1. Investigación

Progesterone (PG) has been shown to have a slowing effect on photoreceptor cell death in mouse models of retinitis pigmentosa when administered orally. The aim of this study was to investigate whether ophthalmically administered progesterone was able to reach neuroretina and thus, the distribution through ocular tissues of different PG formulations was studied. The effect of different initial PG concentration was also investigated. Different formulations with PG in their composition (drops, a corneal/scleral-insert and scleral-inserts) were prepared and assayed. Using whole porcine eyes, the different formulations were topically administered to the ocular surface. Frozen eyes were dissected, the PG in each tissue was extracted in acetonitrile and the amount of PG quantified by UHPLC-MS/MS. Our results show that after topical administration, PG diffuses from the ocular surface and distributes throughout all tissues of the eye. Lower levels of PG were found in sclera, choroid and neuroretina when PG was applied as drops compared to inserts. Our results also show that an increase in the initial PG concentrations applied, resulted in a statistically significant increase in the amounts of PG in aqueous humour, sclera, choroid and neuroretina.

Progesterone (PG) diminishes free radical damage and thus can afford protection against oxidative stress affecting the retina. The therapeutic use of PG is limited because it is a highly hydrophobic steroid hormone with very low solubility in water. This is the main drawback for the therapeutic application of PG at ocular level. The aims of this study were: (i) to analyze if PG causes ocular irritation (ii) to validate a HPLC method to determine PG in ex vivo studies and (iii) to evaluate PG permeation through cornea and sclera. A high performance liquid chromatographic method was developed and validated to detect PG incorporated to β-cyclodextrin using a Waters Sunfire C18 (150 × 4.6 mm) reverse-phase column packed with 5 μm silica particles using a mobile phase consisted of a mixture of acetonitrile (ACN) and pure water 80:20 (v/v), pH 7.4. The limit of detection and the limit of quantification for 50 μL injection of PG were found to be 0.42 and 1.26 μg/mL, respectively. The calibration curve showed excellent linearity over the concentration range (0.5 μg/mL to 100 μg/mL). As proof of concept, ex-vivo experiments to investigate PG permeation through cornea and sclera with vertical diffusion cells were carried out to quantify PG diffusion. Ex vivo experiments demonstrate its applicability to investigate permeation levels of PG from 6.57 ± 0.37 μg/cm2 at cornea and 8.13 ± 0.85 μg/cm2 sclera. In addition, at the end of diffusion studies the amount of PG retained in each tissue was also quantified, and it was 40.87 ± 9.84 μg/cm2 (mean ± SD; n = 6) in cornea and 56.11 ± 16.67 μg/cm2 (mean ± SD; n = 6) in sclera.

Progesterone (PG) affords neuroprotection in degenerative diseases associated to oxidative stress, such as cataracts, age-related macular degeneration, glaucoma, diabetic retinopathy and retinitis pigmentosa. The aim of this project was to develop ocular inserts for delivery of PG to the eye. Different inserts with PG in its composition were formulated and the insert with the best characteristics (59% polyvinyl alcohol, 39% polyvinylpyrrolidone K30 and 2% propylene glycol) was selected for ex vivo studies. Physical characteristics and drug release patterns of the insert were analysed. In vitro diffusion studies revealed a controlled diffusion of progesterone. Ex vivo experiments demonstrated similar trans-corneal and trans-scleral PG diffusion (corneal apparent permeability coefficient 6.46 ± 0.38 × 10-7 cm/s and scleral apparent permeability coefficient 5.87 ± 1.18 × 10- 7 cm/s; mean ± SD; n = 5). However, the amount of PG accumulated in scleras was statistically higher than in corneas (30.07 ± 9.09 μg/cm2 and 15.56 ± 4.36 μg/cm2 respectively). The PG-loaded inserts (55.6 μg/cm2) were thin, translucent, showed no irritancy (HET-CAM test) and were elastic and robust, all suitable properties for its potential use in the treatment of several ocular diseases.