Patricia Loma

Small‐interfering RNAs (siRNAs) as a promising tool for ocular therapy

RNA interference (RNAi) can be used to inhibit the expression of specific genes in vitro and in vivo, thereby providing an extremely useful tool for investigating gene function. Progress in the understanding of RNAi‐based mechanisms has opened up new perspectives in therapeutics for the treatment of several diseases including ocular disorders. The eye is currently considered a good target for RNAi therapy mainly because it is a confined compartment and, therefore, enables local delivery of small‐interfering RNAs (siRNAs) by topical instillation or direct injection. However, delivery strategies that protect the siRNAs from degradation and are suitable for long‐term treatment would be help to improve the efficacy of RNAi‐based therapies for ocular pathologies. siRNAs targeting critical molecules involved in the pathogenesis of glaucoma, retinitis pigmentosa and neovascular eye diseases (age‐related macular degeneration, diabetic retinopathy and corneal neovascularization) have been tested in experimental animal models, and clinical trials have been conducted with some of them. This review provides an update on the progress of RNAi in ocular therapeutics, discussing the advantages and drawbacks of RNAi‐based therapeutics compared to previous treatments.

Diadenosine tetraphosphate induces tight junction disassembly thus increasing corneal epithelial permeability

Here, we have studied the effects of the dinucleotide P1, P4‐Di (adenosine‐5′) tetraphosphate (Ap4A) on corneal barrier function conferred by the tight junction (TJ) proteins and its possible involvement in ocular drug delivery and therapeutic efficiency.

Diadenosine polyphosphates release by human corneal epithelium

Diadenosine polyphosphates are a type of dinucleotides that have been detected in rabbit and human tears. However, their origin and their mechanism of release have not been fully elucidated. In this work we investigated whether the dinucleotides Ap4A and Ap5A can be released from human corneal epithelia as a consequence of shear stress stimuli. In in vitro experiments, concentrations of Ap4A and Ap5A before mechanical stimulus of stratified human corneal epithelial cells were 3.18 ± 0.43 nM and 0.81 ± 0.13 nM, respectively. After shear stimulation, concentrations significantly increased to 12.01 ± 2.19 nM for Ap4A and 2.83 ± 0.41 nM for Ap5A. No significant differences in lactate dehydrogenase activity were detected between non-stimulated stratified human corneal epithelial cells and cells exposed to mechanical shear-stress, indicating that the rise of dinucleotide levels was not due to cell lysis. In in vivo experiments, individuals subjected to a rise in blinking frequency showed a significant increase of Ap4A (∼25-fold when experiment was performed without anaesthetic and 75-fold with anaesthetic) and Ap5A concentration in tears (∼50-fold when experiment was performed without anaesthetic and 125-fold with anaesthetic). Shear-stress stimuli induces Ap4A and Ap5A release from human corneal epithelium, thus explaining the origin of these relevant compounds for the ocular surface biochemistry and physiology.

Topical application of nucleotides increase lysozyme levels in tears

The present work studies the effects of topical application of nucleotides on rabbit tear lysozyme levels. Lysozyme values were determined by the diffusion in Agar method described by van Bijsterveld in 1974, and the protein amount was obtained by measuring the inhibitory halos around a Whatman n°1 paper disc of 5 mm in diameter. The tested nucleotides were UTP, Ap4A and Up4U. These compounds were topically instilled in a single-dose in one eye (with the contralateral eye as a control) and the lysozyme halos were measured along 5 hours. The obtained results showed an increase in the lysozyme concentrations of 67%, 93%, and 119% for UTP, Ap4A, and Up4U, respectively, over the basal levels of lysozyme. For this reason, we suggest these molecules as a potential treatment for the reinforcement of the tear film barrier against ocular infection.

Lactoferrin Levels in Tears are Increased by the Topical Application of Diadenosine Tetraphosphate.

ABSTRACT Purpose: This study was undertaken to determine the effect of the topical application of diadenosine tetraphosphate on lactoferrin levels in rabbit tears. Methods: Diadenosine tetraphosphate was topically instilled in a single-dose, tear samples were collected by micropipette and lactoferrin was measured by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results: The concentration of lactoferrin in rabbit tears was significantly increased 1 h after diadenosine tetraphosphate application, remaining elevated for 3 h more. This effect was blocked by P2 receptors antagonists. Conclusions: Topical application of diadenosine tetraphosphate stimulates the secretion of lactoferrin in rabbit tears through P2 receptor activation.

Beta2 adrenergic receptor silencing change intraocular pressure in New Zealand rabbits

Purpose/aim Glaucoma consists of a group of progressive optic neuropathies that are characterized by degeneration of the optic nerve and irreversible visual filed loss. Elevated intraocular pressure is the only proven treatable risk factor and commercial products used for glaucoma treatment are focused in lowering intraocular pressure. These drugs can have various undesirable side effects and this invites to look for new strategies. The purpose of this work is to study the use of a siRNA (small interfering RNA) to selectively silence beta2 adrenergic receptors and to see whether it reduces IOP (intraocular pressure). Material and methods Topical instillation of beta2 adrenergic receptors small-interfering RNA (siRNA, 25–250 μg) was applied and IOP was measured with a Tonopen XL up to 9 consecutive days. The effect of such siRNA was compared to commercial compounds such as Timoftlol, Trusopt and Xalatan, and it was also analyzed if some anatomical changes occurred by microscopy. Results siRNA designed for beta2 adrenergic receptor induced a reduction of intraocular pressure (IOP) of 30 ± 5%, compared to a control (scrambled siRNA). The results in terms of IOP decrease were similar to that found with commercial compounds but a long-lasting hypotensive action was shown by beta2 adrenergic receptor siRNA treatment as compared to commercial drugs. No apparent side effects were observed in the ocular structures. Conclusion The use of siRNA against the beta2 adrenergic receptors could provide an interesting therapeutic strategy for glaucoma treatment.