The anti-trypanosome drug fexinidazole shows potential for treating visceral leishmaniasis

Abstract

Safer and more effective oral drugs are required to treat visceral leishmaniasis, a parasitic disease that kills 50,000 to 60,000 people each year in parts of Asia, Africa, and Latin America. Here, we report that fexinidazole, a drug currently in phase 1 clinical trials for treating African trypanosomiasis, shows promise for treating visceral leishmaniasis. This 2-substituted 5-nitroimidazole drug is rapidly oxidized in vivo in mice, dogs, and humans to sulfoxide and sulfone metabolites. Both metabolites of fexinidazole were active against Leishmania donovani amastigotes grown in macrophages, whereas the parent compound was inactive. Pharmacokinetic studies with fexinidazole (200 mg/kg) showed that fexinidazole sulfone achieves blood concentrations in mice above the EC(99) (effective concentration inhibiting growth by 99%) value for at least 24 hours after a single oral dose. A once-daily regimen for 5 days at this dose resulted in a 98.4% suppression of infection in a mouse model of visceral leishmaniasis, equivalent to that seen with the drugs miltefosine and Pentostam, which are currently used clinically to treat this tropical disease. In African trypanosomes, the mode of action of nitro drugs involves reductive activation via a NADH (reduced form of nicotinamide adenine dinucleotide)-dependent bacterial-like nitroreductase. Overexpression of the leishmanial homolog of this nitroreductase in L. donovani increased sensitivity to fexinidazole by 19-fold, indicating that a similar mechanism is involved in both parasites. These findings illustrate the potential of fexinidazole as an oral drug therapy for treating visceral leishmaniasis.

Fig. 1. Pharmacodynamic and pharmacokinetic properties of fexinidazole and its metabolites

(A) Effects of drug treatment on the parasite burden of mice infected with L. donovani. Groups of mice (five per group) infected with L. donovani (strain LV9) were dosed with drug vehicle (orally), Pentostam (subcutaneously), miltefosine (orally) or fexinidazole (orally) on day 7 post-infection and for the following four days. On day 14 post-infection, all animals were humanely euthanized and parasite burdens were determined microscopically by examining Giemsa-stained liver smears. Parasite load is expressed in Leishman Donovan Units (LDU): mean number of amastigotes per liver cell × mg liver (29). The inset shows the dose response curve for fexinidazole (ED₅₀ = 11.9 ± 2.3 mg kg⁻¹). (B) Blood concentration of fexinidazole and its metabolites following oral dosing with fexinidazole (200 mg kg⁻¹). The EC₉₉ values of fexinidazole sulfoxide (10,500 ng ml⁻¹) and sulfone (11,500 ng ml⁻¹) for L. donovani (strain LV9) cultured in ex vivo mouse macrophages are shown as dotted lines. Fexinidazole, black circles; sulfoxide, teal; sulfone, maroon. Data are the mean and standard deviation from 3 mice.

Fig. 2. EC₅₀ for combinations of fexinidazole sulfoxide and sulfone against promastigotes

Isobologram shows the EC₅₀ values obtained with combinations of fexinidazole sulfoxide and sulfone against promastigotes of the LdBOB strain of L. donovani. Promastigotes in mid-log growth were incubated with combinations of drug relative to their individual EC₅₀ values. The EC₅₀ values of each combination were determined after 72h. Data are the mean ± SD of triplicate measurements.

Fig. 3. Drug susceptibility of amastigotes overexpressing nitroreductase

Panels A and B show Leishmania donovani (strain LdBOB) amastigotes cultured under axenic conditions, whereas panel C shows amastigotes cultured in macrophages. Data are the mean ± SD of triplicate measurements for panels A and B and duplicate measurements for panel C. Some standard deviations are within the data points. Symbols: open circles, wildtype; closed circles, parasites overexpressing nitroreductase.

Fig. 4. The cytocidal effect of fexinidazole sulfone on L. donovani axenic amastigotes

Fexinidazole sulfone (36 μM, equivalent to 10 times the determined EC₅₀ value) was added to an early-log growth culture of axenic amastigotes from the LdBOB strain of L. donovani (~1 × 10⁶ ml⁻¹). At intervals, the cell density was determined and then samples of culture (500 μl) were removed, washed and resuspended in fresh culture medium in the absence of drug. The viability of washed parasites was monitored for up to 72h following removal from drug exposure and the point of irreversible drug toxicity was determined. Symbols: open circle: no inhibitor; filled circles: fexinidazole sulfone; double dagger, no viable parasites recovered from culture.