Antileishmanial Aminopyrazoles: Studies into Mechanisms and Stability of Experimental Drug Resistance.

Current antileishmanial treatment is hampered by limitations, such as drug toxicity and the risk of treatment failure, which may be related to parasitic drug resistance. Given the urgent need for novel drugs, the Drugs for Neglected Diseases initiative (DNDi) has undertaken a drug discovery program, which has resulted in the identification of aminopyrazoles, a highly promising antileishmanial chemical series. Multiple experiments have been performed to anticipate the propensity for resistance development. Resistance selection was performed by successive exposure of Leishmania infantum promastigotes (in vitro) and intracellular amastigotes (both in vitro and in golden Syrian hamsters). The stability of the resistant phenotypes was assessed after passage in mice and Lutzomyia longipalpis sandflies. Whole-genome sequencing (WGS) was performed to identify mutated genes, copy number variations (CNVs), and somy changes. The potential role of efflux pumps (the MDR and MRP efflux pumps) in the development of resistance was assessed by coincubation of aminopyrazoles with specific efflux pump inhibitors (verapamil, cyclosporine, and probenecid). Repeated drug exposure of amastigotes did not result in the emergence of drug resistance either in vitro or in vivo Selection at the promastigote stage, however, was able to select for parasites with reduced susceptibility (resistance index, 5.8 to 24.5). This phenotype proved to be unstable after in vivo passage in mice and sandflies, suggesting that nonfixed alterations are responsible for the elevated resistance. In line with this, single nucleotide polymorphisms and indels identified by whole-genome sequencing could not be directly linked to the decreased drug susceptibility. Copy number variations were absent, whereas somy changes were detected, which may have accounted for the transient acquisition of resistance. Finally, aminopyrazole activity was not influenced by the MDR and MRP efflux pump inhibitors tested. The selection performed does not suggest the rapid development of resistance against aminopyrazoles in the field. Karyotype changes may confer elevated levels of resistance, but these do not seem to be stable in the vertebrate and invertebrate hosts. MDR/MRP efflux pumps are not likely to significantly impact the activity of the aminopyrazole leads.

In vitro and in vivo pharmacodynamics of three novel antileishmanial lead series.

OBJECTIVES: Three new chemical series (bicyclic nitroimidazoles, aminopyrazoles and oxaboroles) were selected by Drugs for Neglected Diseases initiative as potential new drug leads for leishmaniasis. Pharmacodynamics studies included both in vitro and in vivo efficacy, cross-resistance profiling against the current antileishmanial reference drugs and evaluation of their cidal activity potential. METHODS: Efficacy against the reference laboratory strains of Leishmania infantum (MHOM/MA(BE)/67/ITMAP263) and L. donovani (MHOM/ET/67/L82) was evaluated in vitro on intracellular amastigotes and in vivo in the early curative hamster model. Cidal activity was assessed over a period of 15 days in an in vitro 'time-to-kill' assay. Cross-resistance was assessed in vitro on a panel of L. infantum strains with different degrees of resistance to either antimony, miltefosine or paromomycin. RESULTS: All lead compounds showed potent and selective in vitro activity against the Leishmania strains tested and no cross-resistance could be demonstrated against any of the current antileishmanial drugs. Cidal activity was obtained in vitro for all series within 15 days of exposure with some differences noted between L. donovani and L. infantum. When evaluated in vivo, all lead compounds showed high efficacy and no adverse effects were observed. CONCLUSIONS: The new lead series were shown to have cidal pharmacodynamic activity. The absence of cross-resistance with any of the current antileishmanial drugs opens possibilities for combination treatment to reduce the likelihood of treatment failures and drug resistance.

Structure-activity relationships of 1,4-dihydro-(1H,4H)-quinoxaline-2,3-diones as N-methyl-D-aspartate (glycine site) receptor antagonists. 1. Heterocyclic substituted 5-alkyl derivatives.

A series of 6,7-dichloro-1,4-dihydro-(1H, 4H)-quinoxaline-2,3-diones (1-17) were prepared in which the 5-position substituent was a heterocyclylmethyl or 1-(heterocyclyl)-1-propyl group. Structure-activity relationships were evaluated where binding affinity for the glycine site of the N-methyl-D-aspartate (NMDA) receptor was measured using the specific radioligand [3H]-L-689,560, and functional antagonism was demonstrated by inhibition of NMDA-induced depolarizations of rat cortical wedges. The ability to prevent NMDA-induced hyperlocomotion in mice in vivo was measured for selected compounds. Binding affinity increased significantly if the heterocyclic group, e.g. 1,2,3-triazol-1-yl could participate in accepting a hydrogen bond from the receptor. It was difficult to obtain compounds with adequate aqueous solubility and strategies to improve it were investigated. The most potent compound in this series, 6,7-dichloro-5-[1-(1,2,4-triazol-4-yl)propyl]-1,4-dihydro-(1H, 4H)-quinoxaline-2,3-dione (17) (binding IC50 = 2.6 nM; cortical wedge EC50 = 90 nM), inhibited NMDA-induced hyperlocomotion in mice (6/9 protected at 20 mg/kg iv). Pharmacokinetic parameters, including extent of brain penetration, for 11 and 17 are reported.