A digoxin derivative that potently reduces intraocular pressure: efficacy and mechanism of action in different animal models.

Glaucoma is a blinding disease. Reduction of intraocular pressure (IOP) is the mainstay of treatment, but current drugs show side effects or become progressively ineffective, highlighting the need for novel compounds. We have synthesized a family of perhydro-1,4-oxazepine derivatives of digoxin, the selective inhibitor of Na,K-ATPase. The cyclobutyl derivative (DcB) displays strong selectivity for the human α2 isoform and potently reduces IOP in rabbits. These observations appeared consistent with a hypothesis that in ciliary epithelium DcB inhibits the α2 isoform of Na,K-ATPase, which is expressed strongly in nonpigmented cells, reducing aqueous humor (AH) inflow. This paper extends assessment of efficacy and mechanism of action of DcB using an ocular hypertensive nonhuman primate model (OHT-NHP) (Macaca fascicularis). In OHT-NHP, DcB potently lowers IOP, in both acute (24 h) and extended (7-10 days) settings, accompanied by increased aqueous humor flow rate (AFR). By contrast, ocular normotensive animals (ONT-NHP) are poorly responsive to DcB, if at all. The mechanism of action of DcB has been analyzed using isolated porcine ciliary epithelium and perfused enucleated eyes to study AH inflow and AH outflow facility, respectively. 1) DcB significantly stimulates AH inflow although prior addition of 8-Br-cAMP, which raises AH inflow, precludes additional effects of DcB. 2) DcB significantly increases AH outflow facility via the trabecular meshwork (TM). Taken together, the data indicate that the original hypothesis on the mechanism of action must be revised. In the OHT-NHP, and presumably other species, DcB lowers IOP by increasing AH outflow facility rather than by decreasing AH inflow.NEW & NOTEWORTHY When applied topically, a cyclobutyl derivative of digoxin (DcB) potently reduces intraocular pressure in an ocular hypertensive nonhuman primate model (Macaca fascicularis), associated with increased aqueous humor (AH) flow rate (AFR). The mechanism of action of DcB involves increased AH outflow facility as detected in enucleated perfused porcine eyes and, in parallel, increased (AH) inflow as detected in isolated porcine ciliary epithelium. DcB might have potential as a drug for the treatment of open-angle human glaucoma.

Lens-regulated retinoic acid signalling controls expansion of the developing eye.

Absence of the developing lens results in severe eye defects, including substantial reductions in eye size. How the lens controls eye expansion and the underlying signalling pathways are very poorly defined. We identified RDH10, a gene crucial for retinoic acid synthesis during embryogenesis, as a key factor downregulated in the peripheral retina (presumptive ciliary body region) of lens-removed embryonic chicken eyes prior to overt reductions in eye size. This is associated with a significant decrease in retinoic acid synthesis by lens-removed eyes. Restoring retinoic acid signalling in lens-removed eyes by implanting beads soaked in retinoic acid or retinal, but not vitamin A, rescued eye size. Conversely, blocking retinoic acid synthesis decreased eye size in lens-containing eyes. Production of collagen II and collagen IX, which are major vitreal proteins, is also regulated by the lens and retinoic acid signalling. These data mechanistically link the known roles of both the lens and retinoic acid in normal eye development, and support a model whereby retinoic acid production by the peripheral retina acts downstream of the lens to support vitreous production and eye expansion.

Pharmacologic ciliary body ablation for chronic glaucoma in dogs: A retrospective review of 108 eyes from 2013 to 2018.

OBJECTIVE: To evaluate the long-term outcome and efficacy of intravitreal injection of gentamicin and dexamethasone sodium phosphate (IVGD) or triamcinolone in end-stage glaucoma patients and determine pre-procedure prognostic indicators of success and post-operative complications. PROCEDURE: Medical records were reviewed for 108 dogs (108 eyes) treated with intravitreal gentamicin with or without dexamethasone sodium phosphate or triamcinolone for glaucoma between 2013 and 2018 with 3 months of minimum follow-up. Signalment and clinical findings, including type of glaucoma, pre-procedure intraocular pressure (IOP), chronicity, procedure protocol, and outcome were recorded. Success was defined as an intraocular pressure of ≤25 mm Hg at the time of last re-examination or no ocular hypotensive medications at 3 months or longer post-injection. RESULTS: The overall success rate for pharmacologic ablation was 95%. The success rate for dogs receiving no ocular hypotensive medications was 86%. Seventy-six eyes (70.4%) had primary glaucoma, and 32 eyes (29.6%) had secondary glaucoma. Age at the time of injection had no effect on initial success but did in final success (P =-.03) for dogs requiring repeat injections. Cocker Spaniels required the most repeat 2nd and 3rd injections (3/12 dogs) and (2/4 dogs), respectively. No preoperative variable significantly affected the success rate. The most common complications were phthisis bulbi (59.2%), corneal edema (25.9%), and ulcerative keratitis (22.3%). Uncontrolled IOP resulted in enucleation in two dogs (1.8%). CONCLUSIONS: Pharmacologic ablation has a high overall success rate in lowering IOP to ≤25 mm Hg short-term in blind, glaucomatous canine eyes. Type of glaucoma, pre-procedure IOP, chronicity, and protocol did not affect success.

Effects of Post-Treatment Hydrogen Gas Inhalation on Uveitis Induced by Endotoxin in Rats.

BACKGROUND Molecular hydrogen (H2) has been widely reported to have benefiicial effects in diverse animal models and human disease through reduction of oxidative stress and inflammation. The aim of this study was to investigate whether hydrogen gas could ameliorate endotoxin-induced uveitis (EIU) in rats. MATERIAL AND METHODS Male Sprague-Dawley rats were divided into a normal group, a model group, a nitrogen-oxygen (N-O) group, and a hydrogen-oxygen (H-O) group. EIU was induced in rats of the latter 3 groups by injection of lipopolysaccharide (LPS). After that, rats in the N-O group inhaled a gas mixture of 67% N2 and 33% O2, while those in the H-O group inhaled a gas mixture of 67% H2 and 33% O2. All rats were graded according to the signs of uveitis after electroretinography (ERG) examination. Protein concentration in the aqueous humor (AqH) was measured. Furthermore, hematoxylin-eosin staining and immunostaining of anti-ionized calcium-binding adapter molecule 1 (Iba1) in the iris and ciliary body (ICB) were carried out. RESULTS No statistically significant differences existed in the graded score of uveitis and the b-wave peak time in the Dark-adapted 3.0 ERG among the model, N-O, and H-O groups (P>0.05), while rats of the H-O group showed a lower concentration of AqH protein than that of the model or N-O group (P<0.05). The number of the infiltrating cells in the ICB of rats from the H-O group was not significantly different from that of the model or N-O group (P>0.05), while the activation of microglia cells in the H-O group was somewhat reduced (P<0.05). CONCLUSIONS Post-treatment hydrogen gas inhalation did not ameliorate the clinical signs, or reduce the infiltrating cells of EIU. However, it inhibited the elevation of protein in the AqH and reduced the microglia activation.

Aqueous Extract of Perilla frutescens var. acuta Relaxes the Ciliary Smooth Muscle by Increasing NO/cGMP Content In Vitro and In Vivo.

The leaves of Perilla frutescens var. acuta (PFA) are commonly used as a traditional medicine in Korea, Japan, and China. We previously showed that PFA attenuates eye fatigue by improving visual accommodation through a clinical study. However, detailed mechanisms and chemical compounds have not been studied. In this study, we analyzed the active compounds in an aqueous extract of PFA involved in ciliary muscle relaxation in vitro and in vivo. NMR and MS analyses showed that the PFA extract contained mainly luteolin-7-O-diglucuronide and apigenin-7-O-diglucuronide. The composition after freeze-drying and spray-drying was similar. Freeze-dried PFA (50 µg/mL, 100 µg/mL, and 200 µg/mL) increased nitric oxide and cGMP levels in ciliary muscle cells isolated from the eyes of rats. [Ca2+]i decreased in a dose-dependent manner. Furthermore, Sprague-Dawley rats treated with freeze-dried PFA (200 mg/kg, orally) showed significantly increased cGMP levels compared with the control group and irradiated with white light. Our results suggest that PFA extract has the potential to reduce eye fatigue by relaxing ciliary muscles.

Src Family Kinase Links Insulin Signaling to Short Term Regulation of Na,K-ATPase in Nonpigmented Ciliary Epithelium.

Insulin has been shown to elicit changes of Na,K-ATPase activity in various tissues. Na,K-ATPase in the nonpigmented ciliary epithelium (NPE) plays a role in aqueous humor secretion and changes of Na,K-ATPase activity impact the driving force. Because we detect a change of NPE Na,K-ATPase activity in response to insulin, studies were carried out to examine the response mechanism. Ouabain-sensitive rubidium (Rb) uptake by cultured NPE cells, measured as a functional index of Na,K-ATPase-mediated inward potassium transport, was found to increase in cells exposed for 5 min to insulin. The maximally effective concentration was 100 nM. An intrinsic increase of Na,K-ATPase activity evident as a >2-fold increase in the rate of ouabain-sensitive ATP hydrolysis in homogenates obtained from cells exposed to 100 nM insulin for 5 min was also observed. Insulin-treated cells exhibited Akt, Src family kinase (SFK), ERK1/2, and p38 activation, all of which were prevented by a pI3 kinase inhibitor LY294002. The Rb uptake and Na,K-ATPase activity response to insulin both were abolished by PP2, an SFK inhibitor which also prevented p38 and ERK1/2 but not Akt activation. The Akt inhibitor MK-2206 did not change the Na,K-ATPase response to insulin. The findings suggest insulin activates pI3K-dependent Akt and SFK signaling pathways that are separate. ERK1/2 and p38 activation is secondary to and dependent on SFK activation. The increase of Na,K-ATPase activity is dependent on activation of the SFK pathway. The findings are consistent with previous studies that indicate a link between Na,K-ATPase activity and SFK signaling. J. Cell. Physiol. 232: 1489-1500, 2017. © 2016 Wiley Periodicals, Inc.

Ciliary body thickness changes after preoperative anti-inflammatory treatment in rhegmatogenous retinal detachment complicated by choroidal detachment.

BACKGROUND: The literature is scant on the state of the ciliary body, its role in the development of rhegmatogenous retinal detachment (RRD) complicated by choroidal detachment (CD), and on ciliary body changes following the treatment aimed at resolving concomitant inflammation and choroidal attachment. This study assesses the anatomical position and thickness of the ciliary body and investigates the ciliary body changes after anti-inflammatory pre-vitrectomy treatment in RRD complicated by CD. METHODS: Forty-nine patients (49 eyes) with RRD complicated by CD underwent standard ophthalmological examination (including visual acuity assessment, biomicroscopy, ophthalmoscopy, and ocular tonometry) and ultrasound biomicroscopy of the ciliary body, choroid, and retina both before and following anti-inflammatory pre-vitrectomy treatment. RESULTS: At baseline, all subject eyes had ciliary body edema and detachment extending into the choroid. Ultrasonographic ciliary features included ciliary body edema and disorganization of the supraciliary layer of the pars plana, which was evident by the presence of multiple small oblique fibers. In all subject eyes, the treatment resulted in reattachment of the choroid and the ciliary body as well as a reduction in ciliary body edema (total mean ciliary thickness reduced from 0.83 (0.09) to 0.65 (0.09) mm, with a difference of 0.18 (0.07) mm, P < 0.001). CONCLUSIONS: Preoperative anti-inflammatory treatment in RRD complicated by CD results in restoration of the anatomical position of the ciliary body and a statistically significant reduction in ciliary body edema.

Safety of Using Matrix Metalloproteinase Inhibitor in Experimental Glaucoma Filtration Surgery.

We evaluated the safety of matrix metalloproteinase (MMP) inhibitor in experimental glaucoma filtration surgery in an animal model. Fifteen New Zealand white rabbits underwent an experimental trabeculectomy and were randomly allocated into 3 groups according to the adjuvant agent: no treatment group (n = 5), 0.02% mitomycin C (MMC) soaking group (n = 5), and MMP inhibitor (ilomastat) subconjunctival injection group (n = 5). Slit lamp examination with Seidel testing, pachymetry, and specular microscopy was performed preoperatively and postoperatively. The conjunctiva and ciliary body toxicity were evaluated with scores according to the pathologic grading systems. Electron microscopy was used to examine the structural changes in cornea, conjunctiva, and ciliary body. In the ilomastat-treated group, there was no statistically significant change in central corneal thickness preoperatively and at 28 days postoperatively (P = 0.655). There were also no significant changes in specular microscopy findings over the duration of the study in the ilomastat-treated group. The conjunctival toxicity score was 1 in the control group, 1.5 in the ilomastat-treated group, and 2 in the MMC-treated group. When assessing ciliary body toxicity scores, the ilomastat-treated group score was 0.5 and the MMC-treated group score was 1.5. Transmission electron microscopy did not show structural changes in the cornea and ciliary body whereas the structural changes were noticed in MMC group. A single subconjunctival injection of MMP inhibitor during the experimental trabeculectomy showed a less toxic affect in the rabbit cornea, conjunctiva, and ciliary body compared to MMC.

Pharmacological Actions of Hydrogen Sulfide Donors on Sympathetic Neurotransmission in the Bovine Anterior Uvea, In Vitro.

In the present study, we investigated the effect of three different sources of hydrogen sulfide (H2S) on sympathetic neurotransmission from isolated superfused bovine iris-ciliary bodies. The three agents under consideration were: ACS67, a hybrid of latanoprost and a H2S-donating moiety; L-cysteine, a substrate for endogenous production of H2S and GYY 4137, a slow donor of H2S. We also examined the contribution of prostaglandins to the pharmacological actions of the H2S donors on release of [(3)H]-norepinephrine ([(3)H]NE) triggered by electrical field stimulation. ACS67, L-cysteine and GYY 4137 caused a concentration-dependent inhibition of electrically-evoked [(3)H]NE release from isolated bovine iris-ciliary bodies without affecting basal [(3)H]NE efflux. The cyclooxygenase inhibitor, flurbiprofen enhanced the inhibitory action of ACS67 and L-cysteine on stimulated [(3)H]NE release. Both aminooxyacetic acid, an inhibitor of cystathionine-ß-synthase and glibenclamide, a KATP channel blocker reversed the inhibition of evoked NE release induced by the H2S donors. We conclude that H2S donors can inhibit sympathetic neurotransmission from isolated bovine iris-ciliary bodies, an effect partially dependent on the in situ production of H2S and prostanoids, and is mediated by an action on KATP channels.

The effects of VEGF-A-inhibitors aflibercept and ranibizumab on the ciliary body and iris of monkeys.

PURPOSE: To investigate the effects of intravitreal ranibizumab (Lucentis®) and aflibercept (Eylea®) on the ciliary body and the iris of 12 cynomolgus monkeys with regard to the fenestrations of their blood vessels. MATERIALS AND METHODS: Structural changes in the ciliary body and in the iris were investigated with light, fluorescent, and transmission electron microscopy (TEM). The latter was used to specifically quantify fenestrations of the endothelium of blood vessels after treatment with aflibercept and ranibizumab. Each of the two ciliary bodies treated with aflibercept and the two treated with ranibizumab and their controls were examined after 1 and 7 days respectively. Ophthalmological investigations including funduscopy and intraocular pressure measurements were also applied. RESULTS: Ophthalmological investigations did not reveal any changes within the groups. Both drugs reduced the VEGF concentration in the ciliary body pigmented epithelium. The structure of the ciliary body was not influenced, while the posterior pigmented epithelium of the iris showed vacuoles after aflibercept treatment. Ranibizumab was mainly concentrated on the surface layer of the ciliary epithelium, in the blood vessel walls and the lumen of some of the blood vessels, and in the cells of the epithelium of the ciliary body. Aflibercept was more concentrated in the stroma and not in the cells of the epithelium, but as with ranibizumab, also in the blood vessel walls and some of their lumina, and again on the surface layer of the epithelium. Both aflibercept-and ranibizumab-treated eyes showed a decreased number of fenestrations of the capillaries in the ciliary body compared to the untreated controls. On day 1 and day 7, aflibercept had fewer fenestrations than the ranibizumab samples of the same day. CONCLUSIONS: Both aflibercept and ranibizumab were found to reach the blood vessel walls of the ciliary body, and effectively reduced their fenestrations. Aflibercept might eliminate VEGF to a greater extent, possibly due to a higher elimination of fenestrations in a shorter time. Moreover, the vacuoles found in the iris need further research, in order to evaluate whether they carry a possible pathological potential.