The R enantiomer of the antitubercular drug PA-824 as a potential oral treatment for visceral leishmaniasis

Article abstract of DOI:10.1128/AAC.00722-13

The novel nitroimidazopyran agent (S)-PA-824 has potent antibacterial activity against Mycobacterium tuberculosis in vitro and in vivo and is currently in phase II clinical trials for tuberculosis (TB). In contrast to M. tuberculosis, where (R)-PA-824 is inactive, we report here that both enantiomers of PA-824 show potent parasiticidal activity against Leishmania donovani, the causative agent of visceral leishmaniasis (VL). In leishmania-infected macrophages, (R)-PA-824 is 6-fold more active than (S)-PA- 824. Both des-nitro analogues are inactive, underlining the importance of the nitro group in the mechanism of action. Although the in vitro and in vivo pharmacological profiles of the two enantiomers are similar, (R)-PA-824 is more efficacious in the murine model of VL, with≥99% suppression of parasite burden when administered orally at 100 mg kg of body weight-1, twice daily for 5 days. In M. tuberculosis, (S)-PA-824 is a prodrug that is activated by a deazaflavin-dependent nitroreductase (Ddn), an enzyme which is absent in Leishmania spp. Unlike the case with nifurtimox and fexinidazole, transgenic parasites overexpressing the leishmania nitroreductase are not hypersensitive to either (R)-PA-824 or (S)-PA-824, indicating that this enzyme is not the primary target of these compounds. Drug combination studies in vitro indicate that fexinidazole and (R)-PA-824 are additive whereas (S)-PA-824 and (R)-PA-824 show mild antagonistic behavior. Thus, (R)-PA-824 is a promising candidate for late lead optimization for VL and may have potential for future use in combination therapy with fexinidazole, currently in phase II clinical trials against VL. Copyright

Full text of DOI:10.1128/AAC.00722-13

The R Enantiomer of the Antitubercular Drug PA-824 as a Potential
Oral Treatment for Visceral Leishmaniasis
Stephen Patterson, Susan Wyllie, Laste Stojanovski, Meghan R. Perry, Frederick R. C. Simeons, Suzanne Norval, Maria Osuna-Cabello,
Manu De Rycker, Kevin D. Read, Alan H. Fairlamb
Division of Biological Chemistry and Drug Discovery, Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom
The novel nitroimidazopyran agent (S)-PA-824 has potent antibacterial activity against Mycobacterium tuberculosis in vitro and
in vivo and is currently in phase II clinical trials for tuberculosis (TB). In contrast to M. tuberculosis, where (R)-PA-824 is inac-
tive, we report here that both enantiomers of PA-824 show potent parasiticidal activity against Leishmania donovani, the caus-
ative agent of visceral leishmaniasis (VL). In leishmania-infected macrophages, (R)-PA-824 is 6-fold more active than (S)-PA-
824. Both des-nitro analogues are inactive, underlining the importance of the nitro group in the mechanism of action. Although
the in vitro and in vivo pharmacological profiles of the two enantiomers are similar, (R)-PA-824 is more efficacious in the mu-
rine model of VL, with >99% suppression of parasite burden when administered orally at 100 mg kg of body weight؊1, twice
daily for 5 days. In M. tuberculosis, (S)-PA-824 is a prodrug that is activated by a deazaflavin-dependent nitroreductase (Ddn),
an enzyme which is absent in Leishmania spp. Unlike the case with nifurtimox and fexinidazole, transgenic parasites overex-
pressing the leishmania nitroreductase are not hypersensitive to either (R)-PA-824 or (S)-PA-824, indicating that this enzyme is
not the primary target of these compounds. Drug combination studies in vitro indicate that fexinidazole and (R)-PA-824 are
additive whereas (S)-PA-824 and (R)-PA-824 show mild antagonistic behavior. Thus, (R)-PA-824 is a promising candidate for
late lead optimization for VL and may have potential for future use in combination therapy with fexinidazole, currently in phase
II clinical trials against VL.
isceral leishmaniasis (VL), caused by the protozoan parasite disease, combined with eflornithine infusions, has resulted in cure
V
Leishmania donovani, is the second largest parasitic killer after rates of around 97%, leading to its inclusion on the WHO Essen-
tial Medicines List (8). The success of nifurtimox as part of NECT
prompted the Drugs for Neglected Disease Initiative (DNDi) to
undertake a comprehensive screen of 700 nitroheterocyclic com-
pounds for antiparasitic activity. As a result, the 2-substituted
5-nitroimidazole fexinidazole (Hoe 239), first shown to have an-
titrypanosomal activity almost 30 years ago (9), was rediscovered.
Fexinidazole has now successfully completed phase I clinical trials
for HAT and is now in phase II/III trials (10). To date, nitrohet-
erocyclics have not been widely used in the treatment of leishman-
iasis; however, in our recent studies we demonstrated that fexini-
dazole also has potential as a safe and effective oral drug therapy
for treatment for the visceral disease (11). In light of these find-
ings, fexinidazole is about to enter phase II clinical trials for the
treatment of visceral leishmaniasis in Sudan (http://www.dndi
.org).
malaria, with more than 200 million people currently at risk from
infection. In 95% of cases, death can be prevented by timely and
appropriate drug therapy (1); however, current treatment options
are far from ideal (2). In India, Bangladesh, and Nepal, the epicen-
ter of VL infection with 60% of the world’s reported cases (3),
concerted efforts are being made to eliminate the disease by 2015.
An important element of this strategy involves active case finding
and drug treatment. Antimonial drugs are no longer efficacious in
Asia due to drug resistance (2), and the best currently available
treatments for anthroponotic VL are miltefosine and liposomal
amphotericin B. Undoubtedly, both drugs are vastly superior to
previous therapies, but they also have their limitations. The prin-
cipal drawbacks of miltefosine are its teratogenicity, prolonged
treatment regimen, and high resistance potential which limits its
usefulness in national elimination programs (4). Problems asso-
ciated with liposomal amphotericin B include high treatment
costs and the need for intravenous administration in primary
health care centers (5). In addition, unresponsiveness has been
reported in some Sudanese VL patients (6). Thus, there remains
an urgent need to strengthen the range of treatment options not
only for VL but also for post-kala-azar dermal leishmaniasis
(PKDL), which has a 10 to 20% incidence in VL patients from
India and 50% in the Sudan following apparently effective drug
treatment (7).
As a result of our studies with fexinidazole, we searched the
literature for other nitroimidazoles which may have potential for
Received 9 April 2013 Returned for modification 10 May 2013
Accepted 5 July 2013
Published ahead of print 15 July 2013
Address correspondence to Alan H. Fairlamb (biology queries),
a.h.fairlamb@dundee.ac.uk, or Kevin D. Read (pharmacology queries),
k.d.read@dundee.ac.uk.
In the search for more-effective treatments for the neglected
diseases, there has been renewed interest in the chemotherapeutic
potential of nitroheterocyclic compounds. This interest largely
stems from the success of nifurtimox-eflornithine combination
therapy (NECT) for the treatment of the Gambian form of human
African trypanosomiasis (HAT). Treatment with NECT, consist-
ing of oral nifurtimox, a nitrofuran drug also used against Chagas’
S.P. and S.W. contributed equally.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AAC.00722-13.
Copyright © 2013 Patterson et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution 3.0 Unported license.
doi:10.1128/AAC.00722-13
October 2013 Volume 57 Number 10
Antimicrobial Agents and Chemotherapy p. 4699–4706
aac.asm.org 4699

Patterson et al.
use in the treatment of visceral leishmaniasis. In this process, we
noted the promising antitubercular drug PA-824 (12, 13). This
bicyclic nitroimidazole exhibits potent bactericidal activity
against both replicating and nonreplicating Mycobacterium tuber-
culosis, the causative agent of tuberculosis (TB), and is currently
being tested in phase II clinical trials (14–16). Here, we demon-
strate that in contrast to the TB structure-activity relationship, it is
the R enantiomer of PA-824 that displays the greatest in vitro
100
[I]
Y ϭ
m
1 ϩ
ͩ ͪ
EC50
In this equation, [I] represents inhibitor concentration and m is the
slope factor. Experiments were repeated at least three times, and the data
are presented as the weighted mean plus weighted standard deviation
(22).
All T. cruzi amastigote and HepG2 drug sensitivity data were processed
potency and has promising leishmanicidal activity in vivo. We also using the program IDBS ActivityBase. Raw data were converted into per-
cent inhibition through linear regression by setting the high-inhibition
control as 100% and the no-inhibition control as 0%. Curve fitting was
carried out using the following 4-parameter equation:
demonstrate that the mode of action of (R)-PA-824 does not in-
volve the trypanosomatid type I nitroreductase (NTR) that acti-
vates fexinidazole and nifurtimox.
B Ϫ A
Y ϭ A ϩ
m
MATERIALS AND METHODS
EC50
1 ϩ
Cell lines and culture conditions. The clonal Leishmania donovani cell
line LdBOB (derived from MHOM/SD/62/1S-CL2D) was grown as either
promastigotes or axenic amastigotes in medium specific for each devel-
opmental stage (17). Amastigotes were cultivated at 37°C in 5% CO2, and
promastigotes were grown at 26°C. Parasites were cycled between devel-
opmental stages after a maximum of 7 passages. Transgenic LdBOB par-
asites expressing the Leishmania major nitroreductase (LmjF.05.0660) en-
zyme (11) were cultured under identical conditions.
Procyclic trypomastigotes of Trypanosoma brucei brucei S427 29-13
were grown in SDM-79 medium supplemented with 10% fetal calf serum
(FCS) and hemin (100 mg mlϪ1). T. brucei bloodstream forms (S427
Single Marker) were cultured at 37°C in HMI9T medium (18). Trypano-
soma cruzi Silvio-X10/7 clone A1 epimastigotes were grown in RTH-FCS
at 28°C (19). T. cruzi amastigotes were maintained in Vero cells (African
green monkey kidney cells) cultured in minimal Eagle’s medium (MEM)
supplemented with 1ϫ GlutaMax (Life Technologies) and 10% FCS.
HepG2 cells (hepatocyte carcinoma cells) were grown in MEM supple-
mented with 1ϫ GlutaMax, 1ϫ nonessential amino acids (Life Technol-
ogies), and 10% FCS. All mammalian cells were grown at 37°C, 5% CO2,
in a humidified incubator.
Chemical synthesis of (S)-PA-824 and analogues. (S)- and (R)-PA-
824 were prepared using a four-step sequence from 2-bromo-4-nitroim-
idazole (scheme S1). In addition, (S)-PA-824 was prepared in a single step
as previously described (scheme S2) (20). (S)- and (R)-Des-nitro-PA-824
were prepared in 5 steps according to the method of Singh et al. (13)
(scheme S3). Compound purity was determined by liquid chromatogra-
phy-mass spectrometry (LC-MS), with all compounds found to be of
Ͼ95% purity. For in vivo experiments, compound purity was further
analyzed by ultrahigh-performance liquid chromatography-mass spec-
trometry (UPLC-MS), with all compounds found to be of Ն99% purity.
Optical rotation measurements of (S)-PA-824 were in close agreement
with published values, confirming a high level of optical purity (21). The
optical rotation of (R)-PA-824 was equal and opposite to that of the S
enantiomer, confirming its optical purity. Full experimental protocols
and analytical data for all compounds are given in the supplemental ma-
terial.
In vitro drug sensitivity assays. To examine the effects of test com-
pounds on growth, triplicate cultures were seeded with 1 ϫ 105 parasites
mlϪ1. Parasites (L. donovani promastigotes, T. brucei procyclics, and T.
cruzi epimastigotes) were grown in the presence of drug for 72 h, after
which 50 ␮M resazurin was added to each well (0.5 mM in the case of T.
cruzi epimastigotes) and fluorescence (excitation of 528 nm and emission
of 590 nm) was measured after a further 4 h of incubation. Drug sensitiv-
ities of bloodstream-form T. brucei were determined as previously de-
scribed (22). T. brucei, L. donovani, and T. cruzi epimastigote drug sensi-
tivity data were processed using GRAFIT (version 5.0.4; Erithacus
Software) and fitted to a 2-parameter equation, where the data are cor-
ͩ ͪ
[I]
In this equation, A represents lowest percent inhibition and B repre-
sents highest percent inhibition. If curve definition was poor, the value of
B was fixed to 100.
In-macrophage drug sensitivity assays. In-macrophage drug sensi-
tivity assays were carried out as previously described (11), using mouse
peritoneal macrophages harvested from BALB/c mice.
In-Vero-cell T. cruzi drug sensitivity assays. Details of the T. cruzi
assay will be published elsewhere (unpublished data). Briefly, T. cruzi
trypomastigotes (X10/7) clone A1 were used to infect Vero cells in 384-
well plates (Greiner) and then exposed for 72 h to compounds followed by
fixation, staining with Hoechst 33342, and evaluation of infection using
an Operetta high-content imaging system (PerkinElmer).
HepG2 assays. HepG2 cells (2.5 ϫ 103 per well) were plated in 384-
well white clear-bottomed plates (Greiner) and cultured in minimal Ea-
gle’s medium with 10% FCS for 24 h at 37°C in 5% CO2. Compound
dilution curves were then added to plates directly using a Labcyte Echo
550 acoustic dispenser (25 nl). After compound addition, plates were
incubated for a further 69 h. Resazurin was then added to each well at a
final concentration of 50 ␮M, and fluorescence was measured (excitation
of 528 nm and emission of 590 nm) after 1.5 h of incubation.
Drug combination studies. For isobologram determinations, (R)-
PA-824, (S)-PA-824, and fexinidazole sulfone were tested in fixed ratios
based on their respective EC50s. The EC50s of each combination were
determined after 72 h and plotted as an isobologram (23).
In vivo drug sensitivity. All animal experiments were approved by the
University Ethical Review Committee and performed under the Animals
(Scientific Procedures) Act 1986 in accordance with the European Com-
munities Council Directive (86/609/EEC). Groups of female BALB/c mice
(5 per group) were inoculated intravenously with approximately 2 ϫ 107
L. donovani amastigotes (LV9; WHO designation MHOM/ET/67/HU3)
harvested from the spleen of an infected hamster (24). From day 7 postin-
fection, groups of mice were treated either with drug vehicle only (orally),
with sodium stibogluconate (Pentostam, a gift from GlaxoSmithKline)
(15 mg kg of body weightϪ1 subcutaneously), with miltefosine (12 mg
kgϪ1 orally), or with either (R)- or (S)-PA-824 (30 or 100 mg kgϪ1 orally).
Miltefosine and sodium stibogluconate were administered once daily for 5
days, with (R)- and (S)-PA-824 administered twice daily over the same
period. Drug dosing solutions were prepared fresh each day, and the ve-
hicle for both enantiomers of PA-824 was 10% (vol/vol) dimethyl sulfox-
ide (DMSO), 40% polyethylene glycol 400, and 50% deionized water. On
day 14 postinfection, all animals were humanely euthanized and parasite
burdens were determined by counting the number of amastigotes/500
liver cells (11). Parasite burden is expressed in Leishmania donovani units
(LDU), mean number of amastigotes per liver cell ϫ mg weight of liver
(25).
Determination of PA-824 enantiomer exposure in mice after acute
rected for background fluorescence, to obtain the effective concentration oral dosing. The R and S enantiomers of PA-824 (50 mg kgϪ1) were orally
inhibiting growth by 50% (EC50):
administered to BALB/c mice. Blood samples (10 ␮l) were collected from
4700 aac.asm.org
Antimicrobial Agents and Chemotherapy

PA-824 and Leishmania donovani
TABLE 1 EC50s for PA-824 enantiomers against representative trypanosomatidsa
EC50 (␮M)
(S)-PA-824
(R)-PA-824
Species
Developmental stage
T. brucei
Procyclic
Bloodstream
36.5 Ϯ 2.5
37.8 Ϯ 3.2b
25.0 Ϯ 0.2
14.9 Ϯ 0.7
T. cruzi
Epimastigote
Amastigote (in Vero cell)
3.4 Ϯ 0.03
Ͼ30
0.70 Ϯ 0.08
4.4 Ϯ 0.5
L. donovani
Promastigote
Amastigote (in macrophage)
0.9 Ϯ 0.1
4.92 Ϯ 0.3
0.16 Ϯ 0.03
0.93 Ϯ 0.06
Mammalian (HepG2)
NAc
Ͼ50
Ͼ50
a Results are the weighted means and standard errors of at least three independent experiments, except for HepG2 (n ϭ 2).
b Previously determined value (20).
c NA, not applicable.
the tail vein of each animal into sample storage tubes (Micronic BV) treatment for visceral leishmaniasis prompted us to investigate the
containing deionized water (20 ␮l) at defined intervals postdose and
stored at Ϫ80°C until analysis. The level of each enantiomer in mouse
blood was determined by UPLC-MS/MS (20).
Physicochemicalpropertiesandinvitrodrugmetabolismandphar-
macokinetic (DMPK) parameters. The software package StarDrop by
Optibrium was used to calculate physical parameters, including log P,
molecular weight, and polar surface area (PSA) for (R)- and (S)-PA-824.
The plasma protein binding (PPB) of the enantiomers of PA-824 and its
possibility that other nitroheterocyclic compounds may also have
potential in treating this disease. With this in mind, the antituber-
cular clinical candidate (S)-PA-824, currently in phase II develop-
ment by the TB Alliance, was synthesized and assessed for antil-
eishmanial activity. The potency of (S)-PA-824 was determined in
vitro against L. donovani (LdBOB) promastigotes and against in-
tracellular amastigotes in peritoneal mouse macrophages. (S)-PA-
des-nitro analogues was determined by the equilibrium dialysis method 824 showed antileishmanial activity against both developmental
(22). The aqueous solubility of the enantiomers of PA-824 was measured
using laser nephelometry (BMG Labtech nephelometer) following serial
dilution of DMSO stocks into deionized water to give a final concentra-
tion range of 12 to 250 ␮M, with a final DMSO concentration of 2.5%. The
amount of laser scatter caused by insoluble particulates (relative nephe-
lometry units) was plotted against compound concentration using a seg-
mental regression fit, with the point of inflection being quoted as the
aqueous solubility. To measure the intrinsic clearance (CLint), each enan-
tiomer of PA-824 (0.5 ␮M) was incubated with female CD1 mouse liver
microsomes (Xenotech LLC; 0.5 mg mlϪ1 in 50 mM potassium phosphate
buffer, pH 7.4) and the reaction was started by the addition of excess
NADPH (8 mg mlϪ1). At time zero and then at 3, 6, 9, 15, and 30 min, an
aliquot (50 ␮l) of the incubation mixture was mixed with acetonitrile (100
␮l) to stop the reaction. A structural analogue was then added to all sam-
ples as an internal standard, the samples were centrifuged to sediment
precipitated protein, and the plates were sealed prior to UPLC-MS/MS
analysis using a Quattro Premier XE spectrometer (Waters Corpora-
tion, USA). XLfit (IDBS, United Kingdom) was used to calculate the
exponential decay and consequently the rate constant (k) from the
ratio of peak area of test compound to internal standard at each time
point. CLint was then calculated using the following equation: CLint
(ml minϪ1 [g liver]Ϫ1) ϭ k ϫ V ϫ microsomal protein yield, where V
(ml mg proteinϪ1) is the incubation volume per mg protein added and
microsomal protein yield is taken as 52.5 mg protein per g liver. Vera-
pamil (0.5 ␮M) was used as a positive control to confirm acceptable assay
performance.
stages of the parasite, with EC50s of 0.9 Ϯ 0.1 and 4.9 Ϯ 0.3 ␮M
against promastigotes and amastigotes, respectively (Table 1).
However, (S)-PA-824 was largely inactive against the mammalian
stages of T. brucei and T. cruzi, with EC50s of Ͼ30 ␮M in both
cases. (S)-PA-824 was also found to be inactive (EC50, Ͼ50 ␮M) in
a counterscreen against the mammalian cell line HepG2.
The R enantiomer of PA-284 has been shown to have little or
no activity against Mycobacterium tuberculosis (MIC, Ͼ100 ␮M)
(26); however, as part of a limited hit expansion program, (R)-PA-
824 was synthesized and its antileishmanial activity was assessed.
In direct contrast to that seen in M. tuberculosis, (R)-PA-824
proved to be a ϳ5-fold-more potent inhibitor of L. donovani
growth in vitro than the S-enantiomer candidate, with EC50s of
0.16 Ϯ 0.03 and 0.9 Ϯ 0.1 ␮M against promastigotes and intracel-
lular amastigotes, respectively (Table 1). Indeed, (R)-PA-824 also
showed slightly improved activity against the mammalian stages
of T. brucei and T. cruzi while remaining inactive against the mam-
malian cell line HepG2 (EC50, Ͼ50 ␮M).
Pharmacological profiling of (S)- and (R)-PA-824 and (R)-
des-nitro-PA-824 in vitro. A number of studies have reported
that (S)-PA-824 is well tolerated in mice and can reduce M. tuber-
culosis infection levels after oral dosing (27, 28). To determine if
(R)-PA-824 was similarly suitable for in vivo studies, the plasma
protein binding (PPB), aqueous solubility, and CLint were mea-
sured and compared to those of the S enantiomer. Both enantiom-
ers had good aqueous solubility (Ͼ250 ␮M) and similar low
RESULTS
In vitro sensitivity of L. donovani and related parasites to (S)-
and (R)-PA-824. The promise shown by fexinidazole as an oral mouse PPB, with the fractions unbound for (S)- and (R)-PA-824
October 2013 Volume 57 Number 10
aac.asm.org 4701

Patterson et al.
A
B
40,000
30,000
20,000
10,000
0
400,000
300,000
200,000
100,000
0
EC90 (S)-PA-824
-
EC90 (R)-PA 824
)
)
0)
0
0
250
500
750 1000 1250 1500
Time, min
(15)
(12
m
4 (100)
ine
a
s
st
vehicle (0)
o
f
)-PA-824 (30
)-PA-824 (30)
)-PA82
)-PA-824 (1
S
R
ntrol Pento Milte
Co
S
(
(
(
R
(
Drug treatment (mg kg-1)
FIG 1 In vivo pharmacokinetic properties and efficacy of PA-824 in mice. (A) Blood concentrations of (R)- and (S)-PA-824 following oral dosing at 50 mg kgϪ1
.
The EC90 values of both enantiomers for L. donovani (strain LV9) cultured in ex vivo mouse macrophages and corrected for plasma protein binding are shown
as dashed lines. (S)-PA-824 data are shown in maroon, and (R)-PA-824 data are shown in teal. Data are the means and standard deviations from three mice. (B)
Effects of drug treatment on the parasite burden of mice infected with L. donovani. Mice (five per group) were dosed with drug vehicle (orally), sodium
stibogluconate (Pentostam) (subcutaneously), miltefosine (orally), or (S)-PA-824 or (R)-PA-824 (both orally) for 5 days. Miltefosine and sodium stibogluconate
were administered once daily, and PA-824 enantiomers were administered twice daily.
being 0.21 and 0.22, respectively. The fraction unbound for the following inoculation, parasite burdens in the livers of infected
(R)-des-nitro-PA-824 is 0.45. (R)-PA-824 shows higher mouse mice were determined. Current antileishmanial chemotherapies
microsomal stability than does the S enantiomer (CLint ϭ 0.65 sodium stibogluconate (15 mg kgϪ1) and miltefosine (12 mg
versus 1.6 ml minϪ1 gϪ1). The calculated values for the partition kgϪ1) were included in the study for comparison. Both enantiom-
coefficient, log P (2.7), and polar surface area (91 Å2) were iden- ers of PA-824 were extremely well tolerated by mice throughout
tical for the two enantiomers.
the study, with no overt signs of toxicity. Mice dosed twice daily at
In vivo pharmacokinetic properties of (S)- and (R)-PA-824. 30 mg kgϪ1 with (R)- or (S)-PA-824 demonstrated similar effica-
Blood levels of both enantiomers of PA-824 were determined after cies, with both enantiomers suppressing infection in the murine
oral dosing to establish a suitable dosing regimen for VL efficacy model by approximately 35% compared to untreated controls
studies. Mice dosed with (S)- or (R)-PA-824 at 50 mg kgϪ1 (Fig. 1B). Administering an increased dose of 100 mg kgϪ1did not
showed a maximum concentration in blood after 4 h of 11,600 or improve the efficacy of (S)-PA-824 in vivo, with L. donovani infec-
10,500 ng mlϪ1, respectively (Fig. 1A). Thereafter, blood concen- tion in this group of mice at levels comparable to those seen in
trations decreased [elimination half-life (t1/2), ϳ5 h and 2 h for drug-free animals. An earlier in vivo study, with mice given a range
(S)- or (R)-PA-824, respectively], reaching undetectable levels at a of doses down to 0.4 mg kgϪ1, demonstrated the apparent absence
time point between 8 and 24 h for (R)-PA-824. At 24 h, (S)-PA- of a dose-dependent leishmanicidal effect with the S enantiomer of
824 still had a blood concentration of 900 ng mlϪ1
.
PA-824 (see Fig. S1 in the supplemental material). Interestingly,
(R)-PA-824 has an EC50 of 0.93 ␮M for L. donovani cultured in these findings mirror those seen in clinical trials of (S)-PA-824 in
macrophages (Table 1) with a Hill slope of 1.7, giving an EC90 humans with tuberculosis (29). In stark contrast, treatment with
value of 3.4 ␮M (equivalent to 1,200 ng mlϪ1). After adjusting for (R)-PA-824 at 100 mg kgϪ1 effectively cured the murine model of
plasma protein binding [(R)-PA-824 fraction unbound ϭ 0.22], infection, suppressing infection by 99.9%. Dosing with (R)-PA-
the estimated blood concentration required to exceed the EC90 824 at this level proved to be superior to treatment with current
becomes 5,600 ng mlϪ1. Comparison with the measured levels front-line drugs sodium stibogluconate (41.9% suppression) and
(Fig. 1A) shows that (R)-PA-824 blood levels exceed the EC90 1 h miltefosine (68.7% suppression), underlining the therapeutic po-
after dosing and remain above the EC90 for at least 7 h. Thus, 100 tential of this nitroimidazo-oxazine compound.
mg kgϪ1 twice daily was predicted to provide adequate exposure
(R)-PA-824isnotactivatedby L. majortypeInitroreductase.
for (R)-PA-824. Although (S)- and (R)-PA-824 reach comparable (S)-PA-284 is believed to function as a prodrug which requires
blood levels, the lower potency of (S)-PA-824 (EC90 ϭ 22 ␮M) bioreductive activation prior to exhibiting antitubercular activity
results in a maximal free concentration below EC90 (37,000 ng (13, 30). In M. tuberculosis, this reduction is catalyzed by an un-
mlϪ1) after oral dosing at 50 mg kgϪ1 (Fig. 1A).
usual deazaflavin (F420)-dependent nitroreductase (Ddn). In the
In vivo sensitivity of L. donovani to the enantiomers of PA- absence of a Ddn homologue in the trypanosomatids, we hypoth-
824. The efficacy of both (R)- and (S)-PA-824 was assessed in a esized that reduction of (R)- and (S)-PA-824 in L. donovani may
mouse model of visceral leishmaniasis. Seven days following in- be catalyzed by the NADH-dependent nitroreductase, which has
fection with L. donovani LV9 ex vivo amastigotes, groups of homology to bacterial type I nitroreductases and has already been
BALB/c mice were dosed orally with (R)- or (S)-PA-824 (30 or 100 shown to play a crucial role in the activation of fexinidazole and its
mg kgϪ1) twice daily and for five consecutive days. Fourteen days metabolites (11). To determine whether reduction by this nitrore-
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PA-824 and Leishmania donovani
FIG 2 Susceptibility of NTR-overexpressing LdBOB promastigotes to nifurtimox, (R)-PA-824, and (S)-PA-824. EC50s were determined for nifurtimox (A),
(S)-PA-824 (B), and (R)-PA-824 (C) against WT (open circles) and NTR-overexpressing (closed circles) parasites. EC50s of 5.6 Ϯ 0.16 and 0.2 Ϯ 0.09 ␮M for
nifurtimox, 0.96 Ϯ 0.02 and 0.8 Ϯ 0.06 ␮M for (S)-PA-824, and 0.16 Ϯ 0.003 and 0.11 Ϯ 0.005 ␮M for (R)-PA-824 were determined against WT and
NTR-overexpressing cell lines, respectively. Data are the means of triplicate measurements.
ductase was central to the mechanism of action of PA-824, the a crucial role in the mechanism of action of PA-824 or mediates
potency of both enantiomers was determined against wild-type the binding between PA-824 and its molecular target(s) in L. don-
(WT) parasites and parasites overexpressing the nitroreductase. ovani.
Increased concentrations of nitroreductase in these transgenic
(R)-PA-824 combination therapy. Recent studies have illus-
parasites were confirmed by a 26-fold increase in their sensitivity trated the potential of drug combinations in the treatment of vis-
to nifurtimox (Fig. 2A), a nitrofuran drug known to undergo two- ceral leishmaniasis (32). Multidrug therapy is perceived to have
electron reduction by nitroreductase (31). However, overexpres- several advantages over conventional monotherapy, such as the
sion of nitroreductase in promastigotes did not significantly alter potential for synergistic activity resulting in increased drug effi-
their sensitivity to either (S)-PA-824 (EC50 of 0.96 Ϯ 0.02 and 0.8 cacy and the reduced probability of drug resistance emerging.
Ϯ 0.04 ␮M for wild-type and transgenic parasites, respectively) or With this in mind, we assessed the in vitro interactions of (R)-PA-
(R)-PA-824 (EC50 of 0.16 Ϯ 0.002 and 0.11 Ϯ 0.005 ␮M for wild- 824 in combination with fexinidazole sulfone and (S)-PA-824
type and transgenic parasites, respectively) (Fig. 2B and C). These against L. donovani promastigotes using the fixed-ratio isobolo-
findings suggest that this nitroreductase does not play a crucial gram method (Fig. 3). Isobologram analysis of the potency of
role in the activation of PA-824 in L. donovani.
various (R)-PA-824 and fexinidazole sulfone combinations
In vitro sensitivity of L. donovani to des-nitro-PA-824. To against promastigotes revealed a near-linear relationship, suggest-
determine whether the nitro group was important for the antil- ing that these two compounds act additively in combination (Fig.
eishmanial activity of PA-824, the potencies of (R)- and (S)-des- 3A). However, combinations of (R)- and (S)-PA-824 demon-
nitro-PA-824 were determined against L. donovani promastigotes strated a more complex relationship. As the proportion of (R)-
and intracellular amastigotes. In both assays, both enantiomers of PA-824 in the mixture increases, the two enantiomers become
des-nitro-PA-824 were found to be inactive at the highest concen- progressively more antagonistic. A second experiment gave an
tration tested (50 ␮M), suggesting that the nitro group either plays identical pattern.
FIG 3 Relative EC50s for combinations of (R)-PA-824 with fexinidazole sulfone or (S)-PA-824. Isobolograms show the relative EC50s obtained with combina-
tions of (R)-PA-824 with fexinidazole sulfone (A) or (S)-PA-824 (B) against promastigotes of the LdBOB strain of L. donovani. (A) Absolute EC50s: (R)-PA-824,
0.18 ␮M; fexinidazole sulfone, 8.8 ␮M. (B) Absolute EC50s: (R)-PA-824, 0.10 ␮M; (S)-PA-824, 0.74 ␮M. Data are the weighted means Ϯ standard errors of
duplicate cultures.
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Patterson et al.
FIG 4 Cytocidal effects of PA-824 enantiomers on L. donovani promastigotes. The S (A) and the R (B) enantiomers of PA-824 were added to early-log-phase
cultures of LdBOB promastigotes (ϳ1 ϫ 106 mlϪ1) at concentrations equivalent to 10 times their respective EC50s. At intervals, the cell densities were
determined, and samples of culture (500 ␮l) were removed, washed, and resuspended in fresh culture medium in the absence of drug. The viability of washed
parasites was monitored for up to 72 h following removal from drug exposure, and the point of irreversible drug toxicity was determined (skull and crossbones).
Open circles, no inhibitor; filled circles, presence of drug.
PA-824-mediated cell killing. To determine whether PA-824
In the current study, we have shown that (S)-PA-824 is active
was cytostatic or cytotoxic, we incubated mid-log-phase promas- against L. donovani intracellular amastigotes at a potency compa-
tigotes with either (S)- or (R)-PA-824 at concentrations equiva-
lent to 10 times their respective EC50s (Fig. 4). In both cases,
growth of drug-treated cultures ceased almost immediately, with
cell numbers beginning to decline after 12 h. In cultures treated
with (S)-PA-824, no intact parasites were visible at 36 h (Fig. 4A).
However, cell death was more rapid in parasites treated with the R
enantiomer, with no live parasites remaining at 24 h (Fig. 4B). To
determine the actual point where treated cells completely lost vi-
ability, we washed and subcultured parasites at defined intervals
without drug. No parasites could be recovered at 30 h following
treatment with (S)-PA-824. In comparison, the more rapid cyto-
toxic effect of (R)-PA-824 resulted in parasites losing viability by
24 h. The fact that both enantiomers of PA-824 are leishmanicidal
rather than cytostatic is highly beneficial, since successful drug
therapy with either compound is unlikely to depend on a fully
functional patient immune response (33).
rable to that of fexinidazole sulfone (EC50, 4.9 ␮M versus 5.3 ␮M),
an active metabolite of the VL clinical candidate fexinidazole. In
contrast to the structure-activity relationship for M. tuberculosis,
where the S enantiomer of PA-824 is Ͼ100-fold more active than
the R enantiomer (26), we found that (R)-PA-824 was 5-fold more
potent than the S enantiomer in the L. donovani intramacrophage
assay. Similarly, (R)-PA-824 was also more active against the dis-
ease-relevant stages of T. brucei and T. cruzi but showed no mea-
surable activity in a mammalian cell counterscreen. Both enan-
tiomers of PA-824 were also found to be leishmanicidal in vitro
with good selectivity, good microsomal stability, good aqueous
solubility, and low plasma protein binding.
Both enantiomers were orally bioavailable and were well toler-
ated in mice. The pharmacokinetic profiles of the two compounds
were similar, but only the R enantiomer was predicted to show
complete coverage above the EC90 for the entire duration of treat-
ment (100 mg kgϪ1, orally, twice a day for 5 days). Indeed, under
this treatment regimen (R)-PA-824 almost completely cleared L.
donovani parasites from the livers of infected mice. When either
(R)- or (S)-PA-824 was administered at 30 mg kgϪ1, there was
only a moderate reduction in the observed parasite burden
(ϳ35%). Although we have not established linearity of exposure
with dose for either compound, this modest efficacy is consistent
with the anticipated sub-EC90 free inhibitor blood concentration.
Surprisingly, (S)-PA-824 becomes less efficacious when adminis-
tered at 100 mg kgϪ1. We hypothesize that this unexpected reduc-
tion in potency may be formulation related.
DISCUSSION
The repurposing of existing drugs and clinical candidates for the
treatment of neglected diseases of poverty offers a cost-efficient
approach toward identifying lead compounds for these unprofit-
able indications. This strategy has proven successful in the pursuit
of treatments for VL, with the current oral therapy miltefosine and
the clinical candidate fexinidazole both originally developed for
other indications. The potential for the nitroimidazole fexinida-
zole to treat HAT (34), VL (11), and Chagas’ disease (35) is also an
example of the current resurgence of interest in the potential of
nitro compounds as therapeutics. Encouraged by our findings
with fexinidazole (11), now entering phase II clinical trials against
VL in Africa, we decided to investigate the potential of other ni-
troaromatics against this disease. A search of the literature re-
vealed that the antitubercular nitroimidazole (S)-PA-824 had
been reported to possess an undefined level of activity against L.
donovani axenic amastigotes (36). Both M. tuberculosis and L. don-
ovani reside in macrophages in vivo, and so the investigation of
The target product profile (http://www.dndi.org/) for VL calls
for an oral drug that can be used to treat immunosuppressed pa-
tients. Significantly, (R)-PA-824 is cytocidal rather than cyto-
static, which is advantageous because treatment with this com-
pound would not be dependent on the patient having a fully
functional immune response. In addition, (R)-PA-824 is orally
available. Collectively, our study indicates that (R)-PA-824 repre-
sents a strong lead compound with potential for development as a
antitubercular agents was deemed a particularly appropriate start- much-needed oral treatment for VL. However, it is important to
ing point in the search for potential VL treatments.
note that the S, not the R, enantiomer of PA-824 has advanced into
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PA-824 and Leishmania donovani
clinical trials for the treatment of TB. With this in mind, (R)-PA- ACKNOWLEDGMENTS
824 should be considered a worthy candidate for lead optimiza-
tion and not a classical drug-repurposing project.
We thank Gina MacKay for performing high-resolution mass spectrom-
etry analyses and for assistance with performing nuclear magnetic reso-
nance and other MS analyses, Alastair Pate for data management, Irene
Hallyburton and John Thomas for cell screening assays, Raffaella
Grimaldi for developing the HepG2 assay, and Beatriz Baragana for proof-
reading the manuscript.
A.H.F. is a Wellcome Principal Research Fellow, funded by grants
from the Wellcome Trust (079838, 077705, 092340, and 083481). M.R.P.
is the recipient of a clinical Ph.D. award from the Wellcome Trust
(090665).
Overexpression of the L. major nitroreductase did not alter the
potency of (R)- or (S)-PA-824 against L. donovani promastigotes.
This demonstrates that (R)- and (S)-PA-824 are not activated by
the same type I nitroreductase responsible for the bioactivation of
the antileishmanial nitroimidazole fexinidazole. This is consistent
with the observation that lab-generated strains of nifurtimox-re-
sistant T. brucei (nifurtimox is activated by a type I NTR) were
cross resistant to fexinidazole but showed no change in sensitivity
to (S)-PA-824 (20). However, both (R)- and (S)-des-nitro-PA-
824 were found to be inactive against L. donovani, suggesting that
the nitro group does play a key role in the antileishmanial activity
of this compound series. This result allows for the possibility that
(R)-PA-824 relies on nitro group reduction to exert its antileish-
manial activity. L. donovani does not possess a homologue of the
M. tuberculosis deazaflavin nitroreductase (Ddn), which specifi-
cally reduces (S)-PA-824, but not the R enantiomer (26), and
since Leishmania type I NTR cannot activate (R)-PA-824, this
putative bioreduction would need to be mediated by an as-yet-
unidentified nitroreductase. Alternatively, it is entirely possible
that (R)-PA-824 exerts its activity by inhibiting the function of an
essential protein. If that were the case, then the nitro substituent
could mediate an important binding interaction with the target
protein. It is known that nitro groups can play a role in small-
molecule protein binding by forming hydrogen bonds, by polar-
izing the ␲ system of an aromatic ring, or by reducing the basicity
of an imidazole ring (37). We are currently developing LC-
MS/MS methods in order to monitor the metabolism of PA-824 in
vitro. In preliminary experiments, we have established that PA-
824 disappears over time in supernatants of L. donovani cultures,
suggesting that drug metabolism may be occurring. Further inves-
tigations aimed at determining the mode of action of (R)-PA-824
in Leishmania are under way.
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