Studies on New Approaches to the Chemotherapy of Leishmaniasis

The ability of three different 'physical' drug delivery systems: liposomes, niosomes and starch microparticles, to increase the efficacy of sodium stibogluconate in clearing L. donovani from the liver of mice have been characterised. The efficacy of free sodium stibogluconate in this model has been used as the standard against which to measure the effectiveness of the drug delivery systems. The two vesicular systems; liposomes and niosomes, gave an approximate ten-fold increase in efficacy compared with the free drug. Modifying the properties of the vesicles by varying their cholesterol content and hence their permeability to the encapsulated drug or varying their surface charge by the inclusion of dicetyl phosphate appeared to have little effect on their efficacy as carriers of sodium stibogluconate. Surprisingly, treatment of infected mice with empty vesicles also led to a small decrease in the parasite load. Testing of the vesicular systems against Leishmania infected peritoneal exudate cells (PECs) in vitro gave results similar to those obtained using the in vivo model in that they increased the efficacy of the sodium stibogluconate. Interestingly, L. m. mexicana and L. donovani were equally susceptible to sodium stibogluconate in vesicles. Whereas L. m. mexicana is relatively unaffected by the free drug as with the in vivo model, empty vesicles had an effect on the parasites in exposed PECs, although this effect was difficult to quantify as there was also considerable toxicity to the PECs. The third drug delivery system tested in vivo, starch microparticles, resulted in a 100-fold increase in efficacy compared with the free drug, thus proving much more potent than either of the vesicular systems. This difference could be due to the mechanism of drug release within the host cell. Nevertheless, all three drug delivery systems tested show potential for the treatment of leishmaniasis. Their use ensures that less drug is required, so overcoming the problems of drug toxicity associated with sodium stibogluconate. The efficacy of tubercidin in combination with inhibitors of nucleoside transport in mammalian cells, nitrobenzoylthioinosine (NBMPR) and dipyridamole, in the treatment of experimental leishmaniasis was investigated both in vitro and in vivo. The two nucleoside transport inhibitors, at concentrations which would protect mammalian cells, did not protect promastigotes of L. donovani or L. m. mexicana from the toxic effects of tubercidin. Tubercidin, both alone or in combination with these inhibitors, was toxic to the promastigotes of the two species with approximate LD50s of 0.1 muM and 0.2 muM , respectively, and an approximate MLC of 1 muM for both species. When used alone, tubercidin proved highly toxic to PECs at all concentrations tested (1-10 muM) . These cells were protected from the toxicity of tubercidin by nitrobenzoylthioinosine and dipyridamole. Treatment of infected peritoneal exudate cells with the drug combination did not result in a reduction in total parasite numbers or the number of cells infected. In vivo studies using tubercidin alone or in combination with either dipyridamole or NBMPR confirmed that these inhibitors lessened the toxicity of tubercidin to mice although there was no reduction in the parasite load of infected animals treated with the combination. These results indicate that, unlike the situation reported for malaria parasites, Leishmania does not appear to change the mechanism of uptake of nucleosides into the host cell. Investigations of conditions prevailing in the parasitophorous vacuole of Leishmania infected cells and their importance to parasite survival were carried out by the use of methlyamine, ammonium chloride and dextran sulphate to modify them. The use of methylamine and ammonium chloride to raise the pH of the parasitophorous vacuoles did not affect the growth of L. m. mexicana in PECs but both the growth and survival of L. donovani amastigotes was greatly impaired. In similar experiments dextran sulphate affected the growth of both species of Leishmania equally, but the cause of this effect remains unclear. In a preliminary study, the effect of pH on the uptake and incorporation of adenosine and leucine by different species and stages of Leishmania was also determined. Interestingly, the optimum pH for uptake by metacyclics of L. major (pH 4-5) differed from that of the other promastigotes investigated (pH 6-7) and could represent a preadaption of these forms to life in the lysosomal system of macrophages. Taken together the results of the uptake and incorporation studies and the results of manipulating the lysosomal pH, suggest that L. m. mexicana amastigotes may be adapted for life at a higher pH than L. donovani. This may reflect differences in the survival stratagies of these two species of Leishmania. (Abstract shortened by ProQuest.)