Drug Reservoir Function of Human Amniotic Membrane Miklo ´ s D. Resch, 1 Be ´ la E. Resch, 2 Eszter Csizmazia, 3 La ´ szlo ´ Imre, 1 Ja ´nos Ne ´ meth, 1 Piroska Szabo ´ -Re ´ ve ´ sz, 3 and Erzse ´ bet Csa ´ nyi 3 Abstract Purpose: The aim of the study was the quantitative pharmacokinetic evaluation of drug release from pretreated amniotic membrane (AM) in vitro. Methods: Cryopreserved AM pieces soaked in 3% ofloxacin ophthalmic solution were mounted in vertical Franz-diffusion cell system equipped with autosampler. In vitro release of ofloxacin was determined by quan- titative absorbance measurement carried out with a UV spectrophotometer (wavelength 287 nm). Three groups were created according to the duration of soaking: 60 (Group 1), 120 (Group 2), and 180 (Group 3) minutes. Released amount of ofloxacin pro 1 cm 2 of AM (mg/cm 2 ) was calculated in the period of 1 to 450 min. Results: Ofloxacin was detectable in the acceptor phase 1 min after mounting in all groups. Until 120 min, rapid increase of released ofloxacin could be observed. From 120 to 450 min, the amount of released ofloxacin showed a slower increasing pattern. Released ofloxacin in Group 1 was significantly lower than in Group 2 after 90 min (19.4 – 10.4 mg/cm 2 , 51.6 – 20.7 mg/cm 2 , respectively, P = 0.044). In Group 3, cumulative drug release was higher than in Group at all timepoints. No significant difference could be demonstrated between Groups 2 and 3 at only 1 min timepoint. Conclusion: Significant ofloxacin reservoir capacity of a single human amniotic layer could be demonstrated in vitro. AM acted as an ofloxacin slow release device for upto 7 h in vitro, depending on the duration of pretreatment of AM. Individual pretreatment of AM could increase beneficial effects of AM transplantation, especially in infectious keratitis. Introduction A mniotic membrane (AM) transplantation was found to be beneficial in various ocular surface disease including infectious keratitis and perforating or nonperforating corneal ulcers. 1–4 Topical antimicrobial treatment is essential after AM transplantation in infectious diseases, and ocular phar- macokinetic features of several topical fluorokinolones have been published. 5–8 Besides ocular penetration of topically administered me- dicaments, 9 AM permeability has been recently published. In vitro and animal experiments showed that AM comprises a dual pharmacokinetic impact on topically administered ofloxacin: barrier and drug reservoir function. Kim et al. 10 pointed out the concept of using AM as a slow-release drug reservoir of ofloxacin in an animal model; they investigated AM, tear film, corneal, and aqueous levels of ofloxacin in rabbit eyes after AM transplantation. In our previous works, we described an in vitro model for pharmacokinetic exami- nation of AM. 11 The objective of our study was to quantify the drug reservoir function of human AM applying the model developed for transamniotic pharmacokinetics of ofloxacin, a frequently used broad-spectrum topical antibi- otic in ocular surface disease. 12 Methods AM preparation The research was approved by the Institutional Human Experimentation Committee and adhered to the tenets of the Declaration of Helsinki. AM obtained by elective Cesarean section was separated from the chorion as soon as possible, but latest 1 h after delivery by blunt dissection and rinsed with phosphate-buffered solution (PBS pH: 7.24). AM pieces of 25 mm in diameter (with the epithelial side up) were placed on Porafil cellulose acetate membrane filters of the same size (Machenerey-Nagel, GmbH & Co KG) with a pore diameter of 0.45 mm. AM pieces on filter membranes were frozen at - 20°C. Neither antibiotic nor preservative was added to the medium. 1 Department of Ophthalmology, Semmelweis University, Budapest, Hungary. Departments of 2 Pharmacodynamics and Biopharmacy, and 3 Pharmaceutical Technology, University of Szeged, Szeged, Hungary. JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS Volume 27, Number 4, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/jop.2011.0007 323 323 323