Pinosylvin Extract Retinari™ Sustains Electrophysiological Function, Prevents Thinning of Retina, and Enhances Cellular Response to Oxidative Stress in NFE2L2 Knockout Mice.

Chronic oxidative stress eventually leads to protein aggregation in combination with impaired autophagy, which has been observed in age-related macular degeneration. We have previously shown an effective age-related macular degeneration disease model in mice with nuclear factor-erythroid 2-related factor-2 (NFE2L2) knockout. We have also shown pinosylvin, a polyphenol abundant in bark waste, to increase human retinal pigment epithelium cell viability in vitro. In this work, the effects of commercial natural pinosylvin extract, Retinari™, were studied on the electroretinogram, optical coherence tomogram, autophagic activity, antioxidant capacity, and inflammation markers. Wild-type and NFE2L2 knockout mice were raised until the age of 14.8 ± 3.8 months. They were fed with either regular or Retinari™ chow (141 ± 17.0 mg/kg/day of pinosylvin) for 10 weeks before the assays. Retinari™ treatment preserved significant retinal function with significantly preserved a- and b-wave amplitudes in the electroretinogram responses. Additionally, the treatment prevented thinning of the retina in the NFE2L2 knockout mice. The NFE2L2 knockout mice showed reduced ubiquitin-tagged protein accumulation in addition to local upregulation of complement factor H and antioxidant enzymes superoxide dismutase 1 and catalase. Therefore, the treatment in the NFE2L2 KO disease model led to reduced chronic oxidative stress and sustained retinal function and morphology. Our results demonstrate that pinosylvin supplementation could potentially lower the risk of age-related macular degeneration onset and slow down its progression.

Pharmacokinetic aspects of retinal drug delivery.

Drug delivery to the posterior eye segment is an important challenge in ophthalmology, because many diseases affect the retina and choroid leading to impaired vision or blindness. Currently, intravitreal injections are the method of choice to administer drugs to the retina, but this approach is applicable only in selected cases (e.g. anti-VEGF antibodies and soluble receptors). There are two basic approaches that can be adopted to improve retinal drug delivery: prolonged and/or retina targeted delivery of intravitreal drugs and use of other routes of drug administration, such as periocular, suprachoroidal, sub-retinal, systemic, or topical. Properties of the administration route, drug and delivery system determine the efficacy and safety of these approaches. Pharmacokinetic and pharmacodynamic factors determine the required dosing rates and doses that are needed for drug action. In addition, tolerability factors limit the use of many materials in ocular drug delivery. This review article provides a critical discussion of retinal drug delivery, particularly from the pharmacokinetic point of view. This article does not include an extensive review of drug delivery technologies, because they have already been reviewed several times recently. Instead, we aim to provide a systematic and quantitative view on the pharmacokinetic factors in drug delivery to the posterior eye segment. This review is based on the literature and unpublished data from the authors' laboratory.

Human corneal cell culture models for drug toxicity studies.

In vivo toxicity and absorption studies of topical ocular drugs are problematic, because these studies involve invasive tissue sampling and toxic effects in animal models. Therefore, different human corneal models ranging from simple monolayer cultures to three-dimensional models have been developed for toxicological prediction with in vitro models. Each system has its own set of advantages and disadvantages. Use of non-corneal cells, inadequate characterization of gene-expression profiles, and accumulation of genomic aberrations in human corneal models are typical drawbacks that decrease their reliability and predictive power. In the future, further improvements are needed for verifying comparable expression profiles and cellular properties of human corneal models with their in vivo counterparts. A rapidly expanding stem cell technology combined with tissue engineering may give future opportunities to develop new tools in drug toxicity studies. One approach may be the production of artificial miniature corneas. In addition, there is also a need to use large-scale profiling approaches such as genomics, transcriptomics, proteomics, and metabolomics for understanding of the ocular toxicity.

Long-term topical application of preservative-free prostaglandin analogues evokes macrophage infiltration in the ocular adnexa.

Success of the long-term glaucoma therapy and preservation of the visual function strongly depend on patients' compliance which may be affected by the inconvenience of treatment and its side effects. Recently, introduction of preservative-free anti-glaucoma agents has become an important step towards improved glaucoma care by eliminating the negative effects of preservatives on the eye surface. Although, newly developed eye drop formulations do not contain standard preservatives, they still can be harmful to ocular surface due to other excipients. In this study, we compared tolerability of commercial preservative-free (pf) prostaglandin analogues (pf tafluprost, pf latanoprost and pf bimatoprost) in long-term topical application in rabbits in vivo. We found that after eight weeks treatment, pf latanoprost was the worst tolerated among the tested drops. It expressed increased conjunctival redness and blinking frequency. Furthermore, it caused increased LDH release in the aqueous humour, infiltration of macrophages in the eyelids and visible defects in conjunctival goblet cells. However, we did not detect increased levels of inflammatory markers in the tear fluid or in the aqueous humour. Based on our study, we suspect that these negative effects are related to excipients included in pf latanoprost formulation.

Oxidative stress protection by exogenous delivery of rhHsp70 chaperone to the retinal pigment epithelium (RPE), a possible therapeutic strategy against RPE degeneration.

PURPOSE: To measure the cytoprotective effects of rhHsp70 against oxidative stress and study its cellular uptake, intracellular and intraocular distribution in the retinal pigment epithelium. METHODS: Human retinal pigment epithelial cells (ARPE-19) were pre-treated with rhHsp70 for 24 h, 48 h, and 72 h before being exposed to 1.25 mM hydrogen peroxide. Non-treated cells served as control. We analysed interleukin 6 secretion, cell viability, and cytolysis. Uptake and intracellular distribution of fluorescently labelled rhHsp70 were investigated with flow cytometry and confocal microscopy, respectively. Ocular distribution of radioactively labelled rhHsp70 was followed ex vivo in porcine eyes by micro SPECT/CT. RESULTS: After exposure to hydrogen peroxide, IL-6 secretion decreased by 35-39% when ARPE-19 cells were pre-treated with rhHsp70. Cell viability increased by 17-32%, and cell lysis, measured by the release of lactate dehydrogenase, decreased by 6-43%. ARPE-19 cells endocytosed rhHsp70 added to the culture medium and the protein was localized in late endosomes and lysosomes. Following intravitreal injection into isolated porcine eyes, we found 20% rhHsp70 in the RPE. CONCLUSIONS: Recombinant hHsp70 protein offers protection against oxidative stress. RPE cells take up the exogenously delivered rhHsp70 and localize it in late endosomes and lysosomes. This work provides the basis for a therapeutic strategy to target aggregate-associated neurodegeneration in AMD.

Cationorm shows good tolerability on human HCE-2 corneal epithelial cell cultures.

Preservatives have been for a long time known to cause detrimental effects on ocular surface. Cationorm, a preservative-free compound with electrostatic properties is a novel way to solve the problems encountered with traditional benzalkonium chloride (BAK)-containing eye drops. The aim of this study was to evaluate tolerability of the preservative-free cationic emulsion Cationorm in vitro on corneal epithelial cells. The human corneal epithelial cell (HCE-2) culture line was used to study cellular morphology, cytotoxicity and inflammatory responses after Cationorm diluted 1/10 exposure for 5, 15 and 30 min. Exposures to Systane diluted 1/10 with polyquaternium-1/polidronium chloride 0.001% as preservative, BAK 0.001% or C16 (0.0002%) and normal cell culture medium served as positive and negative references. Cell viability was determined by measuring the release of lactate dehydrogenase (LDH) and mitochondrial dehydrogenase activity was evaluated using 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The possible induction of apoptosis was analyzed by measuring the activity of caspase-3, and Cell Counting Kit-8 (CCK-8) was used to evaluate the number of viable cells after the exposure to test compounds. Furthermore, the tendency of the test compounds to produce inflammatory reaction was determined by analyzing the production of proinflammatory cytokines IL-6 and IL-8, and DNA binding of the p65 subunit of transcription factor NF-κB was measured from cell lysates. HCE-2 cells showed no morphological changes after the exposure to Cationorm, but in cells exposed to BAK, clear cytoplasm vacuolization and loose cell-cell contacts were observed in transmission (TEM) or scanning (SEM) electron microscopic analyses. Cell viability, as measured with the release of LDH, indicated a time dependent increase in LDH expression after exposure to all test compounds but especially with BAK. Moreover, Cationorm and BAK time-dependently decreased the mitochondrial metabolism to 73% with Cationorm and 53% with BAK from that of the control cells after 30 min exposure in MTT assay. BAK was the only test compound having clear adverse effects on the cell number and metabolism in CCK-8 assay. The activity of caspase-3 did not show significant differences between the groups. Inflammatory response after exposure to Cationorm was significantly lower than after exposure to BAK. There were no significant differences in NF-κB activity between the groups. Diluted Cationorm and Systane with polyquaternium-1/polidronium chloride 0.001% showed good tolerability on HCE-2 cells and thereby provide a clear improvement when compared to BAK-containing eye drop formulations.

Endoplasmic reticulum stress in age-related macular degeneration: trigger for neovascularization.

Age-related macular degeneration (AMD) can be classified into two main categories: the atrophic, dry form and the exudative, wet form. The crucial difference between dry and wet AMD is the development of choroidal neovascularization in wet AMD. One fundamental cause of the neovascularization is the increased expression of VEGF (vascular endothelial growth factor) in retinal pigment epithelial cells. Progression of AMD is linked to augmentation of cellular stress, for example, oxidative stress, proteotoxic stress, inflammation and hypoxia. All these conditions can trigger stress in endoplasmic reticulum (ER), which maintains protein quality control in cells. ER stress induces the unfolded protein response (UPR) via IRE1 (inositol-requiring protein-1), PERK (protein kinase RNA-like ER kinase) and ATF6 (activating transcription factor-6) transducers. UPR signaling is a double-edged sword, that is, it can restore cellular homeostasis as far as possible, but ultimately may lead to chronic, overwhelming stress that can cause apoptotic cell death. Interestingly, ER stress is a well-known inducer of angiogenesis in cancer. Moreover, stress conditions associated with the progress of AMD can induce the expression of VEGF. We discuss the role of ER stress in the regulation of neovascularization and the conversion of dry AMD to its wet, detrimental counterpart.

Liquid chromatographic-electrospray ionization mass spectrometric analysis of neutral and charged polyethylene glycols.

Poly(ethylene glycols) (PEGs) are widely used water soluble and biocompatible polymers. PEGs are suitable as paracellular probes in biomembrane permeability studies because they are hydrophilic and various oligomers have defined molecular sizes. In previous studies corneal and conjunctival permeability for neutral PEGs has been measured, and the results were used to calculate the number and size of the cellular pores. In this study we have developed a high-performance liquid chromatographic-electrospray ionization-mass spectrometric method for analysis of neutral PEGs and positively changed amino PEGs simultaneously. The new method is fast, accurate, sensitive and specific for high throughput analysis. The method was used to evaluate the paracellular permeability of PEGs through a corneal epithelial cell culture. Paracellular pores are negatively charged and it was in our interest to characterize the interactions of positive charge and size of the molecules with the paracellular pores.

Simultaneous determination of eight beta-blockers by gradient high-performance liquid chromatography with combined ultraviolet and fluorescence detection in corneal permeability studies in vitro.

A gradient HPLC method with combined ultraviolet and fluorescence detection was developed for the simultaneous determination of eight beta-blockers (alprenolol, atenolol, metoprolol, nadolol, pindolol, propranolol, sotalol and timolol) in corneal permeability studies in vitro. Fluorescence detection with excitation wavelength at 230 nm and emission at 302 nm was selective for six of the compounds, whereas UV detection at 205 nm was able to detect all the compounds. Calibration was performed with fluorescence detection for six compounds from 50 or 200 nM to 3 microM, and with UV detection for all the eight compounds from 100 or 200 nM to 30 microM. With optimized fluorescence detection, detection limits between 0.7 and 1.3 nM (0.035-0.065 pmol per 50 microl injection) were obtained for atenolol, metoprolol, nadolol and sotalol. A mixture of eight beta-blockers was used in cassette dosing permeability studies with a cultured corneal epithelium. The HPLC method revealed marked differences in the permeation between hydrophilic and lipophilic beta-blockers through the corneal epithelial cell culture model.