title: 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.
authors.affiliation: Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, Coimbra, Portugal; Centre of Pharmaceutical Studies, Faculty of Pharmacy of University of Coimbra (CEF/FFUC), Pólo das Ciências da Saúde, Azinhaga de Santa Comba, Coimbra, Portugal.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Sousa
authors.affiliation: Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, Coimbra, Portugal.
authors.forename: Raquel
authors.initials: R
authors.lastname: Varandas
authors.affiliation: Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, Coimbra, Portugal.
authors.forename: Rita C
authors.initials: RC
authors.lastname: Santos
authors.affiliation: Institute of Immunology, Faculty of Medicine, University of Coimbra, Pólo I, Rua Larga, Coimbra, Portugal.
authors.forename: Manuel
authors.initials: M
authors.lastname: Santos-Rosa
authors.affiliation: Institute of Immunology, Faculty of Medicine, University of Coimbra, Pólo I, Rua Larga, Coimbra, Portugal.
authors.forename: Vera
authors.initials: V
authors.lastname: Alves
authors.affiliation: Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, Coimbra, Portugal; CNC- Center for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal.
authors.forename: Jorge A R
authors.initials: JA
authors.lastname: Salvador
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004357:Drug Synergism; D007093:Imidazoles / Q000138:chemical synthesis / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D018314:Leishmania infantum / Q000187:drug effects* / Q000254:growth & development; D008018:Life Cycle Stages / Q000187:drug effects* / Q000502:physiology; D008264:Macrophages / Q000187:drug effects; D051379:Mice; D053978:Pentacyclic Triterpenes; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014315:Triterpenes / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D000094062:Betulinic Acid
chemical_list: C000594003:28-(1H-imidazole-1-yl)-3,28-dioxolup-1,20(29)-dien-2-yl-1H-imidazole-1-carboxylate; C000594002:3-hydroxylupan-29-oxo-28-yl-1H-imidazole-1-carboxylate; D000981:Antiprotozoal Agents; D007093:Imidazoles; D053978:Pentacyclic Triterpenes; D014315:Triterpenes; D010767:Phosphorylcholine; C039128:miltefosine; C002503:betulin; D000094062:Betulinic Acid