AAC Accepted Manuscript Posted Online 1 June 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.00708-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
TXA709: A FtsZ-Targeting Benzamide Prodrug with Improved Pharmacokinetics
2
and Enhanced In Vivo Efficacy against Methicillin-Resistant Staphylococcus
3
aureus
4 5 6
Malvika Kaul,a Lilly Mark,b,c Yongzheng Zhang,b Ajit K. Parhi,b,c Yi Lisa Lyu,a Joan
7
Pawlak,d Stephanie Saravolatz,d Louis D. Saravolatz,d,e Melvin P. Weinstein,f,g Edmond J.
8
LaVoie,c and Daniel S. Pilcha#
9 10
Department of Pharmacology, Rutgers Robert Wood Johnson Medical School, Piscataway, New
11
Jersey, USAa; TAXIS Pharmaceuticals, Inc., North Brunswick, New Jersey, USAb; Department
12
of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers-The State University of
13
New Jersey, Piscataway, New Jersey, USAc; St. John Hospital and Medical Center, Detroit,
14
Michigan, USAd; Wayne State University School of Medicine, Detroit, Michigan, USAe;
15
Departments of Medicine (Infectious Disease)f and Pathology and Laboratory Medicineg,
16
Rutgers Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA
17 18
RUNNING TITLE: ADME/PK Properties and In Vivo Efficacy of TXA709
19 20
#Address correspondence to Daniel S. Pilch,
[email protected].
21
ABSTRACT
22
The clinical development of FtsZ-targeting benzamide compounds like PC190723 has been
23
limited by poor drug-like and pharmacokinetic properties. First-generation prodrugs of
24
PC190723 (e.g., TXY541) resulted in enhanced pharmaceutical properties, which, in turn,
25
led to improved intravenous efficacy as well as the first demonstration of oral efficacy in
26
vivo versus both methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-
27
resistant S. aureus (MRSA). Despite being efficacious in vivo, TXY541 still suffered from
28
sub-optimal pharmacokinetics and the requirement of high efficacious doses. We describe
29
herein the design of a second-generation prodrug (TXA709) in which the Cl group on the
30
pyridyl ring has been replaced with a CF3 functionality that is resistant to metabolic attack.
31
As a result of this enhanced metabolic stability, the product of the TXA709 prodrug
32
(TXA707) is associated with improved pharmacokinetic properties (a 6.5-fold longer half-
33
life and a 3-fold greater oral bioavailability) and superior in vivo antistaphylococcal
34
efficacy relative to PC190723. We validate FtsZ as the antibacterial target of TXA707, and
35
demonstrate that the compound retains potent bactericidal activity against S. aureus
36
strains resistant to the current standard-of-care drugs vancomycin, daptomycin, and
37
linezolid.
38
mammalian cells, establish the prodrug TXA709 as an antistaphylococcal agent worthy of
39
clinical development.
These collective properties, coupled with minimal observed toxicity to
40
INTRODUCTION
41
Bacterial resistance has emerged as a global problem. The Centers for Disease Control and
42
Prevention (CDC) have identified methicillin-resistant Staphylococcus aureus (MRSA) and
43
vancomycin-resistant S. aureus (VRSA) as being two major antibiotic resistance threats (1).
44
Typically, MRSA strains are resistant not only to the penicillins, but also to other classes of
45
antibiotics, including the tetracyclines, the macrolides, the aminoglycosides, and clindamycin (2-
46
4). Current standard-of-care (SOC) drugs for the treatment of MRSA infections are therefore
47
limited to a few drugs, which include vancomycin, daptomycin, and linezolid (3). However,
48
resistance to these SOC drugs is already on the rise, and the clinical utility of these drugs is
49
likely to diminish in the future (3, 5-9).
50
The bacterial protein FtsZ has been identified as an appealing new target for the
51
development of antibiotics that can be used to treat infections caused by multidrug-resistant
52
(MDR) bacterial pathogens (10-14). The appeal of FtsZ as an antibiotic target lies in the
53
essential role the protein plays in bacterial cell division (cytokinesis). Furthermore, FtsZ is
54
prokaryotic-specific with no known eukaryotic homolog.
55
dependent manner to form a ring-like structure (the Z-ring) at midcell that serves as a scaffold
56
for the recruitment and organization of other critical components for proteoglycan synthesis,
57
septum formation, and cell division (15-20).
58
FtsZ self-polymerizes in a GTP-
The substituted benzamide derivative PC190723 has been shown to inhibit bacterial cell
59
division through disruption of FtsZ function (21-24).
PC190723 is associated with potent
60
bactericidal activity against Staphylococcus spp., including MRSA (23, 24). However, the
61
clinical development of this compound has been hindered by poor pharmaceutical and
62
pharmacokinetic properties. We have previously reported the design and characterization of
63
first-generation prodrugs of PC190723 (TXY436 and TXY541) with physico-chemical
64
properties that significantly enhance the ease of formulation in vehicles suitable for in vivo
65
administration (25, 26).
66
efficacious in vivo against MRSA, the doses required for efficacy were high, and their
67
pharmacokinetic properties were sub-optimal (25, 26). Here we describe a second-generation
68
prodrug (TXA709) of an FtsZ-targeting benzamide compound (TXA707) with enhanced
69
metabolic stability, improved pharmacokinetic properties, and superior in vivo efficacy versus
70
MRSA. We provide results that highlight TXA709 as an attractive lead agent for clinical
71
development.
72
MATERIALS AND METHODS
73
Bacterial strains. Methicillin-resistant S. aureus (MRSA) clinical strains (n = 30) were isolated
74
from patients admitted to St. John Hospital and Medical Center (SJHMC) in Detroit, MI (n = 20)
75
or to Robert Wood Johnson University Hospital (RWJUH) in New Brunswick, NJ (n = 10). The
76
MRSA isolates were cultured from blood (n = 26), wound tissue (n = 2), pleural fluid (n = 1),
77
and arm drainage (n = 1). Daptomycin-nonsusceptible S. aureus (DNSSA) isolates (n = 7) were
78
cultured from blood samples collected from patients at SJHMC. Vancomycin-intermediate S.
79
aureus (VISA) isolates (n = 20) were obtained from 20 patients. The VISA isolates were
80
cultured from blood (n = 12), wound tissue (n = 1), bile (n = 1), cerebral spinal fluid (n = 1), and
81
an unknown source (n = 5). Vancomycin-resistant S. aureus (VRSA) isolates (n = 11) were
82
obtained from 11 patients, with these isolates being cultured from wound tissue (n = 8),
83
prosthetic knee drainage (n = 1), urine (n = 1), and a catheter exterior (n = 1). Linezolid-
84
nonsusceptible S. aureus (LNSSA) isolates (n = 6) were cultured from blood (n = 2), sputum (n =
While TXY436 and TXY541 were both orally and intravenously
85
1), and an unknown source (n = 3). The VRSA, 17 of the VISA, and 4 of the LNSSA isolates
86
were provided by the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA)
87
for distribution by BEI Resources, NIAID, NIH. The NARSA website (http://www.narsa.net)
88
describes the location of origin of each isolate.
89
collected at SJHMC, and the remaining two LNSSA isolates were collected at Robinson
90
Memorial Hospital in Ravenna, OH. Methicillin-sensitive S. aureus (MSSA) isolates (n = 10)
91
were cultured from the blood of 10 patients at RWJUH. MRSA Mu3 was a gift from Dr. George
92
M. Eliopoulos (Beth Israel Deaconess Medical Center, Boston, MA) and MSSA 8325-4 was a
93
gift from Dr. Glenn W. Kaatz (John D. Dingell VA Medical Center, Detroit, MI). All other
94
MSSA and MRSA strains were obtained from the American Type Culture Collection (ATCC).
95
Bacillus subtilis FG347 was a gift from Dr. Richard Losick (Harvard University, Boston, MA).
96
Compound synthesis.
The remaining three VISA isolates were
TXY541 and PC190723 were synthesized as described
97
previously (25, 27). TXA707 and TXA709 were synthesized as detailed in the Supplementary
98
Material.
99
Compound stability studies. The conversion of TXA709 to TXA707 in either cation-
100
adjusted Mueller-Hinton (CAMH) broth (Becton, Dickinson and Co.; Franklin Lakes, NJ) or
101
100% filtered mouse serum (Lampire Biological Laboratories, Inc.; Ottsville, PA) at 37 °C was
102
monitored as described previously for the conversion of TXY541 to PC190723 (25). The mouse
103
serum was filtered through a 0.2 µm filter before use.
104
In Vitro susceptibility assays.
In vitro susceptibility assays were conducted with
105
TXA707, TXA709, and PC190723 using vancomycin-HCL, daptomycin, and linezolid as
106
comparator control antibiotics (all obtained from Sigma). Determinations of minimal inhibitory
107
concentrations (MICs) and minimal bactericidal concentrations (MBCs) were conducted in
108
duplicate according to Clinical and Laboratory Standards Institute (CLSI) guidelines (28).
109
Microdilution assays with CAMH broth were used to determine the MICs of all agents. For the
110
daptomycin assays, calcium was added to a final concentration of 50 mg/l. MIC is defined as the
111
lowest compound or drug concentration with no visible growth after 16-24 hours of incubation.
112
MBC is defined as the compound or drug concentration that reduced the number of viable cells
113
by ≥99.9%, as determined by colony counts relative to antibiotic-free controls.
114
Assay for metabolism of TXY541 and TXA709 in the presence of mouse and human
115
hepatocytes. Metabolism experiments were conducted by SAI Life Sciences Ltd. (Pune, India).
116
Mouse hepatocytes, human hepatocytes, and cryopreserved hepatocyte recovery medium
117
(CHRM) were obtained from Invitrogen BioServices India Pvt. Ltd. Cryopreserved hepatocytes
118
were revived in CHRM medium supplemented with Krebs Henseleit Buffer (KHB), pH 7.4
119
(Sigma). A 500 µL cell suspension containing 2 x 106 cells/mL was added to individual wells of
120
24 well plates and incubated at 37 °C for 15 minutes. Duplicate reactions were initiated by
121
adding 500 µL of test compound diluted in KHB (prewarmed at 37 °C), and further incubated at
122
37 °C for 0 and 60 minutes with gentle shaking. The final cell density was 1 x 106 cells/mL, and
123
the final concentration of test compound was 10 µM. Reactions were terminated by adding 1 mL
124
of ice-cold acetonitrile to a final volume of 2 mL. The terminated reaction mixes from each well
125
were transferred to individual tubes and sonicated for 5 minutes prior to centrifugation at 2147 ×
126
g for 15 minutes at 4 °C. A 1.2 mL aliquot of each supernatant was then transferred to a clean
127
tube and the metabolites identified by LC-MS/MS analysis. Testosterone (Sigma) and 7-OH-
128
coumarin (Apin Chemicals Ltd.) were used as positive controls.
129
Pharmacokinetic studies. Pharmacokinetic experiments in male BALB/c mice were
130
conducted by SAI Life Sciences Ltd. (Pune, India) as described previously (25). When used, 1-
131
aminobenzotriazole (ABT) was administered orally at a standard dose 50 mg/kg (29) one hour
132
prior to administration of test prodrug (TXY541 or TXA709) formulated in 10 mM citrate, pH
133
2.6. Plasma concentrations of the prodrug products PC190723 and TXA707 were quantified by
134
LC-MS/MS, with the lower limit of quantitation (LLOQ) being 10.02 and 4.93 ng/mL,
135
respectively.
136
Plasma protein binding studies. Plasma protein binding studies of TXA707 were
137
conducted by SAI Life Sciences Ltd. using human, dog, rat, and mouse plasma. Protein binding
138
was measured via rapid equilibrium dialysis (RED) using an RED device (Thermo Scientific)
139
containing dialysis membrane with a molecular weight cut-off of 8,000 Da. Each plasma type
140
was spiked in triplicate with 5 µM TXA709. Dialysis was then performed with shaking (at 100
141
rpm) for four hours at 37 °C, as per the manufacturer’s recommendation. Following dialysis, the
142
concentration of TXA707 in each well (both plasma and buffer side) was quantified by LC-
143
MS/MS, and the resulting peak area ratios were used to determine the percentages of compound
144
unbound and bound to plasma proteins.
145
S. aureus FtsZ polymerization assay. S. aureus FtsZ was expressed and purified as
146
described previously (30).
Polymerization of S. aureus FtsZ was monitored as described
147
previously (26), with the exception that FtsZ was used at a concentration of 15 µM.
148
Fluorescence microscopy. Fluorescence microscopy studies with B. subtilis FG347
149
were conducted as described previously (26), with the exception that the bacteria were cultured
150
for two hours in the presence of DMSO vehicle or 4 µg/mL TXA707 (8x MIC).
151
Transmission electron microscopy (TEM).
Log-phase MSSA 8325-4 cells were
152
cultured in CAMH broth at 37 °C for 0, 1, 4, or 9 hours in the presence of DMSO (solvent
153
vehicle) or TXA707 at a concentration of 4 µg/mL (8x MIC). At each time point, a 1-mL
154
sample was withdrawn from the culture and centrifuged at 16,000 × g for 3 minutes at room
155
temperature. The supernatant was removed, and the bacterial pellet was washed with 1 mL of
156
phosphate-buffered saline (PBS). The final bacterial pellet was fixed by resuspension in 500 µL
157
of 0.1 M cacodylate buffer (pH 7.2) containing 2.5% gluteraldehyde and 4% paraformaldehyde.
158
The fixed bacterial cells were then post-fixed in buffered osmium tetroxide (1%), and
159
subsequently dehydrated in a graded series of ethanol and embedded in epon resin. Thin sections
160
(90 nm) were cut on a Leica EM UC6 ultramicrotome. Sectioned grids were then stained with a
161
saturated solution of uranyl acetate and lead citrate. Images were captured with an AMT XR111
162
digital camera at 80 Kv on a Philips CM12 transmission electron microscope.
163
Assay for the frequency of resistance (FOR) and identification of resistant ftsZ
164
mutations. FOR studies were conducted as described elsewhere (25), and the ftsZ genes in
165
resistant mutants were sequenced (30).
166
In vivo efficacy assays. Antistaphylococcal efficacy in vivo was assessed in a mouse
167
peritonitis model of systemic infection, as well as in a mouse tissue (thigh) model of infection.
168
The experimental details associated with the in vivo efficacy studies are described in the
169
Supplementary Material.
170
MTT cytotoxicity assay. The cytotoxicity of TXA709 was assessed in human cervical
171
cancer (HeLa) and Madin-Darby canine kidney (MDCK) epithelial cells using a four-day
172
continuous 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as
173
described previously (31), with vehicle and the anticancer drug camptothecin serving as negative
174
and positive controls, respectively. TXA707 was not included in these characterizations, due to
175
its limited solubility at the higher concentrations necessitated by the assay.
176
RESULTS AND DISCUSSION
177
Metabolic attack of the Cl atom on the pyridyl ring of the first-generation prodrug
178
TXY541 results in sub-optimal pharmacokinetics of the active product PC190723. In our
179
previous studies, we demonstrated that the oral and intravenous doses of the prodrug TXY541
180
required for efficacy in mouse models of MRSA and MSSA infection are large (96 to 192
181
mg/kg) due to the rapid elimination and modest oral bioavailability of the active product
182
PC190723 (25). As a first step toward understanding the basis for the rapid elimination of
183
PC190723 upon TXY541 administration, we conducted a metabolic study of TXY541 in the
184
presence of either mouse or human hepatocytes. Several metabolites were observed in the
185
presence of both mouse and human hepatocytes (Fig. 1A). The active product PC190723 was
186
one of these metabolites (M2), detected by mass spectrometry peak analysis at an abundance of
187
≈20% with mouse and ≈30% with human hepatocytes. An even more abundant metabolite (M1)
188
(detected at an abundance of ≈70% with mouse and ≈50% with human hepatocytes) was one
189
resulting from the dechlorination and subsequent monooxygenation of TXY541. Thus, the Cl
190
atom on the pyridyl ring of TXY541 appears to be susceptible to metabolic dehalogenation.
191
Dehalogenation and oxygenation reactions are typically catalyzed by phase I cytochrome
192
P450 (CYP) enzymes. We therefore hypothesized that pretreatment with a CYP inhibitor would
193
retard the elimination of PC190723. To this end, we assessed the impact of pretreatment with
194
CYP inhibitor 1-aminobenzotriazole (ABT) on the pharmacokinetics of PC190723 in mice
195
following administration of TXY541. As shown in Table 1, pretreatment with ABT resulted in
196
an elimination half-life (t½) of 1.95 hours, a value approximately 3.5-times greater than that
197
observed in the absence of ABT (0.56 hours). This increased t½ results from a correspondingly
198
reduced clearance (CL) (13.2 versus 55.1 mL/min/kg). Thus, inhibition of CYP enzymatic
199
activity with ABT retards the elimination of the PC190723 upon administration of the TXY541
200
prodrug.
201
We next sought to determine whether the retarded elimination of PC190723 induced by
202
ABT would also reduce the dose of TXY541 required for antistaphylococcal efficacy in vivo. In
203
the absence of ABT, oral administration of TXY541 required a dose of 128 mg/kg for efficacy
204
(83% survival; 5/6) in mice systemically infected with MSSA ATCC 19636 (Fig. 2A) and a dose
205
of 192 mg/kg for efficacy (100% survival; 6/6) in mice systemically infected with MRSA ATCC
206
43300 (Fig. 2B). By contrast, with ABT pretreatment, the dose of TXY541 required for efficacy
207
(100% survival; 6/6) in both MSSA- and MRSA-infected mice was 64 mg/kg. Pretreatment with
208
the CYP inhibitor therefore reduces the dose of TXY541 required for efficacy by at least 2- and
209
3.5-fold in the MSSA-infected and MRSA-infected mice, respectively.
210
Design of a second-generation prodrug (TXA709) that can circumvent the CYP-
211
mediated dechlorination/oxygenation reactions associated with TXY541. As indicated by
212
our studies described above, the Cl atom on TXY541 is vulnerable to CYP-mediated metabolic
213
attack. Our goal in designing a second-generation prodrug was to substitute the Cl functionality
214
on the pyridyl ring with an electron-withdrawing group that would not undergo dehalogenation.
215
In addition to being resistant to metabolism, the introduced group needed to be hydrophobic in
216
nature, as polar functionalities at this position of the pyridyl ring tend to reduce
217
antistaphylococcal potency (32). One of the most promising second-generation prodrugs to
218
emerge from our design efforts is TXA709 (shown in Fig. 1B) in which the Cl atom present in
219
TXY541 has been replaced with a CF3 functionality. Just as hydrolysis of TXY541 yields the
220
active product PC190723, corresponding hydrolysis of TXA709 yields an active product we
221
denote as TXA707 (also shown in Fig. 1B). In fact, both TXY541 and TXA709 have similar
222
prodrug-to-product conversion kinetics. Fig. 3 shows HPLC chromatograms demonstrating the
223
time-dependent conversion of TXA709 to TXA707 at 37 °C in two different media at pH 7.4,
224
CAMH broth (the media used for culturing S. aureus) and mouse serum. Analysis of the
225
TXA709 peak areas yielded conversion half-lives (7.7 ± 0.1 hours in CAMH broth and 3.0 ± 0.2
226
minutes in mouse serum) of similar magnitude to those previously reported for the conversion of
227
TXY541 to PC190723 (8.2 ± 0.4 hours in CAMH broth and 3.4 ± 0.2 minutes in mouse serum)
228
(25). In other words, substitution of CF3 for Cl on the pyridyl ring does not significantly alter the
229
prodrug-to-product conversion kinetics.
230
The CF3 functionality of TXA709 is resistant to metabolic attack in the presence of
231
mouse and human hepatocytes. To determine if the introduced CF3 functionality of TXA709
232
imparts resistance to metabolism, we examined the metabolism of TXA709 in the presence of
233
mouse or human hepatocytes. With both hepatocyte types, only two metabolites were observed
234
(Fig. 1B), with the major metabolite (M1) being the active product TXA707. Significantly, no
235
metabolic reactions targeting the CF3 group were observed, confirming the metabolic stability of
236
this functionality.
237
TXA709 is associated with improved pharmacokinetic properties relative to
238
TXY541. We next sought to determine whether the metabolic stability of TXA709 would
239
translate to improved pharmacokinetic properties in mice. To this end, we evaluated the plasma
240
pharmacokinetics of the TXA709 prodrug and its TXA707 product following both intravenous
241
(i.v.) and oral (p.o.) administration of TXA709 to mice. TXA709 was not detectable in the
242
plasma at any time point (from 0.08 to 24 hours) following administration by either route. By
243
contrast, the plasma concentration of TXA707 was detectable and quantifiable up to 24 hours
244
following both i.v. and p.o. administration of TXA709. This observation is consistent with the
245
rapid conversion of TXA709 to TXA707 that we observed in the presence of mouse serum at 37
246
°C (Fig. 3).
247
The time-dependent plasma concentrations of TXA707 after both p.o. and i.v.
248
administration of TXA709 are graphically depicted in Fig. S1 of the Supplementary Material.
249
Pharmacokinetic analysis of these data yielded the pharmacokinetic parameters summarized in
250
Table 1. A comparison of these parameters with the corresponding pharmacokinetic parameters
251
for PC190723 following TXY541 administration reveals that TXA707 is associated with an
252
elimination t½ following i.v. administration of TXA709 that is approximately 6.5-times longer
253
than the corresponding t½ of PC109723 following i.v. administration of TXY541 (t½ = 3.65 hours
254
for TXA707 versus 0.56 hours for PC190723). The longer t½ of TXA707 relative to PC190723
255
is due to a correspondingly reduced CL (9.40 mL/min/kg for TXA707 versus 55.11 mL/min/kg
256
for PC190723). In addition, the bioavailability (%F) of TXA707 following p.o. administration
257
of TXA709 was found to be 95%, approximately 3.2-times the corresponding value (%F = 29.6)
258
we have previously shown for PC190723 following p.o. administration of TXY541 (25).
259
To determine whether the observed pharmacokinetic differences between TXA707 and
260
PC190723 might be due to differences in plasma protein binding, we assessed the protein
261
binding properties of TXA707 in mouse, human, rat, and dog plasma. The results of these
262
studies (summarized in Table 2) revealed that TXA707 is ≈86% bound to mouse plasma proteins
263
and ≈91% bound to human, rat, and dog plasma proteins. Significantly, the percentage bound to
264
mouse plasma proteins is nearly identical to that (85.4%) previously reported for PC190723 (32),
265
indicating that the observed pharmacokinetic differences between the two compounds do not
266
reflect differences in plasma protein binding properties. Our collective results therefore indicate
267
that the metabolic stability of the CF3 moiety present in TXA709 confers the second-generation
268
prodrug with markedly improved pharmacokinetic properties relative to TXY541, properties that
269
include an active product with (i) reduced CL, (ii) longer elimination t½, and (iii) enhanced oral
270
bioavailability.
271
TXA709 and TXA707 exhibit potent bactericidal activity versus clinical isolates of
272
MRSA, VISA, VRSA, DNSSA, LNSSA, and MSSA. We evaluated the minimal inhibitory
273
concentrations (MICs) and minimal bactericidal concentrations (MBCs) of TXA709 and
274
TXA707 against a panel of 84 clinical S. aureus isolates, including 30 MRSA, 20 VISA, 11
275
VRSA, 7 DNSSA, 6 LNSSA, and 10 MSSA isolates. The results of these determinations are
276
summarized in Table 3. The modal MIC for TXA707 was 1 µg/mL against all six types of
277
clinical isolates tested (MRSA, VISA, VRSA, DNSSA, LNSSA, and MSSA). We observe an
278
identical modal MIC for PC190723 against the six types of clinical isolates. Thus, the Cl-to-CF3
279
substitution on the pyridyl ring does not diminish antibacterial potency. Note that the modal
280
MIC of TXA707 against the MRSA isolates is similar, if not identical, to the corresponding
281
modal MICs of vancomycin (1 µg/mL), daptomycin (1 µg/mL), and linezolid (2 µg/mL), which
282
were included as comparator control antibiotics representative of current standard-of-care (SOC)
283
drugs for the treatment of MRSA infections. Significantly, TXA707 maintains a modal MIC of
284
1 µg/mL against S. aureus strains that are associated with varying degrees of resistance to
285
vancomycin (VISA and VRSA), daptomycin (DNSSA), or linezolid (LNSSA). Thus, TXA707
286
maintains its antistaphylococcal potency even against strains where current SOC drugs do not.
287
While the prodrug TXA709 also exhibits activity against all 84 S. aureus isolates tested, its
288
modal MICs are 2- to 4-fold higher than those of TXA707. This discrepancy likely reflects the
289
lack of total conversion of TXA709 to TXA707, due to the slow conversion kinetics in CAMH
290
medium (t½ = 7.7 ± 0.1 hours). For all the S. aureus isolates examined, the modal MBCs for
291
TXA707 and TXA709 were similar, if not identical, to the corresponding modal MICs, with the
292
difference between the modal MBCs and MICs never being >2-fold. These results indicate that
293
the antibacterial activity of both compounds against the S. aureus isolates tested is bactericidal in
294
nature.
295
For comparative purposes, we also tested TXA707 and TXA709 for activity versus
296
quality control and laboratory strains of both MSSA and MRSA (two quality control strains and
297
one laboratory strain each), with the results being summarized in Table 4. As expected, the
298
antibacterial potencies of both compounds versus these MSSA and MRSA strains were similar in
299
magnitude (MIC = 0.5 µg/mL for TXA707 and 2-4 µg/mL for TXA709) to those observed
300
versus the clinical isolates described above. These quality control and laboratory strains of S.
301
aureus were used in the in vivo efficacy experiments described below.
302
Validation of the cell division protein FtsZ as the bactericidal target of TXA707. We
303
next sought to establish that the antibacterial activity of TXA707, the active product of the
304
TXA709 prodrug, reflects its ability to target FtsZ. To this end, we conducted the following four
305
series of studies:
306
(i) Impact on FtsZ polymerization. We and others have shown that the antibacterial
307
activities of FtsZ-targeting benzamides like PC190723 result from over-stimulation of FtsZ
308
polymerization dynamics and stabilization of nonfunctional FtsZ polymeric structures (21-24,
309
26, 33, 34). Thus, as a first step in validating FtsZ as the antibacterial target of TXA707, we
310
assessed the impact of TXA707 on the polymerization dynamics of purified S. aureus FtsZ using
311
a microtiter plate-based spectrophotometric assay in which changes in FtsZ polymerization are
312
reflected by corresponding changes in absorbance at 340 nm (A340). Fig. 4A shows the time-
313
dependent A340 profiles of S. aureus FtsZ in the absence and presence of TXA707 at
314
concentrations ranging from 0.5 to 5 µg/mL. Note that TXA707 stimulates FtsZ polymerization
315
in concentration-dependent manner, a similar behavior to that previously demonstrated for
316
PC190723 (21-24, 26, 33, 34). As expected, vancomycin, included as a non-FtsZ-targeting
317
control antibiotic in this assay, did not impact FtsZ polymerization.
318
(ii) Impact on FtsZ Z-ring formation. We also evaluated the impact of TXA707 on FtsZ
319
Z-ring formation in bacteria using fluorescence microscopy. To this end, we used a strain of B.
320
subtilis (FG347) that expresses a GFP-tagged Z-ring marker protein (ZapA) (35). We have
321
previously shown that PC190723 induces a filamentous phenotype in these bacteria, as well as
322
mislocalization of FtsZ from the septal Z-ring at midcell to multiple punctate sites throughout the
323
cell (26, 34). As shown in Fig. 4B, our studies with TXA707 have demonstrated an identical
324
pattern of behavior, consistent with inhibition of cell division by the compound through
325
disruption of FtsZ function.
326
(iii) Impact on septum formation and cell division. As a third approach to establishing
327
TXA707 as an inhibitor of cell division, we also used transmission electron microscopy (TEM)
328
to examine the impact of TXA707 on cell morphology and septum formation in MSSA 8325-4.
329
The precise localization of FtsZ and the penicillin binding proteins (PBPs) to the septum is
330
critical for the proper coordination of septal proteoglycan biosynthesis and cell division in S.
331
aureus (18-20). The FtsZ-targeting activity of PC190723 in S. aureus has been shown to cause
332
the disruption of cell division through mislocalization of both FtsZ and PBP2 (and thus septal
333
biosynthesis) from midcell to the cell periphery (18, 20). Our TEM studies demonstrate that
334
within 1 hour of treatment with TXA707, no septa are observed in the S. aureus bacteria (Fig. 5).
335
Instead, peptidoglycan synthesis appears to have shifted to discrete locations at the cell
336
periphery. In addition, the cells become enlarged by approximately 3-fold over time, eventually
337
leading to cell lysis.
338
(iv) Isolation of resistance mutations that map to the FtsZ coding sequence. Perhaps the
339
most definitive approach for validating a particular bacterial protein as the specific target of an
340
antibacterial agent is through genetic studies demonstrating that mutations in the target protein
341
can result in resistance. In this connection, we used a large inoculum approach in an effort to
342
raise spontaneous mutants of MRSA that are resistant to TXA707. This approach yielded
343
resistant clones at a frequency of ≈3 x 10-8 in MRSA ATCC 43300 and ≈1 x 10-8 in a MRSA
344
clinical isolate from a patient at RWJUH. These FOR values are similar in magnitude to those
345
previously reported for PC190723 in various MSSA and MRSA strains (20, 23, 25). Twenty of
346
the TXA707-resistant clones (10 from each MRSA strain) were isolated, and the ftsZ gene in
347
each clone was sequenced. All 20 resistant clones carried a mutation that mapped to the ftsZ
348
gene. Of the 20 clones, 11 (55%) carried the G196S mutation, 3 (15%) carried the N263K
349
mutation, 3 (15%) carried the G193D nutation, 2 (10%) carried the G196C mutation, and 1 (5%)
350
carried the G196A mutation (Fig. 4C). All of these FtsZ mutations have been previously shown
351
to cause S. aureus resistance to PC190723 at similar relative frequencies (20, 23).
352
mutational analysis is therefore fully consistent with FtsZ being the antibacterial target of
353
TXA707.
Our
354
TXA709 is associated with enhanced oral and intravenous efficacy relative to
355
TXY541 in a mouse systemic (peritonitis) model of MSSA and MRSA infection. Armed
356
with the in vitro susceptibility, FtsZ-targeting, and pharmacokinetic results described above, we
357
next evaluated the in vivo antistaphylococcal efficacy of TXA709 using a mouse peritonitis
358
model of systemic infection with S. aureus.
359
intraperitoneally with a lethal inoculum of MSSA ATCC 19636, against which TXA707 exhibits
360
an MIC of 0.5 µg/mL (Table 4). None of the mice treated p.o. (0/6) or i.v. (0/5) with vehicle
361
alone survived beyond one day post infection (Figs. 6A and 6D). By contrast 50% (3/6) of the
362
mice receiving a p.o. dose of 32 mg/kg TXA709 and 100% (6/6) of the mice receiving a p.o.
363
dose of 64 mg/kg TXA709 survived the MSSA infection. Furthermore, 100% (5/5) of the mice
364
receiving an i.v. dose of 36 or 72 mg/kg TXA709 also survived the MSSA infection. Thus, an
365
i.v. dose of 36 mg/kg TXA709 and a p.o. dose of 64 mg/kg were sufficient for 100% survival of
366
the MSSA infection. Significantly, these efficacious i.v. and p.o. doses of TXA709 are 2- to 2.7-
367
times lower than the corresponding efficacious doses of TXY541 (Fig. 2).
In our initial studies, mice were inoculated
368
In our next set of studies, mice were administered a lethal inoculum of MRSA ATCC
369
43300. None of the infected mice treated p.o. (0/6) or i.v. (0/4) with vehicle alone survived
370
beyond two days post infection (Figs. 6B and 6E). In striking contrast, an i.v. dose of 36 mg/kg
371
TXA709 and a p.o. dose of 48 mg/kg TXA709 resulted in 100% survival (6/6 and 4/4 of the
372
mice treated i.v. and p.o., respectively). Recall that the p.o. dose of TXY541 required for 100%
373
survival in the systemic MRSA 43300 infection model was 192 mg/kg (Fig. 2), 4-times higher
374
than the corresponding efficacious p.o. dose of TXA709 (48 mg/kg). Additional studies with
375
mice infected with MRSA Mu3 revealed efficacious TXA709 doses of 72 mg/kg i.v. and 96
376
mg/kg p.o. (Figs. 6C and 6F). The larger doses of TXA709 required for efficacy against Mu3
377
relative to ATCC 43300 do not reflect a difference in the antibacterial potency of TXA707
378
against the two bacterial strains, as the MIC of TXA707 is 0.5 µg/mL against either strain (Table
379
4). Instead, the reduced efficacy of TXA709 against Mu3 compared to ATCC 43300 may reflect
380
an enhanced virulence on the part of the Mu3 strain, the lethal inoculum for which is 2.5-times
381
lower than that for ATCC 43300 (see Supplementary Material).
382
In the aggregate, our in vivo antistaphylococcal studies demonstrate that TXA709 is
383
associated with enhanced in vivo efficacy relative to TXY541 against both MSSA and MRSA
384
infections.
385
TXA709 is also orally efficacious in a mouse tissue (thigh) model of MRSA infection.
386
Among the pharmacokinetic properties of TXA707 upon i.v. administration of TXA709 was a
387
volume of distribution at steady state (Vd-ss) of 2.02 L/kg (see Table 1). This Vd-ss value is
388
approximately 3-times greater than the total water volume in the mouse (0.7 L/kg), indicating
389
that TXA707 distributes beyond the vascular space and into the tissue. This property is desirable
390
for antistaphylococcal agents, since staphylococcal infections frequently occur in soft tissue. In
391
this connection, we characterized the oral efficacy of TXA709 in a mouse tissue (thigh) model of
392
infection with MRSA ATCC 33591. An oral dose of either 120 or 160 mg/kg TXA709 resulted
393
in a ≈2-log reduction in bacterial CFUs relative to vehicle-treated mice (p ≤ 0.003) at 24 hours
394
post-infection (Fig. 7). Previous studies with PC190723 in a mouse thigh model of MRSA
395
infection yielded only a ≈0.5-log reduction in bacterial CFUs when the compound was
396
administered orally at a dose of 200 mg/kg every 6 hours over a 24-hour period (20). Viewed as
397
a whole, these results indicate that TXA709 is orally efficacious against MRSA not only in a
398
mouse systemic model of infection, but also in a mouse tissue model of infection. Furthermore,
399
the efficacy of orally administered TXA709 against MRSA in the mouse tissue model of
400
infection is significantly greater than that of orally administered PC190723.
401
TXA709 exhibits minimal toxicity to mammalian cells. To probe for any potential
402
mammalian cytotoxicity, we used a four-day tetrazole (MTT)-based assay to assess the
403
cytotoxicity of TXA709 against human cervical cancer (HeLa) and Madin-Darby canine kidney
404
(MDCK) cells. TXA709 was found to be minimally toxic to both cell types, with IC50 values
405
>120 µg/mL. While these IC50 values reflect cell culturing conditions that include the presence
406
of 10% fetal bovine serum, our control studies reveal a modal antistaphylococcal MIC of 2
407
µg/mL for TXA709 in the presence of the same percentage of serum. Thus, the large difference
408
between the mammalian cell IC50s and the antistaphylococcal MICs is due to the targeting
409
specificity of the compound for staphylococci as opposed to serum protein binding effects.
410
In conclusion, TXA709 is a second-generation prodrug of the FtsZ-targeting benzamide
411
compound TXA707.
TXA709 is associated with enhanced metabolic stability, improved
412
pharmacokinetic properties, and superior in vivo antistaphylococcal efficacy (both oral and
413
intravenous) relative to first-generation prodrugs (TXY436 and TXY541) of PC190723. In
414
addition, the TXA707 hydrolysis product of TXA709 maintains potent activity against S. aureus
415
strains (e.g., MRSA, VRSA, DNSSA, and LNSSA) that are resistant to current SOC antibiotics.
416
These characteristics, coupled with minimal cytotoxicity versus mammalian cells, make TXA709
417
an attractive lead compound for development into a clinically useful agent for the treatment of
418
drug-resistant staphylococcal infections.
419
ACKNOWLEDGEMENTS
420
This study was supported by research agreements between TAXIS Pharmaceuticals, Inc. and
421
Rutgers RWJMS (D.S.P. and M.P.W.), Rutgers EMSP (E.J.L.), and SJHMC (L.D.S.). We are
422
indebted to Drs. Glenn W. Kaatz, Richard Losick, and George M. Eliopoulos for providing us
423
with S. aureus 8325-4, B. subtilis FG347, and S. aureus Mu3, respectively. We also wish to
424
thank Raj Patel (Rutgers RWJMS, Core Imaging Lab) for his assistance with the TEM
425
experiments, and Giovanni Divinagracia (RWJUH) for his assistance with the in vitro
426
susceptibility assays.
427
428
REFERENCES
429 430 431
1.
Centers for Disease Control and Prevention. September 2013. Antibiotic Resistance Threats in the United States, 2013, http://www.cdc.gov/drugresistance/threat-report2013/, Atlanta, GA.
432 433
2.
Fair, R. J. and Y. Tor. 2014. Antibiotics and Bacterial Resistance in the 21st Century. Perspect. Med. Chem. 6:25-64.
434 435
3.
Moellering, R. C., Jr. 2012. MRSA: The First Half Century. J. Antimicrob. Chemother. 67:4-11.
436
4.
Otto, M. 2012. MRSA Virulence and Spread. Cell. Microbiol. 14:1513-1521.
437 438 439 440 441 442
5.
Hanaki, H., L. Cui, Y. Ikeda-Dantsuji, T. Nakae, J. Honda, K. Yanagihara, Y. Takesue, T. Matsumoto, K. Sunakawa, M. Kaku, K. Tomono, K. Fukuchi, S. Kusachi, H. Mikamo, T. Takata, Y. Otsuka, O. Nagura, S. Fujitani, Y. Aoki, Y. Yamaguchi, K. Tateda, J. Kadota, S. Kohno and Y. Niki. 2014. Antibiotic Susceptibility Survey of Blood-Borne MRSA Isolates in Japan from 2008 through 2011. J. Infect. Chemother. 20:527-534.
443 444 445 446 447
6.
Ikeda-Dantsuji, Y., H. Hanaki, T. Nakae, Y. Takesue, K. Tomono, J. Honda, K. Yanagihara, H. Mikamo, K. Fukuchi, M. Kaku, S. Kohno and Y. Niki. 2011. Emergence of Linezolid-Resistant Mutants in a Susceptible-Cell Population of Methicillin-Resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 55:24662468.
448 449 450 451 452
7.
Mishra, N. N., A. S. Bayer, C. Weidenmaier, T. Grau, S. Wanner, S. Stefani, V. Cafiso, T. Bertuccio, M. R. Yeaman, C. C. Nast and S. J. Yang. 2014. Phenotypic and Genotypic Characterization of Daptomycin-Resistant Methicillin-Resistant Staphylococcus aureus Strains: Relative Roles of mprF and dlt Operons. PLoS One 9:e107426.
453 454 455 456
8.
Mishra, N. N., J. McKinnell, M. R. Yeaman, A. Rubio, C. C. Nast, L. Chen, B. N. Kreiswirth and A. S. Bayer. 2011. In Vitro Cross-Resistance to Daptomycin and Host Defense Cationic Antimicrobial Peptides in Clinical Methicillin-Resistant Staphylococcus aureus Isolates. Antimicrob. Agents Chemother. 55:4012-4018.
457 458 459 460 461
9.
Saravolatz, L. D., J. Pawlak, L. Johnson, H. Bonilla, L. D. Saravolatz, 2nd, M. G. Fakih, A. Fugelli and W. M. Olsen. 2012. In Vitro Activities of LTX-109, a Synthetic Antimicrobial Peptide, against Methicillin-Resistant, Vancomycin-Intermediate, Vancomycin-Resistant, Daptomycin-Nonsusceptible, and Linezolid-Nonsusceptible Staphylococcus aureus. Antimicrob. Agents Chemother. 56:4478-4482.
462 463
10.
Awasthi, D., K. Kumar and I. Ojima. 2011. Therapeutic Potential of FtsZ Inhibition: A Patent Perspective. Expert Opin. Ther. Patents 21:657-679.
464 465
11.
Lock, R. L. and E. J. Harry. 2008. Cell-Division Inhibitors: New Insights for Future Antibiotics. Nat. Rev. Drug Discov. 7:324-338.
466 467
12.
Sass, P. and H. Brötz-Oesterhelt. 2013. Bacterial Cell Division as a Target for New Antibiotics. Curr. Opin. Microbiol. 16:522-530.
468 469 470
13.
Schaffner-Barbero, C., M. Martin-Fontecha, P. Chacón and J. M. Andreu. 2012. Targeting the Assembly of Bacterial Cell Division Protein FtsZ with Small Molecules. ACS Chem. Biol. 7:269-277.
471 472
14.
Vollmer, W. 2006. The Prokaryotic Cytoskeleton: A Putative Target for Inhibitors and Antibiotics? Appl. Microbiol. Biotechnol. 73:37-47.
473 474
15.
Adams, D. W. and J. Errington. 2009. Bacterial Cell Division: Assembly, Maintenance and Disassembly of the Z ring. Nat. Rev. Microbiol. 7:642-653.
475 476
16.
Bi, E. F. and J. Lutkenhaus. 1991. FtsZ Ring Structure Associated with Division in Escherichia coli. Nature 354:161-164.
477 478
17.
Erickson, H. P., D. E. Anderson and M. Osawa. 2010. FtsZ in Bacterial Cytokinesis: Cytoskeleton and Force Generator All in One. Microbiol. Mol. Biol. Rev. 74:504-528.
479 480
18.
Pinho, M. G. and J. Errington. 2003. Dispersed Mode of Staphylococcus aureus Cell Wall Synthesis in the Absence of the Division Machinery. Mol. Microbiol. 50:871-881.
481 482
19.
Pinho, M. G., M. Kjos and J.-W. Veening. 2013. How to Get (a)round: Mechanisms Controlling Growth and Division of Coccoid Bacteria. Nat. Rev. Microbiol. 11:601-614.
483 484 485 486 487 488 489 490
20.
Tan, C. M., A. G. Therien, J. Lu, S. H. Lee, A. Caron, C. J. Gill, C. Lebeau-Jacob, L. Benton-Perdomo, J. M. Monteiro, P. M. Pereira, N. L. Elsen, J. Wu, K. Deschamps, M. Petcu, S. Wong, E. Daigneault, S. Kramer, L. Liang, E. Maxwell, D. Claveau, J. Vaillancourt, K. Skorey, J. Tam, H. Wang, T. C. Meredith, S. Sillaots, L. Wang-Jarantow, Y. Ramtohul, E. Langlois, F. Landry, J. C. Reid, G. Parthasarathy, S. Sharma, A. Baryshnikova, K. J. Lumb, M. G. Pinho, S. M. Soisson and T. Roemer. 2012. Restoring Methicillin-Resistant Staphylococcus aureus Susceptibility to β-Lactam Antibiotics. Sci .Transl. Med. 4:126ra135.
491 492 493 494
21.
Andreu, J. M., C. Schaffner-Barbero, S. Huecas, D. Alonso, M. L. Lopez-Rodriguez, L. B. Ruiz-Avila, R. Núñez-Ramírez, O. Llorca and A. J. Martín-Galiano. 2010. The Antibacterial Cell Division Inhibitor PC190723 Is an FtsZ Polymer-Stabilizing Agent That Induces Filament Assembly and Condensation. J. Biol. Chem. 285:14239-14246.
495 496 497
22.
Elsen, N. L., J. Lu, G. Parthasarathy, J. C. Reid, S. Sharma, S. M. Soisson and K. J. Lumb. 2012. Mechanism of Action of the Cell-Division Inhibitor PC190723: Modulation of FtsZ Assembly Cooperativity. J. Am. Chem. Soc. 134:12342-12345.
498 499 500 501 502
23.
Haydon, D. J., N. R. Stokes, R. Ure, G. Galbraith, J. M. Bennett, D. R. Brown, P. J. Baker, V. V. Barynin, D. W. Rice, S. E. Sedelnikova, J. R. Heal, J. M. Sheridan, S. T. Aiwale, P. K. Chauhan, A. Srivastava, A. Taneja, I. Collins, J. Errington and L. G. Czaplewski. 2008. An Inhibitor of FtsZ with Potent and Selective AntiStaphylococcal Activity. Science 321:1673-1675.
503 504 505 506 507
24.
Stokes, N. R., J. Sievers, S. Barker, J. M. Bennett, D. R. Brown, I. Collins, V. M. Errington, D. Foulger, M. Hall, R. Halsey, H. Johnson, V. Rose, H. B. Thomaides, D. J. Haydon, L. G. Czaplewski and J. Errington. 2005. Novel Inhibitors of Bacterial Cytokinesis Identified by a Cell-Based Antibiotic Screening Assay. J. Biol. Chem. 280:39709-39715.
508 509 510 511
25.
Kaul, M., L. Mark, Y. Zhang, A. K. Parhi, E. J. LaVoie and D. S. Pilch. 2013. Pharmacokinetics and in Vivo Antistaphylococcal Efficacy of TXY541, a 1Methylpiperidine-4-Carboxamide Prodrug of PC190723. Biochem. Pharmacol. 86:16991707.
512 513 514
26.
Kaul, M., L. Mark, Y. Zhang, A. K. Parhi, E. J. LaVoie and D. S. Pilch. 2013. An FtsZ-Targeting Prodrug with Oral Antistaphylococcal Efficacy in Vivo. Antimicrob. Agents Chemother. 57:5860-5869.
515 516
27.
Sorto, N. A., M. M. Olmstead and J. T. Shaw. 2010. Practical Synthesis of PC190723, an Inhibitor of the Bacterial Cell Division Protein FtsZ. J. Org. Chem. 75:7946-7949.
517 518 519
28.
CLSI. 2009. Methods for Dilution Antimicrobial Sucseptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Eighth Edition, CLSI Document M07-A8, Clinical and Laboratory Standards Institute, Wayne, PA.
520 521 522
29.
Balani, S. K., T. Zhu, T. J. Yang, Z. Liu, B. He and F. W. Lee. 2002. Effective Dosing Regimen of 1-Aminobenzotriazole for Inhibition of Antipyrine Clearance in Rats, Dogs, and Monkeys. Drug Metab. Dispos. 30:1059-1062.
523 524 525
30.
Kaul, M., A. K. Parhi, Y. Zhang, E. J. LaVoie, S. Tuske, E. Arnold, J. E. Kerrigan and D. S. Pilch. 2012. A Bactericidal Guanidinomethyl Biaryl That Alters the Dynamics of Bacterial FtsZ Polymerization. J. Med. Chem. 55:10160-10176.
526 527 528
31.
Qi, H., C.-P. Lin, X. Fu, L. M. Wood, A. A. Liu, Y.-C. Tsai, Y. Chen, C. M. Barbieri, D. S. Pilch and L. F. Liu. 2006. G-Quadruplexes Induce Apoptosis in Tumor Cells. Cancer Res. 66:11808-11816.
529 530 531 532 533 534
32.
Haydon, D. J., J. M. Bennett, D. Brown, I. Collins, G. Galbraith, P. Lancett, R. Macdonald, N. R. Stokes, P. K. Chauhan, J. K. Sutariya, N. Nayal, A. Srivastava, J. Beanland, R. Hall, V. Henstock, C. Noula, C. Rockley and L. Czaplewski. 2010. Creating an Antibacterial with in Vivo Efficacy: Synthesis and Characterization of Potent Inhibitors of the Bacterial Cell Division Protein FtsZ with Improved Pharmaceutical Properties. J. Med. Chem. 53:3927-3936.
535 536
33.
Adams, D. W., L. J. Wu, L. G. Czaplewski and J. Errington. 2011. Multiple Effects of Benzamide Antibiotics on FtsZ Function. Mol. Microbiol. 80:68-84.
537 538 539 540
34.
Kaul, M., Y. Zhang, A. K. Parhi, E. J. LaVoie, S. Tuske, E. Arnold, J. E. Kerrigan and D. S. Pilch. 2013. Enterococcal and Streptococcal Resistance to PC190723 and Related Compounds: Molecular Insights from a FtsZ Mutational Analysis. Biochimie 95:1880-1887.
541 542 543
35.
Gueiros-Filho, F. J. and R. Losick. 2002. A Widely Conserved Bacterial Cell Division Protein That Promotes Assembly of the Tubulin-Like Protein FtsZ. Genes Dev. 16:25442556.
544
545 TABLE 1 Pharmacokinetic parameters of PC190723 and TXA707 following administration of their respective prodrugs (TXY541 and TXA709) to male BALB/c micea Prodrug Dose Administered (mg/kg)
ABT Pretreatment b
Product Measured
tmax (h)
Cmax AUClast t½ (ng/mL) (h•ng/mL) (h)
CL (mL/min/kg)
Vd-ss (L/kg)
%F
TXY541
24 (i.v.)
Yes
PC190723
0.50
7491
28589
1.95
13.20
2.11
NA
TXY541
24 (i.v.)
No
PC190723
0.25
6646
7216
0.56
55.11
2.18
NA
TXA709
24 (i.v.)
No
TXA707
0.50
13794
42299
3.65
9.40
2.02
NA
TXA709
32 (p.o.)
No
TXA707
1.00
9850
53679
2.66
NA
NA
95
a
Parameters were calculated using the sparse sampling mode in the non-compartmental analysis (NCA) module of the Phoenix WinNonlin version 6.3 software package. ABT, 1-aminobenzotriazole; NA, not applicable; i.v., intravenous; p.o., peroral.
b
Mice were pretreated for one hour with 50 mg/kg ABT administered orally.
546 547 548 549 TABLE 2 Protein binding of TXA707 in mouse, human, dog, and rat plasma
550 551
Species
% Protein Bounda
Mouse Human Dog Rat a Values reflect three replicates.
85.9 ± 0.4 90.9 ± 0.5 90.9 ± 0.3 90.8 ± 1.3 the mean ± SD of
TABLE 3 Activities against clinical isolates of MRSA, VISA, VRSA, DNSSA, LNSSA, and MSSAa Isolate and agent
MIC (µg/mL) Range
MBC (µg/mL) Modal
MRSA (n = 30) TXA707 0.25-1 1 TXA709 2-4 2 PC190723 0.5-1 1 VAN 0.5-1 1 DAP 0.5-2 1 LZD 2-4 2 VISA (n = 20) TXA707 0.5-2 1 TXA709 2-4 2 PC190723 0.5-1 1 VAN 4-8 4 DAP 1-8 1 LZD 0.5-2 2 VRSA (n = 11) TXA707 0.5-1 1 TXA709 2 2 PC190723 0.5-1 1 VAN 32->64 >64 DAP 0.25-1 1 LZD 0.5-4 2 DNSSA (n = 7) TXA707 0.5-1 1 TXA709 2-4 2 PC190723 0.5-1 1 VAN 1-2 2 DAP 4 4 LZD 1-2 2 LNSSA (n = 6) TXA707 1 1 TXA709 2 2 PC190723 0.5-1 1 VAN 1-2 1 DAP 0.5-1 0.5 LZD 16-64 16 MSSA (n = 10) TXA707 0.5-1 0.5/1b TXA709 2-4 4 PC190723 0.5-1 1 VAN 1-2 1 DAP 1 1 a VAN, vancomycin; DAP, daptomycin; LZD, linezolid; ND, not for MRSA were determined with 20 isolates. b Bimodal behavior.
Range
Modal
1-2 2-4 0.5-1 0.5-1 0.5-1 8->8
1 4 1 1 1 >8
0.5-2 2-4 0.5-2 4-8 1-16 2->8
1 2 1 4 2 >8
1 2 0.5-1 64->64 0.25-1 8->8
1 2 1 >64 1 >8
1-2 2-4 0.5-1 2 4-8 2->8
1 4 1 2 8 >8
1-2 2-4 1 1-2 0.5-1 32->64
1 2 1 1 0.5/1b >64
1-2 1/2b 4-8 8 1-2 2 1-4 2 ND ND determined. MBC values
552 553 TABLE 4 Activities against reference quality control and laboratory strains of MRSA and MSSA MIC (µg/mL)
Strain
TXA707
TXA709
PC190723
VAN
0.5
4
0.5
2
1
4
1
2
0.5
2
0.5
2
ATCC 19636a
0.5
2
0.5
1
ATCC 29213
0.5
2
1
1
0.5
2
0.5
0.5
MRSA ATCC 43300a b
ATCC 33591 Mu3
a
MSSA
8325-4 a
c
Strains used in the in vivo mouse systemic (peritonitis) infection studies. b Strain used in the in vivo mouse tissue (thigh) infection studies. c Strain used in the TEM studies. 554 555
FIGURE 1
556
557 558 559 560
A
B
FIG 1 Metabolites of the prodrugs TXY541 (A) and TXA709 (B) that are observed upon exposure to either human or mouse hepatocytes for 60 minutes at 37 °C.
FIGURE 2
561
MSSA - ATCC 19636 100
% Survival
80
60
Oral (p.o.) 40
TXY541 (128 mg/kg) TXY541 (64 mg/kg) ABT + Vehicle
20
0
ABT + TXY541 (64 mg/kg)
A 0
1
2
3 Days
4
5
MRSA - ATCC 43300 100
% Survival
80
60
Oral (p.o.) TXY541 (192 mg/kg)
40
TXY541 (64 mg/kg) ABT + Vehicle
20
0 0
562 563 564 565 566
ABT + TXY541 (64 mg/kg)
B 1
2
3 Days
4
5
FIG 2 Impact of pretreatment with 1-aminobenzotriazole (ABT) on the oral efficacy of TXY541 in a mouse peritonitis model of systemic infection with MSSA ATCC 19636 (A) or MRSA ATCC 43300 (B). ABT was administered orally at a dose of 50 mg/kg 1 hour prior to infection.
FIGURE 3
567
CAMH @ 37 °C (t½ = 7.7 ± 0.1 h) 20
0
0 20
TXA709 0.5 min
10
10
0
0
Absorbance @ 296 nm (mAU)
10
10
0
0
0
0
TXA709 7h
20
0
0
20
TXA709 23 h
20 10
0
0
10
10
0
0
10
TXA709 30 min
20
TXA707 23 h
9
TXA709 10 min
10
10
20
TXA709 5 min
10
20 10
TXA709 2 min
20
TXA709 4h
10
TXA709 0.5 min
20
TXA709 2h
20
Serum Alone
10
20
20
569 570 571 572 573 574 575 576 577
20
CAMH Alone
10
568
Mouse Serum @ 37 °C (t½ = 3.0 ± 0.2 min)
11
12
13
Retention Time (min)
14
TXA707 30 min
9
10
11
12
13
14
Retention Time (min)
FIG 3 Reverse-phase HPLC chromatograms of 20 µM TXA709 after the indicated times of incubation at 37 °C in CAMH broth (left) or mouse serum (right). For comparative purposes, the corresponding chromatograms of 20 µM TXA707 after incubation for 23 hours in CAMH (bottom left) or 30 minutes in mouse serum (bottom right) are also presented. The baseline chromatograms of CAMH broth and mouse serum alone are shown at the tops of the left and right panels, respectively. The solid arrows indicate the peaks corresponding to TXA709, while the dashed arrows indicate the peaks corresponding to TXA707. The TXA709-to-TXA707 conversion half-life (t½) in each medium is indicated.
578
FIGURE 4
579 580 581 582
583 584 585 586 587 588 589 590 591 592 593 594 595
FIG 4 (A) Concentration dependence of the impact of TXA707 on the polymerization of S. aureus FtsZ, as determined by monitoring time-dependent changes in absorbance at 340 nm (A340) at 25 °C. Polymerization profiles were acquired in the presence of DMSO vehicle or the indicated concentrations of TXA707. Vancomycin (VAN) was included as a negative (nonFtsZ-targeting) control. Polymerization reactions were initiated by addition of GTP at the time indicated by the arrow. (B) Fluorescence micrographs of B. subtilis FG347 bacteria that express a GFP-tagged, Z-ring marker protein (ZapA). The bacteria were cultured for two hours in the presence of DMSO vehicle (left) or 4 µg/mL TXA707 (8x MIC) (right). The arrows in in the left panel highlight FtsZ Z-rings at midcell. (C) Relative frequency of FtsZ amino acid substitutions conferring resistance to TXA707 among 20 independently isolated TXA707-resistant clones.
596
FIGURE 5
597 598 599 600
FIG 5 Transmission electron micrographs of MSSA 8325-4 bacteria treated with DMSO vehicle or 4 µg/mL TXA707 (8x MIC) for the indicated periods of time.
601
FIGURE 6
602 603 604 605
FIG 6 Oral (A-C) and intravenous (D-F) efficacy of TXA709 in a mouse peritonitis model of systemic infection with MSSA ATCC 19636 (A,D), MRSA ATCC 43300 (B,E), or MRSA Mu3 (C,F). The vehicle was 10 mM citrate (pH 2.6) in all experiments.
606
FIGURE 7
607 608 609
610 611 612 613 614
FIG 7 Oral efficacy of TXA709 in a mouse tissue (thigh) model of infection with MRSA ATCC 33591. The number of CFUs recovered from the infected thighs after 24 hours are shown. Mean CFU reductions resulting from TXA709 treatment were significant (p ≤ 0.003).