University of Groningen Novel therapeutic targets for the treatment of asthma van den Berg, M.P.M.

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Novel therapeutic targets for the treatment of asthma

van den Berg, M.P.M.

DOI:

10.33612/diss.143959597

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van den Berg, M. P. M. (2020). Novel therapeutic targets for the treatment of asthma: Focus on Arginase and TRPA1. University of Groningen. https://doi.org/10.33612/diss.143959597

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Asthma is a chronic airway disease that represents a serious global health problem. People of all ages suffer from asthma and even though it contributes to less than 1% of all deaths in most countries, the majority of these deaths are preventable by proper disease control and prevention of exacerbations (1). The primary burden of asthma lies in its morbidity in both adults and children, thereby imposing a large burden on society, economy, health care systems, and most importantly the quality of life of asthma patients (2).

Asthma treatment is focused on acute relief, symptom control and exacerbation prevention (2). In recent years, the broadening of our understanding of asthma pathology and the realization of the existence of multiple asthma phenotypes and endotypes, led to a shift from universal asthma treatment to a more personalized approach (3). However, as outlined in Chapter 1, there is a substantial group of asthma

patients for whom current treatment options are not sufficiently effective to achieve full control. Patients presenting with Th2-low asthma are often irresponsive to treatment with corticosteroids and other anti-inflammatory drugs (4). For treatment-resistant Th2-high asthmatics novel biologicals, aimed at for example immunoglobin E (IgE), interleukin (IL)-4 receptor, or IL-5/IL-5 receptors, are available, however the efficacy of these biologicals is not always optimal (5), underlining the need for novel therapies. The studies in this thesis explored two potential therapeutic targets, arginase and transient receptor potential ankyrin 1(TPRA1), and identified novel antagonists aimed at these targets.

Arginase as a potential treatment option for asthma

Arginase is the final enzyme of the hepatic urea cycle and catalyzes the formation of urea and ւ-ornithine from ւ-arginine. Downstream metabolism of ւ-ornithine results in the formation of ւ-proline and polyamines, that may contribute to collagen formation and cell proliferation, respectively, and may be involved in the airway remodeling that can be observed in asthma (6, 7). In asthma, arginase expression can be induced by for example the Th2-cytokines IL-4 and IL-13, and by TGF-β (7). Arginase may also contribute to asthma pathophysiology by decreasing the bioavailability of ւ-arginine for nitric oxide synthase (NOS), that uses ւ-arginine for the production of nitric oxide (NO) (7, 8). Under nonpathological circumstances, NO generated by constitutive NOS (cNOS) in airway epithelium and inhibitory nonadrenergic-noncholinergic nerves, plays a protective role in the airways via its bronchodilating and anti-inflammatory effects, and is involved in airway tone (8-10). Allergen exposure in allergic asthma, results in a deficiency in bronchoprotective NO, which underlies among others the induction of allergen-induced airway hyperresponsiveness (AHR) after the early asthmatic reaction (EAR) and airway inflammation after the late asthmatic reaction (LAR) (7). In 2002, our group showed that this allergen-induced loss of cNOS-derived NO is the result of an increased arginase activity (8). In perfused tracheal preparations obtained from allergen-challenged guinea pigs after the EAR, arginase inhibition completely reversed allergen-induced AHR, which could be counteracted by coincubation with a NOS inhibitor. In Chapter 2 we showed

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that this same mechanism applies for the ex vivo lung slice model, that was used for the validation of novel arginase inhibitors. The real proof-of-concept for arginase inhibitors as a potential treatment for allergic asthma came in 2008, when our group demonstrated that inhalation of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) leads to reduced airway sensitivity to inhaled allergen, protects against allergen-induced EAR and LAR and the AHR after these reactions, and decreases airway inflammation (11). Combined with data obtained from asthma patients, showing the likely involvement of arginase in this disease, and polymorphisms ARG1 and ARG2 that are associated with asthma, asthma severity and bronchodilator response, this has led to the interest in arginase as a target for the treatment of allergic asthma (12).

Current status of arginase inhibitors

Currently there are no arginase inhibitors available for clinical use. The reference inhibitors ABH, Nω_{-hydroxy-nor-ւ-arginine (nor-NOHA) and S-(2-boronoethyl)-ւ-cysteine}

(BEC) are often used for research purposes, but show poor pharmacological profiles in

vivo and are therefore not suitable for clinical administration (13). However, as arginase

has shown to play an important role in multiple pathological disorders, including pulmonary and cardiovascular diseases, rheumatoid arthritis, diabetes and cancer (14), interest in arginase as a drug target has emerged and has led to the development of several patented arginase inhibitors (15-19), including the SHK-compound series that were developed by the department of Drug Design (University of Groningen) (patent submitted) and functionally evaluated in the experiments presented in Chapter 2. It

was demonstrated that the novel arginase inhibitors SHK242 and SHK277 were effective inhibitors of both recombinant human arginase 1 and arginase 2 in vitro, protected against allergen-induced airway narrowing in guinea pig lung slices ex vivo and were able to inhibit the development of the EAR and LAR in an in vivo guinea pig model of acute allergic asthma (Chapter 2). Most patented arginase inhibitors are still in early

preclinical testing stages, including SHK242 and SHK277. Only one of the patented arginase inhibitors, INCB001158, has currently made it to a phase I/II clinical trial (20). This is however for its application in cancer that is characterized by the involvement of arginase-secreting myeloid-derived suppressor cells (e.g. colorectal carcinoma, non-small cell lung cancer, basket), and not for the treatment of asthma.

The question remains whether compounds that are effective arginase inhibitors in

vitro, are just as effective in inhibiting arginase in the complex tissue environment of ex vivo and in vivo models. In Chapter 2, we compare the in vitro and ex vivo efficacy

of the highly potent reference arginase inhibitor ABH and the recently patented Mars’ compound (21), and show that a high in vitro efficacy of an inhibitor does not necessarily

translate into a high efficacy in the airways ex vivo. This finding points out an important disparity between in vitro target binding, tissue penetration and cell uptake that each greatly affect compound efficacy ex vivo and in vivo (Chapter 2). The novel arginase

inhibitors SHK242 and SHK277 are at least equally effective in protecting against allergen-induced bronchoconstriction ex vivo and an in acute allergic asthma model in

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vivo as ABH, and more effective in protecting from airway-induced airway narrowing ex vivo than Mar’s compound (Chapter 2). Even though pharmacokinetic data, and

efficacy in chronic asthma models and in human tissue still need to be explored, the initial results are promising for the potential use of these inhibitors for the treatment of asthma.

An often-raised concern for the therapeutic use of arginase inhibitors is their potential toxic side-effects, especially with regard to ammonia detoxification in the liver. However, these concerns were mitigated by the lack of toxic side-effects in animal models of atherosclerosis and hypertension, where both short-term and long-term systemic treatment with arginase inhibitors was investigated (22). The low circulating concentrations of arginase inhibitor after systemic treatment were sufficient to improve vascular function, without inducing significant suppression of liver arginase activity. The absence of these side-effects may be explained by the substantially higher expression levels of arginase in the liver as compared to other organs, such as vessels (22). Besides, asthma and other chronic lung diseases are mostly treated locally by inhalation of nebulized or dry powder-formulated drugs, thereby further reducing the chance of systemic side-effects of arginase inhibitors. Local treatment has the benefit that lower drug doses can be used to achieve alleviation of symptoms compared to systemic treatment. As NO plays a key role in the functioning of vascular endothelium (23), changes to cardiovascular functioning belong to the possible side-effects of the use of arginase inhibitors, even when administered via inhalation, and thus require attention when the safety of arginase inhibitors is evaluated. In animal models of asthma and COPD both systemic and local treatment with arginase inhibitors increases the bioavailability of ւ-arginine and reduces pulmonary symptoms (11, 24-26) (Chapter 2). In Chapter 3 we describe that changes in ʟ-arginine homeostasis, by altered

arginase, NOS and asymmetric dimethylarginine (ADMA) activity and expression, seem to play an important role in several comorbidities of asthma and COPD. Thus, asthma and COPD patients often not only suffer from their chronic lung disease, but are also affected by the occurrence of comorbidities, such as allergic dermatitis and rhinitis, and obstructive sleep apnea syndrome (in asthma) and cardiovascular and cerebrovascular disease, lung cancer and diabetes (in COPD)(27, 28). In both asthma and COPD, the presence of comorbidities can greatly affect disease progression and quality-of-life of the patients (29, 30). Further research should reveal whether local alterations in ʟ-arginine metabolism in the lung may contribute to systemic comorbidities in other organs, if the ʟ-arginine imbalance in systemic comorbidities may affect asthma and COPD severity and if treatment of airway diseases with an arginase inhibitor will also alleviate symptoms associated with comorbidities. Earlier, we found that inhalation of the arginase inhibitor ABH does not only reduce airway inflammation and remodeling, but also right ventricular hypertrophy in a guinea pig model of COPD (25). Furthermore, in a mouse model of allergic asthma, reduction of circulating ʟ-arginine levels resulted in increased AHR (31), and in asthma patients an association can be found between systemic arginase activity, plasma ʟ-arginine and lung function (32). These findings

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indicate that there may indeed be a relation between the effects of local and systemic ʟ-arginine homeostasis, and that restoring the ʟ-arginine balance locally might have an additional benefit of reducing co-occurring symptoms in other organs. In addition, treatment of asthma or COPD with systemic arginase inhibitors could be beneficial for the treatment of comorbidities, but may be less preferred because of side-effects.

Arginase 1 versus arginase 2 and the need for isoenzyme specific arginase inhibitors

In many organisms, including humans, arginase occurs in two isoforms, cytosolic arginase 1 and mitochondrial arginase 2, that are encoded by two distinct genes, located on separate chromosomes, that are differentially expressed throughout the body (33) (Chapter 1).The two arginase enzymes are both involved in ւ-arginine metabolism and,

based on experiments assessing enzyme-substrate binding and substrate conversion, seem to possess almost identical activities in vitro (34). Whether their roles overlap

in vivo, however, is still largely unknown. In both humans and in mouse models,

deficiency of either arginase isoenzyme results in hyperargininemia (35-37). Arg1KO

-mice develop hyperammonemia and die shortly after birth, whereas Arg2KO_{only shown}

mild hypertension at 8-10 weeks of age (37, 38). This striking phenotypic difference indicates differential instead of compensatory roles for the two arginase enzymes. In macrophages, this indeed seems to be the case, as the arginase isoenzymes are differentially expressed depending on macrophage phenotype , where arginase 1 is seen as a marker for M2-macrophages, whereas arginase 2 is associated with M1-macrophages (39).

In humans suffering from autosomal recessive arginase 1 deficiency, arginase 2 expression was found to be upregulated. With an estimated incidence of 1 in 950,000 live births arginase 1 deficiency is a rare disease (40). Arginase 1 deficiency patients exhibit hyperargininemia with spastic paraparesis, progressive neurological and intellectual impairment, persistent growth retardation and infrequent episodes of hyperammonemia (41). Extraneurological symptoms of the disease are rare, and affect mostly the liver and skeletal system. Symptoms in the liver may vary from mild hepatocellular injury with brief episodes of elevated liver transaminases, to mild dysfunction with coagulation anomalies and acute liver failure (42, 43). The first symptoms are normally neurological and typically present in children between 2 and 4 years of age. If left untreated, the disease progresses with gradual developmental regression, however strict dietary intervention (e.g. protein restriction in combination with supplementation of an essential amino acid free of ւ-arginine), ւ-ornithine supplementation and/or the use of nitrogen scavengers, may prevent further neurological deterioration and may lead to partial recovery (41, 42). It is hypothesized that the arginase 2 induction observed in arginase 1 deficiency partially mitigates the degree of hyperargininemia and hyperammonemia (44), which could mean that in humans partial compensation of arginase 1 by arginase 2 is possible.

Also, in the lung the possible distinct involvement of arginase 1 and 2 is still unclear. In asthma patients the expression of both isoenzymes was found to be increased in

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airways and serum (7). Moreover, in a mouse model of allergic asthma arginase 2 was already upregulated in the lungs 3 hours after allergen challenge, where it took 18 hours until the induction of arginase 1 (45). Remarkably, when comparing the induction of arginases in models of allergic asthma that use different antigens in various species and strains, it can be observed that especially arginase 1 expression is universally induced, whereas arginase 2 is only slightly induced or even not induced at all (46). Furthermore,

ARG1 and ARG2 gene polymorphisms are associated with several aspects of asthma;

for example, ARG1 SNPs were found to be linked to AHR severity and responsiveness to inhaled β₂-agonists and corticosteroids, whereas ARG2 SNPs could be linked to risk of asthma, airway obstruction, AHR severity and bronchodilator response to β₂-agonist (47). These findings indeed hint at differential roles for the two arginase enzymes in asthma. Induction of ARG1 is suggested to be the main arginase responsible for to the increased arginase activity that is observed in asthma patients (45, 48). Furthermore, several studies in animal models have indicated that arginase 1 is involved in asthma via substrate competition with NOS (7, 31, 48-50), and increases in Arg1 expression correlated temporally with the development, persistence and resolution of AHR to methacholine induced by IL-13 in mice (49). The role of arginase 2 is more controversial, as both beneficial and detrimental effects of arginase 2 have been suggested (26, 51-54) (Chapter 4).

Xu and colleagues were the first to propose that increased arginase 2 expression might have beneficial effects in asthma (51). In their research they showed that an increased ʟ-arginine metabolism through arginase 2 in the mitochondria leads to enhanced oxidative metabolism, which reduces proinflammatory IL-13-mediated signaling and thereby dampens Th2-drived inflammation in asthma (51). Furthermore, Arg2KO_-mice

exhibit increased airway inflammation in response to allergen challenge and increased mast cell-mediated airway narrowing compared to wildtype animals (51, 53) (Chapter 4).

In Chapter 4 we found a protective role for arginase 2 in mast cells, in which arginase 2

inhibits mast cell differentiation into an allergic phenotype. This mechanism is mediated via the regulation of ʟ-ornithine metabolism by arginase 2, and could underly the protective effect of arginase 2 on mast cell-mediated airway narrowing in a murine ex

vivo model of bronchoconstriction (Chapter 4). Arginase catalyzes the conversion of

ʟ-arginine to ʟ-ornithine, which can be further metabolized to polyamines by ornithine

decarboxylase (ODC) (6, 55). Polyamines are known to be important for cell growth and function (56-58), and ʟ-arginine-derived polyamines, through the functioning of arginase, are suggested to play a role in the proliferation of among others endothelial cells (59), T- and B-cells (60) and blood cord-derived CD34+-cells (61).

Arginase 2 deficiency in mice increased eosinophilic Th2-mediated airway inflammation as well as neutrophilic Th1/Th17-mediated inflammation (53). Therefore, ւ-arginine metabolism by arginase 2 may be involved in Th2-mediated, as well as Th1/Th17-mediated asthma, suggesting the involvement of arginase 2 in different asthmatic phenotypes. This involvement of arginase 2 may be mediated via its role in ʟ-ornithine metabolism (Chapter 4) and/or by interactions with iNOS via TCA cycle activity in

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mitochondria and intracellular L-arginine metabolism, independently of substrate competition (51-53). L-Ornithine formed by arginase 2 in mitochondria is suggested to serve as a precursor for glutamate via ornithine aminotransferase. Subsequently, glutamate transamination via ornithine oxaloacetate, leads to the formation of aspartate, which via argininosuccinate synthetase and argininosuccinate lyase acts as a source for the generation of L-arginine for NO formation by iNOS (51). Common ARG2 polymorphisms associated with lower arginase activity, combined with high fractional exhaled NO (F_ENO) levels, were found to be related to a more severe asthma phenotype (53). Generally, asthmatic patients with high F_ENO suffer from more severe asthma, compared to F_ENO low phenotypes, and are at-risk for exacerbation (52, 62). The high F_ENO level is believed to be the result of iNOS activity (63, 64), as illustrated by increased levels of F_ENO in iNOS overexpressing mice (65), and increased iNOS expression in airway epithelium of asthma patients characterized by high F_ENO (52). Besides increased iNOS expression, F_ENO-high asthmatics were found to have higher airway lactate levels and, counterintuitively, increased expression of arginase 2 in airway epithelium, compared to F_ENO-low asthmatics (52). Bronchial epithelial cells overexpressing iNOS have increased lactate production and decreased glucose oxidation, suggesting that iNOS induces a shift from oxidative glycolysis towards anaerobic glycolysis (52). Co-induction of iNOS and arginase 2 in bronchial epithelial cells still resulted in increased lactate production, but also in an increase in glucose oxidation (52). Xu et al. proposed that the increased arginase 2 levels suppress the inhibitory effect of iNOS-derived NO on glucose oxidative metabolism, suggesting that ւ-arginine metabolism through arginase 2 may partially protect against the adverse effects of NO on mitochondrial respiratory function (52). However, much is still unknown about this complex metabolic role of arginase 2 and iNOS, and how their interaction affects asthma pathophysiology.

Studies in several animal models have shown the involvement of arginase 1 in airway hyper-responsiveness and airway inflammation, and indicate that inhibition of arginase 1 may be a promising approach for the treatment of asthma (11, 31, 48, 66-68). Hitherto there are no arginase 1 selective inhibitors. As the aforementioned findings suggest that arginase 2 may have protective effects, caution is warranted for the use of non-isoenzyme specific arginase inhibitors in asthmatic patients as they may reduce the beneficial effects of arginase 2. This could result in a deterioration of asthmatic symptoms instead of symptom alleviation. However, so far our studies and other studies in animal models of allergic asthma did not show indications for adverse effects by non-selective arginase inhibitors (11, 24) (Chapter 2), not even

when the arginase inhibitor was applied for twelve consecutive weeks (24). Due to its mitochondrial location arginase 2 might be more difficult to reach for arginase inhibitors than the cytosolic arginase 1 (69). Furthermore, it might be that the positive effect of inhibiting arginase 1 simply exceeds the side-effects caused by simultaneous arginase 2 inhibition. However, as ABH does seem to be able to inhibit to arginase 2 activity in mast cells (Chapter 4), an arginase 1 selective inhibitor would still be preferred when

treating asthma. Furthermore, arginase 1 and arginase 2 selective inhibitors would

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greatly aid the study into the differential roles of the two arginase enzymes, thereby creating better understanding of the role of the arginase in asthma.

The development of these isoenzyme selective inhibitors has proven to be a challenging process. As the binding pockets of arginase 1 and arginase 2 show 100% amino acid homology (70), the often-used design strategy for enzyme inhibitors, in which substrate binding in the enzyme binding pocket is mimicked, is not applicable. In a collaborative project with the department of Drug Design (University of Groningen; not part of this thesis), we developed an analogue of the C-terminal region of human arginase 1, the small synthetic peptide (REGNH) that acts as an allosteric arginase inhibitor (34). The C-terminal tail plays an important role in preserving arginase’s trimeric protein assembly that is necessary for the catalytic conversion of ւ-arginine (71, 72). García et al. already showed that manipulation of the C-terminal amino acid composition (e.g. substitution of Arg-308 by alanine, removal of S-shape) of arginase 1 results in loss of activity at physiological pH conditions (71). Even though the inhibitory properties of REGNH in our study were not different for both arginase isoenzymes, this allosteric approach may prove to be the strategy to obtain arginase 1 and/or arginase 2 specific inhibitors, as the C-terminal amino acid sequences of human arginase 1 and arginase 2 do not overlap and the overall peptide homology of the two isoenzymes is only around 60% (34, 70, 73).

TRPA1 as a potential therapeutic target for asthma

TRPA1 is an important sensor for noxious stimuli and tissue damage. In the airways, TRPA1 channels expressed on sensory neurons contribute to respiratory symptoms, such as bronchoconstriction, mucus secretion and reflex cough (74). Furthermore, TPRA1 expressed by non-neuronal cells is found to contribute to airway inflammation (75). Many known exogenous TRPA1 agonist are also able to induce asthma attacks. Moreover, various endogenous TRPA1 inducers are produced in the lungs of asthmatic patients and are involved in asthma pathology (76). Several studies including our own work show the involvement of neuronal TRPA1 channels in asthma (77-79)(Chapter 5), and also non-neuronal TRPA1 is suggested to contribute to asthma pathology

(75)(Chapter 5). Interestingly, TRPA1 agonism by itself was unable to induce airway

narrowing or histamine release in a guinea pig model of allergic asthma (Chapter 5),

suggesting that TRPA1 agonism contributes to bronchoconstriction rather than causing it, as illustrated by the findings that TRPA1 antagonism and deletion do reduce toluene-2,4-diisocyanate-induced AHR and allergen and histamine-induced airway narrowing (80)(Chapter 5).

Many asthmatic patients suffer from cough reflex hypersensitivity (81). TRPA1 channels play a likely role in this, as activation of TRPA1 channels results in the activation of bronchopulmonary C-fibers in both mouse and rat animal models, and induces cough in guinea pigs and healthy human subjects (82-87). The involvement of TRPA1 in so many aspects of asthma pathology (e.g. cough, airway inflammation, mucus secretion and bronchoconstriction), makes it an attractive target for asthma as TRPA1 antagonism may combine bronchoprotective, antitussive and anti-inflammatory effects. The

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attractiveness of TRPA1 antagonists as treatment option for asthma is even further emphasized by the fact that that TRPA1 has been found to be involved in both allergic and non-allergic asthma (88). Interestingly, the involvement of mast cell-expressed TRPA1 may differ per asthma phenotype, as mast cells were found to be crucial in TRPA1 mediated AHR in a mouse model of chemically-induced asthma (80), whereas mast cells were not affected by TPRA1 antagonism in an allergic asthma model in guinea pigs (Chapter 5). However, caution is required when explaining these observed differences

in involvement of mast cell-expressed TRPA1, as species specific differences of mast cells could play a role.

Current status of TRPA1 inhibitors

There is an interest in TRPA1 as a therapeutic target as this channel is suggested to be involved in various diseases, including several pain conditions and cough (89). There are currently no clinically approved TRPA1 antagonists. Nonetheless, the possible effectiveness of TRPA1 inhibitors in respiratory diseases is recognized, as several patents for TRPA1 antagonists in these diseases have been filed (89, 90). Several studies have shown that TRPA1 antagonists are able to alleviate symptoms of asthma. The novel TRPA1 antagonist HC030031 has been found to reduce the LAR and reverse acetylcholine-induced AHR in rodent models of allergic asthma (78, 79), and to improve epithelial barrier integrity in a model of occupational asthma (91). GRC 17536 was effective in decreasing AHR, airway eosinophilia and mucus production in an asthmatic mouse model and inhibited early airway reactivity and eosinophils in a guinea pig model of allergic asthma (92). Moreover, CB-625 reduced the LAR and antigen-induced AHR in a sheep model of asthma (93). In Chapter 5 we present another

novel TRPA1 inhibitor, BI01305834, that is able to protect against allergen-induced AHR after the EAR, and the development of the EAR and LAR in a guinea pig model of acute allergic asthma. Furthermore, in ex vivo guinea pig models, BI01305834 reduces both and histamine-induced airway narrowing and partially reverses allergen-induced bronchoconstriction (Chapter 5). Several TRPA1 antagonists have made it into

clinical trials, mostly for pain-related disorders (90). GRC 17536 made it into phase I/ IIa and phase II inhalation studies for, respectively, mild asthma and refractory chronic cough. In both studies, treatment with GRC 17536 did not improve the clinical outcome, decrease in FEV1 after LAR (asthma) or cough frequency (cough), compared to placebo treatment. However, as these studies remain unpublished in literature, it is unclear whether there was evidence of target engagement (94, 95).

Neuronal-expressed TRPA1 is suggested to react to cold stimuli, however, unlike other TRP family members, TRPA1 is not involved in body temperature regulation at baseline or in response to cold challenge (96, 97). Therefore, TRPA1 inhibitors are not expected to affect body temperature, which was also demonstrated by administration of CB-625 in healthy volunteers (98). BI01305834, the novel TRPA1 antagonist presented in this thesis, shows excellent selectivity against structurally related TRP channels (Chapter 5), cross-reactivity with other TRP channels that do affect body temperature regulation

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is therefore not expected. Also, unintended adverse effects on non-neuronal TRPA1 should be monitored, as TRPA1 antagonism may affect, for example, the channel’s ability to detect bacterial infections. However, TRPA1 deficient mice are viable, fertile, and normal in appearance, and do not exhibit auditory dysfunction or impaired sensitivity to heat and cold (99, 100), and also wild-type mice treated with TRPA1 antagonist show no signs of altered noxious cold sensation or body temperature regulation (96), indicating a healthy phenotype despite TRPA1 deletion or blocking. The abovementioned observations and the fact that the clinical trials with GRC 17536 did not reveal apparent side-effects (94, 95), are promising indications that the possible future application of TRPA1 inhibitors may be safe.

In conclusion

Both arginase and TRPA1 seem promising targets for the treatment of allergic asthma. However, many future steps still need to be taken before arginase and/or TRPA1 inhibitors are available for clinical use. Nevertheless, the findings in this thesis aid to this development process by providing mechanistic insight into the functioning of arginase and TRPA1, and by presenting novel antagonists against these targets:

• The novel compounds SHK242 and SHK277 are potent arginase inhibitors for the potential treatment of allergic asthma, as they are able to inhibit arginases in vitro and protect against allergic airway responses ex vivo and in vivo (Chapter 2).

• The protection of arginase inhibitors against allergic airway responses is primarily mediated by an increase of bronchodilating NO production in the airways, leading to a reduced airway response (Chapter 2).

• A disordered ʟ-arginine homeostasis by changes in arginase, NOS and ADMA activity and expression, does not only play a vital role in the chronic airway diseases, asthma and COPD, but also seems to play an important role in several co-morbidities (Chapter 3).

• Arginase 2 has a protective effect on mast-cell dependent airway narrowing, that may result from the inhibition of mast cell differentiation into an allergic phenotype, which is mediated via the regulation of L-ornithine metabolism. (Chapter 4).

• BI01305834 is a novel TRPA1 antagonist with high potency, that is able to protect against acute allergen-induced hyperresponsiveness and early and late asthmatic reactions in vivo and allergen and histamine-induced airway narrowing ex vivo. Moreover, it reverses allergen-induced bronchoconstriction, independently of inflammation (Chapter 5).

• Both neuronal and non-neuronal TRPA1 play a role in allergen-induced bronchoconstriction (Chapter 5).

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