Guinea Pig Cough Experiments
Animals and pre-screening. For cough studies, animal care and
studies were conducted in alignment with applicable animal welfare
regulations in an AAALAC-accredited facility. The cough studies used
5-10 week-old Dunkin Hartley guinea pigs. The initial study using
intratracheal application of BW-031 under isoflurane anesthesia used
female animals (range of weights of 374-505 g on the day of dosing and
cough challenge), with 9 animals per each of the 4 experimental groups.
The subsequent study using ovalbumin sensitization to induce lung
inflammation used 6 male (416‑580 g) and 6 female (456-557 g) animals
for each experimental group. The number of animals per group was
increased in the ovalbumin sensitization experiments because of the
possibility that variable levels of sensitization might increase
variability in the effectiveness with which BW-031 could enter nerve
terminals. Because preliminary studies showed that some guinea pigs
failed to cough in response to the citric acid challenge, each study
began with 20% more animals than were planned for the protocols and
animals were first pre-screened by inhalation of citric acid (400 mM for
7 minutes, with coughs counted during the 7 minute application and for
10 minutes afterward) and the lowest responders were omitted from the
remaining study protocol. For the intratracheal protocol, animals with
0-1 coughs were omitted; for the ovalabumin sensitization protocol, the
6 animals of each sex with lowest cough counts (0-3 coughs) were
omitted. Pre-screening was performed a minimum of 7 days before the
start of the study protocol to allow animals to recover from any
sensitization produced by the citric acid exposure during the
pre-screening. After pre-screening, the remaining animals were allocated
into each group so that each group had approximately equal group mean
cough counts measured in the pre-screening protocol.
Intratracheal drug administration and citric acid challenge.Animals were dosed via the intratracheal route at a dose volume of 0.5
mL/kg of BW-031 dissolved in saline based on individual bodyweights.
Animals were anaesthetized (3-5% isoflurane/oxygen mix) and secured to
the intubation device by a cord around their upper incisor teeth. A
rodent fiber optic laryngoscope was inserted into the animal’s mouth to
illuminate the posterior pharynx and epiglottis. The tongue was released
and the needle of the dosing device (Penn Century intratracheal aerosol
Microsprayer) guided through the vocal cords into the lumen of the
trachea. Following dosing, the animals were removed from the secured
position and carefully monitored until full recovery. Approximately 1
hour after intratracheal treatment, animals were placed into whole body
plethysmographs connected to a Buxco Finepointe System and exposed to
nebulized 400 mM citric acid for 7 minutes. Cough counts and respiratory
parameters (minute volume) were recorded throughout the 7 minute
exposure period and for 10 minutes following the end of nebulization
period. Determinations of coughs by the Buxco Finepointe system were
confirmed against manual cough counts and this was periodically
re-confirmed. Recovery from the isoflurane anesthesia was complete
within an hour based on the overt behavior of the animals, consistent
with rapid recovery seen using measurements of physiological parameters
(Schmitz et al., 2016);.
Ovalbumin sensitization/challenge. On Day 0 all animals were
sensitized with intraperitoneal and subcutaneous injections of chicken
egg albumin (ovalbumin). Animals were administered 1 mL of a 50 mg/mL
Ovalbumin (Ova) in 0.9% w/v saline solution via the intraperitoneal
route and 0.5 mL of the same solution into 2 separate subcutaneous sites
(1 mL in total divided between the left and right flank). All animals
were administered a single intraperitoneal dose of pyrilamine (15 mg/kg)
at a dose volume of 1 mL/kg approximately 30 min prior to ovalbumin
challenge on Day 14 to inhibit histamine-induced bronchospasm
(Featherstone et al., 1988; Hara et al., 2005). On Day 14 animals were
challenged with aerosolised ovalbumin in 0.9% w/v saline (3 mg/mL) or
0.9% w/v saline for 15 min. Animals were placed in groups in an acrylic
box. 8 mL of ovalbumin in saline was placed in each of two jet
nebulisers (Sidestream®). Compressed air at approximately 6 L/min was
passed through each nebuliser and the output of the nebulisers passed
into the box containing the animals.
Drug administration. On Day 15 approximately 24 hours after the
inflammatory challenge with ovalbumin, animals were placed in a
whole-body plethysmograph (Buxco Finepointe) and dosed with vehicle or
BW-031 by inhalation using an Aeroneb nebulizer (Aerogen) over 60
minutes. Upon the completion of dosing animals were returned to their
home cage for approximately one hour before the cough challenge.
The inhaled dose of BW-031 was calculated according to the algorithm
(Alexander et al., 2008): Inhaled dose (mg/kg) = [C (mg/L) x RMV
(L/min) x D (min)]/BW (kg), where C is the concentration of drug in
air inhaled, RMV is respiratory minute volume, D is the of exposure in
minutes, BW is bodyweight in kg, with RMV (in L/min) calculated as 0.608
x BW (kg)0.852.
Cough/respiratory function measurement. One hour following the
end of vehicle or drug administration on Day 15, the animals were placed
into a whole-body plethysmograph connected to a Buxco Finepointe system.
Animals were then exposed to nebulized 400 mM citric acid for 7 minutes.
Cough counts were recorded throughout the 7 minute exposure period and
for 10 minutes following the end of nebulization period. Animals were
euthanized within approximately 60 min following the end of the cough
challenge recording period by an overdose of pentobarbitone administered
by the intraperitoneal route.
Tissue sampling. Upon euthanasia, 2 mL of blood were sampled
from the descending vena cava from each animal. The blood was allowed to
stand at room temperature for a minimum of 60 minutes but less than 120
minutes to allow the clotting process to take place. Samples were then
centrifuged at 2000 g for 10 minutes at 25°C and the resulting serum was
frozen at -80°C for subsequent analysis of BW-031 concentrations. Also
following termination an incision was made in the neck and the muscle
layers were separated by blunt dissection and the trachea isolated. A
small incision was made in the trachea and a tracheal cannula inserted.
The cannula was secured in place with a piece of thread. The lungs were
then removed and the left lung lobe tied off and removed. The right lung
was lavaged with 3 mL of phosphate buffered saline (PBS) at room
temperature. The PBS was left in the airway for 10 seconds whilst the
organ was gently massaged before being removed, this was repeated twice
further. In total, three lots of 3 mL of PBS was used to lavage the
right lung.
BAL immune cell quantification. A total and differential cell
count of the BAL was performed using the XT‑2000iV (Sysmex UK Ltd). The
sample was vortexed for approximately 5 seconds and analyzed. A total
and differential cell count (including eosinophils, neutrophils,
lymphocytes and mononuclear cells (includes monocytes and macrophages))
was reported as number of cells per right lung per animal.
Liquid Chromatography/Mass Spectrometry (LC/MS). Serum samples
were kept at -80°C until being assayed, at which time they were thawed
at room temperature. Each serum sample was added to 100 µL of 80:20
(acetonitrile:water) solution and the mixture placed in a 1.5 mL
Eppendorf Safe-Lock tubes prefilled with zirconium oxide beads (Next
Advance Inc., Troy, NY). After vortexing for 30 seconds and sonicating
for 10 minutes, the homogenate was centrifuged at 10,000 rpm for 10
minutes. The supernatant was then separated into a new Eppendorf tube to
be stored at -80°C until the time for analysis. To 20 µL of liquid
sample, 10 µL of internal standard (bupivacaine 10 ng/mL in
acetonitrile:water (50:50)) were added, plus 170 µL of methanol chilled
at 4°C. After vortexing for 30 seconds, the mixture was centrifuged at
10,000 rpm for 10 minutes. The supernatant solution was then transferred
to a clean Eppendorf tube and evaporated to dryness under vacuum at 50oC for 40 minutes. The residue was reconstituted with
100 µL of the starting mobile phase, i.e. aqueous 0.1% formic
acid:methanol (90:10), and vortexed for 30 seconds. This solution was
transferred to amber screw neck vials and setup in the refrigerated
autosampler tray of the chromatograph for injection. This whole
procedure was also applied to spiked calibrators and quality control QC
samples used in the quantification and validation methods. At least
three injections were carried out from each vial. Bupivacaine, used as
internal standard, methanol LC/MS grade, and formic acid LC/MS grade
were purchased from Sigma-Aldrich St. Louis, MO, USA). Pure MilliQ water
at 18 MΩ-cm was obtained by reserve osmosis with a Direct-Q3 UV water
purifier (Millipore SAS, France).
The quantification of BW-031 in serum fluid samples was carried out with
an Acquity H Class UPLC chromatographer with a XEVO TQ MS triple
quadrupole mass spectrometer detector (Water Corp., Milford, MA, USA).
The assay used an Acquity UPLC BEH C18 1.7 µm 2.1x100 mm column with a
VanGuard 2.1X 5 mm guard column, both kept at constant
35oC. The mobile phase was ran on a gradient of A:
aqueous 0.1% formic acid and B: methanol starting at time zero with a
A:B proportion of 90:10, at 3 min 10:90, and at 4.2 min 90:10 until the
end of the run at 6 minutes. The flow rate was set at 0.3 mL/min, with
an injection volume of 3 µL, and a post-run organic wash of 5 seconds.
Multiple reaction monitoring (MRM) was used for the mass spectrometry
acquisition in positive electrospray ionization (ESI) mode. The mass
transitions monitored were m/z 263.22→86.02 and m/z 289.09→140.2, for
BW-031 and bupivacaine respectively. The cone voltages were 36V and 30V,
and the collision energies 24eV and 12eV, also respectively. The whole
LC/MS system was controlled by the MassLynx v.4.2 software (Water Corp.,
Milford, MA, USA), including the TargetLynx Application Manager for data
processing and analytes quantification. Good linearity and
reproducibility were achieved in the range of 1–100 ng/mL, and the
precision and accuracy of the method were 2.74% and 98.6%,
respectively. The lower limit of quantification was 1.8 ng/mL.
Cardiotoxicity. Frozen human IPSC-derived cardiomyocytes
(Cor.4U cardiomyocytes) were purchased from Axiogenesis AG (currently
NCardia AG). Cor.4U cells were thawed and plated at a density of 10000
cells/well into 384-well plates that were pre-coated with 10 µg/mL
bovine fibronectin in sterile phosphate buffered saline, pH 7.4. Cor.4U
Culture Medium (Axiogenesis) was used to maintain the cells in culture
for 7 days and was changed daily. Cells exhibited synchronous beating on
day 3 after plating. On day 7 after plating, the medium was changed to
BMCC medium (Axiogenesis). The EarlyTox Cardiotoxicity Kit (Molecular
Devices) was used to measure calcium flux as a proxy for cardiomyocyte
beating activity112. Cor.4U cells were incubated with
the EarlyTox calcium sensitive dye in BMCC media for 1 hour, and then
the plates were transferred to the FDSS700EX plate reader (Hamamatsu
Photonics). The baseline calcium flux was measured for 5 minutes and
then charged local anesthetics dissolved in BMCC media or media alone
were added to the wells using a robot. 10 minutes after compound
treatment, the calcium flux was measured again. All measurements were
performed at 37°C under 95% CO2/5% O2.
Calcium flux parameters were measured using the Hamamatsu Analysis
Software.
Statistical Analysis. Data represent mean ± standard error of
the mean (SEM). Statistical comparisons were performed using GraphPad
Prism 8.0 software with the parameters described in each respective
figure legend. Statistical tests were corrected for multiple comparisons
where appropriate; corrections used for each data set are stated in
figure legends.