Cross-resistance to nitro drugs and implications for treatment of human African trypanosomiasis

Abstract

The success of nifurtimox-eflornithine combination therapy (NECT) for the treatment of human African trypanosomiasis (HAT) has renewed interest in the potential of nitro drugs as chemotherapeutics. In order to study the implications of the more widespread use of nitro drugs against these parasites, we examined the in vivo and in vitro resistance potentials of nifurtimox and fexinidazole and its metabolites. Following selection in vitro by exposure to increasing concentrations of nifurtimox, Trypanosoma brucei brucei nifurtimox-resistant clones designated NfxR1 and NfxR2 were generated. Both cell lines were found to be 8-fold less sensitive to nifurtimox than parental cells and demonstrated cross-resistance to a number of other nitro drugs, most notably the clinical trial candidate fexinidazole (approximately 27-fold more resistant than parental cells). Studies of mice confirmed that the generation of nifurtimox resistance in these parasites did not compromise virulence, and NfxR1 remained resistant to both nifurtimox and fexinidazole in vivo. In the case of fexinidazole, drug metabolism and pharmacokinetic studies indicate that the parent drug is rapidly metabolized to the sulfoxide and sulfone form of this compound. These metabolites retained trypanocidal activity but were less effective in nifurtimox-resistant lines. Significantly, trypanosomes selected for resistance to fexinidazole were 10-fold more resistant to nifurtimox than parental cells. This reciprocal cross-resistance has important implications for the therapeutic use of nifurtimox in a clinical setting and highlights a potential danger in the use of fexinidazole as a monotherapy.

FIG. 1.

Nifurtimox resistance in vitro. (A) Schematic representation of the generation of a nifurtimox-resistant cell line in T. brucei. Each passage of cells in culture (open circle) and loss of a culture due to drug toxicity (skull and crossbones) are indicated. (B) EC₅₀s were determined for nifurtimox (circles) and pentamidine (squares) against WT (open symbols) and NfxR1 (closed symbols) cells, respectively. The curves are the nonlinear fits of data using a two-parameter EC₅₀ equation provided by GraFit (see Materials and Methods). EC₅₀s of 0.95 ± 0.02 and 2.6 ± 0.1 nM were determined for pentamidine against WT and NfxR1 cells, respectively, while values of 2.4 ± 0.1 and 20.1 ± 0.9 μM were determined for nifurtimox. Data are the weighted means ± standard deviations of at least triplicate measurements.

FIG. 2.

Nifurtimox resistance in vivo. Groups of 5 mice were infected with either WT or NfxR1 cells (1 × 10⁴ parasites). Twenty-four hours following infection, WT- and NfxR1-infected mice were either dosed with nifurtimox (100 mg kg⁻¹) via an intraperitoneal injection or left untreated. Data are presented in the form of a Kaplan-Meier survival plot. Animals that succumbed to drug toxicity rather than parasitemia are annotated (skull and crossbones). WT, pink; WT treated with nifurtimox, blue; NfxR1, green; NfxR1 treated with nifurtimox, red.

FIG. 3.

Fexinidazole resistance in vivo. Groups of 5 mice were infected with either WT cells (open circles) or NfxR1 cells (closed circles) (1 × 10⁴ parasites). Twenty-four hours following infection, WT- and NfxR1-infected mice were dosed orally with fexinidazole (25 to 200 mg kg⁻¹). Cured mice were determined to be those which survived to 60 days following inoculation.

FIG. 4.

Blood exposure of fexinidazole and its metabolites. Levels of fexinidazole and two major metabolites (sulfone and sulfoxide) were monitored in blood samples collected from mice at defined intervals, following oral dosing of fexinidazole (200 mg kg⁻¹). The EC₉₉ values of fexinidazole and its sulfoxide and sulfone metabolites, established in vitro, are annotated as solid, dotted, and dashed lines, respectively. Symbols: closed squares, fexinidazole; open circles, sulfoxide; closed circles, sulfone. Data are the means of triplicate measurements.

FIG. 5.

Cross-resistance to nifurtimox in FxR cell lines. EC₅₀s were determined for fexinidazole (circles) and nifurtimox (squares) against WT (open symbols) and FxR1 (closed symbols) cells, respectively. The curves are the nonlinear fits of data using a two-parameter EC₅₀ equation provided by GraFit (see Materials and Methods). In this experiment, the EC₅₀ (and Hill slope) values for fexinidazole are 1.6 ± 0.1 μM (1.6) and 17.8 ± 1.5 μM (1.4) for WT and FxR1 cells, respectively, and for nifurtimox are 1.8 ± 0.1 μM (3.1) and 18.4 ± 0.6 μM (3.9), respectively.