Bio-adhesive polymers containing liposomes for DED treatment

Dry eye disease (DED) is a prevalent and multifactorial pathology of the ocular surface affecting 5-50% of the world’s population.1 The disease is characterized by a loss of homeostasis of the tear film and it may manifest with ocular symptoms such as dryness and burning.2,3 Tear hyperosmolarity and tear film instability constitute the core mechanisms in DED pathophysiology.4 Recently, neutrophil extracellular traps (NETs) have also been postulated to play a role in the pathogenesis of the disease.5,6 Artificial tears constitute the first-line therapy for mild dry eye.7 Nonetheless, most of these ophthalmic formulations in the market supplement only the aqueous phase of the tear film, providing primarily symptomatic relief. Furthermore, artificial tears have drawbacks such as short ocular residence time.8Objectives and ResultsObjective-1: Development of artificial tear formulations containing novel components, with suitable physicochemical properties, optimum tolerance, and extended ocular residence time. Liposomes were utilized to replenish the lipids of the tear film and bioadhesive and/or in situ gelling polymers (hydroxypropyl methylcellulose and/or gellan gum, respectively) to extend the ocular residence time of the formulations. In addition, antioxidants (vitamins A, C, and E), osmoprotectants (levocarnitine and trehalose), or antidessicants (trehalose) were incorporated into the formulations for their potential beneficial effects in DED treatment. The neutral to slightly alkaline pH (7.0–7.6), surface tension (44.6–48.7 mN/m), viscosity (3.6–12.1 mPa.s), slight hypotonicity (222.7–272.7 mOsm/L), and liposome size (191.0–200.1 nm) exhibited by the formulations in the physicochemical characterization were considered suitable for topical ophthalmic administration. Formulations containing gellan gum produced viscoelastic gels with shear-thinning properties and suitable viscosities (2.8–3.0 mPa.s). In vivo tolerance studies (in rabbits) showed that the formulations resulted well tolerated. Objective-2: Development of novel tools to evaluate the efficacy of the artificial tear formulations. An in vivo dry eye model and an ex vivo dry eye model were developed. Dry eye was induced in rabbits with topical administration of the preservative benzalkonium chloride (BAK) at 0.1% for 15 days. Dry eye signs including corneal damage, goblet cell loss, and impairments in the conjunctival epithelium were observed in animals after the BAK treatment. Furthermore, a complete ex vivo porcine anterior eye model (Aston Biological Anterior Eye Model) was optimized by adjusting the irrigation frequency of the eyes with a tear analogue. Objective-3: Efficacy studies of the novel artificial tear formulations. Preliminary studies. Preliminary efficacy studies of one of the novel formulations, composed of an in situ gelling polymer (gellan gum 0.25%), a bioadhesive polymer (hydroxypropyl methylcellulose 0.12%), osmoprotectants (levocarnitine 0.4% and trehalose 1.68%), an antioxidant (vitamin C 0.01%), electrolytes (sodium chloride 0.05% and potassium chloride 0.05%), and borates (boric acid 0.84% and sodium tetraborate anhydrous 0.08%), were performed in the previously mentioned in vivo and ex vivo DED models, over a period of 21 days and 18 hours, respectively. In the in vivo model, the treatment with the artificial tear promoted significant improvements in the cornea and the conjunctiva at 7 days after continued treatment (significantly reduced fluorescein and cytological scores compared with baseline). In the ex vivo model, the formulation improved the ocular surface with two instillations after 18 hours, though the small sample size did not allow statistical analysis. Objective-4: Assessment of the effects of the novel artificial tear formulations on NET formation as a proof of concept. Excessive amounts and impaired clearance of NETs have been observed on the ocular surface of DED patients and linked with the pathogenesis of the disease.5,6 Effects of the novel artificial tear formulations on NET formation (NETosis) were assessed in neutrophils. The formulations did not increase NET formation in unstimulated neutrophils, demonstrating an optimal tolerance. Furthermore, in neutrophils stimulated with a NETosis inducing agent, all of the formulations reduced NET formation significantly.Conclusions1. All of the novel artificial tear formulations developed in this thesis, containing different combinations of liposomes (made up of phosphatidylcholine and cholesterol), a bioadhesive polymer (hydroxypropyl methylcellulose), an in situ gelling polymer (gellan gum), osmoprotectants (levocarnitine and trehalose), antioxidants (vitamins A, C, and E), and other formulation excipients (fructose, electrolytes, and borates) demonstrate suitable physicochemical properties for topical ophthalmic administration and are well tolerated in rabbits.2. Incorporation of gellan gum at 0.25% with electrolytes created artificial tears with in situ gelling properties, capable of producing viscoelastic gels that suggest an extended ocular residence time of the ophthalmic formulations.3. Twice-daily topical ophthalmic administration of the preservative benzalkonium chloride at 0.1% for 15 days induces significant alterations on the ocular surface of rabbits that are indicative of dry eye disease. The experimental dry eye model can be used as a platform to evaluate the efficacy of artificial tears.4. According to the preliminary efficacy studies performed in the in vivo dry eye model, the novel artificial tear containing the in situ gelling polymer gellan gum (0.25%), the bioadhesive polymer hydroxypropyl methylcellulose (0.12%), the osmoprotectants levocarnitine (0.4%) and trehalose (1.68%), the antioxidant vitamin C (0.01%), borates (boric acid 0.84% and sodium tetraborate anhydrous 0.08%), and electrolytes (sodium chloride 0.05% and potassium chloride 0.05%) may be beneficial in DED treatment.