Desarrollo de formulaciones oculares de progesterona para el tratamiento de la retinosis pigmentaria. Development of ocular formulations of progesterone for the treatment of retinitis pigmentosa

Resumo

The term retinitis pigmentosa (RP) is used to describe a group of degenerative diseases, which involve degeneration of the photoreceptor cells of the retina, cones and rods. It is an inheritable, genetic disease which is estimated to affect 1:4,000 people worldwide, approximately 1.5 million people. Initially RP manifests itself by a loss of night vision which is followed by a reduction in peripheral vision and finally central vision. This progressive loss of vision is due to the gradual death of photoreceptor cells. When the rods die as a result of RP progression, oxygen consumption in the retina decreases and therefore, the oxygen concentration in the retina increases. The resulting hyperoxia causes oxidative damage. Treatment with antioxidants to decrease oxidative stress, is considered to be a possible intervention to slow down the degenerative process in RP. In fact, antioxidant treatment in animal models of RP (rd1, rd10 and rds) has been shown to delay photoreceptor cell death. The role of sex steroid hormones and their metabolites, as suppressors of cell death has recently been studied. Progesterone (PG) is a steroid hormone that has been shown in previous studies to inhibit apoptosis and inflammation. It is also able to reduce free radical damage at high concentrations of PG following traumatic brain injury in rats. It may therefore have therapeutic potential for the treatment of various neurodegenerative diseases such as RP. In fact, studies in the rd10 mouse model, have shown that oral administration of PG, 150 mg/kg, from postnatal day 15 to 21 decreases photoreceptor cell death. The mechanism of action of PG treatment remains unclear, but it has been suggested that it could be related to its antioxidant effects in the retina. PG treatment was shown to decrease the amount of lipid peroxidation. Other studies showed similar results in the rd1 mouse model treated with orally administered PG at a lower dose (100 mg/kg) from postnatal day 7 on alternate days. The proposed mechanism for oral administration of PG was the same as in the study conducted in the rd10 mouse model, its antioxidant effects. Oral administration of PG has drawbacks due to the characteristics of the molecule. It is very lipophilic (log P = 3.9), practically insoluble in water and dissolves slowly and incompletely in gastrointestinal fluids. PG is rapidly inactivated when administered orally. It undergoes extensive hepatic and intestinal metabolism and has a plasma half-life of 25 minutes. Its oral bioavailability is limited, which implies that very high concentrations of PG needs to be administered for a small amount of the drug to be able to reach the eye. It is therefore important to study whether it is possible to administer PG topically, locally to the eye, and minimizing the concentration to be administered. In our research, we have studied the incorporation of PG to various formulations and different strategies have been analyzed to assess their possible administration topically to the eye. We have evaluated the use of cyclodextrins, capable of increasing PG solubility and increasing its permeability; as well as encapsulation in polymeric micelles, which facilitate the development of aqueous formulations and increase the permanence of molecules on the ocular surface and their diffusion into the eye.; We have also used ocular inserts, which increase the contact time of the drug with the eye, increasing its bioavailability, and allowing controlled release, precise dosage and less frequent administration. The following specific objectives were set out: (1) to validate a chromatographic method to be able to quantify PG (2) to determine the ex vivo permeability of PG in solution through rabbit cornea and sclera, (3) to elaborate PG micelles that can overcome the solubility and permeability limitations of the drug when intended for the ocular local treatment of RP by assessing the diffusion of the PG-loaded micelles through the cornea and sclera of three animal species (rabbit, pig and bovine) and to study their ocular irritation potential, (4) to develop and characterize an ocular insert to administer PG, by evaluating its ocular irritation potential and to analyze the ex vivo the release of PG from the insert and its diffusion through rabbit cornea and sclera, (5) to determine the ocular biodistribution of PG, using solution and inserts, in pig eyes and (6) to determine the possible therapeutic effect of ocular administration of PG in vivo in rds mice, an experimental model of RP. A high-performance liquid chromatography method to detect PG incorporated into β-cyclodextrin was developed and validated using a Waters Sunfire C18 reversed-phase column (150 × 4.6 mm) packed with 5 μm silica particles and a mixture of acetonitrile (ACN) and pure water 80:20 (v/v) at pH 7.4 as mobile phase. The limit of detection and limit of quantification of PG were 0.42 and 1.26 µg/mL, respectively. The calibration curve showed excellent linearity in the concentration range of 0.5 µg/mL to 100 µg/mL, with a retention time of 3.42 minutes. To analyze the applicability of the method, ex vivo experiments were carried out in diffusion cells to quantify the permeability of PG across rabbit cornea and sclera. The ex vivo experiments demonstrate their applicability to quantify the cumulative amounts of diffused PG per cm2 of membrane (Q = 6.57 ± 0.37 µg/cm2 across the cornea and Q = 8.13 ± 0.85 µg/cm2 in sclera; mean ± SD, n=6), as well as the amounts of PG retained in each tissue, (Q = 40.87 ± 9.84 µg/cm2 in cornea and Q = 56.11 ± 16.67 µg/cm2 in sclera; mean ± SD, n=6). Pluronic F68 (3-4 nm) and Soluplus (52-59 nm) micelles increased PG solubility by one and two orders of magnitude, respectively and exhibited close to 100% encapsulation efficiency. The Soluplus systems showed in situ gelling ability in contrast to the low-viscosity Pluronic F68 micelles. The formulations showed no irritation in the hen's egg test chorioallantoic membrane (HET-CAM) test method. Ex vivo permeability studies were performed in diffusion cells, using eyes from different animal species; cow, pig and rabbit, PG was quantified by HPLC from the samples obtained. PG penetration through rabbit cornea and sclera was faster than through the porcine or bovine cornea, although the differences also depended on the formulation used. PG permeability in pig's eyes was intermediate between the permeability found in rabbit and bovine. Soluplus micelles allowed a higher accumulation of PG in cornea and sclera, while Pluronic F68 micelles provided faster penetration of lower doses of PG. Different inserts were formulated with PG in their composition and their characteristics were analyzed: thickness, weight, translucency, moisture loss and gain, tear resistance, elongation, PG content uniformity, presence of microscopic imperfections and drug release from the insert. The insert with the best characteristics was selected, which was a polymer made up with 59% polyvinyl alcohol, 39% polyvinylpyrrolidone K30 and 2% propylene glycol. Ex vivo diffusion studies were performed with the insert in Franz cells using rabbit cornea and sclera. The quantification of PG in the samples obtained in diffusion studies was performed by HPLC. The study showed sustained diffusion of PG. The diffusion of PG through the cornea and sclera was calculated using a mathematical treatment (chapter 4, equation 7) that considers continuous changes in donor compartment concentrations. The diffusion obtained was similar (corneal Papp = 6.46 ± 0.38 × 10-7 cm/s and scleral Papp = 5.87 ± 1.18 × 10-7 cm/s, mean ± SD, n=5). However, the amount of PG accumulated in the scleras was significantly higher than in the corneas (Q = 30.07 ± 9.09 μg/cm2 and Q = 15.56 ± 4.36 μg/cm2, respectively). The inserts made with PG (55.6 μg/cm2) were thin, translucent, did not cause ocular irritation (HET-CAM test) and were elastic and resilient, properties that make this formulation suitable for potential use in the treatment of ocular diseases. Once the different ophthalmic formulations for topical administration of PG (eye solution and inserts with cyclodextrins and micelles of Soluplus and Pluronic F68 with pure PG) had been studied, the results obtained in ex vivo diffusion studies of PG in rabbit eyes were analyzed together in order to select the best ocular formulation for topical PG administration. To compare all the formulations tested, the data obtained in the previous diffusion studies (solution and micelles) have been used. And re-analysed using the same mathematical treatment used for the sinserts. As the equation is valid for both "sink" (solution and micelles) and "non-sink" (inserts) conditions. The apparent permeability coefficients of the PG were thus calculated and all data obtained were compared. The eye solution showed a higher scleral permeability compared to the other formulations. However, higher permeability coefficients across the cornea were obtained with Pluronic F68 polymeric micelles. Each formulation has its advantages, cyclodextrins can increase the solubility and permeability of PG in eye solution, while polymeric micelles are able to encapsulate hydrophobic drugs such as PG and facilitate the permanence of the formulation on the ocular surface compared to solution because of their higher viscosity. The Pluronic micelles provided higher permeability coefficients than the Soluplus micelles, which may be due to their smaller size (Pluronic, 3 - 4 nm vs. Soluplus, 52 - 59 nm). However, both solution and micelles have the disadvantage that they remain in contact with the ocular membrane for a short time before they are completely removed by blinking. Finally, the PG insert showed lower permeability compared to solution and micelles, but the advantages of eye inserts compared to liquid formulations are numerous. Inserts have higher PG-loading capacity of the insert and is able to provide a sustained release, allowing the effective drug concentration to be guaranteed for a longer period of time, compared to solution and micelles. Drug dosage in the inserts is also more accurate and the risk of side effects might be reduced. In addition, the inserts have a longer shelf life and the addition of preservatives is not necessary. But the main advantage of inserts is that they increase the contact time of the formulation, increasing the availability of the drug. The constant contact of the insert with the ocular membranes suggests that PG would be released and transported in greater amounts to the neuroretina, albeit more slowly than solution or micelles. To investigate whether topically administered PG via the ophthalmic route can reach the neuroretina, its distribution through ocular tissues was studied. The effect of the initial concentration of PG in the formulation was also analysed, as well as the effect of tear clearance. Different formulations of PG were prepared: solution (1 mg/mL), corneo-scleral insert (325.7 μg/cm2) and 3 scleral inserts (32.57, 325.7 and 3257 μg/cm2). The different formulations were administered topically to the ocular surface using whole porcine eyes. After freezing the eye for 12 hours at -80°C, the different anatomical structures of the eye were dissected, and PG was extracted from each tissue fraction in acetonitrile for 12 hours. Quantification of PG in each tissue was performed by UHPLC-MS/MS. The results revealed that, after topical administration, PG diffuses from the ocular surface and it is distributed throughout all ocular structures, including the neuroretina. Lower levels of PG were found in the sclera, choroid and neuroretinal tissues when PG was applied as solution compared to inserts. Only the corneo-scleral insert showed statistically significant differences in the amounts of PG in the cornea compared to solution, probably due to the longer contact time of the drug with the ocular surface when applied by means of the insert. An increase in the initial concentration of PG applied, provided a statistically significant increase in the amounts of PG in the aqueous humour, sclera, choroid and neuroretina. In addition, the results obtained in the simulation of PG elimination by tears, using the ocular insert with or without a constant drip of saline over the cornea, 1.2 µL/min for 24 h, showed there were no statistically significant differences, therefore we can conclude that tears do not affect the amount of PG reaching the neuroretina. As described above, RP triggers photoreceptor death. To study the effect of PG on the death of these photoreceptors, we have studied in an animal model of RP (rds mice) whether topical ocular administration of PG incorporated to cyclodextrins is able to decrease photoreceptor death. In the study, one group of rds mice received one drop of PG-βCD every 12 h for 10 days in one eye, the left eye, while the right eye was left untreated. Another control group of rds mice was treated with saline in the left eye, and again the right eye was left untreated. After treatment, the animals were euthanized on postnatal day 21. TUNEL, GFAP and DAPI staining were performed. Administration of PG-βCD significantly decreased cell death in the retinas of PG-treated rds mice, as well as inflammation. Statistically significant differences were only found in the treated eye, thus revealing that there is no systemic effect from topically administered PG. There seems to be evidence that PG-βCD treatment decreases photoreceptor death in the early stages of RP, which may delay vision loss and decrease inflammation. The data obtained in this work demonstrate that PG can be incorporated into various eye formulations such as eye solution, micelles and inserts. In addition, PG administered topically to the eye, diffuses, permeates and is distributed within the eye in sufficient quantities to reach the neuroretina. It has also been shown that topical ocular administration of PG, reduces photoreceptor death in the rds animal model and thereby delays visual loss. In addition, PG exhibits no toxicity or irritation in HET-CAM studies. Therefore, the data collected here demonstrate for the first time that PG can be administered topically and open the possibility to further investigate these formulations as a new therapeutic strategy for patients with RP, and by extension for patients with other eye diseases associated with oxidative stress such as glaucoma, age-related macular degeneration, macular edema secondary to retinal venous occlusion, cytomegalovirus, retinitis, posterior uveitis and diabetic retinopathy.