Discovery of LFF571: An investigational agent for Clostridium difficile infection

Article abstract of DOI:10.1021/jm201685h

Clostridium difficile (C. difficile) is a Gram positive, anaerobic bacterium that infects the lumen of the large intestine and produces toxins. This results in a range of syndromes from mild diarrhea to severe toxic megacolon and death. Alarmingly, the prevalence and severity of C. difficile infection are increasing; thus, associated morbidity and mortality rates are rising. 4-Aminothiazolyl analogues of the antibiotic natural product GE2270 A (1) were designed, synthesized, and optimized for the treatment of C. difficile infection. The medicinal chemistry effort focused on enhancing aqueous solubility relative to that of the natural product and previous development candidates (2, 3) and improving antibacterial activity. Structure-activity relationships, cocrystallographic interactions, pharmacokinetics, and efficacy in animal models of infection were characterized. These studies identified a series of dicarboxylic acid derivatives, which enhanced solubility/efficacy profile by several orders of magnitude compared to previously studied compounds and led to the selection of LFF571 (4) as an investigational new drug for treating C. difficile infection.

Full text of DOI:10.1021/jm201685h

Article
pubs.acs.org/jmc
Discovery of LFF571: An Investigational Agent for Clostridium
difficile Infection
Matthew J. LaMarche,*,† Jennifer A. Leeds,‡ Adam Amaral,† Jason T. Brewer,† Simon M. Bushell,†
Gejing Deng,‡ Janetta M. Dewhurst,† Jian Ding,† JoAnne Dzink-Fox,‡ Gabriel Gamber,† Akash Jain,∥
Kwangho Lee,† Lac Lee,‡ Troy Lister,† David McKenney,‡ Steve Mullin,‡ Colin Osborne,‡
Deborah Palestrant,§ Michael A. Patane,† Elin M. Rann,† Meena Sachdeva,‡ Jian Shao,† Stacey Tiamfook,‡
Anna Trzasko,‡ Lewis Whitehead,† Aregahegn Yifru,† Donghui Yu,‡ Wanlin Yan,† and Qingming Zhu†
‡
§
†Global Discovery Chemistry, Infectious Disease Area, and Protein Structure Group, Novartis Institutes for Biomedical Research,
250 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
∥Chemical and Pharmaceutical Profiling, Novartis Pharmaceuticals, Cambridge, Massachusetts 02139, United States
S
* Supporting Information
ABSTRACT: Clostridium difficile (C. difficile) is a Gram positive, anaerobic bacterium that infects the lumen of the large
intestine and produces toxins. This results in a range of syndromes from mild diarrhea to severe toxic megacolon and death.
Alarmingly, the prevalence and severity of C. difficile infection are increasing; thus, associated morbidity and mortality rates are
rising. 4-Aminothiazolyl analogues of the antibiotic natural product GE2270 A (1) were designed, synthesized, and optimized for
the treatment of C. difficile infection. The medicinal chemistry effort focused on enhancing aqueous solubility relative to that of
the natural product and previous development candidates (2, 3) and improving antibacterial activity. Structure−activity rela-
tionships, cocrystallographic interactions, pharmacokinetics, and efficacy in animal models of infection were characterized. These
studies identified a series of dicarboxylic acid derivatives, which enhanced solubility/efficacy profile by several orders of mag-
nitude compared to previously studied compounds and led to the selection of LFF571 (4) as an investigational new drug for
treating C. difficile infection.
isolates with increased toxin production.2,3 Annually in the U.S.,
INTRODUCTION
■
there are at least 50 000 admissions, >250 000 cases, and more
than 15 000 deaths due to C. difficile infection.4 The estimated
annual U.S. cost for managing C. difficile infections is con-
servatively estimated at $3.2 billion.5
Therapies for C. difficile infection exist, but they are in-
adequate (e.g., efficacy, tolerability). Vancomycin (5, Figure 1),
metronidazole (not shown), and fidaxomicin6 (6) are used for
C. difficile is a spore forming, anaerobic, Gram positive bacterium.
C. difficile infection results from spore germination in the gut of
infected persons and subsequent toxin production, resulting in
severe inflammation and diarrhea in infected patients. C. difficile
infections often follow broad spectrum antibiotic use that disturbs
normal and beneficial gut flora.1 The prevalence and severity of
C. difficile infections appear to be rising, in part due to changes in
hospital environments, rising numbers of patients with risk factors
for disease (e.g., elderly patients), an increasing use of broad
spectrum antibiotics, and a higher proportion of bacterial
Received: December 14, 2011
Published: February 8, 2012
© 2012 American Chemical Society
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Figure 1. Structures of 1 and natural product-based marketed C. difficile antibiotics 5 and 6.
treating C. difficile infection. While they have comparable initial
response rates, 5 and metronidazole treatments encounter
significant disease relapse.7
Although the in vitro antimicrobial activities of 1 are ex-
cellent, the aqueous solubility was unmeasurable in our assays
(<0.005 nM).11 Also, EF-Tu is located in the cytoplasm, which
presented a particularly formidable drug discovery hurdle, as
soluble derivatives would also need to cross the cell membrane
in order to exert their antibacterial effect, unlike 5 which targets
the cell wall itself. Thus, an increase in aqueous solubility with
the retention of cellular antimicrobial activity represented an
arduous challenge for the optimization effort.
Previously, we described lead finding and optimization activ-
ities associated with 4-aminothiazolyl analogues of 1.12 On the
basis of in vitro and in vivo potency, two distinct carboxylic
acid based antibacterials (2 and 3) were further investigated as
preclinical development candidates for the treatment of infec-
tions caused by C. difficile and other Gram positive organ-
isms.13 A compelling feature of these molecules included the
combination of potent antibacterial activity coupled with im-
proved aqueous solubility through the addition of a single
carboxylic acid functional group. Cocrystallography with EF-Tu
guided our optimization efforts, which focused on amide and
urethane-based aminothiazolyl linkers to orient carboxylic acid
containing side chains toward Arg223 of domain II of EF-Tu
(Figure 2). Since monocarboxylate-containing compounds
(2, 3) retained antibacterial activity and significantly increased
aqueous solubility, we investigated additional solubilizing func-
tional groups and general structure−activity relationship ex-
pansion of this novel class of antibacterials. Given the proximity
of Arg223 and Arg262 within domain II of EF-Tu to the
The discovery of new anti-infective chemical entities is a high
priority in research and development.8 There exists a pressing
need for antibacterial chemical templates that act via novel
mechanisms of action and are potent against clinically resistant
organisms and recalcitrant infections. Historically, natural prod-
ucts have been excellent sources for antibacterial therapies
and medicinal chemistry discovery starting points. Indeed many
marketed antibiotics are natural products themselves or
derivatives thereof (e.g., 5, 6).9
In 1991, Selva and co-workers from Lepetit Research Insti-
tute reported the structure and activity of the natural product
antibiotic 1. This thiopeptide-based secondary metabolite was
isolated from a fermentation broth of Planobispora rosea and
found to inhibit the prokaryotic chaperone elongation factor
Tu (EF-Tu).10 Like 5 and 6, 1 is a macrocyclic natural product
of high molecular weight (1291 Da). However, key structural
differences relative to 5 and 6 include the presence of aromatic
thiazole rings, the dearth of solubilizing functional groups
(e.g., amines, carboxylic acids, sugars), and the mechanism of
action (EF-Tu). The in vitro antibiotic activity of 1 against
C. difficile, as well as a range of Gram positive pathogens includ-
ing methicillin resistant Staphylococcus aureus (MRSA), vancomycin
resistant enterococci (VRE), and group A streptococci, is ex-
cellent, with minimum inhibitory concentrations (MIC) in the
sub μg/mL range.
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Figure 2. Cocrystal of E. coli EF-Tu and carboxylic acid 3.
monocarboxylate-based ligands, we envisioned additional
interactions using additional carboxylates. Thus, we designed,
synthesized, and evaluated di- and tricarboxylic acid bearing
analogues of 1. Herein, we report the structure-guided design,
synthesis, structure−activity relationships, and biological
evaluation of di- and tricarboxylic acid analogues of 1. These
efforts led to the initiation of clinical investigations of the safety
and efficacy of LFF571 (4) for the treatment of C. difficile
infections in humans.
organisms comprised our antibacterial panel: Enterococcus
faecalis, Enterococcus faecium, Staphylococcus aureus, Streptococcus
pyogenes, and C. difficile.15 Streptococcus pneumonia was also
evaluated (not shown) but did not differ significantly from S.
pyogenes. Select compounds were also evaluated in surface
plasmon resonance experiments to determine the Kd and also in
an in vitro transcription/translation assay measuring protein
synthesis inhibition in cell extracts.16 These assays, in conjunc-
tion with the MICs, assisted in the evaluation of cellular uptake
of compounds that lost antibacterial activity. Methyl derivatiza-
tion of the amide linker retained moderate antibacterial activity
in three organisms (MIC of 13: 2 to >64 μg/mL) and also first
demonstrated a method for selective alkylation of the boc pro-
tected urethane nitrogen, which revealed a new area for
medicinal chemistry exploration. Moderate antibacterial activity
was also realized with amides terminating in succinic acid
(MIC of 14, 15: 4 to >32 μg/mL), with a slight stereochemical
preference. Thus, to our surprise, retention of moderate anti-
bacterial activity was permissible while incorporating multiple
ionizable functional groups. Unfortunately, all efforts to im-
prove antibacterial activity by separating the carboxylic acid func-
tional groups failed (16−26). These results included varying
the tether lengths of the second amide side chain (16, 17) and
adding carbocycles, which had previously improved antibacte-
rial activity (18−20).4 Urea-based diacids also initially failed to
retain even moderate antibacterial activity, especially when the
internal nitrogen was substituted (21−23). However, when
carboxylates were positioned on a piperidine-derived urea,
moderate activity was retained (MIC of 24: 0.25−4 μg/mL).
Heterocyclic derivatives also failed to retain moderate
antibacterial activity (25, 26). Of the five target organisms,
C. difficile was the most sensitive to the compounds. In all cases,
the compounds inhibited protein synthesis in bacterial cell
extracts (30−90% at 2 μM, not shown) which compares equally
to 1 (IC50 = 2.0 μM). This indicated that the observed SAR
results from variable cell penetration and not reduced pathway
inhibition. Furthermore, the dicarboxylate analogues that re-
tained moderate activity (14, 15) were profiled in aqueous
solubility assays and displayed a dramatic increase in solubility
RESULTS
■
1 was first subjected to a previously reported acid-catalyzed
rearrangement, basic hydrolysis,14 and acyl derivatization which
afforded acylazide 7 in high overall yield (not shown).3 In order
to evaluate amide, urethane, and urea-linked diacid containing
analogues, three synthetic routes were employed (Scheme 1).
For urea derivatives (e.g., 8), the azide was heated in the pre-
sence of secondary amines and the resulting tertiary urea esters
were selectively alkylated on the internal nitrogen. The ester
protecting groups were then hydrolyzed, which afforded the
ureas (8). For urethane derivatives (e.g., 11), various alcohols
were utilized in the Curtius reaction (e.g., 9, 10), followed by
selective alkylation of the urethane nitrogen and ester hydro-
lysis (e.g., 11). We speculate that the pseudobenzylic nature of
the urethane and urea nitrogens allowed for selective manip-
ulation over other functional groups (amides) within the struc-
turally complex macrocycle. For amide-based diacid derivatives
(e.g., 12), direct alkylation of the amide nitrogen failed, so
the acylazide was heated in tert-butyl alcohol (80 °C), which
furnished the boc-protected aminothiazole (9). Selective
alkylation proceeded at the urethane nitrogen, followed by
boc removal, acylation of the secondary amine, and final ester
hydrolysis (e.g., 12). Alternatively, multiple carboxylic acid
functional groups could be installed in a single side chain
during the Curtius reaction (vide infra).
In order to further improve the aqueous solubility while re-
taining antibacterial activity, di- and tricarboxylic acid analogues
derived from amides, ureas, and urethanes were evaluated for
minimum inhibitor concentrations (MIC, Tables 1−3). Five
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Scheme 1. Synthesis of 4-Aminothiazolyl Diacid Analogues
in neutral buffer (>15 mg/mL). Thus, at the outset of the
diacid investigation, compounds containing a second carboxylic
acid displayed improved aqueous solubility by over an order
of magnitude compared to monoacid containing congeners
(2: ∼0.3 mg/mL); however, the antibacterial activity of the
diacids required further improvement.
In the urethane subseries (Table 2), derivatizing the urethane
nitrogen with carboxylic acid containing functional groups
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Table 1. SAR of Amide and Urea Diacids
retained very potent antibacterial activity (MIC of 27−30, 4:
0.03−2 μg/mL). In addition, carbocyclic derivatives showed
improved potency by an order of magnitude in three out of five
target organisms (MIC of 28−30, 4: 0.03−2 μg/mL), which is
consistent with previous optimization studies of the monoacid
subseries.4 This finding contrasts with the above amide-based
diacid analogues, which failed to retain potent antibacterial activ-
ity (Table 1). Unfortunately, pyridine containing derivatives
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Table 2. SAR of Urethane Diacids
(31), unsaturated congeners (32), heteroatom incorporation
(33, 34), and other carbocyclics (35, 36) failed to improve the
antibacterial activity further, although all retained good to ex-
cellent potency (MIC of 0.03−4 μg/mL). As before, C. difficile
was the most sensitive target organism. In all cases, the com-
pounds inhibited protein synthesis in bacterial cell extracts
(67−86% at 2 μM, not shown) which compares equally to 1
(IC50 = 2.0 μM). This indicated that the SAR results from
variable cell penetration and not reduced pathway inhibition.
The Kd values of select compounds were determined with EF-
Tu·GDPNP. Compound 1 had a Kd of 3 nM, while compound
2 had a higher Kd (43 nM). Compounds 27, 29, and 4 were
equivalent (8 nM, 6 nM, 6 nM, respectively), indicating that
differences in MIC (e.g., in staphylococcus and enterococci) are
most likely the result of variable cell penetration. The most potent
urethane diacid analogues (4, 27−30) were subsequently profiled
in a mouse sepsis model of infection utilizing S. aureus and
E. faecalis.17 The effective doses (ED50) for all five compounds
evaluated in both infection models ranged from 0.8 to 11.0 mg/kg.
Importantly, the in vivo results correlated with the in vitro
MIC findings (E. faecalis ED50 less than S. aureus ED50). In
addition, two of the most potent diacid congeners (4, 27) were
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Table 3. SAR of Urethane Triacids
profiled in solubility assays and retained very high aqueous
solubility at neutral pH (>10 mg/mL). On the basis of the
potent in vitro and in vivo antibacterial results as well as the
substantial aqueous solubility, urethane diacids 4 and 27 were
subsequently selected for further characterization as develop-
ment candidates.
Since the addition of a second carboxylic acid functional
group retained antibacterial activity and improved aqueous sol-
ubility over that of the monoacid class and the natural product
itself, we next evaluated triacid analogues of 1 in order to
determine whether a third anionic group would retain anti-
bacterial activity/cell penetration. Accordingly, the third
carboxylic acid functional group was explored at various points
within the most potent diacid structural templates (e.g., 4, 27).
Similar to the mono- and diacid subseries, in the triacid
subseries, the antibacterial activity was dependent on the
location of the third carboxylate functional group. Indeed,
moderate antibacterial activity was achieved utilizing acyclic
scaffolds containing three carboxylates (Table 3, MIC of 37−39:
0.5 to >32 μg/mL). Again, improved antibacterial activities were
observed in the carbocyclic subseries (e.g., MIC of 40−44:
0.25−8 μg/mL), albeit reduced from the most potent diacid and
monoacid-containing analogues. In particular, maximizing the
distance between the carboxylates improved antibacterial activity
(e.g., 40, 41), and placement within the cyclohexyl ring also
affected activity because 4,4-disubstituted cyclohexanes (42)
proved to be superior to 1,1 disubstituted (43) and 1,2
disubstituted (44) analogues. In all cases, the compounds in-
hibited protein synthesis in bacterial cell extracts (50−79% at
2 μM, not shown), which compares equally to 1 (IC50 = 2.0 μM).
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Figure 3. (a) Cocrystal of E. coli EF-Tu (domain 2 shown) and 4. (b) Protein environment surrounding the diacid side chain of 4.
Figure 4. Characterization of 4, 27, 2, and 5 in the golden Syrian hamster model of C. difficile infection (eight animals per dose group).
This indicated that the SAR results from variable cell pen-
etration and not reduced pathway inhibition. Although some
compounds displayed potent activities across the panel of or-
ganisms tested (e.g., 41, 42), no additional benefit was realized,
especially given the increased structural and synthetic complex-
ity compared to the diacid series.
hamster model of C. difficile infection (Figure 4), which is used
to study treatments for the initial infection and relapse stages
of C. difficile infection.19 In the experiment, clindamycin was
administered on day −1, which cleared the gut flora of the
animals, increasing their susceptibility to C. difficile infection.
On day 0, approximately 1 × 107 CFU of C. difficile were
administered via oral gavage. 5, 27, 2, 4, or vehicle control
(saline) was administered orally once a day from days 1 to 5,
and the animals were observed from days 6 to 21. For com-
pound 2, a dose dependent effect was observed with 0%,
62.5%, and 100% survival in the 0.2, 2, and 20 mg/kg groups,
respectively. For compound 27, there was 0% survival for the
0.2 mg/kg group and 100% for the 2 and 20 mg/kg groups.
For compound 4, a similar effect was observed for the 2 and
20 mg/kg groups. Importantly, all three thiopeptides proved to
be superior at lower doses (2 mg/kg) to 5 (20 mg/kg, 25%
survival). Also importantly, animals that were successfully
treated with 27, 2, 4, or 5 had no detectable levels of C. difficile
toxins A and B in their cecal contents when measured at the
end of the study (via ELISA, day 21). From these experiments
and others, we consider a difference of one animal between
dose groups and vehicle controls as statistically insignificant. All
three thiopeptides (2, 27, 4) had very limited systemic exposure
in the infected hamsters using sparse PK sampling (Table 4),
compared to very high exposure in the gut as measured by
cecum sampling. Thus, in both uninfected rats and infected
hamsters, the thiopeptides had very limited systemic exposure
while maintaining high exposure in the intestinal tract, which is
an ideal profile (i.e., oral nonabsorbed) for treating C. difficile
Cocrystal studies of several diacid congeners and E. coli
EF-Tu were conducted in order to better understand the ob-
served structure−activity relationships.18 The homology be-
tween E. coli EF-Tu and C. difficile EF-Tu is high (86% within
5 Å of the binding site). The macrocyclic conformation of 4
was quite similar compared to previous studies (e.g., 2, 3), with
intermolecular H-bond interactions noted between the
hydroxyphenylalanine moiety of with two residues, Thr228
and Asp216. Further interactions included an additional inter-
molecular hydrogen bond between the amide side chain of 4
and Phe261. Phe218 also formed a T-stack interaction with the
pyridine ring of 4. The urethane aminothiazolyl linker favorably
positions the two acid-containing side chains out to solvent
accessible areas and in proximity to Arg 223 and Arg226
(Figure 3b).
4 was evaluated in rat pharmacokinetic studies and displayed
consistent results with previous experiments.10 Importantly,
very low oral bioavailability was observed (F < 0.1%) result-
ing from low absorption, which was ideal for the dosing
requirements for treating C. difficile infection (i.e., oral non-
absorbed route), a pathogen that infects the lumen of the
large intestine. Therefore, compounds 4 and 27 and a previous
development candidate 2 were evaluated in a golden Syrian
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infections, therapy was initiated 1 and 5 h after inoculation.
Compounds were administered via tail vein bolus injection at several
dose levels to groups of six mice each. Following inoculation the mice
were observed for 5 days. In addition, body temperature was
monitored by electronic microtransponders (Bio Medic Data Systems,
Inc. Seaford, DE) that were implanted in mice subcutaneously prior to
infection. The 50% effective dose (ED50), the dose providing
protection to 50% of mice, and the 95% confidence limits (95% CI)
were calculated from the survival data at day 5 by probit analysis using
the program Systat (SPSS Inc.).
Table 4. Summary of Pharmacokinetics in C. difficile
Infected Hamsters after 5 Days of Oral Dosing with
20 mg/kg Test Compounds
cecum (μM)
1 h 3 h
18 39
plasma (nM)
compd
1 h
3 h
2
BLQ
63.9
44.8
BLQ
BLQ
5.2
a
27
129
72
a
4
61
208
a
2/3 of animals were above the limit of quantitation.
Solubility Measurements. An amount of 1−2 mg of compound
was weighed into 1 mL glass tubes, and a fixed volume of each vehicle
was added to yield approximately 20 mg/mL compound. After initial
mixing using brief vortexing and sonication (5−10 min), samples were
equilibrated by shaking for 24 h at room temperature. After equil-
ibration, the vials were visually examined. If a clear solution was ob-
tained, solubility was reported as >X mg/mL (where X is the starting
concentration in that sample) and the pH of the sample was recorded.
Suspensions or solutions with visible particles were filtered through
0.22 μm PVDF membrane filters. Their pH was recorded, and dis-
solved drug concentration was analyzed using a RP-HPLC assay.
In Vitro Transcription/Translation Assays. The E. coli S30
extract extract system for circular DNA (Promega catalog no. L1020)
was used per recommendation of the manufacturer with slight
modifications. Briefly, 3.5 μL of 286 ng/μLtemplate DNA (pBESTluc)
was mixed with 1.0 μL each of 1 mM “methionineminus” and “cysteine
minus” amino acid mixes, 8 μL of S30 premix, 6 μL of S30 extract, and
0.5 μL of the test agents at 40× final concentration in a total volume
of 20 μL. The reaction mixtures were incubated for 2 h at 37 °C in a
384-well flat-bottom white plate (Corning, catalog no. 3704). The
formation of luciferase was measured by adding equal volume (20 μL)
of Steady-Glo luciferase reagent (Promega catalog no. 27104), and
emitted light was detected with a luminometer (Molecular Devices,
LMaxll plate reader). Kirromycin and puromycin, which are known
to be protein synthesis inhibitors, were used as positive controls.
Ampicillin was used as a negative control. The rabbit reticulocyteTnT
Quick coupled transcription/translation system (Promega catalog no.
L1170) was used as recommended by the manufacturer except that the
assay volumes were scaled down from 50 to 20 μL. Briefly the assay
components consisted of 16 μL of TnT Quick mastermix, 3 μL of
pT7luc at 167 ng/μL, 0.5 μL of methionine, and 0.5 μL of the test
compound at 40× final concentration in a final volume of 20 μL. The
reaction mixtures were incubated at 37 °C for 75 min. Luminescence
was detected as described above.
In Vitro Biotinylation of E. coli EF-Tu. EF-Tu proteins were first
buffer exchanged to 1× PBS by dialysis and then treated with EZ-Link
Sulfo-NHS-LC-LC-Biotin (Pierce). The amount of the cross-linking
reagent, the reaction time, and the temperature were controlled to
allow only one molecule of biotin to cross-link with one molecule of
EF-Tu. Under these conditions, about 25−30% of protein was 1:1
biotinylated. The rest of EF-Tu was not biotinylated (Figure S1) and
was separated from the biotinylated EF-Tu immobilized on
streptavidin (SA) sensor chip by flowing buffer over the chip surface.
Determination of Equilibrium Binding Constant (KD) and
Kinetic Binding Parameters of Thiopeptides by SPR. Biotiny-
lated EF-Tu was immobilized onto a streptavidin (SA) sensor chip
preconditioned with 40 mM NaOH and 1 M NaCl. Immobilization
was controlled to achieve maximum response unit (Rmax) of 50−100.
For standard direct binding studies of thiopeptide binding to the GTP-
bound form of EF-Tu, a running buffer containing 10 mM Tris-HCl,
pH 7.5, 150 mM NaCl, 10 μM GDPNP, 1 mM MgCl2, 1 mM TCEP,
0.05% P20 (GE Healthcare), and 3% DMSO was used. SPR experi-
ments were performed on a Biacore T-100 instrument (GE Healthcare).
Instrument parameters used for EF-Tu/TP interactions were as follows:
flow rate 40 μL/min; contact (association) time = 240 s; dissociation
time = 600−900 s depending on TP’s dissociation rate. All binding
studies were conducted at room temperature. SPR data were analyzed
using BiaEvaluation software (GE Healthcare) to obtain KD.
infection. To our knowledge, this study represents the first
evaluation for the thiopeptide class in the golden Syrian
hamster model of C. difficile infection.
DISCUSSION AND CONCLUSIONS
■
In summary, diacid- and triacid-containing analogues of the
antibiotic natural product 1 were designed, synthesized, and
evaluated for Gram positive bacterial growth inhibition. Several
cycloalkylurethane-based diacid and triacids retained antibacte-
rial activity in vitro across a panel of Gram positive organisms. 4
was selected for further characterization as a development can-
didate on the basis of excellent in vitro and in vivo potency
and very high aqueous solubility. Cocrystallographic studies of
4 demonstrated a similar macrocyclic conformation compared
to previous analogues, and intermolecular protein interactions
within domain 2 of EF-Tu rationalized the observed structure−
activity relationships. 4 was further characterized in oral PK
studies, which demonstrated very low absorption, an ideal
characteristic for its intended use as an oral nonabsorbed
treatment for C. difficile infection. In the golden Syrian hamster
model of C. difficile infection, 4 and other thiopeptides proved
to be superior to 5 at lower doses. Taken together, these studies
culminated in the selection of 4 for clinical development.
Currently, a clinical study evaluating 4 for treating of C. difficile
infections is ongoing.
EXPERIMENTAL SECTION
■
Compound Synthesis and Characterization. Synthetic proce-
dures and compound characterization data are found in the Supporting
Information. Compound purity was assessed by two distinct 20 min
HPLC runs to confirm >95% purity.
MIC Assays. MIC assays were conducted according to broth
microdilution methods described by the Clinical and Laboratory Stan-
dards Institute (CLSI, M07-A8, 2009).15a Bacterial strains included
E. faecalis (ATCC 29212), E. faecium (Prof. Chopra, University of Leeds,
U.K., strain 7130724), S. aureus (MRSA from Prof. Willinger, isolated
from a pharyngeal smear, AKH Vienna A4.018), S. pyogenes (ATCC
BAA-595), C. difficile (ATCC 43255).15b
Mouse Systemic Infection Model. The studies conducted were
approved by the Institutional Animal Care and Use Committee of
the Novartis Institutes for BioMedical Research Inc., Cambridge,
MA. Animals were maintained under controlled conditions with free
access to food and water. Female CD1 mice (21−25 g, Charles River
Laboratories, Wilmington, MA) were used for infections with S. aureus
(ATCC 49951) and E. faecalis (NB04025, a clinical isolate from the
Novartis bacterial collection that is resistant to erythromycin, tetra-
cycline, and gentamicin, courtesy of Dr. B. Willinger, Vienna General
Hospital, Austria). Lethal infections were induced by intraperitoneal
injection of a freshly prepared bacterial suspension of 1 × 108 CFU/
mouse in either 50% sterile rat fecal extract (E. faecalis) or 0.9% NaCl
(S. aureus). The injected bacterial dose corresponded to 10−100 times
the minimal lethal dose as determined from previous lethal dose
titration studies. Therapy for E. faecalis infections was initiated
immediately following the bacterial inoculation, while for S. aureus
Rat Pharmacokinetic Studies. N = 2 or 3, iv PK studies were
performed with male Sprague−Dawley rats weighing 220−270 g that
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are approximately 6−10 weeks old, obtained from Harlan Research
Laboratories (South Easton, MA), each bearing dual implanted jugular
vein cannula. The rats were fasted overnight before use and for 8 h
after dosing. Blood samples were taken into K2-EDTA coated tubes
and then centrifuged to yield plasma sample for analysis by LC−
MSMS. Bioanalysis of rat plasma, from both in-life and protein binding
experiments, were performed by LC−MSMS using a system with the
following configuration: Agilent liquid chromatograph (Santa Clara,
CA), LEAP Technologies CTC-PAL autosampler (Carrboro, NC),
and Applied Biosystems API 4000 mass spectrometer (Framingham,
MA). LC was performed in gradient mode with reversed phase C18
columns (2.1 mm × 30−50 mm × 3.5−5 μm particle size). The
mobile phase A was 0.1% formic acid in water, and mobile phase B was
0.1% formic acid in acetonitrile. Gradients were run from 5% B to
95% B in ∼3.5 min. Plasma samples were protein precipitated with
acetonitrile containing glyburide as the internal standard (Sigma-
Aldrich, St. Louis, MO).
Oral Dosing Solution Formulations. Compound 2. The
concentration used was 10 mg/mL. Ingredients for the amount
for 1 mL (percentage) were as follows: 0.1 N NaOH [250 μL
(25% v/v)], PEG-400 [200 μL (20% v/v)], 10% Cremophor
EL in purified water [250 μL (25% v/v)], purified water
[297 μL (29.7%v /v)], 1 N HCl [3 μL (0.3% v/v)]. The com-
pound was weighed in a vial, and to it was added 0.1 N NaOH
to completely wet the compound. The mixture was stirred for
60 min at 37 °C, and to it was added PEG-400. The mixture
was stirred for 5 min to obtain a yellow solution. To it was
added 10% Cremophor EL solution followed by purified water,
and the mixture was stirred for 5 min (a clear solution results).
The pH of the solution was adjusted to 7.5−8.0 using 1 N HCl.
If the pH is above 8, it was adjusted with an additional amount
of 1 N HCl. Final formulation is a light-yellow clear solution
with pH of 7.5−8.0. Solution is physically stable for at least
24 h at room temperature. Final concentration of Cremophor
EL in formulation is 2.5%. To prepare lower concentrations,
10 mg/mL solution was diluted with pH 7.4 phosphate buffered
saline (0.15 M PBS). The diluted solutions are stable for 24 h at
room temperature.
Compound 27. The concentration was 10 mg/mL. Ingredients
(amount for 1 mL (percentage) were as follows: 0.1 N NaOH [150 μL
(15% v/v)], PEG-400 [200 μL (20% v/v)], 10% Cremophor EL in
purified water [250 μL (25% v/v)], purified water [400 μL (40%
v/v)]. The compound was weighed in a vial, and to it was added 0.1 N
NaOH. The mixture was sonicated/stirred for 20−30 min to
completely dissolve the compound. To it was added PEG-400, and
the mixture was stirred for 5 min. The solution remained clear. To it
was added 10% Cremophor EL solution followed by purified water,
and the mixture was stirred for 5 min. The solution remained clear.
The final pH of the solution was measured and recorded. Final for-
mulation is a light-yellow clear solution with pH of 7.0−8.0. Solution is
physically stable for at least 24 h at room temperature. Final con-
centration of Cremophor EL in formulation is 2.5%. To prepare lower
concentrations, 10 mg/mL solution was diluted with pH 7.4 phos-
phate buffered saline (0.15 M PBS). The diluted solutions are stable
for 24 h at room temperature.
Compound 4. The concentration was 10 mg/mL. Ingredients
(amount for 1 mL) were as follows: 0.1 N NaOH [100 μL (10% v/v)],
PEG-400 [200 μL (20% v/v)], 10% Cremophor EL in purified water
[250 μL (25% v/v)], purified water [450 μL (45% v/v)]. The com-
pound was weighed in a vial. To it was added 0.1 N NaOH, and the
mixture was sonicated/stirred for 20−30 min to completely dissolve
the compound. To it was added PEG-400, and the mixture was stirred
for 5 min. The solution remained clear. To it was added 10% Cremophor
EL solution followed by purified water, and the mixture was stirred for
5 min. The solution remained clear. The final pH of the solution was
measured and recorded. Final formulation is a light-yellow clear
solution with pH of 7.0−8.0. Solution is physically stable for at least
24 h at room temperature. Final concentration of Cremophor EL in
formulation is 2.5%. To prepare lower concentrations, the 10 mg/mL
solution was diluted with pH 7.4 phosphate buffered saline (0.15 M
PBS). The diluted solutions are stable for 24 h at room temperature.
Hamster Pharmacokinetics. The pharmacokinetics of com-
pounds 2, 27, and 4 were determined following 20 mg/kg oral dosing
in infected hamsters using sparse plasma and cecal sampling. Six
hamsters were administered 0.6 mL of each compound at 3.3 mg/mL
in appropriate vehicle once a day for 5 days. At 1 and 3 h after the fifth
dose, three hamsters at each time point were euthanized by CO2
inhalation and both plasma and cecal samples taken. Blood samples
were obtained by cardiac puncture and centrifuged to obtain plasma
(K2-EDTA). The plasma was harvested within 30 min after drawing
the blood and then stored at −80 °C. Cecal contents were weighed
and then frozen at −80 °C after collection. For quantification of
thiopeptides in cecal samples, cecal tissue weight was recorded by
subtracting an average tube weight. To each sample, phosphate
buffered saline (PBS) with 25% acetonitrile was added at 1.5× volume
of the tissue weight (1:2.5 dilution). Samples were sonicated for 10 min
and homogenized with an Omni-TH tissue homogenizer for 30−60 s.
Next the homogenized samples were centrifuged for 10 min at 4000 rpm.
An amount of 5 μL of the resulting cecal supernatant was further diluted
with 45 μL of control cecal homogenate supernatant (1:10 dilution)
resulting in a final dilution of 1:25. In addition, a 10 μL aliquot of con-
trol hamster plasma was added to each cecal sample and standard. The
samples were then extracted by precipitation of proteins with ace-
tonitrile (200 μL) containing 100 ng/mL internal standard (glyburide).
Samples were subsequently vortexed for 5 min followed by
centrifugation at approximately 4000 rpm for 5 min. The resulting
supernatant (∼150 μL) was transferred into a clean 96-well plate, and
50 μL of water was added and vortexed for 2 min.
Hamster Model of C. difficile Infection. Hamsters were kept
under controlled conditions with 12 h dark and 12 h light cycles, 68−
72 °F constant temperature, 50% relative humidity, and 10−15
exchanges of fresh HEPA filter air per hour. Animals were kept in
7.5 in. wide by 6 in. deep cages (Alternative Design Manufacturing,
Silom Springs, AR) with sterilized Bed O-Cob bedding (corn cob) and
free access to water and standard rodent chow (Harlan). Animal were
hamster-golden Syrian (Mesocricetus auratus), Michigan-206 Golden
Syrian wild-type from Harlan, male, 90−110 g, 5−6 weeks old. Studies
described were approved by the Institutional Animal Care and Use
Committee (IACUC) of the Novartis Institutes for BioMedical
Research Inc., Cambridge under protocol number OS 20,061.
C. difficile ATCC 43255 was received from American Type Culture
Collection and stored at −80 °C in Brucella broth supplemented with
20% glycerol. On day −1, all animals received a single subcutaneous
injection of clindamycin (10 mg/kg). At 24 h after clindamycin pre-
treatment, hamsters were infected by oral gavage with approximately
106 CFU of the C. difficile culture. All culture work was completed
within 30 min, and all cultures were back in anaerobic conditions by
this time. Briefly, strains were resuspended from overnight plates (in
reinforced clostridial medium + 1% oxyrase (Oxyrase, Inc., Mansfield,
OH) and diluted to 1 × 107 CFU/mL (OD600 = 1.1−1.3 nm, dilute
1:100). Hamsters were immediately infected with 0.75 mL inoculum
(∼7.5 × 106 CFU/hamster final). An aliquot of inoculum was diluted
1:1000 and spiral plated (100 μL, slow deposition) on TSAB or
RCMA (×2) and incubated at 37 °C anaerobically for determination
of infection titer. Hamsters were administered the test compound
(8 animals per dose level) starting 24 h after infection. Single antibiotic
doses were administered orally and formulated as described above.
Antibiotics were administered 1 time per day and continued for up
5 days. The control group was administered the solution vehicle alone.
Animals were observed two times a day for the duration of the ex-
periment. General observations included signs for mortality and mor-
bidity and for the presence of diarrhea (“wet tail”), overall appearance
(activity, general response to handling, touch, ruffled fur) and
recorded. Animals judged to be in a moribund state will be euthanized.
Criteria used to assign a moribund state are extended periods (5 days)
of weight loss, progression to an emaciated state, prolonged lethargy
(more than 3 days), signs of paralysis, skin erosions or trauma, hunched
posture, and a distended abdomen. Observations continued, with any
deaths or euthanasia recorded for up to 21 days postinfection (for relapse).
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Any animal that died during the observation period was necropsied, and the
contents of their cecums were removed, diluted with an equal volume of
PBS, and frozen at −80 °C until processing (efficacy study). All surviv-
ing animals were euthanized by CO2 inhalation and sampled in a
similar manner as above.
Cecum homogenates are assayed for the presence of C. difficile
yoxins A and B using the Wampole C. difficile Tox A/B II ELISA assay
kit (VWR catalog no. 89013-262) in accordance with the
manufacturer's directions.
Crystallization and X-ray of Compound 4·EF-Tu Complex.
To form the EF-Tu·NVP·4 complex, 10 mg/mL protein (227 μM),
E. coli EF-Tu protein in a buffer containing 50 mM Tris, pH 8, and
50 mM NaCl were incubated with 1 mM compound 4 for 1 h at 4 °C.
The sample was centrifuged at 20000g to remove any resulting
precipitant. Crystallization was carried out using 300 nL of the protein
sample plus 300 nL of crystallization solution containing 0.1 mM Tris,
pH 8.23, 18% PEG3350, 0.2 M MgSO4, using a sitting drop format
and equilibrated against a reservoir of the crystallization solution. The
crystal was flash frozen with liquid nitrogen after being stabilized in a
cryo-buffer containing 0.1 mM Tris, pH 8.23, 18% PEG3350, 0.2 M
MgSO4, 15% ethylene glycol.
X-ray Data Collection. Initial data from a single crystal of the
EF-Tu·4 complex were collected on a ADSC Quantum 210 CCD
detector using synchrotron radiation (λ = 1 Å) at the IMCA-
CAT beamline 17-ID of the Argonne Photon Source. Data were
collected using φ rotations of 0.5°, and 180° of total data were
collected.
ASSOCIATED CONTENT
* Supporting Information
Detailed synthetic procedures and compound characterization
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Telephone: (617) 871-7729. Fax: 617-871-4081. E-mail:
matthew.lamarche@novartis.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank all of the contributors to the EFT project
team.
■
ABBREVIATIONS USED
■
MIC, minimum inhibitory concentration; EC50, 50% effective
dose; G+, Gram positive; MRSA, methicillin resistant Staph-
ylococcus aureus; VRE, vancomycin resistant enterococci;
S. aureus, Staphylococcus aureus; E. faecalis, Enterococcus faecalis;
E. faecium, Enterococcus faecium; S. pyogenes, Streptococcus
pyogenes; EF-Tu, elongation factor Tu
X-ray Data Processing, Structure Determination, and
Refinement. Data from the EF-Tu·4 complex were processed using
the HKL2000 Suite, version 0.95.20 Data processing statistics are
shown in Table 5. Crystals were diffracted to 2.7 Å resolution in the space
group P2(1)2(1)2 with a unit cell of a = 83.303 Å, b = 132.473 Å, c =
37.433 Å, α = β = γ = 90°. Data collection statistics are shown in Table 5.
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resolution range (Å)
total observations
unique reflections
completeness (%)
I/σ
50−2.7 (2.87−2.7)
70876
11365
93.5 (77.2)
20.0 (2.8)
0.099 (0.416)
Rsym
Refinement Parameters
R
work/Rfree
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protein atoms
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92
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89
47.4
Root Mean Squared Deviations from Ideal Value
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1.4
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