Effect of a fixed combination of ripasudil and brimonidine on aqueous humor dynamics in mice

Ripasudil–brimonidine fixed-dose combination (K-232) simultaneously targets three different intraocular pressure (IOP) lowering mechanisms, increasing trabecular meshwork outflow and uveoscleral outflow, and reducing aqueous humor production Vascularly, ripasudil induces transient vasodilation, brimonidine transient vasoconstriction. Investigating effects on IOP, aqueous dynamics, and EVP in mice eyes by microneedle and constant-pressure perfusion methods, and on cytoskeletal and fibrotic proteins changes in HTM cells by a gel contraction assay and immunocytochemistry. Ripasudil, K-232, and brimonidine droplets significantly reduced IOP at 30 min, with K-232 sustaining the effect at 60 min. For EVP, only K-232 exhibited reduced EVP until 60 min after instillation. In vitro, ripasudil inhibited gel contractility and TGFβ2-induced fibrotic changes, whereas brimonidine did not. K-232 significantly lowered IOPs in mice by combining the effects of ripasudil and brimonidine. Brimonidine alone also showed IOP reductions with enhanced outflow facility, and the drug did not interfere with the effects of ripasudil on the trabecular meshwork outflow; K-232 and ripasudil alone both significantly lowered the EVP and enhanced outflow facility, demonstrating that K-232 efficiently reduces IOPs.


Outflow facility measurements
To elucidate the IOP-lowering mechanisms, we measured outflow facilities at 30 and 60 min after drug treatment, corresponding to times when we had observed significant differences between controls and the K-232 group.The values are shown in Fig. 2.There were significant differences between controls and both Rip and

Effects on TGFβ2-induced cytoskeletal and fibrotic changes in HTM cells
We investigated cytoskeletal and fibrotic changes in HTM cells via immunocytochemistry.TGFβ2 treatment increased the expression of fibrotic proteins, such as α-smooth muscle actin (αSMA), and induced cytoskeletal changes characterized by actin bundles with F-actin staining.The TGFβ2-induced cytoskeletal changes were mitigated by treatment with 10 nM Rip.However, treatment with 100 µM Bri did not affect the TGFβ2-induced  www.nature.com/scientificreports/changes.We found similar levels of suppression of cytoskeletal changes to those obtained with ripasudil after Rip/Bri treatments (Fig. 5).In addition, we assessed cell elongation by staining F-actin and analyzing images IN Cell Analyzer 2200 (GE Healthcare).We found that TGFβ2 treatment significantly reduced cell elongation compared to controls.Moreover, the cell elongation reduction by TGFβ2 was significantly suppressed by ripasudil or Rip/Bri treatment but not Bri alone (Fig. 6).By contrast, according to immunostaining experiments, TGFβ2 treatment increased the expression of the fibrotic protein αSMA, but Rip or Rip/Bri treatments inhibited this increase (Fig. 7).Moreover, our Western blot results confirmed that the TGFβ2-induced αSMA expression was suppressed by Rip or Rip/Bri treatments (Fig. 8).

Discussion
We evaluated the IOP-lowering efficacy of a ripasudil-brimonidine fixed-dose combination, and elucidated its IOP-lowering mechanisms.We confirmed that the fixed combination effectively reduces IOPs.In addition, there was significant EVP reduction as well as enhanced outflow facility after K-232 and ripasudil treatments.To the best of our knowledge, this is the first report of an estimated evaluation of EVP measurement in the mouse eye; our results are in accordance with the established mechanisms of ROCK inhibitors.Brimonidine did not alter the effects of ripasudil on the trabecular meshwork outflow pathway when applied simultaneously (both in vivo and in vitro).
As shown in Fig. 1, all tested drugs significantly reduced the IOP 30 min after administration, while ripasudil and K-232 significantly maintained the reduction at 60 min.These results suggest that ROCK inhibitors provide longer sustained IOP-lowering effects than brimonidine after a single dose.Therefore, the fixed dose combination may also offer a long effect.
We evaluated outflow facility and showed that K-232 increased outflow facility significantly after instillation as well as ripasudil did (Fig. 2).This was expected because ROCK inhibitor reduces IOP by enhancing outflow facility 17 .However, 60 min after K-232 and ripasudil administration, when we observed a significant reduction in IOP, the increase in outflow facility was not statistically significant, despite showing an upward trend.This relatively short response, which aligns well with published reports, can be attributed to the small size and thin sclera of mouse eyes, along with physiological differences compared to human eyes.If there are no species differences in the metabolism or binding rate of the drug used and normal mice are used in the experiment, it is known that the turnover rate was about two times higher in mice than in humans [27][28][29] or IOP reduction responses in mice were fairly quickly 30,31 .www.nature.com/scientificreports/ We also found that all products reduced EVPs in the mouse eye (Fig. 3).As far as we know, these data are the first to show that Rho inhibition alone leads to reduced EVPs in mouse eyes.Netarsudil (AR13324), a ROCK inhibitor with norepinephrine transport inhibitory activity, has been reported to lower EVPs in rabbits 32 , and it induces dilation of episcleral vessels in enucleated human eyes, but these effects have been suggested to be mediated mostly by the norepinephrine transport inhibitory activity that causes vasoconstriction 32,33 .The precise mechanisms regulating episcleral circulation remain unclear 34,35 because the interplay between the blood flow and vascular resistance in the episcleral supply arterioles, various arteriovenous anastomoses (AVAs), and muscular veins [36][37][38][39][40][41] is complex.Pharmacologically induced vasodilation and vasoconstriction of the episcleral vasculature can alter the blood flow through AVAs, affecting the EVP, the flow of aqueous into the episcleral veins, and the IOP 40 .Overall vasodilation has been suggested to engorge the AVAs, fill the venules with arterial blood, and increase the EVP, resulting in outflow resistance and IOP elevation 40,42 .By contrast, vasoconstriction has been suggested to reduce the venous perfusion volume and decrease the episcleral venous blood flow, two actions that would enhance the flow of aqueous humor in the veins.
ROCK inhibitors act on the vascular smooth muscle generating distinct vasorelaxant effects: conjunctival hyperemia is often induced by topical ripasudil instillation, peaks at approximately 5-15 min after administration, and generally resolves within 2 h 43 .Although the general vasodilation of the episcleral vasculature (which includes the AVAs) has been suggested to cause increases in both the EVP and IOP, as mentioned above, AVA closure, which reduces blood flow from the arterioles to the venules, may reduce the EVP and IOP 21,40,42 .We hypothesized that a ROCK inhibitor would produce selective vasodilation of the episcleral veins only, without opening the AVAs, and that ROCK inhibitor-induced vasodilation of the episcleral vasculature might reduce the resistance to aqueous outflow and lower the IOP.A study in which the dilation of the sclero-conjunctival vasculature was evaluated using anterior segment-optical coherence tomography angiography (AS-OCTA) supports this hypothesis.In that study, vasodilation associated with ripasudil treatment was primarily caused by selective vasodilation of deep episcleral veins 44 .In addition, the changes in vessel density detected by AS-OCTA were significantly associated with IOP reductions.Moreover, in other studies, topical ripasudil treatment for 1-3 months resulted in dilation of vessels in the scleral vascular plexus 45 , suggesting a possible increase in aqueous-venous drain into the episcleral plexus.In a clinical study, the use of ripasudil in patients with OAG after circumferential incision of the Schlemm's canal by a 360° suture trabeculotomy ab interno produced significant additional IOP reductions, supporting the idea that ripasudil produces distal outflow 46 .The mechanisms of the episcleral vasculature and EVP effects of ROCK inhibitors resulting in IOP reductions remain unclear.Further studies are needed to clarify the association between episcleral vasculature dilation and EVP/IOP.
Brimonidine is a known vasoconstrictor, and it has been shown to decrease the EVP in rabbits 47 ; our results in mice support that finding.In addition to the EVP reduction 30 min after drug administration, it resulted in a slightly increased trend of enhanced outflow facility (Figs. 2 and 3).Its IOP-reduction mechanisms during longterm treatments are due to the suppression of aqueous humor production or enhancement of uveoscleral outflow; fluorophotometry and tomography studies in human eyes have reported that brimonidine does not affect the aqueous flow, outflow facility, or EVP 23,24 .Thus, different results have been obtained in rabbits and humans with respect to blood flow.According to previous reports, α2 receptors have been classified into subtypes A-C 48,49 .These subtypes have been shown to have different effects on blood flow, with subtype 2A decreasing blood flow and subtype 2B increasing blood flow 50 .Subtypes B and C have been shown to be predominant in humans and all three are present in the rabbit 51 , but there are no reports on mice.Taken together, we speculate that the differences in the effects of brimonidine on ciliary blood flow in humans, rabbits, and mice may be related to the differences in the distribution of α2 receptor subtypes in the ciliary vasculature.
In addition, regarding the effects of brimonidine on aqueous outflow and IOP, an acute effect on aqueous dynamics has been reported in human eyes after a single topical drop.The effect is due to brimonidine stimulating α2-receptors within blood vessels of the uvea or ciliary body, causing vasoconstriction, decreases in ciliary blood flow, and decreases in aqueous production 52 .However, it is also reported that even though vasoconstriction may persist overtime, the aqueous flow would be attenuated and return toward its previous value due to the fact that receptors responsible for vasoconstriction may be highly sensitive to pharmacologic stimulation acutely but may lose some of this sensitivity during long-term continued treatment 53 .The suggested mechanisms for the EVP reductions we observed after ripasudil or brimonidine treatments are controversial; therefore, further investigation is needed for clarification.Notably, K-232 treatment led to more sustained EVP reductions than ripasudil or brimonidine treatment alone.In addition, in experiments using HTM cells, we confirmed that brimonidine did not interfere with the trabecular effects of ripasudil.Thus, we speculate that neither brimonidine nor ripasudil hinders the trabecular aqueous outflow or EVP reduction mechanisms of each other.
Our study had some limitations.First, we measured IOPs, outflow facility, and EVPs using a mouse model but did not further investigate aqueous humor production or uveoscleral outflow.Mouse eyes are so small that measuring aqueous humor production or evaluating its detailed outflow is difficult.Future studies should examine the details of aqueous humor production and other outflow pathways.Second, the mechanisms by which ripasudil and K-232 lower EVPs remain unknown.In addition, the results of the present study suggested that brimonidine also may enhanced outflow facility.Although there is a concern that the outflow pathway after TM may be different from normal due to the slightly higher intraocular pressure used in our two-level constant pressure method, there have been several previous studies using similar methods [54][55][56] and studies examining the correlation between IOP values and outflow facility measurements have shown that linearity is maintained within the range of IOP values used in this study 57 .So far, several methods have been reported for the measurement of outflow facility in the past, but there are several factors that can affect the results of each method 58,59 and no uniform method has been established.Therefore, in the present study, we investigated the outflow facility using the method stated, but improving the accuracy of measurement of outflow facility (especially measurement using mice) remains an important issue to be resolved in the future.Third, the mechanisms by which ripasudil and K-232 lower EVPs remain unknown.This is a subject for a future study.Finally, we studied the effects of K-232 on the IOP, outflow facility, and EVP in normal mice using single drops of drug but did not study the effects of K-232 on actual patients with glaucoma.We plan to investigate this in the future.Finally, in this study, in vivo experiments tended not to show significant differences in the later time point.We suspected that individual variability might be a major cause of this, and it could be attributable to the low power of the experiment itself.In particular, we could not follow time-dependent changes in the same individual because of our experimental methods, and it might have made it difficult all the more to detect significant differences.Another reason could be the characteristics of brimonidine as a drug affecting the contralateral eye.Normally, we use the non-treated eye as a control to minimize the influence of individual differences on the test results.However, in the case of brimonidine, the contralateral eye could not be used as a control, therefore the individual variability might have affected on the test results.
In conclusion, K-232 effectively reduced both the IOP and EVP, while enhancing outflow facility, in the eyes of mice.Thus, this combination shows promise as a valuable treatment for glaucoma.

Materials
For in vivo mouse studies, we used the GLA-ALPHA ® combination ophthalmic solution (the fixed-dose combination of 0.4% ripasudil and 0.1% brimonidine, K-232 provided by KOWA Company Ltd.; Aichi, Japan) and the ophthalmic solutions GLANATEC ® (0.4% ripasudil; Rip) and AIPHAGAN ® (0.1% brimonidine; Bri) purchased commercially.For in vitro studies, ripasudil was provided by KOWA Company Ltd. (Aichi, Japan) and brimonidine was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Animals
All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the dictates of our local Animal Use Committee at the University of Tokyo.The protocol for animal experiments used in this study was approved by the University of Tokyo's Animal Ethics Committee.All experiments were performed by relevant named guidelines, regulations, and the ARRIVE guidelines.Male C57BL/6 J (wild type, WT) and ddY (wild type, WT) mice were purchased from Japan SLC (Shizuoka, Japan).The housing temperature was maintained at 21 °C with a 12 h light/dark cycle starting at 6:00 AM.After purchase, all mice had access to food and water ad libitum in a conventional animal room in our laboratory for at least 1 week before the scheduled experimental date.For all experiments, we used 8-to 10-week-old male mice (body weight range, 18-24 g).

Preparation and adding ophthalmic solution
Rip, Bri and K-232 ophthalmic solutions were stored at room temperature.We also prepared a mixture of 0.4% ripasudil and 0.1% brimonidine (Rip/Bri) to use in in vivo studies.Using a micropipette, we applied 3 μL of each eye drop solution or saline (control) topically (in a masked manner) to one eye selected at random.For experiments requiring concomitant use of both drugs, we delivered specific eyedrops at 5 min intervals.

Mouse IOP measurements
IOPs were measured using microneedles 29 .Briefly, the mice were anesthetized using an intraperitoneal injection of a mixture of ketamine (100 mg/kg, Ketalar; DAIICHI SANKYO COMPANY, Tokyo, Japan) and xylazine (9 mg/kg; Seractal, Bayer, Berlin, Germany) prepared at room temperature.We administered anesthesia using a 29-gauge needle.A timer was started immediately after the injection, and we measured IOPs within 4-5 min after administration of anesthesia in all animals.The borosilicate glass microneedle (75-100 μm tip diameter and 1.0 mm outer diameter, 25° bevel angle) was connected to a pressure transducer and the data were sent to a data acquisition and analysis system.We measured IOPs in both eyes of each anesthetized mouse, placing a microneedle in the anterior chamber of the eye.

Outflow facility measurements
To clarify the mechanism by which K-232 reduces IOPs, we measured the outflow facility 30 and 60 min after adding either a drug or saline.We determined the time of measurement as the time at which we found significant IOP reduction.To measure outflow facility, we used a two-level constant-pressure perfusion method expressed as a C value 29,60 .Briefly, an infusion needle identical to the one used for IOP measurements was inserted into the anterior chamber and connected to a reservoir filled with artificial aqueous humor via a pressure transducer.The liquid surface height in the chamber was maintained at 25 or 35 mmHg for a steady-state period of 10 min (H 25 or H 35 , respectively).The inner cross-sectional area of the reservoir (S) was calculated based on the inner diameter.The volumes of artificial aqueous humor infused per min at 25 or 35 mmHg were denoted as V 25 and V 35 .The total outflow facility (C total, μL/min/mm Hg) was calculated based on the following formula: C total = (V 35 − V 25 )/10 = [S(H 35 − H 25 )]/100.The C total was the sum of C conv and C uveo , where C conv is the outflow facility of the pressure-dependent system and C uveo is the uveoscleral system (assumed to be less pressure-dependent).

Episcleral venous pressure measurements in mice
To clarify the mechanism by which K-232 reduces IOPs, we measured EVPs 30 and 60 min after injecting the eyes with either drug or saline.To measure the superior scleral vein pressure, we followed a modified version of a published protocol 29,61 Briefly, we used ddY mice for EVP measurement and a glass needle connected to a transducer and a reservoir bag.After inserting the needle into the anterior chamber of the eye of an anesthetized mouse, we raised or lowered the reservoir bag connected to the transducer to artificially control the pressure in the anterior chamber.Initially, we adjusted the height of the reservoir bag to match the IOP of the mouse, and then we progressively lowered it at a rate of 0.5 mmHg/min while observing the vascular pattern of the surface of the mouse eye under a microscope at 60× magnification.We defined the EVP as the pressure at which venous blood flowed back into Schlemm's canal and recorded its value.
Vol:.( 1234567890 S1) 62 Only well-characterized normal HTM cells, in which Dexamethasone (Dex)-induced myocilin (MYOC) upregulation was confirmed with quantitative qRT-PCR from passages 3 through 5 were used in our studies (Supplementary Figure . S1A).Furthermore, for the HTM cell characterization, Dex-induced MYOC upregulation and immunocytochemistry using antibodies against Aquaporin 1 (AQP-1), Collagen Type IV (COL4A1), Matrix Gla Protein (MGP), tissue inhibitor of metalloproteinase (TIMP)-3, vimentin, and desmin was also performed according to previous reports (Supplementary Fig. S1B) 63,64 .All donor tissues were obtained and managed in line with the guidelines of the Declaration of Helsinki for research involving human tissues.Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and antibiotic-antimycotic solution (100×) (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C and 5% CO2.We used only well-characterized normal HTM cells from passages 3-6 in subsequent experiments.The ripasudil concentrations in aqueous humor 30-60 min after a single adding ripasudil ophthalmic solution (1.0%, w/v) have been reported to be approximately 10 µg/mL (~ 25 µM) in rabbits and 1 µg/mL (~ 2.5 µM) in monkeys 65 .Another study showed that brimonidine levels in aqueous humor 2 h after brimonidine instillation (0.2%, w/v) were 0.647 ± 0.062 µg/mL (~ 1.4 µM) in rabbits 66 and 336.0 ± 276.2 nM in humans 67 .While we are aware of potential interspecies differences in aqueous humor concentrations, we deliberately chose ripasudil concentrations higher than 1 µM and a brimonidine concentration higher than 1 µM for our in vitro experiments with HTM cells, unless otherwise specified.We set a 10 ng/mL concentration of the stimulant TGFβ2 to obtain a positive baseline control for cytoskeletal and fibrotic changes in HTM cells.

Collagen gel contraction assays
We used a Cell Contraction Assay kit (Nitta Gelatin, Osaka, Japan) as described to assess collagen gel contractility by HTM cells 68 .Briefly, we generated a collagen gel matrix in 24-well plates as instructed by the manufacturer.HTM cells were cultured on the collagen gel until confluent.The tops of the collagen gel were stimulated, and the gels were detached from the walls of the culture wells.We photographed the gel areas at 0, 6, 24, 48, 72, and 96 h and analyzed them using ImageJ software (National Institutes of Health, Bethesda, MD, USA).We masked the data inputs for the analysis.Changes in the areas are given as contraction percentages compared to start of the incubation period.We used TGFβ2 stimulation to obtain a positive control for gel contraction and observed its inhibition using each drug alone or drug combinations.

Western blotting
Western blotting was performed as published 69 .Briefly, we collected cell lysates in RIPA buffer (Thermo Fisher Scientific) containing protease inhibitors (Roche Diagnostics, Basel, Switzerland) after treatment.Protein concentrations were determined using a BCA Protein Assay kit (Thermo Fisher Scientific) according to the manufacturer's protocol with bovine serum albumin as a standard.Protein extracts were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories).Then we immersed the membranes in a solution containing primary antibodies overnight at 4 °C.The primary antibodies were anti-alpha smooth muscle actin (1:1000; DAKO) and anti-β-tubulin (1:1000; Wako Pure Chemical Industries, Osaka, Japan).After washing the membranes, we incubated them in a solution with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1:2000-1:5000; Thermo Fisher Scientific) for 1 h at room temperature.The proteins in the membranes were made to react with ECL substrate (Thermo Fisher Scientific), and we placed the membranes in an ImageQuant LAS 4000 mini-imager (GE Healthcare, Chicago, IL, USA) to visualize the bands and quantify them using ImageJ software (ver.1.49, NIH, Bethesda, MD, USA).

Figure 5 .
Figure 5. Effects of ripasudil and brimonidine on cytoskeletal changes in HTM cells.The effects of ripasudil and brimonidine on TGFβ2-induced cytoskeletal changes in HTM cells at 24 h were evaluated via immunocytochemistry.The left panels show cells stained for F-actin.The middle panels show cell nuclei counterstained with 4' ,6-diamidino-2-phenylindole (DAPI; blue).The right panels show merged images.TGFβ2 significantly induced cytoskeletal changes in HTM cells and these changes were suppressed by treatment with either ripasudil or ripasudil/brimonidine.