Microscopic and Biopharmaceutical Evaluation of Emulsion and Self-Emulsifying Oil with Cyclosporine

Abstract

Among the currently available commercial eye drops with cyclosporine A (Cs) there is a lack of long-acting dosage forms and products with a concentration of the drug substance higher than 0.1%, although Cs is widely used in ophthalmology. The aim of the research was to conduct the microscopic and biopharmaceutical evaluation of two formulations, an emulsion (EM) and a self-emulsifying oil (SEO), both with 0.5% of Cs, proposed for use in eye drops, and the comparison of both. SEO eye drops with Cs or any other drug substance are currently not available as marketed products, and the highest concentration of Cs in the ocular emulsion is only 0.1%. The microscopic evaluation of the emulsion and the SEO after emulsification with water was carried out using a high-resolution digital microscopy. The properties of both preparations were compared using the high dynamic range function or optical shadow effect mode. Images in the 3D composition mode were also recorded. The in vivo study of the Cs formulations was performed on male albino rabbits. The eye tolerance of the preparations was assessed using the ocular irritation test, which is a modified Draize test. Placebo carriers (without the drug substance) were also subjected to irritation testing. The concentration of Cs in the tissues (cornea and conjunctiva) and fluids (tear fluid and aqueous humor) of the rabbit eye was determined after multiple instillations of Cs-EM or Cs-SEO. The tested preparations were compared using the digital microscopy technique, which highlights the features of the formulations and eliminates the risk of unnoticeable properties that are difficult to observe in classical optical microscopy. Both tested Cs-loaded formulations are classified as practically non-irritating. There were also no significant differences when testing the placebo carriers. After a topical administration, Cs was widely distributed in all tissues (e.g., in cornea 1.3 ng/mg and 1.0 ng/mg) and fluids of the eye (e.g., in tear fluid 11.6 µg/mL and 4.3 µg/mL), after the administration of Cs-SEO and Cs-EM, respectively. The obtained results allow us to recognize both tested formulations, the emulsion and the self-emulsifying oil with 0.5% Cs content, as carriers safe for ophthalmic use and effective in delivering the drug substance to the structures of the eye.

Keywords: Draize irritation test; cyclosporine; emulsion; ophthalmic delivery; rabbit; self-emulsifying oil (SEO).

Figure 1

Digital microscope images of Cs–EM formulation obtained with HDR function: (a,b,f); 3D mode: (d,e); and optical shadow effect: (g–i). Section: (c) presents the particle size distribution in the tested emulsion obtained via laser diffraction (point 4.3).

Figure 2

Digital microscope images of Cs–SEO formulation obtained with HDR function: (a,b); 3D mode: (d,e); after staining the water and oil phases with hydrophilic and lipophilic dyes, respectively: (f); and optical shadow effect mode: (g–i). Section: (c) presents the particle size distribution in the tested SEO obtained via laser diffraction (point 4.3).

Figure 3

Digital microscope images of the Cs–SEO coalescence process. Green arrows indicate selected places where the oil droplets begin to coalesce and phase separation disappears. Red arrows indicate the progress of changes occurring during coalescence—the direction of reading is from (a–f).

Figure 4

Local ocular reaction observed under the slit lamp after in vivo instillation of SEO with 0.5% Cs (A,B) or emulsion with 0.5% Cs (C,D) for 5 days. Pictures were taken in visible light (A,C) or in Cobalt blue light after fluorescein instillation (B,D).

Figure 5

Biodistribution (n = 8, mean ± SD, *—statistically significant difference) of Cs in rabbit eyes after multiple (7 days, 14 instillations) topical administrations of Cs-loaded (0.5%) emulsion and SEO: (A) cornea, (B) conjunctiva, (C) tear fluid, (D) aqueous humor.