ANNAMYCIN--MECHANISMS OF ACTION

Annamycin (Ann) is a new lipophilic anthracycline antibiotic with 4 distinct structural features compared with the parent compound doxorubicin (Dox), which confer to the molecule a high affinity for lipid membranes and lack of solubility in water. Our initial hypothesis was that because of its distinct physicochemical characteristics, Ann should be easily amenable to be formulated in a wide variety of liposomal carriers and should interact differently with the recognized cellular targets of anthracyclines. Work performed during the initial funding period has confirmed this hypothesis. In addition, we have shown that Ann incorporated in liposomes displays significant lack of cross resistance with Dox in a panel of 4 tumor cells lines and their MDR counterparts and a markedly enhanced in vivo antitumor activity compared with Dox against several murine models including L1210 leukemia, B16 melanoma, M5076 reticulosarcoma, lung Lewis lung carcinoma, and KB/MDR human xenografts in nude mice. Because of its promising antitumor activity profile, initial clinical studies with Ann entrapped in multilamellar vesicles are planned to start at the end of the current funding period at the cellular level, the lack of cross-resistance properties of Ann have been found to be associated with (1) similar drug accumulation and retention in sensitive and resistant cell lines, (2) 20-30 fold higher drug accumulation and retention compared with Dox in resistant cells, (3) lack of P-glycoprotein mediated drug efflux, (4) extent of DNA damage in resistant cells similar to that of Dox in sensitive cells, (5) significant induction of apoptosis in resistant cells, Dox being ineffective. These results indicate significant differences in the way Ann and Dox interact with P- glycoprotein and topoisomerase-II, which are two well recognized cellular targets of anthracyclines. Ann tissue levels after i.v. administration show a preferential distribution to lung and s.c. tumors (6 and 10 fold difference with Dox, respectively), and an ability to cross the blood brain barrier, while heart levels are similar to those achieved with Dox. The great flexibility in chosing the appropriate liposome carrier offers the opportunity of modulating the pharmacokinetics and organ distribution of Ann for specific therapeutic purposes. The main objectives of this competing renewal proposal are (1) to develop and test against human xenografts liposomal formulations of Ann with enhanced tumor targeting properties and low distribution to heart (small unilamellar liposomes containing lipids that avoid recognition of the particles by the RES, thus resulting in a prolonged serum half-life and a preferential distribution to the tumor), (2) to study the pharmacokinetics, in vivo metabolism, and cardiotoxicity of selected liposomal-Ann formulations in rabbits, and (3) to examine in depth the cellular mechanisms of action of Ann at the different cellular targets of anthracyclines: interaction with topoisomerase II, signal transduction pathways, cell membrane interactions, cell cycle regulation, and apoptosis. The results of these studies will be essential in learning how to optimize the use of Ann and in planning its clinical development strategy.