In-vivo Characterization of Sustained Release Timolol Microspheres

Abstract

Introduction: : Background: Although several clinically effective intraocular pressure (IOP) lowering drugs are available for the treatment of glaucoma, patient compliance is often poor leading to suboptimal IOP control and avoidable loss of vision. To address this problem, we recently fabricated microspheres that can deliver timolol maleate as they slowly dissolve and can be delivered by subconjunctival injection. Here we evaluate the in vivo release characteristics and tissue compatibility of the microspheres in rabbit and cat eyes.

Methods: : Timolol microspheres were fabricated using a double emulsion technique a blend of poly(lactic-co-glycolic acid) and poly(D,L-lactic acid). Microspheres were delivered subconjunctivally by a single injection into the eyes of either normal New Zealand White rabbits or cats. Aqueous humor was repeatedly collected for up to three months (rabbits) or 32 days (cats), and vitreous humor was obtained from rabbits after each animal was sacrificed. The concentration of timolol was determined using HPLC/MS at 294 nm. Histochemical and immunohistochemical methods were used to evaluate whether injection of the spheres elicited an inflammatory response in the anterior segment of rabbit eyes.

Results: : The fabricated microspheres have an average size of 16.1 uM (+/- 5.1 uM) and are capable of delivering 21.8 ug of timolol per mg of microspheres over 107 days in vitro. In vivo we were able to detect timolol in the aqueous humor (150 ng/ml) of cats until the end of the experiment (32 days after injection). In rabbits timolol could be detected for thus far for up to 55 days after injection in the aqueous humor (0.3 ng/ml) and up to 62 days in the vitreous humor (151 ng/ml). Gross observation of the eyes at the injection sites did not reveal signs of inflammation, infection, or other side effects in rabbits (0/60 eyes) or cats (0/12 eyes). Histochemical and immunohistochemical examination of rabbit eyes 10 and 21 days after injection did not reveal signs of inflammation. Timolol was undetectable in the serum of all animals and at all times assayed (detection limit = 0.05 ng/ml).

Conclusions: : Our data demonstrate that the sustained delivery of Timolol from a single injection for significant periods of time is feasible. In addition, thus far this delivery method has proven to be safe and well tolerated.

Keywords: drug toxicity/drug effects • injection • intraocular pressure