Intravitreal Fasudil Monotherapy for Treatment of Refractory Diabetic Macular Edema: a Prospective, Interventional Case-series

Purpose: To evaluate the effect of three monthly intravitreal injections of a Rho-associated protein kinase (ROCK) inhibitor (Fasudil, Corporation, Japan) on refractory diabetic macular edema (DME). Study design: Prospective – clinical Methods: This interventional case series included 10 eyes of 10 patients with DME unresponsive to at least six previous intravitreal bevacizumab (IVB) injections. Eligible eyes underwent intravitreal injection of 0.025 mg/0.05 ml Fasudil. Best-corrected visual acuity (BCVA) and central macular thickness (CMT) were evaluated as functional and anatomical response indicators, respectively. Results: Mean age was 60.1±5.1 (range, 53-68) years. Five cases responded to treatment; two with both anatomical and functional response (reduction of CMT from 521 to 395 and from 390 to 301microns and improvement of BCVA from 0.3 to 0.1 LogMAR and 0.6 to 0.4 LogMAR, respectively) and three with only functional improvement (0.7 to 0.4; 0.7 to 0.4; and 0.3 to 0.1 LogMAR). Of note, cases with no signicant CMT change had morphologic improvement of retinal microstructure to some extent. No adverse event was observed during the study period. Conclusion: Monotherapy with intravitreal injection of ROCK inhibitors seems to have moderate visual benets in eyes with DME refractory to IVB. Such effects may be functionally signicant without obvious anatomical improvement. of vision better than 16/20 or macular thickness of less than 300 microns), further injections were suspended. Patients were evaluated for intra- or post-operative complications a day and a week after each intravitreal injection. BCVA and CMT, as obtained via optical coherence tomography (Spectralis SD-OCT, Heidelberg Engineering , Heidelberg, Germany), were evaluated at baseline and four weeks after each injection. Ethics patients informed about the probable risks and benets obtained participants. signed-rank test was evaluate signicance of CMT before after whole intervention period. intravitreal administration in rDME compared to bevacizumab monotherapy (4), but data on ROCK-inhibitors monotherapy in rDME was lacking. An in-vivo study on Kimba mice has shown that ripasudil (a selective ROCK-inhibitor) could suppress the expression of some inammatory cytokines present in rDME, namely, tumor necrosis factor-α (TNF-α), MCP-1, and KC (the murine homolog of interleukin-8), whereas bevacizumab suppressed TNF-α and interleukin-6. Ripasudil alone could bring about a signicant reduction in retinal thickness; bevacizumab co-administration could synergistically enhance this reduction (7). The present preliminary study shows a clinically modest effect of fasudil monotherapy in rDME cases resistant to prior standard treatments with bevacizumab; visual improvements with or without anatomical changes were achieved in 50% of the eyes; however, the changes did not reach statistical signicance. In some cases, the functional and anatomical improvements did not necessarily ensue in parallel, i.e., visual improvements were achieved in three eyes with no signicant CMT reductions. Such discordances between visual improvements and CMT changes have been described by some previous studies as well (10). For instance, plasma kallikrein inhibition in rDME eyes has shown acceptable visual improvements without signicant changes in DME anatomical parameters (11); plasma kallikrein is a serine protease elevated in DME eyes vitreous, capable of increasing vascular permeability and contributing to retinal edema in a VEGF-independent fashion (12). This incongruity may stem from variable edema and ischemia durations with different patterns of inuencing CMT and visual acuity; disorganization of the inner retinal layers, in lieu of CMT changes, is suggested to better correspond with visual acuity modications (10, 13). LogMAR) or worse (n=3) beneted from Fasudil monotherapy in terms of visual function. We injected 0.025mg/0.05mL fasudil intravitreally and detected no adverse events a day and a week after each injection, in line with previous animal studies (14) and our prior experiences (4–6).


Introduction
Diabetic retinopathy is the most prevailing cause of impaired vision and blindness in working-age individuals worldwide; in most cases, diabetic macular edema (DME) is the main reason (1). Optimal therapeutic strategies in managing DME are evolving continuously, and failure of conventional treatments in refractory DME (rDME) underlines the need for new methods. Intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) agents is the mainstay of DME treatment, though it does not carry thorough visual and anatomical success in a signi cant proportion of DME cases (2).
Rho/Rho-associated protein kinase (ROCK) signaling pathway inhibitors are seemingly promising in managing several ophthalmic conditions, including DME (3). Our preliminary experience with combined intravitreal injection of a ROCK inhibitor (Fasudil) and bevacizumab in ve DME patients who had not responded to previous bevacizumab administrations showed signi cant visual and structural improvements within six months (4). In another pilot study with a larger sample size, Fasudil administration as adjunctive therapy in rDME brought about signi cant visual improvement and decreased central macular thickness within four weeks (5). A randomized controlled trial showed that intravitreal injection of Fasudil and bevacizumab could yield more prolonged and more prominent anatomical and visual improvements than bevacizumab alone in center-involved DME (6).
The extent to which monotherapy with ROCK-inhibitors can be of therapeutic value in rDME remains unknown. Therefore, this preliminary interventional case series aimed to investigate the outcome of treatment with Fasudil alone for rDME.

Methods
In this interventional case series, 10 eyes of 10 patients with rDME were included. General inclusion criteria were, i) best-corrected visual acuity (BCVA) of In the total study population, neither BCVA nor CMT values changes were statistically signi cant; mean change ± SD in BCVA was -0.05 ± 0.21 LogMAR (p=0.49) and mean change ± SD in CMT was 60.6 ± 146.5 microns (p=0.674). However, Fasudil administration resulted in a favorable outcome in ve eyes: two with both functional and anatomical improvement (cases No. 1 and 2; see Table-1) and three with functional improvement only (cases No. 4, 7, and 9; see Table-1). As regards case No. 4, in spite of minimal reduction in CMT, morphologic improvement in the foveal contour was noted (see Figure-1). Fasudil administration was discontinued after the rst injection in patient No. 4 because BCVA improved favorably to 0.1 LogMAR after the only injection and patient No. 10 due to the patient's refusal to continue the study. No signi cant improvements were observed in cases No. 3, 5, 6, 8, and 10 (see Table-1).

Discussion
More than half of DME patients respond well to anti-VEGF therapies; however, in ammation in a signi cant proportion of cases does not subside with such therapeutic approaches alone (2). Both VEGF-dependent and -independent pathways are suggested to be involved in DME; in rDME, the latter has more pronounced effects than the former (7). VEGF overexpression in rDME triggers downstream pathways involving Rho/ROCK signaling, capable of independently promoting a retinal in ammatory milieu and disrupting the blood-retinal barrier integrity. The delay between VEGF overexpression and Rho/ROCK pathway upregulation may explain the failure of anti-VEGF therapies in controlling rDME (7). Moreover, Rho/ROCK pathway upregulation promotes in ammation and leukocyte adhesion to the endothelial surface (8) and exerts vasoconstrictive and apoptotic effects on endothelial cells via inhibiting endothelial nitric oxide synthesis (9).
We have previously reported the favorable outcome of combined fasudil and bevacizumab intravitreal administration in rDME compared to bevacizumab monotherapy (4), but data on ROCK-inhibitors monotherapy in rDME was lacking. An in-vivo study on Kimba mice has shown that ripasudil (a selective ROCKinhibitor) could suppress the expression of some in ammatory cytokines present in rDME, namely, tumor necrosis factor-α (TNF-α), MCP-1, and KC (the murine homolog of interleukin-8), whereas bevacizumab suppressed TNF-α and interleukin-6. Ripasudil alone could bring about a signi cant reduction in retinal thickness; bevacizumab co-administration could synergistically enhance this reduction (7).
The present preliminary study shows a clinically modest effect of fasudil monotherapy in rDME cases resistant to prior standard treatments with bevacizumab; visual improvements with or without anatomical changes were achieved in 50% of the eyes; however, the changes did not reach statistical signi cance. In some cases, the functional and anatomical improvements did not necessarily ensue in parallel, i.e., visual improvements were achieved in three eyes with no signi cant CMT reductions. Such discordances between visual improvements and CMT changes have been described by some previous studies as well (10). For instance, plasma kallikrein inhibition in rDME eyes has shown acceptable visual improvements without signi cant changes in DME anatomical parameters (11); plasma kallikrein is a serine protease elevated in DME eyes vitreous, capable of increasing vascular permeability and contributing to retinal edema in a VEGF-independent fashion (12). This incongruity may stem from variable edema and ischemia durations with different patterns of in uencing CMT and visual acuity; disorganization of the inner retinal layers, in lieu of CMT changes, is suggested to better correspond with visual acuity modi cations (10,13).
It is noteworthy that cases without signi cant CMT change had morphologic improvement of retinal microstructure to some extent (Case No. 4). None of the eyes with baseline BCVA of 20/400 (1.30 LogMAR) or worse (n=3) bene ted from Fasudil monotherapy in terms of visual function.
We injected 0.025mg/0.05mL fasudil intravitreally and detected no adverse events a day and a week after each injection, in line with previous animal studies (14) and our prior experiences (4-6).
To summarize, intravitreal fasudil monotherapy in rDME eyes unresponsive to previous conventional treatments is safe and has a moderate bene cial effect in improving visual function, not necessarily accompanied by signi cant anatomical improvement. However, no substantial therapeutic gain may be achieved in patients with severe baseline visual impairments (BCVA<20/400).

Declarations Con icts of interest
Authors declare none.

Funding
No funding was received for the conduction and documentation of this research.