TPT sulfonate, a single, oral dose schistosomicidal prodrug: In vivo efficacy, disposition and metabolic profiling

Article abstract of DOI:10.1016/j.ijpddr.2018.10.004

Treatment of schistosomiasis relies precariously on just one drug, praziquantel (PZQ). In the search for alternatives, 15 S-[2-(alkylamino)alkane] thiosulfuric acids were obtained from a previous research program and profiled in mice for efficacy against both mature (>42-day-old) and juvenile (21-day-old) Schistosoma mansoni using a screening dose of 100 mg/kg PO QDx4. One compound, S-[2-(tert-butylamino)-1-phenylethane] thiosulfuric acid (TPT sulfonate), was the most effective by decreasing female and male worm burdens by ≥ 90% and ≥46% (mature), and ≥89% and ≥79% (juvenile), respectively. In contrast, PZQ decreased mature female and male worm burdens by 95% and 94%, respectively, but was ineffective against juvenile stages. Against 7-day-old lung-stage worms, TPT sulfonate was only effective at twice the dose decreasing female and male burdens by 95 and 80%, respectively. Single oral doses at 400 and/or 600 mg/kg across various developmental time-points (1-, 7-, 15-, 21- and/or 42 day-old) were consistent with the QD x4 data; efficacy was strongest once the parasites had completed lung migration, and female and male burdens were decreased by at least 90% and 80%, respectively. In vitro, TPT sulfonate is inactive against the parasite suggesting a pro-drug mechanism of action. In mice, TPT sulfonate is fully absorbed and subject to rapid, non-CYP-mediated, first-pass metabolism that is initiated by desulfation and yields a series of metabolites. The initially-formed free thiol-containing metabolite, termed TP thiol, was chemically synthesized; it dose-dependently decreased S. mansoni and Schistosoma haematobium motility in vitro. Also, when administered as a single 50 mg/kg IP dose, TP thiol decreased 33-day-old S. mansoni female and male burdens by 35% and 44%, with less severe organomegaly. Overall, TPT sulfonate's efficacy profile is competitive with that of PZQ. Also, the characterization of a parasiticidal metabolite facilitates an understanding and improvement of the chemistry, and identification of the mechanism of action and/or target.

Full text of DOI:10.1016/j.ijpddr.2018.10.004

IJP:DrugsandDrugResistance8(2018)571–586
Contents lists available at ScienceDirect
IJP: Drugs and Drug Resistance
journal homepage: www.elsevier.com/locate/ijpddr
TPT sulfonate, a single, oral dose schistosomicidal prodrug: In vivo efficacy,
disposition and metabolic profiling
Alan R. Wolfea,b, R. Jeffrey Neitzb,c, Mark Burlingamec, Brian M. Suzukib,d, KC Limb,d
Mark Scheidelere, David L. Nelsonf, Leslie Z. Beneta,b, Conor R. Caffreyb,d,∗
,
a Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 533 Parnassus Ave, San Francisco, CA, 94143, USA
b Center for Discovery and Innovation in Parasitic Diseases, University of California, 1700 4th St, San Francisco, CA, 94158, USA
c Department of Pharmaceutical Chemistry, University of California, 1700 4th St, San Francisco, CA, 94158, USA
d Department of Pathology, University of California, 1700 4th St, San Francisco, CA, 94158, USA
e Human First Therapeutics LLC, 9600 Dewitt Drive, Ste 1, Silver Spring, MD, 20910, USA
f Pro-Rectory of Research and Graduate Study, Federal University of the Valleys of Jequitinhonha and Mucuri, 39100-000, Diamantina, MG, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Treatment of schistosomiasis relies precariously on just one drug, praziquantel (PZQ). In the search for alter-
natives, 15 S-[2-(alkylamino)alkane] thiosulfuric acids were obtained from a previous research program and
profiled in mice for efficacy against both mature (> 42-day-old) and juvenile (21-day-old) Schistosoma mansoni
using a screening dose of 100 mg/kg PO QDx4. One compound, S-[2-(tert-butylamino)-1-phenylethane] thio-
sulfuric acid (TPT sulfonate), was the most effective by decreasing female and male worm burdens by ≥ 90%
and ≥46% (mature), and ≥89% and ≥79% (juvenile), respectively. In contrast, PZQ decreased mature female
and male worm burdens by 95% and 94%, respectively, but was ineffective against juvenile stages. Against 7-
day-old lung-stage worms, TPT sulfonate was only effective at twice the dose decreasing female and male
burdens by 95 and 80%, respectively. Single oral doses at 400 and/or 600 mg/kg across various developmental
time-points (1-, 7-, 15-, 21- and/or 42 day-old) were consistent with the QD x4 data; efficacy was strongest once
the parasites had completed lung migration, and female and male burdens were decreased by at least 90% and
80%, respectively. In vitro, TPT sulfonate is inactive against the parasite suggesting a pro-drug mechanism of
action. In mice, TPT sulfonate is fully absorbed and subject to rapid, non-CYP-mediated, first-pass metabolism
that is initiated by desulfation and yields a series of metabolites. The initially-formed free thiol-containing
metabolite, termed TP thiol, was chemically synthesized; it dose-dependently decreased S. mansoni and
Schistosoma haematobium motility in vitro. Also, when administered as a single 50 mg/kg IP dose, TP thiol de-
creased 33-day-old S. mansoni female and male burdens by 35% and 44%, with less severe organomegaly.
Overall, TPT sulfonate's efficacy profile is competitive with that of PZQ. Also, the characterization of a para-
siticidal metabolite facilitates an understanding and improvement of the chemistry, and identification of the
mechanism of action and/or target.
Schistosomiasis
Schistosoma
Anthelmintic
Alkylaminoalkanethiosulfuric acid
Drug metabolism
Drug disposition
1. Introduction
schistosomiasis is a direct contributor to poverty (Hotez et al., 2008;
Utzinger et al., 2011).
Schistosomiasis is a flatworm disease endemic in areas where the
infected snail vectors infest freshwater systems. With over 190 million
people infected (Hotez, 2018) and as many as 700 million at risk (King,
2010), infections can last a lifetime and elicit progressive tissue and
organ damage that results in pain and malaise. The disease detracts
from school attendance and limits the capacity to perform manual
labor, which remains critical in subsistence societies. Thus,
Treatment and control of schistosomiasis relies on one drug, prazi-
quantel (PZQ). Safe and reasonably effective against all the schistosome
species infecting humans, PZQ is the lynchpin for the World Health
Organization's (WHO) strategy of morbidity control via ‘preventative
chemotherapy.’ Promulgation of this strategy has received a recent
boost in interest by national and trans-national agencies that have re-
affirmed a long-lasting and, indeed, expanding commitment to make
∗ Corresponding author. Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of
California San Diego, 9500 Gilman Ave, La Jolla, CA, 92093, USA.
E-mail address: ccaffrey@ucsd.edu (C.R. Caffrey).
https://doi.org/10.1016/j.ijpddr.2018.10.004
Received 26 July 2018; Received in revised form 19 October 2018; Accepted 22 October 2018
Availableonline20November2018
2211-3207/©2018TheAuthors.PublishedbyElsevierLtdonbehalfofAustralianSocietyforParasitology.ThisisanopenaccessarticleundertheCC
BY-NC-NDlicense(http://creativecommons.org/licenses/BY-NC-ND/4.0/).

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
more of PZQ available to more people more often (http://uni-
tingtocombatntds.org/wp-content/themes/tetloose/app/staticPages/
fifthReport/files/fifth_progress_report_english.pdf; Caffrey, 2015). Re-
sistance has yet to be clinically recorded; however, to borrow an in-
vesting axiom, “past performance is no guarantee of future results,” so
we should remain vigilant. Apart for this ever-present concern, PZQ has
a number of often overlooked pharmaceutical and pharmacological
limitations (Caffrey, 2007, 2015). Among these is its inability to ef-
fectively clear immature parasites (Gőnnert and Andrews, 1977; Sabah
et al., 1986; Xiao et al., 1987; Botros et al., 2005; Keiser et al., 2009)
which can grow to maturity after treatment and lay the eggs that are
responsible for pathology and morbidity (Colley et al., 2014). An ideal
new drug would clear the parasite irrespective of developmental stage.
Among the potential alternatives to PZQ that have been reported is
a class of compounds known as alkylaminoalkanethiosulfuric acids
(IUPAC: S-(2-(alkylamino)-1-alkyl) O-hydrogen sulfurothioate; herein
termed ‘thiosulfates’). These originated in the 1960s in the Walter Reed
Army Institute of Research (WRAIR) as putative treatments for radia-
tion sickness (Klayman and Gilmore, 1964; Klayman et al., 1969a, b).
They were subsequently sent to the group of the late José Pellegrino
(Schistosomiasis Research Unit at the Instituto de Ciências Biológicas in
the Universidade Federal de Minas Gerais, Brazil) for in vivo phenotypic
screening (in mice) against mature S. mansoni infections. Of 70 samples
tested, 10 were found to be active (Nelson and Pellegrino, 1976). Based
on these tests, it appeared that derivatives bearing small, branched,
apolar groups bound to the amino nitrogen and without polar groups in
the principal chain of the molecule were more likely to exhibit activity.
Nelson and colleagues subsequently synthesized additional aminoalk-
anethiosulfates with longer carbon chains or phenyl rings in the
principal portion of the molecule (Penido et al., 1994; Moreira et al.,
2000; Moreira et al., 2007) Against mature infections of S. mansoni in
mice, single oral doses of 800 mg/kg decreased worm burdens by
33–61% and 64–100% for male and female worms, respectively (Penido
et al., 1994, 1995, 1999; Moreira et al., 2007).
Since the last communication in 2008 (de Oliveira Penido et al.,
2008), there have been no further reports regarding the anti-schisto-
somal activities of thiosulfates. Prior to closure of the Nelson laboratory
in 2011, the UCSF group obtained the last of 15 thiosulfates (Table 1)
from Dr. Nelson to independently assess their bioactivities against S.
mansoni, including against immature developmental stages which are
less susceptible to PZQ (references above). From these experiments, we
identify one clear lead, S-[2-(tert-butylamino)-1-phenylethane] thio-
sulfuric acid (TPT sulfonate), which comes close to PZQ in being able to
clear adult parasites in vivo and possesses a broader efficacy spectrum
by killing those developmental stages that are less susceptible to PZQ.
In vitro metabolic profiling of TPT sulfonate and its disposition in mice
identify a putative metabolic pathway among which a key, and ap-
parently, non-toxic metabolite responsible for the anti-schistosomal
activity was identified. The identification of this thiosulfate lead and its
cidal principle offers an opportunity for the re-invigorated pursuit of a
compound and/or chemical series that may yield a drug that either
complements or provides an alternative to PZQ.
2. Materials and methods
2.1. Ethics statement
Maintenance and handling of small vertebrate animals were carried
Table 1
Overview of efficacy of alkyl- and aryl-thiosulfates in mice infected with S. mansoni as a function of time of administration (days post-infection).2
Number
R1
R2
R3
Percentage reduction in worm burdens (vs. vehicle controls)
7 dpi
Female
n.t.a
11 dpi
Female
46 totalb
83
21 dpi
Female
62
42 dpi
Male
12
Male
Male
Female
Male
1
2
Ph
Ph
H
H
iPr
60
69
60
37
45
63
46
−8
20
tBu
40
81
89
91
19
5
89
90
95
14
79
3
H
H
H
H
Ph
H
H
H
H
H
H
Ph
H
nBu
nBu
iBu
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
58
−14 total
−12 total
n.t.
32
4
nBu
8
19
5
nHex
nHex
H
nBu
iBu
17
7
31
n.t.
n.t.
22
6
16 total
n.t.
−10
−6
29
7
cHex
iPr
−57
35
48
34
42
0
−6
40
8
nHex
nBu (S)
nHex (S)
nBu (S)
nBu (S)
nHex (R)
H
n.t.
50
−27
5
9
iPr
n.t.
35
−16
−13
21
10
11
12
13
14
15
PZQ
nPr
n.t.
13
nPr
n.t.
52
5
cHex
cHex
sBu (S)
tBu
n.t.
−11
−83
61
−39
33
5
n.t.
−80
72
1
−5
11
n.t.
36
H
n.t.
−4
−9
−9
95
−2
94
44
n.t.
9
iPr, isopropyl; tbu, tert-butyl; nBu, n-butyl; sBu, sec-butyl; cHex, cyclo-hexyl; nHex, n-hexyl. Infections commenced with a sub-cutaneous injection of 150 cercariae in
water (100–150 μL/mouse). Compounds were suspended in 2.5% Kolliphor EL (100–150 μL/mouse; n = 3–6 per group) and administered orally by gavage at
100 mg/kg QDx4 beginning at the days post-infection (dpi) indicated. Worm recovery by perfusion was performed on the days indicated in Table S1. Numbers shown
are the mean male and female worm burden reductions (or increases if the integer is negative). For compounds 1 and 2, more than one experiment was performed at
21 and 42 dpi. Significance relative to vehicle controls was measured using the Student's unpaired t-test with a two-tailed distribution; significance (p < 0.05) is
indicated in bold typeface. See Table S1 for details of each experiment.
a
b
n.t. = not tested.
When it was not possible to accurately sex worms, the total worm burden reductions (or increases) are stated.
572

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
out in accordance with a protocol (AN107779-01) approved by the
Institutional Animal Care and Use Committee (IACUC) of the University
of California San Francisco. UCSF-IACUC derives its authority for these
activities from the United States Public Health Service (PHS) Policy on
Humane Care and Use of Laboratory Animals and the Animal Welfare
Act and Regulations (AWAR).
(Pellegrino and Siqueira, 1956; Duvall and DeWitt, 1967; Colley and
Wikel, 1974; Abdulla et al., 2007) in RPMI 1640 medium containing
50U/l heparin.
Compound efficacy was measured using the primary criterion of
reduction in worm (male and female) burden. The current anti-schis-
tosomal drug, PZQ, was used as a drug control, as described (Abdulla
et al., 2007, 2009). Also, any amelioration of pathology, as evidenced
by decreased liver and spleen weights vs. vehicle controls (Abdulla
et al., 2007), was recorded in Table S1. This Excel workbook also
contains observation notes on worm size and condition upon recovery.
2.2. Synthesis of alkylaminoalkanethiosulfates
These compounds were prepared as previously described (Nelson
et al., 1989; Moreira et al., 2000) by the opening of an epoxide with
excess amine to form the amino alcohol. The amino group was pro-
tected by conversion to its hydrobromide salt, followed by the sub-
stitution of the hydroxyl group by bromine using phosphorus tri-
bromide. Finally, the bromine atom was substituted through the
reaction with sodium thiosulfate to furnish the final product. When the
epoxide was not commercially available, it was prepared from the
corresponding alkene via an epoxidation method described previously
(Venturello et al., 1983). The evaluation of the purity and structure of
the synthesized TPT sulfonate by HPLC, MS and MS/MS is described in
the Supplementary Material.
2.6. Cytotoxicity counter screens
Bovine embryo skeletal muscle (BESM; (Engel et al., 1985), mouse
myoblast (C2C12 (Blau et al., 1985); and human hepatoma HuH-7 cells
(Nakabayashi et al., 1984) were cultured in RPMI 1640 medium sup-
plemented with 10% heat-inactivated fetal calf serum (FCS) and 1%
penicillin/streptomycin at 37 °C in 5% CO2. Cells were seeded into 24-
well plates and grown to 50–60% confluence. Freshly prepared com-
pound in DMSO was added to the cells to yield final concentrations of 5,
10, 20 and 40 μM. Assays were set up in triplicate with DMSO controls.
After 24 h, cells were fixed o/n with an equal volume of PBS containing
10% paraformaldehyde. Cells were then washed in PBS containing
0.1% sodium azide and 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI).
Nuclei were counted across 5–10 fields of view/well using a Zeiss Ax-
iovert 40 C inverted microscope fitted with a 20x objective lens and the
Zeiss filter set 02 (365/420 nm). Total nuclei counts per concentration
were expressed as a percentage relative to DMSO controls.
2.3. S. mansoni life cycle
The acquisition, preparation and in vitro maintenance of S. mansoni
schistosomula (somules or post-infective larvae) and adults have been
described (Abdulla et al., 2009; Štefanić et al., 2010). We employed a
Puerto Rican isolate of S. mansoni that is cycled between Biomphalaria
glabrata snails and male Golden Syrian hamsters (Simonsen Labora-
tories; infected at 4–6 weeks of age) as intermediate and definitive
hosts, respectively.
2.7. Positive ion-mode electrospray tandem MS of TPT sulfonate and its
metabolites in mouse plasma and mouse hepatocyte digests
2.4. Phenotypic screening with S. mansoni somules and adults in vitro
The principal fragments of m/z 290 TPT sulfonate observed using an
API 4000 LC-MS/MS system (SCIEX, Framingham, MA) (see description
under Results) are m/z 234, 135, 120 and 91. Thus, searches for me-
tabolites could be carried out with, for example, m/z 56 neutral loss
scanning (NLS) or m/z 91 precursor ion scanning (PIS). Metabolite
discovery was carried out primarily using m/z 56 NLS, from m/z 100 to
650. Multiple reaction monitoring for quantitation of TPT sulfonate and
its m/z 256 and 176 metabolites used the parent→ daughter ion tran-
sitions m/z 290.1 → 234.1, 256.1 → 200.1 and 176.1 → 120.1, respec-
tively. MS settings were declustering potential (DP), 60 V; collision
energy (CE), 19 eV; collision cell exit potential (CXP), 31 V; entrance
potential (EP), 10 V; collision gas (collisionally activated dissociation,
CAD), 12 psi; curtain gas (CUR), 16 psi; gas 1 (ion source nebulizer gas,
GS1), 30 psi; gas 2 (ion source heater gas, GS2), 40 psi; ion spray
voltage (IS), 5500 V and temperature (TEM), 500 °C.
Phenotypic screens involving in vitro-transformed somules were
performed in 200 μL Basch medium (Basch, 1981) in 96-well plates
(Corning Inc., cat. # 3599), as described (Abdulla et al., 2009; Rojo-
Arreola et al., 2014; Long et al., 2016). Phenotypic screens involving
42-day-old, mixed-sex parasites were performed in 24-well plates
(Corning Inc., cat. # 3544) containing 2 ml Basch medium and 4% FBS
using five worm pairs per well (Abdulla et al., 2009; Long et al., 2016,
2017). Compounds were added in a volume of up to 1 μL DMSO. In-
cubations were maintained for 3 day at 37 °C under 5% CO2. Parasite
responses to chemical insult were observed at various time-points using
a Zeiss Axiovert 40 C inverted microscope and recorded using a con-
strained nomenclature of phenotype ‘descriptors’ (e.g., rounding, de-
generation, overactivity and changes in motility) as described (Abdulla
et al., 2009; Glaser et al., 2015; Long et al., 2016, 2017). WormAssay
(Marcellino et al., 2012), as modified to measure adult schistosome
motility (Long et al., 2017; Weeks et al., 2018), was also employed.
The column employed was a 25 × 0.46 cm Beckman Ultrasphere
5 μm 100 Å C8. The mobile phases were A = 60% and B = 100% me-
thanol/water, containing 0.1% acetonitrile, 0.1% formic acid and
0.16 g/L NH4OAc. The gradient was 0–2 min, 0% B; 2–6 min, linear
ramp to 80% B; 6–7 min, 80% B; 7–8 min, linear ramp to 0% B;
8–12 min, 0% B; all at a flow rate of 0.6 ml/min. Retention times were
5.6, 5.8, 6.2 and 6.4 min for TPT sulfonate and the metabolites at m/z
256, m/z 176 and m/z 210, respectively.
2.5. Murine model of S. mansoni infection
Infections with S. mansoni were initiated by the subcutaneous in-
jection of 150 cercariae in water into 3-5-week-old female Swiss
Webster mice. At various times post-infection, compound was ad-
ministered orally (PO) by gavage once daily for up to four successive
days (QDx4) using 2.5% Kolliphor (Cremophor) EL as the vehicle
(n = 3–6 mice). We considered this treatment regimen as sufficient to
record any compound efficacy given that the desired target product
profile for new-anti-schistosomal drugs calls for short course, pre-
ferably, single-dose, therapy (Nwaka and Hudson, 2006; Caffrey, 2007).
At various time points post-treatment (see experimental details and
results outlined in Table S1), mice were euthanized with an in-
traperitoneal injection of 50 mg/kg sodium pentobarbital and adult
worms were harvested by reverse perfusion of the hepatic portal system
2.8. High mass accuracy MS of TPT sulfonate and the dimer of the thiol
metabolite
To aid in molecular characterization, high mass accuracy mass
spectra (sufficiently accurate to establish atomic composition) were
obtained for samples of TPT sulfonate, the m/z 417 dimer of the syn-
thetic m/z 210 thiol and their daughter ions in positive ion MS/MS
mode. Decomposition products of the m/z 417 dimer were also eval-
uated in MS mode. These spectra were obtained from a linear ion trap-
Orbitrap instrument (LTQ Orbitrap, Thermo Fisher Scientific) with the
573

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
samples being introduced by infusion.
female Swiss Webster mice. TPT sulfonate was formulated in 2.5%
Kolliphor EL and administered orally (PO) by gavage (150 μL) at
100 mg/kg. Blood samples were collected in tubes containing K2EDTA
via retro-orbital bleeding at 0.5, 2.0 and 4.0 h after dosing (n = 3 per
time point).
2.9. Stability of TPT sulfonate in mouse plasma
For comparison with pharmacokinetic results, the stability of
400 μM TPT sulfonate in mouse plasma containing 1% DMSO was
measured at 37 °C using plasma from female Balb/C mice (Biological
Specialty Corp., Colmar PA). Small aliquots of the sample were injected
directly into an Agilent 1100 HPLC using UV detection.
2.13. Extended PK study in mice
To more fully characterize the formation and stability of TPT sul-
fonate and its m/z 256, 210 and 176 metabolites, a larger PK study was
conducted using the contract research organization (CRO), WuXi
AppTec Co. Ltd. (Shanghai). Female Swiss Webster (CFW) mice (∼20 g;
Charles River) that had been fasted overnight were dosed intravenously
(IV) via the tail vein (4 mice, 15 mg/kg) or PO (8 mice, 100 mg/kg).
TPT sulfonate was formulated as a solution in 10% DMSO, 60% PEG-
400, 30% water (IV) or as a suspension in 2% Tween 80 in 0.5% hy-
droxypropyl methylcellulose (PO). For the IV route of administration,
25 μL blood samples were withdrawn after 5, 15, and 30 min, and 1, 2,
4, 8 and 24 h. For the PO route, samples were similarly withdrawn but
omitting the 5 min time point. Blood was collected from the sub-
mandibular or saphenous vein, or via cardiac puncture, and treated
with K2EDTA anticoagulant. Samples were then cooled to 4 °C and
centrifuged for 10 min at 4500×g. The resulting blood plasma was
frozen on dry ice and stored at −80 °C.
2.10. Digestion of TPT sulfonate by mouse microsomes
The susceptibility of TPT sulfonate to digestion by liver microsomal
cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) en-
zymes was evaluated at 37 °C using female CD1 mouse liver (pool of
400) microsomes (Xenotech, Lenexa, KS) in 50 mM potassium phos-
phate, pH 7.4, and 4 μM TPT sulfonate (adding 0.1% DMSO).
For CYP digestion tests, the above assay solution also contained
1.0 mM NADPH as a cofactor (except in the control). All components
except the NADPH were pre-incubated for 5 min before initiating the
reaction. The cysteine protease inhibitor, K11777 (N-Methyl-Pip-F-hF-
VSΦ; (Jacobsen et al., 2000), served as a positive control.
For glucuronidation tests, the above assay solution also contained
1 mM MgCl2, 0.15 mg/ml Brij 58 (a micelle-forming detergent), 5 mM
saccharolactone (β-glucuronidase inhibitor) and 5 mM uridine dipho-
sphate glucuronic acid trisodium salt (UDPGA). All components except
the substrate and UDPGA were pre-incubated for 15 min. Substrate was
then added and after 5 min the reaction was initiated. 1-Napthol served
as a positive control.
Thawed samples were prepared for analysis by combining 8 μL of
plasma with 80 μL of 1:1 methanol:acetonitrile containing 0.1% formic
acid and 200 ng/ml tolbutamide. The mixture was vortexed for 1 min
and centrifuged at room temperature for 15 min at 15,900×g. An ali-
quot (80 μL) of the supernatant was combined with 160 μL of water
containing 0.1% formic acid, vortexed for 10 min and then centrifuged
for 10 min at 3200×g and 4 °C. TPT sulfonate and its m/z 176, 210 and
256 metabolites were then analyzed by LC/MS/MS via their m/z 56
In both assays, 100 μL aliquots of the reaction mixture were re-
moved at various time points, mixed with 100 μL of cold methanol and
placed on dry ice. Samples were then centrifuged for 5 min at 12,000×g
and the supernatants were analyzed with an Agilent 1100 HPLC as
described above.
®
neutral-loss-daughter ions using an ACQUITY UPLC (Waters, Milford
MA) with a 1.7 μm, 2.1 × 50 mm Waters BEH C18 column and an API
4000 mass spectrometer in positive ion electrospray mode. The mobile
phase was a combination of mixture A, consisting of 0.1% formic acid in
19:1 water:acetonitrile, and mixture B, comprising 0.1% formic acid in
1:19 water:acetonitrile. The 1.8-min gradient went from 1% B to 45%
B, with a curve setting of 6, at a flow rate of 0.6 ml/min. Retention
times were 1.15, 0.82, 0.79 and 1.10 min for TPT sulfonate, m/z 256
metabolite, m/z 176 metabolite and m/z 210 metabolite, respectively.
The internal standard used was tolbutamide, using the 271.1 →
155.0 transition. MS settings were CXP, 12 V; EP, 10 V; CAD, 10 psi;
CUR, 20 psi; GS1, 50 psi; GS2, 50 psi; IS, 5500 V; TEM, 500 C. DP and
CE were, respectively, 74 V and 19 eV for m/z 56 neutral loss transi-
tions, and 63 V and 26 eV for the 271.1 → 155.0 tolbutamide transition.
2.11. Digestion of TPT sulfonate by rodent hepatocytes and anti-
schistosomal activity
Rodent hepatocytes were used to further assess the metabolic sta-
bility of TPT sulfonate. Hepatocytes were isolated from a 5-week-old
female Swiss Webster mouse or a 12-week old female Sprague-Dawley
rat and purified by Percoll gradient (Kraemer et al., 1986). Mouse and
rat hepatocytes (3.0 × 106 cells/ml in the experiments used for efficacy
testing) were incubated in Williams' medium E, pH 7.4, containing
4 mM glutamine and 750 μM TPT sulfonate (DMSO final concentration
was 0.16%). Controls comprised (i) hepatocytes incubated without TPT
sulfonate and (ii) 750 μM TPT sulfonate added to Williams' medium E
but without hepatocytes. Incubations were maintained at 37 °C with
shaking and bubbling of 5% CO2:O2. After 30 min, the medium was
withdrawn, centrifuged for 1 min at 12,000×g and frozen at −80 °C
until use. The expected extent of TPT sulfonate digestion in 30 min
(based on the data in Figure S1E) is ∼100% and ∼75% for the mouse
and rat hepatocytes, respectively.
2.14. Software packages
PK data were analyzed using Phoenix WinNonlin 6.2.1 (Certara,
Princeton NJ). Predictions of sites and relative extents of cytochrome P-
450 oxidative metabolism were made with MetaSite 4.0.4 (Molecular
Discovery, Borehamwood, United Kingdom) (Cruciani et al., 2013).
Additional molecular physicochemical and ADME parameter predic-
tions were made with VolSurf+ 1.0.7.1 (Molecular Discovery)
(Cruciani et al., 2000a, 2000b).
Before incubating with adult schistosomes, rat hepatocyte medium
was filtered through a 0.2 μM syringe filter (Millipore). Medium (100,
200, 400 or 700 μL) was added to 200 μL complete Basch medium
containing five adult S. mansoni pairs, or, in one experiment, four adult
S. haematobium males (supplied by Dr. Michael H. Hsieh, then at the
Dept. of Pathology, Stanford University, Palo Alto, CA). Phenotypic
changes in the worms and/or changes in worm motility were assessed
at 2 and 18 h as described above.
3. Results
3.1. Thiosulfates are inactive against S. mansoni in vitro
The 15 aryl- and alkylthiosulfates (structures shown in Table 1)
received from Dr. Nelson were tested for in vitro activity against so-
mules and adult S. mansoni using established protocols (Abdulla et al.,
2009; Rojo-Arreola et al., 2014; Long et al., 2016, 2017; Weeks et al.,
2018). None of the compounds induced obvious phenotypic changes at
2.12. Initial pharmacokinetics (PK) study in mice
The metabolic stability of TPT sulfonate and the identities and
stabilities of the metabolites formed were investigated in 5 week-old,
574

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
Fig. 1. Efficacy of those most potent
compounds of the 15 thiosulfates tested
against mature S. mansoni infections in
mice. Compounds 1, 2 and 8 and PZQ
(A-D respectively) were administered
orally by gavage at 100 mg/kg QDx4 in
150 μL 2.5% Kolliphor EL 42 days post-
infection (dpi) with 150 S. mansoni
cercariae. Means
SD of female
(circle) and male worms (squares) re-
covered by perfusion from mice
(n = 3–6 per group) at 62 (A), 56 (B),
(C) 59 and 60 dpi (D), respectively, are
indicated. Significance relative to ve-
hicle controls for each sex was mea-
sured using the Student's unpaired t-
test with a two-tailed distribution; sig-
nificance (p < 0.05) is indicated by
asterisks. See Table S1 (worksheets 4,
8, 5 and 14, respectively) for further
details on data per mouse.
concentrations of up to 10 μM over 3 days.
3.3. Compound 2 (TPT sulfonate) kills 21 day-old juvenile parasites that
are least sensitive to PZQ
3.2. Screening of thiosulfates against mature S. mansoni infections in mice
identifies one compound (TPT sulfonate) with efficacy similar to that of PZQ
Having established the superior efficacy of TPT sulfonate among the
15 compounds tested at 42 dpi, we next assessed the compounds' effi-
cacy using the same 100 mg/kg QDx4 dosing regimen at 21 dpi – a time
point in the parasite's development at which PZQ is least effective
{Keiser et al., 2009 #1030; Sabah et al. (1986) #38; Gőnnert and
Andrews., 1977 #548}. The data are summarized in Table 1 and the
complete set of data is presented in Table S1. TPT sulfonate was again
the most effective compound whereby female and male worm burdens
were decreased by at least 89% and 79%, respectively, in two separate
experiments (one shown in Fig. 3A). The 2-sec-butyl analog (14) of TPT
sulfonate was the next most effective, decreasing female and male
worm burdens by 72% and 61%, respectively (Table 1; Table S1
worksheet 6). Compound 1 was moderately effective, decreasing fe-
male and male worm burdens by 62% and 60%, respectively (Table 1;
Table S1 worksheet 9). By comparison, PZQ at 100 mg/kg QDx4 was
essentially inactive against 21-day-old parasites (Fig. 3C).
Compound efficacy, i.e., reductions in female and male worm bur-
dens, in a mouse model of S. mansoni infection employed a screening
dose of 100 mg/kg PO QDx4 to target mature 42-day-old parasites. The
data for the 15 compounds are summarized in Table 1. The complete set
of data, as indicated in the appropriate figure legends, regarding
changes in worm burdens, and weights of the liver and spleen (as a
metrics for disease-associated pathology (Abdulla et al., 2007) and re-
ferences therein) is presented in Table S1.
Of the 15 compounds, compounds 1 [(2-isopropylamino)-1-phenyl-
1-ethanethiosulfuric acid)],
2 [(2-tert-butylamino)-1-phenyl-1-etha-
nethiosulfuric acid or TPT sulfonate] and 8 [{2-(isopropylamino)-1-
octanethiosulfuric acid] were the most effective in reducing worm
burdens at 42 days post-infection (dpi) (Table 1; Fig. 1) and of these
TPT sulfonate was the best. Thus, Compound 1 decreased female and
male worm burdens by at least 60% and 37%, respectively, in two se-
parate experiments, (one shown in Fig. 1A). Compound 2 reduced fe-
male and male worm burdens by at least 90% and 46%, respectively, in
two separate experiments (one experiment shown in Fig. 1B). Also,
worms recovered by perfusion after exposure to 2 (TPT sulfonate) were
noticeably shorter and less massive (Fig. 2). Finally, compound 8 de-
creased female and male worm burdens by 50% and 40%, respectively
(Fig. 1C). For comparison, the same QDx4 dosing at 42 dpi with the
current anti-schistosomal drug, PZQ, decreased female and male worm
burdens by 95% and 94%, respectively (Table 1; Fig. 1D).
Focusing now on TPT sulfonate, we tested its efficacy at 100 mg/kg
QDx4 against 7- and 11-day-old S. mansoni in mice, i.e., when the
parasite is in the midst of and has completed its migration through the
lungs, respectively ((Rheinberg et al., 1998) and references therein).
TPT sulfonate was much less effective against 7-day-old lung-stage
parasites, decreasing female burdens by 40% and those of males by 12%
(Table 1; Fig. 3A). In contrast, 11-day-old parasites were markedly
susceptible to TPT sulfonate whereby female and male worm numbers
were decreased by 83% and 81%, respectively (Table 1; Fig. 3B). For
comparison, PZQ at 7 dpi, decreased female and male worm burdens by
58% and 44% (Table 1; Fig. 3C).
575

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
Fig. 2. Effect of TPT sulfonate (com-
pound 2) on worm length and mass.
TPT sulfonate was administered orally
by gavage to mice at 100 mg/kg QDx4
in 150 μL 2.5% Kolliphor EL 42 days
post-infection (dpi) with 150 S. mansoni
cercariae. Vehicle control worms (A)
and those exposed to TPT sulfonate (B)
were recovered by perfusion at 56 dpi.
Worms exposed to TPT sulfonate are
approximately 50% shorter and less
massive. Males can be distinguished
from females by being thicker and paler
in color. Bars = 0.6 cm.
For the series of 15 thiosulfates as a whole, a rudimentary SAR could
significantly increased predicted measures of skin permeability, Caco-2
monolayer permeability and blood-brain barrier permeability (Cruciani
et al., 2000a); p values were < 0.0001, < 0.0001 and 0.0002, respec-
tively. For example, the mean predicted increase in log brain/blood
be demonstrated. Efficacy was greatest among compounds with a
phenyl R1 group placed proximal to the sulfur terminus (compounds 1,
2, 7 and 14) with tert-butyl > isopropyl > sec-butyl > cyclohexyl
being preferred alpha to the amino terminus (R3). Compounds 8–11,
which lack the phenyl R1 group and have noncyclic aliphatic n ≥ 4 R2
and propyl R3 substituents, show a trend for statistically significant
reductions in worm burdens, principally at the 21-day time point. The
similar compound series, 3–6, with bulkier butyl R3 substituents,
showed at best weak and statistically insignificant activity. A cyclo-
hexyl R3 substituent seems to abolish activity.
distribution was 0.075
0.048 (about a 20% increase in the ratio). No
significant correlation with antiparasitic activity (using the average
values for different time points) was found for estimates of properties
such as log P, solubility, permeability, susceptibility to CYP3A4 meta-
bolism or protein binding. Among the molecular descriptors calculated
by VolSurf+, the strongest correlations with efficacy were found for
four that are related to the solubility versus pH profiles (L1LgS –
L4LgS). They represent how closely these profiles correlate with Le-
gendre polynomials of grades 1–4 (useful in distinguishing compounds
similar in solubility but with different pH-dependent profiles, or vice
versa). In these cases, the R value of the correlation was in the
0.71–0.73 range, and the correlation was present in both the phenyl-
bearing and non-phenyl-bearing members of the series. However, given
the large number (128) of molecular properties evaluated, and the
The 3-dimensional configurations and molecular properties of the
thiosulfate series were predicted by VolSurf+ (Cruciani et al., 2000b).
The software indicated the presence of internal hydrogen bonds be-
tween the thiosulfate and the amine. This was true for both the charged
(pH 7.0) and uncharged forms of the molecules. Evaluation of the
properties of the uncharged molecules (which are generally the mem-
brane-crossing species) showed that the presence of internal H-bonds
Fig. 3. Effect of worm age on efficacy of
TPT sulfonate as compared to PZQ in
mice infected with S. mansoni. TPT sul-
fonate (compound 2) was administered
orally by gavage at 100 mg/kg QDx4 in
150 μL 2.5% Kolliphor EL at 7, 21 and
42 (A), and 11 days post-infection (dpi)
(B) with 150 S. mansoni cercariae. PZQ
was administered under the same con-
ditions at 7 and 21 (C), and 42 dpi (D)
with 150 S. mansoni cercariae.
Means
SD of female (circle) and male
worms (squares) recovered from mice
(n = 3–6 per group) 56 (A), 35 (B), 49
(C) and 60 dpi (D) are indicated.
Significance relative to vehicle controls
for each sex was measured using the
Student's unpaired t-test with
a
two-
tailed
distribution;
significance
(p < 0.05) is indicated by asterisks. The
data shown for vehicle and 42 dpi in
panels A and D are the same as those
shown in Fig. 1B and D, respectively,
and are provided here for easier com-
parisons. See also Table S1 (worksheets
2,
8 and 9 for TPT sulfonate, and
worksheets 14 and 15 for PZQ) for de-
tails.
576

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
Fig. 4. Dose-ranging of TPT sulfonate
indicates that mature female S. mansoni
are more susceptible than males. (A)
TPT sulfonate was administered orally
by gavage at the doses (mg/kg) in-
dicated in 150 μL 2.5% Kolliphor EL at
42 days post-infection (dpi) with 150 S.
mansoni cercariae. Means
SD of fe-
male (circle) and male worms (squares)
recovered from mice (n = 3–6 per
group) 56 dpi are indicated. (B) For
comparison, PZQ was administered at
the doses indicated under the same
conditions. Significance relative to ve-
hicle controls for each sex was measured
using the Student's unpaired t-test with
a
two-tailed distribution; significance
(p < 0.05) is indicated by asterisks. See Table S1 (worksheets 10 and 17 for TPT sulfonate and PZQ, respectively) for details.
limited structural diversity in the molecule set, the signifcance and
generality of these correlations is not clear. In contrast, a weaker cor-
relation seen between efficacy and volume of distribution (R = 0.61)
was phenyl-dependent (absent in both the phenyl-bearing and non-
phenyl-bearing molecule sets).
regimen, the 400 and 600 mg/kg single doses of TPT sulfonate were not
significantly effective against 7-day-old lung-stage parasites (≤38%
and ≤32% reductions in female and male burdens, respectively;
Fig. 5A). In contrast, 21 day-old parasites were highly susceptible with
no distinguishable differences between the 400 and 600 mg/kg doses,
i.e., 95 and 91%, and 97 and 95% reductions in female and male bur-
dens, respectively. Likewise, for mature 42 day-old worms, TPT sulfo-
nate was equally effective at the 400 and 600 mg/kg doses, decreasing
female and male burdens by 100 and 85%, and by 100 and 81%, re-
spectively.
3.4. TPT sulfonate is an effective schistosomicide at single oral doses
Having defined the oral efficacy of TPT sulfonate against key de-
velopmental stages of S. mansoni in mice using the initial screening dose
of 100 mg/kg QDx4, we next tested a number of single (100, 200 and
400 mg/kg) and multiple dosing regimens (100 mg/kg QDx2 and
100 mg/kg QDx4) against mature 42-day-old infections to determine
the regimen that produces the greatest reductions in worm burdens. As
indicated in Fig. 4 and consistent with the foregoing data, females were
more susceptible to TPT sulfonate irrespective of dosing regimen. Thus,
the reductions ranged from 83% at the single 100 mg/kg dose to 100%
at the single 400 mg/kg dose, whereas for males, a maximum reduction
of 74% at the single 400 mg/kg dose was measured (Fig. 4A). By
comparison, PZQ decreased female and male worm burdens by 97%
and 78%, and 100 and 97%, respectively at single oral doses of 200 and
400 mg/kg (Fig. 4B). Overall, at the highest single dose of 400 mg/kg,
TPT sulfonate is as effective as PZQ in removing female worms, whereas
male worms are somewhat less susceptible to TPT sulfonate.
As shown in Fig. 5B for the 600 mg/kg dose, one-day-old larvae
(presumably located in the skin (Rheinberg et al., 1998)) were not
susceptible to TPT sulfonate; also, lung-stage parasite burdens at 7 dpi
were non-significantly decreased by 28% (both sexes). In contrast, 15-
day-old, post-lung-migratory worms were highly sensitive as reductions
of 89% and 92% for females and males, respectively, were measured.
3.5. Increasing the QDx4 regimen from 100 mg/kg to 200 mg/kg improves
killing of lung-stage worms
In a final effort to improve efficacy against 7-day-old, lung-stage S.
mansoni, and noting that the original 100 mg/kg QDx4 regimen had
some significant impact on female, but not male worm burdens (Fig. 3;
reductions of 40% and 12%, respectively), we doubled the daily dose of
TPT sulfonate to 200 mg/kg QDx4. This regimen markedly improved
efficacy by decreasing female and male worm burdens by 95 and 80%,
respectively (Fig. 6). Thus, lung-stage worms are susceptible to TPT
sulfonate at the higher dose.
In an attempt to improve the single dose efficacy of TPT sulfonate,
we compared dosing at 400 and 600 mg/kg over a range of time points
(1, 7, 15, 21 and 42 dpi) that spans the entire development of the
parasite (Fig. 5). As noted earlier for the 100 mg/kg QDx4 dosing
Fig. 5. Increasing the single oral dose
of TPT sulfonate can cure mice infected
with S. mansoni: again efficacy depends
on worm age. TPT sulfonate was ad-
ministered orally by gavage at the
doses (mg/kg) indicated in 150 μL
2.5% Kolliphor EL at 7, 21 and 42 days
post-infection (dpi) (A), and 1, 7 and 15
dpi (B) with 150 S. mansoni cercariae.
Means
SD of female (circle) and
male worms (squares) recovered from
mice (n = 3–6 per group) 52 (A) and
44 dpi (B) are indicated. Significance
relative to vehicle controls for each sex
was measured using the Student's un-
paired t-test with a two-tailed distribu-
tion; significance (p < 0.05) is in-
dicated by asterisks. See Table S1
(worksheets 11 and 12 for (A) and (B),
respectively) for details.
577

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
biotransformation by mouse liver microsomes: clearance in the pre-
sence and absence of NADPH was 0.19 and 0.15 ml/h/mg, respectively,
i.e., digestion was very slow and mostly non-CYP mediated (Table 2;
Figure S1B). No significant TPT sulfonate transformation was measured
in the glucuronidation assay (Table 2; Figure S1C).
In contrast to the microsomal assays, TPT sulfonate was readily
metabolized by intact hepatocytes from either mouse or rat (Table 2;
Figures S1D, S1E). The clearance of TPT sulfonate by mouse hepato-
cytes was 1.9 ml/h/million cells (32 μL/min/million cells; Figure S1D).
This value is high relative to those for other compounds in the literature
(Lau et al., 2002). The hepatocyte contents of mouse and rat livers are
known to be similar (Sohlenius-Sternbeck, 2006). If we assume the
microsome content of mouse hepatocytes is also similar to the rat value
(0.37 mg/million cells, based on 61 mg/g and 163 × 106 cells/g (Smith
et al., 2008), the mouse hepatocyte clearance result is equivalent to
5.1 ml/h/mg microsomes, over 20 times the total microsomal clearance
and over 100 times the CYP-mediated or UDP-glucuronosyltransferase-
mediated microsomal clearance (Table 2). Thus, the metabolism of TPT
sulfonate by mouse hepatocytes appears not to be mediated by enzymes
of these classes.
Fig. 6. Increasing the dose of TPT sulfonate from 100 QDx4 to 200 QDx4 sig-
nificantly improves efficacy against 7 day-old lung worms. TPT sulfonate was
administered orally by gavage at the dose 200 mg/kg QDx4 in 150 μL 2.5%
Kolliphor EL at 7 days post-infection (dpi) with 150 S. mansoni cercariae.
Means
SD of female (circle) and male worms (squares) recovered from mice
(n = 3–5 per group) 57 dpi are indicated. Significance relative to vehicle con-
trols for each sex was measured using the Student's unpaired t-test with a two-
tailed distribution; significance (p < 0.05) is indicated by asterisks. See Table
S1 (worksheet 13) for details. Also, compare these data to those shown for
100 mg/kg QDx4 in Fig. 1A.
3.6. Summary of TPT sulfonate's in vivo efficacy
3.8. Transformation of TPT sulfonate by hepatocytes is required for
expression of anti-schistosomal activity, incl. against S. haematobium
In its single, oral dose efficacy, the data so far suggest that: (i) TPT
sulfonate is comparable to PZQ in removing mature 42 day-old female
worms, but it is somewhat less effective against mature males (e.g.,
74–85% worm reductions vs. 97% for PZQ at 400 mg/kg); (ii) TPT
sulfonate is particularly effective against juvenile 21 day-old parasites
(≥91% worm kill), i.e., those parasites that are least responsive to PZQ;
(iii) skin- (1 dpi) and lung-stage worms (7 dpi) are the least susceptible
to TPT sulfonate, and for the later time point, the efficacy measured is
less than that of PZQ and (iv) TPT sulfonate's efficacy markedly im-
proves from 7 to 11 (or 15) dpi, coincident with the completion of
migration of S. mansoni from the lungs and its establishment in the
mesenteric venous system (Rheinberg et al., 1998). Finally, although
lung-stage parasites are less susceptible to TPT sulfonate than older
parasites, they are not refractory because increasing the QDx4 dosing
regimen from 100 to 200 mg/kg markedly improves worm killing. The
data generated here for PZQ at 7, 21 and 42 dpi are consistent with
those previously reported (Gőnnert and Andrews, 1977; Sabah et al.,
1986; Keiser et al., 2009). Regardless of the dosing regimen employed
with TP sulfonate, mice did not exhibit signs of stress, e.g., hunched
posture, agitation, lack of feeding or drinking.
Primary rat hepatocyte cultures in Williams' medium E were in-
cubated for 30 min in the presence or absence of 750 μM TPT sulfonate.
Negative controls comprised either TPT sulfonate incubated in
Williams' medium E but omitting the hepatocytes or hepatocytes in-
cubated in the absence of TPT sulfonate. Medium (100, 200, 400 or
700 μL) was then withdrawn and added to adult S. mansoni in 200 μL
Basch medium. Worms were observed microscopically and motility
measured using WormAssay after 2 and 18 h.
No effects on worms were recorded in the presence of 100 or 200 μL
Williams' medium E conditioned by hepatocytes in the presence of TPT
sulfonate. However, worms incubated with 400 and 700 μL of the same
medium were clearly stressed (Fig. 7). Thus, after 2 h, worm motility
was decreased by approximately 90–95% (Fig. 7A), and males and fe-
males had become uncoupled with an inability to adhere to the floor of
the well. After 18 h, worm motility remained severely depressed and,
observationally, worms were barely moving with tegumental (surface)
damage being also apparent (Fig. 7B, C), a finding that conceivably
would lead to immune-mediated clearance of the worms in vivo as
postulated for PZQ (Andrews, 1985; Brindley and Sher, 1987; Doenhoff
et al., 1987). In a single experiment, the depressed motility and other
phenotypic effects observed microscopically for S. mansoni after 2 and
18 h of incubation in the presence of 400 and 700 μL hepatocyte-con-
ditioned medium were also noted for adult S. haematobium males. For
all experiments, both of the negative control conditions (700 μL
medium) did not alter worm motility or appearance (Fig. 7D, E).
3.7. TPT sulfonate is stable in plasma and to digestion by microsomes, but is
rapidly metabolized by hepatocytes
TPT sulfonate is relatively stable in mouse plasma, degrading by just
over 50% after 40 h at 37 °C (Figure S1A). It is also resistant to
Table 2
Relative rates of TPT sulfonate digestion by mouse microsomes and hepatocytes.
Assay type
k, (h−1
)
Microsomes (mg/ml)
In vitro clearance (ml/h/mg)
Mouse microsomal glucuronidationa
0.07
0.1
3.5
0.02
0.04
0.15
5.1
Mouse microsomal CYPsb
3.33
3.33
0.90d
Mouse microsomes, non-specificc
Mouse hepatocytes
0.5
4.6
In vitro clearance values of TPT sulfonate expressed relative to mouse liver microsome concentration (or microsome content, for hepatocytes). Values of the ex-
ponential decay constant k were obtained by fitting the data to the equation: TPT sulfonate concentration = a*exp(-k*t). In vitro clearance is then given as k/
microsome concentration (the calculation assumes enzyme saturation is negligible). See Figures S1B-S1D for digestion plots.
a
b
c
d
Value with UDPGA and other glucuronidation assay components (see Materials and Methods).
Value with NADPH minus value without.
Without cofactor NADPH or other additions.
Based on the microsome equivalent of rat hepatocytes, 0.37 mg/million cells (Smith et al., 2008).
578

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
Fig. 7. Biotransformation of TPT sul-
fonate by hepatocytes is required for
expression of anti-schistosomal activity
in vitro. (A) Supernatants (100, 200,
400 or 700 μL) from rat hepatocytes
that had been incubated with 750 μM
TPT sulfonate for 30 min were added to
200 μL cultures of adult S. mansoni.
Worm motility was measured with
WormAssay after 2 h (black bars) and
18 h (grey bars). No hep. = a control
whereby TPT sulfonate was incubated
in hepatocyte medium but in the ab-
sence of hepatocytes before transfer of
700 μL of the medium to the parasites.
No TPT-S = a control whereby hepa-
tocytes had been incubated in the ab-
sence of TPT sulfonate before transfer
of 700 μL of the medium to the para-
sites. The means
SD values from a
60 s WormAssay recording from one of
two similar experiments are shown. (B)
and (C) Images of worms incubated
with 400 μL supernatant from rat he-
patocytes that had been incubated with
750 μM TPT sulfonate for 30 min (B) A
coiled female worm 18 h after incuba-
tion: the parasite's head is toward the
center of view. (C) Anterior end of a
male worm 18 h after
a similar in-
cubation: the oral sucker is to the left.
For both images, arrows point to
fraying and blebbing of the surface te-
gument. (D) and (E) Images of a female
and male worm, respectively, in-
cubated with 400 μL supernatant from
rat hepatocytes that had been in-
cubated in the absence of TPT sulfonate
for 30 min. Bars represent 150 μm (B,
D) and 50 μm (C, E), respectively.
3.9. Identification of TPT sulfonate metabolites observed in plasma and
hepatocytes
analogs and the structures of their N-alkyl substituents (with the t-butyl
structure being associated with the greatest potency; see the R3 sub-
stituent in Table 1), it is less likely that metabolites lacking the t-butyl
group contribute to efficacy. Moreover, the observed low rate of CYP
oxidation of TPT sulfonate indicates that metabolites with modified t-
butyl groups are unlikely to form. According to MetaSite, a software
package that accurately predicts phase I metabolic transformations
(Cruciani et al., 2013), the most abundant CYP-mediated modifications
of TPT sulfonate, both overall and at the t-butyl moiety, should be
hydroxylations. No such m/z 306 metabolites were detected either by
the m/z 56 NLS (which would have detected phenyl modifications) or
by the m/z 91, 120, 135 or 234 precursor ion scans (which would have
detected t-butyl modifications). Thus, the t-butyl group is unlikely to be
a site of metabolism and we expect that the m/z 56 NLS technique
should detect the metabolites of most interest.
Plasma from an initial mouse PK experiment (100 mg/kg PO) was
analyzed to identify the metabolites of TPT sulfonate. The analysis was
performed via neutral loss (NLS) and precursor ion scans (PIS) based on
the major daughter ions of TPT sulfonate (m/z 234, 135, 120 and 91;
Fig. 8A). The most useful technique proved to be m/z 56 NLS, em-
ploying the smallest neutral loss associated with the major daughter
ions, which highlights compounds with a t-butyl group (Sassine et al.,
2008), a moiety rare in biochemistry (Bisel et al., 2008). There is no
single MS/MS scanning method that would detect all possible meta-
bolites and m/z 56 NLS would not detect metabolites that are missing
the t-butyl moiety or those with a modified t-butyl group. However,
given the sensitive relationship between efficacy of TPT sulfonate
579

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
Fig. 9. Scheme of putative reactions that generate the major early-eluting
metabolites of TPT sulfonate observed in mouse plasma. Sequential desulfations
form three pairs of compounds with a mass difference of 80 (see Figures S2 and
S3). All of these metabolites except the m/z 270 were also identified in hepa-
tocyte incubations (Fig. 8B). Diagonal arrows represent a possible alternate
pathway in which phenyl oxidation precedes sulfhydryl oxidation.
z 417 thiol dimer (Figures S4 and S5). The absence of matrix in this
sample allowed us to analyze it with a high mass resolution instrument,
the LTQ Orbitrap. In the MS spectrum, we observed monomer and/or
decomposition products with m/z values of 210 and 176, whereas in
the MS/MS spectrum, we observed fragments with m/z 240, 210 and
176. In other words, species of the same mass as three of the TPT sul-
fonate plasma metabolites, representing all of the identified plasma
metabolite structures with no added atoms other than hydrogen,
formed from the sample. In addition, thiol dimer MS/MS fragments of
m/z 208 and 242, representing species with the same masses as the
putative API 4000 inlet decomposition product of the dimer and a
putative hepatocyte metabolite, were also seen. The high accuracy mass
values obtained allowed determination of the atomic compositions of
each of these species. All of these results are consistent with the
structures assigned to metabolites of the same mass.
Fig. 8. Relative ion abundance of TPT sulfonate metabolites from mouse
plasma and a mouse hepatocyte digest. MS/MS peak areas are compared for
metabolites observed in (A) mouse plasma 4 h after 100 mg/kg PO dosing and
in (B) a mouse hepatocyte incubation after 20 min, using 400 μM TPT sulfonate
and 3 × 106 cells/m. Solid colors represent metabolites depicted in Fig. 9;
cross-hatching indicates metabolites differing from those in the figure by two
mass units (possibly representing a gain or loss of a double bond) and vertical
stripes indicate metabolites of unknown structure. Data were obtained via m/z
56 neutral loss scanning from m/z 100 to 320. The m/z 208 compound is not a
metabolite per se, but a decomposition product that forms in the inlet to the API
4000 MS from the m/z 417 thiol dimer.
Further evidence for the metabolite structures was obtained by
analyzing mouse plasma samples using m/z 155 NLS and m/z 91, 120,
135 and 234 precursor ion scanning (PIS). Comparison of the putative
metabolite structures in Fig. 9 and that of m/z 208 (see Figure S5) with
the TPT sulfonate fragment structures in Fig. 8 indicates that the fol-
lowing detections should be impossible (because they require the loss of
a fragment not contained within the metabolite structure): m/z 176 or
190 by m/z 135 PIS, any of the metabolites by m/z 234 PIS, and any
except the m/z 256 or 270 by m/z 155 NLS. Of the 13 detections of one
of these metabolites made using these methods, none fell into the for-
bidden category, consistent with our structural assignments.
M/z 56 NLS of mouse plasma revealed over a dozen metabolites, as
shown in Fig. 8A (a more complete list is presented in Table S2). The six
prominent plasma metabolites moderately retained on a C8 column
have m/z values of 176, 190, 210, 240, 256 and 270 (Figures S2 and
S3). A striking feature of this list is that the m/z 176, 190 and 210
metabolites are related to the m/z 240 and 256 metabolites or to m/z
290 TPT sulfonate, respectively, by a mass loss of 80, a value diagnostic
for loss of SO3. This made it straightforward to relate the structures of
the first three metabolites with their immediate precursors and so re-
veal the major pathways of TPT sulfonate metabolism (Fig. 9). The m/z
256 and 270 precursors can be derived from the putative initially
formed m/z 210 thiol metabolite via sequential oxidations. The plasma
m/z 240 metabolite was deduced to be C12H18NS2 on the basis of its
observed (API 4000) MH+ mass distribution (240.1, 100.0%; 241.1,
13.5%; 242.2, 9.5%, compared to the expected 240.09, 100.0%;
241.09, 13.0%; 242.08, 8.9%, as opposed to the values expected for
The m/z 176 and 256 metabolites have the greatest ion abundances
(based on MS/MS peak area) of those in plasma. Pure standards were
not available for these analyses, and because MS/MS detection effi-
ciency is variable, the actual molecular abundances are uncertain. Some
of the additional metabolites (Table S2) differ from those shown in
Fig. 9, by the mass of a double bond, whereas others have no obvious
relation to the known structures; the most prominent of the latter are
the late-eluting m/z 234 and 209 (Figure S3).
C
12H18NO2S: 240.11, 100.0%; 241.11, 13.0%; 242.10, 4.4%). Dimer-
ization of the m/z 210 thiol would be expected to form an m/z 417
disulfide, and the latter was found in plasma. Reduced glutathione was
evidently either depleted or outcompeted in the location where the m/z
417 dimer formed. We also observed an m/z 208 peak that always
eluted with the m/z 417 dimer, and, thus, appears to be a decomposi-
tion product of the dimer that forms in the API 4000 MS inlet. M/z 208,
therefore, serves as a proxy for the dimer in the API 4000 m/z 56 NLS
analyses.
A plot of the relative peak size of mouse plasma metabolites from
0.5 to 4.0 h after PO dosing does not show strong trends during that
interval in most cases (Figure S6). The only metabolites to show a large
(> 3-fold) and statistically significant increase over this interval were
the m/z 190 (p = 0.04) and 270 (p = 0.01) quinone methides, sug-
gesting that the oxidation of the phenyl moiety is slow relative to the
other biotransformations. Possible reasons for this outcome, beyond
simple formation kinetics, are that quinone methide formation is less
favored in the first pass or that this pathway is upregulated after dosing;
these metabolites might also have longer half-lives than the others. The
Additional evidence to support our structural assignments was ob-
tained from the MS and MS/MS analyses of a sample of the synthetic m/
580

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
Table 3
sulfonate is fully absorbed and that its low bioavailability is due to
Non-compartmental mouse pharmacokinetics of TPT sulfonate.
extensive (≥90%) first-pass metabolism.
The apparent plasma half-life of TPT sulfonate (calculated from the
last three time points in Fig. 10A) was 1.3 h from the PO data and 0.7 h
from the IV data. These values are significantly shorter than those of its
metabolites, consistent with its role as a prodrug (Table 4). The natural
log of plasma concentration vs. time plot for the PO data reaches a
linear terminal phase (R = 0.995). However, the corresponding IV data
is decidedly nonlinear (R = 0.81) with half-lives calculated from ad-
jacent time point pairs starting at 0.04 h and then increasing to over an
hour. The progression indicates that after IV dosing, TPT sulfonate
distributes out of the central compartment into other tissues, becoming
less subject to hepatic elimination as it does so. Reflecting this migra-
tion, the volume of distribution increases from an initial IV value of
0.4 L/kg to a final value of ∼8 L/kg (Table 3). The apparent PO half-life
represents an upper limit for the true terminal plasma half-life, due to
slow GI absorption, whereas the IV value is probably a lower limit since
it doesn't attain a linear phase.
IV (15 mg/kg)
PO (100 mg/kg)
t = 0→last
t = 0→∞
t = 0→last
t = 0→∞
C0 (ng/ml)
Cmax (ng/ml)
V0 (l/kg)
37,200
–
–
710
0.403
0.693
7.9a
Vss (l/kg)
0.984
7.7a
97.6
2560
0.168
–
–
–
Varea (l/kg)
CL (ml/min/kg)
AUC (ng·h/ml)
MRT [+MIT] (h)
F (%)
7.9a
–
7.7a
–
99.6
2510
0.116
–
1430
[1.83]
–
1450
[1.94]
8.5
Abbreviations: V0, time zero volume of distribution = dose/C0; Vss, steady-state
volume of distribution = CL*MRT; Varea, terminal volume of distribution = CL/
terminal k; CL, plasma clearance; AUC, area under the plasma concentration-
time curve; MRT, mean residence time; MIT, mean input time; F, bioavailability.
a
Based on the IV CL and the average of the k values from the last three data
The m/z 210 thiol metabolite had by far the shortest tmax after IV
dosing (Table 4, Fig. 10B), consistent with it being the first metabolite
to form. However, its tmax with PO dosing was among the longest. In
addition, its PO terminal half-life of 6 h was longer than those of the
downstream metabolites analyzed. The larger TPT sulfonate dose given
PO may have led to a greater proportion of thiol dimer formation.
Reversible heterodisulfide formation by the thiol is also likely, but, at
least initially, probably to a lesser extent than homodisulfide formation.
Formation of both types of disulfides probably protects the thiol from
further metabolism. Gradual reduction of the additional dimer back to
thiol monomer could then explain the latter's longer PO tmax. The longer
plasma half-life of the thiol relative to the other metabolites may reflect
that once released into plasma it is less subject to reuptake by the liver
for further metabolism.
points of the IV and PO PK datasets (see Section 3.10).
analyte displaying the most significant downward trend (decreasing
more than 50% in the same interval) was the m/z 290 parent drug
(p = 0.07).
Of the major (> 0.7% abundance) metabolites initially observed in
mouse plasma, all but the m/z 209 and m/z 270 products were also
observed in mouse hepatocyte digests (Fig. 8B; Table S2) along with the
m/z 417 disulfide of the m/z 210 thiol. With the hepatocytes, there is a
higher initial TPT sulfonate concentration than in the plasma, leading
to a higher early concentration of thiol (represented by m/z 210 and m/
z 208 from the dimer). This intermediate might have saturated the
pathway leading to m/z 256 and its downstream m/z 176, 270 and 190
metabolites, resulting in relatively less of these, and relatively more of
the metabolites from other pathways, e.g., m/z 222, 234, and 252. The
hepatocyte metabolites analyzed include those extracted from within
cells.
The kinetics of formation and elimination of the m/z 256 alkenyl
sulfonic acid were generally similar to those of the m/z 176 styrene
metabolite (Table 4; Fig. 10C, D) consistent with formation of the latter
via sequential metabolism, i.e., m/z 256 formation rate-limits m/z 176
formation. Both have tmax values in the 0.5–1.0 h range after IV ad-
ministration and close to 3 h after PO dosing, and terminal half-lives of
about 2 h. The apparent relatively greater AUC of m/z 256 when dosed
PO may reflect a different selectivity for metabolite formation in the
first pass.
3.10. Full mouse PK experiment (IV/PO)
A PK study of TPT sulfonate following IV and PO dosing of mice
indicates that its bioavailability (F) is about 8.5% (Table 3). The plasma
clearance value of almost 100 ml/min/kg approximates the rate of
mouse hepatic blood flow of 90 ml/min/kg; (Davies and Morris, 1993),
indicating that TPT sulfonate is rapidly cleared. Based on the AUC, the
mean residence time is approximately 10 min for the IV data and, in-
cluding mean input time, almost 2 h for the PO data (Table 3). The
rapid metabolism relative to the absorption rate implies flip-flop ki-
netics, i.e., the terminal elimination rate after oral dosing may be
controlled by absorption. The mean PO/IV ratio of metabolite AUCs,
normalized to dose, was 1.03 (Table 4). This indicates that TPT
A puzzling aspect of the m/z 176 metabolite is that, whereas only
one peak was seen for this molecular weight in our initial mouse PK
study (Figure S3C), three peaks were seen in the final study (utilizing a
different chromatographic system; Fig. 10D). These peaks could not be
distinguished from one another on the basis of parent ion m/z (using
NLS) or daughter ion m/z (using a product ion scan). The two main
peaks (at 0.66 and 0.79 min) had very similar plasma time profiles
(Table 4; Fig. 10D). Several isomeric or stereoisomeric structures are
possible for the metabolite and one explanation is that they might have
Table 4
Non-compartmental mouse pharmacokinetics of TPT sulfonate and its metabolites.
Chemical species
tmax (h)
terminal t1/2 (h)
AUC ratio, PO/IV
IV
PO
IV
PO
(normalized to dose)
TPT sulfonate (m/z 290)
m/z 210
–
0.50
4.0
2.0
2.0
4.0
2.0
–
1.3b
5.8b
2.5b
2.4b
2.3b
1.6b
0.085a
0.84c
1.49c
0.98c
1.05c
0.81c
0.083
1.0
0.5
0.5
0.5
–
m/z 256
2.2b
1.7b
1.7b
–
m/z 176, 0.79 min peak
m/z 176, 0.66 min peak
m/z 176, 1.25 min peak
a
b
c
From WinNonlin analysis for t = 0 → ∞
From slope of last 3 data points of ln peak area vs. time plot, for cases with R > 0.98.
From AUC0-8h; mean value for metabolites is 1.03 0.27.
581

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
Fig. 10. Plasma-time profiles of TPT sulfonate and three of its metabolites in mice after IV and oral dosing. Mice were administered TPT sulfonate IV (4 mice, 15 mg/
kg) or PO (8 mice, 100 mg/kg), as described in the Materials and Methods. Mean TPT sulfonate plasma concentrations and metabolite MS/MS peak areas were then
determined. (A) TPT sulfonate (retention time = 1.14 min). (B) The m/z 210 thiol metabolite initially formed from desulfation of TPT sulfonate (retention
time = 1.10 min). (C) The m/z 256 alkenyl sulfonic acid metabolite formed by oxidation of the m/z 210 thiol (retention time = 0.82 min). (D) The m/z 176 2-
phenylethenamine metabolite(s) formed by desulfation of the m/z 256 metabolite. Three retention times were observed for the m/z 176 metabolite, whereas in our
original mouse PK study this m/z was represented by a single peak (see Figure S3C and Discussion). Note: these retentions are from a different LC method than those
in shown in Figure S2 and Figure S3.
co-eluted under our initial but not our final analytical conditions due to
different rates of interconversion. Alternatively, multiple metabolites
might exist that decompose into an m/z 176 ion in the mass spectro-
meter inlet only under the conditions employed in the second (full) PK
study. In our initial study, we observed inlet decomposition of the m/z
417 thiol dimer into m/z 208 and 224. In later work under different
conditions, we sometimes also observed other inlet decompositions,
such as 417 to 361, 417 to 242, 290 to 210 and 224 to 176. Which
chromatographic and/or detection system variables influenced this
process are not clear. Although inlet decomposition of m/z 240 to m/z
176 was evident in the final PK study, this fact did not account for any
of the m/z 176 peaks we analyzed.
Scheme 1. Synthesis of m/z 210 thiol (TPT thiol) and 176 styrene metabolites.
(Scheme 1): synthesis of the m/z 210 component (TP thiol) yielded the
m/z 417 dimer due to spontaneous disulfide bond formation upon
concentration of the reaction mixture.
After 8 h, TP thiol generated a concentration-dependent decrease in
motility of both adult S. mansoni and S. haematobium (Fig. 11A). The
principal microscopic observations were markedly decreased move-
ment, an inability of the worms to adhere to the well floor and the sexes
becoming uncoupled. In contrast, the m/z 176 styrene was inactive
against both schistosome species up to 40 μM after 24 h. In 24 h
counter-toxicity screens with three mammalian cell lines, the TP thiol
was relatively non-toxic up to the highest concentration tested of
40 μM, at which concentration cell nuclei counts were decreased to 86,
78 and 100% of DMSO controls for C2C12, HuH-7 and BESM cells,
respectively (Fig. 11B).
3.11. In vitro identification of the m/z 210 thiol metabolite (TP thiol) as a
cidal principle with low cell toxicity
Mouse metabolism of TPT sulfonate generates the m/z 210 thiol in
the initial step (Fig. 9), whereas the m/z 176 styrene is the metabolite
observed to have the largest LC-MS/MS peak area (a proxy for abun-
dance in the absence of weighed standards or a radiolabel). Both of
these metabolites also form in the transformation of TPT sulfonate by
hepatocytes in vitro (Fig. 9, Table S2). Accordingly, they were synthe-
sized by the CRO WuXi using a synthetic scheme designed by us
582

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
Fig. 11. In vitro bioactivity of the che-
mically synthesized m/z 210 metabo-
lite (TP thiol) against adult S. mansoni
and S. haematobium, and apparent lack
of cell toxicity. (A) Adult S. mansoni
(circles) or S. haematobium (squares)
were incubated for 8 h with different
concentrations of synthesized TP thiol
(as the m/z 417 dimer). Points re-
present the means
SD values across
three wells at each concentration and
one of two experiments is shown. (B)
Counter toxicity screens with various
mammalian cell lines incubated in the
presence of TP thiol were performed as
described in the Materials and Methods. After 24 h, cells were fixed with 10% paraformaldehyde and nuclei stained with DAPI. Nuclei were counted across 5–10
fields of view/well using an inverted microscope fitted with a 20x objective lens. Nuclei counts are expressed as percentages relative to DMSO controls. Points
represent mean counts across a minimum of three wells for each concentration tested and the S.D. value for each mean was ≤20%. Data from one of two experiments
performed are shown.
3.12. Chemically synthesized TP thiol is parasiticidal in vivo
we have not tested the possibility that thiosulfates which lack in vivo
efficacy may generate hepatocyte metabolites with in vitro anti-schis-
tosomal activity.
Sufficient TP thiol (as the corresponding dimer) had been synthe-
sized by WuXi to administer a single 50 mg/kg dose IP in 2.5%
Kolliphor EL to mice harboring 33-day-old infections. Female and male
worm burdens were decreased by 35% and 44% in one experiment,
respectively (Table S1 worksheet 18). All of the worms recovered were
approximately 50% less massive and paler relative to vehicle controls.
Also, infection-related organomegaly was decreased: liver and spleen
weights were 32% and 41% lower compared to those of the vehicle
controls.
The most effective compound to emerge from the initial 100 mg/kg
QD x4 screen was compound 2, TPT sulfonate. TPT sulfonate does not
violate any of the guidelines for drug-like molecules put forward by
Lipinski et al. (2001) or Veber et al. (2002). At single oral doses, the
compound was superior to PZQ against 21-day-old juvenile parasites;
80–90% worm-kill was measured compared to zero kill for PZQ.
Against adult 42-day-old worms at 400 mg/kg, TPT sulfonate was
competitive with PZQ with reductions of 100% and approximately 80%
for female and male worm burdens, respectively, compared to 100% for
both sexes with PZQ. No further worm burden reductions were
achieved at the higher dose of 600 mg/kg TPT sulfonate.
4. Discussion
With the continuing international resolve to make PZQ available to
more communities affected by schistosomiasis, there is concern that
sufficient drug pressure will eventually lead to parasites exhibiting re-
versible or, worse, genetically fixed resistance. In addition, there is a
need to provide a drug that does not suffer the pharmacological
weaknesses of PZQ, chief among which are its lack of cure in the single
dose typically offered and its incomplete efficacy against the parasite, in
particular against 21-28-day-old juvenile worms (Gőnnert and
Andrews, 1977; Sabah et al., 1986; Keiser et al., 2009).
The efficacy of TPT sulfonate also extended to 11- and 15-day-old
juveniles that have completed their migration across the lungs and
taken up residency in the mesenteric venous system (Rheinberg et al.).
However, younger parasites (1-day-old skin larvae and 7-day-old lung-
stage worms) were more resilient. In the case of the lung-stage worms,
twice the initial screening dose, i.e., 200 mg/kg QDx4 was needed to
demonstrate a significant cidal affect. The difference in susceptibility
between skin-/lung-stage somules and juvenile worms in the mesenteric
veins was noted before for one compound (8) whereby a 300 mg/kg
QDx5 dose was 50% efficacious against 24 day-old S. mansoni but in-
effective against the parasites 1–10 dpi (Penido et al., 1994). The reason
for the apparent resilience of skin-/lung-stage worms is unclear but may
lie in the nature of the parasites themselves (e.g., poorer penetration of
the active principle or altered expression of the target(s)) and/or the
availability of the active principle in the relevant tissues.
As possible alternatives to PZQ, Pellegrino, and then Nelson and
colleagues have shown that aliphatic or phenyl-substituted aminoalk-
anethiosulfates kill S. mansoni in a mouse model of infection. Apart
from one study (Penido et al., 1994) with one of the 15 compounds
tested here (compound 8 see below), the emphasis has been on tar-
geting adult worms (Nelson and Pellegrino, 1976; Penido et al., 1994;
Moreira et al., 2007) and the potential efficacy of this compound series
against younger stages of the parasite, including those 21-28 day-old
parasites most refractory to PZQ, has not been investigated. Here, using
an initial screening dose of 100 mg/kg PO QDx4, we found that three of
the 15 thiosulfates tested (compounds 1, 2 and 8) significantly reduced
the burdens of 42-day-old adult and 21-day-old juvenile S. mansoni in
mice. The greater efficacy against mature female worms noted here is
consistent with previous data for these and other thiosulfates (Penido
et al., 1994, 1995, 1999; Moreira et al., 2007) and is extended here to
include females from immature infections.
Compared to the often robust cidal activity measured in vivo, none
of the 15 sulfonates was active against parasites in vitro (somules or
adults) at concentrations of up to 10 μM over 3 days. The result es-
sentially made an in vitro-based development of an SAR impossible.
Previous in vitro tests using S. mansoni adults, newly transformed and
six-day-old lung-stage stage somules with compounds 5, 6, 8, 10, and
13 noted paralysis and tegumental damage for some of the compounds
tested (Penido et al., 1994). However, the studies employed very high
compound concentrations (0.25–1 mM) and the physiological relevance
of the changes reported is unclear. The absence of activity in our in vitro
assays suggested a prodrug mechanism of action, a notion substantiated
for TPT sulfonate by the finding that supernatants from rat hepatocytes
that had been incubated with the compound markedly decreased the
motility of adult S. mansoni and S. haematobium and induced degen-
erative changes.
Examination of the in vivo structural-bioactivity data suggests that
phenyl substitution adjacent to the sulfur of the thiosulfate is associated
with more active compounds (in the 15-member series, the 4 phenyl-
bearing representatives include the 3 most active compounds at 21 dpi).
The identification of active metabolites derived from 2 (see below)
raises the possibility that the SAR observed for this series might be
complicated by a structural effect on the rate of host metabolism in
addition to the influence of structure on the parasite interaction. Also,
Although TPT sulfonate was readily biotransformed by whole he-
patocytes from either mice or rats, it was not oxidized or glucur-
onidated by isolated mouse liver microsomes. These findings suggest
583

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
that metabolic clearance pathways are independent of the two most
common classes of drug-metabolizing enzymes, a desirable feature that
decreases the likelihood of problematic drug-drug interactions, parti-
cularly because the current drug, PZQ, is dispensed to populations with
minimal medical supervision. About a dozen CYP isoforms metabolize
70–80% of all drugs (Zanger and Schwab, 2013) and UGTs are the most
prominent conjugating enzymes in drug metabolism. Together, CYPs
and UGTs account for over 90% of hepatic drug metabolism (Rowland
et al., 2013). For TPT sulfonate, the cytosolic enzymes, aldehyde oxi-
dase and various carboxylesterases might contribute to its conversion to
the active cidal form: sulfatases could also be involved (Coughtrie et al.,
1998; Bojarova and Williams, 2008) Given the broad similarities in
hepatic metabolism across mammals, it is likely that human hepato-
cytes will metabolize TPT sulfonate similarly to rodent hepatocytes.
However, confirming this and identifying the enzyme(s) involved will
require further work.
thiosulfuric acid group of the TPT sulfonate. The mechanisms by which
the active product is generated are not yet clear. Free sulfhydryl groups
are generally disliked in pharmaceutical development (Jaffe, 1986) and
are used much less than amino, hydroxyl or carboxylic acid functional
groups (Carey et al., 2006). They can form protein adducts, often by
displacing a cysteine from a cysteine disulfide residue, as well as other
types of disulfides; thus, their toxicity is increased by GSH depletion.
They can also potentially be oxidized to more reactive sulfenic acid and
sulfinic acid metabolites (Kalgutkar et al., 2005). Complications arising
from non-selective off-target alkylations can confound in vivo experi-
ments unless appropriate controls are in place. In our hands, however,
up to 40 μM TP thiol demonstrated little toxicity toward three different
mammalian cell lines after 24 h. Furthermore, the presence of a free
thiol per se is not an impediment to successful drug development and
there are a number of marketed drugs used for chronic conditions that
bear a free thiol group. These include the angiotensin-converting en-
zyme (ACE) inhibitors, captopril and zofenopril (the latter a pro-drug
that is de-esterified to the active inhibitor, zofenoprilat), thiorphan,
which is the active metabolite of the antidiarrheal racecadotril (acet-
orphan), and tiopronin, which is used primarily for cystinuria. In mice,
these free-thiol drugs are essentially non-toxic in single oral dose tol-
erability tests with LD50 values > 2 g/kg (Imai et al., 1981; Challener,
2001) (http://www.drugfuture.com/toxic/q60-q161.html). Finally, an
erstwhile drug for schistosomiasis, Oltipraz, also contains a free thiol
and has found new life as an antisteatotic agent whereby oral doses of
60 mg/BID over 24 weeks were well-tolerated in a recent randomized
clinical trial (Kim et al., 2017) (www.clinicaltrials.gov: NCT01373554).
A second type of reactive structure, the quinone methide, is present
in the m/z 190 and 270 metabolites of TPT sulfonate. Quinone me-
thides are electrophiles and may react with glutathione and protein
thiol groups (Monks and Jones, 2002) or DNA (Wang et al., 2005).
Drugs that have quinone methide structures or which generate quinone
methide metabolites include the anti-estrogens (such as tamoxifen and
raloxifene) which are used in cancer treatment (Kalgutkar et al., 2005).
Some natural products and food additives, e.g., quercetin and butylated
hydroxytoluene (BHT), are also metabolized to quinone methides
(Dufrasne et al., 2011). Given that some of the drugs associated with
these metabolites can be dosed for periods of years, the possible hazards
of a brief exposure during acute treatment for schistosomiasis are not
clear.
Based on the full mouse PK (IV/PO) study, TPT sulfonate appears to
be fully absorbed when administered orally and is rapidly metabolized
by the liver with over 90% being metabolized in the first pass. To
identify TPT sulfonate metabolite(s), NLS MS/MS analysis was per-
formed with mouse plasma samples taken at various time points up to
4 h post oral dosing and with supernatants withdrawn from rat hepa-
tocytes incubated with TPT sulfonate for up to 30 min. Eight major
plasma metabolites (each with over 1% of total metabolite MS/MS peak
area) were found (Fig. 8) and structures were identified for six of these
(Fig. 9). Seven of the eight were also found in hepatocyte digests
(Fig. 8).
The m/z 210 thiol (TP thiol) and 176 styrene metabolites were se-
lected for synthesis and activity-testing against the parasite in vitro.
They were chosen because the m/z 210 is the first metabolite formed in
the metabolism scheme, and, thus, would be capable of generating all
downstream metabolites, whereas the m/z 176 appears to be most
abundant metabolite. Activity (decreased motility) was only demon-
strated for the TP thiol, a finding that extended to S. haematobium which
is often co-endemic with S. mansoni in Africa. The finding is important
as the current drug, PZQ, is active against all medically relevant
schistosomes. Furthermore, after IP administration, TP thiol was active
in vivo, significantly reducing the male worm burden, a result that is in
line with TP thiol's long plasma half-life (approximately 6 h) in mice
which would favor elimination of the parasite. The in vivo generation of
TP thiol is consistent with the earlier identification of a major disulfide
metabolite after oral administration of 2-(sec-butylamino)-1-octa-
nethiosulfuric acid to mice infected with S. mansoni (Penido et al.,
1995), although cidal activity was not directly linked to the particular
metabolite at that time. Even though TP thiol is isolated as a disulfide
dimer upon chemical synthesis, it would be reduced back to the thiol
monomer under most intracellular conditions (Fig. 9, Table S2). The in
vitro anti-schistosomal activity of TP thiol demonstrates that the para-
site might not be able to desulfate the TPT sulfonate parent drug, but
does not make clear whether the thiol is ultimately the active schisto-
somicide because the parasite's ability to metabolize it further has not
been determined.
A basic target profile has been suggested for new anti-schistosomal
drugs (Nwaka and Hudson, 2006; Caffrey, 2007) into which the current
compound, TPT sulfonate, fits reasonably well. Among the pharmaco-
logical criteria discussed, the compound should: (i) be active against all
major schistosome species infecting humans; (ii) be active against im-
mature flukes against which PZQ has little or no activity; (iii) be orally
active, preferably as a single dose; (iv) belong to a different chemical
class than PZQ; and (v) possess a mechanism of action different from
that of PZQ. Although the last point has yet to be clarified, we might
anticipate a different mechanism of action based on the quite different
chemistries and the gross phenotypic responses of the parasite in vitro to
the TP thiol (appearance of ‘flaccid-paralysis’) and PZQ (tetanic or
constricted paralysis; (Andrews et al., 1983; Andrews, 1985; Pica-
Mattoccia and Cioli, 2004; Abdulla et al., 2009). Attractive attributes of
TPT sulfonate are (i) ease of synthesis which is accomplished in three to
four steps using low-cost reagents and an overall yield of approximately
40%; and (ii) cost, whereby the reagents and solvents necessary for the
laboratory-scale synthesis of 10 mol (2890 g) of TPT sulfonate cost US$
1800.00. If bulk quantities of the reagents are used, the overall cost of
the product would likely be lower.
Looking forward, the identification of a parasiticidal metabolite is
significant as it not only facilitates an understanding of the chemistry
supporting the bioactivity but also allows the convenient in vitro as-
sessment of anti-parasite activity and identification of the mechanism of
action and/or target. The known susceptibility of the schistosome to
perturbation in redox conditions, including depletion of glutathione
levels (Bueding et al., 1982; Mkoji et al., 1990; Williams et al., 2013),
might be a good match for this prodrug's ability to form metabolites
with both oxidative (quinone methides) and reductive (a thiol and
conjugated phenyls) potential, and the capacity to mop up GSH in a
heterodisulfide. We are currently investigating the possible effects on
redox by TP thiol, including whether GSH levels are depleted in the
worm.
In regard of the pursuit of TPT sulfonate/TP thiol as a possible lead
anti-schistosomals, factors for further investigation, apart from defining
the mechanism of action discussed above, include the influence of
chirality on anti-parasite activity and the chemical modification of the
TP thiol scaffold to improve potency and drug-like properties. Among
the alterations being considered is a replacement and/or deletion of the
TP thiol is a free thiol putatively formed by cleavage of the
584

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
thiol to formally confirm its requirement for anti-parasite activity and,
if needed, for modified scaffolds. Also, constraining the central portion
of the molecule by incorporating a heterocycle would conceivably in-
crease the in vivo stability of active forms.
Colley, D.G., Wikel, S.K., 1974. Schistosoma mansoni: simplified method for the production
of schistosomules. Exp. Parasitol. 35, 44–51.
Coughtrie, M.W., Sharp, S., Maxwell, K., Innes, N.P., 1998. Biology and function of the
reversible sulfation pathway catalysed by human sulfotransferases and sulfatases.
Chem. Biol. Interact. 109, 3–27.
Cruciani, G., Baroni, M., Benedetti, P., Goracci, L., Fortuna, C.G., 2013. Exposition and
reactivity optimization to predict sites of metabolism in chemicals. Drug Discov.
Today Technol. 10, e155–165.
5. Conclusions
Cruciani, G., Crivori, P., Carrupt, P.-A., Testa, B., 2000a. Molecular fields in quantitative
structure–permeation relationships: the VolSurf approach. J. Mol. Struct.: Theochem
503, 17–30.
From 15 S-[2-(alkylamino)alkane] thiosulfuric acids that were pro-
filed in mice for oral efficacy against both mature and developing
Schistosoma mansoni, TPT sulfonate emerged as the most effective in
decreasing worm burdens. The efficacy spectrum determined is com-
petitive with that of PZQ. TP sulfonate must be biotransformed to re-
veal its anti-schistosomal activity. In mice, TPT sulfonate is fully ab-
sorbed and subject to rapid, non-CYP-mediated, first-pass metabolism.
The first of many metabolites produced, namely TP thiol, was synthe-
sized and shown to possess anti-schistosomal activity both in vitro and in
vivo. Investigations to understand TP thiol's mechanism(s) of action
and/or target are ongoing, as are efforts to improve its chemistry.
Cruciani, G., Pastor, M., Guba, W., 2000b. VolSurf: a new tool for the pharmacokinetic
optimization of lead compounds. Eur. J. Pharmaceut. Sci.: Official J. Eur. Fed.
Pharmaceut. Sci. 11 (Suppl. 2), S29–S39.
Davies, B., Morris, T., 1993. Physiological parameters in laboratory animals and humans.
Pharm. Res. (N. Y.) 10, 1093–1095.
de Oliveira Penido, M.L., Zech Coelho, P.M., de Mello, R.T., Pilo-Veloso, D., de Oliveira,
M.C., Kusel, J.R., Nelson, D.L., 2008. Antischistosomal activity of aminoalkanethiols,
aminoalkanethiosulfuric acids and the corresponding disulfides. Acta Trop. 108,
249–255.
Doenhoff, M.J., Sabah, A.A., Fletcher, C., Webbe, G., Bain, J., 1987. Evidence for an
immune-dependent action of praziquantel on Schistosoma mansoni in mice. Trans. R.
Soc. Trop. Med. Hyg. 81, 947–951.
Dufrasne, F., Gelbcke, M., Neve, J., Kiss, R., Kraus, J.L., 2011. Quinone methides and their
prodrugs: a subtle equilibrium between cancer promotion, prevention, and cure.
Curr. Med. Chem. 18, 3995–4011.
Conflicts of interest
Duvall, R.H., DeWitt, W.B., 1967. An improved perfusion technique for recovering adult
schistosomes from laboratory animals. Am. J. Trop. Med. Hyg. 16, 483–486.
Engel, J.C., Doyle, P.S., Dvorak, J.A., 1985. Trypanosoma cruzi: biological characterization
of clones derived from chronic chagasic patients. II. Quantitative analysis of the in-
tracellular cycle. J. Protozool. 32, 80–83.
The authors declared that there is no conflict of interest.
Acknowledgements
Glaser, J., Schurigt, U., Suzuki, B.M., Caffrey, C.R., Holzgrabe, U., 2015. Anti-schisto-
somal activity of cinnamic acid esters: eugenyl and thymyl cinnamate induce cyto-
plasmic vacuoles and death in schistosomula of Schistosoma mansoni. Molecules 20,
10873–10883.
We thank Juan C. Engel for advice regarding the counter-toxicity
screening experiments. D. L. Nelson was the recipient of a PVNS fel-
lowship from the Coordenação do Aperfeiçoamento de Pessoal de
Ensino Superior (CAPES). The research reported was supported by a
UCSF Clinical Translational Science Institute T1 Catalyst Award, and
the NIH-NIAID 1R01AI089896 and R21AI107390 awards.
Gőnnert, R., Andrews, P., 1977. Praziquantel, a new board-spectrum antischistosomal
agent. Z. Parasitenkd. 52, 129–150.
Hotez, P.J., 2018. Human parasitology and parasitic diseases: heading towards 2050.
Adv. Parasitol. 100, 29–38.
Hotez, P.J., Brindley, P.J., Bethony, J.M., King, C.H., Pearce, E.J., Jacobson, J., 2008.
Helminth infections: the great neglected tropical diseases. J. Clin. Invest. 118,
1311–1321.
Appendix A. Supplementary data
http://unitingtocombatntds.org/wp-content/themes/tetloose/app/staticPages/
fifthReport/files/fifth_progress_report_english.pdf, 2014.
Imai, K., Hayashi, Y., Hashimoto, K., 1981. Acute toxicological studies of captopril in rats
and mice. J. Toxicol. Sci. 6 (Suppl. 2), 179–188.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ijpddr.2018.10.004.
Jacobsen, W., Christians, U., Benet, L.Z., 2000. In vitro evaluation of the disposition of a
novel cysteine protease inhibitor. Drug Metab. Dispos. 28, 1343–1351.
Jaffe, I.A., 1986. Adverse effects profile of sulfhydryl compounds in man. Am. J. Med. 80,
471–476.
References
Kalgutkar, A.S., Gardner, I., Obach, R.S., Shaffer, C.L., Callegari, E., Henne, K.R., Mutlib,
A.E., Dalvie, D.K., Lee, J.S., Nakai, Y., O'Donnell, J.P., Boer, J., Harriman, S.P., 2005.
A comprehensive listing of bioactivation pathways of organic functional groups. Curr.
Drug Metabol. 6, 161–225.
Abdulla, M.H., Lim, K.C., Sajid, M., McKerrow, J.H., Caffrey, C.R., 2007. Schistosomiasis
mansoni: novel chemotherapy using a cysteine protease inhibitor. PLoS Med. 4 e14.
Abdulla, M.H., Ruelas, D.S., Wolff, B., Snedecor, J., Lim, K.C., Xu, F., Renslo, A.R.,
Williams, J., McKerrow, J.H., Caffrey, C.R., 2009. Drug discovery for schistosomiasis:
hit and lead compounds identified in a library of known drugs by medium-
throughput phenotypic screening. PLoS Neglected Trop. Dis. 3, e478.
Andrews, P., 1985. Praziquantel: mechanisms of anti-schistosomal activity. Pharmacol.
Ther. 29, 129–156.
Keiser, J., Chollet, J., Xiao, S.H., Mei, J.Y., Jiao, P.Y., Utzinger, J., Tanner, M., 2009.
Mefloquine-an aminoalcohol with promising antischistosomal properties in mice.
PLoS Neglected Trop. Dis. 3 e350.
Kim, W., Kim, B.G., Lee, J.S., Lee, C.K., Yeon, J.E., Chang, M.S., Kim, J.H., Kim, H., Yi, S.,
Lee, J., Cho, J.Y., Kim, S.G., Lee, J.H., Kim, Y.J., 2017. Randomised clinical trial: the
efficacy and safety of oltipraz, a liver X receptor alpha-inhibitory dithiolethione in
patients with non-alcoholic fatty liver disease. Aliment Pharmacol. Ther. 45,
1073–1083.
Andrews, P., Thomas, H., Pohlke, R., Seubert, J., 1983. Praziquantel. Med Res Rev 3,
147–200.
Basch, P.F., 1981. Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures
from cercariae and development until pairing. J. Parasitol. 67, 179–185.
Bisel, P., Al-Momani, L., Muller, M., 2008. The tert-butyl group in chemistry and biology.
Org. Biomol. Chem. 6, 2655–2665.
King, C.H., 2010. Parasites and poverty: the case of schistosomiasis. Acta Trop. 113,
95–104.
Klayman, D.L., Gilmore, W.F., 1964. The synthesis of N-substituted 2-aminoethanethio-
sulfuric acids. J. Med. Chem. 7, 823–824.
Blau, H.M., Pavlath, G.K., Hardeman, E.C., Chiu, C.P., Silberstein, L., Webster, S.G.,
Miller, S.C., Webster, C., 1985. Plasticity of the differentiated state. Science 230,
758–766.
Klayman, D.L., Grenan, M.M., Jacobus, D.P., 1969a. Potential antiradiation agents. I.
Primary aminoalkanethiosulfuric acids. J. Med. Chem. 12, 510–512.
Klayman, D.L., Grenan, M.M., Jacobus, D.P., 1969b. Potential antiradiation agents. II.
Guanidinoalkanethiosulfuric acids. J. Med. Chem. 12, 723–725.
Kraemer, M., Vassy, J., Brighton, V., Fuller, S., Yeoh, G., 1986. The effect of dex-
amethasone on transferrin secretion by cultured fetal rat hepatocytes. Eur. J. Cell
Biol. 42, 52–59.
Bojarova, P., Williams, S.J., 2008. Sulfotransferases, sulfatases and formylglycine-gen-
erating enzymes: a sulfation fascination. Curr. Opin. Chem. Biol. 12, 573–581.
Botros, S., Pica-Mattoccia, L., William, S., El-Lakkani, N., Cioli, D., 2005. Effect of pra-
ziquantel on the immature stages of Schistosoma haematobium. Int. J. Parasitol. 35,
1453–1457.
Lau, Y.Y., Sapidou, E., Cui, X., White, R.E., Cheng, K.C., 2002. Development of a novel in
vitro model to predict hepatic clearance using fresh, cryopreserved, and sandwich-
cultured hepatocytes. Drug Metab. Dispos. 30, 1446–1454.
Brindley, P.J., Sher, A., 1987. The chemotherapeutic effect of praziquantel against
Schistosoma mansoni is dependent on host antibody response. J. Immunol. 139,
215–220.
Lipinski, C.A., Lombardo, F., Dominy, B.W., Feeney, P.J., 2001. Experimental and com-
putational approaches to estimate solubility and permeability in drug discovery and
development settings. Adv. Drug Deliv. Rev. 46, 3–26.
Bueding, E., Dolan, P., Leroy, J.P., 1982. The antischistosomal activity of oltipraz. Res.
Commun. Chem. Pathol. Pharmacol. 37, 293–303.
Caffrey, C.R., 2007. Chemotherapy of schistosomiasis: present and future. Curr. Opin.
Chem. Biol. 11, 433–439.
Long, T., Neitz, R.J., Beasley, R., Kalyanaraman, C., Suzuki, B.M., Jacobson, M.P.,
Dissous, C., McKerrow, J.H., Drewry, D.H., Zuercher, W.J., Singh, R., Caffrey, C.R.,
2016. Structure-bioactivity relationship for benzimidazole thiophene inhibitors of
polo-like kinase 1 (PLK1), a potential drug target in Schistosoma mansoni. PLoS
Neglected Trop. Dis. 10, e0004356.
Caffrey, C.R., 2015. Schistosomiasis and its treatment. Future Med. Chem. 7, 675–676.
Carey, J.S., Laffan, D., Thomson, C., Williams, M.T., 2006. Analysis of the reactions used
for the preparation of drug candidate molecules. Org. Biomol. Chem. 4, 2337–2347.
Challener, C., 2001. Chrial Drugs. Ashgate Publishing Ltd., Aldeshot, UK.
Colley, D.G., Bustinduy, A.L., Secor, W.E., King, C.H., 2014. Human schistosomiasis.
Lancet 383, 2253–2264.
Long, T., Rojo-Arreola, L., Shi, D., El-Sakkary, N., Jarnagin, K., Rock, F., Meewan, M.,
Rascon Jr., A.A., Lin, L., Cunningham, K.A., Lemieux, G.A., Podust, L., Abagyan, R.,
585

A.R. Wolfe et al.
IJP:DrugsandDrugResistance8(2018)571–586
Ashrafi, K., McKerrow, J.H., Caffrey, C.R., 2017. Phenotypic, chemical and functional
Chemical and genetic validation of the statin drug target to treat the helminth dis-
characterization of cyclic nucleotide phosphodiesterase 4 (PDE4) as a potential an-
thelmintic drug target. PLoS Neglected Trop. Dis. 11, e0005680.
Marcellino, C., Gut, J., Lim, K.C., Singh, R., McKerrow, J., Sakanari, J., 2012. WormAssay:
a novel computer application for whole-plate motion-based screening of macroscopic
parasites. PLoS Neglected Trop. Dis. 6 e1494.
ease, schistosomiasis. PloS One 9, e87594.
Rowland, A., Miners, J.O., Mackenzie, P.I., 2013. The UDP-glucuronosyltransferases:
their role in drug metabolism and detoxification. Int. J. Biochem. Cell Biol. 45,
1121–1132.
Sabah, A.A., Fletcher, C., Webbe, G., Doenhoff, M.J., 1986. Schistosoma mansoni: che-
motherapy of infections of different ages. Exp. Parasitol. 61, 294–303.
Sassine, A., Martins-Junior, H.A., Lebre, D.T., Valli, F., Pires, M.A., Vega, O., Felinto,
M.C., 2008. An electrospray ionization tandem mass spectrometric study of p-tert-
butylcalix[6]arene complexation with ammonium hydroxide, and ammonium and
sodium ions. Rapid Commun. Mass Spectrom.: RCM (Rapid Commun. Mass
Spectrom.) 22, 385–393.
Mkoji, G.M., Smith, J.M., Prichard, R.K., 1990. Effect of oltipraz on the susceptibility of
adult Schistosoma mansoni to killing by mouse peritoneal exudate cells. Parasitol. Res.
76, 435–439.
Monks, T.J., Jones, D.C., 2002. The metabolism and toxicity of quinones, quinonimines,
quinone methides, and quinone-thioethers. Curr. Drug Metabol. 3, 425–438.
Moreira, L.S., Pilo-Veloso, D., de Mello, R.T., Coelho, P.M., Nelson, D.L., 2007. A study of
the activity of 2-(alkylamino)-1-phenyl-1-ethanethiosulfuric acids against infection
by Schistosoma mansoni in a murine model. Trans. R. Soc. Trop. Med. Hyg. 101,
385–390.
Smith, R., Jones, R.D., Ballard, P.G., Griffiths, H.H., 2008. Determination of microsome
and hepatocyte scaling factors for in vitro/in vivo extrapolation in the rat and dog.
Xenobiotica 38, 1386–1398 the fate of foreign compounds in biological systems.
Sohlenius-Sternbeck, A.K., 2006. Determination of the hepatocellularity number for
human, dog, rabbit, rat and mouse livers from protein concentration measurements.
Toxicol. Vitro 20, 1582–1586.
Moreira, L.S., Piló-Veloso, D., Nelson, D.L., 2000. Synthesis of 2-(alykylamino)-1-phe-
nylethane-1-thiosulfuric acids. potential schistosomicides Quimica Nova 23,
447–452.
Nakabayashi, H., Taketa, K., Yamane, T., Miyazaki, M., Miyano, K., Sato, J., 1984.
Phenotypical stability of a human hepatoma cell line, HuH-7, in long-term culture
with chemically defined medium. Gann 75, 151–158.
Štefanić, S., Dvořák, J., Horn, M., Braschi, S., Sojka, D., Ruelas, D.S., Suzuki, B., Lim, K.C.,
Hopkins, S.D., McKerrow, J.H., Caffrey, C.R., 2010. RNA interference in Schistosoma
mansoni schistosomula: selectivity, sensitivity and operation for larger-scale
screening. PLoS Neglected Trop. Dis. 4 e850.
Nelson, D.L., Pellegrino, J., 1976. Experimental chemotherapy of schistosomiasis XII.
Active derivatives of aminoethanethiosulfuric acids. Rev. Inst. Med. Trop. Sao Paulo
18, 365–370.
Utzinger, J., N'Goran E, K., Caffrey, C.R., Keiser, J., 2011. From innovation to application:
social-ecological context, diagnostics, drugs and integrated control of schistoso-
miasis. Acta Trop. 120 S121-137.
Nelson, D.L., Veloso, D.P., Penido, M.L.O., Cardoso, M.G., Alcântara, A.F.C., 1989.
Synthesis of potential schistosomicides: 2-(N-alkylamino)-1-alkanethiosulfuric acids
and related thiols and disulfides. In: 31st National Organic Symposium, Ithaca, N Y,
pp. 25.
Veber, D.F., Johnson, S.R., Cheng, H.Y., Smith, B.R., Ward, K.W., Kopple, K.D., 2002.
Molecular properties that influence the oral bioavailability of drug candidates. J.
Med. Chem. 45, 2615–2623.
Nwaka, S., Hudson, A., 2006. Innovative lead discovery strategies for tropical diseases.
Nat. Rev. Drug Discov. 5, 941–955.
Venturello, C., Alneri, E., Ricci, M., 1983. A new, effective catalytic system for epox-
idation of olefins by hydrogen peroxide under phase-transfer conditions. J. Org.
Chem. 48, 3831–3833.
Pellegrino, J., Siqueira, A.F., 1956. A perfusion technic for recovery of Schistosoma
mansoni from experimentally infected Guinea pigs. Rev. Bras. Malariol. Doencas Trop.
8, 589–597.
Wang, P., Song, Y., Zhang, L., He, H., Zhou, X., 2005. Quinone methide derivatives:
important intermediates to DNA alkylating and DNA cross-linking actions. Curr. Med.
Chem. 12, 2893–2913.
Penido, M.L., Coelho, P.M., Nelson, D.L., 1999. Efficacy of a new schistosomicidal agent
2-[(methylpropyl)amino]-1-octanethiosulfuric acid against an oxamniquine resistant
Schistosoma mansoni isolate. Mem. Inst. Oswaldo Cruz 94, 811–813.
Penido, M.L., Nelson, D.L., Vieira, L.Q., Coelho, P.M., 1994. Schistosomicidal activity of
alkylaminooctanethiosulfuric acids. Mem. Inst. Oswaldo Cruz 89, 595–602.
Penido, M.L., Nelson, D.L., Vieira, L.Q., Watson, D.G., Kusel, J.R., 1995. Metabolism by
Schistosoma mansoni of a new schistosomicide: 2-[(1-methylpropyl)amino]-1-octa-
nethiosulphuric acid. Parasitology 111 (Pt 2), 177–185.
Weeks, J.C., Roberts, W.M., Leasure, C., Suzuki, B.M., Robinson, K.J., Currey, H.,
Wangchuk, P., Eichenberger, R.M., Saxton, A.D., Bird, T.D., Kraemer, B.C., Loukas,
A., Hawdon, J.M., Caffrey, C.R., Liachko, N.F., 2018. Sertraline, paroxetine, and
chlorpromazine are rapidly acting anthelmintic drugs capable of clinical repurposing.
Sci. Rep. 8, 975.
Williams, D.L., Bonilla, M., Gladyshev, V.N., Salinas, G., 2013. Thioredoxin glutathione
reductase-dependent redox networks in platyhelminth parasites. Antioxidants Redox
Signal. 19, 735–745.
Pica-Mattoccia, L., Cioli, D., 2004. Sex- and stage-related sensitivity of Schistosoma
mansoni to in vivo and in vitro praziquantel treatment. Int. J. Parasitol. 34, 527–533.
Rheinberg, C.E., Mone, H., Caffrey, C.R., Imbert-Establet, D., Jourdane, J., Ruppel, A.,
1998. Schistosoma haematobium, S. intercalatum, S. japonicum, S. mansoni, and S.
rodhaini in mice: relationship between patterns of lung migration by schistosomula
and perfusion recovery of adult worms. Parasitol. Res. 84, 338–342.
Rojo-Arreola, L., Long, T., Asarnow, D., Suzuki, B.M., Singh, R., Caffrey, C.R., 2014.
Xiao, S.H., Yue, W.J., Yang, Y.Q., You, J.Q., 1987. Susceptibility of Schistosoma japonicum
to different developmental stages to praziquantel. Chin Med J (Engl) 100, 759–768.
Zanger, U.M., Schwab, M., 2013. Cytochrome P450 enzymes in drug metabolism: reg-
ulation of gene expression, enzyme activities, and impact of genetic variation.
Pharmacol. Ther. 138, 103–141.
586