Quinolone amides as antitrypanosomal lead compounds with In Vivo activity

Article abstract of DOI:10.1128/AAC.01757-15

Human African trypanosomiasis (HAT) is a major tropical disease for which few drugs for treatment are available, driving the need for novel active compounds. Recently, morpholino-substituted benzyl amides of the fluoroquinolone-type antibiotics were identified to be compounds highly active against Trypanosoma brucei brucei. Since the lead compound GHQ168 was challenged by poor water solubility in previous trials, the aim of this study was to introduce structural variations to GHQ168 as well as to formulate GHQ168 with the ultimate goal to increase its aqueous solubility while maintaining its in vitro antitrypanosomal activity. The pharmacokinetic parameters of spray-dried GHQ168 and the newly synthesized compounds GHQ242 and GHQ243 in mice were characterized by elimination half-lives ranging from 1.5 to 3.5 h after intraperitoneal administration (4 mice/compound), moderate to strong human serum albumin binding for GHQ168 (80%) and GHQ243 (45%), and very high human serum albumin binding (>99%) for GHQ242. For the lead compound, GHQ168, the apparent clearance was 112 ml/h and the apparent volume of distribution was 14 liters/kg of body weight (BW). Mice infected with T. b. rhodesiense (STIB900) were treated in a stringent study scheme (2 daily applications between days 3 and 6 postinfection). Exposure to spray-dried GHQ168 in contrast to the control treatment resulted in mean survival durations of 17 versus 9 days, respectively, a difference that was statistically significant. Results that were statistically insignificantly different were obtained between the control and the GHQ242 and GHQ243 treatments. Therefore, GHQ168 was further profiled in an early-treatment scheme (2 daily applications at days 1 to 4 postinfection), and the results were compared with those obtained with a control treatment. The result was statistically significant mean survival times exceeding 32 days (end of the observation period) versus 7 days for the GHQ168 and control treatments, respectively. Spray-dried GHQ168 demonstrated exciting antitrypanosomal efficacy.

Full text of DOI:10.1128/AAC.01757-15

AAC Accepted Manuscript Posted Online 2 May 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.01757-15
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1
2
Quinolone amides as anti-trypanosomal lead compounds with in vivo activity
3
Georg Hiltensperger,a Nina Hecht,a Marcel Kaiser,b,c Jens-Christoph Rybak,a Alexander Hoerst,a
Nicole Dannenbauer,d Klaus Müller-Buschbaum,d Heike Bruhn,e Harald Esch,a
Leane Lehmann,a Lorenz Meinel,a Ulrike Holzgrabea#
4
5
6
7
Universität Würzburg, Institut für Pharmazie und Lebensmittelchemie, Am Hubland, 97074
Würzburg, Germanya; Swiss Tropical and Public Health Institute, Parasite Chemotherapy,
Socinstr. 57, 4002 Basel, Switzerlandb; Universität Basel, Peterplatz 1, 4003 Basel, Switzerlandc;
Universität Würzburg, Institut für Anorganische Chemie, Am Hubland, 97074 Würzburg,
Germanyd; Universität Würzburg, Institut für Molekulare Infektionsbiologie, Josef-Schneider-
Straße 2/Bau D15, 97080 Würzburg, Germanye.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
#Corresponding author: Prof. Dr. Ulrike Holzgrabe, +49-931-3185461,
ulrike.holzgrabe@uni-wuerzburg.de
G.H. and N.H. contributed equally to this work.
Supplemental material for this article may be found at http://aac.asm.org/
Running title: Anti-trypanosomal quinolone amides
22 Keywords: Trypanosoma; quinolone amides; formulation; in vivo activity; cytotoxicity;
23 pharmacokinetics; metabolism
Page 1 of 34

24
25
26
27
ABSTRACT
Human African Trypanosomiasis (HAT) is a major tropical disease with few available drugs for
treatment driving the need for novel active compounds. Recently, morpholino-substituted benzyl
amides of the fluoroquinolone-type antibiotics were identified as compounds highly active
28 against Trypanosoma brucei brucei. Since the lead compound GHQ168 was challenged by poor
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
water solubility in previous trials, the aim of this study was to introduce structural variation to
GHQ168 as well as formulating GHQ168 with the ultimate goal to increase the aqueous
solubility while maintaining in vitro anti-trypanosomal activity. Pharmacokinetic parameters in
mice for spray-dried GHQ168 and the newly synthesized compounds GHQ242 and GHQ243
were characterized by an elimination half-life ranging from 1.5 to 3.5 hours after i.p.
administration (4 mice / compound), moderate to strong human serum albumin binding for
GHQ168 (80 %) and GHQ243 (45 %) and very high binding for GHQ242 (> 99 %). For the lead
compound GHQ168, an apparent clearance was 112 mL/h and an apparent volume of
distribution 14 L / kg body weight (BW). Mice infected with T.b.rhodesiense (STIB900) were
treated in a stringent study scheme (2 daily applications between days 3-6 post infection).
Exposure to spray dried GHQ168 in contrast to control resulted in 17 versus 9 and statistically
significant differences in mean survival days, respectively. Insignificant results were obtained
between control and GHQ242 and GHQ243. Therefore, GHQ168 was further profiled in an
early-treatment scheme (2 daily applications at days 1-4 post infection) against control, resulting
in statistically significant mean survival times exceeding 32 days (end of the observation period)
versus 7 days. Spray dried GHQ168 demonstrated exciting anti-trypanosomal efficacy.
Page 2 of 34

45
46
47
INTRODUCTION
Human African Trypanosomiasis (HAT), also known as sleeping sickness, is caused by
Trypanosoma brucei rhodesiense (T.b. rhodesiense) and T.b. gambiense. It is widely spread in
48 Eastern and Southern Africa (T.b. rhodesiense) and in Western and Central Africa (T.b.
49
50
51
52
53
54
55
56
57
58
59
60
61
gambiense). In spite of currently decreasing numbers of new infections in humans, these regions
are threatened by a large reservoir of parasites in cattle, horses and other non-mammalian
species, creating an imminent risk of the next outbreak of HAT (1). In addition, a rapid
development of resistance can be observed (2). Moreover, as the available drugs, such as
suramine, pentamidine, melarsoprol or eflornithine, are either active against one of the two
stages of HAT only, acute or chronic form, or exhibit dangerous adverse events, new anti-HAT
drugs are urgently needed. Thus, it is important - perhaps indispensable - to rigorously expand
the arsenal of potent and safe antiparasitic compounds with demonstrated efficacy for HAT (3).
Currently, two new drugs are in clinical trials, i.e. the 5-nitroimidazole fexinidazole (phase II/III)
and the oxaborole SCYX-6759 (phase I) with promising initial results (4, 5). However,
significant hurdles remain to be solved before these new drug candidates may become
commercially available.
By means of a medium throughput screening campaign we recently discovered a new group of
62 anti-trypanosomal compounds derived from the fluoroquinolones, the latter being active against
63
64
65
66
67
most gram-positive and gram-negative bacteria species, but not against T. b. brucei (6-9). The
replacement of the carboxylic acid functionality, which is important for the inhibitory activity
towards many classes of bacterial topoisomerases, by a benzyl amide group resulted in a novel
compound library. The newly synthesized compounds are active towards T.b. brucei and T.b.
rhodesiense at nanomolar concentrations (10) without cell toxicity as assessed in macrophages.
Page 3 of 34

68
69
70
71
72
73
74
Structure-activity studies revealed the quinolone carrying a butyl chain in position N1, an N-
benzyl group at the amido function in position 3 and a morpholino ring in position 7, to be the
most active compound, referred to as GHQ168 (Fig. 1).
Initial biological studies hinted to the influence of the active compound GHQ168 (compound 29
in ref. 10) on the morphology of the mitochondria and on the hindered segregation of the
kinetoplast (10). In contrast to quinolone carboxylic acids being active against the trypanosoma
topoisomerase II (from T.b. brucei) (7, 8, 11), we could show that the corresponding amides do
75 not affect the topoisomerase in trypanosoma (10). Taken together, the mode of action of
76
77
78
79
80
81
82
83
84
85
86
87
88
89
GHQ168 remains to be elucidated.
Even though GHQ168 has favorable structural properties (logP < 5 (GHQ168: 3.8), molecular
mass < 500 g/mol (GHQ168: 437.5 g/mol), 5 hydrogen bond donors (GHQ168: 1) and 10
hydrogen bond acceptors (GHQ168: 7)), predicting oral bioavailability (11), this compound
showed very poor aqueous solubility. Thus, in order to overcome the solubility problem in the
mice experiments, the compound was administered intra-peritoneally (i.p.) as a lipid formulation.
However, these previous experiments had to be stopped due to low tolerance of the vehicle by
the test animals (10).
Within this study, we aimed at the development of fluoroquinolone amide compounds with
improved solubility by chemical modification. Furthermore, we developed a formulation for
GHQ168 with the same goal of meeting the necessary solubility requirements. The compounds
were characterized with regard to their physicochemical properties, in vitro anti-trypanosomal
activity, and in vitro toxicity. Three of the synthesized compounds were selected and analyzed
with regard to intestinal absorption (Caco-2 model) for a feasibility assessment of future oral
Page 4 of 34

90
dosage forms. Furthermore, these three compounds were profiled for in vivo pharmacokinetics
91 (PK) and in vivo anti-trypanosomal activity in infected mice.
92
93 MATERIAL AND METHODS
94
95
96
Materials. Reference substances (used for the determination of lipophilicity,
permeability, in vitro activity, and metabolism), excipients and reagents were purchased from
Sigma Aldrich, Taufkirchen, Germany, and were of analytical or pharmaceutical grade unless
97 noted otherwise. Poly(methacrylic acid-co-methyl methacrylate) (Eudragit® L100, approx.
98
99
Mw=125 000 g/mol) for formulation preparation was from Evonik, Essen, Germany. Liquid
chromatography columns were obtained from Phenomenex, Aschaffenburg, Germany (Synergi
MAX-RP, Kinetex, Luna) and from MZ-Analytical, Mainz, Germany (Inertsil ODS-2).
PLTK4710 ultrafiltration membrane for albumin binding was purchased from Millipore,
100
101
102 Schwalbach, Germany. Caco-2 cells were from ATCC, PET-membrane was from Greiner Bio-
103
104
105
106
One, Frickenhausen, Germany; Dulbecco's Modified Eagle Medium (DMEM) high glucose with
L-glutamine, non-essential amino acids and penicillin / streptomycin solution were from
GibcoLife, Darmstadt, Germany. For cytotoxicity assays, all cells were purchased from ATCC,
Wesel, Germany, RPMI medium from GibcoLife, Darmstadt, Germany, and Fetal calf serum
107 (FCS) from PAA GE Healthcare, Freiburg, Germany. For metabolism and mutagenesis
108
109
110
111
112
experiments, NADP sodium salt was obtained from Applichem, Darmstadt, Germany, MS-grade
solvents from VWR, Ismaning, Germany, and all other chemicals including cell culture medium
and supplements were from Sigma-Aldrich, Taufkirchen, Germany or Roth, Karlsruhe Germany
unless specified otherwise. For metabolic stability testing, S9-fraction was purchased from MP
Biomedicals, Illkirch-Graffenstaden, France; Bradford Reagent was from BioRad, Munich,
Page 5 of 34

113
114
115
116
117
Germany. For mutagenicity testing fetal calf serum was purchased from Invitrogen, Karlsruhe,
Germany and culture flasks and tissue culture dishes were from Greiner Bio-One, Frickenhausen,
Germany. For plasma stability assessment, human plasma was obtained from Bayerisches Rotes
Kreuz, Munich, Germany. For the analysis of blood samples, solvents were purchased from
Fisher Scientific, Schwerte, Germany.
118
119
120
121
Synthesis. The synthesis of GHQ168 was performed according to (10). The synthesis of
the other related salt compounds presented herein (GHQ237, GHQ242, GHQ243, GHQ250,
GHQ232, GHQ215) can be found in SI. The stoichiometry of the substances and the respective
counterion was determined using CHN-analysis.
122
Formulation. A solid dispersion was produced using a Nano Spray Dryer B-90 (Büchi,
123 Essen, Germany). GHQ168 (0.2 %; w/V) and Eudragit® L100 (2.0 %; w/V) were dissolved in
124
125
126
127
128
methanol, resulting in a 1+10 (w/w) spray dried formulation. Spray drying was performed with
the head in vertical position through 7 µm mesh size in an air flow set at 150 L/min, inlet
temperature of 60 °C, outlet temperature of 34 °C, pressure of 52 mbar and at a spray rate of
100 %. The resulting white, amorphous nanoparticles were stored in a desiccator over silica gel
at room temperature.
129
130
131
132
133
Solubility. A preliminary determination for the fast and approximate assessment of
kinetic solubility was achieved by diluting DMSO stock solutions of desired compounds (1 mM,
20 mM) into PBS buffer (pH 7.4) in order to give aqueous solutions with concentrations of 10,
100, 500, 1000, 1500, 2000, 2500, 3000, 4000 and 5000 µM (n=1). Precipitation was detected
microscopically (SZ-PT, Olympus, Shinjuku, Japan).
Page 6 of 34

134
135
136
137
138
139
140
141
142
In addition, a more precise determination of kinetic solubility was conducted for those
compounds applied in the efficacy studies. “Kinetic solubility” refers to the apparent solubility of
a metastable state, at which in spite of the fact that the chemical potential of the compound in
solution exceeds the chemical potential of the solid compound, precipitation is so slow that a
(kinetic) solubility exceeding the equilibrium solubility may be observed.
As described above, dilution series were prepared for each compound but with closer
concentration steps (GHQ168: 5, 10, 15, 20, 25, 30, 35 µg/mL; GHQ242: 150, 200, 250, 300,
350, 400, 450, 500 µg/mL; GHQ243: 50, 100, 125, 150, 175, 200, 250, 300 µg/mL; n=3, each)
so as to end in a maximum of 2.5 % residual DMSO content. The detection was accomplished
143 nephelometrically (NEPHEOLOstar BMG, Ortenberg, Germany) using 96-well plates with a flat
144
145
146
147
148
149
150
151
152
153
154
155
156
bottom (Greiner Bio-One, Frickenhausen, Germany). The temperature was set to 37 °C, laser
intensity was 80 % and laser beam focus was 2.20 mm. The gain was adjusted to 60 (GHQ168,
GHQ242) and 75 (GHQ243), respectively, and the measurement time per well was 0.1 s. The
mean result of three dilution series was determined. Two replicate measurements of the same
solutions were performed (time frame 30 minutes) and SD was calculated from the means of
three replicate measurements over time.
In contrast to the kinetic solubility, the thermodynamic solubility (also called “equilibrium”
solubility) describes a thermodynamically stable state which might take its time to be generated
but will be maintained when environmental conditions remain unchanged. The determination of
thermodynamic solubility of GHQ168 was conducted by dosing solid substance (in excess) into
2 mL Eppendorf vials followed by a dissolution into PBS buffer (pH 7.4). Throughout the assay,
continuous shaking (800 rpm) and a constant temperature (37 °C) were maintained (Eppendorf
Thermomixer; Eppendorf AG, Hamburg, Germany). Samples were taken after 10 min, 30 min,
Page 7 of 34

157
158
159
160
161
60 min, 120 min and 1200 min followed by centrifugation (13.000 rpm, 1 min; Micro 2416,
VWR International, Darmstadt, Germany) and the HPLC-UV (Jasco, Groß-Umstadt, Germany)
analysis of the supernatant (column: Synergi MAX-RP 80Å, 4 µm, 150 x 4.6 mm; mobile phase:
acetonitrile/water 72/28; temperature: 40 °C, flow rate: 1.2 mL/min; injection volume: 20 µl;
detection: 280 nm) was performed. Solubility determination was performed in triplicate.
162
163
164
165
166
167
168
169
170
171
X-ray diffractometry. X-ray powder diffractograms were recorded on a X-ray powder
diffraction (XRPD) (Bruker D8 Discover, Karlsruhe, Germany) using a copper tube operating at
40 kV and 40 mA. A focusing Goebel mirror was installed in the primary beam path (slit:
0.6 mm, axial soller: 2.5°). For the secondary beam path, no slit was applied and axial soller was
set to 2.5°. Detection was done using a LynxEye® (Bruker AXS) 1D-detector. The investigation
was performed in ‘Coupled Two Theta/Theta’ mode, with a 2Θ range of 5 – 45°, a step width of
0.025° and 1.0 seconds measurement time per step. Data collection and processing was
conducted with the software packages DIFFRAC.Suite (V2 2.2.690, Bruker AXS 2009-2011)
and DIFFRAC.EVA (Version 3.0, Bruker AXS 2010-2013). Method details for single crystal
structure analysis can be found in the supplemental material.
172
173
174
175
Scanning electron microscopy (SEM). The scanning electron microscopy (JSM-7500F,
Jeol, Japan) was performed with an accelerating voltage of 2.0 kV and a 1000-fold magnification
at a working distance of 8.6 mm. Prior to the examination, the samples were sputter coated with
gold.
176
177
178
Physicochemical parameters. LogP data were recorded using HPLC-UV (C18 reversed-
phase Inertsil ODS-2, 5 µm, 150 × 4.6 mm; mobile phase: phosphate-buffer 10 mM (pH 7.4) /
methanol (containing 0.02 % N,N-dimethylhexylamine) = 30/70; temperature: 30 °C; flow: 1.5
Page 8 of 34

179
mL/min; injection volume: 40 µl; detection wavelength = 254 nm) as described previously (10).
180 Compounds with known logP values (2-phenylethanol, benzene, N,N-dimethylaniline, toluene,
181
182
183
184
185
186
chlorbenzene, ethylbenzene, biphenyl and anthracene) served as reference substances. The
capacity factor (k’) that correlates with logP values, can be derived from the compounds
retention times (tR) and dead time (t0) by the following equation: k´= (tR – to)/to.
Experimental determination of pKA was conducted for GHQ168, GHQ242 and GHQ243 on a
Sirius T3 instrument (Sirius, Forest Row, UK). Due to its low aqueous solubility the pKA
determination of GHQ168 was conducted from a DMSO stock solution (10 µM) using the assay
187 type “fast UV-metric pKA” (pKA assessment is accomplished by the identification of changes in
188
189
the absorption profile due to sample ionization). For GHQ242 and GHQ243 the solid compounds
(0.43 mg - 0.56 mg) were dosed into the instrument and the assay type “pH-metric pKA”
190 (potentiometric pKA assessment using a pH-electrode) was applied. 1.5 mL of potassium
191
192
193
194
chloride solution (0.15 M) was added to the compounds and temperature was maintained at
25 °C throughout the assay. The titrations were conducted over the pH range of 2-12, starting at
low pH values (acidification prior to titration with hydrochloric acid 0.5 M; titration with
potassium hydroxide solution 0.5 M).
195
196
197
Differential Scanning Calorimetry (DSC). Melting point determinations were
performed using a DSC 8000 Instrument (Perkin Elmer, Waltham, USA). Scanning rate was set
to 20 K/min over the range of -50 °C to 300 °C.
198
199
200
Serum Albumin binding. In principle, albumin binding was determined in vitro by
means of the continuous titration methods as described herein (12). In brief, the self-made
instrument consists of a low-pressure HPLC pump (Bischoff, Leonberg, Germany), an injection
Page 9 of 34

201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
valve (Rheodyne, Alsbach, Germany) for injection of the human serum albumin (HSA) solution
into the system, the ultrafiltration cell and an UV detector (Knauer, Berlin, Germany). The
ultrafiltration cell contains the PLTK4710 ultrafiltration membrane with a molecular weight cut-
off of 30 kDa. HSA and the test compounds were dissolved in a 0.03 M phosphate buffer (pH
7.4) containing 0.1 M NaCl for the simulation of physiological conditions. In the case of
GHQ168, the substance was dissolved in dimethylformamide (1 mg/mL) and 420 µl of this
solution was diluted with 2 mM polysorbate 20 in buffer to 100.0 mL. The concentration of the
drug solutions was within the range of 10 to 20 mg/L. Initially, the drug solution was
continuously pumped through the cell and the measured absorption curves were plotted versus
time. The system was rinsed with buffer and subsequently the albumin solution (40 mg/mL) was
injected into the system. The ultrafiltration membrane retains the protein in the cell. The drug
solution is pumped again through the ultrafiltration cell and the absorbance is measured again.
The interaction between drug and HSA present in the cell leads to a shift of the second curve to
the right compared to the first recorded curve. The size of the area between the two drug curves
is proportional to the amount of drug bound to the HSA.
216
Permeability through Caco-2 monolayers. The evaluation of the permeability was in
217 principle performed as reported previously (13). In brief, Caco-2 cells with passage number 51
218 were thawed, passaged four times and seeded with 1.3 x 105 cells/cm2 on 24 well cell culture
219
220
221
222
223
inserts with PET-membrane, pore size 0.4 µM. The cells were cultivated in DMEM high glucose
with L-glutamine, non-essential amino acids and penicillin / streptomycin solution for 23 days at
37 °C and 5 % CO2. Transepithelial electric resistance (TEER) in DMEM was monitored three
times a week during medium change (electrode: EVOM2, World Precision Instruments, USA).
Prior to the permeability experiment, the cells were washed twice with Hanks balanced salt
Page 10 of 34

224
225
226
227
228
solution (HBSS), 25 mM Hepes pH 7.4 and TEER was measured in order to assess the
monolayer integrity (cells with TEER values < 200 Ohm*cm2 were excluded from the
experiment). For the permeability testing, 150 µl of 20 µM GHQ168, GHQ242 and GHQ243,
respectively, in HBSS / 0.5 % DMSO were added to the apical compartment and 600 µl of HBSS
were added into the basolateral compartment followed by gentle shaking for 1 h at 37 °C, 5 %
229 CO2. The cells were washed with HBSS and the integrity of the monolayer was reconfirmed by
230
231
232
233
234
235
TEER measurement. Fluorescein isothiocyanate dextran was used as non-permeable negative
control and propranolol HCl as high-permeable positive control. For each compound, at least
three independent Caco-2 cell assays were conducted within one day. The concentration of
fluorescein isothiocyanate dextran was determined fluorimetrically (Perkin Elmer, Luminescence
Spectrometer LS50B, Excitation WL: 490 nm, Emission WL: 514 nm). All other compounds
were analyzed by LC/MS/MS using the analytical method described under “Pharmacokinetics”.
236
237
In vitro activity. See SI: The methods as described in Räz et al., Baltz et al.,
Papadopoulou et al., Larson et al. and Muth et al. (14-17) were applied.
238
239
Cytotoxicity See SI.
240
241
242
243
244
Metabolism and Mutagenesis. Phase I metabolism by cytochrome-P450 dependent
monooxygenase of GHQ168 was investigated in rat liver microsomes. Microsomes were
prepared by ultracentifugation (100000 g, 60 min, 4 °C) from commercially available S9-fraction
of Aroclor 1254 treated male Sprague-Dawley rats. After resuspension in HEPES buffer (25 mM
HEPES, 100 mM NaCl, 1.5 mM disodium EDTA, 1 mM dithiothreitol, and 10% Glycerol, pH
Page 11 of 34

245
246
247
248
7.4) protein concentration was determined with Bradford Reagent yielding 7 mg/mL, and
microsomes were stored at −80 °C until use.
Microsomal incubations (final volume 500 µl) at 37 °C for 30, 60, and 90 min contained
GHQ168 (100 µM, 1 % DMSO), microsomal protein (1 mg/mL), 0.1 M phosphate buffer (pH
249 7.4), and NADPH-generating system. The NADPH-generating system was prepared from
250
251
252
253
254
255
256
257
isocitrate (10 mM), isocitrate dehydrogenase (0.05 U), MgCl2 (4 mM), NADP (1 mM) and was
preincubated for 5 min at 37 °C prior addition to the incubation system. Control incubations
were carried out under the same conditions with heat-deactivated microsomes.
After the incubation period, the reaction was stopped by addition of ethyl acetate. Then, 7-(4-
acetylpiperazin-1-yl)-N-(2,4-dichlorobenzyl)-6-fluoro-1-(2-fluorophenyl)-4-oxo-1,4-
dihydroquinoline-3-carboxamide, which is structurally similar to GHQ168, was added as internal
standard and the reaction mixture was extracted three times with ethyl acetate. After evaporation
of the solvent, the residues were dissolved in 100 µl acetonitrile and subjected to HPLC with
258 photodiodearray detection (LaChrome, Merck Hitachi, Darmstadt, Germany) to determine rate of
259
260
261
262
263
264
GHQ168 decrease (column: Kinetex 2.6 µm C18, 100 x 3 mm; mobile phase A: water, 0.5 %
formic acid, mobile phase B: acetonitrile, 0.5 % formic acid; gradient: 38 % B for 6 min,
gradually increasing to 60 % B within 44 min; room temperature; flow rate: 0.2 mL/min;
injection volume: 10 µl, detection: 282 nm). M/z ratios of metabolites were obtained by HPLC
mass spectrometry (Agilent LC/MSD G1946D single quadrupol mass spectrometer, Agilent
Technologies, Waldbronn, Germany) in full scan mode using positive ionization, fragmentor
265 voltage 70 V, capillary voltage 4000 V and full scan range m/z 100 - 500.
266
267
Three independent microsomal incubations were performed (new vial of microsomes and fresh
solution of test compound).
Page 12 of 34

268
269
270
271
Mutagenicity of GHQ168 was assessed in the hypoxanthine-guanine phosphoribosyltransferase
(HPRT) assay in the V79 Chinese hamster fibroblast cell line (18). V79 cells were cultured in
DMEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 % FCS
referred to as DMEM complete. The HPRT assay was performed as described previously (19).
272 Briefly, 1.5x106 V79 cells were seeded in 175 cm2 cell culture flasks containing 20 mL DMEM
273 complete. After 24 h, the medium was changed (day 0) and cells were treated with different
274 concentrations of GHQ168 or 1 µM 4-nitroquinoline-N-oxide (NQO; positive control) or 1 %
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
DMSO (negative control) for 24 h. A total of 1x106 cells were subcultured in fresh medium
directly after treatment (day 1) and again on day 4.
On days 1, 4, and 6, the number of viable cells and cells with disintegrated cell membrane were
counted with a CASY Model DT electronic cell counter (Schaerfe, Reutlingen, Germany) as a
reference point for cytotoxicity and proliferation.
On day 6, cells with mutations at the Hprt gene locus were selected by growing cells in DMEM
complete and 7 µg 6-thioguanine (6-TG)/mL using three tissue culture treated dishes (diameter:
145 mm) with 1x106 cells per dish. To determine the plating efficiency (PE) on days 1 and 6,
cells were grown in the absence of 6-TG (500 cells per 100 mm dish, three dishes). After 1 week,
cells were fixed with ethanol and stained with methylene blue (0.5 % in methanol). Colonies
were counted and the PE, i.e. the number of colonies per number of seeded cells, and the mutant
frequency, i.e. the number of colonies/(number of seeded cells x PE on day 6), were calculated.
Three independent HPRT tests were performed (new batch of cells and fresh solution of test
compound).
Plasma stability. Plasma stability assay was performed according to Di et al. (20). In
brief, a 1 mM DMSO stock solution of the selected compound was added to human plasma, 1:2
Page 13 of 34

291
292
293
294
295
296
297
diluted with PBS buffer (pH = 7.4) to achieve a final compound concentration of 10 µM. The
mixture was incubated at 37 °C for 2 h. Within that time aliquots of 100 µl were taken at t =
0 min (reference), 30 min, 60 min and 120 min. All samples were diluted with 300 µl
acetonitrile, centrifuged (3000 rpm for 15 min) and the supernatant (200 µl) was analyzed by
means of HPLC (column: Synergi MAX-RP 80Å, 4 µm, 150 x 2.00 mm; mobile phase:
ACN/10 mM NH4OAc buffer (pH = 4); gradient: 10 % ACN increasing to 90 % in 7 min, 90 %
ACN for 2 min, 90 % ACN decreasing to 10 % ACN in 2 min, 10 % ACN for 2 min;
298 temperature: 40 °C, flow rate: 0.4 mL/min; injection volume: 30 µl; detection: 281 nm
299
300
301
(GHQ242) and 278 nm (GHQ243)). The resulting peak areas of each aliquot were divided
through the peak area of the reference sample (t = 0) to calculate the percentage decrease.
Stability of each compound was measured in duplicate.
302
PBPK modelling and pharmacokinetics. Prior to the in vivo efficacy studies, in silico
303 physiologically based pharmacokinetic (PBPK) modelling (21) was conducted using Simcyp
304 software package (Simcyp, Sheffield, UK) in order to identify a suitable dose and study design.
305
306
307
The study design (e.g. duration, dosing interval) and the prediction of the plasma levels were
performed on a mouse model. The parameters calculated experimentally to enable determination
of a relevant PK study design took into account molar mass, pKa, logP and fraction unbound in
308 plasma. The volume of distribution (VD) was calculated by the software according to the
309
310
311
corrected Poulin and Theil method (22-25). Clearance (Cl) was calculated from the predicted
volume of distribution normalized per kg body weight (VD) according to the formula: ꢀꢁ = ꢂꢃ ×
ꢄꢅ × weight of mouse; with ke being the elimination rate constant as derived experimentally
Page 14 of 34

312
313
314
315
316
317
318
319
320
321
322
from in vitro metabolic stability assays. The weight of a mouse was set to be 25 g and
simulations were conducted assuming first order elimination kinetics.
Analysis of mice blood samples was performed using a Triple Quad™ 5500 LC/MS/MS system
from AB SCIEX (Darmstadt, Germany) (column: Luna 3 µm C18 (2), 100 Å, 75 x 3 mm
reversed phase column; mobile phase: methanol (800 mL), LC/MS-water (200 mL), ammonium
formate (0.636 g) and formic acid conc. (1.25 mL) (pH 4.5); room temperature; injection
volume: 3 µl; detection: 438.1 Da - 331.0 Da (GHQ168), 439.1 Da - 331.0 Da (GHQ242),
453.1 Da - 72.0 Da (GHQ243) and 494.2 Da - 368.9 Da (glibenclamide; internal standard),
turbospray ionization in the positive mode). Dwell time was set to 150 msec. Nitrogen was used
both as nebulizing and drying gas (validation data on the analytical method: Table S3A-F and
Fig. S6A-C in the supplemental material).
323
324
325
326
327
328
329
In vivo efficacy studies. The T.b. rhodesiense STIB900 strain is a derivative of the
STIB704 strain isolated from a patient in Ifakara, Tanzania, in 1982 (26). The STIB900 acute
mouse model mimics the first stage of the disease in which the trypanosomes are localized in the
hemolymphatic system. Prior the to efficacy studies a donor female NMRI mouse was inoculated
intraperitoneally with 1 x 104 bloodstream forms of STIB900. In the efficacy studies four female
NMRI mice were used per experimental group. Each mouse was inoculated i.p. with 104
bloodstream forms of STIB900. For that, heparinized blood from the donor mouse with
330 approximately 5x106 /mL parasitemia was suspended in phosphate-saline-glucose (PSG) to
331
332
333
334
obtain a trypanosome suspension of 4×104 /mL. Each mouse used for the experiments was
injected with 0.25 mL trypanosome suspension. Two efficacy studies were conducted: in a first
study, compound administration began on day 3 post infection (stringent model; test compounds:
GHQ168, GHQ242, GHQ243) and in a second study, compound administration began 1 day post
Page 15 of 34

335
336
337
infection (early-treatment model, test compound: GHQ168). Besides this, both studies were
designed equally (route of administration: i.p., dosing interval: 12 h, study duration: 4 days,
single dose: GHQ168 3.5 mg/kg; GHQ242: 22.9 mg/kg; GHQ243: 21.9 mg/kg; fluid intake with
338 dose: 0.1 mL/10 g body weight).
339
340
341
342
343
344
345
For in vivo administration, GHQ168 spray dried particles and crystalline GHQ242 and GHQ243,
respectively, were dissolved in the following delivery vehicle prior to i.p. application (GHQ168:
Glucose 5 % (w/V), polysorbate 80 1 % (w/V), PBS buffer (pH 7.4); GHQ242/GHQ243:
Glucose 5 % (w/V), polysorbate 80 1 % (w/V), DMSO 10 % (V/V), PBS buffer (pH 7.4)).
Four mice served as infected-untreated controls. They were not injected with the vehicle alone
since it is established in the lab that these vehicles do not affect parasitemia nor the mice (data
not shown). All mice were monitored for parasitemia by microscopic examination of tail blood
346 twice a week until day 32 post infection and afterwards once per week until day 60. The time to
347
348
349
350
351
352
353
354
355
356
parasite relapse was recorded to calculate the mean relapse time in days (MRD) after infection.
Mice were euthanized after parasitemia relapse detection (at least 2 trypanosomes per field of
view). The mean survival days (MSD) of the treated groups and the control group were
compared using an unpaired t-test; level of significance was defined as α = 0.05 (Minitab®
17.2.1). Mice that died during the treatment but without parasites in the blood are excluded from
the calculation of MSD and MRD as the reason of death is not related to parasitemia. Surviving
and aparasitemic mice at day 60 (stringent model) or at day 32 (early-treatment model) were
considered cured (endpoint of the model) and euthanized.
During the efficacy study in the stringent mouse model, blood samples were collected from tail
blood for subsequent LC/MS/MS analysis at predetermined time points (1 h and 4 h after the 7th
357 treatment and 16 h after the 8th treatment) in order to assess pharmacokinetics (Table S4 in the
Page 16 of 34

358
359
360
361
supplemental material). In vivo efficacy studies in mice were conducted at the Swiss Tropical
and Public Health Institute (Basel) according to the rules and regulations for the protection of
animal rights ("Tierschutzverordnung") of the Swiss "Bundesamt für Veterinärwesen". They
were approved by the veterinary office of Canton Basel-Stadt, Switzerland.
362 Statistics. Statistical tests (in metabolism and mutagenesis studies) were performed using
363
364
OriginPro®9.1 (OriginLab corp., Northampton, MA, USA). Multiple data sets were analyzed by
ANOVA with post hoc comparison by Scheffé. Pairwise comparisons were performed by
365 Student’s t-test. Statistical significance of evidence was postulated for p < 0.05.
366
RESULTS
367
368
369
370
Solubility in water. The thermodynamic solubility of GHQ168 was 0.005 ±
0.001 µg/mL as assessed after equilibration for 20 h in PBS buffer and at conditions described
under “methods and materials”. The kinetic solubility was 15 ± 1 µg/mL, i.e. exceeding the
thermodynamic solubility by a factor of 3000. Therefore, GHQ168 was practically insoluble in
371 water (23), driving the need for a solubility improvement before in vivo efficacy studies
372
373
commenced. Solubility improvement was addressed by (i) formulation approaches and (ii)
structural modification.
374
375
376
377
The formulation strategy was fueled by the coplanar stacking of GHQ168 within its crystal (Fig.
2) with the crystalline nature being corroborated by XRPD (Fig. S1A in the supplemental
material). These highly ordered structures may allow for a low free enthalpy state, consequently
demanding high work for molecular escape from the crystal. The resulting high melting points of
378 the crystal (171 °C) reflect these patterns and favor the low solubilities for GHQ168.
379
Consequently, a formulation strategy was deployed pushing GHQ168 to higher free enthalpy
Page 17 of 34

380
381
states as compared to the state of the crystal. For that, GHQ168 was spray dried into a polymer,
thereby embedding the drug in an amorphous state (Fig. S1B in the supplemental material)
382 within microparticles (see scanning electron micrographs: Fig. S2 in the supplemental material).
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
In addition to the amorphous presentation, spray dried formulations massively increased the
surface area of the compound as compared to the compact arrangement within the crystal
typically increasing the dissolution rate based on simple Fickian diffusion considerations. The
methacrylic acid polymer used for embedding the compound was selected to readily dissolve at a
pH exceeding approximately 5.5, thereby gradually releasing the molecularly dispersed drug
when exposed to or exceeding this pH threshold (27).. Stability studies demonstrated the physical
stability of this formulation for one year of storage by absence of (re-)crystallization (no later
time points were assessed; Fig. S1B in the supplemental material). Spray drying of GHQ168
resulted in white nanoparticles of a size ranging below 350 nm which was confirmed by
subsequently performed DLS experiments (Fig. S3 in the supplemental material) in order to
monitor the pattern in solution over time. Furthermore, smooth surface, spherical form, and
narrow size distribution were illustrated with the help of scanning electron micrographs (Fig. S2
in the supplemental material). The overall spray drying process yielded an efficacy of about 90
%. The maximum solubility (supersaturated state) in PBS buffer was determined as 32.9 ±
1.6 µg/mL after 30 minutes and decreasing to 7.3 µg/mL ± 0.7 after 6 hours (Fig. 3), thereby
transiently exceeding the thermodynamic solubility of GHQ168 by a factor > 6000. Physical
mixtures of GHQ168 with the methacrylic acid polymer did not impact the solubility profile,
indicating that amorphization is essential to improve the solubility and corroborating the
401 formulation hypothesis in that breaking the crystalline forces by amorphization is instrumental to
402 increase the pharmaceutical properties of GHQ168 (Fig. 3).
Page 18 of 34

403
404
The chemical strategy targeted a solubility improvement by structural modification of GHQ168
(Fig. 4) as follows:
405 1) The introduction of basic moieties (tertiary amines) allowed salt formation and the resulting
406
407
408
409
410
GHQ243 and GHQ250 were presented as oxalate salts with a stoichiometry (API:oxalate) of 1:1
and 1:1.5, respectively.
2) The introduction of a polar substituent, i.e. the replacement of a phenyl moiety with a pyridine
ring, resulted in GHQ242 and GHQ237, again presented as oxalate salts with a stoichiometry
(API:oxalate) of 1:1.5 and 1:0.5, respectively.
411 3) The formation of the amidine of GHQ168 resulted in GHQ232, presented as a hydrochloride.
412
413
414
415
416
417
418
419
420
421
422
423
424
425
4) The introduction of an additional carbonyl group in position 2 resulted in GHQ215, an acidic
moiety.
Details of the synthesis pathways are described in the supplemental material (including the
experimental details). The introduction of a basic functionality in position 7 - which is essential
for the activity of the topoisomerase inhibitors in clinical treatment (25) - always resulted in a
substantially decreased activity against T. b. brucei and along with a lower selectivity index (SI;
calculated as 50% of the cytotoxic concentration on Vero cells divided by the IC50 of the
compound for T. cruzi cells) (10). Thus, this position was not considered for the introduction of a
basic substituent.
With exception of GHQ215 the solubility of the compounds was enhanced by structure
variations, particularly for the oxalate salts GHQ242, GHQ243 and GHQ250, as preliminarily
assessed by light microscopy (Table 1). However, all new compounds were less active against
T.b. brucei, and, therefore, displayed a decreased selectivity index (Table 1), which was already
reported previously (10). The potentiometric determination of the pKA of GHQ168 did not result
Page 19 of 34

426
427
428
429
430
in a measurable value between pH 2-12. After introduction of basic moieties the pKA values of
GHQ242 and GHQ243, the candidates chosen for in vivo efficacy studies, were at 5.25 ± 0.05
and 6.86 ± 0.06, respectively (data not shown). We detailed the preliminary, microscopic
assessment of the solubility (Table 1) nephelometrically with an aqueous kinetic solubility of
274 ± 18 µg/mL and 152 ± 1 µg/mL for GHQ242 and GHQ243, respectively. Both had a
431 statistically significant and improved kinetic solubility as compared to GHQ168 (15 ± 1 µg/mL).
432
Albumin binding, metabolism, plasma stability, mutagenicity, trans-epithelial
433 transport. The albumin binding was determined by a continuous titration method (12, 28).
434
435
436
437
438
439
440
441
442
443
Whereas GHQ168 and GHQ243 bound to albumin at 81 % and 46 %, respectively, GHQ242 was
completely bound to albumin exceeding 99 % (Table 1). Since GHQ168 was found to be the
most active compound in vivo (see below), the oxidative metabolism of GHQ168 was studied.
The HPLC-MS full scan analysis revealed 4 peaks (m/z 454, 412, and twice 348) not observed in
control reactions. The m/z results suggested metabolites resulting from ring-hydroxylation, β-
oxidation, and N-dearylation, respectively. Although linear NADPH generation and activity of
microsomal enzymes for 90 minutes was assessed (data not shown), the decrease in GHQ168
concentration did not differ significantly after 30, 60, and 90 min (4.7 + 0.9%), respectively,
indicating enzyme inhibition by GHQ168 or one of its metabolites. Concerning the plasma
stability, no degradation was observed throughout 2 h for GHQ168, GHQ242, and GHQ243.
444
445
446
447
The mutagenicity of GHQ168 was assessed in the HPRT assay in cultured V79 cells. In light of
GHQ168-mediated inhibition of microsomal enzyme activity and the absence of metabolites
with a structural alert for mutagenicity (such as catechols) the HPRT assay was performed in the
absence of a metabolically activating system. GHQ168 concentrations ranged from 2 to 47 µM,
Page 20 of 34

448
449
450
451
452
453
454
455
456
457
458
significantly decreasing plating efficiency and cell numbers at 47 µM and 16-47 µM,
respectively, following previously described protocols (29). Whereas the known mutagen NQO
caused a significant increase in the mutant frequency to 186 ± 42 compared to control cells (9 ±
3), the mutant frequency was not affected by treatment with any concentration of GHQ168.
In light of future profiling of the compounds, GHQ168, GHQ242, and GHQ243 were tested for
transepithelial transport. GHQ168, GHQ242, and GHQ243 had a comparable flux through Caco-
2 cell monolayers (as an in vitro model for intestinal absorption) to the positive control
Propranolol-HCl (Fig. S4 and Table S2 in the supplemental material). Based on the previously
demonstrated low solubility and the good permeability as observed with the Caco-2 monolayer
assay, GHQ168 can in absence of in vivo information only preliminarily be categorized as
Biopharmaceutics Classification System (BCS) class II (low solubility, high permeability)..
459
460
461
462
Cell toxicity and in vitro efficacy. GHQ168 as well as the new derivatives were
characterized for their physicochemical properties (logP, kinetic solubility, mp) as well as
albumin binding and their activity towards T.b. brucei, T.b. rhodesiense (72 h), and cytotoxicity
(muscle cells, macrophages, kidney cells, and hepatocytes) which are displayed in Table 1.
463 Based on the solubility results and demonstrated in vitro efficacy, spray-dried GHQ168, and the
464
oxalate salts of GHQ242 and GHQ243 were selected for in vivo efficacy studies.
465
466
467
468
469
Pharmacokinetics. Blood samples were collected during the efficacy study in the
stringent mouse model from the surviving mice at three time points and analyzed by LC/MS/MS
(Fig. 5 and Fig. S7 in the supplemental material). Blood concentrations were about 10 fold
higher for GHQ168 (delivered from the spray dried formulation), and GHQ243 as compared to
GHQ242. An unusual time response of the blood concentration was observed among mice for
Page 21 of 34

470
471
472
473
474
475
476
GHQ242. Two of the four mice tested within the GHQ242 group had higher blood
concentrations at a subsequent time point as compared to a previous one (Fig. S7 in the
supplemental material; data for these 2 mice is highlighted by arrows). The half-life of GHQ168,
and GHQ243 were comparable, ranging from approximately 1.5 to 3.5 hours (Table 2).
Pharmacokinetic outcome for GHQ242 was more heterogeneous (vide supra) such that we
cannot decide whether the blood concentrations analysed 16 h after application were due to
metabolism of the compound or due to flip-flop kinetics. In fact, the half-life reported for
477 GHQ242 may reflect metabolism or may reflect another step such as drug release from a
478
479
480
481
482
483
precipitate formed during i.p. injection. As the obtained data was insufficient to clarify this
question (i.v. data would be required), we did not calculate the apparent clearance or the apparent
volume of distribution for GHQ242 as in absence of a clear assignment of the terminal half life
to elimination for this compound, possible conclusions may very well be prone to
misinterpration. However, the apparent clearance and the apparent volume of distribution were
similar for GHQ168 and GHQ243 (Table 2).
484
In vivo efficacy studies. The dose and its regimen were simulated through PBPK
485 modelling prior to in vivo studies (Fig. S5A-S5C in the supplemental material). Two in vivo
486
487
488
489
490
models were used, referred to as the ‘stringent mouse model’ with 8 doses administered twice
daily throughout days 3-6 post infection and the early-treatment model, with 8 doses twice daily
throughout days 1-4. All three tested compounds showed in vivo activity in comparison to the
untreated control group (Fig. 6A), especially GHQ168. All infected mice treated with compound
GHQ168 were free of parasites for 10 days (tail blood examinations; microscopy test) and
491 relapsed on day 14. One mouse treated with GHQ168 died after the 5th treatment without
492
parasitemia. Test animals treated with compounds GHQ242 and GHQ243 did not show
Page 22 of 34

493
494
495
496
497
498
499
500
parasitemia on day 7 but relapsed on day 10 (GHQ242) and day 14 (GHQ 243), respectively,
post infection. The mean survival days (MSD) of mice within the stringent mouse model and
medicated with GHQ168, GHQ242, GHQ243, and control were 17, 14, 12, and 9 days,
respectively (Table 3), meaning that statistically significant differences were observed for
GHQ168 versus control (p < 0.001), whereas insignificant results were obtained for GHQ242
and GHQ243 compared to control. Based on this outcome and corroborated by the in vitro
microbiological assays, GHQ168 was further profiled in the early-treatment model (Fig. 6B).
GHQ168 administration prevented parasitemia all animals (Fig. 6B). However, one mouse died
501 after the 5th dose and two mice died after the 8th dose but without external signs of toxicity or
502
503
504
505
506
parasitemia. The MSD of the three remaining mice were > 32 days (end of study; all surviving
mice were free of parasites as tested on tail blood). This resulted in statistically significant
differences in the MSD for GHQ168 versus control (p < 0.001). A similar efficacy of
melarsoprol (2 mg/kg) and pentamidine isethionate (5 mg/kg) was reported in previous studies
for surviving mice (29). However, in this study, the mice were infected with T.b. brucei.
507
508
509
510
511
512
513
514
DISCUSSION
In a previous study, a morpholino substituted quinolone amide (Fig. 1) was identified as a highly
active compound towards T.b. brucei and T.b. rhodesiense (10). However, this substance was
practically insoluble in aqueous media (Table 1), thereby challenging in vivo studies. Thus, the
aim of the study reported here was to overcome the solubility limitations and to characterize the
compound class concerning physicochemical and biological properties as well as profiling these
in in vivo pharmacokinetic and efficacy experiments for the first time.
Page 23 of 34

515
516
517
518
519
The solubility can be enhanced by means of an appropriate formulation (Fig. 3) as well as by
structural variations of the hit/lead compound (Fig. 4). Especially the introduction of amino
functions as well the replacement of phenyl rings with pyridine rings resulted in compounds of
lower lipophilicity hence increased solubility, and the oxalate salts of the active bases were
particularly increasing the solubility. However, the solubility enhancement by these structural
520 modifications was achieved at the expenses of activity, i.e. a decrease of IC50 values of at least
521
522
523
524
tenfold towards T.b. brucei (Table 1). Since the cytotoxicity, determined in L6 cells,
macrophages, kidney cells and hepatocytes (Table 1), was found to be almost equal or even
higher, the selectivity deteriorated. Nevertheless, compounds GHQ242 and GHQ243 were
considered for in vivo studies because both substances showed (i) rather high activity towards
525 T.b. rhodesiense (with the IC50 being at 2 nM and 18 nM for GHQ242 and GHQ243 as
526
527
528
529
530
531
determined after 72 hours), (ii) low cytotoxicity, and (iii) sufficient solubility as determined by
PKPD modelling. Since GHQ168 still ranked among the most active compounds with an IC50
(72 h) value of 1 nM against T.b. rhodesiense a formulation was developed improving the kinetic
solubility by a factor of 6000 (Fig. 3), thereby opening this promising compound to efficacy
studies. The spray drying technology has been applied industrially for decades and in the
meantime became a well understood process easily allowing scale up from developmental batch
532 sizes. The spray drying of GHQ168 was a robust and efficient process facilitating the
533
534
535
536
amorphization of the drug substance whilst maintaining stability in Eudragit L100. In order to
avoid blood concentration-time levels beyond the therapeutic window, the corresponding doses
for the in vivo studies for intraperitoneal administration as well as the study design were
predicted by means of the PBPK simulations.
Page 24 of 34

537
538
539
540
541
542
543
544
545
546
547
548
Since pharmacokinetic properties govern the activity of drugs, the most important properties
were evaluated. Whereas GHQ168 and GHQ243 exhibited a high and moderate human serum
albumin binding (81 % and 46 %) an almost exhaustive albumin binding was found for
GHQ242. Therefore, the fraction unbound was very low for GHQ242 and providing that the
dissociation kinetics are slow (not determined) this pattern may potentially challenge the use of
this compound in the future, at least for parenteral use. Interestingly, the pharmacokinetic profile
of GHQ242 reflected analogous observations to those found for albumin binding. In spite of the
fact that the same dose (mol) was applied as compared to GHQ168 and GHQ243, PK
concentrations were roughly 10 fold lower upon i.p. administration. Possible explanations for
this low PK concentration profile include (i) local precipitation upon i.p. injection (suggested by
the fluctuating PK profile – vide infra), or (ii) extensive binding to extracellular matrix, and other
surfaces at the site of injection and/or at other sites (suggested by the albumin binding – vide
549 supra), or (iii) both. One may speculate that the initially paradox observation in that some blood
550
551
552
553
554
555
556
557
558
559
concentrations following GHQ242 exceeded results from previous sampling reflect slow drug
release from precipitated drug following i.p. injection. Further studies including pharmacokinetic
profiles following i.v. administration are required to detail these findings. However, in light of
the possibility of GHQ242 precipitation at the injection site, the half-life calculated from the
pharmacokinetic profile (Fig. 5 and Fig. S7 in the supplemental material) might not reflect
metabolism (i.e. elimination half-life) but slow release of molecules from its precipitate at the
injection site (half-life of molecules being released from the precipitate). With that in mind, we
did not calculate the apparent clearance and apparent volume of distribution for GHQ242, as due
to missing i.v. data we were unable to clearly assign the terminal half-life to metabolism or drug
release kinetics from a possible precipitate formed upon administration. These pharmacokinetic
Page 25 of 34

560
561
562
563
564
data sets indicated that the successful development of GHQ242 as a parenteral form is more
risky as compared to GHQ168, or GHQ243. However, GHQ242 may be successfully presented
as an oral form, thereby overcoming the possible local precipitation upon injection and as
suggested by the permeability results from the Caco-2 monolayer studies (Fig. S4 in the
supplemental material).
565 The target site of these compounds is still unknown. Previously performed studies point to the
566 kinetoplast as a possible target by impacting kinetoplast segregation by these quinolone amide
567 derivatives (10). However, sensitivity measurements with acriflavin-induced dyskinetoplastic T.
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
b. brucei revealed a decrease of the IC50 value, but the IC50 is still in the low micromolar range
of concentration (data not shown), indicating that the compounds are still highly active and may
address another lethal target. This remains to be elucidated.
In contrast to available quinolones marketed today, GHQ168 and its related molecules differ in
that the free carboxyl group of the marketed quinolones is derivatized, therefore not addressing
the topoisomerase target in trypanosoma. Due to the absence of the carboxyl group in GHQ168,
this compound might be associated with less or no side effect issues due to complexation of
Mg2+ and Ca2+; this will need to be experimentally confirmed in future studies.
The small number of animals exposed to these novel quinolones is a limitation of this study.
Therefore, positive trends may be assumed from the data sets but larger studies need to
corroborate the findings for final conclusion. As pointed out before, no final conclusion can be
drawn regarding the pharmacokinetic profile for GHQ242 as flip-flop kinetics may have
occurred due to a possible precipitation of the i.p. administered molecule. Lastly, solubility
challenges for GHQ168 as observed during the serum albumin binding studies render the
outcome of this experiment for this compound questionable.
Page 26 of 34

583 The in vivo studies suggested a statistically significant impact of GHQ168 on mean survival as
584
585
compared to the untreated control in both efficacy studies (Fig. 6A, 6B). However, statistical
calculations based on the small data set are critical and and require carefully interpretation.
586 Future studies need to confirm the evidence provided here within. Following i.p. administration
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
of GHQ168, this test substance kept them free from parasites over a period of 14 days on average
(stringent mouse model) and over more than 32 days (easy to cure model). However, some mice
died during the treatment period without parasitemia. Although no external signs of toxicity were
observed in the efficacy studies (and in the PK studies), future studies need to detail the cause of
these events. GHQ242 and GHQ243 had a positive trend on the MSD but without significant
outcome as compared to control (Fig. 6A). This observation suggested, that GHQ242’s ten times
higher in vitro activity (T.b. rhodesiense, Table 1) compensated for its 10 times lower exposure
(AUC, Table 3) as compared to GHQ243.
In conclusion, the quinolone amide skeleton was found to be a promising starting point for the
development of highly active anti-trypanosomal compounds. This study identified a lead with
promising pharmacokinetic properties and efficacy, in that curing of T.b. rhodesiense infected
mice was achieved. The development of even more active compounds and compounds with
greater solubility by variation of the skeleton is in progress as well as the search for the detailed
mode of action.
.
Page 27 of 34

603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
ACKNOWLEDGMENTS
We thank Elena Katzowitsch and Tobias Ölschläger (Institute for Molecular Infection Biology,
University of Würzburg) for the cytotoxicity screening, Jennifer Rath and Antje Fuss (Medical
Mission Institute, Würzburg) for testing the compounds against T.b. brucei and Alena Zikova
(University of South Bohemia, Czech Budejovice, Czech Republic) for the dyskinetoplast
experiments, Annette Albrecht (Institute of Pharmacy and Food Chemistry, University of
Wuerzburg) for testing GHQ168 in the HPRT assay, Marius Gareis (Institute of Pharmacy and
Food Chemistry, University of Wuerzburg) for performing the studies on oxidative metabolism
of GH168 and Lena Lauber for the support in Caco-2 assays. V79 cells were kindly provided by
H. Glatt, German Institute of Human Nutrition, Potsdam, Germany. We gratefully acknowledge
the Deutsche Forschungsgemeinschaft (SFB 630) and the Bayerische Forschungsstiftung (Grant
‘Springs and Parachutes’) for financial support as well as ACC GmbH (Analytical Clinical
Concepts GmbH, Leidersbach) for financial support, support in statistics (Volker Guth) and in
analytical work (Martin Barkworth).
The authors declare no conflict of interest.
Page 28 of 34

620
References
621
622
623
624
625
626
627
628
629
630
1.
2.
3.
Franco JR, Simarro PP, Diarra A, Jannin JG. 2014. Epidemiology of human African
trypanosomiasis. Clin Epidemiol 6:257-275.
Stich A, Ponte-Sucre A, Holzgrabe U. 2013. Do we need new drugs against human
African trypanosomiasis? The Lancet Infectious Diseases 13:733-734.
Pena I, Pilar Manzano M, Cantizani J, Kessler A, Alonso-Padilla J, Bardera AI,
Alvarez E, Colmenarejo G, Cotillo I, Roquero I, de Dios-Anton F, Barroso V,
Rodriguez A, Gray DW, Navarro M, Kumar V, Sherstnev A, Drewry DH, Brown
JR, Fiandor JM, Julio Martin J. 2015. New compound sets identified from high
throughput phenotypic screening against three kinetoplastid parasites: an open resource.
Sci Rep 5:8771.
631 4.
http://www.dndi.org/diseases-projects/diseases/hat.html. (access 23rd January, 2015).
Maser P, Wittlin S, Rottmann M, Wenzler T, Kaiser M, Brun R. 2012. Antiparasitic
agents: new drugs on the horizon. Curr Opin Pharmacol 12:562-566.
Bringmann G HV, Holzgrabe U, Stich A. 2003. Antitrypanosomal
naphthylisoquinoline alkaloids and related compounds. Pharmazie 58:343-346.
Nenortas E, Kulikowicz T, Burri C, Shapiro TA. 2003. Antitrypanosomal activities of
fluoroquinolones with pyrrolidinyl substitutions. Antimicrob Agents Chemother 47:3015-
3017.
632
633
634
635
636
637
638
639
640
5.
6.
7.
8.
Keiser J, Burri C. 2001. Evaluation of quinolone derivatives for antitrypanosomal
activity. Tropical Medicine & International Health 6:369-389.
Page 29 of 34

641
642
643
9.
Ma X, Zhou W, Brun R. 2009. Synthesis, in vitro antitrypanosomal and antibacterial
activity of phenoxy, phenylthio or benzyloxy substituted quinolones. Bioorg Med Chem
Lett 19:986-989.
644 10.
645
Hiltensperger G, Jones NG, Niedermeier S, Stich A, Kaiser M, Jung J, Puhl S,
Damme A, Braunschweig H, Meinel L, Engstler M, Holzgrabe U. 2012. Synthesis and
structure-activity relationships of new quinolone-type molecules against Trypanosoma
brucei. J Med Chem 55:2538-2548.
646
647
648
649
650
651
652
653
654
655
656
657
658
11.
12.
Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. 2001. Experimental and
computational approaches to estimate solubility and permeability in drug discovery and
development settings. Adv Drug Deliv Rev 46:3-26.
Heinze A, Holzgrabe U. 2006. Determination of the extent of protein binding of
antibiotics by means of an automated continuous ultrafiltration method. Int J Pharm
311:108-112.
13.
14.
Hubatsch I, Ragnarsson EG, Artursson P. 2007. Determination of drug permeability
and prediction of drug absorption in Caco-2 monolayers. Nat Protoc 2:2111-2119.
Muth M, Hoerr V, Glaser M, Ponte-Sucre A, Moll H, Stich A, Holzgrabe U. 2007.
Antitrypanosomal activity of quaternary naphthalimide derivatives. Bioorg Med Chem
Lett 17:1590-1593.
659 15.
660
Larson EM, Doughman DJ, Gregerson DS, Obritsch WF. 1997. A new, simple,
nonradioactive, nontoxic in vitro assay to monitor corneal endothelial cell viability.
Invest Ophthalmol Vis Sci 38:1929-1933.
661
662
663
16.
Papadopoulou MV, Bloomer WD, Lepesheva GI, Rosenzweig HS, Kaiser M,
Aguilera-Venegas B, Wilkinson SR, Chatelain E, Ioset JR. 2015. Novel 3-
Page 30 of 34