Journal of Ocular Pharmacology and Therapeutics

The pharmacological characteristics of levobetaxolol, a single active isomer of betaxolol, were determined and compared with activities of other β-adrenoceptor antagonists. Levobetaxolol (43-fold β₁-selective) exhibited a higher affinity at cloned human β₁ (K_i = 0.76 nM) than at β₂ (K_i = 32.6 nM) receptors, while dextrobetaxolol was much weaker at both receptors. Levobetaxolol potently antagonized functional activities at cloned human β₁ and β₂ receptors, and also at guinea pig atrial β₁, tracheal β₂ and rat colonic β ₃ receptors (IC₅₀s = 33.2 nM, 2970 nM and 709 nM, respectively). Thus, levobetaxolol was 89-times β₁-selective (vs β₂). Levobetaxolol (K_i = 16.4 nM) was more potent than dextrobetaxolol (K_i = 2.97 μM) at inhibiting isoproterenol-induced cAMP production in human non-pigmented ciliary epithelial cells. Levobunolol and (l)-timolol had high affinities at β₁ and β₂ receptors but were considerably less β₁-selective than levobetaxolol. Levo-, dextro- and racemic-betaxolol exhibited little or no affinity, except at sigma sites and Ca²⁺-channels (IC₅₀s > 1 μM), at 89 other receptor/ligand binding sites. Levobetaxolol exhibited a micromolar affinity for L-type Ca²⁺-channels. In conscious ocular hypertensive cynomolgus monkeys, levobetaxolol was more potent than dextrobetaxolol, reducing intraocular pressure by 25.9 ± 3.2% at a dose of 150 μg/eye (n = 15–30). Quantitative [³H]levobetaxolol autoradiography revealed high levels of binding to human ciliary processes, iris, choroid/retina, and ciliary muscles. In conclusion, levobetaxolol is a potent, high affinity and β₁-selective IOP-lowering β-adrenoceptor antagonist.

Experiments were undertaken to determine the role played by nitric oxide (NO) in basal ocular blood flow in the anterior aspect of the eye. Subsequent studies focused on existence of autoregulatory mechanisms and on the potential involvement of NO. Cats were anesthetized with pentobarbital (36 mg/kg, i.p.). A femoral artery and vein were cannulated for measuring blood pressure and for drug administration, respectively. Anterior segment blood flow was measured in a continuous fashion from the long posterior ciliary artery (LPCA) using ultrasonic flowmetry and from the anterior choroid using laser-Doppler flowmetry. A needle was placed into the anterior chamber, and autoregulatory mechanisms were studied by decreasing ocular perfusion pressure via stepwise elevations of IOP. Non-selective inhibition of NO synthase with L-NAME (20 mg/kg, i.v.) significantly decreased basal blood flow from both sites. L-NAME (5 mg/kg, i.v.) was without effect as was D-NAME (25 mg/kg, i.v.). Increasing IOP produced a linear decrease on LPCA blood flow indicating absence of autoregulation. In contrast, stepwise elevation of IOP produced a delayed, non-linear response in the anterior choroid suggestive of a strong autoregulatory response. Neither response to elevated ocular perfusion pressure was further altered by inhibition of NO synthase with L-NAME (20 mg/kg, i.v.). The results confirm previous reports that nitric oxide plays a pivotal role in maintenance of basal ocular blood flow. Autoregulation was not seen in the LPCA. In contrast, there was a clear autoregulatory response in the anterior choroid, although neither response was altered by inhibition of NO synthase.

Mitomycin-C has recently become an adjunct medication for inhibition of fibroblast proliferation in glaucoma filtering procedures. Prolonged postoperative ocular hypotony has been a frequent complication of trabeculectomy with mitomycin-C. In order to characterize the hypotony mechanism, we compared the toxic effects of mitomycin-C on cultured rabbit ciliary process cells and trabecular meshwork cells. The results indicate that mitomycin-C has a more marked effect on ciliary process cells on ³H-thymidine uptake than on trabecular meshwork cells at concentrations ranging from 10^-1 to 10^-5 mg/ml after 3-, 5- and 60-min treatment, respectively. The living cells after mitomycin-C treatment were estimated with MTT assay that was converted tetrazolium dye of living cells only into insoluble purple formazan crystals within mitochondria. In the presence of mitomycin-C for 3, 5, and 60 min, the cellular MTT values in ciliary process cells were more decreased than in trabecular meshwork cells. Depolarization of the trabecular meshwork cells with 50 mM KCl led to an increase in intracellular calcium concentration, whereas application of mitomycin-C at 10^-3 mg/ml resulted in decrease of KCl-induced intracellular calcium increase. Mitomycin-C (10^-3 mg/ml) decreased cAMP concentration in ciliary process cells following 3- and 5-min treatment; however, it did not significantly affect the cellular cAMP concentration after only a 1-min exposure. Mitomycin-induced marked ladder pattern of DNA fragmentation was observed in ciliary process tissues after treatment with 10^-1 mg/ml of mitomycin-C for 3 and 5 min. However, the DNA pattern in trabecular meshwork tissues was not obviously affected by mitomycin-C. These findings from our results indicate that mitomycin-induced ocular hypotony may result from damage to both ciliary process and trabecular meshwork tissues.

The changes of vitreous pH values under acute glaucoma status have never been reported. In this study, we measured the changes of vitreous pH values in an acute glaucoma rabbit model. Under ketamine and xylazine anesthesia, the anterior chamber of the right eye of New Zealand white rabbits was cannulated and connected to a polygraph for continuous intraocular pressure (IOP) monitoring and a mini-pump for persistent injection of viscoelastic substance, Healon GV. The measurement of vitreous pH was done by inserting a pH probe via a third opening through the sclera into the vitreous. Different IOP levels were maintained by varied amounts of Healon GV injections into the anterior chamber. The changes of vitreous pH values following the intracameral injections were recorded and evaluated. The changes of vitreous pH values were minimal when IOPs were maintained at lower than 35 mmHg. Vitreous pH values decreased significantly from 7.32 to less than 7.03, when the IOPs were kept at 70 mmHg or higher. However, the decrease of pH values was reversible if IOP was held at 70 mmHg for less than 10 minutes and then reduced to normal level at once. In addition, with MTT viability assay, it was noted that the decrease in vitreous pH was associated with a higher percentage of retinal cell death.

The effect of endothelins on corneal endothelial cells is not well understood. We have investigated the effects of endothelin-1 (ET-1), endothelin-2 (ET-2) and endothelin-3 (ET-3) on bovine corneal endothelial cellular proliferation and the secondary messenger changes in cells in the presence of ET-1. It was found that the ³H-thymidine uptake was enhanced by ET-1 significantly, whereas ET-2 and ET-3 had no effect. ET-1 remarkably affects the increase of corneal endothelial cells on ³H-thymidine, ³H-leucine, and ³H-uridine uptakes in a dose-dependent manner. The 50% effective concentrations (EC₅₀) for ET-1, as measured by ³H-thymidine uptake, ³H-uridine uptake, and ³H-leucine uptake were 10^-8.78 M, 10^-8.53 M and 10^-8.04 M, respectively. It was found that endothelin-1 increased intracellular calcium concentration by using the method of preloading with Fura-2-AM and assaying with spectrophotometry. The cellular IP_l, IP₂, and IP₃ were also stimulated in the presence of ET-1. Moreover, ET-1 enhanced the basal cellular cAMP and cGMP concentrations in corneal endothelial cells in a dose-dependent manner. Immunofluorescent staining revealed that ET-1 increased the fibronectin protein concentration and changed protein distribution in corneal endothelial cells. These findings indicate that endothelin-1 increases in cell proliferation and biological changes may be involved in changing intracellular calcium mobility, increasing intracellular phosphoinositides, enhancing intracellular cGMP and cAMP accumulation, and fibronectin protein synthesis.

The incidence of recurrent vitreous hemorrhage of proliferative diabetic retinopathy following posterior vitrectomy ranges from 29% to 75% in reported series. Fluid-gas exchange and vitreous cavity lavage are the popular methods of treating this kind of recurrent hemorrhage. The fluid-gas exchange cannot offer clear vision immediately after the procedure. To improve the function of the classic vitreous cavity lavage, we designed a volume homeostatic fluid-fluid exchanger - Chen's I/A device. Tissue plasminogen activator (t-PA) is a protease that preferentially converts fibrin-bound plasminogen to the active proteolytic enzyme, plasmin. It has been clinically and experimentally proven effective in lysis of postvitrectomy blood clot and fibrin formation. When the blood clot is formed in the vitreous cavity, intravitreal injection of t-PA can convert plasminogen to plasmin and remove the clot. From July 1999 to January 2000, ten eyes of postvitrectomy diabetic vitreous hemorrhage (PDVH) were collected. In each case, 4 days after intravitreal injection (IVI) of t-PA (30 μg), vitreous cavity lavage was performed with Chen's I/A device. Of these cases, 8 eyes (80%) experienced an immediate clearing of the vitreous cavity. Early complications included anterior hyaloid fibrovascular proliferation (2 eyes) and postoperative intraocular pressure elevation (3 eyes). On the basis of the results of this study, our conclusion is that volume homeostatic vitreous cavity lavage, combined with intravitreal injection of t-PA, is an excellent method for treatment of postvitrectomy diabetic vitreous hemorrhage but, in cases of PDVH with iris rubeosis, the advantage of this procedure is uncertain.

The development of aldose reductase inhibitors for the treatment of diabetic complications, such as cataract and retinopathy, has been of intense interest in the pharmaceutical community for the last 20 years. To date, aldose reductase inhibitors have been synthetically developed from leads obtained from in vitro screening studies. Recently, we have observed that mammalian tissues contain intrinsic inhibitors of aldose reductase, which may be used as potential drugs for treating diabetic complications with potentially less side effects than synthetic aldose reductase inhibitors. Intrinsic inhibitor(s) of aldose reductase have been observed in the methanolic extracts from rat and human kidneys and bovine lenses that were subjected to a number of chromatographic techniques, including counter current chromatography, flash chromatography, gel filtration and high pressure liquid chromatography. This inhibition results from a direct interaction between the inhibitor and enzyme. The intrinsic inhibitor, present in the lipophilic fraction of human kidney and bovine lens extracts, can easily penetrate into the lens to inhibit sugar alcohol formation. Intraperitoneal injection of partially purified bovine lens extract inhibited lens polyol formation in young rats fed 50% galactose diet.

N-acetyl-p-benzoquinone imine (NAPQI), a semiquinone metabolite of acetaminophen, produces cataract in mice. Naphthalene is biotransformed to the cataractogenic metabolite 1,2-naphthoquinone (NQ). Intracameral injection of NAPQI elicits a rapid increase in free intracellular Ca²⁺ in the lens epithelium and calpain activation before lens opacification begins. In order to test whether the cellular response is a common feature of quinone-induced cataracts, we injected in this work 1,2-naphthoquinone (NA) in the anterior chamber of mouse eye and followed cellular responses in the lens prior to opacity development. A marked rise in free intracellular Ca²⁺ in the lens epithelium and concurrent activation of calpain were observed within 1 hr after NQ injection preceding lens opacity development. These results support the suggestion that Ca²⁺ release and calpain activation are involved in the mechanism of quinone-induced cataractogenesis.

The delivery of drugs to the retina from solution or from prolonged release forms is considered in terms of pharmacokinetics, interference with vision, and toxicity and safety. It is concluded that visual loss will be the limiting factor in determining the useful life of a drug administered as a microdispersion. Because microparticles can be lost from the vitreous by diffusion, a condensed depot of drug can give a greater lifetime. However, it needs to be anchored outside the visual pathway which requires an intraocular operative procedure. Attaching a reservoir to the external sclera with a diffusion-controlled entry through a tube to the vitreous is suggested as having more flexibility than other systems.