Interaction Between and on , Pupil Diameter, and the Aqueous- Outflow Pathway

Reiko Yamagishi-Kimura,1 Megumi Honjo,1 Takashi Komizo,2 Takashi Ono,2 Akiko Yagi,2 Jinhee Lee,2 Kazunori Miyata,2 Tomokazu Fujimoto,3 Toshihiro Inoue,3 Hidenobu Tanihara,3 Junko Nishida,1 Takatoshi Uchida,1 Yuka Araki,1 and Makoto Aihara1 1Department of Ophthalmology, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan 2Miyata Eye Hospital, Miyazaki, Japan 3Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan

Correspondence: Makoto Aihara, PURPOSE. To explore interactions between pilocarpine and the ROCK inhibitor, ripasudil, on Department of Ophthalmology, Uni- IOP and pupil diameter in human eyes, and morphological and functional changes in outflow versity of Tokyo School of Medicine, tissues in vitro. 7-3-1 Hongo Bunkyo-ku, Tokyo, 113– 8655, Japan; METHODS. IOP and pupil diameter were measured after pilocarpine and/or ripasudil, which [email protected]. were topically applied in healthy subjects. Human (HTM) cells were Submitted: January 18, 2018 used in a gel contraction assay, for the evaluation of phosphorylation of myosin light chain Accepted: February 28, 2018 and cofilin, and immunostaining for cytoskeletal proteins. Porcine ciliary muscle (CM) was used in a CM contraction assay. The permeability of human Schlemm’s canal endothelial (SCE) Citation: Yamagishi-Kimura R, Honjo cells was evaluated by measuring transendothelial electrical resistance and fluorescein M, Komizo T, et al. Interaction be- tween pilocarpine and ripasudil on permeability. intraocular pressure, pupil diameter, RESULTS. Both pilocarpine and ripasudil significantly reduced IOP in human eyes, but and the aqueous-outflow pathway. pilocarpine interfered with ripasudil-induced IOP reduction when concomitantly introduced. Invest Ophthalmol Vis Sci. Ripasudil significantly inhibited gel contraction, TGFb2-induced stress fiber formation, a- 2018;59:1844–1854. https://doi.org/ smooth muscle actin expression, and phosphorylation of both myosin light chain and cofilin 10.1167/iovs.18-23900 in HTM cells. Pilocarpine reduced these effects, significantly inhibited the ripasudil-induced HTM cell responses to TGFb2 stimulation, and increased the permeability in SCE cells. In CM, ripasudil inhibited pilocarpine-stimulated contraction, but ripasudil did not have significant effects on pilocarpine-induced .

CONCLUSIONS. Pilocarpine interfered with the direct effects of ROCK inhibitor on the conventional outflow pathway leading to IOP reduction and cytoskeletal changes in trabecular meshwork cells, but did not affect the relaxation effect of the ROCK inhibitor. It is therefore necessary to consider possible interference between these two drugs, which both affect the conventional outflow. Keywords: ciliary muscle, conventional outflow, pilocarpine, ripasudil, trabecular meshwork

ntraocular pressure (IOP) reflects the balance between The M3 receptor is also expressed by human TM (HTM) I aqueous humor production and outflow through the cells,7 wherein pilocarpine functions to activate Gq and the conventional pathway via the trabecular meshwork (TM), and phospholipase C pathway.8 Thus, pilocarpine-induced morpho- the uveoscleral pathway via the to the supracho- logical changes in TM cells, in turn, may alter the conventional roidal space. In glaucomatous eyes, IOP becomes elevated outflow features. when outflow resistance increases, mainly in the TM. Recently, the ROCK inhibitor, ripasudil (Glanatec; Kowa Pilocarpine, a parasympathetic M3 receptor agonist, rapidly Company, Nagoya, Japan), has been launched in Japan; another increases conventional outflow attributable to indirect dilation ROCK inhibitor, Roclatan (Aerie, Durham, NC, USA) is now in of the flow space in the TM and Schlemm’s canal (SC).1–4 The clinical trials and Rhopressa ( 0.02%; Aerie) has been longitudinal fibers of the ciliary muscle (CM) are anchored to recently approved by the Food and Drug Administration as a the choroid, and pull on tendons extending into conventional new IOP-lowering drug for glaucoma patients. ROCK inhibitors outflow tissues, terminating in the TM and the inner walls of were first reported to reduce rabbit IOP by relaxing the TM the SC.3,5 Thus, pilocarpine-induced contraction of the CM followed by expansion of the intertrabecular space via indirectly increases the space within the conventional pathway. disruption of actin bundles,9,10 although other mechanisms, The mechanism of pilocarpine-induced contraction involves including degradation of the extracellular matrix and an drug binding to the CM via the M3 receptor followed by increase in giant vacuole numbers within the SC, may also be activation of Rho-kinase (ROCK) and phospholipase C.6 in play.11,12

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To date, ROCK inhibitors have been shown to exert additive Preparation of Human TM and Monkey SCE Cells, effects when given together with analogs, beta- and Drug Concentrations blockers, and carbonic anhydrase inhibitors, in efforts to increase uveoscleral outflow or decrease aqueous humor Primary human TM (HTM) cells and primary monkey SCE cells production,13–15 but there have been no reports on the were isolated from human donor eyes or monkey eyes and 16–18 interaction between the ROCK inhibitor and pilocarpine, both characterized as described previously. Only well-charac- of which increase the conventional outflow by different terized normal HTM cells from passages 3 to 8 and SCE cells mechanisms. Pilocarpine triggers CM contraction and expands from passages 3 to 5 were used in subsequent studies. the intertrabecular space within the SC,7,8 whereas ROCK The ripasudil concentrations in aqueous humor 30 to 60 inhibitors relax TM cells and the CM.9,10 Thus, as M3 and minutes after a single instillation of ripasudil ophthalmic ROCK are both present in the TM and the CM, the two drugs solution (1.0%, wt/vol) were approximately 10 lg/mL in the rabbit and 1 lg/mL in the monkey, corresponding to may exert opposite actions on these tissues, affecting the 19 balance between contraction and relaxation, and in turn, approximately 25 lM and 2.5 lM, respectively. A previous modulating outflow and/or IOP reduction. report showed that the pilocarpine level in aqueous humor of the rabbit eye 30 to 60 minutes after 2% (wt/vol) pilocarpine In the present study, we explored the interactions between 20 pilocarpine and a ROCK inhibitor in terms of the outflow instillation was 2.53 to 3.72 lg/mL, or approximately 92 lM. pathway. We first evaluated the IOP-lowering effect and the In addition, we have preliminarily evaluated the concentrations pupil diameter of a ROCK inhibitor, ripasudil, and the of 1, 10, 30, 50, and 100 lM for both ripasudil and pilocarpine additional effect of pilocarpine in healthy human subjects. in in vitro studies and investigated the dose-response and We then studied the morphological and functional changes in maximal effects of each drugs, and then we determined the human TM cells, human SC endothelial (SCE) cells, and the appropriate concentration of each drug. Although among- porcine CM in vitro. species differences in aqueous humor concentrations may be in play, we chose final ripasudil concentrations between 10 and 50 lM, and a pilocarpine concentration of 100 lM for in vitro studies with cells, unless otherwise specified. METHODS Subjects Collagen Gel Contraction Assay Using TM Cells This study was approved by the ethics committee of the Collagen gel contraction assays were performed using a Cell University of Tokyo and Miyata Eye Hospital, and adhered to Contraction Assay Kit (Nitta Gelatin, Inc., Osaka, Japan) as the tenets of the Declaration of Helsinki. All subjects provided described previously.21 Ripasudil to final concentrations of 1, written informed consent before participation in the study. 10, and 100 lM, or/and pilocarpine to final concentrations of A total of 20 healthy volunteers were recruited, and all 1, 10, and 100 lM, were added to the tops of the collagen gel subjects underwent a comprehensive ophthalmic examination. lattices, and gel areas were photographically recorded. The The criteria for inclusion of healthy subjects were as follows: at dose-dependent effects of ripasudil and pilocarpine were least 18 years of age; an IOP between 10 and 21 mm Hg; recorded at 1, 2, 3, 18, 24, and 48 hours, and the additive normal anterior chamber depth with an open angle; and no effect of 10 lM ripasudil and 100 lM pilocarpine was also family history of glaucoma, ophthalmic diseases, surgery, or investigated at 24, 48, 72, and 120 hours, and analyzed with systemic diseases. The right eye of each subject was used for the aid of ImageJ software (http://imagej.nih.gov/ij/; provided analyses of the IOP and pupil diameter. in the public domain by the National Institutes of Health, Bethesda, MD, USA). The -fold area changes in the different IOP and Pupil Diameter Measurements After treatment groups were recorded in bar graphs. Administration of Pilocarpine and the ROCK Immunostaining of Cultured HTM Cells Inhibitor, Ripasudil, in Healthy Volunteers Immunostaining of cultured HTM cells was performed as The study design for IOP reduction was a single blind described previously.9,21 After serum starvation for 24 hours, comparative study of four regimens for 20 subjects with a drugs were added to final concentrations of 50 lM ripasudil, single drop for one eye. The study was performed over four 100 lM pilocarpine, and 50 lM ripasudil þ 100 lM pilocarpine, visits with a minimum interval of 6 days between each visit. and the cells were washed with PBS, and then stimulated with IOP and pupil diameter were measured before (9 AM) and at 2 5 ng/mL TGFb2 (mimicking the pathogenic development of (11 AM), 4 (1 PM), and 8 hours (5 PM) after drug instillation. primary open-angle glaucoma).22 To identify actin bundles and The IOP and pupil diameter were measured using a Goldmann cytoskeletal changes, the slides were incubated with anti-alpha tonometer after instillation of topical anesthesia and an autoref- smooth muscle actin (aSMA) antibody (DAKO, Tokyo, Japan), keratometer (Tomey Corporation, Nagoya, Japan), respectively. phalloidin-rhodamine (Sigma-Aldrich Corp., St. Louis, MO, Three measurements were obtained, and the average IOP was USA), and anti-vinculin antibody (Sigma-Aldrich Corp.). After recorded. The pupil diameter was measured in a dark room. washing and incubation with the secondary antibody Alexa The subjects were divided into four groups and one of four Fluor 488 (Invitrogen, Waltham, MA, USA), the slides were regimens was randomly allocated for each group, with four imaged under a fluorescence microscope (BZ-9000; KEYENCE, regimens completed during the four visits. The four regimens Osaka, Japan). included the baseline IOP variation without any eye drops as a control group. The study subjects were treated with 0.4% Western Blotting ripasudil ophthalmic solution (R-group: Glanatec), 2% pilocar- pine ophthalmic solution (P-group: Sanpilo; Santen Pharma- The expression of aSMA, and phosphorylation status of the ceutical, Osaka, Japan), or a combination of 0.4% ripasudil and myosin light chain (MLC) and cofilin were determined by 2% pilocarpine ophthalmic solution (R/P-group). One of the Western blotting, using mouse monoclonal antibody against researchers (TK) allocated four regimens for each group. The aSMA, rabbit polyclonal antibodies against phospho-MLC (Cell examiners were all blinded to the allocated regimens. Signaling Technology, Danvers, MA, USA) or phosphor-cofilin

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(Cell Signaling Technology), as previously described.23 HTM three independent experiments. A difference was considered cells were cultured in six-well dishes and, after serum statistically significant at a P < 0.05. starvation for 24 hours, drugs were added to final concentra- tions of 10 or 50 lM ripasudil; 100 lM pilocarpine; and 10, 30, or 50 lM ripasudil þ 100 lM pilocarpine; the cells were RESULTS washed with PBS and then stimulated with 5 ng/mL TGFb2for 24 hours. b-tubulin served as the loading control. All IOP Reduction by Ripasudil and Pilocarpine in membranes were stripped of antibodies using Western blot Healthy Volunteers stripping solution and incubated with mouse monoclonal antibody b-actin (1:1000), and subsequently with H goat anti- Twenty healthy volunteers (3 males and 17 females) were mouse IgG antibody (1:2000) as a loading control. Densitom- included in the study. The mean age was 40.8 6 11.6 years etry of scanned films was performed using ImageJ 1.49, and (mean 6 SD). Baseline IOP was 14.9 6 2.28 mm Hg (mean 6 SD). No eyes showed adverse effects to the treatments. results are expressed relative to the loading control (b-tubulin). Time course of IOP reduction from the baseline IOP at 0 hour without any eye drops (control group) of control and CM Contraction Assay three groups is shown in Figure 1A. After instillation, among CM contraction was measured as described previously.24 We three groups of R, P, and R/P, IOP reduction from the control used fresh porcine eyes within 2 hours after enucleation, and group was significant in R at 2 hours (P ¼ 0.0033) and in R/P at ciliary muscle strips were excised according to the methods 4 hours (P ¼ 0.0288). At 2 hours after instillation, which was described previously.9,24 The dissected ciliary muscle strips the time point of the peak IOP reduction by ripasudil, a (approximately 8 3 2 3 2 mm) were placed in 10-mL tissue significant difference of IOP reduction was observed only baths, and filled with pre-aerated Krebs’ bicarbonate solution between R- and P-groups (P ¼ 0.0174), but not between R- and at 378C. The upper end of the preparation was tied to an R/P-groups, and also not between P- and R/P-groups (P ¼ isometric transducer and preloaded with 100 mg. The strips in 0.1017 and P ¼ 0.4553, respectively). Comparison between tissue baths were allowed to equilibrate for at least 1 hour. groups at 2 hours showed the significant difference between After equilibration, we confirmed the contractile response of control and R- (P < 0.0001), control and R/P- (P < 0.0019), and CM by 106 M carbachol. After that, the tissue bath was rinsed R- and P-groups (P ¼ 0.0005). At 4 hours after instillation, twice with Krebs-Henseleit physiologic solution and equili- which was the time point of the peak IOP reduction by brated again for 1 hour. The strips that sowed stable tone for pilocarpine, no significant IOP reduction was observed among 100 mg were used in further experiment. Mean isometric force R-, P-, and R/P-groups. Comparison between groups at 4 hours measurements were expressed as values relative to those of the showed the significant difference between control and R- (P ¼ responses at the maximum pilocarpine concentration (105 0.0093), control and P- (P ¼ 0.0208), and control and R/P- M). Submaximal contraction was obtained with 10 lM groups (P < 0.0001). Collectively, additional instillation of pilocarpine in CM strips. Following contraction by pilocarpine, pilocarpine on ripasudil did not cause a significant additional the relaxation responses to ripasudil were obtained in a IOP reduction; on the contrary, pilocarpine compromised the cumulative manner in CM strips. Relaxation responses were ripasudil-induced IOP reduction at 2 hours after instillation in expressed as percentages of the maximum effect (100%) healthy human volunteers. elicited by pilocarpine in each strip. Pupil Diameter Changes Induced by Ripasudil and Measurement of Monolayer Transendothelial Pilocarpine in Healthy Volunteers Electrical Resistance (TEER) and Permeability The baseline pupil diameter was 5.59 6 0.66 mm. Ripasudil Measurement of monolayer TEER and permeability of FITC- did not affect the pupil diameter when compared with dextran (average molecular weight, 4000; Sigma-Aldrich Corp.) baseline. Pilocarpine caused significant miosis from 2 hours of SCE cells were performed according to a previously after instillation, and addition of ripasudil did not abrogate the described method.12,18 After serum starvation overnight, SCE miosis induced by pilocarpine (Fig. 1B). cells were treated with 50 lM ripasudil, 100 lM pilocarpine, or a mixture of both. TEER was measured at 1, 4, 6, and 24 hours Human TM-mediated Collagen Gel Contraction after stimulation, and FITC-dextran permeability was measured We used the collagen gel contraction assay to explore the dose- at 24 hours. Each experiment was performed at least four dependence of ripasudil and pilocarpine on HTM contraction. times. Ripasudil at 10 and 100 lM significantly suppressed contrac- tion of the collagen gel in a dose-dependent manner, especially Statistics between 24 and 48 hours (Fig. 2A). However, pilocarpine did not show any significant effect up to 48 hours (Fig. 2B). The summary statics of the continuous variables of the healthy Therefore, we next explored the interaction and additional volunteers and experimental results are presented as means 6 prolonged effects of 10 lM ripasudil and 100 lM pilocarpine SDs. The estimates of the mean using a mixed-effect model up to 120 hours. Pilocarpine did not abrogate the relaxation by were presented as means estimate with 95% confidence ripasudil on TM-mediated collagen gel contraction (Fig. 2C). intervals. Statistical analysis was performed with the aid of SAS Ver9.4.and JMP Pro 11 software (SAS Institute, Inc., Cary, NC, USA). The changes from baseline of the IOPs and pupil Effects of Ripasudil and Pilocarpine on TGFb2- diameters in each group were evaluated using the mixed- induced Cytoskeletal Rearrangements and effects model. The significance level of alpha ¼ 0.05 was used Colocalization of aSMA to Stress Fibers in HTM in all statistical tests. In experimental results, Student’s t-test in Cells combination with a Bonferroni post hoc test was used for between-group comparisons. Dunnet’s multiple comparisons HTM cells were stained with an anti-aSMA antibody to evaluate test was used as a post hoc test following ANOVA to compare the extent of the epithelial-mesenchymal-transition-like phe- more than two groups. All data represent the means of at least nomenon (indicated by a green color). Phalloidin-rhodamine

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FIGURE 1. IOP reduction and changes in pupil diameter in healthy volunteers by ripasudil and pilocarpine. The effects of both ripasudil and pilocarpine on IOP reduction (A) and pupil diameter (B) were investigated in 20 healthy volunteers after 2, 4, and 8 hours of instillation. The estimates of the mean using a mixed-effect model are presented as means estimate with 95% confidence intervals. C-group, control; R-group, ripasudil; P-group: pilocarpine; R/P-group, ripasudil and pilocarpine (n ¼ 5 in each group). *P < 0.05 or **P < 0.01 compared with baseline using the mixed-effects model.

was used to highlight the F-actin cytoskeleton (red), an anti- etal changes, compared with those observed after addition of vinculin antibody (green) to reveal focal adhesions, and 40,6- ripasudil alone. diamidino-2-phenylindole (DAPI) to identify the nucleus (blue). Western blotting was used to detect aSMA in efforts to In line with a previous report, and our HTM collagen gel explore this interaction between ripasudil and pilocarpine in contraction assay results, ripasudil inhibited TGFb2-dependent terms of the TGFb2-dependent myofibroblast-like transition of actin stress fiber formation without compromising cell viability HTMcells(Fig.3B).TGFb2 significantly induced aSMA (data not shown).25 Pilocarpine did not affect TGFb2-mediated expression (an increase of approximately 340%; P < 0.05), actin bundle polymerization. As previously observed,26,27 in whereas 50 lM ripasudil significantly suppressed such the context of the TGFb2-dependent myofibroblast-like transi- expression (P < 0.05), but concomitant addition of ripasudil tion of HTM cells, TGFb2 induced significant increases in aSMA and pilocarpine mitigated the reduction in TGFb2-induced and vinculin accumulation, and in the numbers of actin stress aSMA expression induced by ripasudil alone (Fig. 3B, lower fibers, compared with the control values (Figs. 3A, 4). panel). Ripasudil completely inhibited the TGFb2-induced Ripasudil completely blocked the increases in aSMA and recruitment of vinculin to the ends of actin stress fibers, but vinculin accumulation; however, pilocarpine did not signifi- concomitant addition of ripasudil and pilocarpine mitigated cantly affect TGFb2-induced cytoskeletal changes. Concomi- the reduction in TGFb2-induced vinculin accumulation in- tant addition of ripasudil and pilocarpine slightly reduced the duced by ripasudil alone (Fig. 4, arrowheads). Together, the TGFb2-induced myofibroblast-like transition, and the cytoskel- data suggest that pilocarpine compromises the effects of

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FIGURE 2. Human TM-mediated collagen gel contraction. The dose-dependent effects of ripasudil (A) and pilocarpine (B) on TM-mediated contraction were investigated using a collagen gel contraction assay. The additive effect of 10 lM ripasudil and 100 lM pilocarpine was also investigated (C). Affected gel areas were photographed at 1, 2, 3, 18, 24, and 48 hours for (A) and (B) and at 24, 48, 72, and 120 hours for (C). Data are expressed as means 6 SD (n ¼ 4 per time point). *P < 0.05 or **P < 0.01; control versus each group; Dunnett’s test.

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FIGURE 4. Effect of ripasudil and pilocarpine on TGFb2-induced cytoskeletal rearrangements in HTM cells. After serum starvation for 24 hours, cultured TM cells were pretreated with 50 lM ripasudil, 100 lM pilocarpine, or 50 lMripasudilþ 100 lM pilocarpine for 1 hour, and then stimulated with 5 ng/mL TGFb2 for 24 hours before immunostaining for vinculin (green)andF-actin.White arrowheads: vinculin staining. Cell nuclei were counterstained with DAPI (blue). Scale bar:60lm.

ripasudil on TGFb2-induced cytoskeletal rearrangements in, and the myofibroblast-like transition of, HTM cells.

Effects of Ripasudil and Pilocarpine on MLC and Cofilin Phosphorylation The MLC and cofilin play crucial roles in regulating dynamic rearrangements of the cytoskeleton. Phosphorylation of these proteins stabilizes actin filaments and triggers the formation of stress fibers. To explore the details of how pilocarpine compromises the effects of ripasudil on the cytoskeletal FIGURE 3. Effect of ripasudil and pilocarpine on TGFb2-induced aSMA changes in HM cells observed by immunostaining, we used expression and colocalization of aSMA and stress fibers in HTM cells. After serum starvation for 24 hours, cultured TM cells were pretreated Western blotting to detect phosphorylated MLC (p-MLC) and with 50 lM ripasudil, 100 lM pilocarpine, or 50 lM ripasudil þ 100 lM phosphorylated cofilin (p-cofilin). As shown in Figure 5, TGFb2 pilocarpine, for 1 hour and then stimulated with 5 ng/mL TGFb2 for 24 induced significant phosphorylation of MLC (an increase of hours before immunostaining for aSMA (green) and F-actin (red)(A). approximately 250%; P < 0.01) compared with the control, Cell nuclei were counterstained with DAPI (blue). Scale bar:100lm. and 10 lM and 30 lM ripasudil significantly inhibited this The upper panel shows representative Western blots of aSMA in (B). induction (P < 0.05, P < 0.01, respectively). However, Lower panel in (B) is a relative aSMA level, in which the data are shown pilocarpine had no effect on MLC phosphorylation, and as means 6 SE, n ¼ 3. *P < 0.05, compared with control; Student’s t-test. concomitant ripasudil (10 lM) and pilocarpine (100 lM)

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FIGURE 6. Effect of ripasudil on pilocarpine-induced contraction of CM. Contraction of porcine CMs was isometrically measured using a force-length transducer. Ripasudil (1–100 lM) was gradually added to the bath. Mean isometric force measurements were expressed relative to those of the maximal pilocarpine-induced response (at 105 M of the drug). Relaxation responses are expressed as percentages of the maximum effect (100%) elicited by pilocarpine in each strip. *P < 0.01 compared with control; Student’s t-test.

Effect of Ripasudil on Pilocarpine-induced Contraction of CM It is well known that pilocarpine induces CM contraction. When ripasudil was added to muscle strips precontracted with pilocarpine (105 M), muscle contraction was inhibited in a concentration-dependent manner (Fig. 6). Ripasudil at both 10 and 100 lM significantly inhibited contraction compared with the control value (P < 0.01, t-test).

Effects of Ripasudil and Pilocarpine on Barrier Function and Permeability of SCE Cell Monolayers To evaluate the barrier function and permeability of the SCE cell monolayer, we measured TEER. Following treatment with 50 lM ripasudil, FITC-dextran permeability was significantly increased at 24 hours after treatment. Addition of 100 lM pilocarpine slightly attenuated this increased FITC-dextran permeability induced by ripasudil treatment (Fig. 7A). We then evaluated the effects of ripasudil and pilocarpine on TEER, and found that TEER was significantly decreased by 50 lM ripasudil treatment in a time-dependent manner until 4 hours after drug treatment (Fig. 7B). Although treatment with pilocarpine alone induced no significant difference in TEER when compared with the control, the decrease of TEER by ripasudil was FIGURE 5. Effect of ripasudil and pilocarpine on TGFb2-induced significantly attenuated by concomitant treatment of ripasudil phosphorylation of the MLC and cofilin in HTM cells. HTM cells were and pilocarpine at 4 hours after treatment (Fig. 7B, # denotes P pretreated with 10 lMor30lM ripasudil, 100 lM pilocarpine, or 10 < 0.05 ripasudil only versus ripasudil/pilocarpine). These lMor30lM ripasudil þ 100 lM pilocarpine, for 1 hour, and then observations suggest that simultaneous administration of stimulated with 5 ng/mL TGFb2 for 24 hours. The effects of ripasudil pilocarpine inhibited the ripasudil-induced decrease in barrier and pilocarpine on MLC phosphorylation are shown in (A), and those function of the cultured SCE cell monolayers. The Table on cofilin phosphorylation in (B). The upper panels show represen- summarizes the interactions between these two drugs. tative Western blots of p-MLC, p-cofilin, and b-tubulin. (A) p-MLC and p- cofilin. Data are shown as means 6 SE, n ¼ 3. *P < 0.05, **P < 0.01 compared with control; Student’s t-test. DISCUSSION We first explored the interactions between two antiglaucoma downregulated p-MLC production. Thus, pilocarpine compro- drugs with different mechanisms of action, but the same target, mised the ripasudil-induced effect (Fig. 5A). Similar results which are used to increase conventional outflow to reduce were observed when phosphocofilin levels were assayed, but IOP, and that cause morphological changes in TM, SC, and CM. the differences were not significant (Fig. 5B). Pilocarpine induces CM contraction via the muscarinic M3

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receptor, expanding the conventional outflow space, whereas ROCK inhibitors relax TM cells to open the meshwork. However, both target molecules (the M3 receptor and ROCK) are present not only in the CM but also in TM cells.7,28–30 Thus, when conventional outflow must be enhanced, the balance between trabecular tissue contraction and relaxation induced by both drugs is of great clinical and physiological interest. Both pilocarpine and ripasudil significantly reduced IOP,but no additive effect was observed in human subjects, and pilocarpine interfered with the IOP reduction by ripasudil at the peak IOP reduction (Fig. 1A). Additionally, ripasudil did not have any effect on the miosis induced by pilocarpine (Fig. 1B). The expression of ROCK in iris tissue has not been clarified so far; however, the levels of mRNAs for ROCK and ROCK substrates can differ depending on the tissues, as the levels of mRNA for ROCK in the CM were reportedly lower than that in the TM.30 Therefore, it is considered that ripasudil significantly lowered IOP, but did not affect miosis caused by pilocarpine. Concomitant instillation of pilocarpine and cytochalasin B (a cytoskeleton-targeting drug also effecting morphological changes in TM cells) has been previously shown to result in no additive effect on outflow in cynomolgus monkeys.31 We observed interference effects on IOP reduction by the concomitant application of these two clinically available drugs in human eyes, but future studies are required to confirm this result, which may prevent the ineffective application of multiple glaucoma drugs in clinical situations. Pilocarpine is not currently used as often as in the past because of the high frequency of ocular and systemic side effects.32 Excessive accommodation and miosis triggering transient headache, myopia, and/or dimness are attributable to contraction of the sphincter ciliary and pupillary muscles. However, several recent surgical procedures to augment the physiological outflow through the TM to the SC, including FIGURE 7. Effects of ripasudil, pilocarpine, and ripasudil/pilocarpine on FITC-dextran permeability and transendothelial electrical resistance minimally invasive glaucoma surgery (MIGS) or Trabectome 33,34 in SCE cell monolayers. (A) Ripasudil increased the permeability of have been noteworthy. After these procedures, including FITC-dextran in SCE cell monolayers. The increase of permeability by the traditional trabeculotomy procedure, pilocarpine is often ripasudil was slightly attenuated (P ¼ 0.192 ripasudil versus ripasu- prescribed in an attempt to induce miosis and avoid peripheral dilþpilocarpine with Student’s t-test) by concomitant treatment of anterior synechia.35 However, it has been reported that ROCK ripasudil and pilocarpine. Data are expressed as mean 6 SD (n ¼ 4–5/ inhibitors can dilate the SC and reduce episcleral venous each column). *Indicates P < 0.05; control versus the ripasudil-treated pressure, which could be beneficial after reconstructive group using Student’s t-test. (B) Ripasudil decreased TEER significantly, surgery of the physiological outflow pathway, including MIGS and the decrease of TEER by ripasudil was significantly attenuated by 36 concomitant treatment of ripasudil and pilocarpine at 4 hours (P ¼ and trabeculotomy. Therefore, ophthalmologists may have 0.044). Data are expressed as mean 6 SD (n ¼ 4–5/column). * and ** the opportunity to prescribe both types of eye drops in indicate P < 0.05 or P < 0.01 when comparing the control versus the glaucoma patients. However, if similar results as in the present ripasudil group using Student’s t-test. # indicates P < 0.05 when study are clinically observed, care should be taken to use these comparing ripasudil only versus ripasudil þ pilocarpine. drugs together, with concerns about a reduction of IOP because of interference in contraction of the sphincter ciliary and pupillary muscles.

TABLE. Summary of Interactions Between Ripasudil and Pilocarpine

Measurements Specimens Measurements Ripasudil Pilocarpine Interference

IOP Human IOP reduction þþ þ þ PD Human PD change þþ CM Porcine Contraction of CM þþþ* TM cells Human Contraction of TM þþ Cytoskeletal changes aSMA þ þ vinculin þ þ p-MLC þ þ p-cofilin þ þ SCE cells Monkey Barrier function þ þ permeability þ þ þþ, significant interaction; þ, slight interaction; , no interaction; PD, pupil diameter. * Ripasudil relaxed CM contraction induced by pilocarpine.

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To investigate why the two drugs interfere in IOP reduction, studies have found that TGFb2-induced changes in the we next explored the effects of these drugs on TM fibrogenic and contractile properties of HTM cells are pathophysiology, including cytoskeletal rearrangements and mediated in part by activation of Rho and Rho-kinase, which myofibroblast-like transition. TGFb2wasusedtomimic may explain the pathobiology of IOP elevation in glaucoma glaucoma of HTM cells.37 As shown in Figure 2, collagen gel patients.27,37,43,44 Because ROCK inhibitors regulate Rho/ contraction by HTM cells was completely blocked by the ROCK-dependent phosphorylation of the MLC kinase, TGFb- ROCK inhibitor but not pilocarpine, which also did not or pilocarpine-induced contraction of the TM or CM may be abrogate the ripasudil effect. Next, we explored TGFb2- almost completely inhibited by a ROCK inhibitor. ROCK induced cytoskeletal rearrangements (including actin stress inhibitors are known to relax the smooth muscles; therefore, fiber assembly, vinculin recruitment, and myofibroblast-like we assumed if such an inhibitor relaxes the CM, iris sphincter transition). TGFb2-induced cytoskeletal and fibrotic changes muscles might be affected by the drug. However, the ROCK were completely abrogated by the ROCK inhibitor (Fig. 3). inhibitor did not affect the pupil diameter in human eyes (Fig. Pilocarpine induced cell contraction and did not affect the 1B). One reason for this may be that the local doses afforded by TGFb2-dependent contraction, cytoskeletal rearrangements, or eye drops may differ from the doses used in vitro. Another myofibroblast-like transition of HTM cells, even when added reason may be a difference in pharmacological affinity of the with ripasudil (Figs. 3, 4). ROCK inhibitor for TM and CM, and iris sphincter muscles. The cytoskeleton is the key framework for many cell Pilocarpine reportedly produces a greater TM contraction than activities, and cytoskeletal alterations may effectively control a CM contraction. CM contraction depends almost entirely on aqueous humor outflow resistance.37 To clarify the molecular calcium, but TM contraction uses both calcium-dependent and mechanisms of such cytoskeletal interference, and drug dose- calcium-independent pathways.45,46 Additionally, higher levels dependency, we explored the phosphorylation status of the of mRNAs for ROCK and ROCK substrates were found in the MLC and cofilin, both of which play crucial roles in TM when compared with CM,30 and Y-27632 is known to have cytoskeletal regulation: phosphorylation stabilizes actin fila- a faster and more potent effect on TM cells when compared ments and triggers stress fiber formation. Ripasudil significantly with CM.47 The detailed expression of ROCK in iris tissue and inhibited TGFb2-dependent MLC and cofilin phosphorylation, effects of ROCK inhibitor on iris sphincter muscles are unclear, correlating with the decrease in actin stress fiber levels evident and further studies will be needed. on immunostaining. In contrast, pilocarpine per se did not Our study had several limitations. First, we studied only exert any significant effect, but compromised the effects of 10 short-term responses to the drugs. Both pilocarpine and ROCK lM ripasudil on TGFb2-dependent MLC and cofilin phosphor- inhibitors can affect the extracellular matrix,18,36,42,48 so a ylation when concomitantly applied (Fig. 5). However, no longer period of evaluation after dosing is required. Second, significant interference was apparent between 30 lM ripasudil the doses we used in vitro may differ from those in the eye. and pilocarpine. Thus, dose levels must be carefully considered Thus, some serious issues may yet require attention. Third, we in clinical situations. only evaluated the IOP and pupil diameter in healthy We also assessed the effect of the ROCK inhibitor on volunteers, with relatively lower baseline IOP. Tissue responses pilocarpine-induced contraction of CM. As was noted in TM to pilocarpine or ROCK inhibitors may differ in the presence cells, higher concentrations of ripasudil completely relaxed CM and absence of , and such future studies contraction (Fig. 6). Ripasudil has been reported to bind would be of particular interest. Finally, in vitro studies allowed strongly to melanin, and significantly higher ripasudil levels us to observe the cellular functions of conventional outflow (approximately 16-fold those in the aqueous humor) have been tissues. In the future, optical coherence tomography should be observed in the iris-ciliary body of pigmented rabbits, even used to analyze the in vivo interactions of the TM, SC, and CM. after a single instillation.19 Therefore, it is possible that In conclusion, the interactions of two glaucoma drugs, relaxation induced by ripasudil overcomes CM contraction pilocarpine and a ROCK inhibitor, both affecting the conven- induced by pilocarpine. The actual drug concentrations may tional outflow pathway, were explored using in vivo IOP differ in vivo; further studies are required to determine measurements in human eyes and in vitro cell and tissue whether our in vitro observations are relevant in vivo. culture studies. Pilocarpine and the ROCK inhibitor did not The SCE is known to be an important ocular component additively reduce IOP. Pilocarpine inhibited the effects of the producing outflow resistance against aqueous humor in the ROCK inhibitor in TM cells, SCE cells, and CM. These findings conventional outflow route, and junctional protein complexes suggest that modification of Rho/ROCK signals may be a in SCE cells create a barrier against aqueous humor outflow. feasible means by which to increase conventional outflow. The Rho/ROCK signaling pathway is reportedly involved in 12,18 regulating SCE permeability. In this study, to evaluate the Acknowledgments barrier function of SCE, we measured FITC-dextran permeabil- ity and TEER in a confluent SCE cell monolayer, and observed Supported by the Japan Society for the Promotion of Science that treatment with ripasudil increased the permeability and 15K10854 (MH). decreased the TEER when compared with control levels (Fig. Disclosure: R. Yamagishi-Kimura, None; M. Honjo, None; T. 7), which was consistent with previous reports.12,18 As Komizo, None; T. Ono, None; A. Yagi, None; J. Lee, None; K. expected, pilocarpine did not affect permeability or TEER, Miyata, None; T. Fujimoto, None; T. Inoue, None; H. Tanihara, which is reasonable because pilocarpine is known to expand None; J. Nishida, None; T. Uchida, None; Y. Araki, None; M. SC by the contraction of the CM in the eye.38,39 However, we Aihara, None found that a decrease of TEER induced by ripasudil was significantly attenuated by concomitant treatment with pilo- References carpine. Although the precise mechanisms should be further explored, this observation may be important for clinical 1. Flocks M, Zweng HC. Studies on the mode of action of practice because it suggests that pilocarpine may have effects pilocarpine on aqueous outflow. Am J Ophthalmol. 1957;44: against SCE cells without the involvement of CM contraction. 380–386; discussion 387–388. Collectively, our results suggest that signals downstream of 2. Barany EH. The mode of action of pilocarpine on outflow TGFb2 and M3 are shared (at least in terms of Rho activation) resistance in the eye of a primate (Cercopithecus ethiops). in HTM cells and CM, and possibly in SCE cells.40–42 Many Invest Ophthalmol. 1962;1:712–727.

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