EFFECT OF INTRAVITREAL TRIAMCINOLONE IN DIABETIC MACULAR EDEMA UNRESPONSIVE TO INTRAVITREAL BEVACIZUMAB SOHEE JEON, MD,*† WON KI LEE, MD, PHD‡ Purpose: To evaluate the efficacy of intravitreal triamcinolone injection in diabetic macular edema unresponsive to intravitreal bevacizumab. Methods: Patients with diabetic macular edema unresponsive to at least three monthly intravitreal bevacizumabs were included. At least 2 months after the last intravitreal bevacizumab, intravitreal triamcinolone was performed after obtaining an aqueous humor sample. Multiplex cytokine array was used to assay vascular endothelial growth factor, interleukin (IL)-2, IL-6, IL-8, tumor necrosis factor-a, and transforming growth factor-b2. Bestcorrected visual acuity and central subfield thickness were evaluated from Month 0 to 3. Results: Twenty eyes were enrolled. The mean best-corrected visual acuity was 47.1 ± 18.9 letters at baseline, and significantly increased to 53.3 ± 19.7 letters at 1 month (P = 0.002) and 52.4 ± 19.1 letters at 2 months (P = 0.041). These visual gains were not sustained at 3 months (50.9 ± 18.6; P = 0.204). A decrease in central subfield thickness more than 11% of baseline occurred in 12 eyes at 1 month. Multivariate analysis showed that intraocular levels of IL-8 (b = 0.538 P = 0.006) was an independent factor for anatomic response at 1 month. Conclusion: Intravitreal triamcinolone has a role in patients who are unresponsive to intravitreal bevacizumab over the short-term. Elevated intraocular IL-8 levels were related to the efficacy. RETINA 34:1606–1611, 2014
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nearly 30 years, focal/grid laser photocoagulation has been the standard treatment for DME because the early treatment diabetic retinopathy study (ETDRS) showed beneficial effects of laser in DME.3 Vision has failed to improve in a substantial number of patients, however, after focal/grid laser photocoagulation. Peribulbar4 or intravitreal5–8 injection of corticosteroids is most frequently used as primary or adjuvant medical treatment for DME. Many patients, however, experience an elevation of intraocular pressure (IOP) and cataract progression after treatment, and the efficacy on DME is not satisfactory over long-term; a 2-year result of a multicenter randomized clinical trial conducted by Diabetic Retinopathy Clinical Research Network (DRCR.net) showed that the focal/grid laser is a better treatment than intravitreal triamcinolone (IVTA) in eyes with DME involving the fovea with visual acuity between 20/40 and 20/320.8 Newer drugs targeting vascular endothelial growth factor (VEGF) have been introduced, based on the fact
iabetic macular edema (DME) is a major complication of diabetic retinopathy (DR) and is the most frequent cause for preventable blindness in working adults in many countries. A large meta-analysis has estimated the overall prevalence of any DR to be 34.6%, and the prevalence of DME was 6.8%. There are approximately 93 million people with DR and 21 million with DME worldwide.1,2 Various therapeutic approaches have been used to minimize or delay visual loss from DME. For From the *Catholic High-Performance Cell Therapy Center, College of Medicine, The Catholic University of Korea, Seoul, Korea; †Department of Medical Life science, College of Medicine, The Catholic University of Korea, Seoul, Korea; and ‡Department of Ophthalmology, Seoul St Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea. None of the authors have any financial/conflicting interests to disclose. Reprint requests: Won Ki Lee, MD, PhD, Department of Ophthalmology, Seoul St Mary’s Hospital, College of Medicine, The Catholic University of Korea, #505 Banpo-Dong, Seocho-Gu, Seoul 137-701, Korea; e-mail: [email protected]
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that VEGF is a major mediator of increased retinal permeability in DME.9 Many large multicenter clinical trials have shown promising anatomic and functional outcomes from anti-VEGF in various protocols and intervals.10–15 Anti-VEGF is associated with better visual outcomes and lower adverse events at two years compared with triamcinolone.10 Therefore, anti-VEGF treatment has rapidly gained popularity in DME treatment around the world. A substantial number of patients remain, however, unresponsive to multiple injections of anti-VEGF. The safety and efficacy of ranibizumab in diabetic macular edema (RESOLVE) study showed that approximately 10% of patients enrolled in the ranibizumab arm show the same or decreased visual acuity at 12 months.13 Intraocular cytokines have been extensively studied to evaluate their contribution to DME and indicate that VEGF and various inflammatory cytokines aggravate DME.16–22 In addition, there is an increased awareness that several intraocular inflammatory cytokines are further upregulated after anti-VEGF treatment, potentially because of compensatory reactions in response to decreased VEGF.23–25 Based on these findings, we hypothesized that DME patients who are unresponsive to multiple injections of anti-VEGF might have a different intraocular cytokine milieu from other DME patients, suggesting a possible role for corticosteroids in these patients. Therefore, in this study, we evaluated the efficacy of IVTA in patients who were unresponsive to repeated anti-VEGF and further investigated their intraocular cytokine milieu in relation to the efficacy of IVTA.
Patients and Methods The study was a prospective, open-label, uncontrolled nonrandomized interventional case series performed on patients with DME unresponsive to intravitreal bevacizumab (IVB) with or without focal laser photocoagulation. Institutional Review Board/ Ethics Committee approval was obtained before recruitment. The study protocol adhered to the tenets of the Declaration of Helsinki. All participants signed an informed consent form after a detailed explanation of the study design, associated surgical procedures for scientific purposes, and adjuvant imaging procedures. Among the patients with DME seen between January 2012 and June 2012 at Seoul St Mary’s hospital, we prospectively enrolled patients who were unresponsive to IVB. Inclusion criteria were: 1) at least three previous IVB treatments with monthly intervals, with or without focal laser photocoagulation; and 2) changes in central subfield thickness (CST)
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detected by spectral domain optical coherence tomography (OCT3; Carl Zeiss Meditec, Inc, Dublin, CA) less than 11% (the reliability limit for real change determined in the DRCR.net study26) of preinjection thickness. Exclusion criteria were: 1) study eyes with a history of IVTA; 2) eyes mainly with focal leakage from extrafoveal microaneurysm, which was treatable with focal laser; 3) eyes with pharmacological intervention in the fellow eye within 3 months; 4) IOP $ 25 mmHg at baseline; 5) any ocular disease other than DR; 6) history of major surgery; or 7) media opacity hindering retinal examination. Intravitreal triamcinolone was performed under sterile condition at least 2 months after the last IVB to avoid the residual effects of IVB. Before the injection of triamcinolone acetonide, undiluted aqueous humor sample (50–100 mL) was obtained through a limbus using a 30-gauge needle and stored at −70°C until analysis. A dose of 4 mg in 0.1 mL triamcinolone acetonide was injected through the pars plana at 3.5 mm from the limbus using a 30-gauge needle. A comprehensive ocular examination including ETDRS best-corrected visual acuity (BCVA), IOP measurement, slit-lamp examinations, color fundus photography, macular cube 512 · 128 scan by spectral domain optical coherence tomography, and FA were performed before IVTA. ETDRS BCVA, IOP, slitlamp examination, and spectral domain optical coherence tomography were evaluated monthly to month 3. Best-corrected visual acuity was measured at 4 m with standard ETDRS protocols using a testing chart transilluminator (Lighthouse International, New York, NY). Visual acuity was scored as the total number of letters read correctly. Aqueous samples were analyzed using suspension array technology (xMAP; Luminex Corp, Austin, TX). Capture bead kits (Beadlyte; Upstate Biotechnology, Lake Placid, NY) were used for the detection of interleukin (IL)-2, IL-6, IL-8, tumor necrosis factor-a, transforming growth factor-b2, and VEGF. The undiluted samples were incubated overnight. Kits were run according to the manufacturer’s instructions. Standard curves for each cytokine (in duplicate) were generated using the reference cytokine concentrations supplied with the kit. All incubation steps were performed at room temperature and in the dark to protect the beads from light. Samples were read on the suspension array system. Statistical Analysis The Wilcoxon signed-rank test was used to analyze the changes in CST and BCVA after IVTA during the follow-up. Demographic characteristics, such as age,
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sex, duration of diabetes, total number of serial antiVEGF injections, intervals between last injection (months), and intraocular cytokine levels including VEGF, IL-2, IL-6, IL-8, transforming growth factorb2, and tumor necrosis factor-a were used as dependent variables for anatomic response at 1 month. Intraocular levels of VEGF, IL-2, IL-6, IL-8, transforming growth factor-b2, and tumor necrosis factor-a were logtransformed, because none displayed a normal distribution as assessed using the Shapiro–Wilk test. An anatomic response was calculated as follows: Anatomic response ¼ 1− CSTfollow-up =CSTbaseline · 100 Statistical analyses were performed using the statistical software (SPSS for Windows, version 13.0; SPSS Science, Chicago, IL). A P value ,0.05 was considered as statistically significant. Results Twenty eyes (20 patients) were enrolled in this prospective study. The baseline characteristics are summarized in Table 1. The mean age was 60.8 ± 6.7 years, and the mean duration of diabetes was 13.2 ± 8.9 years. Intravitreal bevacizumab was performed a mean of 4.2 ± 1.1 times, and the last IVB was performed 2.7 ± 1.5 months before IVTA. Among the patients, 10 eyes were pseudophakic, and 16 eyes had a history of focal laser photocoagulation. Figure 1 shows the changes in CST and BCVA during the follow-up period. The mean CST was 465.5 ±131.9 mm at baseline, and significantly Table 1. Baseline Characteristics of Study Population (n = 20) Gender, male Age, years Duration of diabetes, years Insulin treatment, yes Hypertension, yes* HbA1c, % Number of previous IVB, times Time interval from last IVB, months History of cataract operation, yes History of focal laser treatment, yes
10 (50.0) 60.8 ± 6.7 13.2 ± 8.9 13 (65.0) 15 (75.0) 7.4 ± 1.8 4.2 ± 1.1 2.7 ± 1.5 10 (50.0) 16 (80.0)
Values are represented in frequency (percentage) for categorical variables, mean, and standard deviation for continuous variables. *Hypertension was defined using the average of 2 blood pressure readings with cut points of systolic blood pressure $ 130 mmHg, diastolic blood pressure $ 85 mmHg, or hypertension medication use. HbA1c, glycated hemoglobin.
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decreased to 380.5 ± 126.0 mm at 1 month (P , 0.001) and 402.1 ± 140.2 mm at 2 months (P = 0.004) after IVTA. The difference was not significant at 3 months (411.8 ± 130.6; P = 0.057). A decrease in CST more than 11% of baseline occurred in 12, 10, and 9 eyes at 1, 2, and 3 months, respectively. In 12 eyes showing a decrease in CST more than 11% at 1 month, the mean CST was reduced by 28% compared with baseline value (463.2 ± 133.8 mm at baseline and 325.9 ± 91.8 mm at 1 month; P = 0.02). The mean BCVA was 47.1 ± 18.9 letters at baseline, and significantly increased to 53.3 ± 19.7 letters at 1 month (P = 0.002) and 52.4 ± 19.1 letters at 2 months (P = 0.041). These visual gains were not sustained at 3 months (50.9 ± 18.6; P = 0.204). At baseline, the median VEGF level was 6.14 pg/ mL (range, 3.2–41.96). The values of each cytokine are shown in Table 2. The effects of demographic characteristics and intraocular cytokines on anatomic response were examined by univariate and multivariate regression analysis. Simple regression analysis revealed that duration of diabetes (P = 0.030) and intraocular levels of IL-8 (P = 0.041) were related to anatomic response at 1 month. Multivariate regression analysis showed that intraocular levels of IL-8 (b = 0.538 P = 0.006) was an independent factor for anatomic response at 1 month after IVTA, after adjusting for age, sex, and total number of serial anti-VEGF injections (Figure 2). No serious adverse event, such as endophthalmitis or uncontrolled IOP despite medical treatment, was observed throughout the study. Of 20 eyes, 5 eyes (25.0%) experienced IOP higher than 21 mmHg during the follow-up, but the IOP was controlled with topical eye drops.
Discussion This study showed beneficial effects of IVTA on DME unresponsive to multiple IVBs for at least 2 months. It is notable that IVTA resulted in a mean reduction in CST of 28% at 1 month in 12 of 20 eyes, considering that they have shown no response to conventional IVB treatment. Triamcinolone reduces vascular permeability by downregulating VEGF expression,27,28 inhibiting the arachidonic acid pathway,29 and increasing the activity and/or the density of the tight junctions of the retinal capillary endothelial cells.30 In addition, triamcinolone inhibits osmotic swelling in retinal glial cells by releasing endogenous adenosine,31,32 and causes vasoconstriction and decreased hydrostatic pressure, which improves Starling’s equilibrium.33 The anti-inflammatory effects of
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Fig. 1. The changes in CST and BCVA during the follow-up period. A. Baseline CST was decreased significantly at Month 1 and 2 after IVTA injection (P , 0.001 and 0.001), but the significance was diminished at Month 3 (P = 0.085). B. The BCVA improved significantly at Month 1 and 2 (P = 0.003 and 0.019), but these visual gains were not sustained at Month 3 (P = 0.109).
IVTA also contribute to resolve DME by inhibiting proinflammatory cells, and pro-angiogenic and inflammatory mediators, such as IL-6 and IL-8.34,35 The effect of IVTA in our patients seemed largely because of the anti-inflammatory effect, in addition to its antipermeability effect, because the efficacy was associated with elevated levels of aqueous IL-8. IL-8 is associated with increased vascular permeability and is elevated in patients with DR and DME.19–22 IL-8 is a potent proinflammatory chemokine that induces the accumulation of neutrophils along the vascular wall.21 A central role for leukostasis in mediating retinal vascular damage has been described.36 Of interest, the VEGF levels were low in our patients at the time of IVTA, at least 2 months after the last IVB, which might be associated with repeated IVBs. Funk et al23 also observed that patients responding well to anti-VEGF therapy still presented disease activity and needed regular retreatment despite constantly minimal VEGF levels. This may suggest that other upstream or parallel pathways of the VEGF signaling pathway may be important for development of DME and that VEGF alone is not the sole etiologic factor responsible for DME development.37 Several cytokines like IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) have been proposed Table 2. Baseline Intraocular Levels of Each Cytokine (n = 20) Cytokine, pg/mL VEGF TGF-b2 IL-2 IL-6 IL-8 TNF-a
Values 6.14 516.44 0.98 6.67 12.77 0.87
(3.20–41.96) (111.32–1,849.97) (0.34–2.70) (0.22–81.70) (1.42–33.28) (0.65–2.75)
Values are represented as median (range). TGF, transforming growth factor; TNF, tumor necrosis factor.
to be implicated in the development of DME.17–19,23 Results of these reports somewhat differ in which cytokines show increased expression and are associated with the disease activity or treatment response. Rho et al19 observed that recurrence of DME after IVB was associated with elevated aqueous IL-6, but not VEGF. IL-6 is a cytokine that functions widely throughout the inflammatory cascade and is known to induce acute phase reactions and increase vascular permeability.18 However, another study was unable to replicate the finding of increased aqueous IL-6 in patients with DME. In that study, significantly increased expression of IL-8 and MCP-1 were demonstrated at baseline, which was not affected by IVB therapy.23 Differences between the studies could be related to ethnicity or underpowering due to small sample sizes. Large interindividual differences in DR stage, duration of DME, and systemic condition could be another explanation. Particularly, in the eyes treated by anti-VEGF drug, compensatory increases in other inflammatory mediators might be resulted from a sudden decrease in VEGF, and the extent of increase would be different according to the span of anti-VEGF treatment, number of injection, and elapsed time from the last injection.23–25,37,38 In this study, the duration of diabetes was associated with the anatomic response in univariate regression analysis. Long duration of diabetes is one of the most important risk factors for development of DME.39 We speculate that the long-standing diabetes continuously disrupts balance between prosurvival neutrophils and inflammatory mediators, leading to chronic inflammatory response in retinal endothelial and neural cells. We used “at least 3 monthly IVB” and “less than 11% of reduction in CST” for the definition of unresponsive DME in this study. More prolonged IVB treatment might result in more favorable responses in
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2014 VOLUME 34 NUMBER 8
acuity and reduced macular thickness; however, these effects were not sustained for more than 3 months. The efficacy was associated with elevated levels of aqueous IL-8, which stress the role of suppression of inflammatory mediators in addition to VEGF in these patients. Further studies with larger number of patients and longer follow-up period are required to clarify the longterm efficacy of IVTA in patients with DME showing poor responses to conventional anti-VEGF therapy. In addition, a comparative study on the differences in the intraocular cytokine levels between anti-VEGF responder and nonresponder is warranted to elucidate the roles of specific cytokines in the pathogenesis of DME and to get a rationale of individualized treatment using steroid. Fig. 2. The effect of intraocular cytokines on changes in early treatment diabetic retinopathy study (ETDRS) letters at Month 3. IL-8 was an independent factor for visual gain at 3 months after IVTA injection, after adjustment of age, and duration of diabetes (b = 0.841, P , 0.001).
some cases. Also, the absence of progression might be a sign of efficacy. Nonetheless, in Ranibizumab for Edema of the Macula in Diabetes (READ-2) study, 4 ranibizumab injections over a span of 6 months resulted in a mean reduction in excess foveal thickness of approximately 50% and an average improvement in BCVA of 7.4 letters.11 DRCR net study showed that most of the overall improvement in mean visual acuity within the ranibizumab-treated groups occurred by the 8-week study visit. The benefits of ranibizumab were observed as early as 7 days after treatment initiation, signifying a very rapid effect of the drug in DME.15 Therefore, patients in our series showing no anatomic and functional improvement at all with a mean of 4.2 monthly IVBs could be considered as candidates of “nonresponder” in real clinical practice. In addition, the study results provide meaningful information on this group, although some of them would not be true nonresponders. Our study has a number of inherent limitations including few patients, short follow-up period, and noncomparative study design. It is not clear whether continuous IVTA in our patients would show a beneficial effect over the long-term and better results than continuous IVB. Treatment benefits of IVTA would be often limited by the common side effects of IOP elevation and cataract formation. Nonetheless, our study results suggest that IVTA can limit the opportunity for accumulated fluid to persist in refractory DME to multiple IVBs and may have a role as an adjuvant treatment to IVB. In conclusion, IVTA had beneficial effects on DME unresponsive to multiple IVBs in improved visual
Key words: bevacizumab, diabetic macular edema, triamcinolone. References 1. Cheung N, Mitchell P, Wong TY. Diabetic retinopathy. Lancet 2010;376:124–136. 2. Yau JW, Rogers SL, Kawasaki R, et al; Meta-Analysis for Eye Disease (META-EYE) Study Group. Global prevalence and major risk factors of diabetic retinopathy. Diabetes Care 2012;35:556–564. 3. Early Treatment Diabetic Retinopathy Study Group. Photocoagulation for diabetic macular edema: ETDRS report no. 4. Int Ophthalmol Clin 1987;27:265–272. 4. Chew E, Strauber S, Beck R, et al. Randomized trial of peribulbar triamcinolone acetonide with and without focal photocoagulation for mild diabetic macular edema: a pilot study. Ophthalmology 2007;114:1190–1196. 5. Martidis A, Duker JS, Greenberg PB, et al. Intravitreal triamcinolone for refractory diabetic macular edema. Ophthalmology 2002;109:920–927. 6. Gillies MC, Sutter FK, Simpson JM, et al. Intravitreal triamcinolone for refractory diabetic macular edema: two-year results of a double-masked, placebo-controlled, randomized clinical trial. Ophthalmology 2006;113:1533–1538. 7. Gillies MC, McAllister IL, Zhu M, et al. Intravitreal triamcinolone prior to laser treatment of diabetic macular edema: 24month results of a randomized controlled trial. Ophthalmology 2011;118:866–872. 8. Diabetic Retinopathy Clinical Research Network. A randomized trial comparing intravitreal triamcinolone acetonide and focal grid photocoagulation for diabetic macular edema. Ophthalmology 2008;115:1447–1449. 9. Hippenstiel S, Krull M, Ikemann A, et al. VEGF induces hyperpermeability by a direct action on endothelial cells. Am J Physiol 1998;274:L678–L684. 10. Diabetic Retinopathy Clinical Research Network Writing Committee, Elman MJ, Bressler NM, Qin H, et al. Expanded 2-year follow-up of ranibizumab plus prompt or deferred laser or triamcinolone plus prompt laser for diabetic macular edema. Ophthalmology 2011;118:609–614. 11. Nguyen QD, Shah SM, Heier JS, et al; READ-2 Study Group. Primary end point (six months) results of the ranibizumab for edema of the macula in diabetes (READ-2) study. Ophthalmology 2009;116:2175–2181.
IVTA IN DME UNRESPONSIVE TO BEVACIZUMAB JEON AND LEE 12. Nguyen QD, Shah SM, Khwaja AA, et al; READ-2 Study Group. Two-year outcomes of the ranibizumab for edema of the macula in diabetes (READ-2) study. Ophthalmology 2010; 117:2146–2151. 13. Massin P, Bandello F, Garweg JG, et al. Safety and efficacy of ranibizumab in diabetic macular edema (RESOLVE Study): a 12-month, randomized, controlled, double-masked, multicenter phase II study. Diabetes Care 2010;33:2399–2405. 14. Mitchell P, Bandello F, Schmidt-Erfurth U, et al; RESTORE Study Group. The RESTORE study: ranibizumab monotherapy or combined with laser versus laser monotherapy for diabetic macular edema. Ophthalmology 2011;118:615–625. 15. Nguyen QD, Brown DM, Marcus DM, et al; RISE and RIDE Research Group. Ranibizumab for diabetic macular edema: results from 2 phase III randomized trials: RISE and RIDE. Ophthalmology 2012;119:789–801. 16. Lim JW, Han JR. Aqueous humour levels of vascular endothelial growth factor and erythropoietin in patients with diabetic macular oedema before and after intravitreal erythropoietin injection. Clin Experiment Ophthalmol 2011;39:537–544. 17. Funatsu H, Noma H, Mimura T, et al. Association of vitreous inflammatory factors with diabetic macular edema. Ophthalmology 2009;116:73–79. 18. Funatsu H, Yamashita H, Ikeda T, et al. Vitreous levels of interleukin-6 and vascular endothelial growth factor are related to diabetic macular edema. Ophthalmology 2003;110: 1690–1696. 19. Roh MI, Kim HS, Song JH, et al. Effect of intravitreal bevacizumab injection on aqueous humor cytokine levels in clinically significant macular edema. Ophthalmology 2009;116:80–86. 20. Suzuki Y, Nakazawa M, Suzuki K, et al. Expression profiles of cytokines and chemokines in vitreous fluid in diabetic retinopathy and central retinal vein occlusion. Jpn J Ophthalmol 2011; 55:256–263. 21. Ghasemi H, Ghazanfari T, Yaraee R, et al. Roles of IL-8 in ocular inflammations: a review. Ocul Immunol Inflamm 2011; 19:401–412. 22. Elner SG, Strieter R, Bian ZM, et al. Interferon-induced protein 10 and interleukin 8. C-X-C chemokines present in proliferative diabetic retinopathy. Arch Ophthalmol 1998;116:1597–1601. 23. Funk M, Schmidinger G, Maar N, et al. Angiogenic and inflammatory markers in the intraocular fluid of eyes with diabetic macular edema and influence of therapy with bevacizumab. Retina 2010;30:1412–1419. 24. Forooghian F, Kertes PJ, Eng KT, et al. Alterations in the intraocular cytokine milieu after intravitreal bevacizumab. Invest Ophthalmol Vis Sci 2010;51:2388–2392. 25. Jeon S, Lee WK. Intravitreal bevacizumab increases intraocular interleukin-6 levels at 1 day after injection in patients with proliferative diabetic retinopathy. Cytokine 2012;60:535–539. 26. Diabetic Retinopathy Clinical Research Network, Krzystolik MG, Strauber SF, Aiello LP, et al. Reproducibility of macular thickness
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and volume using Zeiss optical coherence tomography in patients with diabetic macular edema. Ophthalmology 2007; 114:1520–1525. Fischer S, Renz D, Schaper W, Karliczek GF. In vitro effects of dexamethasone on hypoxia-induced hyperpermeability and expression of vascular endothelial growth factor. Eur J Pharmacol 2001;411:231–243. Matsuda S, Gomi F, Oshima Y, et al. Vascular endothelial growth factor reduced and connective tissue growth factor induced by triamcinolone in ARPE19 cells under oxidative stress. Invest Ophthalmol Vis Sci 2005;46:1062–1068. Doukas J, Hechtinan HB, Shepro D. Endothelial-secreted arachidonic metabolites modulate polymorphonuclear leukocyte chemotaxisand diapedesis in vitro. Blood 1988;71: 771–779. Antonetti DA, Wolpert EB, DeMaio L, et al. Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin. J Neurochem 2002;80:667–677. Uckermann O, Kutzera F, Wolf A, et al. The glucocorticoid triamcinolone acetonide inhibits osmotic swelling of retinal glial cells via stimulation of endogenous adenosine signaling. J Pharmacol Exp Ther 2005;315:1036–1045. Wurm A, Iandiev I, Hallborn M, et al. Purinergic receptor activation inhibits osmotic glial cell swelling in the diabetic rat retina. Exp Eye Res 2008;87:385–393. Paques M, Krivosic V, Girmens JF, et al. Decreased venoustortuosity associated with resolution of macular edema after intravitreal injection of triamcinolone. Retina 2005;25: 1099–1101. Hurme M, Siljander P, Anttila H. Regulation of interleukin-1 beta production by glucocorticoids in human monocytes: the mechanism of action depends on the activation signal. Biochem Biophys Res Commun 1991;180: 1383–1389. Waage A, Bakke O. Glucocorticoids suppress the production of tumour necrosis factor by lipopolysaccharide-stimulated human monocytes. Immunology 1988;63:299–302. Joussen AM, Poulaki V, Le ML, et al. A central role for inflammation in the pathogenesis of diabetic retinopathy. FASEB J 2004;18:1450–1452. Owen LA, Hartnett ME. Soluble mediators of diabetic macular edema: the diagnostic role of aqueous VEGF and cytokine levels in diabetic macular edema. Curr Diab Rep 2013;13: 476–480. Jonas JB, Jonas RA, Neumaier M, et al. Cytokine concentration in aqueous humor of eyes with diabetic macular edema. Retina 2012;32:2150–2157. Klein R, Knudtson MD, Lee KE, et al. The Wisconsin Epidemiologic Study of Diabetic Retinopathy XXIII: the twentyfive-year incidence of macular edema in persons with type 1 diabetes. Ophthalmology 2009;116:497–503.
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nearly 30 years, focal/grid laser photocoagulation has been the standard treatment for DME because the early treatment diabetic retinopathy study (ETDRS) showed beneficial effects of laser in DME.3 Vision has failed to improve in a substantial number of patients, however, after focal/grid laser photocoagulation. Peribulbar4 or intravitreal5–8 injection of corticosteroids is most frequently used as primary or adjuvant medical treatment for DME. Many patients, however, experience an elevation of intraocular pressure (IOP) and cataract progression after treatment, and the efficacy on DME is not satisfactory over long-term; a 2-year result of a multicenter randomized clinical trial conducted by Diabetic Retinopathy Clinical Research Network (DRCR.net) showed that the focal/grid laser is a better treatment than intravitreal triamcinolone (IVTA) in eyes with DME involving the fovea with visual acuity between 20/40 and 20/320.8 Newer drugs targeting vascular endothelial growth factor (VEGF) have been introduced, based on the fact
iabetic macular edema (DME) is a major complication of diabetic retinopathy (DR) and is the most frequent cause for preventable blindness in working adults in many countries. A large meta-analysis has estimated the overall prevalence of any DR to be 34.6%, and the prevalence of DME was 6.8%. There are approximately 93 million people with DR and 21 million with DME worldwide.1,2 Various therapeutic approaches have been used to minimize or delay visual loss from DME. For From the *Catholic High-Performance Cell Therapy Center, College of Medicine, The Catholic University of Korea, Seoul, Korea; †Department of Medical Life science, College of Medicine, The Catholic University of Korea, Seoul, Korea; and ‡Department of Ophthalmology, Seoul St Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea. None of the authors have any financial/conflicting interests to disclose. Reprint requests: Won Ki Lee, MD, PhD, Department of Ophthalmology, Seoul St Mary’s Hospital, College of Medicine, The Catholic University of Korea, #505 Banpo-Dong, Seocho-Gu, Seoul 137-701, Korea; e-mail: [email protected]
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that VEGF is a major mediator of increased retinal permeability in DME.9 Many large multicenter clinical trials have shown promising anatomic and functional outcomes from anti-VEGF in various protocols and intervals.10–15 Anti-VEGF is associated with better visual outcomes and lower adverse events at two years compared with triamcinolone.10 Therefore, anti-VEGF treatment has rapidly gained popularity in DME treatment around the world. A substantial number of patients remain, however, unresponsive to multiple injections of anti-VEGF. The safety and efficacy of ranibizumab in diabetic macular edema (RESOLVE) study showed that approximately 10% of patients enrolled in the ranibizumab arm show the same or decreased visual acuity at 12 months.13 Intraocular cytokines have been extensively studied to evaluate their contribution to DME and indicate that VEGF and various inflammatory cytokines aggravate DME.16–22 In addition, there is an increased awareness that several intraocular inflammatory cytokines are further upregulated after anti-VEGF treatment, potentially because of compensatory reactions in response to decreased VEGF.23–25 Based on these findings, we hypothesized that DME patients who are unresponsive to multiple injections of anti-VEGF might have a different intraocular cytokine milieu from other DME patients, suggesting a possible role for corticosteroids in these patients. Therefore, in this study, we evaluated the efficacy of IVTA in patients who were unresponsive to repeated anti-VEGF and further investigated their intraocular cytokine milieu in relation to the efficacy of IVTA.
Patients and Methods The study was a prospective, open-label, uncontrolled nonrandomized interventional case series performed on patients with DME unresponsive to intravitreal bevacizumab (IVB) with or without focal laser photocoagulation. Institutional Review Board/ Ethics Committee approval was obtained before recruitment. The study protocol adhered to the tenets of the Declaration of Helsinki. All participants signed an informed consent form after a detailed explanation of the study design, associated surgical procedures for scientific purposes, and adjuvant imaging procedures. Among the patients with DME seen between January 2012 and June 2012 at Seoul St Mary’s hospital, we prospectively enrolled patients who were unresponsive to IVB. Inclusion criteria were: 1) at least three previous IVB treatments with monthly intervals, with or without focal laser photocoagulation; and 2) changes in central subfield thickness (CST)
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detected by spectral domain optical coherence tomography (OCT3; Carl Zeiss Meditec, Inc, Dublin, CA) less than 11% (the reliability limit for real change determined in the DRCR.net study26) of preinjection thickness. Exclusion criteria were: 1) study eyes with a history of IVTA; 2) eyes mainly with focal leakage from extrafoveal microaneurysm, which was treatable with focal laser; 3) eyes with pharmacological intervention in the fellow eye within 3 months; 4) IOP $ 25 mmHg at baseline; 5) any ocular disease other than DR; 6) history of major surgery; or 7) media opacity hindering retinal examination. Intravitreal triamcinolone was performed under sterile condition at least 2 months after the last IVB to avoid the residual effects of IVB. Before the injection of triamcinolone acetonide, undiluted aqueous humor sample (50–100 mL) was obtained through a limbus using a 30-gauge needle and stored at −70°C until analysis. A dose of 4 mg in 0.1 mL triamcinolone acetonide was injected through the pars plana at 3.5 mm from the limbus using a 30-gauge needle. A comprehensive ocular examination including ETDRS best-corrected visual acuity (BCVA), IOP measurement, slit-lamp examinations, color fundus photography, macular cube 512 · 128 scan by spectral domain optical coherence tomography, and FA were performed before IVTA. ETDRS BCVA, IOP, slitlamp examination, and spectral domain optical coherence tomography were evaluated monthly to month 3. Best-corrected visual acuity was measured at 4 m with standard ETDRS protocols using a testing chart transilluminator (Lighthouse International, New York, NY). Visual acuity was scored as the total number of letters read correctly. Aqueous samples were analyzed using suspension array technology (xMAP; Luminex Corp, Austin, TX). Capture bead kits (Beadlyte; Upstate Biotechnology, Lake Placid, NY) were used for the detection of interleukin (IL)-2, IL-6, IL-8, tumor necrosis factor-a, transforming growth factor-b2, and VEGF. The undiluted samples were incubated overnight. Kits were run according to the manufacturer’s instructions. Standard curves for each cytokine (in duplicate) were generated using the reference cytokine concentrations supplied with the kit. All incubation steps were performed at room temperature and in the dark to protect the beads from light. Samples were read on the suspension array system. Statistical Analysis The Wilcoxon signed-rank test was used to analyze the changes in CST and BCVA after IVTA during the follow-up. Demographic characteristics, such as age,
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sex, duration of diabetes, total number of serial antiVEGF injections, intervals between last injection (months), and intraocular cytokine levels including VEGF, IL-2, IL-6, IL-8, transforming growth factorb2, and tumor necrosis factor-a were used as dependent variables for anatomic response at 1 month. Intraocular levels of VEGF, IL-2, IL-6, IL-8, transforming growth factor-b2, and tumor necrosis factor-a were logtransformed, because none displayed a normal distribution as assessed using the Shapiro–Wilk test. An anatomic response was calculated as follows: Anatomic response ¼ 1− CSTfollow-up =CSTbaseline · 100 Statistical analyses were performed using the statistical software (SPSS for Windows, version 13.0; SPSS Science, Chicago, IL). A P value ,0.05 was considered as statistically significant. Results Twenty eyes (20 patients) were enrolled in this prospective study. The baseline characteristics are summarized in Table 1. The mean age was 60.8 ± 6.7 years, and the mean duration of diabetes was 13.2 ± 8.9 years. Intravitreal bevacizumab was performed a mean of 4.2 ± 1.1 times, and the last IVB was performed 2.7 ± 1.5 months before IVTA. Among the patients, 10 eyes were pseudophakic, and 16 eyes had a history of focal laser photocoagulation. Figure 1 shows the changes in CST and BCVA during the follow-up period. The mean CST was 465.5 ±131.9 mm at baseline, and significantly Table 1. Baseline Characteristics of Study Population (n = 20) Gender, male Age, years Duration of diabetes, years Insulin treatment, yes Hypertension, yes* HbA1c, % Number of previous IVB, times Time interval from last IVB, months History of cataract operation, yes History of focal laser treatment, yes
10 (50.0) 60.8 ± 6.7 13.2 ± 8.9 13 (65.0) 15 (75.0) 7.4 ± 1.8 4.2 ± 1.1 2.7 ± 1.5 10 (50.0) 16 (80.0)
Values are represented in frequency (percentage) for categorical variables, mean, and standard deviation for continuous variables. *Hypertension was defined using the average of 2 blood pressure readings with cut points of systolic blood pressure $ 130 mmHg, diastolic blood pressure $ 85 mmHg, or hypertension medication use. HbA1c, glycated hemoglobin.
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decreased to 380.5 ± 126.0 mm at 1 month (P , 0.001) and 402.1 ± 140.2 mm at 2 months (P = 0.004) after IVTA. The difference was not significant at 3 months (411.8 ± 130.6; P = 0.057). A decrease in CST more than 11% of baseline occurred in 12, 10, and 9 eyes at 1, 2, and 3 months, respectively. In 12 eyes showing a decrease in CST more than 11% at 1 month, the mean CST was reduced by 28% compared with baseline value (463.2 ± 133.8 mm at baseline and 325.9 ± 91.8 mm at 1 month; P = 0.02). The mean BCVA was 47.1 ± 18.9 letters at baseline, and significantly increased to 53.3 ± 19.7 letters at 1 month (P = 0.002) and 52.4 ± 19.1 letters at 2 months (P = 0.041). These visual gains were not sustained at 3 months (50.9 ± 18.6; P = 0.204). At baseline, the median VEGF level was 6.14 pg/ mL (range, 3.2–41.96). The values of each cytokine are shown in Table 2. The effects of demographic characteristics and intraocular cytokines on anatomic response were examined by univariate and multivariate regression analysis. Simple regression analysis revealed that duration of diabetes (P = 0.030) and intraocular levels of IL-8 (P = 0.041) were related to anatomic response at 1 month. Multivariate regression analysis showed that intraocular levels of IL-8 (b = 0.538 P = 0.006) was an independent factor for anatomic response at 1 month after IVTA, after adjusting for age, sex, and total number of serial anti-VEGF injections (Figure 2). No serious adverse event, such as endophthalmitis or uncontrolled IOP despite medical treatment, was observed throughout the study. Of 20 eyes, 5 eyes (25.0%) experienced IOP higher than 21 mmHg during the follow-up, but the IOP was controlled with topical eye drops.
Discussion This study showed beneficial effects of IVTA on DME unresponsive to multiple IVBs for at least 2 months. It is notable that IVTA resulted in a mean reduction in CST of 28% at 1 month in 12 of 20 eyes, considering that they have shown no response to conventional IVB treatment. Triamcinolone reduces vascular permeability by downregulating VEGF expression,27,28 inhibiting the arachidonic acid pathway,29 and increasing the activity and/or the density of the tight junctions of the retinal capillary endothelial cells.30 In addition, triamcinolone inhibits osmotic swelling in retinal glial cells by releasing endogenous adenosine,31,32 and causes vasoconstriction and decreased hydrostatic pressure, which improves Starling’s equilibrium.33 The anti-inflammatory effects of
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Fig. 1. The changes in CST and BCVA during the follow-up period. A. Baseline CST was decreased significantly at Month 1 and 2 after IVTA injection (P , 0.001 and 0.001), but the significance was diminished at Month 3 (P = 0.085). B. The BCVA improved significantly at Month 1 and 2 (P = 0.003 and 0.019), but these visual gains were not sustained at Month 3 (P = 0.109).
IVTA also contribute to resolve DME by inhibiting proinflammatory cells, and pro-angiogenic and inflammatory mediators, such as IL-6 and IL-8.34,35 The effect of IVTA in our patients seemed largely because of the anti-inflammatory effect, in addition to its antipermeability effect, because the efficacy was associated with elevated levels of aqueous IL-8. IL-8 is associated with increased vascular permeability and is elevated in patients with DR and DME.19–22 IL-8 is a potent proinflammatory chemokine that induces the accumulation of neutrophils along the vascular wall.21 A central role for leukostasis in mediating retinal vascular damage has been described.36 Of interest, the VEGF levels were low in our patients at the time of IVTA, at least 2 months after the last IVB, which might be associated with repeated IVBs. Funk et al23 also observed that patients responding well to anti-VEGF therapy still presented disease activity and needed regular retreatment despite constantly minimal VEGF levels. This may suggest that other upstream or parallel pathways of the VEGF signaling pathway may be important for development of DME and that VEGF alone is not the sole etiologic factor responsible for DME development.37 Several cytokines like IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) have been proposed Table 2. Baseline Intraocular Levels of Each Cytokine (n = 20) Cytokine, pg/mL VEGF TGF-b2 IL-2 IL-6 IL-8 TNF-a
Values 6.14 516.44 0.98 6.67 12.77 0.87
(3.20–41.96) (111.32–1,849.97) (0.34–2.70) (0.22–81.70) (1.42–33.28) (0.65–2.75)
Values are represented as median (range). TGF, transforming growth factor; TNF, tumor necrosis factor.
to be implicated in the development of DME.17–19,23 Results of these reports somewhat differ in which cytokines show increased expression and are associated with the disease activity or treatment response. Rho et al19 observed that recurrence of DME after IVB was associated with elevated aqueous IL-6, but not VEGF. IL-6 is a cytokine that functions widely throughout the inflammatory cascade and is known to induce acute phase reactions and increase vascular permeability.18 However, another study was unable to replicate the finding of increased aqueous IL-6 in patients with DME. In that study, significantly increased expression of IL-8 and MCP-1 were demonstrated at baseline, which was not affected by IVB therapy.23 Differences between the studies could be related to ethnicity or underpowering due to small sample sizes. Large interindividual differences in DR stage, duration of DME, and systemic condition could be another explanation. Particularly, in the eyes treated by anti-VEGF drug, compensatory increases in other inflammatory mediators might be resulted from a sudden decrease in VEGF, and the extent of increase would be different according to the span of anti-VEGF treatment, number of injection, and elapsed time from the last injection.23–25,37,38 In this study, the duration of diabetes was associated with the anatomic response in univariate regression analysis. Long duration of diabetes is one of the most important risk factors for development of DME.39 We speculate that the long-standing diabetes continuously disrupts balance between prosurvival neutrophils and inflammatory mediators, leading to chronic inflammatory response in retinal endothelial and neural cells. We used “at least 3 monthly IVB” and “less than 11% of reduction in CST” for the definition of unresponsive DME in this study. More prolonged IVB treatment might result in more favorable responses in
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acuity and reduced macular thickness; however, these effects were not sustained for more than 3 months. The efficacy was associated with elevated levels of aqueous IL-8, which stress the role of suppression of inflammatory mediators in addition to VEGF in these patients. Further studies with larger number of patients and longer follow-up period are required to clarify the longterm efficacy of IVTA in patients with DME showing poor responses to conventional anti-VEGF therapy. In addition, a comparative study on the differences in the intraocular cytokine levels between anti-VEGF responder and nonresponder is warranted to elucidate the roles of specific cytokines in the pathogenesis of DME and to get a rationale of individualized treatment using steroid. Fig. 2. The effect of intraocular cytokines on changes in early treatment diabetic retinopathy study (ETDRS) letters at Month 3. IL-8 was an independent factor for visual gain at 3 months after IVTA injection, after adjustment of age, and duration of diabetes (b = 0.841, P , 0.001).
some cases. Also, the absence of progression might be a sign of efficacy. Nonetheless, in Ranibizumab for Edema of the Macula in Diabetes (READ-2) study, 4 ranibizumab injections over a span of 6 months resulted in a mean reduction in excess foveal thickness of approximately 50% and an average improvement in BCVA of 7.4 letters.11 DRCR net study showed that most of the overall improvement in mean visual acuity within the ranibizumab-treated groups occurred by the 8-week study visit. The benefits of ranibizumab were observed as early as 7 days after treatment initiation, signifying a very rapid effect of the drug in DME.15 Therefore, patients in our series showing no anatomic and functional improvement at all with a mean of 4.2 monthly IVBs could be considered as candidates of “nonresponder” in real clinical practice. In addition, the study results provide meaningful information on this group, although some of them would not be true nonresponders. Our study has a number of inherent limitations including few patients, short follow-up period, and noncomparative study design. It is not clear whether continuous IVTA in our patients would show a beneficial effect over the long-term and better results than continuous IVB. Treatment benefits of IVTA would be often limited by the common side effects of IOP elevation and cataract formation. Nonetheless, our study results suggest that IVTA can limit the opportunity for accumulated fluid to persist in refractory DME to multiple IVBs and may have a role as an adjuvant treatment to IVB. In conclusion, IVTA had beneficial effects on DME unresponsive to multiple IVBs in improved visual
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