IN-VITRO LIFE-CYCLE OF PENTAMIDINE-RESISTANT AMASTIGOTES - STABILITY OF THE CHEMORESISTANT PHENOTYPES IS DEPENDENT ON THE LEVEL OF RESISTANCE INDUCED

Using a continuous drug pressure protocol, we induced pentamidine resi stance in an active and dividing population of amastigote forms of Lei shmania mexicana. We selected in vitro two clones with different level s of resistance to pentamidine, with clone LmPENT5 being resistant to 5 mu M pentamidine, while clone LmPENT20 was resistant to 29 mu M pent amidine. Resistance indexes (50% inhibitory concentration [IC50] after drug pressure/IC50 before drug pressure) of 2 (LmPENT5) and 6 (LmPENT 20) were determined after drug selection, Both resistant clones expres sed significant cross-resistance to diminazene aceturate and primaquin e, Pentamidine resistance was net reversed by verapamil, a calcium cha nnel blocker known to reverse multidrug resistance (A. J. Bitonti, et al., Science 242:1301-1303, 1988; A. R. C. Safa et al., J. Biol. Chem. 262:7884-7888, 1987), No difference in the in vitro infectivity for r esident mouse macrophages was observed between the wild-type clone (cl one LmWT) and pentamidine-resistant clones. During in vitro infectivit y experiments, when the life cycle was performed starting from the int ramacrophagic amastigote stage, the drug resistance of the resulting L mPENT20 amastigotes was preserved even if the intermediate promastigot e stage could not be considered resistant to 20 mu M pentamidine. In t he same way, when a complete developmental sequence of L. mexicana was achieved axenically by manipulation of appropriate culture conditions , the resulting axenically grown LmPENT20 amastigotes remained pentami dine resistant, whereas LmPENT5 amastigotes lost their ability to resi st pentamidine, with IC(50)s and index of resistance valued close to t hose for the LmWT clone. These results strongly indicate that the leve l of pentamidine tolerated by resistant amastigotes after the life cyc le was dependent on the induced level of resistance. This fact could b e significant in the in vivo transmission of drug-resistant parasites by Phlebotominae. Particular attention should be given to the finding that the emergence of parasite resistance is a potential risk of the u se of inadequate doses as therapy in humans.