The current study aimed to evaluate the efficacy of the novel TRPA1 antagonist BI01305834 in guinea pig models of allergic asthma. In the in vivo studies, we demonstrate that TRPA1 antagonism with BI01305834 protects against allergen-induced AHR after the EAR, and the development of the EAR and LAR. TRPA1 antagonism did not inhibit allergen-induced airway inflammation. The results obtained from the lung slice experiments show that TRPA1 antagonism protects against both allergen and exogenous histamine-induced airway reactions, as is in line with our observations in the in vivo model. Furthermore, using tracheal strips the bronchodilating properties of BI01305834 in response to allergen-induced constriction were confirmed. Together, this suggests that AHR, EAR and LAR are inhibited by TRPA1 antagonism irrespective of the inflammatory response in a partially histamine-dependent manner.
The in vivo guinea pig model of allergic asthma used in this study offers a unique chance to test the efficacy of BI01305834 in a whole organism. In airway pharmacology perspective, guinea pigs are superior experimental animals compared to mice and rat as they better resemble human airway physiology (Canning and Chou, 2008). 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 (Meurs et al., 2006). In the current study, the TRPA1 antagonist BI01305834 was able to reduce the AHR after the EAR. Unfortunately, no AHR after the LAR was observed in the current model and the effect of BI01305834 could therefore not be evaluated. However, the magnitude of the EAR and LAR themselves was reduced in the presence of protective effect of BI01305834. This is in line with previously reported findings by Raemdonck et al, showing that allergen challenge leads to activation of TRPA1 channels on sensory nerves during the LAR in rats, which resulted in enhanced cholinergic reflex bronchoconstriction (Raemdonck et al., 2012). These results are further supported by the fact that in guinea pigs the anticholinergic tiotropium was also able to reduce the EAR and LAR without affecting inflammatory cell infiltration in the BAL (Smit et al., 2014).
TRPA1 antagonists have been shown to alleviate asthma symptoms in different animal models of asthma. The novel TRPA1 antagonist HC030031 was shown to improve epithelial barrier integrity in a toluene diisocyanate-induced model of occupational asthma (Yao et al., 2019). Furthermore, in OA-induced asthma models in mice and rat, HC030031 was able diminish the LAR (Raemdonck et al., 2012) and reverse the AHR to acetylcholine, albeit at relatively high concentrations (Caceres et al., 2009). In vitro-potency assays show that BI01305834, the TRPA1 antagonist used in this study, is a more potent inhibitor of TRPA1-mediated calcium flux than HC030031, demonstrated by IC50-values for TRPA1 of 0.05 µM and 6.2–7.5 µM, respectively (McNamara et al., 2007, Eid et al., 2008).
As a second aim, we investigated how TRPA1 antagonism could alleviate asthma symptoms in guinea pig models of allergic asthma. Lung slices of allergen-sensitized guinea pig were used as they allow for investigation of functional airway responses in the lung tissue (Ressmeyer et al., 2006). In contrast to the in vivo model, the lung microenvironment represented by the lung slice model enabled us to study the effect of TRPA1 inhibition on airway smooth muscle contraction in a more direct manner, as well as the contribution of mast cells. An additional advantage of the lung slices is that many lung slices can be obtained from one animal, and therefore multiple conditions can be tested in the same animal (Martin et al., 1996). The results obtained in the OA-induced experiments confirmed the protective and bronchodilating effect of BI01305834 observed in vivo, in particular on OA-induced airway narrowing, and in smaller extent in the preconstricted trachea strips. More modest inhibitory effects were seen on exogenous histamine-induced airway narrowing. The exogenous histamine used in the lung slices induces airway narrowing by directly affecting the airway smooth muscle. In contrast, the OA-induced airway narrowing is based on the release of contractile mediators in response to allergen provocation, by for example mast cells. These mediators will in turn affect the contractility of the airway smooth muscle. As the allergen-induced smooth muscle contractility is a secondary effect of OA-provocation in the lung slice, this could explain why a relatively smaller maximal effect in airway narrowing is induced by OA in comparison to the full airway closure that is induced by exogenous histamine. Furthermore, the mix of mediators released from the mast cell includes not only histamine, but also serotonin, TxA2 and cysteinyl leukotrienes, all of which have been shown to contribute to the functional airway narrowing response (Ressmeyer et al., 2006, Santing et al., 1994). AITC was unable to induce airway narrowing or histamine release in the lung slices. Similar findings were reported in a mouse model of chemical induced asthma, where AITC was unable to induce AHR in toluene-2,4-diisocyanate-sensitized mice while TRPA1 blockage or knock-out prevented the development of AHR (Devos et al., 2016). Together this suggests that TRPA1 agonism by itself is not enough to induce bronchoconstriction, whereas TRPA1 antagonism is able to prevent allergen-induced airway narrowing.
The mast cell is an important player in allergen-induced airway narrowing, and an inflammatory cell type key to asthma pathophysiology. Upon antigen binding to their surface receptors mast cells release pro-contractile mediators including histamine, thereby inducing airway smooth muscle contraction (Bradding et al., 2006). Interestingly, mast cells express TRPA1 channels (Yang et al., 2015) and are able to induce sensitization of sensory nerves (Yu et al., 2009). Furthermore, TRPA1 affects mast cell degranulation (Hermanns et al., 2009). In the lung slices, we observed a small decrease in histamine release by the highest concentration antagonist that may play a role in the protective effect of BI01305834. However, it does not seem likely that mast cell-mediated effects and especially allergen-induced histamine release by mast cells, can fully explain the alleviation of asthma symptoms after TRPA1 antagonism as observed in vivo. As we observed only small effects and these results do not completely comply with the results obtained in the OA-induced airway narrowing experiment where 1 µM of BI01305834 was already able to achieve maximal protection. In the in vivo study, the free fraction of BI01305834 was calculated to be 244 ± 50 nM at time of OA challenge after administration of 1 mg.kg− 1 BI01305834. In the lung slice model concentrations of 0.01, 0.1, 1 and 10 µM BI01305834 were used. This means that the results obtained with 10 µM BI01305834 in the lung slices are supraphysiological compared with the effects seen in vivo. As we measured the IC50 for cytotoxicity in U937 cells to be 640 µM, we do not expect the observed results on histamine-induced airway narrowing and histamine release after administration of 10 µM BI01305834 are related to toxicity. Rather, we expect this to be an off-target effect induced by the supraphysiological dose of the compound. This indicates that the protective effect of BI01305834 on allergen-induced changes in asthma models was probably not mediated via TRPA1 channels on mast cells, as both in vivo and ex vivo these effects were observed at lower concentrations already.
We know from previous experiments in our lab that there is no effect of atropine on basal airway tone in the guinea pig lung slice. Furthermore, spontaneous neurotransmitter release from guinea pig tracheal preparations (measured by HPLC) is not detectable; electric field stimulation is necessary to achieve such release (de Haas et al., 1999). This essentially rules out the possibility that neurotransmitter release will interfere in this setting. An explanation for the observed protective effects of BI01305834 as being primarily mediated via TRPA1 channels expressed on nerves is therefore quite unlikely in this setting. However, as we observed a small, but protective effect of TRPA1 antagonism on exogenous histamine-induced airway narrowing in the lung slice, this suggests that TRPA1 channels expressed on other cell types in the lung, in particular airway smooth muscle cells, may also contribute to the protective effect of TRPA1 antagonism and histamine may be involved in this mechanism. Histamine receptor ligation activates among others phospholipase C, resulting in the generation of intracellular inositol-(1,4,5)-triphosphate (IP3) via the hydrolysis of phosphatidylinositol-(4,5)-biphosphate (PIP2) (Dai et al., 2007, Bessac and Jordt, 2008). The release of intracellular calcium induced by IP3 is thought to be involved in the activation of TRPA1 (Wang et al., 2008). As airway smooth muscle cells express calcium permeable TRPA1 channels (Nassini et al., 2012) and histamine stimulation of smooth muscle results in intracellular calcium peaks required for constriction, this suggests a direct role for TRPA1 in smooth muscle contraction independent of neuronal contribution. It is possible that also other mediators are involved in the TRPA1-mediated effect on bronchoconstriction. Grace et al (2014) suggested a role for prostaglandin E2 (PGE2) and bradykinin in TRPA1-related nerve activation (Grace et al., 2012). Furthermore, bradykinin is known to stimulate the release of 15-hydroxyeicosatetraenoic acid (15-HETE) and PGE2 by bronchial epithelial cells (Salari and Chan-Yeung, 1989). From studies into the role of TRPA1 in pain, it is known that TRPA1, the histamine H1-receptor and 15-HETE work synergistically to induce nociception (Fischer et al., 2017). This suggests a direct role for TRPA1 in smooth muscle contraction independent of neuronal contribution.
Inflammation is an important part of asthma pathology and sensory nerve activation may also contribute to this by inducing neurogenic inflammation (Grace et al., 2014). Against expectations and in contrast with current literature, we did not observe an anti-inflammatory effect of TRPA1 antagonism in our in vivo guinea pig model. Thus, BI01305834 did not affect inflammatory cells in the lavage fluid or IL-13 gene expression in lung homogenates. Substance P and neurokinin A were measured in the lavage fluid as mediators of neurogenic inflammation but were below detection limit (data not shown). In part, the fact that TRPA1 antagonism did not affect inflammation in the current study might be explained by the different allergen exposure protocols that were used in the animal models. Caceres et al challenged mice for three consecutive days (Caceres et al., 2009), whereas the guinea pigs in our study were challenged only once. Previously it was shown that in an in vivo guinea pig model of chronic asthma multiple allergen challenges will result in a more developed allergic response possibly resulting in differential outcomes after similar treatments, as was shown for budesonide and tiotropium in the current model (Bos et al., 2007). Both were not effective in inhibiting inflammation in the acute model, but do inhibit inflammation in the chronic model using twelve OA challenges as compared to one OA challenge in the acute model (Kistemaker et al., 2016). Testing BI01305834 in a chronic asthma model might therefore be a more suitable way to test its effect on airway inflammation. Nevertheless, it is also possible that BI01305834 inhibits AHR and allergen-induced asthmatic responses without effecting inflammation.
Airway inflammation is important for the development of AHR (Meurs et al., 2008). However, other mechanisms are also involved as AHR persists in the absence of inflammation. Here, there might be an important role for airway nerves, as vagotomy or anticholinergic treatment inhibits AHR in experimental models (McAlexander et al., 2015, McAlexander and Carr, 2009, Santing et al., 1995). It is increasingly recognized that neural plasticity and increased nerve activity contribute to many symptoms of asthma including AHR and cough (Kistemaker and Prakash, 2019). Both animal studies and human biopsy studies suggest that the airway neural network is more dense in asthma (Drake et al., 2018, Aven et al., 2014). Enhanced activity of sensory nerves via increased excitability or lowering of activation threshold and enhanced transmission may further contribute to asthma symptoms (Undem and Taylor-Clark, 2014). For example, OA challenge in rats induces hypersensitivity of pulmonary C-fibers with increased baseline activity and an increased response to the TRPV1 agonist capsaicin (Zhang et al., 2008). Together, this further supports nerve targeting approaches such as TRPA1 antagonism as a treatment for asthma.
Currently there are no clinically approved TRPA1 antagonist available. However, many preclinical studies show the potential for TRPA1 antagonism in asthma and other airway disorders, including chronic obstructive pulmonary disease and cough (Mukhopadhyay et al., 2016). Based on the results reported in this study, BI01305834 shows to be an effective bronchodilator in allergen-induced asthmatic reactions. Existing bronchodilators are β-adrenoreceptors agonists, muscarinic acetylcholine receptor antagonists or xanthines (Cazzola and Matera, 2014). Although they improve the quality of life of many patients, most of the times the usage of current bronchodilators does not lead to full symptom relief. Interestingly, TRPA1 antagonists are a good potential additional treatment option as they combine bronchoprotective activity and antitussive activity, and may have anti-inflammatory effects as well. LABAs and tiotropium most certainly are good bronchoprotectors, but have limited effects on cough. The anti-inflammatory effects of LABAs are limited (Price et al., 2014), whereas anti-inflammatory effects of LAMAs have been noted in preclinical models (Koarai and Ichinose, 2018, Kistemaker and Gosens, 2015), but are still under investigation in the clinical setting. Thus, alleviation of both bronchoconstriction, inflammation and cough by a single target may be of benefit for patients with severe asthma. Furthermore, TRPA1 antagonist may be beneficial in treatment-resistant and non-allergic asthma as well, as cough responses are shown to be unrelated to AHR, airflow obstruction and treatment with inhaled corticosteroid (Edwards et al., 2017).