IN VITRO OCULAR BIOCOMPATIBILITY OF NOVEL HP-β-CD ACETAZOLAMIDE COMPLEXES

JAVIER A. CALLES, MARIA J. MORA, ANTONIO LOPEZ-GARCÍA, SANTIAGO D. PALMA, ENRIQUE M. VALLÉS, MARCELA R. LONGHI, GLADYS E. GRANERO, YOLANDA DIEBOLD

Congreso; Xth Spanich-Portuguese Conference on Controlled Drug Delivery; 2013

INTRODUCTION Cyclodextrins (CDs) are a group of natural products formed during bacterial digestion of starch. These cyclic oligosaccharides consist of (α-1,4)-linked α-D glucopyranose units with a hydrophilic outer surface and a lipophilic central cavity1. CDs are able to solubilize many lipophilic water-insoluble drugs otherwise is hard to formulate in aqueous eyedrop solutions2,3. Acetazolamide (ACZ) is a carbonic anhydrase inhibitor used orally for the reduction of intraocular pressure (IOP) in patients suffering from glaucoma4. The two major problems that hinder the topical effectiveness of ACZ are its poor aqueous solubility (w0.7 mg/ml) and a low corneal permeability coefficient of 4.1x10-6 cm/s5. In order to enhance the ocular bioavailability of ACZ, a multicomponent complex with hydroxypropyl-ß-cyclodextrin (HP-ß-CD) and triethanolamine (TEA) was prepared for ocular topical application. The aim of this work was to evaluate the in vitro ACZ delivery performance and the bio-compatibility between ocular surface cells and ACZ complexes as a first step in the design of a topical DDS for ocular administration. MATERIALS AND METHODS The inclusion complexes were obtained by dissolving precisely weighed quantities of ACZ, HPβCD and / or TEA, in 1:1 and 1:1:1 molar ratios, in Milli Q water. The solutions were sonicated, filtered, frozen and then lyophilized. The human corneal epithelial cell line (HCE)6 was used to determine whether films were biocompatible after 24 h exposure in terms of: i) Cell viability and ii) Cell proliferation. To evaluate potencial cytotoxic effects of complexes, the XTT test was used with 0,001% Benzalkonium chloride as cytotoxicity positive control [n=3]. Viability was mesured immediatly after complex removal. To measure cell proliferation rate alterations, the Alamar Blue test was used [n=3]. Cells also were observed by optical microscope to determine morphological alterations. The in vitro release of ACZ for all the formulations was determined using the membrane diffusion technique. The diffusion cell is comprised of two compartments separated by a non-limiting cellulose ester membrane (0.22 mm pores) soaked for 24 h in the diffusion medium, which does not constitute as a barrier against ACZ diffusion. The donor compartment was filled with 2.0 ml of the formulation as a suspension or solution, containing 5 mg of ACZ or an accurately measured amount of ACZ complex or physical mixture. The receiver compartment (bottom) was filled with 10.5 ml pH 7.4 phosphate buffered saline. At various time intervals up to 5 h, aliquots were withdrawn from the receiver side and replaced with an equal volume of fresh phosphate buffer. Simultaneous complexation and salt formation with TEA significantly increased the HP-β-CD solubilizing power for ACZ by forming drug/HP-β-CD/TEA multicomponent system, enhancing the ACZ availability in aqueous HP-β-CD solutions. Furthermore, significantly better results with formulations containing HP-β-CD and TEA from the release studies establish TEA as an effective additive for the corneal transport of ACZ in aqueous eye drops.