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.