Title of study:

Effects of Ranibizumab to delay or regression non-proliferative diabetic retinopathy(NPDR) with DME assessed by microaneurysm changes: A pilot study Objectives Diabetic retinopathy (DR) is a major cause of visual impairment. Anti-vascular endothelial growth factors have demonstrated therapeutic benefits in diabetic macular edema (DME). We aimed to prospectively analyze the effects of early intensive treatment using intravitreal ranibizumab (IVR) injections in nonproliferative diabetic retinopathy patients with macular edema.

Primary objective:

To investigate other efficacy endpoints including other visual acuity, anatomical change in mild-to-moderate NPDR with DME after intravitreal Ranibizumab injection from baseline through 6 months after treatment.

Secondary objectives:

To compare microvascular changes assessed by microaneurysm counts and perifoveal non-perfusion area changes and safty in eyes of mild-to-moderate NPDR with DME after intravitreal Ranibizumab injection from baseline through 6 months after treatment.

Title of study:

Effects of Ranibizumab to delay or regression non-proliferative diabetic retinopathy(NPDR) with DME assessed by microaneurysm changes: A pilot study

Study Rationale:

Diabetic retinopathy is the leading disease that causes acquired vision loss after 20 by making diabetic macular edema and neovascularization. In recent young generation, as prevalence of type 2 diabetes is growing, the burden of sight-threatening retinopathy is increasing on trend.1 Pathologically, angiogenesis is a main cause that destroys the structure of the eye and induces the visual function disorder as VEGF playing an important role in increasing the migration and proliferation of endothelial cells and increasing the permeability of the blood vessels.2,3 VEGF is made from the endothelial cells of retinal tissue, perivascular cells, pigment endothelial cells by hypoxia. And hypoxic condition of intraocular tissues is a key regulator of intra ocular angiogenesis by VEGF, the balance between VEGF and angiogenesis inhibitors determines the neovascular proliferation in diabetic retinopathy.4 VEGF is also inducing the expression of cell-to-cell contact molecule (intracellular adhesion molecule-1, ICM-1) and the adhesion of leukocytes to help the inflammatory response5, as mediator which destroys the blood retinal barrier, affecting the protein of tight junctions, making a microaneurysm and increasing permeability of capillary, that makes the liquid leakage and macular edema.5,6 Microanuerysm is the earliest clinical manifestations, the saccular local lesion that perivascular cells protruding in damaged areas on the capillary wall. According to Stitt AW et al9, diabetic microaneurysm is non-functioning extrusion of the vascular system from the deep part of inner retinal capillary plexus.

It is sometimes disappeared by being blocked with blood clots, on the other hand, new microanerysm is occurred in the other vascular bed structure.

Through these changes, the investigators know the course of a diabetic retinopathy and it is known that the generation rate of microaneurysm is associated with the clinically significant progression of macular edema (CSME) in mild-to-moderate nonproliferative diabetic retinopathy .10,11 In addition, the number of microanerysm is an important prognostic indicator which can estimate the progression or regression of diabetic retinopathy, as predicting whether becoming better or worse in diabetic retinopathy.12 Kohner and Sleightholm13 describe this concept at first time in 1986, its association with the number of microvascular flow and the severity of diabetic retinopathy. In recent years it is reported that measuring the number of microaneurysm and the turnover rate associated with the appearance or disappearance of microaneurysm, are predictors in progression of diabetic retinopathy and macular edema.14 To delay the progression of diabetic retinopathy and to improve macular edema, the laser photocoagulation have been the important role.15 Although the laser photocoagulation have had treatment effect in the diabetic retinopathy by reducing the amount of VEGF in micraneurysm and by degenerating the neovascularization after laser therapy, there was a problem that has many limitations - peripheral visual field defects, night blindness, progression of macular edema etc., and that the disease does not cured in a good time because of limits of laser therapy due to cataract, vitreous hemorrhage and turbidity. As an alternative method to solve these limitations, there is an anti-VEGF therapy.6 According to a previous study result, intraocular injection of Bevacizumab inhibits occurring of neovascularization by blocking the VEGF receptor .17 Recently, several studies have been reported that when injected intravitreal anti-VEGF, macular edema is improved and neovascularization is inhibited, by reducing the leakage of neovascularization.3 Especially, Leicht SF et al18 reported the number of microaneurysms and turnover rate in NPDR(non-proliferative diabetic retinopathy) patients injected with Ranibizumab. And the result showed entire number of the microaneurysms and turnover rate are decreased, which could be mean the regression of diabetic retinopathy and it could decide the therapeutic effect.

On this study, through the fluorescein fundus angiography, the average number of microaneurysms was significantly decreased after intravitreal injection of anti-VEGF therapy (p<0.05). The decrease of 35.70±24.79% in the treatment group was statistically higher than 13.95±38.21% in the control group of the fellow eye (p<0.05).

The result is found because decreased concentration of intravitreal VEGF inhibits the progression of diabetic retinopathy, such as endothelial cell proliferation and endothelial cell damage on retinal capillary and perivascular cells.

Sjølie AK et al12 reported up-regulation of VEGF occuring microaneurysms causes endothelial cell proliferation and inflammation and effusion reaction, so anti-VEGF is effective in early diabetic retinopathy. But there is less effectiveness in late diabetic retinopathy as it reaches the non-changing point. Also Kohner EM et al19 reported diabetic retinopathy lesions are reversible and could be delayed in early diabetic retinopathy.

So far, changes of microaneurysms in late diabetic retinopathy is uncertain, and if there would be a finding according to anti-VEGF therapy, the investigators could get a clue of surrogate marker which represents treatment results in diabetic retinopathy.

This study was designed to find the clinical evaluation and reduction rate through fluorescein angiography as microaneurysm examination tools to research NPDR with DME treatment results assessed by microaneurysm counts, timely monitored with anti-VEGF therapy.

Objectives To evaluate the effects of intravitreal Ranibizumab injection on microvascular changes in eyes of mild-to-moderate NPDR with DME.

Primary objective:

To investigate other efficacy endpoints including other visual acuity, anatomical change in mild-to-moderate NPDR with DME after intravitreal Ranibizumab injection from baseline through 6 months after treatment.

Secondary objectives:

To compare microvascular changes assessed by microaneurysm counts and perifoveal non-perfusion area changes and safty in eyes of mild-to-moderate NPDR with DME after intravitreal Ranibizumab injection from baseline through 6 months after treatment.

Primary and secondary endpoints:

Primary endpoint:

To compare other visual acuity, anatomical changes after intravitreal Ranibizumab injection from baseline through 6 months after treatment.

i) The changes in best corrected visual acuity (BCVA) using ETDRS chart. ii) The central macular thickness - Circle Diameters : 1 mm ETDRS by spectralis OCT ; Heidelberg Engineering.

Secondary endpoints:

To compare microaneurysmal changes and perifoveal non-perfusion area and safty after intravitreal Ranibizumab injection from baseline through 6 months after treatment.

i) The total number of microaneurysm by fundus photo using Retmarker DR(version 1.0.2) software.

ii) The microaneurysm formation rate : Number of new MAs detected/month. iii) The microaneurysm disappearance rate : Number of MAs that resolved/month. iv)The microaneurysm turnover. v) Perifoveal non-perfusion area in FAG (mm²) using ImageJ software (version 1.52a) by FAG image.

vi) Safety parameters : Systemic adverse events (MI, CVA, etc), Ocular adverse events (retinal detachment, RPE tear, endophthalmitis, uveitis, vitreous hemorrhage, subretinal hemorrhage, cataract , IOP elevation, etc).

Methodology:

single center, prospective, interventional, one arm, pilot study

Evaluation Participants will be evaluated with a full ocular examination at each visit (VA measurement, tonometry, slit lamp exam). Fluorescein angiography (FA) will be performed at baseline, at 3 months after baseline and at the last visit(6 months after baseline), and OCT will be performed monthly (baseline and 1, 2, 3, 4, 5, 6 months from baseline).

The MAs and perifoveal non-perfused areas in individual retinas were evaluated at 6 months using fundus photography and FA imaging. The Retmarker (version 1.0.2 by Retmarker Ltd, Coimbra, Portugal) software was used for automatic measurement and analysis of changes in number and extent of MAs on fundus photographs and to calculate the total number and turnover of MAs.

Perifoveal non-perfused area was estimated using the early FA image (from each of the three examinations) in which both the vascular arch and the non-perfused area were clearly visualized. Subsequently, the ImageJ software (version 1.25a 23/04/2018 by ImageJ, USA) was used for scaling each image to 200 µm and for equalizing the contrast and sensitivity of each picture to the maximum possible extent, by auto-adjustment of brightness and contrast. The raw red-green-blue (RGB) images were then converted to 8-bit images, with the threshold set for optimal visualization of the non-perfused area. The same threshold was applied to all images of the same patient.

Result analysis The investigators compare the differences between at baseline, at 3 month, and at 6 month. Statistical analyses will be performed using SPSS ver.18.0 (SPSS Inc., Chicago, Il, USA). Kolmogorov-Smirnov test was applied to test for normality of sample group data. the paired T-test was used for comparative statistical analysis of these parameters. (BCVA, CRT, total number of MAs, MA formation rate, MA disappearance rate, MA turnover, and perifoveal non-perfused area)

Inclusion Criteria:

• Patients (Male & female) ≥40 years of age
• Type 2 DM
• Best corrected visual acuity ≥ 20/200 (Snellen equivalent using Early Treatment Diabetic Retinopathy Study chart)
• central retinal thickness of ≥300 µm on optical coherence tomography
• nonproliferative diabetic retinopathy (NPDR) with diabetic macular edema

Exclusion Criteria:

• proliferative diabetic retinopathy
• Vitreous hemorrhage
• previous history of vitreoretinal surgery, post-cataract operation status (≤4 months before participation in this study)
• prior treatment with anti-VEGF drugs, intraocular corticosteroids, and/or retinal laser application
• Uncontrolled hypertension.
• Uncontrolled glaucoma.
• If both eyes met the study inclusion criteria, the more severely affected eye was selected

Totally, 25 eyes of 25 patients (13 males, 12 females, mean age 63.80±11.45 years; range: 41-80 years) were included in the study

Twenty-five eyes of 25 type 2 diabetes mellitus patients with DME were included between August 2016 and February 2019. For 6 months, patients were administered 0.5-mg IVR injections monthly.

Ocular evaluation, including best-corrected visual acuity (BCVA; using the Early Treatment Diabetic Retinopathy Study chart), central retinal thickness (CRT; using optical coherence tomography), fundus photography, and fluorescein angiography, was performed for all participants. Results obtained at baseline were compared to those observed after 6 months.