
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Fig. 3. List of novel drugs based on mechanisms of action in diabetic macular edema: 1. Anti-VEGF, 2. Anti-inflammatory, 3. Non-Steroidal anti-inflammatory drugs (NSAIDs), 4. Inhibitors of multiple growth factors, 5. Integrin inhibitors, 6. Cytokine and chemokine inhibitors, 7. Others.
compared to the standard of care. A phase II trial using the VEGF DARPin (abicipar pegol) (Allergan) in wet ARMD patients showed improvement in vision and decreases in central retinal thickness in a way very similar to that of monthly ranibizumab injections (Campochiaro et al., 2013). The data indicated that abicipar can be injected every 12 weeks following loading doses. A phase III clinical trial for abicipar for wet AMD is in progress now. A Phase 2 clinical trial, PALM, has evaluated abicipar pegol in DME patients, and the drug met its study endpoints and the efficacy of abicipar was shown in all three treatment groups (molecularpartners.com).
0.3 mg ranibizumab in DME patients (Brown et al., 2013). 3.3. Extended delivery of anti-VEGF therapy Monthly anti-VEGF injections are difficult for patients to comply with. Different Long – acting delivery systems such as biodegradable implants, biodegradable microspheres, encapsulated cells, and gene therapy are currently under investigation. A phase 1 study testing refillable, non-biodegradable, long-term delivery implant (Genetech) of ranibizumab has been completed on 20 patients with wet age-related macular degeneration (ARMD). The implant is placed under the conjunctiva in the pars plana, and requires a 3.2 mm surgical incision without sutures. The implant is refilled when needed intervals. The results demonstrated improvement in visual acuity of 10 letters throughout 1 year (Rubio, 2014). A phase II clinical trial (Study of the Efficacy, 2017) is now being done in wet ARMD patients to study the efficacy, safety and pharmacokinetics of three different formulations of ranibizumab delivered via the ranibizumab port delivery system compared with the standard of care of intravitreal injections of ranibizumab (ClinicalTrials.gov NCT02510794). As there is a more consistent pharmacokinetic profile with fewer peaks and troughs, sustained delivery of anti-VEGF drugs offers better long-term visual outcomes and disease modification, and thus the treatment burden may be decreased.
4. Steroids 4.1. Current steroid therapy Steroid injections suppress proinflammatory chemokines, and inhibit leukostasis and phosphorylation of cell-junction proteins. The DRCR protocol I trial has shown the efficacy of intravitreal triamcinolone in DME (DRCR Network, 2010). There has been visual improvement and reduction in central retinal thickness with triamcinolone in a similar way to that of ranibizumab up to 24 weeks, but after that period the effect of triamcinolone drops due to high rates of cataract formation and elevation of intraocular pressure (IOP). In a subgroup analysis of pseudophakic patients, the triamcinolone plus laser group was found to be superior to the laser treatment alone group in terms of vision improvement, and equivalent to the ranibizumab group. Sustained release corticosteroids were developed in effort to decrease the number of injections a patient with DME requires. The dexamethasone implant (Ozurdex; Allergan, Inc.) is a biodegradable copolymer that releases the dexamethasone over a 6 month-period and
3.4. VEGF designed ankyrin repeat protein (DARPin) Designed ankyrin repeat proteins (DARPins) are genetically engineered antibody mimetic protein that show highly specific and highaffinity target protein binding. Its small size, high potency, and long intra-vitreal half-life offers the potential for less frequent injections 4
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danazol in these patients.
the implant can be placed in the office, using a 23-gauge needle. The safety and efficacy of the implant has been evaluated in multiple phase II studies, which found that the implant is effective in improving BCVA and in decreasing vascular leakage. In the MEAD Study, dexamethasone intravitreal implant resulted in visual improvement of > 15 letters in 22% patients (0.7 mg) and 18% (0.35 mg) compared with 12% in the sham group (Boyer et al., 2014). Cataract formation was seen in up to 68% of treated patients, and increases in IOP could be managed with medication, or no therapy. The implant is currently FDA approved for use of treatment of DME. The newer generation of flucinolone acetonide implant (Iluvien, Alimera Sciences) is a non-biodegradable microimplant containing 190 μg of drug, with release of 0.2 μg of drug per day. In the randomized Fluocinolone Acetonide for macular Edema (FAME) study, there was significant vision improvement in DME patients (Campochiaro et al., 2011). This implant is now FDA approved for treatment of DME in patients who have not previously demonstrated IOP spikes when using steroids.
5. Cytokine and chemokine Inhibitors 5.1. Angiopoietin-2 inhibitor Angiopoietins are a family of growth factors that bind to endothelial receptor tyrosine kinases, also known as Tie receptors (Rangasamy et al., 2011). When activated, Tie receptors play a role in vascular development and function. Angiopoietin (Ang)-1 and -2 share 60% amino acid identity and bind with similar affinity to Tie-2. The activity of Tie2 is differentially regulated by the two ligands. Ang-1 is a strong agonist of the Tie-2 receptor, and Ang-2 acts as an agonist or antagonist in a context-dependent manner. Ang-2 is an endogenous weak, partial agonist that competes with Ang-1, thereby suppressing Tie2 phosphorylation. Ang-2 is predominantly expressed in endothelial cells, stored in vesicles known as Weibel-Palade bodies, and is rapidly released in response to specific stimuli. Emerging evidence indicates that Ang-2 is upregulated in response to hyperglycemia and plays an important role in the alteration of the BRB in diabetes. Increased Ang-2 leads to decreased phosphorylation of Tie-2 which results in increased retinal vascular permeability (Rangasamy et al., 2011). We previously showed that the up-regulation of proteinases in microvascular endothelial cells by Ang-2 may be an important early response during the development of retinal neovascularization (Das et al., 2003). Many systemic conditions (sepsis, acute lung injury, systemic capillary leak syndrome, disseminated intravascular coagulation and acute kidney injury) with systemic vascular leakage have been reported to be associated with high serum Ang-2 levels. In summary, the benefits of angiopoietin inhibition in diabetic macular edema include decrease in vaso-permeability as well as decrease in inflammation in the retina by decreasing leukocyte adhesion to retinal vessel wall, decreasing cell adhesion molecule, ICAM-1, decreasing chemokines, CCL2 and CCL5 in retinas, and decreasing monocyte trafficking and microglia activation in retinas (Nitta et al., 2015). There are three ongoing clinical trials ongoing targeting angiopoietins in DME patients. A small molecule, AKB-9778 (Aerpio Therapeutics) is an inhibitor of an enzyme, vascular endothelial protein tyrosine phosphatase (VE-PTP) that stimulates phosphorylation of Tie2 in vascular endothelial cells and suppresses VEGF-induced leakage. Levels of both angiopoietin 2 and VE-PTP are increased by hypoxia, accentuating VEGF-mediated retinal neovascularization and vascular leakage. Inhibitors of VE-PTP (AKB-9778) could activate Tie-2 receptors and thus provide a strategy of inhibiting retinal vascular leakage (Campochiaro et al., 2015). The TIME-2 study found that subcutaneous injections of AKB-9778 combined with VEGF inhibitor (ranibizumab) causes a significantly greater reduction in DME than that seen with suppression of VEGF alone (Campochiaro et al., 2016). One advantage of this drug is its subcutaneous administration route that can be easily done by diabetic patients themselves. A phase II trial, BOULEVARD (Roche, Basel, Switzerland) is using an approach of targeting both VEGF and Ang-2 by using a bispecific IgG1 monoclonal antibody (RO6867461) that has one arm binding to VEGF and the other arm binding to Ang-2. Participants are randomized into each arm group (1:1:1): ranibizumab group 0.3 mg, intravitreal injection of R06867461 1.5 mg, and R06867461 6 mg (Clinicaltrials.govBOULEVARD). The study will have duration of 28 weeks. A similar approach is being followed in a phase II trial, RUBY (Regeneron) using a co-formulation of nesvacumab (REGN910) and aflibercept (Clinicaltrials.gov-RUBY). The nesvacumab is a fully human, monoclonal antibody that binds to Ang-2, and aflibercept is a VEGF trap molecule, that binds to VEGF. The advantage of using an inhibitor of Ang-2 is that it also blocks the inflammatory cascade in addition to its beneficial effect of inhibiting the retinal vascular permeability.
4.2. Combination of steroid + anti-VEGF therapy A Phase II DRCR randomized clinical trial is investigating whether the addition of steroid (dexamethasone) to an anti-VEGF treatment regimen (ranibizumab) in eyes with persistent center-involved DME despite anti-VEGF treatment increases visual acuity and decreases DME in the short term, compared with continued anti-VEGF treatment alone (ranibizumab) (DRCR.net). Furthermore, this phase II study is being conducted to provide information for a Phase III trial. 4.3. Triamcinolone acetonide with aflibercept A phase II trial, TANZANITE study recently evaluated the safety and efficacy of a single suprachoroidal injection of, triamcinolone acetonide (Zuprata; Clearside Biomedical, Alpharetta, GA) injectable suspension, given along with an intravitreal injection of aflibercept compared to aflibercept alone in subjects with retinal vein occlusion (RVO). Results demonstrated that 78% of patients in the combination arm required no additional treatments over 3 months compared with 30% in the control arm (clearsidebio.com). Based on the results of this trial, an open-labeled multi-center Phase I/II trial (HULK) has been started using the same combination therapy in DME patients. 4.4. Betamethasone microspheres Betamethasone is a steroid used for a number of diseases including rheumatoid arthritis and systemic lupus erythematosus, dermatitis and psoriasis, allergic conditions such as asthma and angioedema. A phase II/III clinical trial evaluated the efficacy and safety of sub-tenon injection of biodegradable, sustained release betamethasone microspheres (DE-102; Santen Pharmaceutical Co.) in DME patients (clinicaltrials.gov). The study was completed but results are not yet published. 4.5. Danazol Danazol is an orally administered steroid that is already being used to treat other conditions, mainly endometriosis. It is a synthetic steroid analog of a 17-a-ethinyl testosterone, that when given at extremely low doses, is able to strengthen cell-cell junctions and reduce capillary permeability. A safety and efficacy study of oral danazol for the treatment of DME was recently completed (Ampio pharmaceuticals) (clinicaltrials.gov). The study evaluated the efficacy of ultra-low dose danazol (Optina) vs placebo in DME patients. In addition, there was a significant reduction in central retinal thickness compared to those in the placebo group (Singer, Orlando, & Baro-Or, 2016). There was no side effect related to insulin resistance and hyperglycemia in using 5
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activates PPK into catalytically active PKal. PKal further activates bradykinin (BK), which in turn activates the BK B2 receptor (B2R). Both the B2R and B1R receptors are highly expressed in the retina. Specifically, retinal expression of B1R is increased in diabetes. It is speculated that hemorrhage, activated platelets, and unfolded proteins activate the KKS in diabetic retinopathy (Liu & Feener, 2013). Vitreous proteomics analysis in advanced stages of diabetic retinopathy have shown increased levels of plasma KK system components including plasma kallikrein, coagulation factor XII, and high molecularweight kininogen (Kita et al., 2015). A Phase I study (Kalvista Pharmaceutical, Cambridge, MA) studied the safety and tolerability of a novel plasma kallikrein inhibitor, intravitreal KVD001, in patients with DME (clinicaltrials.gov-KVD001). Promising results with increases in VA and decreases in CRT lasting up to 12 weeks have been reported, and based on the outcome, the company is planning a phase II trial in DME in 2017.
5.2. Interleukin Inhibitors IL-6 is an important cytokine that has been consistently elevated in vitreous of DME patients. Intravitreal injection of an antibody against interleukin-6 potentially inhibits choroidal neovascularization in an animal model (Schmidt, Tisdale, Lowden, Kovalchin, & Furfine, 2014). There are two clinical trials investigating the blockage of IL-6 in DME patients. The first trial involves an IL-6 antibody (EBI-031; Eleven Biotherapeutics, Roche), a humanized monoclonal antibody that potentially binds IL-6 and inhibits all known forms of IL-6 cytokine signaling in the eye (ir.elevenbio.com). The second study, READ-4 trial will investigate the safety, tolerability and efficacy of ranibizumab and intravenous tocilizumab alone and in combination in DME patients (Clinicaltrials.gov-READ-4). Tocilizumab, a humanized monoclonal antibody against IL-6 receptor, is an immunosuppressant drug that is used in treatment of rheumatoid arthritis and systemic juvenile idiopathic arthritis. 6. Inhibitors of adhesion molecules
8. Inhibitors of multiple growth factors
6.1. Integrin
Squalamine is a small molecule anti-angiogenic drug that counteracts multiple growth factors in the cascade of angiogenesis including VEGF, platelet- derived growth factor, (PDGF) and basic fibroblast growth factor (Sills et al., 1998). It does not block all VEGF mediated inflammatory pathways, but it inhibits mitogen-activated protein kinase, p38 inflammatory and vascular endothelial-cadherin and other signaling pathways. This compound was discovered in tissues of dogfish sharks that have anti-angiogenic and antiviral properties. A phase II study (IMPACT) in wet ARMD patients demonstrated that more than twice the proportion of patients on combination of squalamine lactate eye drops (0.2%, OHR-102; Ohr Pharmaceutical, New York, NY) and ranibizumab achieved ≥3 line gains in visual acuity at 9 months compared with the ranibizumab alone (clinicaltrials.govsqualamine). A phase II trial using squalamine eye drops is currently ongoing in DME patients.
Integrins are cell-surface receptors that regulate attachments among cells and between cells. In a broad sense, they control the way in which cells communicate with each other in the extracellular matrix. In the retina, integrins are located on endothelial cells of blood vessels and serve to interact with growth factors and regulate their functions. The pathologic process of retinal angiogenesis is largely dependent on specific integrins like αvβ3, αvβ5, and α5β1. An integrin antagonist, Luminate (ALG-1000) (Allegro Ophthalmics, San Juan Capistrano, CA) is an oligopeptide that binds to multiple integrin receptor sites (αvβ3, αvβ5, and α5β1) and can inhibit integrins in multiple pathways. This antagonist also binds to α3β1 that mediates attachment of vitreous to the retinal interface, and induces vitreolysis. Liquefying the vitreous and inducing a posterior vitreous detachment lowers the VEGF levels and prevent them from continuing to accumulate over the macula, and thus helps in reducing the retinal vascular leakage in DME. Recently, the DEL MAR phase IIb clinical trial demonstrated that Luminate (1.0 mg) was non-inferior to intravitreal bevacizumab injections in terms of visual acuity gains and decreases in central retinal thickness and patients needed half the number of injections (allegroeye.com-DEL MAR). A larger, phase II study is currently evaluating the safety and exploring the efficacy of Luminate compared to that of bevacizumab injection and focal laser photocoagulation.
9. Conclusions Currently, anti-VEGF drugs are the first line of treatment in centerinvolving diabetic macular edema. Once the focal/grid laser therapy as the standard treatment for DME, is now being reserved only for noncenter-involving macular edema. Over the last ten years, we have seen the rapid, wide use of anti-VEGF drugs in treatment of many retinal diseases including diabetic macular edema. No doubt, anti-VEGF therapy is an effective therapy in DME. But compared to other angiogenic diseases like proliferative diabetic retinopathy and wet macular degeneration, the effect of anti-VEGF drugs is much less robust in DME patients. Multiple, frequent monthly anti-VEGF injections are needed in DME patients, and a significant number of patients poorly respond to these anti-VEGF drugs. DME appears to be a heterogeneous disease with good responders (33–45%) to anti-VEGF drugs on one end of the spectrum, poor responders on the other end of the spectrum, and many intermediate responders in between. In the latter two groups, probably factors other than VEGF are responsible for alteration of the BRB. The cytokine/chemokine profile of DME patients varies from one to another. No biomarkers are currently available to predict good, poor, or intermediate responders. Probably, genetic factors play an important role in this anti-VEGF responsiveness. Anti-VEGF therapy will still be the first line of treatment in DME patients in the coming years. However, we will see combination therapies of novel inhibitors targeting the molecules beyond VEGF in the coming years in management of diabetic retinopathy. Also, novel drug delivery systems using nanotechnology, sustained-release delivery implants, and stem cell therapy are on the horizon.
6.2. Vascular adhesion protein (VAP-1) Vascular adhesion protein-1 is an endothelial adhesion molecule that is expressed on the surface of endothelial cells. It is involved in leukocyte transmigration during inflammation. In a diabetic rat model, orally given VAP-1 inhibitor prevented increased retinal vascular leakage (Inoue et al., 2013). ASP8232 (Astellas; Northbrook, IL) is an orally administered VAP-1 inhibitor that is currently being studied in DME patients in the phase II VIDI trial (clinicaltrials.gov-VIDI trial). The study involves 3 intervention arms: ASP8232 + sham injections, ASP8232 + ranibizumab injections, placebo + ranibizumab injections. 7. Inhibitors of kallikrein-kinin (KK) system The kallikrein-kinin system (KKS) contributes to the vascular inflammatory response associated with hereditary angioedema (Liu & Feener, 2013). The KKS also mediates vascular hyperpermeability, leukostasis, cytokine production, and retinal thickening in rodent models of diabetic retinopathy. The proinflammatory effects of the KKS are mediated by plasma kallikrein (PKal), a protease derived from a circulating zymogen named plasma prekallikrein (PPK). Factor XII 6
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