Antitrypanosomal Activity of Fexinidazole, a New Oral Nitroimidazole Drug Candidate for Treatment of Sleeping Sickness
ABSTRACT
Fexinidazole is a 5-nitroimidazole drug currently in clinical development for the treatment of human sleeping sickness (human African trypanosomiasis [HAT]), caused by infection with species of the protozoan parasite Trypanosoma brucei. The compound and its two principal metabolites, sulfoxide and sulfone, have been assessed for their ability to kill a range of T. brucei parasite strains in vitro and to cure both acute and chronic HAT disease models in the mouse. The parent molecule and both metabolites have shown trypanocidal activity in vitro in the 0.7-to-3.3 μM (0.2-to-0.9 μg/ml) range against all parasite strains tested. In vivo, fexinidazole is orally effective in curing both acute and chronic diseases in the mouse at doses of 100 mg/kg of body weight/day for 4 days and 200 mg/kg/day for 5 days, respectively. Pharmacokinetic data indicate that it is likely that the sulfoxide and sulfone metabolites provide most, if not all, of the in vivo killing activity. Fexinidazole and its metabolites require up to 48 h exposure in order to induce maximal trypanocidal efficacy in vitro. The parent drug and its metabolites show no in vitro cross-reactivity in terms of trypanocidal activity with either themselves or other known trypanocidal drugs in use in humans. The in vitro and in vivo antitrypanosomal activities of fexinidazole and its two principal metabolites provide evidence that the compound has the potential to be an effective oral treatment for both the T. b. gambiense and T. b. rhodesiense forms of human sleeping sickness and both stages of the disease.
INTRODUCTION
Human African trypanosomiasis (HAT), also know as sleeping sickness, is caused by two subspecies of the protozoan parasite Trypanosoma brucei and is transmitted through the bite of infected tsetse flies. In west and central Africa, T. b. gambiense is responsible for the chronic form of the disease, whereas T. b. rhodesiense is responsible for a more acute form of the disease in eastern Africa. Poor and neglected populations living in remote rural areas of sub-Saharan Africa are at risk for HAT, and in 2006, it was estimated that 50,000 to 70,000 individuals were infected (35). In recent years, the reported HAT cases have decreased to approximately 10,000 (29, 36), with over 95% of the reported cases due to T. b. gambiense infection.
There are four drugs currently registered for use against sleeping sickness. Pentamidine and suramin are used against the hemolymphatic stage (stage 1) of the disease, while melarsoprol and eflornithine (DFMO) are used against stage 2 of the disease, when the parasites have invaded the central nervous system (CNS). The disease is fatal if left untreated. The drugs currently in use are unsatisfactory due to cost, toxicity, poor oral bioavailability, long treatment, and lack of efficacy. Melarsoprol treatment is highly toxic, and up to 5% of the second-stage patients treated with melarsoprol die of a reactive encephalopathy. Eflornithine treatment requires four daily intravenous infusions over 14 days, meaning that this therapy is expensive and logistically difficult in rural clinics. The only advance in the last 25 years has been the introduction of nifurtimox-eflornithine combination therapy (NECT) (25). Despite the reduced toxicity and treatment duration of NECT compared to those of melarsoprol or eflornithine, the requirement for 7 days of intravenous administration is still a limitation.
The aim of the present study was to characterize the antitrypanosomal activity of the 5-nitroimidazole drug candidate fexinidazole and its two principal metabolites, fexinidazole sulfoxide and fexinidazole sulfone, using phenotypic in vitro and in vivo screening. Fexinidazole is targeted for the treatment of HAT, currently in phase I clinical studies, and had been in preclinical development as a broad-spectrum antimicrobial agent during the 1970s when the in vivo efficacy in the T. b. brucei strain GVR35 mouse CNS model of HAT was first demonstrated (14).
Some of the data presented here have previously been published in summary form (33).
MATERIALS AND METHODS
Materials.
Fexinidazole (1-methyl-2-{[p-(methylthio)phenoxy]methyl}-5- nitroimidazole) manufactured under good manufacturing practice conditions (Axyntis), its sulfoxide and sulfone derivatives [1-methyl-2-(4-methylsulfonyl phenoxymethyl)-5-nitro imidazole and 1-methyl-2-(4-methylsulfonyl phenoxymethyl)-5-nitro imidazole] at laboratory grade (Axyntis), and nifurtimox (Bayer) were provided by the Drugs for Neglected Diseases Initiative (DNDi), pentamidine isethionate and diminazene aceturate were purchased from Sigma-Aldrich, and melarsoprol (Aventis) was provided by the WHO. The chemical structures of the experimental drug fexinidazole and the two metabolites fexinidazole sulfoxide and fexinidazole sulfone have previously been published (33).
All other reagents were of standard laboratory grade and purchased from commercial suppliers.
Preparation of compounds.
For in vitro studies, compounds were dissolved in 100% dimethyl sulfoxide (DMSO) and diluted in culture medium prior to being assayed. The maximum DMSO concentration in the in vitro assays was 1%.
For in vivo studies, the compounds were dissolved in DMSO and further diluted with distilled water to a final DMSO concentration of 10%, unless stated otherwise. In some studies, fexinidazole was prepared in an optimized suspension medium for oral administration comprising 5% (wt/vol) Tween 80 and 0.5% (wt/vol) Methocel in water, which has previously been shown to maximize absorption of the drug (33).
Parasites and cell culture conditions.
(i) T. b. rhodesiense.
The T. b. rhodesiense STIB900 strain is a derivative of the STIB704 strain isolated from a patient in Ifakara, Tanzania, in 1982 (5). STIB900mel and STIB900pent are melarsoprol- and pentamidine-resistant lines, respectively, which were generated by growing STIB900 in increasing subcurative drug concentrations (3).
(ii) T. b. gambiense.
(iii) T. b. brucei.
The T. b. brucei strains used include BS221, a derivative of the S427 strain isolated in Uganda in 1960 (7), AT1KO, a P2 transporter knockout of the BS221 strain (21), and STIB950mdr, which is a derivative of the CP 2469 strain isolated in 1985 from a cow in Hakaka, Soakow District, Somalia (15). The GVR35 strain was isolated from a wildebeest in the Serengeti in 1966 (primary isolate S10) (13).
T. b. rhodesiense and T. b. brucei parasites were cultured at 37°C under a humidified 5% CO2 atmosphere in minimum essential medium (MEM) with Earle's salts, supplemented according to the protocol of Baltz et al. (2) with the following modifications: 0.2 mM 2-mercaptoethanol, 1 mM Na-pyruvate, 0.5 mM hypoxanthine, and 15% heat-inactivated horse serum as a supplement. T. b. gambiense strains were grown in HMI-9 medium (11) supplemented with 15% heat-inactivated fetal bovine serum (FBS) and 5% human serum. To ensure maintenance of a log growth phase, parasites were subcultured into fresh medium at appropriate dilutions every 2 to 3 days.
In vitro growth inhibition assays.
The compounds were tested in an AlamarBlue serial drug dilution assay (27) in order to determine the 50% inhibitory concentrations (IC50s).
Serial drug dilutions were prepared in 96-well microtiter plates containing the appropriate culture medium as described above for each parasite strain, and wells were inoculated with either 2,000 bloodstream forms for the T. b. rhodesiense or T. b. brucei assay or 10,000 trypanosomes for the T. b. gambiense assay. Cultures were incubated for 70 h at 37°C under a humidified 5% CO2 atmosphere. After this time, 10 μl of resazurin (12.5 mg resazurin [Sigma] dissolved in 100 ml phosphate-buffered saline) was added to each well. The plates were incubated for an additional 2 to 4 h for T. b. rhodesiense and T. b. brucei and an additional 6 to 8 h for T. b. gambiense isolates. The plates were read in a SpectraMax Gemini XS microplate fluorescence scanner (Molecular Devices) using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. The IC50s were calculated by linear regression (12) from the sigmoidal dose inhibition curves using SoftmaxPro software.
In vitro dynamic assays.
T. b. rhodesiense (STIB900) was seeded in clear 96-well V-bottom plates at a density of 10,000 parasites per well in 100 μl medium and incubated for 1, 6, and 24 h with serially diluted test compounds. One plate was prepared for each time point. At the designated time point, a plate was spun at 650 relative centrifugal force (RCF) for 5 min to sediment the parasites. The supernatant was removed, and 100 μl of warmed MEM was added to each well to resuspend the parasites. The wash process was repeated four more times. After the washing procedure, the parasites were resuspended in 100 μl medium and transferred into new culture plates and further incubated. After a total of 70 h of incubation, resazurin was added and trypanocidal activities (IC50 and IC90 values) were determined as described for the in vitro growth inhibition assays.
In vitro combination assays.
Drug combination studies were performed as previously described (10). Initially, the IC50s of the test drugs alone were determined. Subsequently, drug solutions were diluted with culture medium to an initial concentration of 10 times the predetermined IC50. The solutions were combined in ratios of 1:3, 1:1, and 3:1. Single and combination drug solutions were then introduced into 96-well plates, and the parasites were cultured as described above. The IC50s of the drugs alone and in combination were determined as described above. For data interpretation, the IC50s of the drugs in combination were expressed as fractions of the IC50s of the drugs alone. These data were expressed as fractional inhibitory concentrations (FICs) for drug A and drug B.
Isobolograms were constructed by plotting the FIC of drug A (FIC-A) against that of drug B (FIC-B) for each of the three drug ratios, with concave curves indicating synergism, straight lines indicating addition, and convex curves indicating antagonism. To obtain numeric values for the interactions, results were expressed as the sum FICs (ΣFICs) of FIC-A and FIC-B. Cutoff ranges were determined by mixing the same drug at various ratios and accounting for experimental variation. Changes in FIC values indicate the nature of the interactions as follows: synergism, ΣFIC < 0.5; indifferent, ΣFIC = 0.5 to 4.0; and antagonism, ΣFIC > 4 (8, 23). Mean ΣFICs were used to classify the overall nature of the interaction.
In vivo experiments.
Adult female NMRI mice (Harlan Laboratories, The Netherlands) weighing between 20 and 25 g at the beginning of the study were housed under standard conditions with food pellets and water ad libitum. All protocols and procedures used in the current study were reviewed and approved by the local veterinary authorities of the Canton Basel-Stadt, Switzerland.
T. b. rhodesiense (STIB900) acute mouse model.
The STIB900 acute mouse model mimics the first stage of the disease. Experiments were performed as previously described (32), with minor modifications. Female NMRI mice were infected intraperitoneally (i.p.) with 104 T. b. rhodesiense (STIB900) bloodstream forms. Experimental groups of four mice were treated i.p. or orally (per os [p.o.]) with compounds on four consecutive days from days 3 to 6 postinfection. A control group was infected but remained untreated. The tail blood of all mice was checked for parasitemia up to 60 days postinfection. Surviving and aparasitemic mice at day 60 were considered cured and were euthanized. The day of relapse of the animals (including the cured mice) was recorded (as >60), and data were expressed as the mean day of relapse (MRD).
T. b. brucei (GVR35) mouse CNS model.
The GVR35 mouse CNS model mimics the second stage of the disease. Five female NMRI mice per experimental group were inoculated i.p. with 2 × 104 T. b. brucei (GVR35) bloodstream forms. Treatment (i.p. or p.o.) with compound was given on five consecutive days from days 21 to 25 postinfection. Some experimental groups were treated twice daily with a time interval of 7 to 8 h. In all experiments with fexinidazole, a control group was treated on day 21 with a single intraperitoneal dose of diminazene aceturate at 40 mg/kg of body weight, which is subcurative since it clears the trypanosomes only in the hemolymphatic system and not in the CNS, leading to a subsequent reappearance of trypanosomes in the blood (13). Parasitemia was monitored twice per week in the first 5 weeks after treatment, followed by once a week up to 180 days postinfection. Surviving and aparasitemic mice at day 180 were considered cured and were euthanized. The day of relapse of the animals (including the cured mice) was recorded (as >180) to calculate the MRD.
RESULTS
In vitro activity of fexinidazole and its primary metabolites against African trypanosomes.
Fexinidazole and its sulfoxide and sulfone metabolites and the reference drugs melarsoprol, pentamidine, eflornithine, nifurtimox, and the veterinary compound diminazene aceturate have been assessed for in vitro efficacy against T. brucei subspecies isolates (Table 1). Fexinidazole showed in vitro trypanocidal activity against all tested T. brucei subspecies and strains in the range of 0.7 to 3.3 μM (0.2 to 0.9 μg/ml). The fexinidazole sulfoxide and sulfone metabolites were slightly more potent but within the same order of magnitude as the parent compound. Fexinidazole and its sulfoxide and sulfone metabolites showed activity comparable to that of eflornithine and nifurtimox but were considerably less potent than the three other drugs tested.
Table 1.
| Parasite | Strain | IC50 (μM)a | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Fexinidazole (279.3) | Fexinidazole sulfone (295.3) | Fexinidazole sulfoxide (311.3) | Melarsoprol (398.3) | Pentamidine (592.7) | Eflornithine (236.7) | Nifurtimox (287.3) | Diminazene (515.5) | ||
| T. b. rhodesiense | STIB900 (wild type) | 2.17 ± 0.29 | 1.44 ± 0.22 | 1.64 ± 0.36 | 0.011 ± 0.003 | 0.002 ± 0.0003 | 8.58 ± 2.7 | 1.09 ± 0.33 | 0.009 ± 0.002 |
| 5.56 ± 1.9b | 3.2 ± 0.15b | 3.2 ± 0.44b | |||||||
| STIB900mel | 2.66 ± 0.57 | 1.26 ± 0.51 | 1.16 ± 0.29 | 0.092 ± 0.028 | 0.095 ± 0.035 | ND | ND | 0.019 ± 0.002 | |
| STIB900pent | 2.71 ± 0.87 | 1.16 ± 0.39 | 1.48 ± 0.75 | 0.043 ± 0.022 | 0.058 ± 0.019 | ND | ND | 0.011 ± 0.004 | |
| T. b. brucei | BS221 (wild type) | 2.38 ± 0.88 | 1.63 ± 0.92 | 1.49 ± 0.61 | 0.013 ± 0.004 | 0.002 ± 0.0003 | ND | ND | 0.005 ± 0.001 |
| BS221 AT1KO | 1.33 ± 0.21 | 0.56 ± 0.04 | 0.85 ± 0.32 | 0.034 ± 0.003 | 0.008 ± 0.002 | ND | ND | 0.060 ± 0.016 | |
| STIB950mdr | 2.44 ± 0.99 | 0.99 ± 0.34 | 1.21 ± 0.14 | 0.038 ± 0.011 | 0.002 ± 0.0002 | ND | ND | 0.062 ± 0.05 | |
| T. b. gambiense | STIB930 | 1.84 ± 1.13 | 0.91 ± 0.27 | 0.94 ± 0.39 | 0.012 ± 0.005 | 0.016 ± 0.001 | 2.85 ± 0.98 | 2.24 ± 0.66 | 0.021 ± 0.009 |
| DAL 898R | 1.01 ± 0.36 | 0.76 ± 0.30 | 1.03 ± 0.13 | 0.009 ± 0.002 | 0.002 ± 0.0002 | ND | ND | 0.014 ± 0.001 | |
| K3048 | 0.95 ± 0.19 | ND | ND | 0.032 ± 0.012 | 0.084 ± 0.015 | 7.63 ± 2.5 | 0.99 ± 0.12 | 0.076 ± 0.03 | |
| 45R | 2.47 ± 1.59 | 0.95 ± 0.47 | 1.24 ± 0.60 | 0.033 ± 0.011 | 0.069 ± 0.044 | 9.98 ± 2.4 | 1.06 ± 0.38 | 0.074 ± 0.033 | |
| 40R | 2.61 ± 1.03 | 0.67 ± 0.35 | 0.95 ± 0.33 | 0.032 ± 0.006 | 0.088 ± 0.024 | 11.4 ± 5.8 | 1.46 ± 0.20 | 0.12 ± 0.02 | |
| 349Pi | 1.07 ± 0.14 | ND | ND | 0.043 ± 0.011 | 0.066 ± 0.012 | 16.7 ± 3.6 | 0.78 ± 0.19 | 0.043 ± 0.025 | |
| 349R | 3.31 ± 0.88 | ND | ND | 0.033 ± 0.015 | 0.095 ± 0.012 | 22.8 ± 13.9 | 2.73 ± 0.66 | 0.064 ± 0.031 | |
| 130R | 2.37 ± 1.14 | ND | ND | 0.055 ± 0.023 | 0.074 ± 0.011 | 9.4 ± 2.19 | 1.34 ± 0.17 | 0.051 ± 0.013 | |
a
Values are the means ± standard deviations for 3 to 5 cultures. Values in parentheses are molecular weights. ND, not determined.
b
IC90 values.
In vivo efficacy of fexinidazole in an experimental model of acute infection with African trypanosomes.
Fexinidazole showed dose-related efficacy in the T. b. rhodesiense (STIB900) acute mouse model at intraperitoneal (i.p.) doses of 20 to 50 mg/kg/day and oral (per os [p.o.]) doses of 25 to 100 mg/kg/day given on four consecutive days, with 100 mg/kg/day p.o. being 100% curative (Table 2). In a separate experiment, the two fexinidazole metabolites were less potent than fexinidazole when administered i.p. or orally in the acute model of infection. Fexinidazole sulfoxide cured one of four infected mice at a dose of 50 mg/kg/day i.p. and two mice at 100 mg/kg/day p.o. Fexinidazole sulfone was not effective at 50 mg/kg/day i.p. and cured one mouse at a dose of 100 mg/kg/day p.o.
Table 2.
| Compound | Dose (mg/kg)a | Route | No. of cured mice/no. of infected mice | Mean day of relapse |
|---|---|---|---|---|
| Control | 0/12 | 8.75b | ||
| Fexinidazole | 20 | i.p. | 0/4 | 11 ± 2 |
| Fexinidazole | 50 | i.p. | 4/4 | >60 |
| Fexinidazolec | 25 | p.o. | 0/4 | 12 ± 2 |
| Fexinidazolec | 50 | p.o. | 1/4 | >27 |
| Fexinidazolec | 100 | p.o. | 4/4 | >60 |
| Fexinidazole sulfoxide | 50 | i.p. | 1/4 | >24.5 |
| Fexinidazole sulfoxide | 100 | p.o. | 2/4 | >38.25 |
| Fexinidazole sulfone | 50 | i.p. | 0/4 | 11 ± 2 |
| Fexinidazole sulfone | 100 | p.o. | 1/4 | >31.5 |
| Melarsoprol | 4 | i.p. | 4/4 | >60 |
a
Doses were given on four consecutive days, and 10% DMSO was used as a vehicle.
b
Mean survival days after infection of untreated control animals. The value given is the average for three experiments.
c
Data from reference 33.
In vivo efficacy of fexinidazole in an experimental model for chronic infection with African trypanosomes involving brain infection.
Fexinidazole was shown to be effective in the GVR35 mouse model, which mimics the advanced and fatal stage of the disease, when parasites have disseminated into the brain (Table 3). At i.p. doses of 50 mg/kg given twice per day (b.i.d.) or p.o. doses of 100 mg/kg also given twice per day for five consecutive days, all mice were cured; at single doses of 200 mg/kg/day p.o. for five consecutive days, 7 of 8 mice were cured, and at single doses of 100 mg/kg/day p.o., 3 of 5 mice (DMSO-water vehicle) and 2 of 8 mice (Tween 80-Methocel vehicle) were cured. In another experiment using the same vehicle, fexinidazole was compared to nifurtimox at a dose range of 50 to 200 mg/kg/day p.o. given for 5 days. While fexinidazole resulted in a partial cure at 100 mg/kg/day (2/8 mice cured) and almost a complete cure at 200 mg/kg/day (7/8) (33), nifurtimox had no curative effect at any dose tested. Significant levels of fexinidazole and the sulfoxide and sulfone metabolites can be detected in mice treated using the same protocol and assessed for plasma drug levels after day 5 (33). The plasma levels of both fexinidazole sulfoxide and fexinidazole sulfone following 5 days of once-per-day oral treatment with fexinidazole were found to be in the same range as that shown to kill all parasites in vitro, indicating that these compounds probably provided the bulk of the trypanocidal activity of the administered parent compound.
Table 3.
| Compound | Dose (mg/kg)a | Route | Vehicle | No. of cured mice/no. of infected mice | Mean day of relapse |
|---|---|---|---|---|---|
| Fexinidazole | 50 | i.p. | DMSO-water | 1/5 | 73.8 |
| Fexinidazole | 50 b.i.d. | i.p. | DMSO-water | 5/5 | >180 |
| Fexinidazole | 100 | p.o. | DMSO-water | 3/5 | >127 |
| Fexinidazole | 100 b.i.d. | p.o. | DMSO-water | 11/15 | >156.5f |
| Fexinidazoleb | 50 | p.o. | Methocel-Tween 80c | 0/8 | 41.3 ± 9 |
| Fexinidazoleb | 100 | p.o. | Methocel-Tween 80 | 2/8 | >82.1 |
| Fexinidazoleb | 200 | p.o. | Methocel-Tween 80 | 7/8 | >163.8 |
| Nifurtimox | 50 | p.o. | Methocel-Tween 80 | 0/8 | 31.0 ± 2 |
| Nifurtimox | 100 | p.o. | Methocel-Tween 80 | 0/8 | 31.0 ± 2 |
| Nifurtimox | 200 | p.o. | Methocel-Tween 80 | 0/8 | 37.4 ± 5 |
| Diminazene | 40d | i.p. | DMSO-water | 0/24 | 49.8 ± 6g |
| Eflornithine | 2%e | p.o. | Water | 0/4 | 76.3 ± 8h |
| Melarsoprol | 5 | i.p. | Propyleneglycol-water | 0/5 | 57.6 ± 14i |
| Melarsoprol | 10 | i.p. | Propyleneglycol-water | 1/5 | >103.4i |
| Melarsoprol | 15 | i.p. | Propyleneglycol-water | 4/5 | >180i |
a
Doses were given on five consecutive days unless indicated otherwise.
b
Data from reference 33.
c
An optimized suspension medium for oral administration comprising 0.5% (wt/vol) Methocel and 5% (wt/vol) Tween 80 in water to maximize absorption. These data have previously been published and are reproduced here for comparative purposes (33).
d
A single dose of diminazene was given.
e
A 2% solution of eflornithine provided in drinking water for 10 days.
f
Mean result for 3 separate experiments (n = 15).
g
Mean result for 5 separate experiments (n = 24).
h
Data from 1 experiment (n = 4).
i
Representative data from 1 experiment (n = 5 per group).
In vitro dynamic results.
In order to better understand the in vitro trypanocidal activity of fexinidazole and the sulfoxide and sulfone metabolites, pulse incubation experiments were performed and IC50 and IC90 values were determined following compound washout at various time points after exposure. The results are shown in Fig. 1. A 48-h period of exposure to the compounds is required to produce activities similar to those in the standard 72-h assay, indicating that maximum killing effectiveness requires up to 48 h of exposure to the drugs.
Fig. 1.
In vitro drug combination results.
Although NECT is currently the only available drug combination therapy to treat HAT, the development of resistance to existing therapies is making the potential use of combination therapies increasingly relevant. Data on the in vitro interaction of possible combinations have been proposed to support such development options (30). Fexinidazole and the biologically active sulfoxide and sulfone metabolites have been assessed in combination with several drugs currently available to patients. All drug combination studies were performed at three different ratios (1:3, 1:1, and 3:1) using the fixed-ratio isobologram method (10), and the data were analyzed using the IC50 results. Fexinidazole combined with its sulfoxide and sulfone metabolites as well as the combination of sulfoxide and sulfone showed indifferent effects. The combination of fexinidazole or either of its metabolites with melarsoprol, eflornithine, or pentamidine also resulted in an indifferent effect. These data indicate that there are no cross-reactivities between these compounds, which would preclude their use in, albeit unlikely, combination therapies.
DISCUSSION
Only four drugs are registered for HAT treatment. Pentamidine and suramin are used against the early stage of the disease, while treatment of the second stage depends on melarsoprol, eflornithine, and the recently introduced nifurtimox-eflornithine combination therapy (NECT). Melarsoprol is an arsenical compound and is highly toxic with severe adverse effects (18). In addition, there have been alarming reports of treatment failures with both melarsoprol and eflornithine, which until recently have been the only available drugs for second-stage treatment (1), and it is hoped that the broad implementation of the NECT regimen may avert the further development of eflornithine resistance. New safe and effective drugs with simplified dosing regimens are urgently needed. Ideally, such new treatments would be effective in both acute and chronic disease stages. Such new treatment options would largely simplify disease management and, importantly, avoid the painful lumbar puncture procedure currently required for distinguishing between disease stages.
Fexinidazole has recently been identified as a promising new drug candidate for the treatment of HAT (33), and data presented here provide in vitro and in vivo profiling of the antitrypanosomal efficacy of fexinidazole and its two primary metabolites, sulfoxide and sulfone.
Fexinidazole and the sulfoxide and sulfone metabolites were tested in vitro alongside reference drugs against a panel of African trypanosomes of T. brucei spp. (Table 1), which included sensitive and resistant wild-type strains, laboratory-induced melarsoprol- and pentamidine-resistant and P2-transporter knockout strains, and new field isolates. The data showed that there is no evidence of innate resistance to fexinidazole or the two metabolites within any of the strains tested, as all IC50s were in similar ranges and varied by less than a factor of 4. The new T. b. gambiense strains showed reduced IC50s for pentamidine, but this is unlikely to indicate resistance in the field given the higher blood levels and long terminal half-life of the drug found in patients after standard treatment (4).
Fexinidazole showed in vivo efficacy in both the acute mouse model and, more importantly, the chronic mouse model with established brain infection. In the STIB900 acute mouse model, fexinidazole demonstrated 100% efficacy at an i.p. dose of 50 mg/kg/day and an oral dose of 100 mg/kg/day, both given for 4 days (Table 2). While a fexinidazole dose of 50 mg/kg/day i.p. was fully effective, the sulfoxide only partially cured with the same dose and route of administration and the sulfone was ineffective. After oral administration at a dose of 100 mg/kg/day, both the sulfoxide and sulfone metabolites were only partially effective, whereas fexinidazole cured 100% of the animals. Although no pharmacokinetic data are currently available to formally demonstrate oral absorption of the sulfoxide or sulfone metabolite in mice, it may be that neither is as readily absorbed as fexinidazole via the oral route. However, it is apparent that even when the i.p. route of administration, which should maximize the systemic bioavailability of the compounds, was used, neither metabolite was as effective as the parent fexinidazole in this acute model of disease. In addition, it is unlikely that protein binding could account for the lack of effectiveness of the metabolites when given orally, and while fexinidazole is highly protein bound in plasma (93% in mice and 95% in humans), neither metabolite is highly protein bound, at least in human plasma (26% and 42%, respectively, for the sulfoxide and sulfone metabolites) (data not shown). Overall, these data support the view that fexinidazole itself, acting as a biologically active prodrug while rapidly being metabolized to the sulfoxide and sulfone metabolites in all animals tested (33), is likely to be a more useful compound for oral treatment than either of its two metabolites given alone.
In 1983, Jennings and Urquhart reported that fexinidazole, given in combination with suramin, cured a T. brucei CNS infection in mice (14). We have tested fexinidazole as monotherapy in the GVR35 mouse model of stage 2 HAT involving brain infection using two different vehicle formulations (Table 3). Using the optimized Methocel-Tween 80 vehicle, fexinidazole showed a dose-related increase in efficacy and cured 7 of 8 infected mice at a single oral daily dose of 200 mg/kg/day for 5 days. In comparison, nifurtimox was ineffective in the GVR35 mouse model up to a dose of 200 mg/kg/day for 5 days. It is of interest to note that the presumed trough levels of the two metabolites after 24 h are reported to be around 1 μg/ml (33), which would allow for a daily dosing schedule to be maintained with systemic drug levels near those required to kill the parasite in vitro. Clearly, in this model, the drug levels in the CNS are of key importance, and while no data are available from the experiments presented, published data indicate that, in mice, brain levels of fexinidazole, sulfoxide, and sulfone are approximately 0.8, 5, and 1 μg/ml, respectively, at 60 min post-oral dosing with fexinidazole (33). Further experiments are under way to more fully assess the brain levels of the compounds in mice at different times. While the most effective oral dose of 200 mg/kg may seem high, fexinidazole is well-tolerated in laboratory animals at significantly higher doses (32), and although no data are available for mice regarding a no-toxic-effect level, a 50% lethal dose of >10,000 mg/kg has been reported (data not shown).
It is important to note that, of the drugs currently in clinical use, only melarsoprol has been shown to be effective in this experimental stage 2 HAT model.
Pulse incubation of T. b. rhodesiense with fexinidazole and the sulfoxide and sulfone metabolites shows that a 48-h period of exposure is required to produce irreversible effects on trypanosomal survival for all three compounds (Fig. 1). This result has implications for in vivo efficacy, as it suggests that plasma or cerebrospinal fluid (CSF) concentrations may need to be maintained at or above optimal trypanocidal concentrations for >48 h to achieve elimination of all parasites. As discussed above, it is apparent, at least in mice, that while plasma levels of fexinidazole may not be maintained at a sufficient killing concentration, both the sulfoxide and sulfone metabolites are present in plasma and in the brain at concentrations sufficient to kill all parasites. In addition, the data indicate that a 5-day dosing schedule would ensure sufficient trough levels of these metabolites at 24 h to maintain effective killing concentrations in plasma. Concentrations in the brain reach several micrograms per ml 1 h after oral application (33), but information on the persistence of fexinidazole and its metabolites is not available. It can be assumed that the metabolites and mainly sulfone are responsible for the trypanocidal effect in the brain. The CSF is often used as a surrogate for the brain since it is accessible without the need to kill the animal (6). Thus, these data provide support for the observations in both mouse models that oral treatment with fexinidazole for 4 days (acute model) or 5 days (CNS model) can achieve a cure. This time-dose relationship has previously been described for diamidines such as diminazene aceturate, which is able to kill trypanosomes after a short exposure time of 15 min at 1 μg/ml (16), while other trypanocidal agents (e.g., trybizine hydrochloride) with an in vitro potency similar to or greater than that of diminazene aceturate require a much longer exposure time, >8 h, at 10 μg/ml to lead to death of the parasites (17).
Fexinidazole and the sulfoxide and sulfone metabolites have similar in vitro trypanocidal activities (Tables 1 and 2) (33). The in vivo activity of fexinidazole is likely to be due to the concerted action of the three molecules. The in vitro combination studies performed support this hypothesis. All combinations of fexinidazole and its metabolites were investigated using the fixed-ratio isobologram method (10). The IC50s for fexinidazole, sulfoxide, and sulfone in combination did not differ from that of each drug alone, resulting in indifferent mean ΣFIC values between 1 and 1.4 for the combinations.
In several foci, melarsoprol treatment failures have reached 30% of those treated (19, 22, 28, 31), and treatment failures of up to 16% with eflornithine have recently been reported (1, 24). A strategy to prevent the development of resistance is the use of drugs in combination, and the introduction of nifurtimox-eflornithine combination therapy (NECT) is an important development in the treatment of T. b. gambiense infections (25). The rationale behind combination treatments in general is that the likelihood of developing resistance to a single drug is relatively high but much lower with a drug combination (34). Although in vitro cross-resistance studies have yet to be fully validated as predictive of human drug resistance, a recently published study on the cross-resistance of fexinidazole and its sulfoxide and sulfone metabolites in a nifurtimox-resistant T. b. brucei strain supports the approach of utilizing chemically unrelated drug combinations (30). Those same authors also showed that resistance against fexinidazole could easily be generated in the laboratory, thus underlining the potential need for a combination partner for fexinidazole. In the present study, fexinidazole and its sulfoxide and sulfone metabolites were tested in vitro in combination with three existing drugs, pentamidine, melarsoprol, and eflornithine. All combinations resulted in indifferent mean ΣFIC values. This observation supports the proposition that fexinidazole could be a candidate for combination with existing drugs that are currently acceptable treatments, such as pentamidine, eflornithine, and NECT, or more likely with other new drug candidates that may become available in the future.
In conclusion, the data presented in this paper demonstrate that fexinidazole and its sulfoxide and sulfone metabolites rapidly formed in vivo are effective at killing the parasites responsible for human African trypanosomiasis. Fexinidazole is effective in both acute and chronic mouse models of HAT at doses and dosing regimens which are expected to be practicable for human treatment. Time-dose studies indicate that effective drug levels need to be maintained for at least 48 h, and interaction data show that there is no cross-inhibition between fexinidazole and the sulfoxide or sulfone metabolite or other chemically unrelated treatment modalities. Overall, these data provide evidence that fexinidazole has the potential to be an effective oral treatment for both T. b. gambiense and T. b. rhodesiense forms of human sleeping sickness and both stages of the disease.
ACKNOWLEDGMENTS
These studies were funded via a grant from the DNDi, who received financial support from the following donors for this work: the Department for International Development (DFID) of the United Kingdom, the German Agency for Technical Cooperation (GTZ), Médecins Sans Frontières (MSF), the Ministry of Foreign and European Affairs of France, the Spanish Agency for International Cooperation and Development, and a Swiss foundation.
None of the donors had any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Information & Contributors
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Published In
Antimicrobial Agents and Chemotherapy
Volume 55 • Number 12 • December 2011
Pages: 5602 - 5608
PubMed: 21911566
Copyright
© 2011 American Society for Microbiology.
History
Received: 23 February 2011
Returned for modification: 18 May 2011
Accepted: 5 September 2011
Published online: 11 November 2011
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Antitrypanosomal Activity of Fexinidazole, a New Oral Nitroimidazole Drug Candidate for Treatment of Sleeping Sickness
. Antimicrob Agents Chemother
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