Tolouei et al.: Leishmanicidal Activity of Films Containing Paromomycin… Introduction Cmore than 88 countries are at risk of urrently 350 million individuals in leishmaniasis. Recently due to increase in CL incidence rate and co- infection of leishmaniasis with HIV, more attention is demanded to the neglected disease. Approximately 90% of all CL cases are reported from eight countries including Iran (1). However using antimony needs multiple injections, accompanies with side effects and the efficacy of antimony is very low especially in CL lesions induced by Leishmania tropica and resistant to anti- mony is reported (2-4). Aminoglycoside antibiotics such as paromomycin showed to be effective against Leishmania parasites and promising results generated in phase 3 clinical trials (5-8). Topical therapy against CL if available is ideal since such topical therapy dramatically improves the patients' compliance. Currently topical formulation of paromomycin and gentamicin is in phase 3 clinical trials (9) (Personal communication with Alan Magill and Max Grogl). In this study, a physical form of film containing paromomycin 15% and gen- tamicin 0.5% was prepared. The anti Leishmania activities of the films were as- sessed in vitro and in vivo. Material and Methods Parasite Leishmania major strain (MRHO/IR/75/ER) used in this experiment is the same strain which was used for leishmanization and preparation of experimental Leishmania vac- cine (10,11). Drug films The films were prepared at School of Phar- macy, Isfahan University of Medical Sci- ences using paromomycin 15% and gen- 61 tamicin 0.5% incorporated into ethyl cellu- lose and Hydroxyl Propyl Methyl cellulose (HPMC). The films contain of backing layer, drug reservoir, rate controlling membrane, and adhesive layer. Direct compression and solvent casting methods are used to make films containing paromomycin and gen- tamicin with permeation enhancer in poly- meric matrices. Physical properties of the prepared films such as flexibility, stability, thickness, and apparent uniformity were checked. Releasing study was done by put- ting the films on Bacillus subtilis culture and growth inhibition zone was assessed and compared with standard curve of discs con- taining the same amount of paromomycin. In vitro experiments Leishmania promastigotes were harvested at stationary phase using a set of modified NNN medium without overlaid liquid layer. The plates were sealed with parafilm and incubated at 26 ± 1°C for 7-10 days, the sur- face of the plates were checked for the growth of the promastigotes. Three types of culture plates were prepared triplicity as fol- low; paromomycin and gentamicin incorpo- rated into ethyl cellulose and HPMC film plates, placebo film plates and control media with no film. The same number of promas- tigotes was cultured in the abovementioned three types of plates. After 4-5 days, the plates were checked for the growth of the promastigotes by direct microscopy and the number of motile and non-motile promas- tigotes was determined. Viability of promas- tigotes was also evaluated using trypan blue. In vivo experiments Female 6 -8 weeks old Balb/c mice were purchased from Pasteur Institute of Iran. The mice were inoculated at the ramp of the tail with 2×106 L. major promastigotes harvested