Rapid assessment of ocular drug delivery in a novel ex vivo corneal model

Abstract

Drug delivery by topical application has higher patient acceptance and lower morbidity than intraocular injection, but many ophthalmic treatments are unable to enter the eye or reach the posterior segment after topical application. The first stage towards posterior segment delivery after topical application is ocular surface penetration and existing models are in vivo or use large quantities of tissue. We therefore developed a novel ex vivo model using discs of porcine and human cornea and sclera (5 mm diameter) to assess penetration of a candidate neuroprotective siRNA. siRNA against caspase 2 or control solutions of known penetrance were applied to the corneal epithelial surface and trans-corneal penetration and corneal adsorbance measured at fixed time points. To demonstrate that leakage did not occur, we applied dextran blue, which should not penetrate the intact cornea and did not do so in our model. Fluorescein penetration (0.09%) was less than rhodamine B (6.98%) at 60 min. siCASP2 penetration was 0.01% by 60 min. When the applied siCASP2 was washed off after 2 min, (representing lacrimal drainage) 0.071% penetrated porcine cornea by 60 min and 0.0002% penetrated human cornea and 0.001% penetrated human sclera. Our ex vivo model rapidly and cost-effectively assesses transcorneal penetration of candidate topical therapies, allowing rates of trans-corneal penetration for potential therapies such as siRNA to be evaluated with small quantities of human or animal tissue. Ocular penetration of topically-applied drugs remains a pharmaceutical challenge. Small interfering ribonucleic acid (siRNA) molecules have the potential to treat a wide range of ophthalmic pathologies 1 , generating an interest in agents to enhance their penetration after topical application 2. In particular, siRNA against caspase-2 (siCASP2) prevents retinal ganglion cell death and is in Phase 3 clinical trials for non-arteritic anterior ischaemic optic neuropathy (NCT02341560; ClinicalTrials.gov) 3, 4. Current application of siRNA requires intraocular injection, which is invasive, carries a risk of infection and is less acceptable to patients than other methods of drug delivery. Diffusion of compounds through the eye after topical application is hindered by the presence of anatomical barriers including the tear film, cornea, conjunctiva, sclera, choroid, aqueous, lens and vitreous 5. Having passed through these barriers, compounds are additionally cleared by vascular or aqueous drainage 5, 6. The bloodaqueous and the blood-retinal barriers provide challenges for systemic drug delivery in addition to systemic toxicity 7-9. As a result, after initial topical application approximately 1/100,000 of compounds such as steroids including prednisolone acetate will reach the back of the eye 5, 10. The therapeutic efficacy of such dilutions may not be sufficient for biologically relevant treatment effects. However, topical delivery offers the potential for noninvasive treatment of intra-ocular diseases with greater patient acceptance and reduced compl...

Determining the effect of ocular chemical injuries on topical drug delivery

“…Both alkali and acid injury significantly altered corneal barrier integrity as measured by TEER, consistent with previous reports (Guimera et al., 2012 ; Fukuda & Sasaki 2016 ; Uematsu et al. 2016 ; Kaluzhny et al., 2018 ; Begum et al., 2020 ). Microscopy of alkali-injured corneas demonstrated significant structural abnormalities of the epithelium and stroma and the drop in TEER most likely reflects immediate epithelial damage after acid or alkaline injury.…”

“…Corneal preparations were carried out as previously described (Begum et al., 2020 ). Briefly, porcine eyes were procured un-scalded and within two hours of death from Dissect Supplies (Birmingham, UK).…”

“…We therefore aimed to define the effect of acid and alkaline corneal injury on corneal absorption (modeling drug retention in the cornea) and permeability, defined as penetration across the cornea of dexamethasone and to reference these effects to polar/non-polar compounds in our ex vivo model of corneal penetration (Begum et al., 2020 ).…”

“…Ocular chemical injury (OCI) is an emergency requiring immediate action and treatment and can be caused by contact with either acid, such as sulfuric acid (battery acid), or alkali, such as calcium hydroxide (e.g. in cement) (Blanch et al, 2018). As acid and alkaline products are commonplace in the home and at work, OCI are common, comprising 11.5-22.1% of all ocular injuries (Wagoner, 1997;Sharma et al, 2018), with an annual incidence of 5.1-5.6 cases per 100,000 population in the USA and UK (White et al, 2015;Ghosh et al, 2019).…”

Development and Evaluation of Polyvinylpyrrolidone K90 and Poloxamer 407 Self-Assembled Nanomicelles: Enhanced Topical Ocular Delivery of Artemisinin

“…The apparent permeability coefficient was calculated using the following formula: where, Δ Q /Δ t is the flux across the cornea (ng/min), A is the diffusion area exposed, Co is the starting concentration of the artemisinin in a donor region, and 60 is regarded as the minute-to-second factor. The corneal flux was derived from the path of the regression line, which was drawn for the amount of drug ( Q ) penetrated by time ( t ) [ 32 , 33 ].…”