In vitro IC50 assessment
HEK cells overexpressing human TRPA1 were pre-incubated for 2 hours with Calcium 6 dye (Molecular Devices) in HBSS [+ CaCl2/MgCl2] + 20 mM HEPES + 0.1% BSA. BI01305834 was applied 30 minutes prior to stimulation in DMSO (final concentration 0.1%) and cells stimulated with 5 µM supercinnamaldehyde. Calcium flux was measured as fluorescence using FLIPR. Concentration response curves were plotted and IC50-values calculated using GraphPad Prism.
Compliance with requirements for studies using animals
Male Dunkin-Hartley guinea pigs (Envigo, NL) weighing approximately 500 g and 650-850 g were used in this study for in vivo and ex vivo experiments respectively. The animals were group-housed in individual cages in climate-controlled animal quarters, and given water and food ad libitum, while a 12-h on/12-h off light cycle was maintained. In airway pharmacology perspective, guinea pigs are superior experimental animals compared to mice and rat as they better resemble human airway physiology . The specific model used in this study allows for measurements on conscious, unrestrained animals and importantly, it also enables the monitoring of the full EAR and LAR . All protocols described were approved by the University of Groningen Committee for Animal Experimentation (license AVD105002016492 and AVD10500201581).
Experimental protocol guinea-pig in vivo studies
The experimental protocol is depicted in Figure 1.
Animals were actively IgE sensitized to OA (Sigma-Aldrich) by injecting 1.0 ml of an allergen solution containing 100 μg.ml-1 OA and 100 mg.ml-1 Al(OH)3 in saline. Of this 1.0 ml, 0.5 ml was injected i.p., while another 0.5 ml was divided over seven s.c. injection sites in the proximity of lymph nodes in the paws, lumbar regions and neck, as described previously . One week after sensitization, the animals for the in vivo study were surgically provided with a balloon catheter in the thoracic cavity, as outlined below. Animals were treated via oral gavage with the TRPA1 antagonist BI01305834 on t=-0.5h before ovalbumin challenge, t=5.5h after challenge and t=23.5h after challenge (figure 1). These time points were chosen based on previous assessments in this model, showing that AHR after the EAR is best measured at t=6h and AHR after the LAR is best measured at t=24h . To allow for target binding, the TRPA1 antagonist was administered 30 min prior to these measurement time points.
In the pilot study, animals received 0.1, 1 or 10 mg.kg-1 BI01305834. In the main study, animals were treated with 1 mg.kg-1 BI01305834. In the vehicle-treated groups, 7 animals were included in the saline-challenged group and 11 in the OA-challenged group. In the groups treated with BI01305834, 7 animals were included in the saline-challenged groups and 13 in the OA-challenged group. Group sizes were initially designed to be equal in size and calculated using airway eosinophilia as the primary read-out parameter, with alpha=0.05, sigma=20 and mu1-mu2=30 (based on known parameters from previous studies). However, some animals were lost during surgery or suffered from extensive inflammation as a result of the surgery, and therefore had to be excluded from analysis. Vehicle/OA and BI01305834/OA groups were complimented with animals from pilot study to increase power for statistical analysis.
Measurement of lung function
Animals underwent surgery to install an intrapleural balloon catheter to measure lung function and EAR and LAR. Lung function was assessed by online measurement of pleural pressure (Ppl) under conscious and unrestrained conditions as described previously [33,35]. Before and during surgery animals were anaesthetized by inhalation of a mixture of N2O (500 ml.min-1)/O2 (500 ml.min-1)/3-5% isoflurane. 0.015 mg.kg-1 buprenorphine was administered by intramuscular injection before surgery for epi- and postoperative analgesia.
In short, a small fluid-filled latex balloon-catheter was surgically implanted inside the pleural space. The free end of the cannula was driven subcutaneously to the neck of the animal, where it was permanently exposed. Via an external saline-filled cannula the intrapleural balloon-catheter was connected to a pressure transducer (TXX-R,Viggo-Spectramed, Bilthoven, Netherlands) and Ppl was continuously measured using an online computer system.
Using a combination of flow measurement with a pneumotachograph, implanted inside the trachea, and pressure measurement with the intrapleural balloon-catheter, it was previously shown that changes in Ppl are linearly related to changes in airway resistance and hence can be used as a sensitive index for allergen- and histamine-induced bronchoconstriction. In this way, airway function can be monitored continuously, while the animals are unaware of the measurements being taken and without the use of anaesthetics, as these function as TRPA1 agonists in guinea pigs .
Airway function measurements were carried out in a specially designed 9 l perspex cage in which the guinea pigs could move freely, as described previously . A DeVilbiss nebulizer (type 646) driven by an airflow of 8 l.min-1 provided the aerosol with an output of 0.33 ml.min-1. All provocations were preceded by an adaptation period of 30 min, followed by a control provocation with saline, lasting 3 min.
In order to reduce stress-induced effect on Ppl-measurements, animals were first habituated to the experimental procedures in four training sessions. For the first session the animals underwent a control provocation with saline, lasting 3 min. During the second session, the animals’ intrapleural balloon-catheter was connected to the pressure transducer via the external saline-filled canula. Again, the animals underwent a control provocation. During the third and fourth training session, the animals were connected to the pressure transducer and histamine-provocations (PC100-measurements) were performed. In order to assess the airway reactivity to inhaled histamine, subsequent provocations with increasing concentration steps (6.25, 12.5, 25, 50, 75, 100, 125, 150, 175, 200 and 250 µg.ml-1) in saline were performed. Histamine provocations lasted maximally 3 min and were separated by 8 min intervals. Animals were challenged until Ppl was increased by more than 100% above baseline. The provocation concentration of histamine causing a 100% increase of Ppl (PC100) was derived by linear intrapolation of the concentration-¬Ppl curve and was used as an index for airway reactivity towards histamine. Ppl returned to baseline within 15 min after the last histamine provocation.
In order to determine histamine responsiveness before allergen challenge, animals underwent a PC100-measurement to histamine 24h before allergen challenge. Histamine provocations were performed as during training sessions.
Allergen provocations were performed by inhalation of OA in saline. The OA inhalation was discontinued when the first signs of respiratory distress were observed and an increase in Ppl of more than 100% was reached. First a 0.05% OA-solution in saline was used. When this dose was insufficient to induce respiratory distress within 3 min, a 0.1% OA solution in saline was used. OA-dose was calculated as exposure to µg OA x minute. After OA-challenge, animals were left connected to the pressure transducer to allow for measurement of lung function over time.
AHR induced by ovalbumin was assessed by exposing the guinea pigs to histamine, in line with the PC100-measurement to histamine 24h before allergen challenge. AHR in response to histamine was measured 6h after OA-challenge (after the EAR) and 24h after OA-challenge (after the LAR). AHR was assessed as a ratio of histamine responsiveness pre- and post- ovalbumin challenge, both after the EAR and the LAR.
In addition to the airway responsiveness to histamine, the magnitude of the EAR and LAR was quantified, by assessing the area under the curve of the plearal pressure curve over time. This are measurements of baseline pleural pressure after ovalbumin exposure, measurements are taken every 5 minutes and the area under the curve from 1-6h is calculated for the magnitude of EAR, and 9-24h for the magnitude of LAR.
Animals were anaesthetized by inhalation of a mixture of N2O (500 ml.min-1)/O2 (500 ml.min-1)/ 5% isoflurane 25h after OA challenge. Under terminal anaesthesia, blood samples were drawn by cardiac puncture into EDTA tubes. Blood samples were centrifuged at 4500 rpm for 5 min at 4°C and the plasma aspirated and stored at -80°C prior to analysis of BI01305834. The trachea was exposed and cannulated, and the lungs were lavaged gently using 5 ml of sterile saline at 37°C, followed by three subsequent aliquots of 8 ml of saline. The recovered lavage samples were kept on ice, and centrifuged at 200 g for 10 min at 4°C. The pellets were combined and resuspended to a final volume of 1.0 ml in phosphate-buffered saline (PBS) and total cell numbers were counted using a Casy Cell Counter (Model TT, Innovatis). For cytological examination, cytospin-preparations were stained with May-Grünwald and Giemsa stain. A cell differential was performed by counting at least 400 cells in duplicate in a blinded fashion, as described previously .
Lung homogenates were prepared by pulverizing the tissue under liquid nitrogen and total RNA was extracted using the NucleoSpin® RNA isolation kit (Macherey-Nagel, #740955.250) according to the manufacturer's instructions. Total RNA concentrations were determined with a NanoDrop ND-1000 spectrophotometer. Equal amounts of total mRNA were then reverse transcribed (Promega, #A3500), and cDNA was subjected to real-time qPCR (Westburg, Leusden, The Netherlands). Real time qPCR was performed with SYBR green as the DNA binding dye (Roche, #04913914001) on an Illumina Eco Real-Time PCR system, with denaturation at 94°C for 30 seconds, annealing at 59°C for 30 seconds and extension at 72°C for 30 seconds for 40 cycles followed by 10 minutes at 72°C. Real-time qPCR data were analysed using LinRegPCR analysis software  and GAPDH was used as a reference gene. Interleukin (IL)-4, IL-5 and IL-13 gene expression was assessed. Gene expression was presented as relative gene expression (N0) compared to GAPDH. The specific forward and reverse primers used are listed in Table 1.
Table 1. Primers used for qRT-PCR analysis.
Guinea pig IL-4
Forward – GGG TGC AAC CAC CAC ACC TT
Reverse – TGG ACC CTG GGG ATC AGC AA
Guinea pig IL-5
Forward – TAC ACA AGG GGA AGC TCT GG
Reverse – CCA GTT TGG TCT CAG CCT TC
Guinea pig IL-13
Forward – TCA CCC AGG ATC AGA AGA CC
Reverse – CCA CCT CGA TCT TGG TGT CT
Guinea pig GAPDH
Forward – AGA TGG TGA AGG TCG GAG TG
Reverse – GAC GAG CTT CCC ATT CTC AG
IL: interleukin; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.
Exposure to BI01305834
Blood serum concentrations of BI01305834 were determined by liquid chromatography / mass spectrometry using a 2.1 x 50 mm column, 5 µm, 100 Å at 40°C, using a mobile phase of water containing 0.1% Formic Acid (A) and acetonitrile containing 0.1% Formic Acid (B) at a flow rate of 400 µL.min-1.
Precision-cut lung slices
Precision-cut lung slices were prepared as described previously [38,39] at least four weeks after sensitization. Sensitization was performed as described above. For the preparation of lung slices, animals were anaesthesized by intradermal injection of 100 mg.ml-1 ketamine containing 10-4 M isoproteranol and 5 mg.ml-1 diazepam. After loss of reflexes, animals were euthanized by intracardial injection with pentobarbital (Euthasol 20%, Produlab Pharma, Raamsdonksveer, the Netherlands) and exsanguinated. In order to fill the lungs with a low melting agarose solution (1.5% final concentration (Gerbu Biotechnik GmbH, Wieblingen, Germany) containing isoproterenol (1 µM) in CaCl2 (0.9 mM), MgSO4 (0.4 mM), KCl (2.7 mM), NaCl (58.2 mM), NaH2PO4 (0.6 mM), glucose (8.4 mM), NaHCO3 (13 mM), Hepes (12.6 mM), sodium pyruvate (0.5 mM), glutamine (1 mM), MEM-amino acids mixture (1:50), and MEM-vitamins mixture (1:100), pH = 7.2)) the trachea was cannulated. To solidify the agarose, ice was placed on the lungs for at least 30 min. Afterwards, the lungs were removed and placed on ice. Tissue cores with a diameter of 15 mm were prepared of the separate lobes. In cold medium consisting of CaCl2 (1.8mM), MgSO4 (0.8 mM), KCl (5.4 mM), NaCl (116.4 mM), NaH2PO4 (1.2 mM), glucose (16.7 mM), NaHCO3 (26.1 mM), Hepes (25.2 mM), pH = 7.2 and isoproterenol (1 µM)) the cores were sliced into 500 µM slices with a tissue slicer (CompresstomeTM VF- 300 microtome, Precisionary Instruments, San Jose CA, USA). The lung slices were incubated in a humid atmosphere under 5% CO2/95% air at 37°C. Lung slices were washed every 30 min, 3 times with medium containing isoproterenol and once with medium only to wash out the isoproterenol and kept overnight.
Ex vivo airway narrowing studies
Lung slices were used for OA and histamine induced airway narrowing studies. In order to do this, lung slices were placed in 1 ml medium and fixed with a plastic ring. After 30 min pre-treatment with the TRPA1 antagonist BI01305834 (0.01, 0.1, 1 or 10 µM) or the vehicle (10% cyclodextrin) OA (10-5-102 µg.ml-1) or histamine (10-8-10-2 M) dose-response curves were established. Using a microscope (Eclipse, TS100; Nikon) time-lapse images (1 frame per 2 s) of the lung slices were captured. The airway luminal area was quantified using image acquisition software (NIS-elements; Nikon) and expressed as percentage of basal area, as described previously [39,40].
Lung slices were used for OA-induced histamine determinations, with separate slices for each condition individually. BI01305834 and vehicle pre-treated lung slices were challenged with OA (10 µg.ml-1) for 5 min. Untreated and unchallenged lung slices were used as a control for spontaneous histamine release. Supernatant was collected and lung slices were transferred to ice-cold acetic acid solution (0.08 M) and homogenized by sonication (10 s; 60 pulses)(Vibra Cell; Sonics, Newton, USA). Afterwards, the sonicated samples were centrifuged for 30 min at 15000 rpm and 4°C. Histamine levels of original supernatant and homogenized lung slice supernatant were assessed by liquid chromatography in combination with isotope dilution tandem mass spectrometry (LC-MS/MS). Histamine-d4 (Toronto Research Chemicals) was used as internal standard. Inter-assay imprecision (n = 20 days) was < 2.9 % at three different levels (60, 986, 3873 nM, respectively) and limit of quantification was 3.0 nM. Histamine release was calculated as percentage of total histamine present in both supernatant and slice. Data of histamine released by BI01305834 pre-treated lung slices was normalized to histamine released by vehicle pre-treated lung slices.
Ex vivo bronchodilator studies
The OA-sensitized animals were sacrificed by experimental concussion followed by rapid exsanguinations. The trachea was removed from the larynx to the bronchi and rapidly placed in a Krebs–Henseleit (KH) solution (NaCl (117.50 mM), KCl (5.60 mM), MgSO4 (1.18 mM), CaCl2 (2.50 mM), NaH2PO4 (1.28 mM), NaHCO3 (25.0 mM) and D-glucose (5.50 mM), pH = 7.4) at 37°C, gassed with 95% O2 and 5% CO2. Using surgical wire, single guinea pig open-ring tracheal strips were connected to an isometric force-displacement transducer (Grass FT03) and the resting tension was adjusted to 0.5 g. After a 60 min equilibration period with three washes, strips were preconstricted with 20 and 40 mM KCl, followed by maximal relaxation established by the addition of (-)-isoproterenol (0.1 µM). After three additional washouts, tracheal preparations were pre-contracted with OA (0.1 μg.ml-1) or histamine (1 μM). Cumulative concentration-response curves were constructed using BI01305834 (0.001-10 μM). After washout, basal tone was re-assessed using isoproterenol (10 μM). Tension on tracheal strips was calculated as percentage of 40 mM KCl-induced constriction and expressed as percentage of preconstricted state.
Randomization and blinding
Animals and slices were randomly assigned to different treatments. The experimenter could not be blinded as animals were individually treated with saline or OA and the solubility of the antagonist was limited. Data analysis and histamine determination was done in a blinded manner.
Data and analysis
Data are presented as mean ± S.E.M. Data was only subjected to statistical analysis with a minimum group size of n=5 separate experiments or animals.
All declared group sizes are the number of independent values, statistical analysis was done using these independent values. Outliers were included in data presentation and analysis. The data were checked for normality using D’Agostino’s K-squared test. For in vivo data normality could not be demonstrated and thus a non-parametric approach was used. Statistical evaluation of differences of in vivo data was performed using a Mann-Whitney U test or One-way nonparametric Kruskal-Wallis ANOVA with Dunnett’s post-hoc test where appropriate compared to saline and OA control groups. In order to reduce unwanted sources of variation, data of ex vivo airway narrowing and relaxation studies were normalized to initial airway size and preconstriction, respectively. Histamine determinations were normalized to control. Statistical evaluation of differences of ex vivo data was performed using One- or Two-way ANOVA with Dunnett’s post-hoc test where appropriate. Post-hoc tests were run only if F achieved P<0.05 and there was no significant variance inhomogeneity. Differences were considered to be statistically significant when p<0.05. Statistical analysis was performed with GraphPad Prism 5.0 software or Sigmaplot 13 for PCLS dose-response curves.