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. 2025 Dec 31;16:4124. doi: 10.1038/s41598-025-34244-4

Pluripotent epigenetic regulator OBP-801 attenuates fibrosis and maintains lower intraocular pressure in a rabbit PRESERFLO MicroShunt surgery model

Yusuke Koga 1, Toru Ikushima 1, Nao Hiramoto 1, Kengo Yoshii 2, Naoki Murata 1, Atsushi Mukai 1, Yuji Yamamoto 1, Hiroki Mieno 1, Yasuo Urata 3, Morio Ueno 1,✉, Chie Sotozono 1
PMCID: PMC12858950  PMID: 41476143

Abstract

The inhibition of fibrosis in filtering blebs is indispensable for lowering intraocular pressure (IOP) after PRESERFLO MicroShunt surgery (PMS). We previously reported the anti-fibrotic effect of OBP-801 (OBP) on a filtering bleb in a rabbit glaucoma filtration surgery model using a 22-gauge cannula. In this study, we investigated the effect of OBP after PMS in rabbit eyes for the development of a new practically applicable anti-fibrotic treatment. This study involved 19 Japanese white rabbits that underwent PMS in the right eye, with those eyes divided into 3 groups: (1) intraoperative subconjunctival injection of 0.02% mitomycin-C (MMC) (n = 6), (2) postoperative instillation of 100 nM OBP eye drops (n = 7), or (3) a balanced salt solution (BSS) control (n = 6). Bleb morphology and IOP were monitored for 12 weeks postoperative, with the bleb tissues then undergoing evaluation of fibrosis and Western blot analysis. A lower postoperative IOP was maintained in the OBP-group eyes, and at 12 weeks postoperative, the IOP was significantly lower in that group than in the BSS and MMC groups (p < 0.01). OBP-treated eyes showed no adverse effects and reduced levels of alpha-smooth muscle actin and collagen deposition, thus suggesting that OBP is a promising candidate for improving surgical outcomes post PMS.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-34244-4.

Keywords: PRESERFLO MicroShunt, OBP-801, Fibrosis, Bleb scaring, Glaucoma filtration surgery

Subject terms: Drug development, Glaucoma

Introduction

Glaucoma is a neurodegenerative disease characterized by the progressive dysfunction and death of retinal ganglion cells and their axons, primarily due to elevated intraocular pressure (IOP). It is the leading cause of irreversible blindness worldwide, as it reportedly afflicts approximately 3.61 million people with blindness and nearly 4.14 million people with visual impairment1.

To date, the only treatment for glaucoma is a lowering of the elevated IOP. Recently, the PRESERFLO MicroShunt (Santen Pharmaceutical Co., Ltd., Osaka, Japan), an 8.5 mm and 70 µm lumen, minimally-invasive, glaucoma drainage device that is safe and effective for not only lowering IOP, but also for reducing the number of IOP-lowering medications used, has been developed2–8. Currently, mitomycin C (MMC) is widely used during PRESERFLO MicroShunt surgery (PMS) to prevent postoperative subconjunctival fibrosis. However, it has been reported that 6.1- 44.4% of patients develop postoperative scarring in the filtration bleb that requires needling surgery2–4,7, suggesting that MMC does not adequately inhibit postoperative scarring in bleb tissues. In addition, there are certain risks of complications when MMC is used, such as hypotony maculopathy and leaky blebs that increase susceptibility to endophthalmitis, which subsequently leads to surgical failure9.

It is well known that histone deacetylase inhibitors (HDACi) potently target excessive fibrosis10, and reports have indicated that the anti-fibrotic action of HDACi will be clinically applied for the treatment of lung, kidney, and heart disease11,12. From the aspect of wound healing following tissue injury, it was previously reported that HDACi stopped corneal opacity after photorefractive keratectomy in a rabbit model13.

OBP-801 (OBP) is a novel and potent HDACi that was identified through a p21 promoter–reporter screen14. Preclinical studies have demonstrated its antitumor efficacy against a wide range of solid tumors, including rhabdomyosarcoma, renal cell carcinoma, bladder cancer, ovarian cancer, neuroblastoma, and breast cancer15–20. These effects are mediated through histone hyperacetylation, cell-cycle arrest, induction of apoptosis17–19, and in some contexts, enhancement of immune responsiveness21. Furthermore, in a Phase Ia clinical trial involving patients with advanced solid tumors, OBP showed good tolerability, with no dose-limiting toxicities observed in the lower dose cohorts and a short plasma half-life without accumulation22. In addition, OBP is also a novel pluripotent epigenetic repressor of diverse genes involved in multiple steps of tissue inflammation and wound healing23. Moreover, OBP showed potent HDACi activity at a very low concentration, such as 1 nM; i.e., levels that are 1/1000-times lower than the standard levels thus far reported for HDACi, suberoylanilide hydroxamic acid, valproic acid (VPA) and trichostatin A24–27. In our previous investigations, we found that OBP comprehensively inhibits fibrosis, scarring, and angiogenesis in retinal pigment epithelial cells28. Furthermore, we previously reported that OBP eye drops inhibited postoperative scarring and maintained the filtration bleb in a rabbit glaucoma filtration surgery (GFS) model using a 22-gauge (G) cannula23.

Thus, the aim of this present study was to investigate the effect of OBP on filtering blebs post PMS in rabbit eyes for the development of a new practically applicable antifibrotic treatment other than MMC.

Results

Histone acetylation was induced by OBP in conjunctival fibroblast

We previously reported that administering 100 nM of OBP in a rabbit GFS model effectively inhibited fibrosis23. To confirm the effect of HDACi on histone acetylation, we showed that OBP at a concentration of 10 nM increased acetylated histone levels in in vitro cultured human conjunctival fibroblasts (Figs. S1, S7). Furthermore, we investigated whether or not histones are acetylated in in vivo conjunctival tissues, and performed western blot analysis using conjunctival tissues from naïve (non-surgical) eyes after twice-daily topical instillation of OBP (100 nM) for 1 week. Untreated conjunctival tissues from naïve eyes were used as a control. As shown in Fig. 1, at 1 week after a twice-daily topical administration of OBP at a concentration of 100 nM, conjunctival tissues demonstrated significantly higher levels of acetylation of histone H3 (Lys9) compared to the non-treatment control (P = 0.02).

Fig. 1.

Fig. 1

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Expressions of histone H3 and acetyl-histone H3 in rabbit conjunctival tissues. Western blot analysis was performed using conjunctival tissues from naïve (non-surgical) eyes after twice-daily topical instillation of OBP-801 (100 nM) for 1 week. Untreated conjunctival tissues from naïve eyes were used as a control. OBP treatment significantly increased the acetylation levels of histone H3 compared with the control group. Column and error bars indicate the mean ± SD (OBP group, n = 3; control group, n = 3).

OBP induced unscarred blebs with normal vascular appearance

To examine the effect of a 100-nM OBP treatment in the morphology of the filtering bleb, we observed the filtering blebs for 12 weeks postoperative, and compared the findings among three groups: (1) eyes treated with 100 nM OBP eye drops, (2) those treated with subconjunctival injections of 0.02% MMC, and (3) those treated with balanced salt solution (BSS) (BSS Sterile Irrigating Solution; Alcon, Fort Worth, TX, USA) eye drops as a negative control (Table 1). Swogger et al.29 reported that subconjunctival injections of MMC can be more effective for IOP reduction and fibrosis inhibition in the filtering bleb than the standard method of a sponge application of MMC on the bare sclera. MMC is also administered via subconjunctival injection in GFS procedures, such as used for the XEN Gel Implant (AbbVie Inc., Chicago, IL, USA)30. Hence, we administered MMC via subconjunctival injection in this current study based on its established use in both clinical settings and previous rabbit glaucoma model investigations. Eyes treated with BSS gradually became flat at 4-days postoperative. Conjunctival hyperemia in the superficial layers peaked at 4 days postoperative, and gradually disappeared by 2 weeks postoperative. By 4 weeks postoperative, large vessels appeared in the deeper layers of the conjunctiva, yet at 3 weeks postoperative, no major change in the appearance of bleb conjunctival tissue was observed. Finally, eyes treated with BSS were associated with a flat, yet not avascular, scarred bleb at 12 weeks postoperative (Figs. 2 and S2). In eyes treated with OBP, conjunctival hyperemia in the superficial layers peaked at 4 days postoperative, as in the case of the BSS group, and gradually disappeared by 1 week postoperative. Unlike the eyes that were treated with BSS, eyes treated with OBP did not show the emergence of large vessels in the deeper layers of the conjunctiva, and were found to be associated with diffuse filtering blebs of normal conjunctival appearance and without scarring at 12 weeks postoperative (Figs. 2 and S3). Conversely, eyes treated with MMC exhibited marked chemosis at 1 day postoperative. The conjunctival blood vessels began to disappear at 4 days postoperative, and became completely avascular at 2 weeks postoperative. The filtration blebs were associated with a thin-walled and cystic bleb with encapsulation by scar tissue at 4 weeks postoperative. After that, the filtering blebs gradually shrank via subconjunctival scarring, with the invasion of the conjunctival vessels and blebs being completely replaced with scar tissue at 12 weeks postoperative (Figs. 2 and S4).

Table 1.

The rabbit-model PRESERFLO MicroShunt surgery model treatment groups.

Timing (days)
Groups Treatment Concentration Volume Routes 0 1 2 3 4 5 6 7
BSS BSS 80 μL ED ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯
OBP OBP 100 nM 80 μL ED ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯
MMC MMC 0.02% 100 μL SI ◯
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BSS balanced salt solution, ED eye drop, MMC mitomycin C, OBP OBP-801, SI subconjunctival injection.

Fig. 2.

Fig. 2

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(a) Postoperative macroscopic photographs showing the time-course changes of the filtering bleb in the rabbit eyes. OBP (100 nM) and BSS were administered topically once before surgery and twice daily thereafter for 1 week, while MMC (0.02%) was administered by a 100 µL subconjunctival injection only at 30 min before surgery. Eyes treated with BSS (top row) developed flat blebs with conjunctival hyperemia initially, followed by large vessel formation and scarring by 12 weeks postoperative. OBP-treated eyes (middle row) showed diffuse, functional blebs with a normal vascular appearance and minimal scarring throughout the 12 weeks time course. In contrast, MMC-treated eyes (bottom row) developed thin-walled, avascular, cystic blebs with encapsulation and subsequent scarring. The white dotted outlines mark the bleb margins for all groups at each time-point. Black arrows in the BSS and black arrowheads in the MMC images indicate representative conjunctival vessels. (b) Quantitative analysis of bleb area (scale 0–4: 0 = no bleb, 1 = very small bleb, 2 = small bleb, 3 = medium bleb, and 4 = large bleb) at 1 day and 1-, 4-, 8-, and 12 weeks postoperative. The OBP group maintained a larger bleb area over time. Compared with the BSS group, the OBP group had a significantly larger bleb area from 4 weeks through 12 weeks postoperative. In comparison with the MMC group, the bleb area was larger in the MMC group at 1-week postoperative, yet decreased thereafter. At 12 weeks postoperative, the bleb area in the MMC group was significantly smaller than that in the OBP group. (c) Quantitative analysis of bleb vascularity (scale 1–4: 1 = avascular, 2 = normal vascularity, 3 = mild vessel inflammation, and 4 = moderate or severe inflammation). The OBP group remained approximately stable and did not differ significantly from the BSS group, whereas the MMC group was significantly more avascular than the OBP group at 1- and 4 weeks postoperative. However, at 12 weeks postoperative, vascularity in MMC group significantly increased and was higher than that in the OBP group. Line plots present group means ± SD (n = 6, 7, and 6 for the BSS group, OBP group, and MMC group, respectively).

In addition, we quantified bleb morphology (area and vascularity) according to the scoring system developed by Fujimoto et al., which is based on the Moorfields Bleb Grading Scheme (n = 6, 7, and 6 for BSS, OBP, and MMC, respectively)31,32. Compared with BSS, the OBP group had a significantly larger bleb area from weeks 4 through 12 (p = 0.048, 0.008, and 0.02, respectively). In comparison with MMC, the bleb area was significantly larger in MMC at week 1 (p = 0.01) but decreased thereafter; at week 12 the MMC bleb area was significantly smaller than that of OBP (p = 0.004) (Fig. 2b). For bleb vascularity, OBP remained approximately stable and did not differ significantly from BSS, whereas MMC was significantly more avascular than OBP at weeks 1 and 4 (p = 0.001 and 0.02, respectively) and at week 12 vascularity in MMC significantly increased (p = 0.01) (Fig. 2c). Using a linear mixed-effects model, bleb area differed significantly between OBP and BSS across the time course (p < 0.001), whereas no difference was detected between OBP and MMC (p = 0.884). For bleb vascularity, the model identified a significant difference between OBP and MMC (p = 0.007), while OBP and BSS did not differ (p = 0.107).

OBP enabled IOP lowering until 12-weeks post PMS

Since the morphology of the filtering bleb is related to postoperative IOP, we examined whether or not OBP treatment has effects that maintain the reduction of IOP after PMS. A linear mixed-effects model of the longitudinal IOP data showed significant overall differences across the time course (OBP vs. BSS, p < 0.001; OBP vs. MMC, p < 0.001). IOP in the rabbit eyes treated with BSS remained low for approximately 1-week postoperative, and then gradually increased. On the other hand, IOP in the rabbit eyes treated with OBP remained low up to 12 weeks postoperative. IOP in the rabbit eyes treated with MMC remained low for approximately 4 weeks postoperative, then gradually rose to the highest IOP among the 3 groups at 12 weeks postoperatively (Fig. 3). In per-time-point comparisons, IOP was maintained lower by OBP than by BSS at weeks 4, 8, and 12 (p = 0.005, 0.049, and 0.014, respectively), and lower than by MMC at weeks 8 and 12 (p = 0.002 and 0.001, respectively). No significant difference was found between IOP in the rabbit eyes treated with BSS and those treated with MMC at 12-weeks postoperative. As supplemental information, a post hoc power analysis of the 12-week between-group contrasts, BSS vs OBP estimated at Cohen’s d = 3.73 with power of 99.9%, and MMC vs OBP estimated at d = 4.95 with power of 99.9%.

Fig. 3.

Fig. 3

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Graph showing postoperative intraocular pressure (IOP) in the rabbit PRESERFLO™ MicroShunt surgery (PMS) models. OBP (100 nM) and BSS were administered topically once before surgery and twice daily thereafter for 1 week, while MMC (0.02%) was administered as a single 100-µL subconjunctival injection at 30 min before surgery. A linear mixed-effects model of the longitudinal IOP data showed significant overall differences across the time course (OBP vs. BSS, p < 0.001; OBP vs. MMC, p < 0.001). IOP in the rabbit eyes treated with BSS remained low for approximately 1-week postoperative, and then gradually increased. On the other hand, IOP in the rabbit eyes treated with OBP remained low up to 12 weeks postoperative. IOP in the rabbit eyes treated with MMC remained low for approximately 4 weeks postoperative, then gradually rose to the highest IOP among the three groups at 12-weeks postoperatively. In per-time-point comparisons, OBP maintained lower IOP than BSS at 4-, 8-, and 12 weeks postoperative (p = 0.005, 0.049, and 0.014, respectively), and lower IOP than MMC at 8- and 12-weeks postoperative (p = 0.002 and 0.001, respectively). No significant difference was observed between BSS- and MMC-treated groups. Data are presented as mean ± standard deviation (n = 6, 7, and 6 for the BSS group, OBP group, and MMC group, respectively). *p < 0.05 for BSS vs. OBP, †p < 0.05 for MMC vs. OBP. NS = Not significant.

OBP inhibited the expression of fibrous deposits

Next, to investigate whether or not the differences in postoperative IOP were due to fibrosis of the filtering bleb tissue we conducted histological analysis of the tissue. At 12 weeks postoperative, hematoxylin and eosin (H&E) staining of the bleb tissue in the eyes treated with BSS revealed fibrous deposition in the conjunctival tissue, and immunohistochemical staining revealed the presence of a small number of α-smooth muscle antibody (α-SMA)-positive cells in the filtering blebs. The α-SMA-positive cells beneath the conjunctival epithelium coincided with conjunctival vessels, and the expression of collagen (COL) type 1 (COL-1) was observed in the subconjunctival bleb tissue, thus suggesting fibrosis in the filtering blebs (Fig. 4a–c). On the other hand, H&E staining of the bleb tissues at 12 weeks postoperative in the eyes treated with OBP showed only mild fibrous deposition in the tissue, and immunohistochemical staining revealed that there was no existence of α-SMA-positive cells in the filtering blebs. The α-SMA-positive cells beneath the conjunctival epithelium coincided with conjunctival vessels, and sparse expression of COL-1 was observed in the bleb tissue, thus suggesting that the OBP treatment effectively suppressed fibrosis (Fig. 4d–f). In contrast, in the eyes treated with MMC, H&E staining of the bleb tissues at 12 weeks postoperative showed the densest form of fibrous deposition in the subconjunctival tissue. Immunohistochemical staining revealed cells strongly α-SMA-positive in the bleb tissue and massive expression of COL-1, thus suggesting pronounced fibrosis in the tissue (Fig. 4g–i). To further assess these histological findings, we quantitatively analyzed the immunofluorescence staining results measuring mean gray values of α-SMA and COL-1 signals (n = 3, 4, and 3 for BSS, OBP, and MMC, respectively). The OBP group showed significantly lower mean gray values of COL-1 compared with the BSS group (p = 0.010) and MMC group (p = 0.027), and also significantly lower values of α-SMA compared with the BSS group (p = 0.017) and MMC group (p = 0.022). No significant differences were observed between the BSS and MMC groups for either marker (Fig. 4j, k).

Fig. 4.

Fig. 4

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Microscopic analysis of bleb tissues of the rabbit PRESERFLOTM MicroShunt Surgery (PMS) models at 12 weeks postoperative (i.e., final examination). OBP (100 nM) and BSS were administered topically once before surgery and twice daily for 1 week postoperative, while MMC (0.02%) was administered as a single 100 µL subconjunctival injection at 30 min before surgery. The images show representative sections from the bleb tissues in each group. Hematoxylin and eosin (H&E) staining (top row), immunofluorescence staining for α-smooth muscle actin (α-SMA) (middle row), and collagen (COL) type 1 (COL-1) (bottom row). OBP decreased COL deposition and reduced expressions of α-SMA and COL-1 in bleb tissue after PMS, whereas COL deposition and expressions of α-SMA and COL-1 were increased in the MMC-treated rabbit bleb tissues. Scale bars: 200 μm. Asterisks indicate bleb tissues. (j, k) Quantification of mean gray values of α-SMA (j) and COL-1 (k) staining in each group (n = 3, 4, and 3 for the BSS group, OBP group, and MMC group, respectively). Data are presented as mean ± SD. *p < 0.05.     

Next, the bleb tissue was analyzed by western blotting at 12 weeks postoperative. The expression level of α-SMA in the MMC-group tissue was higher than that in the BSS-group tissue. Conversely, the expression of α-SMA was downregulated in the OBP-group tissue. Moreover, the expression level of COL-1 in the OBP-group tissue was lower than that in the BSS-group and MMC-group tissue, yet no statistically significant difference was observed. The expression levels of both α-SMA and COL-1 in the OBP-group tissue were extremely low, and without variation (Fig. 5).

Fig. 5.

Fig. 5

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Western blot analysis of α-SMA and COL-1 in the bleb tissues. Western blot analysis was performed at 12-weeks postoperative in the BSS, OBP, and MMC groups. OBP (100 nM) and BSS were administered topically once before surgery and twice daily for 1 week postoperative, while MMC (0.02%) was administered as a single 100-µL subconjunctival injection at 30 min before surgery. Although the differences did not reach statistical significance, OBP treatment reduced the expression levels of α-SMA and COL-1 compared with those in the BSS- and MMC-treated eyes. Column and error bars indicate the mean ± SD (n = 3).

Safety of PMS and OBP eye drops

To investigate the safety of PMS, longitudinal slit-lamp examinations were performed. In all three groups, no leakage of aqueous humor, endophthalmitis, or cataract was observed. To elucidate if OBP eye drops caused drug toxicity, we further conducted slit-lamp examinations with fluorescein staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay analyses using sacrificed corneal tissue. Slit-lamp examination revealed no corneal epithelial damage, corneal opacity, or corneal edema in all rabbits. In addition, no corneal TUNEL-positive cells, which indicate apoptotic cells, were found in all groups (Fig. 6).

Fig. 6.

Fig. 6

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Representative images of in vivo corneal fluorescence staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of corneas sacrificed at 12 weeks postoperative. OBP (100 nM) and BSS were administered topically once before surgery and twice daily for 1 week postoperative, while MMC (0.02%) was administered as a single 100-µL subconjunctival injection at 30 min before surgery. In addition, a no-surgery control (NSC) group was included for comparison. Irrespective of the treatment group, including the NSC group, no fluorescence-stained corneal epithelial damage or TUNEL-positive apoptotic cells was found in any of the rabbits. Scale bars: 200 μm.

Discussion

The findings in this study revealed that OBP is effective in inhibiting fibrosis of the bleb tissue and maintaining lower IOP for 3 months post PMS in rabbit eyes. Although various agents have been reported to be effective in inhibiting postoperative fibrosis following GFS in a rabbit model33–38, our findings demonstrate for the first time that OBP can be a new, practically-applicable antifibrotic treatment to maintain decreased IOP and inhibit bleb scarring, thus making it an excellent alternative to MMC post PMS.

In the clinical setting, MMC is topically applied, followed by washing with saline, to inhibit postoperative fibrosis of the filtration bleb in PMS2–4,7. In addition, in cases of GFS, 5-FU is sometimes administered via subconjunctival injection to inhibit postoperative fibrosis39. We previously reported that when OBP is topically administered as eye drops, it is as effective as subconjunctival injections for preventing postoperative fibrosis23. In this current study, our findings confirmed that acetylation of histone H3 was significantly increased in the conjunctival tissue at 1-week post topical administration. Furthermore, administering OBP as eye drops also effectively prevented fibrosis of the filtration bleb for 3 months after PMS. OBP is effective even when administered via eye drops, thus providing practical advantages over MMC, which is limited to intraoperative application only. This makes it possible to easily add OBP post surgery. Therefore, in cases where there are strong postoperative fibrosis and a high risk of scarring at the filtration bleb, OBP can be used for a longer period. Moreover, if fibrosis reoccurs, the treatment can easily be resumed due to its administration as eye drops, allowing for a flexible application tailored to individual cases. 5-FU is usually administered after surgery via a subconjunctival injection, which is more invasive.

While MMC has antifibrotic effects, formation of avascular areas and thinning of the walls of the filtration bleb can also occur, which ultimately leads to leakage of aqueous humor from the bleb and an increase in the risk of infection post GFS. Fujimoto et al.32 also reported the formation of avascular areas following PMS with MMC in rabbit eyes, which is consistent with the results in this study. Although no postoperative infections were observed in either study, the risk remains. In contrast, the filtration blebs in the OBP-treated group in this study were flat, not avascular, and non-scarred, thus more closely resembling normal conjunctiva. In addition, our findings demonstrated that there were no corneal epithelial defects or toxicity to the corneal stroma in the OBP group, and indicate that OBP contributes to a lower risk of postoperative infection and lessens the risk of scarring, thus displaying its potential for safe use.

In regard to systemic safety, it has been generally reported that topical ophthalmic medications rarely result in clinically meaningful systemic accumulation because they are designed to minimize systemic absorption, with some variability depending on the drug40. As OBP was administered in this study via topical eye drops at a nanomolar concentration (i.e., 100 nM), we believe that even if one drop was absorbed systemically, the plasma levels would remain below the detectable limit. Furthermore, the findings in a Phase Ia clinical trial of OBP in patients with advanced solid tumors demonstrated that no dose-limiting toxicities were observed in the low-dose cohort (i.e., 1.0 mg/m2), and that OBP exhibited a very short plasma half-life (i.e., 0.2–0.7 h) without accumulation, thus supporting the plausibility of its safe use as an ophthalmic formulation22.

The PM is composed of styrene-b-isobutylene-b-styrene, a highly biocompatible material, and reportedly reduces the likelihood of conjunctival scarring in rabbit eyes41. However, it was reported that PMS without antifibrotic treatment resulted in the deposition of thick, disorganized collagen fibers in the filtering bleb by 28 days after surgery27. In this present study, immunohistochemical staining of the filtration bleb tissues at 12-weeks postoperative similarly detected higher levels of COL-1 expression and α-SMA-positive cells in the BSS group compared to the OBP group, with western blot analysis confirming an increase in those proteins, consequently leading to a gradual increase in IOP in the BSS group. These findings suggest the necessity of using antifibrotic agents when performing PMS.

Fujimoto et al.32 reported that even when MMC is used in conjunction with PMS on rabbit eyes, immunohistochemical staining of the filtration bleb from 2- to 12 weeks postoperative showed deposition of α-SMA and COL-1, which is consistent with our findings that the MMC group exhibited more deposition of α-SMA and COL-1 compared to the OBP group, and western blot analysis also showed substantial expression of these proteins. The remodeling caused by excessive fibrosis may have induced the recurrence of elevated IOP post surgery, as evidenced by our findings where the MMC group exhibited a rise in IOP in the later postoperative period.

It was recently reported that the combined therapy of 0.03% VPA and low-dose MMC (0.1 mg/mL) is more effective than high-dose MMC (0.4 mg/mL) in inhibiting fibrosis of the filtering bleb at 28 days after PMS26,27. Like OBP, 0.03% VPA is an HDACi that suppresses fibrosis, however, the results in those previous studies were short-term (i.e., 28 days postoperative), and the authors did not clearly elucidate the reduction of IOP post surgery, which is a very important outcome. Moreover, VPA must be administered via subconjunctival injection at every postoperative follow-up. OBP, on the other hand, can be administered via eye drops at a much lower concentration of 100 nM, and it not only suppresses fibrosis but also maintains IOP reduction for 3-months postoperative, thus demonstrating its potential as an effective and less invasive antifibrotic agent for PMS.

In our findings, while preoperative subconjunctival injection of MMC led to an increase in IOP at 12-weeks post PMS, the pre- and postoperative topical administration of OBP contributed to maintaining IOP reduction in the rabbit-model eyes, similar to the results in our previous report involving a rabbit GFS model23. In another previous report involving rabbit eyes, the findings indicated that at 12 weeks after surgery, PMS combined with 0.04% MMC maintained IOP reduction comparable to the level in the GFS model32. In that study, MMC was applied and then washed off with saline, thus possibly weakening the assessment of its efficacy compared to a subconjunctival injection of 0.02% MMC. In the clinical setting, there are sometimes cases in which IOP increases due to scarring of the filtration bleb even with the application of MMC post GFS, and we posit that the MMC group in this current study may have replicated such cases.

Limitations

It should be noted that this study had several limitations. First, the sample size in this exploratory preclinical study was small. For the IOP endpoint, a post hoc power analysis of the 12-week intergroup comparisons indicated high power at the observed effect sizes. However, post hoc power analysis depends on the observed effect sizes. With a small sample size, the precision of effect estimates decreases, so the results should be interpreted with caution. Second, steroids were not used, even though they commonly are used after PMS. Postoperative glucocorticoids are understood to act primarily upstream by suppressing the inflammatory cascade including cytokine release and leukocyte infiltration, thereby contributing to the reduction of postoperative fibrosis and scarring42. At present, and to the best of our knowledge, there have been no studies evaluating the effects of OBP in the presence of postoperative glucocorticoids. However, in our previous report we modeled an inflamed postoperative environment in vitro using transforming growth factor beta (TGF-β) 2 with or without tumor necrosis factor alpha (TNF-α) and found that OBP (i.e., 0.5–1 nM) almost completely prevented TGF-β–driven myofibroblast transition in human primary conjunctival fibroblasts, suppressing α-SMA and COL-I, -III, and -XVI23. In that study, OBP also reduced induction of the lysyl oxidase (LOX)/LOX-like (LOXL) family - key extracellular matrix cross-linking enzymes - indicating restraint of both matrix production and matrix maturation even under pro-inflammatory stimulation, and our findings demonstrated in vivo downregulation of TGF-β3, COL1A, and LOX, and reduced α-SMA and COL-1 proteins in bleb tissue. Since these mechanisms are likely complementary, we speculate that combining OBP with conventional postoperative steroid therapy may provide additive or even synergistic antifibrotic effects, which should be evaluated in future studies. Third, systemic safety was not comprehensively assessed. Although previous clinical data suggest favorable tolerability and topical ophthalmic dosing typically yields minimal systemic exposure, this current study did not include formal systemic toxicology. We observed no local ocular toxicity or serious systemic adverse events, such as sudden death; however, further systemic organ assessments and exposure profiling are warranted to support future clinical development of OBP as an ophthalmic formulation. Fourth, corneal fluorescence and TUNEL staining were only evaluated at 12-weeks postoperative, i.e., when tissue responses were expected to be stable. Early postoperative changes were not assessed, as they may largely reflect surgical invasion and acute inflammation rather than the direct effects of the administered agents, which could complicate the interpretation of drug-related toxicity. Further studies are needed to confirm both local and systemic safety following topical ocular administration.

Conclusions

The findings in this study revealed that OBP, a pluripotent epigenetic regulator, attenuates fibrosis of the filtering bleb and maintains IOP reduction for 12 weeks post PMS in rabbit eyes, and suggest that a PMS and OBP combined treatment could be an ideal GFS to maintain IOP reduction with a healthy wound-healing process. For clinical application, the long-term effect of the combination of OBP and steroid requires further investigation.

Methods

PMS rabbit model

Adult female Japanese white rabbits (2.5–3.0 kg in weight) were obtained from Shimizu Laboratory Supplies (Kyoto, Japan) and used for the PMS rabbit-model experiments performed in this study. PM devices were provided by Santen Pharmaceutical. For adaptation to the laboratory environment, all rabbits were kept in separate cages in the laboratory for 1 week prior to undergoing PMS. On the day of the operation, PMS was performed on the right eye of each rabbit under general anesthesia with intramuscular injections of a mixture of 50 mg/kg ketamine hydrochloride (Daiichi Sankyo Propharma Co., Ltd., Tokyo, Japan) and 10 mg/kg xylazine hydrochloride (Bayer Yakuhin, Ltd., Osaka, Japan), combined with a topical administration of oxybuprocaine eye drops (Santen Pharmaceutical).

PMS was performed as follows. First, a fornix-base conjunctival incision was made in the upper quadrant, either superior-temporally or superior-nasally. Approximately 3 mm from the limbus, a scleral pocket of about 1.2 mm was created using a MVR Lance 20G (MANI, Inc., Utsunomiya, Japan). From within that scleral pocket, a pathway for device insertion was created toward the anterior chamber angle using a 25 G needle (Nipro Corporation, Settsu, Japan) through which a PM was then inserted. Properly inserted, the PM protruded 2 or 3 mm into the anterior chamber without contacting the iris or cornea, with its fins positioned to fit within the scleral pocket. Finally, the Tenon’s capsule and conjunctiva were sutured with 10–0 nylon (MANI) (Fig. 7a, b). At the end of surgery, 1 drop of 1% atropine sulfate (Nitten Pharmaceutical Co., Ltd., Nagoya, Japan) and an ointment of 0.1% betamethasone sodium phosphate and 0.35% fradiomycin sulfate (Shionogi & Co., Ltd., Osaka, Japan) was applied. Each surgery was performed by a single experienced surgeon (T.I). All evaluation of subjective/qualitative grading measures, such as bleb appearance and the histology of the rabbits that underwent PMS, were carried out by persons other than the surgeon who performed the operations in a masked fashion.

Fig. 7.

Fig. 7

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Schematic diagrams illustrating the rabbit PRESERFLOTM MicroShunt Surgery (PMS) method (a, b). Briefly, a limbal conjunctival incision was first made in the upper quadrant, either superior-temporally or superior-nasally. Next, a PM was inserted into anterior chamber approximately 3 mm from the limbus. The Tenon’s capsule and conjunctiva are then fixed with a 10–0 nylon suture.

Agents and administration

A 250 μM (20 mg/ 20 ml) stock solution and 10 μM sub-stock solution of OBP was prepared by dissolving it in dimethyl sulfoxide, and it was subsequently stored at -30 °C. OBP was then dissolved at 100 nM in BSS . According to our previous reports, OBP at a concentration 10 or 100 times higher than that resulting in the in vitro suppression of α-SMA and COL-1 was administered.

The stock MMC solution (Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan) was dissolved in BSS at 0.2%, and then diluted to 0.02% at the time of use. BSS and OBP were administered as eye drops, and MMC was administered via subconjunctival injection in a well-tolerated applied regimen without serious adverse events. Rabbits undergoing PMS were classified into the following 3 treatment groups: (1) BSS group (n = 6 rabbits that received 4 eye drops of 20 μL of BSS at 30 min before surgery and twice daily for 7 days postoperative); (2) OBP group (n = 7 rabbits that received 4 eye drops of 20 μL of 100 nM OBP solution at 30 s intervals at 30 min before surgery and twice daily for 7 days postoperative); (3) MMC group (n = 6 rabbits that received a 100 μL subconjunctival injection of 0.02% MMC solution at 30 min before surgery).

Clinical evaluation of the effects in the PMS rabbit-model

To assess the safety and efficacy, the anterior segment of the eyes, bleb appearance, and IOP were evaluated prior to PMS and at 1-day and 1-, 4-, 8-, and 12 weeks postoperative. We quantified bleb morphology according to the scoring system developed by Fujimoto et al., which is based on the Moorfields Bleb Grading Scheme and was applied to the rabbit PMS model31,32. In specific, the bleb area was graded on a 5-point ordinal scale (i.e., 0 = no bleb, 1 = very small bleb, 2 = small bleb, 3 = medium bleb, and 4 = large bleb) and vascularity was graded on a 4-point ordinal scale (i.e., 1 = avascular, 2 = normal vascularity, 3 = mild vessel inflammation, and 4 = moderate or severe inflammation). The scores were obtained longitudinally at 1-day and 1-, 4-, 8-, and 12 weeks postoperative, and were independently evaluated by two ophthalmologists using blinded bleb-photograph data (n = 6, 7, and 6 for BSS, OBP, and MMC, respectively). IOP was measured in both eyes (3 times per eye) of each rabbit with a tonometer (iCare TONOVET; iCare Finland Oy, Vantaa, Finland), with the median value of the 3 measurements then calculated43.

Histological and immunohistological evaluation

At 12 weeks after PMS, each rabbit was euthanized with an overdose of pentobarbital sodium (100 mg/kg), administered intravenously through the marginal ear vein using a sterile syringe and a 27-gauge needle. Both eyes were enucleated, and the bleb tissues were sectioned. Conjunctival tissue obtained from the non-operated contralateral eye of each rabbit was used as a control. The tissues were stained with H&E stain. Immunofluorescence staining for α-SMA, a marker of myofibroblasts, using mouse anti-α-SMA antibody (Sigma-Aldrich, St. Louis, MO, USA), and COL-1 using mouse anti-COL-1 antibody (Thermo Fisher Scientific, Waltham, MA, USA), was performed. Images were obtained using a dual-sensor, charge-coupled device (CCD), microscope digital camera (DP80; Olympus Corporation, Tokyo, Japan) for the H&E staining and a confocal laser-scanning microscope (FV-1200; Olympus Corporation) for the immunofluorescence staining. Immunofluorescence images were imported into FIJI (ImageJ, version 1.54f., National Institutes of Health, Bethesda, MD, USA). The staining signals of α-SMA and COL-1 were selected using the polygon selection tool, and the images were converted to 8-bit grayscale. Mean gray values were measured, and the averages ± standard deviation (SD) were calculated from representative images for each group (n = 3, 4, and 3 for BSS, OBP, and MMC, respectively).

Western blot analysis

For the analysis of bleb tissues, each rabbit was sacrificed at 12-weeks postoperative and treated with the above-described procedures. The isolated bleb tissue was put into a 1.5-mL tube and frozen in liquid nitrogen. The frozen tissue was cut into small pieces (5 mm × 5 mm in size), and lysed with radioimmunoprecipitation assay buffer containing a protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific). The tissue was then thoroughly ground with Fisherbrand™ Pellet Pestles™ (Thermo Fisher Scientific) and sonicated at 30s intervals for 5 min and rotatory mixed for several hours. Next, the supernatant was collected by centrifugation at 10,000 × g for 20 min at 4 °C, and the protein concentration was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). The blocking and detection reaction were performed using the iBind Western System (Thermo Fisher Scientific) after sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 4–12% gel. The primary antibodies, mouse anti-histone H3 (1:1000) (MAB Institute, Nagano, Japan), mouse anti-acetyl histone H3 (Lys8) (1:200) (MAB Institute), mouse anti-α-SMA (1:500) (Sigma-Aldrich), mouse anti-COL-1 (1:500, Thermo Fisher Scientific), and mouse anti-GAPDH (1:2000) (Medical & Biological Laboratories Co., Inc., Tokyo, Japan) antibodies were used, and goat anti-mouse IgG HRP conjugate (1:1000) (Southern Biotech, Birmingham, AL, USA) antibody was used as a secondary antibody. The protein bands were made visible with the Western BLoT chemiluminescent substrate series; Quant, Hyper, or Ultra Sensitive HRP Substrate (TaKaRa Bio Inc., Shiga, Japan). Luminescence was observed using ImageQuant LAS-3000 (FUJIFILM, Tokyo, Japan), a CCD camera system. Semi-quantification of the density of each band was conducted using the Image J software program (1.54i; National Institutes of Health, Bethesda, MD, USA) with GAPDH as the reference protein.

Statistical analyses

Descriptive statistics are expressed as mean ± SD of the mean of at least 3 independent experiments. Relative intensity of Western blot analysis and the mean gray value of immunofluorescence images were analyzed by the t-test. Longitudinal changes in IOP, bleb area and vascularity were analyzed using a linear mixed-effects model with treatment (OBP, BSS, MMC) and time as fixed effects and rabbit as a random intercept. Time points were baseline, 1 day, 1, 4, 8, and 12 weeks for IOP, and 1 day, 1, 4, 8, and 12 weeks for bleb area and vascularity. For between-group comparison at each time point, we used Dunn’s multiple comparisons test. This was an exploratory preclinical study. Therefore, a priori sample-size calculations were not performed because effect sizes and variance estimates were unavailable at the design stage. For the IOP endpoint at 12-weeks postoperative, we conducted a post hoc power analysis for three pairwise comparisons (i.e., OBP vs. BSS, OBP vs. MMC, and BSS vs. MMC), estimating power from the observed mean differences, SDs, and group sizes. To account for the three comparisons, we applied a Bonferroni-adjusted correction: α = 0.0167.

The statistical analyses were conducted using the R software environment (The R Foundation for Statistical Computing, Vienna, Austria), and a p value of < 0.05 was considered statistically significant.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (1.2MB, pdf)

Acknowledgements

The authors wish to thank Prof. Junji Hamuro for the invaluable comment in this study, and Mr. John Bush for his invaluable assistance in reviewing the manuscript, and Dr. Makoto Ikeya and Dr. Souta Motoike for the excellent technical transfer. This study was supported by a Grant-in-Aid (No. 20K18352) obtained from the Japanese Society of the Promotion of Science.

Author contributions

Ueno M designed the research. Koga Y, Hiramoto N, Yamamoto Y, Mukai A, Murata N and Ikushima T conducted the experiments. Koga Y, Hiramoto N, Yoshii K, and Ueno M performed data analysis and interpretation. Urata Y provided materials. Koga Y, Hiramoto N, Yoshii K, and Ueno M wrote the manuscript. Mieno H and Sotozono C proofed and revised the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by a Grant-in-Aid (No. 20K18352) obtained from the Japanese Society of the Promotion of Science.

Data availability

All data generated or analyzed during this study are included in this published article (and in its Supplementary Information files).

Declarations

Competing interests

Y.U. is the President and CEO of Oncolys BioPharma, Inc., the manufacturer of OBP. M.U. received research support from Oncolys BioPharma, Inc. and from Santen Pharmaceutical Co., Ltd. The other authors have no real or potential conflicts of interest to declare.

Ethical approval

All procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the study protocols were approved by the Animal Use Committee of Kyoto Prefectural University of Medicine, Kyoto, Japan (approval no. B2021-105). The study is reported in accordance with the ARRIVE guidelines.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

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Supplementary Materials

Supplementary Material 1 (1.2MB, pdf)

Data Availability Statement

All data generated or analyzed during this study are included in this published article (and in its Supplementary Information files).

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