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Purpose: To analyze the efficacy, safety, and accessibility of netarsudil 0.02% in patients with glaucoma (suspect, open or closed) at a safety-net academic medical center, Boston Medical Center (BMC). Methods: Retrospective chart review of patients prescribed netarsudil 0.02% for uncontrolled glaucoma at BMC between December 2017 and September 2019. Outcome measures included change in intraocular pressure (IOP) from baseline and evaluation of adverse events (AEs). Results: One hundred thirty patients (60% severe stage) were analyzed. The IOP reduction from baseline was about 3 mmHg. Fifty-four patients (42%) experienced an AE (eg, conjunctival hyperemia). Thirty-eight patients (29%) started netarsudil 0.02% in lieu of laser or surgery. Ninety-nine patients (71%) required prior authorization for insurance coverage of netarsudil 0.02%. Ten patients (7%) were unable to obtain netarsudil 0.02% due to issues with insurance coverage. Conclusion: Netarsudil 0.02% yielded significant IOP reduction in our cohort, however, to a smaller degree compared with prior studies that bore equivocal IOP reduction regardless of baseline IOP. Conjunctival hyperemia was the most common AE. In a limited number of patients, netarsudil 0.02% was not covered by insurance.

Notch3 mutations cause Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), which predisposes to stroke and dementia. CADASIL is characterised by vascular dysfunction and granular osmiophilic material (GOM) accumulation in cerebral small vessels. Systemic vessels may also be impacted by Notch3 mutations. However vascular characteristics and pathophysiological processes remain elusive. We investigated mechanisms underlying the peripheral vasculopathy mediated by CADASIL-causing Notch3 gain-of-function mutation. We studied: (i) small arteries and vascular smooth muscle cells (VSMCs) from TgNotch3R169C mice (CADASIL model), (ii) VSMCs from peripheral arteries from CADASIL patients, and (iii) post-mortem brains from CADASIL individuals. TgNotch3R169C vessels exhibited GOM deposits, increased vasoreactivity and impaired vasorelaxation. Hypercontractile responses were normalised by fasudil (Rho kinase inhibitor) and 4-phenylbutyrate (4-PBA; endoplasmic-reticulum (ER) stress inhibitor). Ca2+ transients and Ca2+ channel expression were increased in CADASIL VSMCs, with increased expression of Rho guanine nucleotide-exchange factors (GEFs) and ER stress proteins. Vasorelaxation mechanisms were impaired in CADASIL, evidenced by decreased endothelial nitric oxide synthase (eNOS) phosphorylation and reduced cyclic guanosine 3′,5′-monophosphate (cGMP) levels, with associated increased soluble guanylate cyclase (sGC) oxidation, decreased sGC activity and reduced levels of the vasodilator hydrogen peroxide (H2O2). In VSMCs from CADASIL patients, sGC oxidation was increased and cGMP levels decreased, effects normalised by fasudil and 4-PBA. Cerebral vessels in CADASIL patients exhibited significant oxidative damage. In conclusion, peripheral vascular dysfunction in CADASIL is associated with altered Ca2+ homoeostasis, oxidative stress and blunted eNOS/sGC/cGMP signaling, processes involving Rho kinase and ER stress. We identify novel pathways underlying the peripheral arteriopathy induced by Notch3 gain-of-function mutation, phenomena that may also be important in cerebral vessels.

AIM: To investigate the effects of a selective inhibitor of Rho-associated kinase (ROCK), Y-27632, on inbred Wuzhishan porcine corneal endothelial cells (PCECs) in vitro and in vivo studies. METHODS: Primary PCECs were trypsinized from Wuzhishan miniature porcine corneal tissues. The optimal concentration of Y-27632 on PCECs was determined through MTT and 5-ethynyl-2’-deoxyuridine (EdU)-labeling assays. Seven New Zealand rabbits were used as a corneal endothelial dysfunction model, and a PCECs suspension supplemented with Y-27632 was injected into the anterior chamber of the rabbits. The progression of rabbit corneal opacity and edema were observed by slit lamp examination. The rabbits were sacrificed, and rabbit globes were enucleated for trypan blue-alizarin red staining, hematoxylin-eosin staining, and immunofluorescence analysis. RESULTS: Administration of 100 μmol/L Y-27632 facilitated PCECs’ proliferation obviously. The rabbit corneas injected with PCECs suspension and 100 μmol/L Y-27632 were restored to transparency significantly after 14d. CONCLUSION: The 100 μmol/L Y-27632 treatment improves PCECs’ proliferation significantly. And our results suggest that Y-27632 and PCECs can be used to treat corneal endothelial dysfunction.

In this study, we investigated the effects of emotional stress and physical stress using the social defeat stress (SDS) model in mice. Male C57BL/6 J mice were attacked by male non-retired ICR mice for 10 min daily for 10 days (physical stress; PS), while the other cohort of mice witnessed the defeat (emotional stress; ES). As a result, both PS and ES mice exhibited decreased social behavior in the social interaction test (SIT) and increased immobility in the forced swim test (FST). Interestingly, only ES mice exhibited decreased sucrose preference, and only PS mice exhibited decreased time spent in the open arms in the elevated plus-maze test. ES mice did not exhibit increased levels of corticosterone and epinephrine after a single stress exposure, but showed a decrease in plasma CXCL16 levels 1 month after stress exposure. Finally, a RhoA/Rho kinase inhibitor, fasudil, which has been reported to attenuate the effects of chronic stress, suppressed the increased immobility in the FST in PS mice, but not in ES mice. These results demonstrate that, although ES and PS mice shared many characteristics, the effects of emotional stress are not identical to those of physical stress in mice.

Purpose: To evaluate efficacy and safety of ripasudil for 1 year in addition to or replacing existing treatment regimens. Methods: We retrospectively reviewed the medical records for 128 eyes of 12...

ZusammenfassungGlaukom ist in Deutschland die zweithäufigste Ursache irreversibler Erblindungen. Es stehen verschiedene therapeutische Ansätze zur Verfügung. Neben der Verbesserung der vaskulären Perfusion und der Reduktion des oxidativen Stresses steht als zentrale Therapieoption nach wie vor die Senkung des Augeninnendrucks im Vordergrund. Dafür kommen verschiedene Wirkstoffklassen mit unterschiedlichem Wirkmechanismus und Nebenwirkungsprofil zur Anwendung.

Intra-procedural myocardial ischemia as an iatrogenic complication still remains a critical issue in contemporary interventional cardiology. The aim of this study was to examine the usefulness of fasudil, a selective Rho-kinase inhibitor, for percutaneous coronary intervention (PCI)-related myocardial ischemia.Among 448 PCI sessions performed between October 2015 and December 2017, we retrospectively examined 36 patients (69.0 ± 9.1 [SD] yrs., M/F 26/10) who underwent intracoronary administration of fasudil during a procedure to resolve myocardial ischemia that was resistant to intracoronary nitrate administration.The refractory myocardial ischemia was caused by distal embolization (69%), enhanced vasoconstriction at distal site of chronic total occlusion (11%), coronary spasm (11%), and coronary dissection (8%), most of which occurred immediately after balloon or stent dilatation. Intracoronary fasudil significantly improved corrected TIMI frame count (from 37 [30-56] to 24 [12-36]) and TIMI flow grade (from 2 [1-2.5] to 3 [2-3]) (both P 0.001). Finally, 86% of all subjects successfully obtained TIMI flow grade 3 at the end of the procedure. Intracoronary fasudil tended to be more effective in patients with an attenuated plaque detected by intravascular ultrasound. Importantly, among the 19 elective cases, fasudil successfully prevented 17 patients from developing post-procedure myocardial infarction. Although fasudil-induced transient hypotension requiring a vasopressor was noted in 22% of the subjects, no other adverse effects were noted.These results indicate that fasudil is a useful and safe therapeutic option for PCI-related myocardial ischemia refractory intracoronary nitrate.

Mayuresh Naik,1 Monika Kapur,2 VishnuSwarup Gupta,1 HarinderSingh Sethi,3 Kartikeya Srivastava3 1Department of Ophthalmology, H.I.M.S.R & H.A.H. Centenary Hospital, New Delhi, India; 2Department of Ophthalmology, School of Medical Sciences & Research, Sharda University, Greater Noid, Uttar Pradesh, India; 3Department of Ophthalmology, V.M.M.C & Safdarjung Hospital, New Delhi, 110029, IndiaCorrespondence: Mayuresh NaikDepartment of Ophthalmology, H.I.M.S.R & H.A.H. Centenary Hospital, Room No. 3 of Eye OPD, 1st Floor of OPD Building, Near GK-2, Alaknanda, New Delhi, 110062, IndiaTel +91-8287344576Email mayureshpnaik@gmail.comPurpose: To elucidate the use of Ripasudil in patients of advanced glaucoma on maximally tolerated medical therapy who could not be offered the option of surgery due to the global pandemic lockdown.Materials and Methods: Only patients with primary open angle glaucoma (POAG), who had a cup-disc ratio (CDR) of 0.9 or a near total cupping on maximum tolerated medical therapy for at least 4 weeks and yet could not meet the target IOP were included. Target IOP was defined as ≤ 12 mm Hg. A total of 30 patients were enrolled. All patients in study cohort were started on E/D Ripasudil BD. Patients were followed up at 1 week, 2 weeks, 4 weeks and then monthly for 6 months for their best corrected visual acuity (BCVA), intraocular pressure (IOP), disc changes (slit lamp biomicroscopy), perimetry, and retinal nerve fibre layer analysis using optical coherence tomography (OCT-RNFL).Results: Mean pre-treatment IOP on five drugs was 18.3 ± 2.1 mm Hg (range 14 to 22mmHg) on maximally tolerated medical therapy. At 1 week follow-up, mean post-treatment IOP was 15.1 ± 1.7 mm Hg (range 12 to 18mmHg) and at 2 week follow-up, mean post-treatment IOP was 12.5 ± 1.9 mmHg (range 10 to 16mmHg). Thus, target IOP ≤ 12mmHg was attained in 28 patients at 2 weeks. This target IOP was maintained throughout the 6 months of follow-up period. Of the 2 patients who could not meet target IOP, 1 patient needed rearrangement of their fixed-drug-combinations to achieve target IOP at 4 weeks. The second patient required unfixing of all fixed-drug-combinations to achieve target IOP at maximally tolerated medical therapy at 6 weeks.Conclusion: Ripasudil not only provides a better IOP control but also has a high safety profile even when started as an add-on drug to already-existing yet inadequate maximally tolerated medical therapy.Keywords: advanced glaucoma, ripausdil, ROCK1 inhibitor, maximally-tolerated-medical-therapy, COVID-19 lockdown

The insertion of mouse renin gene (Ren-2) into the genome of normotensive rats causes a spontaneous rise of blood pressure (BP), leading to an angiotensin II (Ang II)-dependent form of hypertension in transgenic (mRen-2)27 rats (TGR). However, enhanced sympathetic BP component was demonstrated in heterozygous TGR aged 20 weeks. In the present study we used another model, i.e. Cyp1a1-Ren-2 transgenic rats (iTGR) in which hypertension can be induced by natural xenobiotic indole-3 carbinol (I3C) added to the diet. We investigated whether the development of high blood pressure (BP) in 5-month-old iTGR animals fed I3C diet for 10 days is solely due to enhanced Ang II-dependent vasoconstriction or whether enhanced sympathetic vasoconstriction also participates in BP maintenance in this form of hypertension. Using acute sequential blockade of renin-angiotensin system (RAS), sympathetic nervous system (SNS) and NO synthase (NOS) we have demonstrated that the observed gradual increase of BP in iTGR fed I3C diet was entirely due to the augmentation of Ang II-dependent BP component without significant changes of sympathetic BP component. Thus, the hypertension in iTGR resembles to that of homozygous TGR in which high BP was entirely dependent on Ang II-dependent vasoconstriction. Moreover, our measurements of acute BP response to Rho kinase inhibitor fasudil in animals subjected to a combined blockade of RAS, SNS and NOS indicated the attenuation of basal calcium sensitization in both iTGR and homozygous TGR.

Glaucoma leads to irreversible blindness. Numerous anti-glaucoma eye drops have been developed. Unfortunately, many patients with glaucoma still suffer from progressive visual disorders. Recently, ripasudil hydrochloride hydrate, a selective Rho-associated protein kinase inhibitor, was launched for the treatment of glaucoma. However, adverse events, such as conjunctival hyperemia, are often noted in clinical trials using healthy subjects. Therefore, we investigated the onset, offset, and kinetic changes of conjunctival hyperemia induced by ripasudil ophthalmic solution in patients with open-angle glaucoma or ocular hypertension who had already been treated with anti-glaucoma eye drops other than ripasudil. Conjunctival hyperemia was evaluated by both clinical grading by 3 ophthalmic physicians and pixel coverage of conjunctival blood vessels determined by conjunctival hyperemia-analyzing software. Conjunctival hyperemia appeared within 10 min post-instillation in most of the participants. Clinical grade and pixel coverage increased significantly 10 min post-instillation and then decreased. In most of the participants, hyperemia resolved within 2 h. Median conjunctival hyperemia offset was 90 min. A tendency of monotonic increase was observed between clinical grade and pixel coverage. Taken altogether, hyperemia induced by ripasudil was transient in glaucoma patients who had already been treated with anti-glaucoma eye drops other than ripasudil.

Objective We previously found that mirabegron exerts a relaxant effect in the presence of the β3 -adrenoceptor antagonist SR58894A during carbachol-induced contraction in human and pig detrusor. The aim of this study was to explore the possible mechanism underlying the relaxant effects of mirabegron using detrusor smooth muscle. Methods Human tissue was obtained from urinary bladders of patients undergoing radical cystectomy at Kyushu University and Harasanshin Hospital. Pig tissue was obtained from an abattoir. Tension force (organ bath experiments) was measured in intact or permeabilised (α-toxin or β-escin) detrusor smooth muscle strips. The contribution of cAMP-dependent signaling and the inhibition of Ca2+ sensitization to the relaxant effects of mirabegron were characterized using 1 μM SR58894A, 100 μM SQ22536 (an adenylyl cyclase inhibitor), 10 μM H-89 (a protein kinase [PK] A inhibitor), 10 μM Y-27632 (a selective Rho kinase inhibitor), and 10 μM GF-109203X (a selective PKC inhibitor). Results 30 μM Mirabegron impaired carbachol (0.03-1 μM)-induced contraction in human detrusor smooth muscle. SR58894A only partially attenuated the relaxant effects of mirabegron in human and pig detrusor strips precontracted with 1 μM carbachol. In α-toxin-permeabilized detrusor strips, tension force at 1 μM [Ca2+ ]i was decreased by mirabegron in a concentration-dependent manner. The relaxant effect of mirabegron was only slightly attenuated by H-89 and not significantly affected by SQ22536. Y-27632 potentiated the relaxation response to mirabegron, but attenuated responses to cAMP; GF-109203X had little effect. Mirabegron but not cAMP had a notable relaxant effect in the pig detrusor smooth muscle permeabilized with β-escin. Conclusions Mirabegron-induced relaxation of pig and human detrusor smooth muscle occurs via both a β3 -adrenoceptor/cAMP-dependent and -independent pathway.

Multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), and other neurodegenerative diseases of central nervous system (CNS) disorders are serious human health problems. Rho-kinase (ROCK) is emerging as a potentially important therapeutic target relevant to inflammatory neurodegeneration diseases. This is supported by studies showing the beneficial effects of fasudil, a ROCK inhibitor, in inflammatory neurodegeneration diseases. MS is an autoimmune disease resulting from inflammation and demyelination in the white matter of the CNS. It has been postulated that activation of Rho/ROCK causes neuropathological changes accompanied with related clinical symptoms, which are improved by treatment with ROCK inhibitors. Therefore, inhibition of abnormal activation of the Rho/ROCK signaling pathway appears to be a new mechanism for treating CNS diseases. In this review, we extensively discussed the role of ROCK inhibitors, summarized the efficacy of fasudil in the MS conventional animal model of experimental autoimmune encephalomyelitis (EAE), both in vivo and in vitro, and highlighted the mechanism involved. Overall, the findings collected in this review support the role of the ROCK signaling pathway in neurodegenerative diseases. Hence, ROCK inhibitors such as fasudil can be novel, and efficacious treatment for inflammatory neurodegenerative diseases.

Background Cardiac sympathetic hyperinnervation after myocardial infarction (MI) is associated with a high incidence of lethal arrhythmia. Erythropoietin-producing hepatoma interactor B2 (EphrinB2), a diffusible axonal chemorepellent that can induce growth cone collapse and axon repulsion of several neuronal populations, is crucial in neurodevelopment during disease development and progression. However, whether EphrinB2 could inhibit cardiac sympathetic hyperinnervation after MI remains unclear. Methods and results A rat model of MI was developed by left anterior descending coronary artery ligation. EphrinB2 expression was markedly increased in the infarcted border at 3 days after MI. Downregulation of EphrinB2 by intramyocardial injection of lentivirus carrying EphrinB2-shRNA significantly increased sympathetic hyperinnervation along with downregulated RhoA expression. In contrast, injection of EphrinB2-overexpressing lentivirus markedly upregulated EphrinB2, concomitant with inhibition of sympathetic sprouting and upregulated RhoA expression, accompanied by decreased incidence of ventricular arrhythmias (VAs). However, co-administering EphrinB2-overexpressing lentivirus and Fasudil (Rho kinase inhibitor) nearly abolished the inhibition of nerve sprouting effect. Additionally, EphrinB2 expression did not affect nerve growth factor level in the infarcted heart. Conclusions Overexpression of EphrinB2 may ameliorate MI-induced sympathetic hyperinnervation and further reduce the incidence of VAs, at least in part by activating RhoA-mediated axonal retraction.

Glucocorticoids are widely used as an ophthalmic medication. A common, sight-threatening adverse event of glucocorticoid usage is ocular hypertension, caused by dysfunction of the conventional outflow pathway. We report that netarsudil, a rho-kinase inhibitor, decreased glucocorticoid-induced ocular hypertension in patients whose intraocular pressures were poorly controlled by standard medications. Mechanistic studies in our established mouse model of glucocorticoid-induced ocular hypertension show that netarsudil both prevented and reduced intraocular pressure elevation. Further, netarsudil attenuated characteristic steroid-induced pathologies as assessed by quantification of outflow function and tissue stiffness, and morphological and immunohistochemical indicators of tissue fibrosis. Thus, rho-kinase inhibitors act directly on conventional outflow cells to prevent or attenuate fibrotic disease processes in glucocorticoid-induced ocular hypertension in an immune-privileged environment. Moreover, these data motivate the need for a randomized prospective clinical study to determine whether netarsudil is indeed superior to first-line anti-glaucoma drugs in lowering steroid-induced ocular hypertension.

This study aimed to investigate the efficacy of the rho-kinase (ROCK) inhibitor ripasudil administered as ophthalmic solutions for chronic visual acuity impairment induced by Fuchs’ endothelial corneal dystrophy (FECD). Twelve patients with FECD and glaucoma (20 eyes) treated with eye drops (0.4% ripasudil) twice a day were investigated. The control group comprised 12 patients with FECD only (i.e., without glaucoma) (20 eyes). Ripasudil was used during the 18 months of the observation period. All participants had grade 6 dystrophy according to the modified Krachmer grading scale. The mean logMAR visual acuity was improved at 3, 6, 12, and 18 months after ripasudil administration. LogMAR changes 18 months after administration were 0.181 ± 0.170 in the ripasudil group and were significantly superior (P = 9.5 × 10−4) to those in the control group (−0.057 ± 0.106). Central corneal thickness (CCT) 18 months after ripasudil administration/CCT before administration were 0.945 ± 0.029 in the ripasudil group and significantly superior (P = 2.4 × 10−7) compared with those in the control group (1.040 ± 0.032). ROCK inhibitors can be effective against visual acuity impairment and corneal edema in FECD. They can be more effective when administered during the waiting period before corneal endothelium transplantation.

International audience; Rho-associated kinase (ROCK) activation was shown to contribute to microvascular closure, retinal hypoxia, and to retinal pigment epithelium (RPE) barrier disruption in a rat model of diabetic retinopathy. Fasudil, a clinically approved ROCK inhibitor, improved retinal perfusion and reduced edema in this model, indicating that ROCK inhibition could be a promising new therapeutic approach for the treatment of diabetic retinopathy. However, due to its short intravitreal half-life, fasudil is not suitable for long-term treatment. In this study, we evaluated a very potent ROCK1/2 inhibitor (BIRKI) in a depot formulation administered as a single intravitreal injection providing a slow release for at least four weeks. Following BIRKI intravitreal injection in old Goto-Kakizaki (GK) type 2 diabetic rats, we observed a significant reduction in ROCK1 activity in the retinal pigment epithelium/choroid complex after 8 days and relocation of ROCK1 to the cytoplasm and nucleus in retinal pigment epithelium cells after 28 days. The chronic ROCK inhibition by the BIRKI depot formulation restored retinal pigment epithelial cell morphology and distribution, favored retinal capillaries dilation, and reduced hypoxia and inner blood barrier leakage observed in the diabetic retina. No functional or morphological negative effects were observed, indicating suitable tolerability of BIRKI after intravitreous injection. In conclusion, our data suggest that sustained ROCK inhibition, provided by BIRKI slow-release formulation, could be a valuable treatment option for diabetic retinopathy, especially with regard to the improvement of retinal vascular infusion and protection of the outer retinal barrier.

Background and aims: Expansion and morphological dysregulation of the bronchial vascular network occurs in asthmatic airways. Interleukin (IL) -17 and Rho-kinase (ROCK) are known to act in inflammation control and remodeling. Modulation of Rho-kinase proteins and IL-17 may be a promising approach for the treatment of asthma through the control of angiogenesis. Our objective was to analyze the effects of treatment with anti-IL17 and/or Rho-kinase inhibitor on vascular changes in mice with chronic allergic pulmonary inflammation. Methods: Sixty-four BALB/c mice, with pulmonary inflammation induced by ovalbumin were treated with anti-IL17A (7.5/µg per dose, intraperitoneal) and/or Rho-kinase inhibitor (Y-27632-10 mg/kg, intranasal), 1 h before each ovalbumin challenge (22, 24, 26, and 28/days). Control animals were made to inhale saline. At the end of the protocol, lungs were removed, and morphometric analysis was performed to quantify vascular inflammatory, remodeling, and oxidative stress responses. Results: Anti-IL17 or Rho-kinase inhibitor reduced the number of CD4+, CD8+, dendritic cells, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, Rho-kinase 1 and 2, transforming growth factor (TGF-β), vascular endothelial growth factor (VEGF), nuclear factor (NF)-KappaB, iNOS, metalloproteinase (MMP)-9, MMP-12, metalloproteinase inhibitor-1 (TIMP-1), FOXP-3, signal transducer and activator of transcription 1 (STAT1) and phospho-STAT1-positive cells, and actin, endothelin-1, isoprostane, biglycan, decorin, fibronectin and the collagen fibers volume fraction compared with the ovalbumin group ( p + and IL-17, TGF-β, and phospho-STAT1-positive cells and endothelin-1 in the vessels ( p Conclusion: We observed no difference in angiogenesis after treatment with Rho-kinase inhibitor and anti-IL17. Although the treatments did not show differences in angiogenesis, they showed differences in the markers involved in the angiogenesis process contributing to inflammation control and vascular remodeling. The reviews of this paper are available via the supplemental material section.

To describe 3 cases of corneal clearance after the use of topical rho-kinase inhibitor, netarsudil, in the setting of endothelial cell dysfunction in comparison to one case without corneal clearance after the use of netarsudil.Four patients presenting to a busy academic clinical corneal practice with visual complaints from corneal edema secondary to endothelial cell dysfunction were treated with topical netarsudil one drop daily in the affected eye.Corneal clearance was observed in 1) a case of peripheral corneal edema in the setting of iridocorneal endothelial syndrome after 4 weeks on netarsudil, 2) a case of corneal edema in the setting of early penetrating keratoplasty graft failure after 2-week use of netarsudil, and 3) a case of corneal edema in the setting of chronic penetrating keratoplasty graft failure after 4-week use of netarsudil. Corneal clearance was not observed in a case of corneal edema in the setting of pseudophakic bullous keratopathy from previous complicated intraocular lens exchange surgery with placement of an anterior chamber intraocular lens after the use of netarsudil for 12 weeks.Addition of topical rho-kinase inhibitor in the form of netarsudil can result in corneal clearance in a variety of certain cases of endothelial cell dysfunction, not previously documented in the literature.

PURPOSE To investigate corneal clearance time using a topical rho-kinase inhibitor, netarsudil, after descemetorhexis without endothelial keratoplasty (DWEK). METHODS Twenty eyes from 10 patients with Fuchs endothelial corneal dystrophy had DWEK with cataract surgery. For the first eye of each participant, netarsudil was administered immediately after surgery until corneal clearance. For the second eye, netarsudil was withheld 2 weeks beyond the time for corneal clearance of the first eye and then administered only if corneal edema was still present. Interpatient and intrapatient comparisons were made for pachymetry, endothelial cell count, intraocular pressure, and time to corneal clearance. RESULTS Intrapatient comparison demonstrated no significant difference in preoperative pachymetry (P value 0.58), endothelial cell counts (P value 0.97), and intraocular pressure (P value 0.46) between eyes treated with netarsudil immediately after DWEK and those with delayed netarsudil use. Average time for corneal clearance in eyes treated with netarsudil immediately after surgery was 4.6 ± 1.7 weeks, which was significantly shorter than eyes not treated with netarsudil immediately at 8 ± 1.9 weeks (P < 0.01). Corneal clearance occurred in eyes between 1 and 2 weeks after addition of netarsudil as a "rescue" drop. Interpatient comparison demonstrated significantly greater endothelial cell counts in eyes treated with netarsudil immediately compared with eyes with a delay in netarsudil use (P = 0.05). CONCLUSIONS Netarsudil significantly reduces the time to corneal clearance after DWEK. Furthermore, increased endothelial cell counts in eyes with immediate netarsudil use versus delayed netarsudil use suggests that the immediate perioperative period is crucial in cell regeneration and migration.

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 β-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.

Glucocorticoids are widely used as an ophthalmic medication. A common, sight-threatening adverse event of glucocorticoid usage is ocular hypertension, caused by dysfunction of the conventional outflow pathway. We report that netarsudil, a rho-kinase inhibitor, rapidly reversed glucocorticoid-induced ocular hypertension in patients whose intraocular pressures were uncontrolled by standard medications. Mechanistic studies in our established mouse model of glucocorticoid-induced ocular hypertension show that netarsudil both prevented and reversed intraocular pressure elevation. Further, netarsudil reversed characteristic steroid-induced pathologies as assessed by quantification of outflow function and tissue stiffness, and morphological and immunohistochemical indicators of tissue fibrosis. Thus, rho-kinase inhibitors act directly on conventional outflow cells to efficaciously prevent or reverse fibrotic disease processes in glucocorticoid-induced ocular hypertension. These data motivate a novel indication for these agents to prevent or treat ocular hypertension secondary to glucocorticoid administration, and demonstrate the antifibrotic effects of rho-kinase inhibitors in an immune-privileged environment.

Purpose. We sought to explore the effects of intravitreal injection of the Rho-kinase inhibitor Y-27632 in a rodent model of nonarteritic anterior ischemic optic neuropathy (rAION). Methods. The rAION model was established by using laser-induced photoactivation of intravenously administered Rose Bengal in rats. The rats received intravitreal injections of Y-27632 or PBS 1, 3, and 6 days after rAION induction. Optical coherence tomography (OCT) was performed at 2 days and 4 weeks after induction. Visual evoked potential (VEP) was used to evaluate the visual function at 4 weeks. Brn3a immunofluorescence staining of surviving RGCs and apoptosis assays of RGCs were performed at 4 weeks. Results. Optic nerve head (ONH) width was significantly reduced in the Y-27632 group compared with that in the PBS group at 2 days after induction (p<0.05). At 4 weeks, the P1 amplitude of flash-VEP (FVEP) in the Y-27632 group was significantly higher than that of the PBS group (p<0.05). The RGC densities in the central and midperipheral retinas in the Y-27632 group were significantly higher than those in the PBS group (p<0.05). Furthermore, there was a significant decrease in apoptotic RGCs in the Y-27632 group than in the PBS group (p<0.05). Conclusions. Intravitreal injection of Y-27632 had neuroprotective effects on ONH edema, RGC survival, and visual function preservation in rAION.

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. 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. 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)]. 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.

Adriamycin (ADR)-induced chronic heart injury (CHI) is a serious complication of chemotherapy. The present study was designed to assess the ability of fasudil, a Rho kinase inhibitor, to prevent ADR-induced CHI. Forty male 6-week-old C57BL6 mice were randomly divided into the following four groups: (1) control group, (2) CHI induced by adriamycin (ADR group), (3) CHI plus low dose fasudil (ADR + L group), and (4) CHI plus high dose fasudil (ADR + H group). Animals from groups 2–4 received ADR (2.5 mg/kg, i.p.) once a week for 8 weeks, and the control group received saline. Meanwhile, the animals in groups 3–4 received 2 mg/kg/day or 10 mg/kg/day fasudil, respectively. After measurement of cardiac functions, blood samples were collected for biochemical assays. The hearts were excised for histological, immunohistochemistry and western blot study, respectively. Adriamycin produced evident cardiac damage revealed by cardiac functions changes: decreased left ventricular fractional shortening (FS), left ventricular ejection fraction (EF), increased left ventricular volume, cardiac injury marker changes (increased creatine kinase, lactate dehydrogenase), antioxidant enzymes activity changes (decreased superoxide dismutase), and lipid peroxidation (elevated malondialdehyde) to the control group. Fasudil treatment notably ameliorated ADR-induced cardiac damage, restored heart function, suppressed cell apoptosis and senescence, ameliorated redox imbalance, and DNA damage. Fasudil has a protective effect on ADR-induced chronic heart injury, which partially attributed to its antioxidant, anti-apoptotic effects of inhibiting the RhoA/Rho kinase (ROCK) signaling pathway.

A 43-year-old man was diagnosed with acute myocardial infarction (AMI) due to multivessel coronary vasospasm. Accordingly, two coronary vasodilators were administered, and he was discharged without an angina attack. However, from the following day, he reported frequent chest pain and was re-hospitalized. Despite adding multiple coronary vasodilators, it was difficult to completely suppress the angina attack. He also demonstrated hypereosinophilia from the onset of AMI, and his eosinophil count gradually increased up to 6,238 /μL. After corticosteroid administration was started, the vasospasm was completely controlled, and his eosinophil count normalized. He remained free from angina attacks for two years with corticosteroid therapy.

Introduction New open-angle glaucoma (OAG) and ocular hypertension (OHT) therapies that reduce treatment burden and improve outcomes relative to currently available agents are needed. Netarsudil, a novel Rho kinase inhibitor approved by the US Food and Drug Administration, reduces intraocular pressure (IOP) by increasing trabecular outflow. Two phase 3 superiority studies compared a fixed-dose combination (FDC) of netarsudil and the prostaglandin latanoprost with each active component for IOP-lowering efficacy. Methods Pooled efficacy and safety data were analyzed from MERCURY-1 and -2 studies in patients with OAG or OHT. Patients instilled one drop of netarsudil (0.02%)/latanoprost (0.005%) FDC (n = 483), netarsudil (0.02%, n = 499), or latanoprost (0.005%, n = 486) into each eye once-daily between 20:00 and 22:00. IOP was measured at 08:00, 10:00, and 16:00 at weeks 2, 6, and the primary endpoint at month 3. Results Baseline mean diurnal IOP was 23.6, 23.6, and 23.5 mmHg in netarsudil/latanoprost FDC, netarsudil, and latanoprost groups, respectively. Mean diurnal IOP in each group was 15.3, 18.1, and 17.5 mmHg at week 2, 15.7, 18.4, and 17.4 mmHg at week 6, and 15.8, 18.4, and 17.3 mmHg at week 12. The netarsudil/latanoprost FDC met criteria for superiority compared with each active component (p, Plain Language Summary In patients with open-angle glaucoma (OAG) or ocular hypertension (OHT), treatment to lower intraocular pressure (IOP) is needed to prevent optic nerve damage and vision loss. Many patients do not achieve sufficient IOP lowering with a single drug. The use of multiple IOP-lowering agents, which may have different dose regimens, can be complex and reduce patient adherence. Combination treatments that incorporate multiple agents are available, but until recently none included a prostaglandin analogue, the most widely prescribed first-line therapy. A once-daily, fixed-dose combination (FDC) therapy (ROCKLATAN®) has been approved in the USA that contains the prostaglandin analogue latanoprost and netarsudil, a novel Rho kinase inhibitor. The netarsudil/latanoprost FDC demonstrated efficacy and safety in lowering IOP among patients with OAG and OHT in the 12-month MERCURY-1 and the 3-month MERCURY-2 clinical trials. To better characterize efficacy and safety, we pooled and analyzed the data from each trial. The pooled data support the findings of the individual studies: Efficacy: 1. Netarsudil/latanoprost FDC demonstrated statistical superiority to the individual components netarsudil and latanoprost in decreasing IOP at all time points assessed over 3 months. 2. Nearly twice as many patients receiving FDC achieved at least a 30% reduction from baseline in IOP, as recommended by the American Academy of Ophthalmology for a first-line treatment, compared to the IOP reduction achieved by netarsudil and latanoprost monotherapy. Safety: No new safety signals were identified. Netarsudil/latanoprost FDC was associated with no treatment-related serious adverse events, minimal systemic adverse events, and manageable ocular adverse events. Electronic supplementary material The online version of this article (10.1007/s12325-020-01277-2) contains supplementary material, which is available to authorized users.

Background Coronary vasospasm after coronary artery bypass grafting (CABG) is a rare but potentially lethal complication. It is often refractory to several vasodilators. We report a case of refractory coronary vasospasm relieved by fasudil injection. Case presentation A 74-year-old woman who had three instances of in-stent stenosis at the left anterior descending artery (LAD) was referred for CABG treatment. Preoperative coronary angiography showed 90% in-stent stenosis of the proximal LAD and 75% stenosis of the diagonal branch. We performed a left internal thoracic artery (LITA)-LAD bypass and a right internal thoracic artery (RITA) diagonal branch bypass. After anastomosis, transit time flow measurement revealed poor blood flow of LITA-LAD bypass even after re-anastomosis. We performed coronary angiography and detected a vasospasm in the native coronary arteries, which was not relieved using conventional vasodilators (calcium channel blockers, isosorbide dinitrate, and nicorandil) However, we were able to relieve the coronary vasospasm by administering fasudil (a Rho kinase inhibitor) injection without causing systemic hypotension. Conclusions Fasudil may be an important vasodilator, especially in cases of coronary vasospasm after CABG.

BACKGROUND Excessive Rho/Rho-kinase pathway activation occurs subsequent to stroke. We examined the neuroprotective effects of pre- and posttreatment with fasudil (a Rho-kinase inhibitor) in a rat transient spinal cord ischemia-reperfusion model under normothermic conditions. METHODS After approval by our animal research committee, male Sprague-Dawley rats were assigned to 1 of 6 groups: pre- and postcontrol (C); pre- and postfasudil (F); and pre- and postsham (S). Fasudil (10 mg/kg) or normal saline was administered intravenously over 30 minutes before ischemia in the pre-F or pre-C groups, and over 30 minutes after reperfusion in the post-F or post-C groups. Sham groups were not subjected to ischemia. Ischemia was induced by aortic occlusion using a balloon catheter combined with hypotension for 10 minutes. Neurologic deficit scores (NDS; 0-8 points) were assessed 1, 7, and 14 days after ischemia, and then histopathologic outcomes were assessed. RESULTS NDS 7 and 14 days after ischemia in the pre-F group (median [range]; 3.5 [2-6] and 2.5 [0-6]) were lower than those in the pre-C group (5.5 [4-7] and 4.5 [4-6]; P = .046 and P = .049), whereas NDS in the post-F group and in the post-C group were not different. The numbers of intact neurons in the gray matter in the pre- and post-F groups (mean ± standard deviation [95% confidence interval]: 25 ± 7 [20-30] and 16 ± 5 [12-19]) were greater than those in the pre- and post-C groups (11 ± 5 [7-14] and 9 ± 3 [7-11]; P < .001 and P = .002). The number of intact neurons in the post-F group (16 ± 5 [12-19]) was lower than the number in the post-S group (26 ± 2 [24-29]; P < .001). The percentages of vacuolation in the white matter in the pre- and post-F groups (21.5 ± 8.4 [15.5-27.5] and 13.6 ± 7.4 [8.3-18.9]) were lower than those in the pre- and post-C groups (43.7 ± 10.4 [36.3-51.1] and 40.6 ± 12.3 [31.8-49.4]; P < .001 and P < .001). CONCLUSIONS Our results demonstrated that intravenous fasudil administered before ischemia improved both neurologic and histopathologic outcomes even 14 days after ischemia, while fasudil administered postinsult improved histopathologic outcomes only in normothermic rats. Fasudil may be a relevant pretreatment paradigm for planned procedures at risk for spinal cord ischemia.

The inability to propagate human prostate epithelial cells indefinitely has historically presented a serious impediment to prostate cancer research. The conditionally reprogrammed cell (CRC) approach uses the combination of irradiated J2 mouse fibroblasts and a Rho kinase inhibitor such as Y27632 to support the continuous culture of cells derived from most epithelial tissues, including the prostate. Due to their rapid establishment and overall ease of use, CRCs are now widely used in a variety of basic and preclinical settings. In addition, CRCs were successfully used to clinically treat respiratory papillomatosis. Although both normal and tumor-derived prostate CRCs have been used to study the basic biology of prostate cancer and to test new therapies, certain limitations exist. We have previously reported that prostate CRCs form functional prostate glands when implanted under the mouse renal capsule. However in conventional culture, the prostate CRCs exist in an adult stem-like, transient amplifying state and consequently do not adequately recapitulate several important features of a differentiated prostate epithelium. To address these limitations, we previously described a transwell dish-based model that supported the culturing of prostate CRCs and the collection of cells and cell extracts for molecular and genetic analyses. Using normal and tumor-derived prostate CRCs, we describe the combined effects of the multi-dimensional transwell platform and defined culture media on prostate cellular proliferation, differentiation and signaling.

Introduction In the pathogenesis of sepsis-induced acute lung injury (ALI), the crosstalk between inflammation and coagulation plays a pivotal role. The aim of this study was to investigate the role of Rho kinase (ROCK) inhibitor in alleviating pulmonary inflammation and coagulation in lipopolysaccharide (LPS)-induced acute lung injury (ALI) models. Methods In the in vivo study, mice were randomized to four different groups: Control, Y-27632 (Y), LPS, and LPS + Y-27632 (LPS + Y). ALI was induced by intranasally administering LPS (10 μg in 50 μL PBS). Y-27632 (10 mg/kg body weight,) was injected intraperitoneally at 18 h and 1 h before LPS challenge. Mice were euthanized at 3 h or 8 h post LPS challenge (N = 8 per group). In the in vitro study, human pulmonary microvascular endothelial cells (HPMECs) were incubated with LPS alone (1 μg/mL) or in combination with 10 μM Y-27632 or 50 μM BAY11-7082. Cells were pretreated with the inhibitors 30 min before exposure to LPS. Three hours later, cells were isolated for subsequent analysis. Results The myeloperoxidase (MPO) activity and fibrinogen deposits in the lung tissue significantly decreased and the lung damage in ALI mouse was attenuated. Pretreatment with Y-27632 markedly reduced the LPS-induced expression of interleukins 1β and 6, and the activation of nuclear factor (NF)-κB. Furthermore, ROCK inhibitor treatment antagonized the expression of tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 in lung tissue and HPMECs. Conclusions ROCK inhibition protects against the endotoxin-induced pulmonary inflammation and coagulation via NF-kappaB pathway modulation.

11 Aim Obstructive sleep apnea increases in diabetes and morbid obesity. We tested a hypothesis that circulating autoantibodies in adult type 2 diabetes which increase in association with morbid obesity are capable of causing long-lasting neuronal depolarization and altered calcium release in mouse atrial cardiomyocytes. 12 Methods Protein-A eluates from plasma of 14 diabetic obstructive sleep apnea patients and 17 age-matched diabetic patients without sleep apnea were tested for effects on depolarization and neurite out growth in N2a mouse neuroblastoma cells. The mechanism of autoantibody-mediated neurite outgrowth inhibition was investigated in co-incubation experiments of diabetic obstructive sleep apnea autoantibodies with specific antagonists of G-protein coupled receptors or the RhoA/Rho kinase signaling pathway. Following long-term storage of the protein-A eluates (to allow spontaneous proteolysis and IgG subunit dissociation), plasma autoantibodies from diabetic obstructive sleep apnea, cancer or control patients were compared for enhancement of inhibitory effects on endothelial cell survival. Size exclusion chromatography performed (in the presence or absence of a specific membrane type 1-matrix metalloproteinase inhibitor) was used to characterize the IgG autoantibody subunit(s) or fragments associated with peak neurotoxicity in diabetic obstructive sleep apnea. 13 Results Diabetic obstructive sleep apnea (n = 14) autoantibodies caused a significant increase (P = 0.01) in membrane depolarization in N2a mouse neuroblastoma cells compared to control diabetic patients (n = 15) not suffering with obstructive sleep apnea. Process extension in N2A mouse neuroblastoma cells was significantly inhibited (P = 0.01) by diabetic obstructive sleep apnea (n = 9) autoantibodies compared to effects from identical 10 μg/mL concentrations of control diabetic autoantibodies in patients without obstructive sleep apnea. Ten micromolar concentrations of SCH-202676, a G-protein coupled receptor antagonist (n = 5) or ten micromolar concentration of Y27632, a selective Rho kinase inhibitor (n = 6), each significantly prevented (P < 0.001) neurite outgrowth inhibition by diabetic obstructive sleep apnea autoantibodies. Autoantibodies in representative patients with obstructive sleep apnea and either atrial fibrillation or left ventricular hypertrophy evoked acute large increases in intracellular Ca2+ in HL-1 mouse atrial cardiomyocytes. The magnitude of intracellular Ca2+ release was dose-dependently significantly correlated to the electrocardiographic Cornell voltage-duration product. Gel filtration of diabetic obstructive sleep apnea autoantibodies revealed peak neurotoxicity associated with MWs corresponding to IgG light chain dimer(s), monomers or half-light chains as well as a novel ∼ 5.5 kD putative light chain fragment. 14 Conclusions These results suggest that diabetic obstructive sleep apnea autoantibodies may induce strong depolarization in neuronal cells and alter Ca2+ signaling in atrial cardiomyocytes consistent with a role in pathophysiology in subsets of diabetic obstructive sleep apnea having co-morbid atrial fibrillation or another clinically significant cardiac rhythm disturbance.

The current study was designed to investigate the potential beneficial therapeutic outcome of Rho kinase inhibitor (fasudil) against hypercholesterolemia-induced myocardial and vascular injury in rabbits together with diet modification. Sixteen male rabbits were randomly divided into four groups: normal control group which received standard rabbit chow, hypercholesterolemic control group, and treated groups which received cholesterol-rich rabbit chow (1.5% cholesterol) for 8 weeks. Treated groups received either fasudil (100 mg/kg/day) or rosuvastatin (2.5 mg/kg/day) starting from the ninth week for further 4 weeks with interruption of the cholesterol-rich chow. Biochemical assessment of serum cholesterol, triglyceride, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and myocardial oxidative/antioxidant biomarkers malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH), besides biochemical assessment of serum nitric oxide (NO), creatine kinase (CK), and lactate dehydrogenase (LDH) activities and serum total antioxidant capacity (TAC), was conducted. Serum vascular cell adhesion molecule 1 (VCAM-1) and serum Rho-associated protein kinase 1 (ROCK-1) were also evaluated along with histopathological examination of aorta specimens. Fasudil administration significantly decreased serum cholesterol, triglyceride (TG), and LDL and significantly increased serum HDL, with concomitant decrease in serum CK and LDH activities, NO, and restoration of serum TAC. Myocardial MDA significantly declined; SOD activity and GSH contents were restored. Serum ROCK-1 and VCAM-1 levels significantly declined as well. Vascular improvement was confirmed with histopathological examination, which revealed normal aortic intema with the absence of atheromas. Fasudil has promising anti-atherogenic activity mediated primarily via alleviation of hypercholesterolemia-induced oxidative stress and modulation of inflammatory response.

In the last years there have been many developments regarding corneal diseases and treatments.

Aims: IL17 and Rho-kinase may play a critical role in regulating inflammatory and remodeling responses in inflammatory lung diseases. Objectives: To study the effects of anti-IL17 and/or Rho-kinase inhibitor treatments on vascular changes in mice with chronic allergic inflammation. Methods: we used 64 BALB/c mice with pulmonary inflammation induced by ovalbumin that were treated with anti-IL-17A (7.5/µg/dose-ip) and/or Rho-kinase inhibitor (Y-27632-10mg/kg-intranasal) administered 1h before each ovalbumin challenge (22, 24, 26 and 28/days). Control animals were inhaled with saline. At the end of protocol, lungs were removed, morphometric analysis was performed to quantity vascular inflammatory, remodeling and oxidative stress responses. Results: OVA-Rhoi and OVA-anti-IL17 decreased all parameters (CD4+, IL5+, IL13+, IL17, Rho-Kinase2, NFκB, TIMP1, MMP9, MMP12, iNOS, FOXP3 positive cells/104μm2, collagen fibers and PGF2α contents) compared to OVA(p Conclusions: Anti-IL17 or Rho-kinase inhibitor modulate vascular inflammation, remodeling and oxidative stress responses. The association of these drugs does not potentiate the control of these responses compared to isolated treatments in this asthma model. Financial Support: FAPESP, CNPq, LIM20-HC-FMUSP.

Novel pharmacological approaches are needed to improve outcomes of patients with idiopathic pulmonary hypertension. Rho-associated protein kinase (ROCK) inhibitors have shown beneficial effects in preclinical models of pulmonary arterial hypertension (PAH), because of their role in the regulation of pulmonary artery vasoconstrictor tone and remodeling. We compared a ROCK inhibitor, Y-27632, for the first time with the dual endothelin receptor antagonist, macitentan, in a monocrotaline-induced rat pulmonary hypertension model. Different methods (echocardiography, hemodynamics, histology of right ventricle and pulmonary vessels, and circulating biomarkers) showed consistently that 100 mg/kg daily of Y-27632 and 10 mg/kg daily of macitentan slowed the progression of PAH both at the functional and structural levels. Treatments started on day 14 after monocrotaline injection and lasted 14 days. The findings of all experimental methods show that the selective ROCK inhibitor Y-27632 has more pronounced effects than macitentan, but a major limitation to its use is its marked peripheral vasodilating action.

Introduction: Glaucoma refers to a heterogeneous group of optic neuropathies characterized by an enhanced intraocular pressure (IOP). To date, there are six available different drug classes for the treatment of glaucoma and ocular hypertension: β-adrenergic antagonists, prostaglandins, carbonic anhydrase inhibitors, α2-selective adrenergic, muscarinic agonists and rho kinase inhibitors, which act by reducing the production or increasing the drainage of aqueous humor. Areas covered: This manuscript reviews patent US2018244666A1, that describes the synthesis of novel potential rho kinase and monoamine transporters inhibitors with a benzamide or isoquinoline amide scaffolds, and patent US2018256595A1 that investigates the long-term treatment of glaucoma or ocular hypertension with ripasudil, a rho kinase inhibitor. Expert opinion: A variety of netarsudil congeners were synthesized as rho kinases inhibitors in patent US2018244666A1. Results of patent US2018256595A1 showed that ripasudil is among the best candidates for glaucoma long-term treatment, as IOP continuously dropped over the course of the treatment.

We measured the effect of Rho-kinase on inflammation and mucus hypersecretion in the airways of mouse models of asthma. Additionally, we aimed to determine if these effects were the result of JNK 1/2-AP1 pathway inhibition.We sensitized and challenged female C57BL/6 mice using house dust mites (HDM) followed by treatment with an inhibitor of Rho-kinase. Lung tissue was harvested to evaluate inflammation and mucus secretion in the airways of asthma mice. Cytokine expression in broncho-alveolar lavage fluid (BALF) was established by ELISA and airway responsiveness, and was determined by the invasive lung function test. JNK1/2, p-JNK1/2, AP-1, and p-AP-1 protein expression was determined by Western blot analysis. Asthma model mice that were treated with Rho-kinase inhibitor showed a significantly decrease in inflammation score, inflammatory cells, and airway responsiveness. Additionally, we found that IL-13 expressions in BALF and mucus secretion were decreased in HDM-challenged mice treated with Rho-kinase inhibitor. Furthermore, Rho-kinase inhibitor treatment decreased the expression of JNK1/2 and AP-1 phosphorylation. Our findings indicated that the Rho-kinase inhibitor decreased HDM-induced mucus secretion as well as airway inflammation in asthma mice through regulation of the JNK1/2-AP-1 pathway.

The present study aimed to evaluate the cardioprotective effects of a Rho‑kinase inhibitor, Y‑27632, and the underlying mechanisms. A rat model of myocardial ischemia‑reperfusion (I/R) injury was generated by ligation of the coronary artery, and global ischemia of isolated rat hearts was conducted using the Langendorff system. Staining with triphenyltetrazolium chloride (TTC) and hematoxylin and eosin was performed to analyze the myocardial infarct size and histopathological alterations of the I/R‑induced rat heart. In addition, coronary flow, myocardial contractility and an electrocardiogram were analyzed. The effects of Y‑27632 on inflammatory cytokines and cardiac enzymes in the serum were assessed by ELISA. The expression of apoptosis‑ and inflammation‑associated proteins was also analyzed via western blotting. Rats in the Y‑27632 group exhibited alleviated myocardial I/R injury according to TTC staining and histopathological diagnosis. Additionally, Y‑27632 restored the ST segment. The data of coronary flow and myocardial contractility in isolated rat hearts indicated that Y‑27632 improved heart function following I/R. The levels of inflammatory cytokines and cardiac enzymes in the serum were downregulated by Y‑27632. The mitogen‑activated protein kinase (MAPK) and nuclear factor (NF)‑κB signaling pathways were inhibited by Y‑27632. Furthermore, apoptosis‑associated protein expression in rats and the isolated hearts was effectively inhibited by Y‑27632. In conclusion, the findings of the present study indicated that Y‑27632 attenuated myocardial injury via inhibiting the activation of the MAPK and NF‑κB signaling pathways; thus, apoptosis and the inflammatory response were suppressed.

Background and aim Mucosal healing is an important clinical goal in patients with inflammatory bowel disease. Recently, short-chain fatty acids (SCFAs) have been reported to have multifaceted effects to host. However, the effects of SCFAs on wound healing in intestinal epithelial cells are unclear. In the present study, we investigated the effects of acetate, one of the major SCFAs, on the wound healing of murine colonic epithelial cells. Methods Young adult mouse colonic epithelial cells were used to determine the effect of acetate using wound healing assay. Mitogen-activated protein kinase and Rho kinase inhibitor were used to elucidate intracellular signal of wound healing treated with acetate. Meanwhile, Rho activation assays were utilized to measure Rho activation levels. To assess in vivo effects, C57B6 mice with dextran sodium sulfate for 7 days were treated with enema administration of acetate for 7 days. Body weight, disease activity index, colon length, and mucosal break ratio in histology were examined. Results Acetate enhanced wound healing and fluorescence intensity of actin stress fiber compared with control. These effects were canceled with pretreatment of c-Jun N-terminal kinase (JNK) inhibitor or Rho kinase inhibitor. Furthermore, JNK inhibitor reduced the activation of Rho induced by acetate. In the dextran sodium sulfate-induced colitis model, the mice with enema treatment of acetate significantly exhibited recovery. Conclusions In this study, we demonstrated that acetate promoted murine colonic epithelial cell wound healing via activation of JNK and Rho signaling pathways. These findings suggested that acetate could have applications as a therapeutic agent for patients with intestinal mucosal damage, such as inflammatory bowel disease.

Purpose The purpose of this study was to investigate the IOP-lowering effects of the ITRI-E-212, a new Rho-associated protein kinase (ROCK) inhibitor. ITRI-E-212 improved fluid outflow through the trabecular meshwork and reduced IOP with transient and mild conjunctival hyperemia. ITRI-E-212 can potentially be developed into new antiglaucoma agents. Methods ITRI-E-212 was selected from more than 200 amino-isoquinoline structures because of its adequate solubility and drug-loading percentage in eye drops. ITRI-E-212 has less than 50% inhibitory concentration (IC50) against ROCK2. The in vitro kinase inhibition was evaluated using the ADP-Glo kinase assay. A comprehensive analysis of the kinase inhibitor selectivity of ITRI-E-212 was performed using the KINOMEscan methodology. The IOP-lowering effect and tolerability of ITRI-E-212 were investigated in normotensive and ocular hypertensive rabbits. The pharmacokinetics study was performed in vivo in the aqueous humor (AH), and hyperemia was assessed. Results ITRI-E-212 showed high in vitro inhibitory activity against ROCK2 and high specificity against AGC kinases. The mean IOP-lowering effect of ITRI-E-212 in normotensive and ocular hypertensive models was 24.9% and 28.6%, respectively; 1% ITRI-E-212 produced notable reductions in IOP that were sustained for at least 6 hours after each dose once per day. Only transient, mild hyperemia was observed. The compound extracted from the AH reached 78.4% ROCK2 kinase inhibition at 1 hour after dose administration and was sustained for 4 hours. Conclusions ITRI-E-212 is a novel and highly specific ROCK2 inhibitor with the ability to lower IOP in animal models. It has favorable pharmacokinetic and ocular tolerability profiles with only minimal conjunctival hyperemia.

Fuchs corneal dystrophy (FD) is a common cause of endothelial keratoplasty. Recently, a series of FD cases treated with Descemet stripping only (DSO) demonstrated recovery of the central endothelium without transplantation of donor cells. Ripasudil, a rho kinase inhibitor, has been shown to promote corneal endothelial wound healing in animal models. This study prospectively evaluated the use of ripasudil in patients undergoing DSO for FD.Enrolled patients underwent DSO with or without cataract surgery, performed by 1 surgeon. On the first postoperative day, patients were assigned to topical ripasudil 0.4% (Glanatec) 4 times a day for 2 months or no ripasudil and followed up monthly for the first 6 months and then at 9 and 12 months after surgery. Endothelial cell density (ECD) and pachymetry were evaluated at each postoperative visit.Eighteen patients were enrolled, including 8 women and 1 man in each group. Overall, patients who underwent DSO with ripasudil recovered vision more quickly (4.6 vs. 6.5 weeks, P0.01). In addition, the ripasudil group had a statistically significantly higher average ECD at 3, 6, and 12 months. The patients in the DSO observation group had a 10% decrease in peripheral ECD when comparing counts before surgery with counts 12 months after surgery (P0.05). In the DSO ripasudil group, there was no significant difference between peripheral ECD at preoperative baseline versus 12 months after surgery.DSO with topical rho kinase inhibitors may be an alternative treatment for patients with FD and a peripheral ECD greater than 1000 cells/mm.

Cell based therapy, as an alternative to conventional donor corneal transplantation, is anticipated to provide a less invasive and more effective therapeutic modality for corneal endothelial dysfunction. The proliferative capability of human corneal endothelial cells is severely controlled, thus making the establishment of optimal conditions for the cultivation of human corneal endothelial cells a critical aspect for the clinical application of a cell based therapy. Recently, successful cultivation protocol of human corneal endothelial cells for clinical application which includes the use of Rho kinase (ROCK) inhibitor and the inhibition of transforming growth factor beta signaling were reported to promote the cell proliferation and to support human corneal endothelial cell cultures that show high cell density. Animal experiments showed that a cell-injection therapy combined with the use of a ROCK inhibitor promotes cultivated corneal endothelial cell adhesion onto the posterior cornea, ultimately resulting in the recovery of corneal transparency. A pilot clinical study was recently initiated in Japan to investigate the efficacy of a cell-injection therapy for patients with bullous keratopathy.

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 p p p p 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.

PURPOSE To evaluate the efficacy of rho-associated protein kinase inhibitor, AR-12286 topical solution, for its effect in eyes with exfoliation syndrome (XFS) and ocular hypertension (OHT) or exfoliative glaucoma (XFG) and examine any lasting effect on intraocular pressure (IOP) after discontinuation. METHODS Prospective, double-masked, randomized, interventional study. Patients with XFS and OHT or XFG were enrolled. The study eyes were treated once daily with AR-12286, randomized to 0.5% or 0.7% for 24 weeks. Visits included baseline, 1, 4, and 12 weeks after drug initiation; at 12 weeks AR-12286 was discontinued for 1 week and was resumed at week 13. At the week 24 visit, AR-12286 was discontinued, and a final reexamination was performed at week 25. RESULTS Ten patients were treated. Mean baseline IOP was 25±2.4 mm Hg, mean IOP was reduced to 19.1±2.3 mm Hg at 1 week (P

WOS: 000379095300009, PubMed ID: 27160814, Objectives: Rho-kinases (ROCKs), are one of the dynamic structures of the actin cytoskeleton and they mediate different biological processes, including regulation of calcium sensitivity of smooth muscle contraction. The activation of Rho A/ROCK system is thought to be effective on the termination time of the pregnancy process. The aim of this study, was to investigate in vitro effects of the ROCK enzyme inhibitors, clinically available fasudil hydrochloride, and a new promising inhibitor AS1892802, on the contractions of isolated pregnant rat myometrium. Study design: Term pregnant Wistar albino rats (n = 12), weighing 200-220 g, were used in this study. Myometrial tissues obtained from rats were dissected into four full-thickness longitudinal muscle strips and then myometrial tension was recorded isometrically. The inhibitory effects of cumulative concentrations of AS1892802 and of fasudil hydrochloride in the presence and absence of ODQ (guanylate cyclase inhibitor), L-NAME (nitric oxide synthase inhibitor) and L-NNA (endothelial nitric oxide synthase inhibitor) on oxytocin-induced myometrial contractions were measured, and values for -log10 EC50 (pD(2)) and mean maximal inhibition (E-max) were compared. Results: Both ROCK inhibitors, AS1892802 and fasudil hydrochloride starting from the concentrations of 10(-6) M reached statistical significance on contraction amplitude and frequency of myometrial strips (p < 0.05). The inhibition of the amplitude and frequency of myometrial contractions was antagonized with ODQ (10(-5) M; only amplitude), L-NAME (3 x 10(-5) M) and L-NNA (10(-5) M) (p < 0.05). Conclusion: These results suggest that fasudil hydrochloride and AS1892802 may contribute to the development of new tocolytic drugs. We conclude that AS1892802 and fasudil hydrochloride perform this inhibitory effect partially through ROCK inhibition and the NO/cGMP pathway. (C) 2016 Elsevier Ireland Ltd. All rights reserved., Cumhuriyet University Scientific Research Project (CUBAP, Sivas, Turkey) [T-587], This study was supported by Cumhuriyet University Scientific Research Project T-587 (CUBAP, Sivas, Turkey).

Introduction It remains controversial whether statins have a beneficial effect on pulmonary arterial hypertension (PAH). This study is intended to evaluate whether statin, co-administered with Rho-kinase inhibitor, could enhance its efficacy. Although Rho-kinase inhibitors, including fasudil, have been reported to improve pulmonary hypertension in experimental and clinical studies, the combination of these agents has not been tested in the treatment of pulmonary hypertension (PH). Material and methods The effects of such a regimen on hemodynamics, right ventricle hypertrophy, and Rho-associated protein kinase (ROCK) activity in experimental monocrotaline (MCT)-induced pulmonary hypertension were examined. Fourteen days after monocrotaline injection (60 mg/kg), male rats were treated orally for another 14 days with fasudil (15 mg/kg per day), or with a combination of fasudil + rosuvastatin (10 mg/kg per day). Results The drug combination reversed the MCT-induced increase in right ventricle pressure (RVP) and reduced right ventricular hypertrophy (RV/LV + S ratio) more than Rho kinase inhibitor alone. The simultaneous administration of fasudil and rosuvastatin caused a further decrease of RhoA kinase activity in isolated lung tissues as compared to fasudil alone. Conclusions The results indicate that rosuvastatin intensifies the beneficial effects of Rho-kinase inhibitor on the Rho/Rho-kinase pathway and such a combination may represent an option for the treatment of pulmonary arterial hypertension.