Sodium stibogluconate (Pentostam) inhibition of glucose catabolism via the glycolytic pathway, and fatty acid beta-oxidation in Leishmania mexicana amastigotes.

The biochemical mechanism of action of antimony (Sb) in pentavalent form complexed to gluconic acid (sodium stibogluconate)--the drug of choice for the leishmaniases--has been only slightly investigated. We recently reported that, in stibogluconate-exposed Leishmania mexicana amastigotes, there is a dose-dependent decrease in the ATP/ADP ratio [Berman et al., Antimicrob. Agents Chemother. 27, 916 (1985)]. To investigate mechanisms by which ADP phosphorylation to ATP might be inhibited, stibogluconate-exposed amastigotes were incubated with [14C]glucose, fatty acid, or acetate, and 14CO2 production was determined. In organisms exposed to 500 micrograms Sb/ml, formation of 14CO2 from [6-14C]glucose and [1-14C]palmitate was inhibited 69 and 67% respectively. In comparison, formation of 14CO2 from [1-14C]glucose and [2-14C]acetate was inhibited less than 15%. These results suggest that glucose catabolism via glycolytic enzymes and fatty acid beta-oxidation, but not glucose metabolism via the hexosemonophosphate shunt or the citric acid cycle, is specifically inhibited in stibogluconate-exposed Leishmania mexicana amastigotes. Inhibition of these pathways suggests a mechanism for the inhibition of ADP phosphorylation previously reported.

Pharmacokinetics of pentavalent antimony (Pentostam) in hamsters.

Pentavalent antimony (Sb) is the classical treatment for visceral and cutaneous leishmaniasis. We investigated Sb levels in serum, liver, spleen, and skin of hamsters administered therapeutic dosages of Sb (600 and 300 mg Sb/kg). Single administration of Sb was more effective against hepatic parasites than dividing the same total dose into multiple administrations, which suggests that for elimination of hepatic parasites in vivo, peak Sb concentration is more important than total area-under-the-curve levels. Serum Sb declined with an initial half-life of 1 hr. Skin Sb levels (352 micrograms Sb/g 1 hr after 600 mg Sb/kg) were initially higher than liver levels (77 micrograms Sb/g) or splenic levels (156 micrograms Sb/g), but levels were comparable (7-24 micrograms Sb/g) in the three organs by 8 hr after dosing. The generally comparable levels of Sb in the skin and in the visceral organs support the present clinical practice of administering the same dosage of Sb for cutaneous and visceral leishmaniasis.

Activity of pentamidine-containing human red cell ghosts against visceral leishmaniasis in the hamster.

A recent approach to the chemotherapy of visceral leishmaniasis has been the encapsulation of clinical agents within macrophage directed carriers such as liposomes. Because in mammals Leishmania are obligate intramacrophage microorganisms, injection of an encapsulated drug should deliver large quantities of drug to the organisms, thus decreasing both the number of drug administrations needed for cure and drug toxicity. Drugs contained within red cell ghosts have been used clinically to treat other macrophage disorders. We encapsulated the clinical antileishmanial agent pentamidine within human red cell ghosts and administered it to hamsters infected with Leishmania donovani. The ED50 and ED90 of single injections of this preparation were 231-240 times lower than that of the positive control drug, sodium stibogluconate (Pentostam), and essentially all parasites could be eliminated by 2.5 mg encapsulated drugs/kg. Against splenic parasites, the ED50 was 195 times lower than that of antimony, although only 80% of parasites were eliminated by the highest doses of encapsulated drug (2.5-6.4 mg/kg). The difference in liver vs. splenic parasite killing is probably related to the greater uptake of encapsulated drug by the liver (11-14 micrograms/g tissue) vs. the spleen (2-3 micrograms/g). If activity in this model is comparable to activity in humans, these results suggest that a single injection of a preparation consisting of ghosts of a patient's own red cells and the amount of pentamidine in one standard dosage (4 mg/kg) would eliminate 80%-100% of L. donovani from the spleen and liver.

Uptake, distribution, and oxidation of fatty acids by Leishmania mexicana amastigotes.

Fatty acid uptake, distribution, and beta-oxidation were investigated in Leishmania mexicana amastigotes. The uptake of radiolabeled palmitic, stearic, and oleic acids was similar, reaching 3-6 nmol/10(8) cells in 2 min and 8-12 nmol/10(8) cells in 60 min. The percent of radiolabeled fatty acid that was esterified in the form of triglycerides or phospholipids increased from less than 25% at 2 min to 65-86% at 60 min. The dehydrogenase(s) in an amastigote granule fraction were unusual in that the Vmax for long-chain substrates (0.95-1.6 delta Abs units/min-mg protein) approximated the Vmax for short-chain substrates (0.82-2.0 delta Abs units/min-mg protein), and the Km for long-chain substrates was high (approximately 250 microM), in contrast to data for a mammalian liver mitochondrial fraction. The high Vmax and Km for long-chain substrates suggest a biochemical mechanism for the postulated high utilization of fatty acids as an energy source for amastigotes. Although the primary anti-leishmanial agent, Sb in the form of Pentostam, inhibited oxidation of palmitic acid to CO2 by intact organisms, Sb did not significantly inhibit fatty acid uptake or esterification by organisms, or beta-oxidation by the granule fraction, and the mechanism of action of Sb remains unclear.

Antileishmanial activity of human red blood cells containing formycin A.

Formycin B is the most active antileishmanial agent in vitro because it is metabolized by the parasites to formycin A phosphates. The in vivo use of formycin B may be limited by its toxicity to humans due to the slight similar metabolism of the drug in human cells. The obligatory intramacrophage localization of Leishmania in man suggests that encapsulation of drugs within macrophage-directed carriers, such as human red blood cells (RBCs), might enhance the therapeutic-toxic ratio. Since uncharged formycin B would be poorly metabolized by the RBC and would diffuse from the carrier, RBCs were incubated with formycin A so that approximately 90% of the formycin A was taken up by the cells, phosphorylated by RBC enzymes to charged formycin A triphosphate, and retained in the cells. In vitro, 81% of Leishmania-infected macrophages phagocytized IgG-coated RBCs containing this active form of formycin B, and multiplication of organisms within macrophages could be suppressed by approximately 80%. The 50% effective dose of the formycin A-RBC formulation was 0.02 microM, whereas the 50% effective dose of unencapsulated drug was 0.84 microM. This report of in vitro activity of human RBCs containing formycin A and coated with IgG indicates that this formulation should be tested for antileishmanial activity in vivo.

Semiautomated assessment of in vitro activity of potential antileishmanial drugs.

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.