The Evaluatıon of The Effıcacy of Adalımumab in Refractory Non-Infectıous Uveıtıs Wıth Ultra-Wıdefıeld Fundus Fluoresceın Angıography

Aim: To investigate the ecacy of adalimumab in the cases with refractory non-infectious uveitis and evaluate retinal vascular leakage changes on ultra-wideeld fundus uorescein angiography. Methods: Twenty-three patients with refractory uveitis were included in study. Results: Forty-four eyes of 23 patients with non-infectious uveitiswere evaluated. Clinically active inammation was present in 19 eyes (43.18%), while 25 (56.8%) were inactive. The mean drug burden was a 9.91±3.78 (5-21) in baseline, 7.3±4.25 at third and 8.0±4.71 at sixth month (p=0.022). The mean choroidal thickness was 256.65±43.63 μm in baseline, 240.49±36.73 μm at third and 224.81±34.91 μm at sixth month (p ≤ 0.05). In terms of leakage extend, leakage was initially present in a mean of 2.95±4.55 clock hour quadrants, 2.41±3.91 at thirdand 1.76±3.44 at sixth month (p<0.001). Conclusion: Adalimumab was found to be effective in establishing inammation control by reducing drug burden, controlling retinal vascular leakage and choroidal inammation in refractory uveitis. especially evaluating retinal vascular and optic discleakages in posterior segment have been investigated in a few studies.The purpose of the present study was to investigate retrospectively the ecacy of adalimumab in retinal vascular leakage, and controlling vascular and choroidal inammation in patients with non-infectious uveitis, which is refractory to conventional immune suppressive therapy, by evaluating the posterior segment and peripheral retina with ultra-wideeld fundus uorescein angiography. 10h Grade1 Statistically signicant correlation was observed between extent and grade of leakage at baseline (r = 0.98, p < 0.001). The choroidal thickness exhibited signicant correlation with extent and grade of leakage (r = 0.31, p = 0.04, and r = 0.37, p = 0.016, respectively). At the beginning of treatment, optic disk staining was observed in eight eyes, compared to four eyes at thirdmonth and ve eyes at the sixth month. No signicant difference was determined in terms of optic disc staining between the visits (Chi square, p = 0.074). However, signicant correlation was observed between optic disc staining and extent and grade of leakage (r = 0.73, p < 0.001, and r = 0.68, p < 0.001, respectively). Signicant correlation was also determined at the third month of treatment between extent of leakage and optic disc staining, and between extent of leakage and leakage grade (r = 0.537, p < 0.001, and r = 0.952, p < 0.001, respectively). signicant correlation observed at the third month of treatment between choroidal thickness and extent or grade of leakage (r = 0.18, p = 0.24 and r = 0.23, p = 0.14, respectively). Signicant correlation was observed between extent of leakage and optic discstaining atthe sixth month of treatment (r = 0.661, p < 0.001). signicant correlation was observed between extent and grade of leakage at the sixth month of treatment (r = 0.984, p < 0.001). No signicant correlation observed at sixth month between choroidal thickness and extent or grade of leakage (r = 0.21, p = 0.19 and r = 0.24, p = 0.12, respectively). Macular leakage observed at in ve eyes (one eye in three patients two eyes in patient).


Introduction
Uveitis is a sight-threatening intraocular in ammation andour understanding of the pathogenesis of the disease, other than infectious causes, is still limited. It is responsible 5-20% of legal blindness in developed countries. [1,2] Systemic autoimmune diseases such as Behçet disease, sarcoidosis, spondyloarthropathies, and Vogt-Koyanagi-Harada syndrome or unclassi able idiopathic uveitis constitute non-infectious uveitis. [3,4] The main objective in the treatment of uveitis is to keep the intraocular in ammation under control, to prevent recurrences, to establish full remission, and to protect the vision. [5] Steroids are used as the rst therapeutic choice in active in ammations.However,severe ocular and systemic side-effects may develop with long-term steroid use. Since recurrences are common under steroid monotherapy, conventional immunosuppressive agents are employed in long-term treatment. Conventional immunosuppressive agents such as azathioprine, cyclosporine, and methotrexate can be used aloneor in combination in treatment. However, some patients are resistant to treatment, and the disease cannot be brought under control, despite the use of combination therapies. [5] TNF-α is a cytokine produced from macrophages and neutrophils that contributes to in ammation in immune diseases, including non-infectious uveitis.
Increased TNF-α levels have been reported in both serum and aqueous humor in uveitis patients, and this elevation being correlated with disease activity. [6] Due to the pivotal role of TNF-α in in ammation, inhibiting its activity may be an effective strategy in the treatment of uveitis.Anti-TNF agents are of proven e cacy in non-infectious uveitis, and are today widely employed. [7][8][9][10] These agents are the most important choicein posterior/intermediate uveitis and panuveitis refractory to conventional treatment, and reduce exposure to and dependence on steroids. They make a positive contribution to treatment in severe cases, and prevent permanent loss of vision. Adalimumab (Humira, AbbVie, Chicago, IL, USA) is a completely human monoclonal IgG1 TNF-α antibody and the rst drug approved for the treatment of rheumatoid arthritis by the US Food and Drug Administration (FDA) in 2002.
Following two randomized placebo-controlled trials, VISUAL-I and VISUAL-II, adalimumab has now been approved for use in non-infectious intermediate, posterior and panuveitis in adults by the European Medicines Agency and the FDA . [9,10] The e cacy of adalimumab in non-infectious uveitis has frequently been investigated in terms of degree of clinically detectable ocular in ammation (anterior chamber and vitreous in ammation) and attack frequency. The e cacy of adalimumab in uveitis and in ammation control has been proved in several studies, although especially evaluating retinal vascular and optic discleakages in posterior segment have been investigated in a few studies.The purpose of the present study was to investigate retrospectively the e cacy of adalimumab in retinal vascular leakage, and controlling vascular and choroidal in ammation in patients with non-infectious uveitis, which is refractory to conventional immune suppressive therapy, by evaluating the posterior segment and peripheral retina with ultra-wide eld fundus uorescein angiography.

Materials And Methods
The patients who were resistant to conventional immunosuppressive therapy and started on adalimumab therapy at the Ophthalmology Department Uvea Unit, Medical Faculty, Karadeniz Technical University,Turkey, between April 2017 and March 2020 were included in the study. All patients were receiving prednisolone and immunomodulator (azathioprine, cyclosporine, methotrexateand/or interferon) drug combination. Patients who still had signs of active uveitis despite receiving single or combined therapy were considered as resistant and were included in the study. The study protocol was approved by the ethical committee of medical faculty, and written informed consent was obtained from all patients. The study was conducted in accordance with the Helsinki declaration.
Patients receiving adalimumab therapy for at least six months and attending regular follow-ups were enrolled. All patients underwent full protein derivative (PPD) tests, complete blood count, and kidney and liver function tests at baseline.Patients diagnosed with latent tuberculosis, de ned as a protein-puri ed derivative skin conversion involving an induration of 5 mm in the absence of radiographic or clinical disseminated or pulmonary disease ndings, received anti-tuberculosis prophylaxis at least three weeks before the rst dose of adalimumab. Magnetic resonance imaging of the brain was performed in all cases with pars planitis in order to exclude demyelinating disease.
Adalimumab was administered via the subcutaneous route at a dose of 40 mg at two-week intervals. The in ammatory status of each eye at baseline was classi ed as either 'active' (based on clinical ndings of at least one active in ammatory chorioretinal or retinal vascular lesion, an anterior chamber cell grade of 1 + or higher or vitreous haze grade of 1 + or higher, according to the Standardization of Uveitis Nomenclature (SUN) Working Group and adapted National Eye Institute criteria) or as 'inactive' (corresponding to eyes without active in ammatory chorioretinal or retinal vascular lesions in addition to an anterior chamber cell grade and vitreous haze grade of 0.5 + or less). [11,12] For the analysis of systemic outcomes, patients who have one or two active eyes at baseline were classi ed as 'active', while individuals in whom both eyes were quietat baseline were classi ed as 'inactive'.
Patients were evaluated at baseline and at third month and sixth month. Best corrected visual acuity(BCVA), slit-lampanterior segment and vitreous examination, intraocular pressure measurement, and comprehensive fundoscopy examination including posterior vitreous assessment were performed at each examination. Visual acuity was assessed using a Snellen chart and was converted into a logarithm of minimum angle of resolution (logMAR) for analysis. Ultra-wide eld fundus uorescein angiography (FFA) images were taken using an Optos device (OptosCalifornia, PLC, Dunfermline, UK). FFA was performed on all visits, and the staining onthe head of the optic disk, retinal vascular leakage, and ischemia were evaluated. Optic nerve hyper uorescence was investigated on late-phase images.The peripheral retina was de ned as the region from the equator to the ora serrata. Retinal Patients' immunosuppressive drug burdens were calculated according to the immunosuppressive agents as described by Nuseenblattet al. [13] Patients were asked about side-effects occurring during treatment, and complete blood count and serum sampletests were repeated at two-month intervals.

STATISTICAL ANALYSIS
Statistical analyses were performed on SPSS 21.0 (Statistical Package for Social Science) for Windows. Normality tests were performed for all variables.
Compatibility with normal distribution was assessed using the Shapiro-Wilk test. The matched t test was applied for normally distributed dependent groups, and the Mann-Whitney U test for non-normally distributed dependent groups. Time-dependent changes in the same parameters at repeated measurements were analyzed using analysis of variance (ANOVA). Descriptive statistics for continuous variables exhibiting normal distribution were expressed as mean ± standard deviation, while those not exhibiting normal distribution were expressed as median, maximum, and minimum values and were reported together. p values < 0.05 were regarded as signi cant.

Results
Forty-four eyes of 23 patients with non-infectious uveitis, 13 (56.5%) women and 10 (43.5%) men, were included in the study. One eye of one patient was excluded from the study because of neovascular glaucoma and another patient because of phthisis bulbi.Behçet uveitis was diagnosedin 14 (60.9%) patients, sarcoidosis in two (8.7%), and idiopathic uveitis in seven (30.4%) patients. All patients were classi ed anatomically based on International Uveitis Study Group criteria. Panuveitis was present in 10 (43.5%) cases, posterior uveitis in four (17.4%), intermediate uveitis in ve (21.7%), and posterior and uveitis in four (17.4%). The mean duration of uveitis was 5.26 ± 5.16 years. Etiologies and locationsof uveitis, and drug therapies prior to adalimumab are shown in Table 1. observed in leakageextend between the visits (p < 0.001). However, the grade of leakage decreased signi cantly during the study (p < 0.001). The changes in leakage extentand grades are shown in Table 2.  Adalimumab therapy was well-tolerated by patients, although local side-effects such as pain in the injection site, rash and itching were frequently seen.
Pulmonary thromboembolism was observed in one patient. We think that this embolism is secondary to the vasculitis seen in Behçet's disease. These were regarded as the mild risk group and were started on anticoagulant therapy, while adalimumab therapy was maintained. shown to lower both the incidence and severity of intraocular in ammation. [14] Several studies have shown the e cacy of the TNF-α inhibitors such as in iximab and adalimumab in non-infectious uveitis. [15][16][17] The VISUAL I and VISUAL II studies showed the effectiveness of adalimumab in active and inactive refractory non-infectious uveitis and reported that it reduced the frequency of in ammation relapses in uveitis patients with a broad range of uveitic conditions. [9,10] The effects of TNF-α antagonist on visual acuity, attack frequency, and anterior chamber and vitreous in ammation have frequently been researchedin the literature, but their e cacy against retinal vascular and choroidal in ammation has not been evaluated in detail. Studies have shown the e cacy of in iximab against retinal vasculitis, but information concerning the vascular effect of adalimumab is limited. Retinal and optic discpathology developing in association with retinal and choroidal in ammation, particularly in panuveitis and posterior uveitis,lead to vision loss in uveitis. Keino et al. [18] reported that retinal and optic disk damage occurring in patients with Behçet's disease was not solely associated with acute in ammation attacks, but also potentially with chronic in ammation capable of causing background vascular leakage.
With the introduction of the ultra-wide eld angiographies, it has become possible to identify retinal peripheral leakages in clinically active or inactive patients.In this way, evaluating the presence and extent of retinal pathologies, diagnosis, treatment planning and follow-up can be done more accuratelyin the patients with uveitis.Several studies have reported that active vasculitis ndings can be determined in the early period with wide-angle angiographies, and that treatment can thus be arranged early, with better potential outcomes. [19,20] In the present study, we investigated the effect of adalimumab therapy, not only in clinically identi able uveitis attacks, but also particularly on retinal vascular structuresand choroidal involvement.
Keino et al. [18] evaluated the effectiveness of in iximab on retinal vascular leakage and optic disc leakage in the patientswith Behçet disease,and reported 79% improvement in retinal vascular and optic discleakage at 12-month follow-up. Pirani et al. [21] reported a statistically signi cant decrease in vasculitis in the four retinal quadrants at 12-month follow-up with adalimumab therapy (p = 0.01).Fabiani et al. [22] investigated the e cacy of adalimumab in patients with Behçet uveitis and reported that vasculitis was initially present in 22 (55%) patients, and decreasedto eight (20%) patients at three months and to one (2.5%) at 12 months.Again, in another study of Fabiani et al. [23] evaluated the e cacy of adalimumab and in iximab in treatment of recalcitrant retinal vasculitis, it was found that there was no difference between adalimumab and in iximab therapy, and both provided a signi cant reduction in retinal vasculitis ndingsat 12-month follow-up (p < 0.001). Sharma et al. [24] reported that remission was achieved in 88.23% of patients with in iximab therapy on recalcitrant retinal vasculitisat six-month follow-up. In another study, Keino et al. [25] reported that background vascular leakage and optic discleakage in inactiveeyes decreasedwith in iximab treatment in Behçet patients over four-year follow-up.Since we could not nd detailed studies in the literature similar to our study evaluating the severity and extent of vasculitis, retinal vascular leakage, we could not make a comparison in this respect.In our study, the presentof the retinal leakage was evaluated in clock hour quadrants, andwe found a signi cant decrease in the extent and grade of leakage at 3 and 6 months (p < 0.001)with treatment.Our results are compatible with the American Academy of Ophthalmology's recommendation for the use of anti-TNF-α as a rst-line therapy in severe uveitis attacks in patients with Behçet disease. [26 ] Pirani et al. [21] reported that choroidal thickness decreased from 236 µm to 208.75 µm following adalimumab therapy. In the present study, choroidal thickness signi cantly decreased from 256.65 ± 43 µm to 224.81 ± 34.91 µm at six-month treatment. In our study, we found a statistically signi cant decrease in choroidal thickness at the 3rd and 6th months with treatment (p = 0.005).
Lee et al. [7] reported no marked improvement in vision levels following adalimumab therapy. In a study of 131 patients, Diaz Llopis et al. [4] reported that vision levels remained unchanged over six months in 75.4% of patients, while improving from 0.39 ± 0.244 logMAR to 0.26 ± 0.39 logMAR in 21.3% at the end of six-month follow-up.Durrani et al. [27] reported no improvement in vision levels at three-and six-month follow-ups after adalimumab therapy. In contrast to these studies, Bawazeer et al. [8] reported improved visual acuity of three lines or more in 17 put of 21 patients at an average follow-up of 10.8month .In our study, we did not nd any statistically signi cant changein visual acuity during follow-up (p = 0.07).
An another important issue in patients with uveitis is use of steroids. Because they need to be used for a long time and serious side effects may occur.
The ability to control in ammation with other agents will reduce both the drug load and dose and durationof steroids.The great majority of patients in the present study had Behçet uveitis and were frequently in receipt prednisolone and azathioprine therapy. While, the mean initial dosage of Prednisolone in this study was 17.5 mg /day (4-64 mg), it was 7.11 mg at the sixth month. Prednisolone therapy was discontinued entirely in four patients at three months and in eight patients at six months. In one patient havingrecurrence, orally Prednisolone at 1 mg/kg was restarted at the sixth month. Since severe ocular and systemic side-effects maydevelop with long-term steroid use, prednisolone therapy is used for as short a time as possible, and steroid sparing agents that suppress in ammation are then used. In addition, the drug load decreased from 9.91 ± 3.78 initially to8.0 ± 4.71 at the end of 6 months (p = 0.022). This is important in terms of reducing possible complications of the drugs.
Kempen et al. [28] reported uncorrelation agreement between central macular thickness measured using OCT and macular leakage in fundus angiography, and stated that these two methods are not a substitute but complementary methods in evaluating macular pathology. They particularly recommended the use of OCT due to its non-invasive and inexpensive nature. [28] Similarly, Ossewaarde-van Norel et al. [29] reported that macular leakagewas detected in 34 (30%) out of 112 eyes with uveitic macular edema using FFA, while no edema was detected using OCT. In the present study, macular leakage was detected at FFAin ve eyes, while no edema was detected at OCT.
Although serious side effects of the drugs used in the treatment of uveitis are an important problem, adalimumab therapy was well-tolerated by patients, with side-effects consisting of local erythema, pain, hemorrhage, and rash in the injection sitein the present study. Systemic side-effects developed were leukopenia in two patients and pulmonary thromboembolism in one. One patient with suspected embolism secondary to Behçet disease-related vasculitis was started on anticoagulant therapy. Additionally, adalimumab therapy was maintained, rather than being stopped, in these patients. None of our patients developed severe side-effects such as viral or bacterial infection, demyelinating neurological disease, tuberculosis activation, or tumor development.
The limitations of this study are that it is retrospective and includes a small number of patients. The e cacy of adalimumab therapy was evaluated during only six-month follow-up, and its effectiveness or failure of treatment were not evaluated in terms of long-term outcomes.
In conclusion, this study shows that adalimumab therapy is effective in the cases withpersistentactive and inactive uveitis in terms of controllingin ammation, reducing the drug burden, and controlling retinal vascular leakage and choroidal in ammation. Controlling of these pathologies in uveitis will provide a better visual prognosis in patients.Adalimumab therapy appears to be particularly effective in cases of refractory retinal vasculitis due to uveitis, although further longer-term studies with larger case numbers are needed to evaluate treatment failure and resistance development over long-term follow-up.

Declarations
The Authors declares that there is no con ict of interest. There is no funding support for this manuscuript.
Ethical approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent: Informed consent was obtained from all individual participants included in the study.