Journal of Medicinal Chemistry
Article
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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dx.doi.org/10.1021/jm201685h | J. Med. Chem. 2012, 55, 2376−2387