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The leishmaniases consist of visceral and cutaneous syndromes present in 30 endemic regions of the world. Miltefosine (hexadecylephosphocholine) is the first oral agent that is effective and tolerated for both visceral and cutaneous disease in several endemic regions, and represents a major advance in the treatment of these diseases.

Visceral leishmaniasis (kala-azar) results from parasitization of the macrophages of the liver, spleen, and the rest of the visceral reticuloendothelial system with Leishmania donovani. Pentavalent antimony is the drug of choice for leishmaniasis chemotherapy; amphotericin B (AmB) is active but is rarely used, because of drug toxicity. AmB encapsulated within macrophage-directed carriers (liposomes) has been used to treat humans with systemic mycoses complicating neoplastic diseases; dosages of up to 5 mg of encapsulated AmB per kg per day for greater than 14 days are without apparent kidney or liver toxicity. In the present work, greater than 99% of L. donovani parasites were eliminated from the liver and spleen of infected hamsters by one administration of 1.5 to 11 mg of liposome-encapsulated AmB (L-Amb) per kg. A total of 98 to 99% of hepatosplenic parasites were eliminated from squirrel monkeys by three administrations of 4 mg of L-AmB per kg. L-AmB was 170 to 750 times as active as antimony in hamsters, and approximately 60 times as active as antimony in monkeys. The demonstration that apparently nontoxic human dosages of L-AmB eliminate essentially all hepatosplenic parasites in hamster and primate models suggests that this preparation should be considered for clinical trial against kala-azar.

To facilitate in vitro studies of the immunology of human leishmaniasis, we developed a method of growing pathogenic Leishmania in human monocyte-derived macrophages. After 6 days of incubation, adherent mononuclear cells were infected with Leishmania donovani amastigotes obtained from infected hamster spleen cells or with L. tropica amastigotes obtained from infected BALB/c tissue mouse footpad. Forty-eight percent of the macrophages were initially infected, with a mean of 3.0 amastigotes per infected macrophage. After 6 days of incubation, 59% of macrophages were infected and contained 8.8 amastigotes per infected macrophage, representing 2.9-fold multiplication. Electron microscopy revealed the presence of dividing parasites within phagolysosomes. These observations indicate that Leishmania survive and multiply within human monocyte-derived macrophages despite fusion of secondary lysosomes with the parasitophorous vacuole.

Monocytes of healthy, full-term newborns were isolated from cord blood, and functional and biochemical activities were quantitated. The yield of monocytes per milliliter of cord blood was 60% greater than that from the peripheral blood of healthy adults. Placental monocytes were initially less well spread than cells from adults, but no other morphological differences were noted. During 4 days of in vitro cultivation, placental monocytes secreted lysozyme at a constant rate, lost peroxidase activity, and increased 5'-nucleotidase activity 15- to 25-fold. Similar findings were obtained with monocytes from adults. Placental monocytes also displayed Fc and complement receptor activity. Ingestion and intracellular multiplication of Toxoplasma gondii were identical in normal placental and adult monocytes. Furthermore, toxoplasma multiplication was significantly inhibited by cells from both sources when the monocytes were preincubated with supernatants prepared from sensitized lymphocytes and toxoplasma antigen. Monocytes from newborns were competent cells in terms of the specific functions and activities we examined.

We have compared the in vitro activity of six agents against macrophage-contained Leishmania tropica amastigotes determined by the conventional Giemsa staining procedure, with the activity determined by the semiautomated assessment of incorporation of radiolabeled uracil into the nucleic acid of the organisms. Although the mean 50% effective dose of Pentostam by Giemsa staining (4.1 micrograms/ml) was somewhat higher than that by uracil incorporation (2.8 micrograms/ml), the ED50S for the other two clinical agents (pentamidine, 0.035 versus 0.037 micrograms/ml; amphotericin B, 0.67 versus 0.70 micrograms/ml) and for three promising experimental agents (ketoconazole, 11.3 versus 11.3 micrograms/ml; the 8-aminoquinoline WR 6026, 1.6 versus 1.5 micrograms/ml formycin B, 0.018 versus 0.017 micrograms/ml) were virtually identical. The radiolabeling technique has several advantages over the Giemsa staining procedure. These include the need for relatively few macrophages, rapid and objective data generation, and viability of the test organism being measured. The successful application of the radiolabeling technique to at least six different chemical classes of compounds suggests that it would be useful for the routine assessment of antileishmanial activity in vitro.

Pentavalent antimonial agents such as sodium stibogluconate (Pentostam; Burroughs Wellcome Co., London, United Kingdom) are the drugs of choice for the treatment of leishmaniasis, but their biochemical mechanisms of action are virtually unknown. The viability of Leishmania mexicana (WR 227) promastigotes and amastigotes was decreased 40 to 61% by a 4-h exposure to 500 micrograms of Sb (in the form of stibogluconate) per ml. Such exposure also resulted in a 51 to 65% decrease in incorporation of label into DNA, RNA, and protein; a 56 to 65% decrease in incorporation of label into purine nucleoside triphosphate; and a 34 to 60% increase in incorporation of label into purine nucleoside monophosphate and diphosphate. It is postulated that the apparent decrease in ATP and GTP synthesis from ADP and GDP contributes to decreased macromolecular synthesis and to decreased Leishmania viability. Further experiments suggested that inhibition of glycolysis and the citric acid cycle may partially explain the inability to phosphorylate ADP.

The activity of purine analogs against Leishmania tropica in human monocyte-derived macrophages in vitro was determined. Formycin B, formycin A, formycin B and A monophosphate, and formycin A triphosphate all had 50% effective doses of 0.02 to 0.04 microM and eliminated 90% of organisms at less than or equal to 0.5 microM. Allopurinol ribonucleoside was much less active: the 50% effective dose was 76 to 190 microM, and 90% of organisms were not eliminated at the highest dose tested (190 microM). 7-Deazainosine had a low 50% effective dose (0.2 microM), but only 80% of organisms were eliminated at 4 microM. Thio derivatives were as active as or less active than the parent compounds. These data suggest that certain inosine analogs are much more active than others against macrophage-contained Leishmania spp. such as are found in human lesions. However, because toxicity to the human macrophage hosts generally paralleled antileishmanial activity, the more active compounds might also be more toxic to human cells. The activity of 3-deazaguanosine (50% effective dose, 3.6 microM) in this model suggests that guanosine derivatives may have potential as antileishmanial agents.

2,4-Diaminoquinazoline analogs of folate were assessed as antileishmanial agents and as dihydrofolate reductase inhibitors. Against Leishmania major in human macrophages in vitro, two compounds with tertiary amines attached directly to the quinazoline ring were remarkably active. The 50% effective doses were in the picogram per milliliter range (12 to 91 pg/ml), and the in vitro therapeutic indices were approximately 10(5). These compounds were 1,000 times more active on an absolute basis and had a 100 times more favorable therapeutic index than any compound previously tested in this model. Antileishmanial activity was not correlated with activity against Leishmania mexicana promastigote reductase, which suggests that folate utilization in general, rather than reductase activity specifically, was being inhibited.