FormalPara Key Summary Points

Why carry out the study?

The ripasudil–brimonidine fixed-dose combination (RBFC; K-232) is the first combination topical therapy containing ripasudil (a Rho kinase inhibitor) and brimonidine (an α2-adrenoceptor agonist), with demonstrated ability to lower intraocular pressure (IOP) among various other pharmacologic effects.

This single-center, prospective, randomized, open-label, blinded endpoint, crossover study evaluated the pharmacologic effects of a ripasudil–brimonidine fixed-dose combination on IOP, conjunctival hyperemia, and corneal endothelial morphology in healthy volunteers compared with ripasudil or brimonidine alone.

What was learned from the study?

RBFC significantly reduced IOP from baseline (reductions were greater than with ripasudil and brimonidine alone); RBFC and ripasudil were associated with mild conjunctival hyperemia (scores were lower with RBFC than with ripasudil), and transient morphologic changes in corneal endothelial cells were also observed.

RBFC had a stronger efficacy and similar safety profile compared with the single agents; a combination of each agent’s pharmacologic profile was observed in that of RBFC.

Introduction

Glaucoma is characterized by functional and/or structural abnormalities in the optic nerve associated with intraocular pressure (IOP)-related changes [1], and is the leading cause of irreversible blindness worldwide [1, 2].

On the basis of randomized clinical trial data [3,4,5,6,7,8,9,10,11], the only treatment approach proven to preserve visual function and slow the rate of visual deterioration is the lowering of IOP [12,13,14]. International guidelines recommend initiating medical treatment with monotherapy, with the addition of a second drug considered when the first choice of monotherapy is well tolerated and lowers the IOP but the target IOP is not reached [12,13,14]. However, as multidrug regimens for the treatment of glaucoma pose several important clinical problems, such as poor adherence and inconvenience, fixed-dose combination therapy, when available, is preferable to instillation of two separate agents [12,13,14]. Indeed, approximately 40% of Japanese patients with newly diagnosed glaucoma discontinue topical therapy within the first year of treatment [15], with the medication regimen being one of the significant barriers to treatment compliance [16]. Further, real-world data indicate that progression of glaucoma is significantly reduced when treatment adherence is increased [17].

The ripasudil–brimonidine fixed-dose combination (RBFC; development code K-232) is the first combination topical therapy containing ripasudil hydrochloride hydrate (ripasudil 0.4%), a Rho-associated coiled-coil containing protein kinase (Rho kinase; ROCK) inhibitor, and brimonidine tartrate 0.1% (brimonidine), an α2-adrenoceptor agonist. The efficacy and safety of RBFC was previously demonstrated in two multicenter, randomized, phase 3 clinical trials, in which RBFC significantly reduced IOP in patients with primary open-angle glaucoma or ocular hypertension and IOP levels of at least 18 mmHg [18]. Ripasudil reduces IOP by increasing aqueous humor outflow via the conventional outflow pathway through modulation of the trabecular meshwork cell cytoskeleton, production of extracellular matrix, and Schlemm’s canal endothelial cell permeability [19,20,21,22]. Brimonidine is a potent agonist of α2-adrenergic receptors, which reduces IOP by suppressing aqueous humor production and promoting uveoscleral outflow [23, 24]. The pharmacologic actions of ROCK inhibitors include modulating the motility, contractility, adhesion, and shape of most cell types, which can lead to smooth muscle relaxation and morphological changes in corneal endothelial cells in addition to IOP reduction [19, 25, 26]. The actions of brimonidine include ciliary body vasoconstriction [27] and pupillary miosis [24].

In the current study, we evaluated the pharmacologic effects of the RBFC on IOP, conjunctival hyperemia, corneal endothelial cell morphology, pupil diameter, and pharmacokinetics compared with ripasudil or brimonidine alone in healthy volunteers.

Methods

Study Design

This was a single-center, prospective, randomized, open-label, blinded endpoint (PROBE) study, with a 3 × 3 crossover design.

The study was conducted at the Medical Corporation Heishinkai OPHAC Hospital in compliance with the principles of the Helsinki Declaration of 1964, and its later amendments and Good Clinical Practice guidelines, and the protocol was prospectively approved by the institutional review board of the participating site. Written informed consent was obtained from all subjects prior to study entry. This study is registered with the Japan Registry of Clinical Trials (Registration No. jRCT2080225220).

Subjects

The study enrolled healthy men, aged 20–35 years, who had an IOP of at least 15.0 mmHg in either eye (measured by noncontact tonometry) and a body mass index (BMI) of 17.6–26.4 kg/m2 at screening. Individuals were excluded if they had ocular diseases (except myopia, hyperopia, or astigmatism), best-corrected visual acuity of less than 20/25, Shaffer classification grade 0–2, difficulty in measuring IOP using a Goldmann applanation tonometer, corneal endothelial cell density of less than 2000 cells/mm2, cornea guttata, or corneal thickness of 620 μm or more in either eye. In addition, the use of drugs (including over-the-counter drugs), supplements, ophthalmic laser surgery, ophthalmic surgery, other nasolacrimal duct surgical procedures, and the wearing of contact lens were prohibited during the study period. Full exclusion criteria are provided in Table S1 in the Supplementary Material.

Randomization and Interventions

Eligible subjects who provided written informed consent were enrolled in administration phase 1 (Supplementary Material Fig. S1). Subjects underwent three consecutive administration phases (8 days of dosing followed by a drug-free period of at least 5 days) with RBFC, ripasudil, or brimonidine. An individual independent from the K-232 Clinical Study Group (see Appendix in the Supplementary Material) and sponsor used the permutation block method to randomly assign subjects (1:1:1) to one of three groups based on treatment order: group A received RBFC → ripasudil → brimonidine; group B received ripasudil → brimonidine → RBFC; and group C received brimonidine → RBFC → ripasudil (Supplementary Material Fig. S1). Treatment was masked throughout the study period for the investigator, sub-investigator, and sponsor. During each administration phase, one drop of the study drug was instilled into both eyes twice daily, in the morning (09:00) and evening (21:00) on each dosing day. Subjects were admitted to the hospital on the day prior to the first dose of study drug for each administration phase (day − 1) and discharged on day 9 after 8 days of twice-daily dosing (on day 8, morning dosing only) from day 1 (Supplementary Material Fig. S1).

Endpoints

The change from baseline in IOP (ΔIOP) with the RBFC compared with ripasudil or brimonidine was assessed.

Safety was assessed by the number and percentage of adverse events (AEs) and adverse drug reactions (ADRs), as well as the change in ophthalmologic parameters concerning the severity of conjunctival hyperemia, corneal endothelial cell morphology, and pupil diameter.

The pharmacokinetic properties of the RBFC were assessed after instillation by measuring the plasma concentrations, peak plasma concentrations (Cmax), area under the plasma concentration–time curve from time 0 to last observation (AUC0–τ), time to Cmax (tmax), and elimination half-life (t1/2) of unchanged ripasudil, its major metabolite (M1), and unchanged brimonidine.

Outcome Measures

IOP was measured in both eyes with a Goldmann applanation tonometer before and 1, 2, 4, 6, 9, and 12 h after the morning instillation on days 1 and 8.

The incidence of AEs and ADRs was collected for safety assessment.

The cornea, palpebral and bulbar conjunctivae, anterior chamber, lens, and iris were examined before and 15 min, 1, 2, 4, 6, 9, and 12 h after the morning instillation on days 1 and 8 using a slit lamp microscope. The palpebral and bulbar conjunctivae were evaluated for hyperemia using four grades: “–” = no findings or minimal findings but physiologic changes; “+” = several vasodilatations; “2+” = multiple vasodilatations or limbal vasodilatation; “3+” = vasodilatation in the whole conjunctiva (Supplementary Material Fig. S2) [28,29,30,31].

Assessments of corneal endothelial cell morphology and density and corneal thickness were performed before and 1, 2, 4, 6, 9, and 12 h after the morning instillation on days 1 and 8. Corneal endothelial cell morphology and density were examined by a noncontact specular microscope. Automated image capture and corneal cell count using the software associated with the device was performed. Corneal endothelial cell morphology was evaluated using noncontact specular microscopy and classified into one of four grades or as “undeterminable”: grade 0 = normal endothelial cell morphology (cells appear as white polygons with black borders) with no findings similar to guttae (corneal guttae findings are black irregularly shaped cells with white borders); grade 1 = many endothelial cells have clear borders, but at least 10% are partially blackened out with findings similar to guttae; grade 2 = endothelial cell borders are indistinct, with many having findings similar to guttae; grade 3 = endothelial cell borders cannot be identified; undeterminable = not applicable to any of grades 0–3 (Supplementary Material Fig. S3). Central corneal thickness was measured using Scheimpflug tomography.

Pupil diameter was measured using a Haab’s pupilometer before and 2, 4, 6, 9, and 12 h after morning instillation on days 1 and 8.

Visual acuity, fundoscopy, physiology (height, weight, BMI, blood pressure, heart rate, and temperature), and laboratory tests (hematologic, biochemical, and urinalysis) were also performed.

For the pharmacokinetic analysis, blood samples were collected before the morning instillation on days 1, 6, and 7. On day 8, blood samples were collected before the morning instillation and 5, 15, and 30 min, and 1, 1.5, 2, 3, 4, 5, 6, 9, and 12 h after the morning instillation.

Statistical Analysis

The sample size of 18 subjects (six in each group) was determined as a reasonable number of subjects to provide adequate pharmacokinetic information of the RBFC, ripasudil, and brimonidine in each administration phase. No hypothesis testing was performed for power calculation as this was not a confirmatory study.

Descriptive statistics were used to summarize the study outcomes. Continuous variables were presented as the number of subjects (percentage), mean ± standard deviation (SD), and median unless otherwise noted.

For the ΔIOP at each time point, the least squares (LS) mean and the LS mean ± standard error (SE) difference between the RBFC and ripasudil or brimonidine were determined using a marginal model that included the pre-dose IOP, preceding treatment, current study drug, and administration phase as fixed effects and had a compound symmetry (CS) covariance structure for subjects. The inferences for degrees of freedom were performed on the basis of the Kenward–Roger approach [32]. Change from baseline in conjunctival hyperemia grade in each administration phase (post hoc analysis) was evaluated using the same marginal model as for ΔIOP, with the preceding treatment, current study drug, and administration phase included as the fixed effects. Change from baseline in pupil diameter in each administration phase was evaluated by Wilcoxon signed rank test.

The two-sided significance level was 0.05 and the two-sided confidence interval (CI) of the safety endpoints was 95%. All statistical analyses were conducted at Kowa Company, Ltd. using SAS, version 9.4 (SAS Institute, Cary, NC, USA).

Results

Subject Disposition and Characteristics

The study was conducted from June 13 to September 20, 2020; consent was obtained from 50 healthy male volunteers, and 18 subjects were assigned in total, six to each group (A, B, or C). All assigned subjects received at least one dose of the study drug, and 17 completed the study (Fig. 1). The subject demographics and characteristics are summarized in Table 1. At enrollment, subjects had a mean ± SD age of 26.2 ± 3.3 years and IOP of 14.1 ± 2.1 mmHg.

Fig. 1
figure 1

Disposition of subjects. aRipasudil–brimonidine fixed-dose combination → ripasudil → brimonidine. bRipasudil → brimonidine → ripasudil–brimonidine fixed-dose combination. cBrimonidine → ripasudil–brimonidine fixed-dose combination → ripasudil. dOne subject in group B had a fever on day 2 in administration phase 1 and a COVID-19 infection could not be ruled out; treatment was discontinued at the discretion of the investigator (ripasudil–brimonidine fixed-dose combination and brimonidine were not administered). COVID-19 coronavirus disease 2019

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Table 1 Subject demographics and characteristics
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Change in IOP

With the RBFC, mean ± SD IOP decreased from 12.7 ± 2.4 mmHg at baseline to 9.1 ± 2.8 mmHg and 9.0 ± 2.0 mmHg at 1 h post-instillation on days 1 and 8, respectively, after which the IOP-lowering effect of the RBFC diminished over time (Fig. 2). A significantly greater ΔIOP was observed with RBFC versus ripasudil at 1 h post-instillation on day 1 (LS mean ± SE difference, − 1.0 ± 0.3 [P = 0.006]). RBFC also significantly reduced IOP compared with brimonidine at 1, 2, and 6 h post-instillation on day 1 (LS mean ± SE difference, − 2.0 ± 0.3 [P < 0.001], − 1.4 ± 0.4 [P = 0.006], and − 1.2 ± 0.5 [P = 0.024], respectively) and at 1 and 2 h post-instillation on day 8 (LS mean ± SE difference, − 1.6 ± 0.5 [P = 0.005] and − 1.2 ± 0.6 [P = 0.046], respectively; Fig. 2).

Fig. 2
figure 2

Change in intraocular pressure over time on days 1 and 8. The difference in the adjusted mean between ripasudil–brimonidine fixed-dose combination and ripasudil or brimonidine at each time point was determined using a marginal model. All values are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs. baseline in each administration phase; ††P < 0.01 vs. ripasudil; ‡P < 0.05, ‡‡P < 0.01, ‡‡‡P < 0.001 vs. brimonidine. n = 18 at day 1 for ripasudil; n = 17 at days 1 and 8 for ripasudil–brimonidine fixed-dose combination and brimonidine and at day 8 for ripasudil. IOP intraocular pressure, n number of patients, SD standard deviation

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Safety

AEs were observed in all 17/17 subjects (100.0%) during RBFC treatment, 18/18 (100.0%) with ripasudil, and 12/17 (70.6%) with brimonidine. ADRs were reported in 17/17 subjects (100.0%) with RBFC, 18/18 (100.0%) with ripasudil, and 10/17 (58.8%) with brimonidine.

Conjunctival hyperemia was the most common AE, occurring in 17/17 subjects (100.0%) with RBFC, 18/18 (100.0%) with ripasudil, and 10/17 (58.8%) with brimonidine, and the most common ADR, occurring in 100.0% of subjects with RBFC, 100.0% with ripasudil, and 8/17 (47.1%) with brimonidine (Supplementary Material Table S2). All cases of conjunctival hyperemia were mild and recovered without treatment, and most cases occurred transiently post-instillation, with the exception of one subject with ripasudil and two with brimonidine.

With the exception of conjunctival hyperemia and morphologic changes in corneal endothelial cells, no other clinically meaningful changes were observed in ophthalmic, physiological, or laboratory tests.

Conjunctival Hyperemia

Both palpebral and bulbar conjunctival hyperemia showed a transient increase in severity with RBFC or ripasudil, peaking at 15 min post-instillation and then decreasing over time. By visual inspection, palpebral conjunctival hyperemia was less severe and had a lower incidence with RBFC than with ripasudil at most time points (Fig. 3a), and bulbar conjunctival hyperemia was less severe and had lower incidence with RBFC than with ripasudil at 1 h post-instillation on day 1 (Fig. 3b).

Fig. 3
figure 3

Hyperemia scores for a palpebral and b bulbar conjunctival hyperemia after the first and the last instillations. The number of cases of conjunctival hyperemia is shown for each evaluation criteria (–, +, 2+, 3+; Supplementary Material Fig. S2). n = 18 at day 1 for ripasudil; n = 17 at days 1 and 8 for ripasudil–brimonidine fixed-dose combination and brimonidine and at day 8 for ripasudil. h hour, L left eye, n number of patients, R right eye

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A post hoc analysis examined the differences in the effects on hyperemia between RBFC and ripasudil. This analysis found significant differences in the mean grade scores of both eyes for palpebral and bulbar conjunctival hyperemia between RBFC and ripasudil at some time points. The post hoc analysis indicated a carryover effect for palpebral conjunctival hyperemia; therefore, this endpoint was evaluated by t test to compare RBFC with ripasudil or brimonidine in administration phase 1. For palpebral conjunctival hyperemia, mean ± SD grade scores were significantly lower with RBFC versus ripasudil at 15 min (0.7 ± 0.8 vs. 1.4 ± 0.6, respectively) and 1 h (0.3 ± 0.4 vs. 0.9 ± 0.7, respectively) post-instillation on day 1 (LS mean ± SE difference, − 0.9 ± 0.4 [P = 0.028] and −  0.8 ± 0.3 [P = 0.011], respectively for group comparisons; Fig. 4a). For bulbar conjunctival hyperemia, mean ± SD grade scores were significantly lower with RBFC versus ripasudil at 1 h (0.4 ± 0.5 vs. 0.9 ± 0.5, respectively) post-instillation on day 1 (LS mean ± SE difference, − 0.6 ± 0.1 [P < 0.001]; Fig. 4b). Mean grade scores for palpebral and bulbar conjunctival hyperemia in both eyes were lower with brimonidine alone than with RBFC (Fig. 4a, b).

Fig. 4
figure 4

Change in clinical grade score for a palpebral conjunctival hyperemia and b bulbar conjunctival hyperemia. The change in score from baseline in each administration phase using quantified criteria for evaluation of conjunctival hyperemia (– = 0, + = 1, 2+ = 2, 3+ = 3; Supplementary Material Fig. S2) and were analyzed using a marginal model. Clinical grade scores for palpebral conjunctival hyperemia were evaluated in administration phase 1. All values are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs. baseline in each administration phase; †P < 0.05, †††P < 0.001 vs. ripasudil; ‡P < 0.05, ‡‡‡P < 0.001 vs. brimonidine. n = 18 at day 1 for ripasudil; n = 17 at days 1 and 8 for ripasudil–brimonidine fixed-dose combination and brimonidine and at day 8 for ripasudil. SD standard deviation

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Corneal Endothelial Cells

Changes in corneal endothelial cell morphology were observed after instillation of RBFC or ripasudil, which resolved to grade 0 in all subjects at 6 h post-instillation (Fig. 5). Some subjects developed grade 1 or 2 morphologic changes after instillation with RBFC or ripasudil, but none developed grade 3 changes. No morphologic changes were observed with brimonidine.

Fig. 5
figure 5

Morphologic changes in corneal endothelial cells after first and last instillations of study drug (grade evaluation). The evaluation criteria for corneal endothelial cell morphology were grade 0–3 and undeterminable (Supplementary Material Fig. S3), and the number of subjects in each grade are shown over time. h hour, L left eye, R right eye

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The corneal endothelial cell density, which was automatically measured by the noncontact specular microscope, appeared to be reduced in cells with morphologic changes; however, cell density returned to pre-instillation values after the morphologic changes resolved. There were no clinically significant changes in corneal thickness and no remarkable findings on corneal examination.

Pupil Diameter

Mean pupil diameter decreased significantly from baseline after instillation with brimonidine at 2, 4, and 12 h post-instillation on day 1 and at 2, 4, and 6 h post-instillation on day 8. Similar reductions in mean pupil diameter were only observed with ripasudil at the first point post-instillation on day 1. No significant changes were observed with RBFC (Fig. 6). There were no clinically relevant changes with any of the three treatments, and no subjects developed abnormal light responses.

Fig. 6
figure 6

Change in pupil diameter after the first and the last instillations in the a left eye and b right eye. All values are presented as mean ± SD. *P < 0.05, **P < 0.01 vs. baseline in each administration phase (Wilcoxon signed-rank test). n = 18 at day 1 for ripasudil; n = 17 at days 1 and 8 for the ripasudil–brimonidine fixed-dose combination and brimonidine and at day 8 for ripasudil. SD standard deviation

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Pharmacokinetics

After instillation of RBFC, the Cmax points of unchanged ripasudil and brimonidine (0.3461 ng/mL and 33.751 pg/mL, respectively) were reached at 0.083 h, and that of the ripasudil metabolite M1 (0.7379 ng/mL) was reached at 0.500 h. Plasma concentrations then rapidly decreased, with the t1/2 of unchanged ripasudil, M1, and unchanged brimonidine being 0.325, 3.096, and 1.903 h, respectively. The AUC0–τ of unchanged ripasudil, M1, and unchanged brimonidine was 0.1064 ng·h/mL, 2.4998 ng·h/mL, and 59.983 pg·h/mL, respectively (Supplementary Material Table S3).

Discussion

In this randomized, 3 × 3 crossover study of healthy male volunteers, the RBFC was associated with a significant decrease in mean IOP from baseline at 1 h post-instillation on days 1 and 8, decreasing from 12.7 to 9.1 mmHg and 9.0 mmHg, respectively, which is close to the episcleral venous pressure (8 mmHg) [33]. These reductions in IOP with RBFC were significantly greater than those observed with each individual agent (ripasudil or brimonidine). Previously, two phase 3 randomized clinical trials in patients with primary open-angle glaucoma or ocular hypertension demonstrated that RBFC had a greater IOP-lowering effect than ripasudil or brimonidine alone over 8 weeks of treatment [18]. These results suggest that the combination of ripasudil and brimonidine has an additive effect due to the three mechanisms of action, i.e., the enhancement of aqueous outflow via the conventional outflow [19,20,21,22] and uveoscleral outflow pathways, and the inhibition of aqueous humor production [23, 24].

Studies in healthy adults and patients with glaucoma have reported transient conjunctival hyperemia with ripasudil therapy that peaks 5–15 min after instillation [28,29,30, 34,35,36]. Given that one of the main actions of ROCK inhibitors is vascular smooth muscle relaxation [26], it has been suggested that the conjunctival hyperemia associated with ripasudil treatment is due to this pharmacologic effect, although an allergic response to ripasudil in some patients may cause conjunctival hyperemia. Topical brimonidine, on the other hand, reportedly causes vasoconstriction in the ciliary body [27], thus having the opposite effect on blood vessels to that of ripasudil, which may explain the slightly reduced conjunctival hyperemia observed with RBFC.

In a previous study in healthy subjects, Lee and colleagues found that the mean score for conjunctival hyperemia was significantly lower with combined instillation of ripasudil and brimonidine than with ripasudil alone at 2 h after instillation [37]. In this study, subjects received a single instillation of four patterns of ripasudil and brimonidine (no instillation, ripasudil alone, brimonidine alone, and a combination of ripasudil and brimonidine), with a gap of at least 1 week between instillations [37]. This suggests that, when used in combination with ripasudil, brimonidine suppresses ripasudil-associated conjunctival hyperemia. Similarly, our study found that both palpebral and bulbar conjunctival hyperemia transiently increased in severity with the RBFC and ripasudil, peaking at 15 min post-instillation and thereafter decreasing over time. The incidence, severity, and duration of palpebral and bulbar conjunctival hyperemia appeared to be lower with the RBFC than with ripasudil, similar to that observed in the previous study [37]. It is possible that the vasoconstrictive effects of brimonidine prevent the conjunctival hyperemia caused by the vasodilatory effects of ripasudil.

Ripasudil has been reported to cause transient changes in corneal endothelial cell morphology within a few hours after instillation in healthy volunteers [25] and in patients with glaucoma [38] that recovered 6 h after instillation. Our study also observed for the first time transient morphologic changes in corneal endothelial cells after RBFC instillation. Following instillation of RBFC, corneal endothelial cell density also appeared to be reduced for several hours, but returned to baseline levels over time. Because of the morphological impact of topical ROCK inhibitor therapy on cell boundaries of corneal endothelial cells, it is difficult to accurately evaluate cell density by a noncontact specular microscope with automated analysis. Therefore, it is important to be aware of the elapsed time post-instillation when evaluating corneal endothelial cell density.

Brimonidine has been shown to have a moderate miotic effect in healthy volunteers due to sympathetic α2-adrenergic receptor stimulation [39, 40]. On the other hand, ROCK inhibitors reportedly have a mydriatic effect in rabbits [41] and no effect on pupil diameter in healthy volunteers [42]. In the study by Lee et al., the pupil diameter significantly decreased after combined instillation of ripasudil and brimonidine or instillation of brimonidine alone, whereas no change in pupil diameter was found with ripasudil monotherapy [37]. In our study, mean pupil diameter was significantly decreased after brimonidine instillation at some time points; however, no significant changes in pupil diameter were observed following RBFC instillation. Hence, the miotic effect of RBFC was considered to be small.

Limitations

The main limitation of this study was its short duration (8 days) and that it was conducted in a small number of healthy adults. Therefore, a long-term study in a large population of patients with glaucoma or ocular hypertension is necessary to confirm the findings of this study. Moreover, this study was not placebo controlled and the comparisons were made between active drug groups.

Conclusion

The RBFC provided significantly greater reductions in IOP with 8 days of short-term instillation compared with each agent as monotherapy in healthy adult volunteers. Although RBFC instillation was associated with transient conjunctival hyperemia, which peaked at 15 min, the incidence and severity of hyperemia was lower than after instillation of ripasudil alone. Transient morphologic changes in corneal endothelial cell were observed for up to several hours after instillation, after which the morphology returned to normal. RBFC had no significant effect on pupil diameter, and the safety profile of RBFC was similar to that of the individual agents. Pharmacokinetic analyses demonstrated rapid elimination of unchanged ripasudil, ripasudil metabolite M1, and unchanged brimonidine from the plasma following topical instillation of RBFC.