Synthesis and characterization of a safe and effective hyaluronic acid-hydrocortisone conjugate for topical ophthalmic administration in Dry Eye Disease treatment

Dry Eye Disease (DED) is a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film and by inflammation. Among the possible treatments, an option is represented by corticosteroids, such as hydrocortisone (Hyc), which are drugs with anti-inflammatory and immunomodulatory activity. However, they are indicated only for short-term therapy, since their chronic use and permeation to the anterior chamber of the eye is associated with the manifestation of some ocular side effects, such as an increase in intraocular pressure (which can lead to glaucoma) and cataract. Considering that DED treatment often requires a chronic therapy to manage symptoms, an unmet medical need for innovative formulations with significant anti-inflammatory activity and minimized side effects is present. In this thesis work, two new hyaluronic acid-hydrocortisone conjugates have been designed, synthesized, characterized and formulated in eye drops for the treatment of Dry Eye Disease. The rationale for the administration of hydrocortisone conjugated to HA is to limit the permeation of the drug beyond the cornea, maintaining the anti-inflammatory effect on the surface of the cornea, but avoiding the side effects of long-term use. Specifically, the aim of this thesis is the synthesis and characterization of a hyaluronic acid-hydrocortisone (HA-Hyc) conjugate with a degree of molar substitution (DSmol) between 5% and 15% mol/mol, stable in the eye drops formulation and effective in preventing or reducing the corneal permeation of hydrocortisone. At first, hydrocortisone 21-mesylate and hydrocortisone 21-iodide were synthesized and then they were both reacted with the tetrabutylammonium salt of HA, giving a HA functionalized with hydrocortisone through an ester bond. Another derivative was synthesized via click chemistry, using a copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction. For this reaction, a 21-azide derivative of hydrocortisone was synthesized and then further reacted with a HA propargyl amide derivative, to obtain a HA functionalized with hydrocortisone through a 1,2,3-triazole ring. After the chemical synthesis, the derivatives were characterized via 1H NMR, the DSmol was determined both via 1H NMR and with UV/Vis spectroscopy and the unreacted hydrocortisone derivative was determined with HPLC-UV/Vis. The HA-Hyc derivatives were successfully obtained and those with an appropriate DSmol were formulated in eye drops. The formulations prepared with the ester derivative obtained from the synthesis with Hyc 21-mesylate and with the click derivative appeared transparent and were compatible with the sterilizing filtration. The stability data of the tested formulations showed that the ester derivative was completely hydrolyzed after 2 months at 25 °C once formulated in eye drops. On the contrary, the click HA-Hyc derivative did not release hydrocortisone when stored at 55 °C for 30 days. An impurity appeared over time, but being below the identification and qualification threshold of the ICH Q3B(R2), it was not necessary to identify and qualify it. In the end, the corneal permeation of the click HA-Hyc derivative was tested in an in vitro/ex-vivo model using Franz cells and compared with a control of hydrocortisone sodium phosphate. The experiment was performed using synthetic membranes or porcine corneas. In both cases the conjugation proved to be effective in reducing the transcorneal permeation of the active principle in comparison to the control. In conclusion, the click HA-Hyc conjugate has been selected as the best candidate because it meets the desired features: it has a DSmol between 5% and 15% mol/mol, it is compatible with the sterilization by filtration, it is stable during the stability study in accelerated condition and it reduces hydrocortisone permeation to the anterior chamber of the eye.