In Vitro and In Vivo Evaluation of Aminopeptidase Inhibitors as Antimicrosporidial Therapies

Microsporidia of the genus Encephalitozoon are emerging obligate intracellular pathogens in immunocompromised hosts. To date, there have been few advances in the treatment of microsporidiosis, and a limited number of drugs have been used to treat microsporidial infections with varied success. Two agents, albendazole and fumagillin, show activity in vitro and in limited clinical applications. Fumagillin, an angiogenesis inhibitor, has been used topically to treat ocular infections of E. intestinalis and E. hellem, however it is systemically toxic to humans at therapeutic doses. In comparison, TNP-470, a derivative of fumagillin, had a relatively low toxicity in an athymic mouse model, and was able to reduce replication of the Encephalitozoon spp. and Vittaforma corneae in vitro. To date, antimicrosporidial agents have been evaluated primarily on an empirical basis. Several in vitro systems for measuring antimicrosporidial activity have been described. A variety of animal models describe Encephalitozoon spp. infections in squirrel monkeys, rabbits, and a variety of genetically modified, immunosuppressed or immunodeficient mice. The time course for development of a fatal E. cuniculi infection in athymic and SCID mice is predictable and reproducible, while infection in immunocompetent mice is typically asymptomatic. The E. cuniculi infected athymic mouse model has been primarily used for immunological and host-parasite interaction studies, and no well-defined model for drug efficacy studies has been described. However, the athymic murine model has been used on a limited basis to investigate the efficacy of TNP-470 and albendazole. Proteases are increasingly investigated as targets for therapeutic intervention for infectious diseases through the development of low-toxicity inhibitors. Aminopeptidase inhibitors have shown promise in vitro against several protozoan infections. Bestatin, [(2S,2R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-Leucine, a modified dipeptide aminopeptidase inhibitor, is a reversible inhibitor with many analogues including a nitrated semisynthetic compound, nitrobestatin, [2S, 3R]-3-amino-2-hydroxy-4-[-4-nitrophenyl]-butanoyl-L-Leucine. The naturally occurring modified tripeptide, amastatin ([(2S, 2R)]-3-amino-2-hydroxy-5-methylhexanoyl]-Val-Val-Asp-OH), is also a potent protease inhibitor. This study evaluated the antimicrosporidial activity and toxicity of selected aminopeptidase inhibitors in vitro and in vivo using an E. cuniculi infected