A Treat-and-Extend Regimen of Intravitreal Brolucizumab for Exudative Age-Related Macular Degeneration Refractory to Aflibercept: A 12-Month Result

We aimed to investigate whether a treat-and-extend regimen of intravitreal brolucizumab (6.0 mg/0.05 mL) is effective for eyes with exudative age-related macular degeneration (AMD) refractory to aflibercept for 12 months. Sixty eyes from 56 patients receiving brolucizumab for exudative AMD refractory to aflibercept were included. Patients received a mean of 30.1 aflibercept administrations for a mean 67.9-month follow-up. All patients exhibited exudation on optical coherence tomography (OCT) despite regular 4–8 weeks of aflibercept administration. Visit 1 was scheduled at the same interval from the last aflibercept injection to the baseline. The treatment interval was extended or shortened by 1–2 weeks depending on the presence or absence of exudation on OCT. After switching to brolucizumab, the follow-up interval significantly extended at 12 months (before switching: 7.6 ± 3.8 weeks vs. at 12 months: 12.1 ± 6.2 weeks, p = 1.3 × 10−7). Forty-three percent of the eyes achieved a dry macula at 12 months after switching. However, the best-corrected visual acuity did not improve at any visit. Morphologically, the central retinal thickness and subfoveal choroidal thickness significantly decreased from baseline at 12 months (p = 3.6 × 10−3 and 1.0 × 10−3, respectively). Switching to brolucizumab can be considered to extend the treatment interval in eyes with exudative AMD refractory to aflibercept.


Introduction
Age-related macular degeneration (AMD), one of the leading causes of blindness in developed countries, accounts for 8.7% of all cases of blindness worldwide and the projected number of people affected by this disease has been estimated to increase to 288 million in 2040 worldwide [1][2][3][4][5][6]. Advanced AMD is classified into two subtypes, exudative AMD originating from macular neovascularization (MNV), and geographic atrophy, also known by the recent term "complete retinal pigment epithelium (RPE) and outer retinal atrophy" through optical coherence tomography (OCT) [7,8]. AMD is a chronic inflammatory eye disease with a combination of genetic and environmental factors and its hallmark is known as drusen [9,10]. Conventional drusen size is associated with the progression and development of advanced AMD [11]. In addition to drusen size and RPE abnormalities, the drusen type is associated with the progression of the advanced AMD subtype, including the MNV types [12][13][14][15][16][17]. In Asians, geographic atrophy is a rare phenotype of advanced AMD, and MNV is exclusively seen in daily clinical practice. The growing number of patients with exudative AMD and medical expenses has become a social problem and burden in the developed countries [18][19][20][21].
Several cytokines were identified in the tissue of macular neovascularization, including pigment epithelium-derived factor and vascular endothelial growth factor (VEGF) [22]. Among these cytokines, VEGF is a key factor in the development and progression of Table 1. Baseline characteristics of the patients with exudative age-related macular degeneration treated with anti-vascular endothelial growth factor agents and switched to brolucizumab.

Number of Eyes 60
Age 76.2 ± 7.  Figure 1 shows the mean treatment interval before and after switching to brolucizumab. After switching to brolucizumab, the mean treatment interval at 12 months was significantly longer than that before switching (7.6 ± 3.8 weeks at baseline, and 12.1 ± 6.2 weeks at 12 months, p = 1.3 × 10 −7 ). Twenty-six eyes (43.3%) did not show any extension in the interval at 12 months. Twenty-seven (45.0%) eyes showed an extended interval of 4 weeks or more at 12 months. After switching, the patients received a mean of 6.15 ± 2.40 injections during 12 months, which was significantly fewer than those during 12 months before switching (mean of 6.87 ± 2.92 injections, p = 0.045). (Left) At 12 months, the mean treatment interval significantly extended from 7.6 ± 3.8 weeks to 12.1 ± 6.2 weeks (p = 1.3 × 10 −7 ). (Right) The mean logMAR BCVA was 0.44 ± 0.39 at baseline and was maintained to 0.45 ± 0.43 at 12 months after switching (p = 0.72). BCVA, bestcorrected visual acuity; logMAR, logarithm of minimal angle resolution. Figure 1 shows the change in the mean best-corrected visual acuity (BCVA) before and after switching. Mean logarithm of the minimal angle resolution (logMAR) BCVA was maintained from 0.44 ± 0.39 at baseline to 0.45 ± 0.43 at 12 months after switching (p = 0.72). To investigate the correlation between visual outcomes and changes in the treatment intervals, we divided the patients into 3 groups according to the length of the extended treatment intervals (0 weeks, 1-3 weeks, and 4 weeks or more). The results showed no significant difference in mean BCVA from baseline to 12 months in each group ( Figure  2). The mean central retinal thickness (CRT) on swept-source optical coherence tomography (SS-OCT) significantly decreased from 313 ± 145 µm at baseline to 272 ± 165 µm at 12 months after switching (p = 3.6 × 10 −3 , paired t-test, Figure 3). The mean subfoveal choroidal thickness (SCT) on SS-OCT also significantly decreased from 210 ± 93 µm at baseline to 193 ± 48 µm at visit 3 after switching (p = 1.0 × 10 −3 , paired t-test, Figure 3). Figures 4 and 5 show the mean change of CRT/SCT on SS-OCT according to the length of the extended treatment intervals. Mean CRT significantly decreased from baseline to 12 months in the patients with extended intervals of ≥4 weeks ( Figure 4). Mean SCT significantly decreased in the patients with extended intervals of 0 weeks and ≥4 weeks ( Figure 5).  Figure 1 shows the change in the mean best-corrected visual acuity (BCVA) before and after switching. Mean logarithm of the minimal angle resolution (logMAR) BCVA was maintained from 0.44 ± 0.39 at baseline to 0.45 ± 0.43 at 12 months after switching (p = 0.72). To investigate the correlation between visual outcomes and changes in the treatment intervals, we divided the patients into 3 groups according to the length of the extended treatment intervals (0 weeks, 1-3 weeks, and 4 weeks or more). The results showed no significant difference in mean BCVA from baseline to 12 months in each group ( Figure 2). The mean central retinal thickness (CRT) on swept-source optical coherence tomography (SS-OCT) significantly decreased from 313 ± 145 µm at baseline to 272 ± 165 µm at 12 months after switching (p = 3.6 × 10 −3 , paired t-test, Figure 3). The mean subfoveal choroidal thickness (SCT) on SS-OCT also significantly decreased from 210 ± 93 µm at baseline to 193 ± 48 µm at visit 3 after switching (p = 1.0 × 10 −3 , paired t-test, Figure 3). Figures 4 and 5 show the mean change of CRT/SCT on SS-OCT according to the length of the extended treatment intervals. Mean CRT significantly decreased from baseline to 12 months in the patients with extended intervals of ≥4 weeks ( Figure 4). Mean SCT significantly decreased in the patients with extended intervals of 0 weeks and ≥4 weeks ( Figure 5).            Table 2 shows the number of patients showing exudation on SS-OCT before and after switching. At 12 months, 26 out of 60 eyes (43.3%) achieved a dry macula. Among the remaining 34 eyes with exudation, 25 eyes showed SRF, 6 eyes showed IRF, and 3 eyes showed both SRF and IRF. Figure 6 shows a representative case of switching to brolucizumab.   Table 2 shows the number of patients showing exudation on SS-OCT before and after switching. At 12 months, 26 out of 60 eyes (43.3%) achieved a dry macula. Among the remaining 34 eyes with exudation, 25 eyes showed SRF, 6 eyes showed IRF, and 3 eyes showed both SRF and IRF. Figure 6 shows a representative case of switching to brolucizumab.  Best-corrected visual acuity in his right eye was improved from 0.15 logarithm of minimum angle resolution (logMAR) from the baseline to 0.10 loMAR at 12 months. The interval between the last aflibercept injection and the baseline was 5 weeks, which was extended to 8 weeks at 12 months. Best-corrected visual acuity in his right eye was improved from 0.15 logarithm of minimum angle resolution (logMAR) from the baseline to 0.10 loMAR at 12 months. The interval between the last aflibercept injection and the baseline was 5 weeks, which was extended to 8 weeks at 12 months.

Adverse Events
During the study period, adverse events were seen in 12 eyes (20%). Among 12 eyes, 2 eyes showed mild anterior chamber cells and keratic precipitates, and 7 eyes showed mild posterior IOI including vitreous opacity and anterior vitreous cells. Topical steroids were administered to these patients with mild anterior or posterior IOI, and the IOIs were immediately resolved. Intravitreal brolucizumab injection was discontinued, and the patient was switched to aflibercept again from the next visit. Floaters were seen in 3 eyes (5%) of 3 patients, and these patients underwent funduscopic examination with pupil dilation; however, fundus examination showed no sign of IOI, and no additional treatment was undertaken. The symptoms resolved spontaneously in these patients.

Discussion
In the present study, we investigated whether intravitreal brolucizumab (6.0 mg) was effective for exudative AMD refractory to aflibercept using a treat-and-extend regimen for 12 months. Before the initiation of brolucizumab therapy, the follow-up interval of all patients was equal to or less than 8 weeks. The patients had a mean of 30 aflibercept injections at regular 4-8-week (mean 7.6 weeks) intervals before switching to brolucizumab. At 12 months from baseline, a dry macula was achieved in almost half of the eyes that switched to brolucizumab. Visit 1 was scheduled at the same interval between the last aflibercept injection and the baseline; a dry macula was achieved in 26.7% of eyes at visit 1, suggesting that one session of intravitreal injection of brolucizumab was more effective than aflibercept from a morphological point of view.
Although study design, treatment regimen, and study duration were different, several studies have investigated the efficacy of brolucizumab for persistent neovascular AMD [46][47][48][49][50][51][52][53][54]. Recently, Ueda-Consolvo et al. reported an 18-month follow-up study of switching to brolucizumab from aflibercept in 42 eyes with exudative AMD [50]. They reported that treatment intervals were significantly extended from 7.4 ± 1.4 weeks to 11.6 ± 2.6 weeks for type 1 macular neovascularization and from 6.9 ± 1.3 weeks to 11.7 ± 3.1 weeks for polypoidal choroidal vasculopathy. They also reported that IOI occurred in 16.7% (7/43) of study eyes. Similarly, the results in this study showed the significant extension of treatment intervals from 7.6 ± 3.8 weeks to 12.1 ± 6.2 weeks at 12 months, and IOIs occurred in 15.0% (9/60) of study eyes. These results also support the efficacy of brolucizumab for exudative AMD refractory to aflibercept and IOIs appeared in almost one out of six eyes among Japanese patients. The incidence of IOIs in these studies was relatively higher than that in a large cohort study conducted in the United States [55]. Although the exact reason was unknown, the differences in race, including pigmentation of RPE and the size of the study population, might affect the results.
Brolucizumab is a novel VEGF inhibitor with a molecular mass of 26 kDa, less than commercially available ranibizumab (48 kDa) and aflibercept (97-115 kDa). Owing to its high solubility, brolucizumab can be concentrated up to 120 mg/mL [29]. Therefore, the binding affinity of brolucizumab to VEGF is greater than that of aflibercept. Thus, brolucizumab has a therapeutic advantage because of its prolonged effect. This study's treatment interval significantly extended from the first to the last interval at 12 months (from 6.15 ± 2.40 to 6.87 ± 2.92, p = 0.045). Several studies have reported the efficacy of brolucizumab for refractory exudative AMD in short-term results in the real-world [41,49,[56][57][58][59][60][61][62]. In the present study, 45.7% (26 eyes) could not extend the treatment interval during 12-month follow-up. This means that almost half of poor responders to aflibercept therapy did not respond to a treat-and-extend regimen of brolucizumab. Although we can select Pharmaceuticals 2023, 16, 562 9 of 15 various VEGF inhibitors with the advent of brolucizumab, we must recognize that treatment resistance concerns remain as well as safety concerns. and we must work on how to treat these patients.
In the present study, BCVA was almost similar between baseline and at 12 months, although half of the eyes achieved a dry macula. In a previous study, Enríquez et al. investigated the efficacy of switching to brolucizumab for neovascular AMD refractory to the prior anti-VEGF agents because of persistent fluid. They reported that there was no difference in mean VA of the patients with neovascular AMD prior to starting brolucizumab compared with after brolucizumab injections or at the final study evaluation [53]. The FLUID study revealed that the presence of SRF less than 200 µm at the fovea was tolerable concerning BCVA improvement in patients with neovascular AMD treated with ranibizumab [63]. However, the previous study period was relatively short (24 months). Therefore, assuming a long-standing visual prognosis in eyes with exudative AMD, complete resolution of SRF is preferable when treated with anti-VEGF agents. Further studies are needed to confirm these questions.
About SCT after treatment for exudative age-related macular degeneration, three monthly ranibizumab administrations induced a decrease in SCT by 6%, three monthly aflibercept administrations induced a decrease in SCT by 12-16% and photodynamic therapy (PDT) induced a decrease SCT by 20% [64][65][66][67][68]. A recent study reported that subfoveal choroidal thickness significantly decrease by 20% after 3 monthly brolucizumab injections, [69] suggesting 3 monthly administration of brolucizumab has a similar effect on choroidal thickness to PDT. The effect of brolucizumab on choroidal thickness might be associated with greater strength of exudation resolution.
This study had several limitations. Major limitations are the small number of patients and the retrospective nature of the study. To validate the present conclusions, a longerduration study and/or a large-scale prospective study are needed. In addition, we only focused on the anatomical/functional outcomes after the administration of brolucizumab and did not evaluate specific molecules or immunological parameters. Therefore, we could not discuss the preceding pathologies at the molecular level.
In summary, treat-and-extend regimen using brolucizumab is effective for improving the anatomical outcomes in eyes with exudative AMD refractory to aflibercept.

Participants
A retrospective medical chart review was performed on consecutive patients with exudative AMD treated with 6.0 mg brolucizumab, initiating a treat-and-extend regimen from 1 September 2020 to 1 June 2021 in the Macula Clinic, Department of Ophthalmology, University of Yamanashi Hospital. This retrospective study was approved by the Institutional Review Board of the University of Yamanashi (approval number: 2485 approval date: 13 July 2021) and was conducted in accordance with the tenets of the Declaration of Helsinki. Written informed consent for treatment was obtained from all patients.
The inclusion criteria were as follows: (1) eyes with neovascular AMD or PCV, (2) eyes showing exudation including subretinal or intraretinal fluid despite regular intravitreal aflibercept (2.0 mg/0.05 mL) injections equal to or less than 8-week intervals, (3) eyes that completed the follow-up of at least 12 months from switching.
The exclusion criteria were as follows: (1) eyes with retinal angiomatous proliferation, myopic choroidal neovascularization, or choroidal neovascularization secondary to angioid streaks; (2) treatment-naïve eyes, and (3) eyes treated with brolucizumab using other treatment regimens including regular injections or as-needed injections; (4) eyes within a 12-month follow-up period; (5) eyes receiving other treatment including cataract surgery, sub-tenon triamcinolone acetonide injection during the study period.

Diagnosis
The diagnosis of neovascular AMD or PCV was made on multimodal imaging by comprehensive examination including color fundus photography using TRC-50DX (Topcon, Tokyo, Japan), SS-OCT (DR-1/Atlantis, Topcon, Tokyo, Japan), and fluorescein/indocyanine green angiography (FA/ICGA) using a confocal laser scanning system (HRA-2; Heidelberg Engineering, Dossenheim, Germany) as previously described [70]. Briefly, PCV showed aneurysmal dilation with or without branching vascular network on ICGA, and retinal pigment epithelium protrusion was observed corresponding to the aneurysmal orange red lesion on SS-OCT. The diagnosis of neovascular AMD was also made using angiography and SS-OCT. Neovascular AMD showed classic or occult choroidal neovascularization on FA without aneurysmal dilation on ICGA. Type 1 or 2 neovascularization was observed on SS-OCT. In eyes with Type 1MNV, subretinal hyperreflective materials were seen beneath the RPE on OCT, and in eyes with Type 2 MNV, hyperreflective materials were seen above the RPE on OCT. An OCT scan pattern consisted of both vertical and horizontal scans with a 9-mm length centering at the fovea.

A Treat-and-Extend Regimen
The detailed flow-chart of the treat-and-extend regimen is shown in Figure 7. All patients had a treatment history of multiple regular aflibercept administrations for at least 12 months. At baseline, an intravitreal injection of brolucizumab (6.0 mg/0.05 mL) was administered in all eyes. The first visit (visit 1) was scheduled at the same interval between the last aflibercept injection and the baseline. The second visit (visit 2) was extended by 1-2 weeks based on one (Y.S) of the doctors' discretion if there was no exudation including subretinal fluid and intraretinal fluid on SS-OCT during the first visit. If exudation was observed on SS-OCT, the interval between visits 1 and 2 was retained. Similarly, the next visits were extended by 1-2 weeks if there were no exudations including subretinal and intraretinal fluid on SS-OCT during the previous visits. If exudation was observed on SS-OCT, the next interval was shortened by 1-2 weeks, as the interval was extended at the previous visit. None of the intervals were designed to be shortened to less than the first interval. A "12-month visit" is defined as a 52-week visit or the visit first exceeding 52 weeks. All of the patients were instructed to make an immediate call in case of any abnormal symptoms after injection. Patients who reported symptoms of floater or blurred vision after the intravitreal injection were immediately seen in our hospital.

Follow-Up Examination
At every visit, all patients underwent BCVA measurement using a Landolt chart, intraocular pressure measurement, slit-lamp biomicroscopy with or without 78D lens, color fundus photography, and SS-OCT using DR-1/Atlantis. Scan signal strength equal to or more than 6 was applied to all.

Statistical Analysis
Statistical analyses were performed using the Statflex 7 software (Artec Co., Ltd., Osaka, Japan). BCVA measured on a Landolt chart was converted into logMAR for statistical analyses. The paired t-test was used to determine the significance of the difference between the values before and after treatment. Statistical significance was set at p-value less than 0.05. interval. A "12-month visit" is defined as a 52-week visit or the visit weeks. All of the patients were instructed to make an immediate call in mal symptoms after injection. Patients who reported symptoms of flo sion after the intravitreal injection were immediately seen in our hospit  Author Contributions: All authors contributed to the study's conception and design. Material preparation was performed by Y.F., M.M., A.S., Y.K. and Y.S. Data collection and analysis were performed by W.K. and Y.S. The first draft of the manuscript was written by W.K. The supervision was performed by K.K. and all authors commented on previous versions of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding:
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Institutional Review Board Statement:
This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Institutional Review Board of the University of Yamanashi (Date 13 July 2021/No. 2485).
Informed Consent Statement: Written informed consent was obtained from all participants during the study period. The authors affirm that human research participants provided informed consent for the publication of the images in Figure 3.

Data Availability Statement:
The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors have no relevant financial or non-financial interests to disclose.