Antileishmanial activity of semisynthetic lupane triterpenoids betulin and betulinic acid derivatives: synergistic effects with miltefosine

Abstract

Leishmaniasis is a neglected tropical disease (NTDs), endemic in 88 countries, affecting more than 12 million people. The treatment consists in pentavalent antimony compounds, amphotericin B, pentamidine and miltefosine, among others. However, these current drugs are limited due to their toxicity, development of biological resistance, length of treatment and high cost. Thus, it is important to continue the search for new effective and less toxic treatments. The anti-Leishmania activity of sixteen semisynthetic lupane triterpenoids derivatives of betulin (BT01 to BT09) and betulinic acid (AB10 to AB16) were evaluated. Drug interactions between the active compounds and one current antileishmanial drug, miltefosine, were assessed using the fixed ratio isobologram method. In addition, effects on the cell cycle, apoptosis/necrosis events, morphology and DNA integrity were studied. The derivatives BT06 (3β-Hydroxy-(20R)-lupan-29-oxo-28-yl-1H-imidazole-1-carboxylate) and AB13 (28-(1H-imidazole-1-yl)-3,28-dioxo-lup-1,20(29)-dien-2-yl-1H-imidazole-1-carboxylate) were found to be the most active, with IC50 values of 50.8 µM and 25.8 µM, respectively. Interactions between these two compounds and miltefosine were classified as synergistic, with the most effective association being between AB13 and miltefosine, where decreases of IC50 values to 6 µM were observed, similar to the miltefosine activity alone. AB13 induced significant morphological changes, while both derivatives produced anti-proliferative activity through cell cycle arrest at the G0/G1 phase. Neither of these derivatives induced significant apoptosis/necrosis, as indicated by phosphatidylserine externalization and DNA fragmentation assays. In addition, neither of the derivatives induced death in macrophage cell lines. Thus, they do not present any potential risk of toxicity for the host cells. This study has identified the betulin derivative BT06 and the betulinic acid derivative AB13 as promising molecules in the development of new alternative therapies for leishmaniasis, including those involving combined-therapy with miltefosine.

Figure 1. Structures of betulin derivatives and IUPAC nomenclature.

Figure 2. Structures of betulinic acid derivatives and IUPAC nomenclature.

Figure 3. Effects of betulin (A) and betulinic acid (B) derivatives on Leishmania infantum promastigotes viability.

Cultures of log-phase promastigotes (2×10⁶ cells.ml⁻¹) were incubated at 26°C for 24 h at different drug concentrations. Values are expressed as means and SEM.

Figure 4. Effects of combinations of betulin and betulinic acid derivatives with miltefosine on Leishmania infantum promastigotes viability.

(A) Betulin derivative BT06; (B) Betulinic acid derivative AB13. Values are expressed as means and SEM. Cultures of log-phase promastigotes (2×10⁶ cells.ml⁻¹) were incubated at 26°C for 24 h at different compound concentrations.

Figure 5. Comparison of the effects of combinations between BT06 and miltefosine with the effects of BT06 alone.

Cultures of log-phase promastigotes (2×10⁶ cells.ml⁻¹) were incubated at 26°C for 24 h. Significance level of *p<0.05, **p<0.01 and ***p<0.001.

Figure 6. Comparison of the effects of combinations between AB13 and miltefosine with the effects of AB13 alone.

Cultures of log-phase promastigotes (2×10⁶ cells.ml⁻¹) were incubated at 26°C for 24 h. Significance level of *p<0.05, **p<0.01 and ***p<0.001.

Figure 7. Isobologram analysis for the combinations between BT06 and miltefosine (A) and AB13 and miltefosine (B).

The line indicates synergy, additivity or antagonism when the points are located below, on or above the line, respectively. (a) derivative and 2 µM miltefosine; (b) derivative and 4 µM miltefosine; (c) derivative and 6 µM miltefosine.

Figure 8. Optical microscopy observation of Leishmania infantum promastigotes in the absence and presence of the betulinic acid derivative AB13.

Hanging dropin phase contrast (A, B, magnification 200×) and Giemsa staining (C, D, magnification 1000×). Control cells incubated with DMSO at 26°C for 24 h (A, C). Cells exposed to the AB13 derivative at 26°C for 24 h (B, D).

Figure 9. Representative cell cycle histograms of Leishmania infantum.

Promastigotes were incubated at 26°C for 24 h in the absence (A and C) or presence of betulin derivative BT06 (B) and betulinic acid derivative AB13 (D) at IC₅₀ concentrations. Propidium iodide staining was performed and samples were analyzed by flow cytometry.