Efficacy of Anti-VEGF Agents in the Treatment of Age-Related Macular Degeneration

Efficacy of Anti-VEGF Agents in the Treatment of Age-Related Macular Degeneration Marilita M. Moschos Abstract- Purpose: To evaluate by OCT and mf-erg the macular function in eyes with CNV due to ARMD before and after intravitreal use of bevacizumab and ranibizumab. Methods: Eighteen eyes with subfoveal CNV due to ARMD were studied before and after intravitreal use of bevacizumab with MFERG and OCT. The post follow up was three months. Also fifteen eyes with CNV due to ARMD were studied with OCT and mferg before, during and at the end of the, a year after the first injection of ranibizumab. Results: Three months after with bevacizumab, OCT showed a real resolution of the subretinal fluid. The electrical responses in the fovea and parafovea remained the same or slightly improved in some cases. After the use of ranibizumab visual acuity increases significantly by time (p=0.005). A borderline negative association between foveal thickness and time with decrease of the mean foveal thickness for one time increment was found. The retinal response density of mferg show a significant increase in ring 1, remained almost unchanged in ring 2, whereas in ring 3 there is some evidence of increase not statistically significant. Conclusion: The intravitreal use of bevacizumab may provide anatomical correlates that support the concept of disease amelioration but the functional improvement of the macula three months after is not obvious. The intravitreal use of ranibizumab may provide some anatomical and electrophysiological amelioration of the macula. However, randomized long-term clinical trials are needed to determine the potential clinical benefit of bevacizumb and ranibizumab. Keywords: Age related macular degeneration (ARMD), bevacizumab (Avastin), ranibizumab, multifocal Electroretinogram (MFERG), Optical Coherence Tomography (OCT). I. INTRODUCTION Choroidal neovascularisation (CNV) is a leading cause of loss of central vision in developed countries.[1] The neovascularisation is originated from the choroidal blood vessels and grows through Bruch s membrane usually at multiple sites into the sub-retinal pigmented epithelial space and disrupts the anatomy of the retinal pigment epithelium photoreceptors complex.[2,3,4] The important role of the vascular endothelial growth factor (VEGF) in the pathogenesis of exudative ARMD has been documented in many studies [5,6,7] and new therapeutic strategies consider the application of VEGFbinding substances to reduce the activity of VEGF and thus to prevent progression of exudative ARMD. Bevacizumab binds all biologically active forms of VEGF, as ranibizumab does. It is currently approved for use in humans but is labeled for cancer [7]. In our study we offered offlabel intravitreal use of bevacizumab to patients with CNV due to ARMD not expected to respond to conventional methods of [8]. The aim of our nonrandomized prospective study was to 190

record the electroretinographic changes of the foveal, parafoveal and perifoveal area by means of mferg [9] in eyes with subfoveal CNV concurrently with OCT changes before and after intravitreal use of bevacizumab of ranibizumab and to assess objectively its efficacy. II. MATERIALS and METHODS We studied 2 groups of patients Group A includes 18 eyes of 18 consecutive patients with CNV secondary to ARMD treated with off-label intravitreal bevacizumab. All eyes had large occult or minimally classic lesions. In these cases no benefit would be expected by using any alternative method of. The study protocol was approved by the local institutional review board of our hospital and all the patients signed a written consent statement particularly in the regard to the offlabel use of intravitreal use of bevacizumab. The mean age of the patients was 72.28 years (range 52-84), (SD 8.31) (Table 1). Ten patients were males and eight females. Best corrected visual acuity was measured by means of standard Snellen chart at baseline, 1 month and 3 months after. The procedure was carried out in the operation theatre. Topical proparacaine hydrochloride 0.5% was applied to the ocular surface followed by preparation with 5% povidone iodine. Visual acuity and MFERG monitored the functional response to. The anatomical improvement was assessed by OCT central macular thickness measurement at 1 and 3 months after in injection. Group B includes 15 patients (15 eyes) with CNV due to ARMD were treated with intravitreal use of ranibizumab. All eyes had predominantly classic, minimal classic or occult CNV. Nine patients were female and six male with a mean age of 70.6 (SD 5.94). All eyes were injected monthly for the first 3 months, followed by doses every three months for a year with 0.3 mg of intravitreal ranibizumab. Thus during the 12-months study, a total of six ranibizumab injections were given. Intravitreal injection procedure has been described previously.[16] On presentation all patients underwent a complete ophthalmic examination, including measurement of best corrected visual acuity, fundus examination, intraocular pressure measurement, fluorescein angiography, OCT scan and mf-erg recording. At subsequent injection visits, subjects underwent a preinjection safety evaluation. OCT and mf-erg assessment was evaluated at the 1st, 3rd, 6th, 9th and 12th month after the first injection of ranibizumab. Best corrected visual acuity was measured by means of LogMar equivalent at baseline, 1, 3, 6, 9 and 12 months after the first injection of ranibizumab. Optical Coherence Tomography (OCT) OCT examination was performed with the OCT model 3000. (Carl Zeiss Meditec, Stratus OCT). The retinal mapping software was used, calculating the averaged retinal thickness of the central ring. All eyes were scanned in a radial spoke pattern centered on the foveola with scan length of 6mm. Multifocal-ERG For the recording of the mf-erg, the EP- 1000 model (Tomey, Nagoya, Japan) was used and the recording protocol followed was according to the ISCEV guidelines for basic mf-erg. Statistical analysis Quartiles, mean and standard deviation were calculated in each time period in order to describe the distribution of amplitude, latency, foveal thickness and visual acuity. Linear mixed effects models were used to analyze the data. Differences in longitudinal amplitude and latency data over time were evaluated among foveal (ring1), parafoveal (ring 2) and perifoveal (ring 3) area. The interaction term (type of ring*time) was included to consider differential effects in the rate of change over time in each ring. Foveal thickness and visual acuity were measured only in the foveal area (ring 1). Kolmogorov- 191

Smirnov test evaluated the assumption of normality. We tested for quadratic term of time, but it was not found significant in none of the statistical models. Restricted likelihood (REML) was used for maximization. All statistical tests were two sided; p-value<0.05 was considered statistically significant. The analyses were conducted with Stata statistical software (version 10) and graphs with MLwiN. III. RESULTS Table 1 shows the changes of VA during the 3 months follow-up period after the injection with bevacizumab. No significant difference was found between VA at baseline and 3 months after (P=0.2261) (Table 1). Table 2 shows the changes of foveal thickness during the 3 months follow-up period after with bevacizumab. Statistical analysis revealed an extremely significant difference between foveal thickness at baseline and 1 month after (P<0.001). No difference was found 3 months after (P=0.0592). It is interesting that 3 months after, in 8 cases the foveal thickness (case no 1, 9, 10, 11, 12, 14, 15, 16 and 17) was lower than before, in 4 cases (case no 5, 8, 13 and 18) was higher and in the remaining 5 cases (case no 2, 3, 4, 6 and 7) remained almost unchanged. Concerning the mf-erg changes in area 1 no difference was found between baseline and 3 months (P=0.1285) (Table 2). Statistical analysis revealed a very significant difference between retinal response density of area 2 at baseline and 1 month after (P<0.01). This difference remained significant 3 months after (P<0.05) (Table 2 and Fig 1). It should be stressed that 3 months after, the retinal response density of area 1 improved more than 1nV/deg² in 8 cases (case no 4, 5, 9, 10, 13, 15 and 17), decreased in 2 cases (case no 8 and 12) and remained almost stable in the remaining 8 cases. The retinal response density of area 2 recorded by MFERG improved of more than 1nV/deg² in 4 cases (case no 3, 5, 7 and 8) and remained almost unchanged in the remaining 14 cases. On the other hand a total of 15 patients (15 eyes) treated with ranibizumab were included in the study. Figure 2 shows graphical presentations of the mean values of retinal response density (Figure 2A), latency (Figure 2B), foveal thickness (Figure 2C) and VA (Figure 2D) in each time measurement (Figure 3, 4). Several models of the linear mixed effects analysis are presented in Table 3. In Model 1 retinal response density was considered as the outcome variable whereas time and ring as the explanatory ones. There was a highly significant interaction between ring and time, indicating differences in the rate of change of amplitude over time among the three rings. Specifically, one time unit increase was found to increase significantly the mean retinal response density in ring1 by 5.13 nv/deg 2 (95% CI: 2.45, 7.80). However, mean retinal response density remained almost unchanged over time in ring 2, whereas for ring 3, there was some evidence for increase over time but the result was of borderline significance (p=0.063). In Model 2 latency was the outcome variable whereas time and ring the explanatory variables. At baseline measurement the mean levels of latency were not found to differ significantly among ring 1, ring 2 and ring 3. Time did not seem to have any effect in latency levels with mean latency remaining almost unchanged throughout the twelve months of the period in all the three rings. With respect to foveal thickness which was measured by OCT, the linear mixed effects analysis (Model 3, Table 3) showed a borderline inverse association between foveal thickness and time (p=0.065) with a decrease of mean foveal thickness by 8.46 µm (95% CI: -17.44, 0.52) for one time unit increase. Lastly, according to Model 4 (Table 3), a highly significant time effect was identified, with mean visual acuity increasing significantly by time (p=0.005). 192

Concerning retinal response density, its significant positive association with time remained. Amplitude was not found to be associated with foveal thickness (p=0.887) whereas a borderline positive association was identified between amplitude and visual acuity (OR: -13.79, 95% CI: -28.20, 0.63). Concerning latency, its relationship with time remained statistically non-significant even after adjustment for foveal thickness and visual acuity. Foveal thickness and visual acuity seemed to be unrelated to latency. Visual acuity, after adjustment for foveal thickness, increased significantly by time. However foveal thickness was not found to be associated with visual acuity (p=0.482). IV. DISCUSSION The of choroidal neovasularization in ARMD has changed enormously during the last decade. While 10 years there was only Argon laser coagulation and later photodynamic therapy with verteporfin, today we have several options for CNV.[1] Results from PIER study indicate that ranibizumab administered on a schedule of monthly dosing for three months followed by quarterly dosing provides significant visual acuity benefit to patients with all angiographic subtypes of CNV. Unfortunately, there are no data available at six and nine months to allow assessment of duration of action of ranibizumab in the individual subjects and the temporal association with retinal thickness. In this retrospective study 18 eyes with CNV due to ARMD were treated with intravitreal bevacizumab. Our results show that there are anatomical correlates to support the concept of disease improvement. This is mainly the decrease of macular thickness as measured by OCT in an extremely significant degree (P<0.001) the first month after. On the contrary the mean visual acuity improved only by 0.03 the first month after and by 0.02 three months after. Also the MFERG improvement did not follow the decrease of macular thickness and is significant the first month after (P<0.05). However MFERG results, three months after, show a little rise of the electrical activity of area 2 (P<0.05), which may be attributed to the decrease of the macular edema more than to the improvement of the macular function [9, 10]. Our results from the intravitreal use of ranibizumab show a significant increase of the visual acuity and the retinal response density of the foveal area (ring1) during, whereas in ring 2 and 3 the retinal response density remained almost unchanged. Also, the latency remained unchanged during. Concerning with the foveal thickness a borderline non statistically significant decrease was identified. Finally, visual acuity and retinal response density of the foveal area seemed to be unrelated with foveal thickness. This may be explained by the fact that the macular edema is only a parameter that may affect visual acuity and the electrical activity of the macula. Consequently, increase or decrease of macular thickness does not necessarily reflect the course of the visual acuity as supported.[11] Atrophy of the retina, particularly of the photoreceptors, atrophy of the pigment epithelium and scarring are all unmeasured parameters which influence vision. While anti-vegf s are revolutionary, the frequency of use of the drugs still needs to be determined. Two previous studies investigated the effects of anti-vegf (bevacizumab) on retinal function by means of mf-erg in ARMD patients before and after intravitreal use of bevacizumab.[10] They found that neuroretinal function did not change in most of the patients compared to baseline values. Feigl s preliminary results in a small number of patients receiving ranibizumab confirm the above mentioned studies.[12] However we consider that randomized longterm clinical trials are needed to determine more accurately the potential clinical benefit of intravitreal anti-vegf. REFERENCES 193

1. Kroll P, Meyer CH (2006) Which is best for AMD patients. Br J Ophthalmol. 90:128-130. 2. Chang TS, Freund KB, de la Cruz Z, Yannouzi LA, Green WR (1994) Clinicopathological correlation of choroidal neovascularization demonstrated by indocyanine green angiography in a patient with retention of good vision for almost four years. Retina. 14:114-124. 3. Green WR, Wilson DJ (1996) Choroidal neovascularisation. Ophthalmology. 93:1169-1176. 4. Green WR, Ehger C (1993) Age related macular degeneration histopathologic studies. The 1992 Lorenz E. Zimmerman Lecture. Ophthalmology. 100:11519-1535. 5. Ambati J, Ambati BK, Yoo SH, Lanchulev S, Adamis AP (2003) Agerelated macular degeneration: etiology, pathogenesis and therapeutic strategies. Surv Ophthalmol. 48:257-293. 6. Zarbin MA (2004) Current concepts in the pathogenesis of age-related macular degeneration. Arch Ophthalmol. 122:598-614. 7. Adamis AP, Shima DT (2005) The role of vascular endothelial growth factor in ocular health and disease. Retina. 25:111-118. 8. Avery RL, Pieramici DJ, Rabena MD et al (2006) Intravitreal bevacizumab (Avastin) for neovascular age-related macular degeneration. Ophthalmology. 113:363-372 e5. 9. Sutter EE, Tram D (1992) The field topography of ERG components in man. I. The photopic luminance response. VBision res. 32:433-466. 10. Moschos MM, Brouzas D, Apostolopoulos M, Koutsandrea Chr, 11. Loukianou E, Moschos M (2007) Intravitreal use of bevacizumab (Avastin) for choroidal neovascularization due to ARMD: a preliminary multifocal ERG and OCT study. Doc Ophthalmologica. 114:37-44. 12. Lipski A, Bornfeld N, Jurklies B (2007) Multifocal electroretinography in patients with exudative AMD and intravitreal with pegaptamib sodium. Retina. 27:864-872. 13. Feigl B, Greaves A, Brown B (2007) Functional outcome after multiple s with ranibizumab in neovascular age-related macular degeneration beyond visual acuity. Clin Ophtalmology. 167-175. 194

Table 1. Clinical data of patients with CNV due to ARMD before, 1 and 3 months after with bevacizumab. No Age VA* before VA* 1 month after 1 66 0.2 0.2 0.15 2 67 0.25 0.25 0.2 3 65 0.1 0.1 0.1 4 75 0.3 0.3 0.25 5 75 0.05 0.1 0.05 6 83 0.9 0.9 0.9 7 63 0.05 0.05 0.05 8 70 0.9 0.9 0.9 9 70 0.05 0.05 0.05 10 84 0.1 0.15 0.1 11 71 0.05 0.1 0.1 12 80 0.45 0.45 0.4 13 80 0.45 0.5 0.5 14 73 0.2 0.2 0.5 15 84 0.05 0.05 0.05 16 74 0.05 0.1 0.1 17 52 0.15 0.3 0.3 18 70 0.1 0.2 0.15 Mean value 72.28 0.24 0.27 0.25 SD 8.31 0.27 0.26 0.267 *Visual VA* 3 months after acuity 195

Table 2. OCT and MFERG recordings of area 1 and 2 before, 1 month and 3 months after with bevacizumab. The mean foveal thickness measured by OCT is expressed in µm. The retinal response densities measured by MFERG are expressed in nv/deg². No Before 1 month after 3 months after Before 1 month after 3 months after OCT OCT OCT Area 1 Area 2 Area 1 Area 2 Area 1 Area 2 1 458 342 351 1.5 0.39 2.08 0.53 2.08 0.5 2 224 204 215 2.95 2.49 2.97 0.62 2.85 2.05 3 259 235 246 0.92 1.1 5.21 3.1 3.5 2.5 4 205 187 287 7.53 5.66 7.23 5.44 7.15 5.15 5 248 242 238 1.23 0.32 2.3 1.69 2.3 1.8 6 308 265 190 14.25 1.34 14.96 1.78 14.5 1.65 7 205 198 210 2.89 0.98 3.91 3.22 3.15 2.68 8 205 202 215 12.32 5.76 14.32 7.92 11.15 7.82 9 279 250 256 1.66 0.89 3.65 0.82 3.65 0.85 10 289 241 245 1.98 2.4 3.45 2.66 3.2 2.46 11 309 246 278 1.69 2.72 2.76 2.98 1.95 2.56 12 348 267 270 6.23 0.47 2.29 1.37 2.25 1.31 13 254 250 280 1.33 2.61 2.65 2.67 2.58 2.55 14 407 287 305 4.25 1.9 4.32 2.24 4.06 2.25 15 504 298 229 3.24 5.32 7.56 5.39 6.7 5.22 16 290 232 250 4.28 1.32 4.33 1.56 3.95 1.49 17 306 257 234 3.41 2.02 5.68 3.11 5.38 2.1 18 275 221 291 1.99 2.43 4.87 3.54 4.76 3.15 Average 298.5 245.78 255 4.09 2.23 5.25 2.92 4.73 2.67 STDEV 84.41 38.37 39.47 3.79 1.73 3.77 1.81 3.35 1.77 196

Figure 2. Mean values in the foveal area (ring 1) in each time measurement* for: (A) amplitude of mfvep, (B) latency of mfvep, (C) foveal thickness, and (D) visual acuity of patients treated with ranibizumab Figure A Figure B Figure C Figure D *Time measurement: 1: baseline 2: 1 month after first 3: 3 months after first 4: 6 months after first 5: 9 months after first 6: 12 months after first 197

Table 3: Linear mixed effects analysis for the association between over time and amplitude (Model 1), latency (Model 2), foveal thickness (Model 3) and visual acuity (Model 4) of patients treated with ranibizumab Category or Increment Coefficient 95% CI P-value Model 1: Amplitude Intercept Ring 1 103.99 (88.41, 119.58) - Ring 2 76.40 (60.81, 91.98) 0.001 Ring 3 52.28 (36.69, 67.86) <0.0001 Time 1 time unit increase in Ring 1 5.13 (2.45, 7.80) - 1 time unit increase in Ring 2 0.50 (-2.17, 3.17) 0.008 1 time unit increase in Ring 3 1.90 (-0.78, 4.57) 0.063 Model 2: Latency Intercept Ring 1 42.64 (40.29, 44.99) - Ring 2 41.03 (38.68, 43.38) 0.131 Ring 3 41.71 (39.36, 44.06) 0.382 Time 1 time unit increase in Ring 1-0.002 (-0.38, 0.38) - 1 time unit increase in Ring 2-0.17 (-0.55, 0.21) 0.461 1 time unit increase in Ring 3-0.11 (-0.49, 0.27) 0.641 Model 3: Foveal thickness Intercept 442.07 (363.12, 521.03) - Time 1 time unit increase -8.46 (-17.44, 0.52) 0.065 Model 4: Visual acuity Intercept 0.23 (0.17, 0.29) - Time 1 time unit increase -0.01 (-0.009, -0.002) 0.005 Ring 1 is the reference group. All P-values derived from the comparison of each category with the reference group. 198

Proceedings of the World Medical Conference Figure 1. MFERG of case 2, at presentation (A) and 3 months later (B). The 3-dimension topographic plot of MF-ERG 3 months after remains pathological and the ERG traces in area 1 and 2 do not show any amelioration. Figure 3. OCT recordings, 3-dimension topographic plot and mf-erg traces dimension topographic plot of a case at presentation (A), 6 months (B) and 1 year after (C) with ranibizumab. The pre OCT showed an increase of central retinal thickness and mf-erg revealed a decrease of electrical activity of the fovea and perifovea area. At the end of the follow-up period (C) the retinal thickness of the fovea was decreased and the mf-erg was ameliorated. 199

Figure 4. OCT recordings, 3-dimension topographic plot and mf-erg traces dimension topographic plot of a case at presentation (A), 6 months (B) and 1 year after (C) with ranibizumab. The pre OCT showed an increase of central retinal thickness and a decrease of electrical activity of the fovea and perifovea area. At the end of the follow-up period (C) the retinal thickness of the fovea was still increased and the mf-erg revealed a craterlike depression of the foveal and parafoveal area.. 200