MERCURY-3: a randomized comparison of netarsudil/latanoprost and bimatoprost/timolol in open-angle glaucoma and ocular hypertension.

PURPOSE   : To compare the efficacy and safety of the fixed-dose combination (FDC) of netarsudil 0.02%/latanoprost 0.005% ophthalmic solution (NET/LAT; Roclanda®) with bimatoprost 0.03%/timolol maleate 0.5% (BIM/TIM; Ganfort®) ophthalmic solution in the treatment of open-angle glaucoma (OAG) and ocular hypertension (OHT). METHODS: MERCURY-3 was a 6-month prospective, double-masked, randomized, multicenter, active-controlled, parallel-group, non-inferiority study. Patients (≥ 18 years) with a diagnosis of OAG or OHT in both eyes that was insufficiently controlled with topical medication (IOP ≥ 17 mmHg in ≥ 1 eye and < 28 mmHg in both eyes) were included. Following washout, patients were randomized to once-daily NET/LAT or BIM/TIM for up to 6 months; efficacy was assessed at Week 2, Week 4, and Month 3; safety was evaluated for 6 months. Comparison of NET/LAT relative to BIM/TIM for mean IOP at 08:00, 10:00, and 16:00 h was assessed at Week 2, Week 6, and Month 3. Non-inferiority of NET/LAT to BIM/TIM was defined as a difference of ≤ 1.5 mmHg at all nine time points through Month 3 and ≤ 1.0 mmHg at five or more of nine time points through Month 3. RESULTS: Overall, 430 patients were randomized (NET/LAT, n = 218; BIM/TIM, n = 212), and all received at least one dose of study medication. Efficacy analyses were performed at Month 3 on 388 patients (NET/LAT, n = 184; BIM/TIM, n = 204). NET/LAT demonstrated non-inferiority to BIM/TIM, with a between-treatment difference in IOP of ≤ 1.5 mmHg achieved at all time points and ≤ 1.0 mmHg at the majority of time points (six of nine) through Month 3. Mean diurnal IOP during the study ranged from 15.4 to 15.6 mmHg and 15.2 to 15.6 mmHg in the NET/LAT and BIM/TIM groups respectively, with no between-group statistically significant difference. No significant differences were observed in key secondary endpoints. No serious, treatment-related adverse events (AEs) were observed, and AEs were typically mild/moderate in severity. The most common treatment-related AEs were conjunctival hyperemia (NET/LAT, 30.7%; BIM/TIM, 9.0%) and cornea verticillata (NET/LAT, 11.0%; BIM/TIM, 0%). CONCLUSIONS: Once-daily NET/LAT was non-inferior to BIM/TIM in IOP reduction in OAG and OHT, with AEs consistent with previous findings. NET/LAT offers a compelling alternative FDC treatment option for OAG and OHT.

Role of Rho Kinase in Regulating Arterial Stiffness in Anesthetized Rabbits.

The effects of Rho kinase inhibitors fasudil and ripasudil on arterial stiffness were assessed using anesthetized rabbits, where the aortic ß and femoral ß were measured as a stiffness parameter at each arterial region. Intravenous administration of fasudil and ripasudil dose-dependently decreased blood pressure and femoral vascular resistance and increased femoral arterial blood flow, which appeared according to their in vitro potencies for Rho kinase inhibition. Both drugs increased the aortic ß but decreased the femoral ß at hypotensive doses. These results suggest that the inhibition of Rho kinase contributes to reducing elastic recoil in the aorta and an increase in compliance in the femoral artery. In addition, the Rho kinase-associated Ca2+-independent mechanism of arterial vascular smooth muscle contraction may be essential in the regulation of femoral arterial stiffness.

Favorable effect of ripasudil use on surgical outcomes of microhook ab interno trabeculotomy.

PURPOSE: We have previously demonstrated that prolonged use of glaucoma medications was associated with a poor surgical outcome of ab interno trabeculotomy (µTLO). Given that almost all types of glaucoma eye drop either enhance the drainage through the uveoscleral pathway or reduce aqueous humor production, we hypothesized that prolonged use of these medications might cause disuse atrophy of the conventional pathway. In contrast, ripasudil increases the conventional outflow and eventually shows a favorable outcome of µTLO. This study aimed to evaluate the effect of ripasudil use on µTLO outcomes. METHOD: The medical charts of 218 patients who underwent µTLO were analyzed retrospectively. We compared the 1-year outcome between ripasudil users versus nonusers by using propensity score matching. We set the covariates as age, sex, glaucoma types, preoperative intraocular pressure (IOP), the mean deviation values of visual field tests, the presence or absence of concomitant cataract surgery, trabecular meshwork incision range, the presence or absence of any glaucoma medication except ripasudil and duration of glaucoma medical therapy. Success was defined as a postoperative IOP between 5 and 21 mmHg, a ≥ 20% IOP reduction from baseline, and no additional glaucoma surgery at postoperative 1 year. RESULT: Fifty-seven patients each were allocated to the ripasudil users or nonusers. The 1-year success rates were 74% in ripasudil users and 51% in nonusers (p = 0.01). Kaplan‒Meier survival curves also showed that the ripasudil users had a higher survival distribution (p = 0.01). CONCLUSION: The patients who took ripasudil showed a favorable 1-year outcome of µTLO.

Effectiveness of Netarsudil versus Brimonidine in Eyes already Being Treated with Glaucoma Medications at a Single Academic Tertiary Care Practice: A Comparative Study.

Background: Rho kinase inhibitors, such as netarsudil, are a relatively new class of medications recently introduced into the market for the treatment of glaucoma, the leading cause of irreversible blindness in the world. Previous clinical trials have studied netarsudil's efficacy when used as a first- or second-line agent but limited studies have investigated its effectiveness in the real world where it is more commonly used as a third, fourth, or fifth agent in combination with other topical medications. Equally important, prior studies have not compared its effectiveness to its peer medications in these settings. Objective: To compare intraocular pressure (IOP) lowering after initiation of netarsudil or brimonidine therapy in patients with glaucoma using >2 medications for IOP management. Methods: A chart review of 369 eyes from 279 patients followed at a single academic tertiary practice was performed with an institutional review board waiver of consent to compare IOP lowering after prescription of netarsudil (n = 176) versus brimonidine (n = 193) as a third, fourth, or fifth IOP-lowering agent. Patients were identified by querying the electronic medical record for those with a glaucoma-related diagnosis who were prescribed either medication. Five sequential IOP measurements were obtained to determine the mean change in IOP before and after treatment (ΔIOP = mean IOP4,5 - mean IOP1,2,3). A multilevel linear mixed-effects model assessed the influence of medication (independent variable) on ΔIOP (dependent variable). Additional independent variables of interest included the number of glaucoma medications at baseline, age, sex, glaucoma type and severity, race, and pretreatment IOP. Bootstrap analysis was performed to remove sampling bias and confirm mixed-effects model findings. Kaplan-Meier survival analysis evaluated the probability of requiring additional intervention within 3 years following the date of medication prescription. Results: The unadjusted mean (SD) ΔIOP for netarsudil and brimonidine was -2.20 (4.11) mm Hg and -2.21 (3.25) mm Hg, respectively (P = 0.484). The adjusted linear mixed-effects models and bootstrap analysis demonstrated that there was no statistical difference in IOP-lowering effectiveness between the medications. Netarsudil and brimonidine failed to adequately control IOP at similar rates with 42% and 47% probabilities of survival respectively by the 3-year follow-up (P = 0.520). Conclusions: When escalating pharmacologic therapy, the IOP-lowering effect of netarsudil appeared to be similar to that produced by brimonidine. (Curr Ther Res Clin Exp. 2023; 84:XXX-XXX).

Rho Kinase Inhibitor Y27632 Improves Recovery After Spinal Cord Injury by Shifting Astrocyte Phenotype and Morphology via the ROCK/NF-κB/C3 Pathway.

Spinal cord injury (SCI) usually results in loss or reduction in motor and sensory functions. Despite extensive research, no available therapy can restore the lost functions after SCI. Reactive astrocytes play a pivotal role in SCI. Rho kinase inhibitors have also been shown to promote functional recovery of SCI. However, the role of Rho kinase inhibitors in reactive astrocytic phenotype switch within SCI remains largely unexplored. In this study, astrocytes were treated with proinflammatory cytokines and/or the Rho kinase inhibitor Y27632. Concomitantly the phenotype and morphology of astrocytes were examined. Meanwhile, the SCI model of SD rats was established, and nerve functions were evaluated following treatment with Y27632. Subsequently, the number of A1 astrocytes in the injured area was observed and analyzed. Eventually, the expression levels of nuclear factor kappa B (NF-κB), C3, and S100A10 were measured. The present study showed that the Rho kinase inhibitor Y27632 improved functional recovery of SCI and elevated the proliferation and migration abilities of the astrocytes. In addition, Y27632 treatment initiated the switch of astrocytes morphology from a flattened shape to a process-bearing shape and transformed the reactive astrocytes A1 phenotype to an A2 phenotype. More importantly, further investigation suggested that Y27632 was actively involved in promoting the functional recovery of SCI in rats by inhabiting the ROCK/NF-κB/C3 signaling pathway. Together, Rho kinase inhibitor Y27632 effectively promotes the functional recovery of SCI by shifting astrocyte phenotype and morphology. Furthermore, the pro-regeneration event is strongly associated with the ROCK/NF-κB/C3 signal pathway.

Response to netarsudil in goniotomy-treated eyes and goniotomy-naïve eyes: a pilot study.

PURPOSE: To compare the intraocular pressure (IOP)-lowering effects of netarsudil on goniotomy-treated eyes versus goniotomy-naïve control eyes. METHODS: Retrospective cohort study of 70 eyes from 49 adult glaucoma patients treated with netarsudil. Thirty-five eyes received sectoral goniotomy using Kahook Dual Blade (KDB) combined with cataract surgery with minimum of 3 months prior to netarsudil treatment. Thirty-five eyes in the control cohort received only cataract surgery prior to netarsudil. Primary outcome was treatment success, defined as ≥ 20% decrease in IOP at minimum 1 month follow-up. Secondary outcome measures included percent of IOP reduction, adverse effects of medication, medication discontinuation rate, and relationship between KDB goniotomy response and netarsudil response. RESULTS: Eighty-three percent of KDB-treated eyes achieved netarsudil treatment success compared to 54% of control eyes (P = .012). IOP reduction was 30.3 ± 16.2% (IQR 21-38%) in KDB-treated eyes and 19.4 ± 12.4% (IQR 9.2-30.8) in control eyes (P = .007). History of prior KDB increased the likelihood of success to netarsudil treatment compared to eyes without prior KDB, regardless of surgical response to KDB (odds ratio 4.51, 95% CI 1.34-15.14, P = .015). The overall rate of adverse effects of netarsudil was 42%, most commonly reported as conjunctival hyperemia, allergy, and blurred vision. CONCLUSIONS: Netarsudil had a greater IOP-lowering effect in eyes treated with prior goniotomy and may serve as a promising adjunctive ocular hypotensive agent to further reduce IOP in eyes with prior goniotomy.

Intraocular pressure-lowering efficacy and ocular safety of Rho-kinase inhibitor in glaucoma: a meta-analysis and systematic review of prospective randomized trials.

PURPOSE: To evaluate the intraocular pressure (IOP)-reducing efficacy and safety of Rho-kinase inhibitor (RKI). METHODS: Published studies in PubMed and EMBASE were searched on March 20, 2021. Study selection and data extraction were performed according to PRISMA. Meta-analysis of the IOP-lowering effect was performed with the bivariate random-effects model, with studies categorized into 2 classes: RKI versus placebo and RKI versus another medication. The main outcome was the difference in IOP reduction between RKI and non-RKI groups. Subgroup analysis of adjunctive RKI efficacy and additional review of its major ocular adverse events (AE) were also performed. RESULTS: Ten (2.6%) out of 391 studies were retrieved. In the RKI versus placebo class, RKI showed greater IOP reduction after 4-8 weeks (mean difference = - 1.69 mmHg [- 2.22, - 1.16], P < 0.001). In the RKI versus another medication class, IOP reduction by RKI was noninferior to timolol 0.5% twice-daily after 4-8 weeks (mean difference = 0.39 mmHg [0.01, 0.76], P = 0.043) and 12 weeks (mean difference = 0.48 mmHg [0.11, 0.85]; P = 0.011). In the subgroup analysis, the mean difference in IOP reduction by adjunctive RKI and placebo was - 1.42 mmHg (P < 0.001). The most common ocular AE of RKI was conjunctival hyperemia (19-65%), followed by conjunctival hemorrhage (6-20%) and cornea verticillata (13-26%). CONCLUSIONS: With a treatment duration of 1-3 months, RKI showed effective IOP reduction noninferior to timolol as monotherapy and as adjunctive therapy. Our results suggested RKI be a reliable IOP control medication; however, its higher incidence of some ocular complications should be attended to.

Netarsudil for the Treatment of Open-Angle Glaucoma and Ocular Hypertension: A Literature Review.

OBJECTIVE: To evaluate netarsudil's role as first-line therapy for the treatment of open-angle glaucoma (OAG) and ocular hypertension (OHT). DATA SOURCES: A literature search utilizing MEDLINE and CINAHL was performed using netarsudil and AR-13324 as keywords. Studies published from January 1970 to September 2020 were eligible. STUDY SELECTION AND DATA EXTRACTION: For inclusion, articles were required to be published in English and participants enrolled in phase I, II, or III clinical trials. Articles were excluded if netarsudil was coformulated with another medication. Preclinical research, case reports, case series, review articles, citations without an abstract, and newsletters were excluded. LITERATURE REVIEW: The search retrieved 97 unique citations; 90 results were excluded, and 7 studies were included for analysis. RELEVANCE TO PATIENT CARE AND CLINICAL PRACTICE: In all, 20 years elapsed between the Food and Drug Administration's approvals of distinct medications to treat OAG. Existing first-line therapies target the uveoscleral pathway, which is responsible for a small amount of aqueous humor outflow. Rho kinase inhibitors target the trabecular pathway, which is responsible for 90% of aqueous humor outflow; thus, Rho kinase inhibitors may significantly reduce intraocular pressure and improve clinical outcomes for patients with OAG or OHT. CONCLUSIONS: Evidence demonstrates that netarsudil is inferior to prostaglandin analogues and noninferior to topical ß-blockers in the treatment of OAG and OHT. Hyperemia is a common adverse drug reaction, which often resolves after medication discontinuation. Additional phase III clinical trials and evidence-based guidelines are necessary to determine netarsudil's position in OAG and OHT management.

Effects of 0.02% netarsudil ophthalmic solution on intraocular pressure of normotensive dogs.

OBJECTIVES: To evaluate the effect of QD or BID 0.02% netarsudil ophthalmic solution (Aerie Pharmaceuticals) on intraocular pressure (IOP) in normotensive dogs and to describe any adverse effects. ANIMALS STUDIED: Normotensive Labrador retriever dogs were included in this study: 10 received netarsudil in one eye and artificial tears in the contralateral eye QD, and 10 received netarsudil in one eye and artificial tears in the contralateral eye BID. PROCEDURES: Intraocular pressure curves were acquired over a 3-day acclimation period, 5-day dosing period (QD or BID-10 dogs/group), and 3-day recovery period. Toxicity was assessed daily using slit-lamp biomicroscopy and the semiquantitative preclinical ocular toxicology scoring system. RESULTS: Once-daily dosing did not lower IOP over the entire 5-day dosing period (95% CI 0.1 to -0.9 mm Hg, P = .20) or on the last day of dosing (95% CI 0.4 to -0.9 mm Hg, P = .65). Twice-daily dosing resulted in a statistically significant, but clinically unimportant, IOP reduction over the entire 5-day dosing period (-0.6 mm Hg; 95% CI 0.05 to -1.1 mm Hg, P = .02) and on the last day of dosing (-0.9 mm Hg; 95% CI 0.2 to -1.5 mm Hg, P = .003). Adverse events were limited to transient mild-to-moderate conjunctival hyperemia during the dosing phase in eyes receiving netarsudil vs control (P < .0001). CONCLUSIONS: Netarsudil 0.02% ophthalmic solution twice daily resulted in a small, statistically significant, but clinically unimportant, IOP reduction in normotensive dogs. Future studies should investigate efficacy in glaucomatous dogs.

Intraocular pressure-lowering effects of ripasudil, a rho-kinase inhibitor, and selective laser trabeculoplasty as adjuvant therapy in patients with uncontrolled glaucoma.

OBJECTIVES: To compare the intraocular pressure (IOP)-lowering effects of ripasudil, a rho-kinase inhibitor, and selective laser trabeculoplasty (SLT) as adjuvant therapy in Japanese glaucoma patients and to identify the factors associated with treatment success. METHODS: We performed a retrospective medical chart review of patients with glaucoma who received ripasudil or SLT as an adjuvant therapy. We collected data on 65 eyes (65 patients) with primary open-angle glaucoma, normal-tension glaucoma, or exfoliation glaucoma with at least 12 months of follow-up. IOP and number of glaucoma medications at 0, 1, 3, 6, 9, and 12 months were compared between and within groups. A repeated-measures mixed model was used to perform statistical analysis. We also investigated factors associated with treatment success, which was defined as ≥ 20% reduction in IOP at all follow-up periods, using univariate and multivariate logistic regression analysis. RESULTS: Significant IOP reduction was observed at all time-points after treatment in the ripasudil group (n = 33) and in the SLT group (n = 32), with no statistically significant difference between the groups before or after treatment. Patients in the SLT group used more anti-glaucoma medications before treatment, but fewer during follow-up, than those in the ripasudil group. Regardless of treatment, higher baseline IOP was associated with treatment success [crude odds ratio: 1.21 (95% confidence interval: 1.06-1.38), adjusted odds ratio: 1.37 (95% confidence interval: 1.06-1.77)]. CONCLUSIONS: Adjuvant SLT or ripasudil in patients with inadequately controlled glaucoma both reduced IOP to a similar degree, but SLT contributed to reducing the number of medications used.

Synergistic effects of Rho kinase inhibitor Y-27632 and Noggin overexpression on the proliferation and neuron-like cell differentiation of stem cells derived from human exfoliated deciduous teeth.

Stem cells from human exfoliated deciduous teeth (SHEDs) are highly proliferative, clonogenic, and multipotent stem cells with a neural crest cell origin. This property could be a desirable option for potential therapeutic applications. In this study, we focus on the effects of Rho kinase inhibitors Y-27632 and Noggin on the proliferation of SHEDs and their differentiation into neuron-like cells. SHEDs were extracted from 10 samples of deciduous teeth obtained from healthy children aged from 5 to 10. The passaged SHEDs were transfected with Noggin, Y-27632, or their combination. By means of MTT and colony formation assays, the effects of Y-27632 and Noggin on cell viability and colony formation were detected. Cellular morphology and neurosphere formation were observed under a microscope. Y-27632 transfection in SHEDs showed enhanced cell viability, colony formation, and neurosphere formation indicating that Y-27632 could promote cell proliferation of SHEDs. Furthermore, we observed that the SHEDs treated with Noggin in combination with Y-27632 displayed typical neuron-like cell morphology and reticular processes. Noggin or Y-27632 alone or in combination induced obviously increased NSE, Nestin, and GFAP levels, which were highest in SHEDs treated with the combination of Noggin and Y-27632. These findings suggest that Y-27632 promotes the proliferation of SHEDs, and Y-27632 and Noggin in combination have a synergistic effect on promoting differentiation of SHEDs into neuron-like cells.

Acute lung injury: The therapeutic role of Rho kinase inhibitors.

Acute lung injury (ALI) is a pulmonary illness with high rates of mortality and morbidity. Rho GTPase and its downstream effector, Rho kinase (ROCK), have been demonstrated to be involved in cell adhesion, motility, and contraction which can play a role in ALI. The electronic databases of Google Scholar, Scopus, PubMed, and Web of Science were searched to obtain relevant studies regarding the role of the Rho/ROCK signaling pathway in the pathophysiology of ALI and the effects of specific Rho kinase inhibitors in prevention and treatment of ALI. Upregulation of the RhoA/ROCK signaling pathway causes an increase of inflammation, immune cell migration, apoptosis, coagulation, contraction, and cell adhesion in pulmonary endothelial cells. These effects are involved in endothelium barrier dysfunction and edema, hallmarks of ALI. These effects were significantly reversed by Rho kinase inhibitors. Rho kinase inhibition offers a promising approach in ALI [ARDS] treatment.

Discovery of a phenylpyrazole amide ROCK inhibitor as a tool molecule for in vivo studies.

Structure-activity relationship optimization on a series of phenylpyrazole amides led to the identification of a dual ROCK1 and ROCK2 inhibitor (25) which demonstrated good potency, kinome selectivity and favorable pharmacokinetic profiles. Compound 25 was selected as a tool molecule for in vivo studies including evaluating hemodynamic effects in telemeterized mice, from which moderate decreases in blood pressure were observed.

Dose-dependent effects of netarsudil, a Rho-kinase inhibitor, on the distal outflow tract.

PURPOSE: To characterize the effects of netarsudil on the aqueous humor outflow tract distal to the trabecular meshwork (TM). We hypothesized that netarsudil increases outflow facility in eyes with and without circumferential ab interno trabeculectomy (AIT) that removes the TM. METHODS: Sixty-four porcine anterior segment cultures were randomly assigned to groups with (n = 32) and without circumferential AIT (n = 32). Cultures were exposed to 0.1, 1, and 10 µM netarsudil (N = 8 eyes per concentration). For each concentration, IOP and vessel diameters were compared with their respective pretreatment baselines. Outflow tract vessel diameters were assessed by spectral-domain optical coherence tomography (SDOCT) and rendered in 4D (XYZ time series). RESULTS: Netarsudil at 1 µM reduced IOP both in eyes with TM (- 0.60 ± 0.24 mmHg, p = 0.01) and in eyes without TM (- 1.79 ± 0.42 mmHg, p < 0.01). At this concentration, vessels of the distal outflow tract dilated by 72%. However, at 0.1 µM netarsudil elevated IOP in eyes with TM (1.59 ± 0.36 mmHg, p < 0.001) as well as in eyes without TM (0.23 ± 0.32 mmHg, p < 0.001). Vessels of the distal outflow tract constricted by 31%. Similarly, netarsudil at a concentration of 10 µM elevated IOP both in eyes with TM (1.91 ± 0.193, p < 0.001) and in eyes without TM (3.65 ± 0.86 mmHg, p < 0.001). At this concentration, outflow tract vessels constricted by 27%. CONCLUSION: In the porcine anterior segment culture, the dose-dependent IOP changes caused by netarsudil matched the diameter changes of distal outflow tract vessels. Hyper- and hypotensive properties of netarsudil persisted after TM removal.

The protective effect of fasudil against acrylamide-induced cytotoxicity in PC12 cells.

Acrylamide (ACR), a vinyl monomer that has multiple chemical and industrial applications, is a neurotoxic agent in human and animal. Fasudil is a potent Rho-kinase inhibitor which exhibits neuroprotective effects in some neuronal degenerative disorders. In this study, the potential protective effect of Fasudil on ACR-induced cytotoxicity in PC12 cells was evaluated. Our results showed that ACR increased the level of intracellular reactive oxygen species (ROS) and consequently upregulated the Bax/Bcl-2 ratio and significantly elevated the level of caspase-3 and 9 proteins in PC12 cells. Interestingly, pretreatment with Fasudil protected PC12 cells against ACR-induced toxicity mainly through the reduction of ROS production and modulation of proteins which involved in apoptosis pathway. Fasudil down-regulated the Bax/Bcl-2 ratio and the levels of caspase-3 and 9 proteins in cells exposed to ACR. In conclusion, the neuroprotective effect of Fasudil against ACR-induced toxicity in PC12 cells appears to be mediated through inhibition of ROS production and modulation of apoptosis.

Vitamin A-coupled liposomal Rho-kinase inhibitor ameliorates liver fibrosis without systemic adverse effects.

AIM: Rho-kinase (ROCK) inhibitor could ameliorate liver fibrosis by suppressing hepatic stellate cell (HSC) activation. However, because systemic administration of ROCK inhibitor causes serious adverse effects, we developed a drug delivery system selectively delivering ROCK inhibitor to HSCs. Here, we examined whether our developed vitamin A (VA)-coupled liposomal ROCK inhibitor reduced liver fibrosis in rats without causing systemic adverse effects. METHODS: LX-2 HSCs were analyzed for morphological changes and the expression of profibrotic proteins. The inhibitory effects of VA-coupled liposomal ROCK inhibitor on liver fibrosis were confirmed in a rat model of liver fibrosis induced by i.p. injection of carbon tetrachloride. The degree of liver fibrosis, biochemical changes, and survival rates were also investigated. RESULTS: Vitamin A-coupled liposomal ROCK inhibitor had an effect at approximately 1/100 the amount of the free ROCK inhibitor for inhibiting the activation of LX-2 cells and caused significant decreases in the expression levels of α-smooth muscle actin (SMA) and transforming growth factor (TGF)-ß1. The degree of liver fibrosis was suppressed by treatment with VA-coupled liposomal ROCK inhibitor, and the expression of α-SMA and TGF-ß1 in liver tissues was also significantly suppressed. In addition, serum levels of alanine aminotransferase and hyaluronic acid were significantly reduced, and there was no decline in kidney function, which has been noted as a systemic adverse effect of ROCK inhibitor. Furthermore, VA-coupled liposomal ROCK inhibitor improved survival rates in rats with liver fibrosis. CONCLUSION: Vitamin A-coupled liposomal ROCK inhibitor efficiently suppressed liver fibrosis without causing systemic adverse effects.

Systemic Rho-kinase inhibition using fasudil in mice with oxygen-induced retinopathy.

PURPOSE: To investigate the influence of the selective Rho-kinase (ROCK) inhibitor, fasudil, on the mRNA level of proinflammatory factors and the retinal vascular development in mice with oxygen-induced retinopathy (OIR). METHODS: C57BL/6J mice underwent standard protocol for OIR induction from postnatal days 7 to 12. Subsequently, they received a daily intraperitoneal injection of fasudil or sodium chloride from P12 to P16. Analyses were performed using vascular staining on retinal flat mounts, RNA expression by qPCR, and immunohistochemistry on paraffin sections. RESULTS: On retinal flat mounts, the proportion of avascular area and tuft formation did not differ between the fasudil and NaCl group. Immunohistochemical staining revealed a less intense staining with inflammatory markers after fasudil. Nevertheless, there were no differences on RNA level between the two groups. CONCLUSIONS: In conclusion, our findings support that daily systemic application of fasudil does not decrease retinal neovascularization in rodents with oxygen-induced retinopathy. The results of our study together with the controversial results on the effects of different ROCK inhibitors from the literature makes it apparent that effects of ROCK inhibition are more complex, and further studies are necessary to analyze its potential therapeutic effects.

Effects of newly synthetized isoquinoline derivatives on rat uterine contractility and ROCK II activity.

Protein kinases have an important role in signal transduction in the cellular system via protein phosphorylation. RhoA activated Rho-kinases have a pivotal role in the regulation of smooth muscle contraction. ROCK I and ROCK II phosphorylate myosin-phosphatase and myosin-kinase, which induces contraction in the myometrium. Several studies have investigated the affinity of isoquinoline alkaloids (HA-1077, H1152P) to Rho-kinases, and these compounds notably inhibited the Ca2+-independent process. We measured the efficiency of 25 original, newly synthesized isoquinoline derivatives for the Rho-kinase activity using Rho-associated kinase activity assay and determined their effects on the non-pregnant, 20-day pregnant and parturient rat myometrial contraction in vitro. The IC50 values of 11 from among the 25 derivatives were significantly lower on the oxytocin-induced non-pregnant rat uterine contraction compared with Y-27632 and fasudil, although their maximal inhibitory effects were weaker than those of Y-27632 and fasudil. We measured the effects of 11 isoquinoline molecules with significant IC50 values on ROCK II activity. We found two isoquinolines out of 11 compounds (218 and 852) which decreased the active ROCK II level similarly as Y-27632. Then we found that 218 and 852 relaxed the 20th-day pregnant and parturient rat uterus with greater potency as compared with fasudil. The majority of the synthesized isoquinoline derivatives have uterus relaxant effects and two of them significantly suppress the Rho-kinase mediated myosin light chain phosphorylation. Our results may suggest that the isoquinoline structure has a promising prospect for the development of new and effective inhibitors of uterine contractions in preterm birth.

[Acute effects of intravenous fasudil with different dosage on patients with congenital heart defects and severe pulmonary arterial hypertension].

Objective: To compare the acute hemodynamic effects of intravenous fasudil with different dosage on patients with congenital heart defects (CHD) and severe pulmonary arterial hypertension (PAH). Methods: Sixty patients (37±17 years old) with CHD and PAH were consecutively enrolled. All patients underwent heart catheterization. The patients were randomly divided into two groups: the regular dosage group and the large dosage group. At initiation and 30 min after intravenous fasudil(30 mg and 60 mg respectively), the following hemodynamic parameters were measured and calculated: right atrial pressure(RAP), pulmonary arterypressure(PAP) , systemic artery pressure (SAP), pulmonary capillary wedge pressure(PCWP) , pulmonary vascular resistance(PVR) and systemic vascular resistance( SVR), cardiac index (CI) and artery oxygen saturation (SaO(2)). Results: Compared with pre-medication, both mPAP and PVR tended to reduce significantly in the regular dosage group and the large dosage group: mPAP from (63.7±8.6)to (58.3±8.5)mmHg(P<0.01) and from (62.9±8.8) to(55.1±7.8)mmHg (P<0.01), respectively; PVR from(9.9±4.3)to (7.7±3.9) Wood(P<0. 01) and from (9.5±4.9)to(6.1±4.8)Wood(P<0.01); CI tended to increase significantly in the two groups: from (2.9±0.9) to (3.1±1.1) L·min(-1)·m(-2)( P<0.05) and from(3.0±0.8) to (3.5±1.6) L·min(-1)·m(-2)( P<0.05), respectively . Compared with the regular dosage group, both mPAP and PVR tended to reduce significantly in the large dosage group: mPAP (8.2±1.8) vs (4.2±1. 0)mmHg (P<0.05); PVR(3.7±1.1) vs (2.1±0.8 ) Wood (P<0.05) .Meanwhile , there was no significant difference in CI, SAP, SVR and SaO(2) between the two groups. Conclusion: Fasudil could improve the acute hemodynamic effects of patients with CHD and severe PAH, especially in the large dosage group.

Additive effects of the Rho kinase inhibitor Y-27632 and vardenafil on relaxation of the corpus cavernosum tissue of patients with erectile dysfunction and clinical phosphodiesterase type 5 inhibitor failure.

OBJECTIVES: To evaluate the expression of the Rho/Rho-associated protein kinase (ROCK) pathway in the corpus cavernosum of patients with severe erectile dysfunction (ED) compared with healthy human corpus cavernosum, and to test the functional effects of two Rho kinase inhibitors (RKIs) on erectile tissue of patients with severe ED, which did not respond to phosphodiesterase type 5 inhibitors (PDE5Is). PATIENTS AND METHODS: Human corpus cavernosum samples were obtained after consent from men undergoing penile prosthesis implantation (n = 7 for organ bath experiments, n = 17 for quantitative PCR [qPCR]). Potent control subjects (n = 5) underwent penile needle biopsy. qPCR was performed for the expression of RhoA and ROCK subtypes 1 and 2. Immunohistochemistry staining against ROCK and α smooth muscle actin (αSMA) was performed on the corpus cavernosum of patients with ED. Tissue strips were precontracted with phenylephrine and incubated with 1 µm of the PDE5I vardenafil or with DMSO (control). Subsequently, increasing concentrations of the RKIs azaindole or Y-27632 were added, and relaxation of tissue was quantified. RESULTS: The expression of ROCK1 was unchanged (P > 0.05), while ROCK2 (P < 0.05) was significantly upregulated in patients with ED compared with controls. ROCK1 and ROCK2 protein colocalized with αSMA, confirming the presence of this kinase in cavernous smooth muscle cells and/or myofibroblasts. After incubation with DMSO, 10 µm azaindole and 10 µm Y-27632 relaxed precontracted tissues with 49.5 ± 7.42% (P = 0.1470 when compared with vehicle) and 85.9 ± 10.3% (P = 0.0016 when compared with vehicle), respectively. Additive effects on relaxation of human corpus cavernosum were seen after preincubation with 1 µm vardenafil. CONCLUSION: The RKI Y-27632 causes a significant relaxation of corpus cavernosum in tissue strips of patients with severe ED. The additive effect of vardenafil and Y-27632 shows that a combined inhibition of Rho-kinase and phosphodiesterase type 5 could be a promising orally administered treatment for severe ED.