Ex Vivo Phenotypic Screening of Two Small Repurposing Drug Collections Identifies Nifuratel as a Potential New Treatment against Visceral and Cutaneous Leishmaniasis

Abstract

Leishmaniases are vector-borne neglected diseases caused by single-celled parasites. The search for new antileishmanial drugs has experienced a strong boost thanks to the application of bioimaging to phenotypic screenings based on intracellular amastigotes. Mouse splenic explants infected with fluorescent strains of Leishmania are proven tools of drug discovery, where hits can be easily transferred to preclinical in vivo models. We have developed a two-staged platform for antileishmanial drugs. First, we screened two commercial collections of repurposing drugs with a total of 1769 compounds in ex vivo mouse splenocytes infected with an infrared emitting Leishmania infantum strain. The most active and safest compounds were scaled-up to in vivo models of chronic Leishmania donovani visceral leishmaniasis and Leishmania major cutaneous leishmaniasis. From the total of 1769 compounds, 12 hits with selective indices >35 were identified, and 4 of them were tested in vivo in a model of L. donovani visceral leishmaniasis. Nifuratel, a repurposed synthetic nitrofuran, when administered orally at 50 mg/kg bw once or twice a day for 10 days, caused >80% reduction in the parasitic load. Furthermore, the intralesional administration of nifuratel in a model of cutaneous leishmaniasis by L. major produced the parasitological cure. From the previous results we have deduced the great capacity of mouse splenic explants to identify new hits, a model which could be easily transferred to in vivo models, as well as the potential use of nifuratel as an alternative to the current treatment of cutaneous leishmaniasis.

Miltefosine and Nifuratel Combination: A Promising Therapy for the Treatment of Leishmania donovani Visceral Leishmaniasis

“…Moreover, the estimated half-life of NFT was barely 3 h, thus indicating that the compound had practically been eliminated from plasma after 11 h. For this reason, we opted to use a 12 h dosing regimen during the 10 days of the experiment. This pattern proved to be safe throughout the trial as previously described [ 26 ]. With respect to MTF, snapshot pharmacokinetics were performed at doses of 3 mg/kg and 5 mg/kg, choosing 10 mg/kg bw once a day during the 10 days of the trial (1/4 of the dose used with MTF40, which served as positive control).…”

“…To define the in vivo oral doses of the NFT/MTF combination, we used the empirical data obtained in a previous study carried out in Balb/c mice in our laboratory, in which the dose of 50 mg/kg/d bid/10 days produced a significant reduction in the total parasite load and in the thymus, liver and spleen [ 26 ]. In addition, individual pharmacokinetic results obtained in previous studies for NFT and MTF in mice and humans after oral administration under similar conditions, were helpful to know the plasma concentrations of both drugs.…”

“…Recently, our group screened two commercial collections of anti-infective agents, which included 1769 repurposing drugs. For this purpose, a phenotypic platform consisting of splenic explants from Balb/c mice infected with an infrared fluorescent L. donovani strain [ 25 ] allowed us to identify 43 compounds with antileishmanial activity at <1 μM final concentration [ 26 ]. Among the selected hits, several compounds with a nitroheterocyclic structure were identified as the most potent, including nifurtimox, nitrofurantoin, nitrofurazone, PA-824, fexinidazole and, surprisingly, the synthetic nitrofurantoin derivate Nifuratel (NFT), the latter being marketed as the active ingredient in several treatments against most agents that cause genital urinary tract infections [ 27 , 28 ].…”

“…Among the selected hits, several compounds with a nitroheterocyclic structure were identified as the most potent, including nifurtimox, nitrofurantoin, nitrofurazone, PA-824, fexinidazole and, surprisingly, the synthetic nitrofurantoin derivate Nifuratel (NFT), the latter being marketed as the active ingredient in several treatments against most agents that cause genital urinary tract infections [ 27 , 28 ]. NFT administered orally to Balb/c mice infected with L. donovani was shown to be effective, since it reduced the parasite load by more than 80% in a model of VL [ 26 ]. The mechanism of action of NFT is not well understood.…”

Anti-trypanosomatid drug discovery: progress and challenges

“…Many of the more-high-throughput primary assays lack the detailed physiological relevance 1 , 10 . Various approaches to improve relevance are being explored: replacement of tissue-culture cell lines with more-relevant complex cell systems (for example, primary human host cells 74 , 75 , stem cell-derived host cells 76 and explant models 74 , 75 ); improved assay set-up (many viability assays cannot distinguish between cytostatic and cytocidal compounds as starting pathogen density is below the detection limit, and methods that overcome this have been reported 77 , 78 ); and alternative assay platforms to detect activity against key rare populations such as persisters (for example, washout outgrowth assays 23 ). Often the more relevant models are challenged by limited throughput and/or long duration.…”