Leishmania donovani develops resistance to drug combinations

Abstract

Drug combinations for the treatment of leishmaniasis represent a promising and challenging chemotherapeutic strategy that has recently been implemented in different endemic areas. However, the vast majority of studies undertaken to date have ignored the potential risk that Leishmania parasites could develop resistance to the different drugs used in such combinations. As a result, this study was designed to elucidate the ability of Leishmania donovani to develop experimental resistance to anti-leishmanial drug combinations. The induction of resistance to amphotericin B/miltefosine, amphotericin B/paromomycin, amphotericin B/Sb(III), miltefosine/paromomycin, and Sb(III)/paromomycin was determined using a step-wise adaptation process to increasing drug concentrations. Intracellular amastigotes resistant to these drug combinations were obtained from resistant L. donovani promastigote forms, and the thiol and ATP levels and the mitochondrial membrane potential of the resistant lines were analysed. Resistance to drug combinations was obtained after 10 weeks and remained in the intracellular amastigotes. Additionally, this resistance proved to be unstable. More importantly, we observed that promastigotes/amastigotes resistant to one drug combination showed a marked cross-resistant profile to other anti-leishmanial drugs. Additionally, the thiol levels increased in resistant lines that remained protected against the drug-induced loss of ATP and mitochondrial membrane potential. We have therefore demonstrated that different resistance patterns can be obtained in L. donovani depending upon the drug combinations used. Resistance to the combinations miltefosine/paromomycin and Sb(III)/paromomycin is easily obtained experimentally. These results have been validated in intracellular amastigotes, and have important relevance for ensuring the long-term efficacy of drug combinations.

Figure 1. Stability of resistance to drugs in L. donovani promastigote lines.

(A) The stability of resistance with respect to the initial EC₅₀ (black columns) is determined at 1 (gray columns) and 4 months (white columns) after removal of the drug pressure. The results shown are the average of three independent experiments ± SD. Significant differences versus the initial EC₅₀ were determined using Student's t-test (*: p<0.05; **: p<0.01; ***: p<0.005; ****: p<0.001). (B) Results from panel A expressed in terms of the resistance index. The horizontal black line represents a resistance index of 1 equivalent to a completely loss of resistance. ^a: line that lose resistance after 1 month in a drug-free medium; ^b: line that maintain similar initial resistance levels after 4 months in a drug-free medium.

Figure 2. Stability of cross-resistance to drugs in L. donovani promastigote lines.

(A) The stability of cross-resistance at 1 month after removal of drug pressure (gray columns), relative to initial EC₅₀ (black columns), was determined. The results are the average of three independent experiments ± SD. Significant differences versus the initial EC₅₀ were determined using Student's t-test (*: p<0.005). (B) Results from panel A expressed in terms of resistance index. The horizontal black line represents a resistance index of 1 equivalent to a completely loss of resistance. ^a: line that lose resistance after 1 month in a drug-free medium.

Figure 3. Thiol levels in L. donovani promastigote lines.

Log-phase promastigotes from WT and resistant lines were labelled with 2 µM CellTracker (black columns), and fluorescence was analyzed as described in the Materials and Methods section. Promastigotes were depleted of thiols by treatment with 3 mM BSO (a γ-glutamylcysteine synthetase inhibitor) for 48 h at 28°C (white columns). The results are the average of three independent experiments ± SD. All lines show significant differences with respect to WT (*: p<0.001) and when comparing singly versus doubly-resistant lines using Student's t-test (^a: p<0.05 A vs. AS; ^b: p<0.005 A vs. AP; ^c: p<0.001 A vs. AM; ^d: p<0.001 M vs. AM or MP; ^e: p<0.05 P vs. AP; ^f: p<0.001 P vs. MP or SP; ^g: p<0.001 S vs. AS or SP). The black line indicates the threshold at which the thiol levels have been modified with respect to WT.

Figure 4. Effect of anti-leishmanial drugs on ATP synthesis and ΔΨm in resistant L. donovani promastigote lines.

(A) ATP levels were measured using CellTiter-Glo. (B) ΔΨm was measured by determining the accumulation of Rh123 (0.5 µM) in WT and resistant lines. In both cases, promastigote log-phase cultures were left untreated (controls, black columns) or exposed to 0.2 µM AmB (white columns), 25 µM MLF (oblique lines columns) for 3 h, 2 mM Sb^III (gray columns) for 8 h, or 10 µM FCCP as a depolarization control (light gray columns) for 10 min. Measurements are expressed in arbitrary luminescence (panel A) or fluorescence (panel B) units ± SD from three independent experiments and show significant differences (^a: p<0.05; ^b: p<0.01; ^c: p<0.005; ^d: p<0.001) when comparing the corresponding control values for each line with itself after treatment. A comparison of the different untreated lines with untreated WT lines also shows significant differences (*: p<0.05; **: p<0.005; ***: p<0.001).