Introduction
The histamine H
4 receptor (H
4R) is one of the four known subtypes of histamine receptors (H
1R–H
4R). This receptor is a member of class A G-protein coupled receptors (GPCRs). On the molecular level the activation of H
4R by histamine results in the liberation of Gα
i/o subunits, subsequent inhibition of adenylyl cyclase activity, and decreased concentration of cAMP. The downstream signals of this pathway involve changed activity of protein kinase A and transcription of genes regulated by cAMP-responsive elements [
1,
2]. Strong increase in the phosphorylation of mitogen-activated protein kinase associated with H
4R stimulation was also reported [
3]. Moreover, the Gβγ subunit of G-protein induces calcium
2+ mobilization through phospholipase C activation [
4]. An increased concentration of calcium
2+ leads to actin polymerization enabling the cells with an expression of H
4R to change the cell shape and to migrate into the site of inflammation [
5]. Research into GPCRs reveal that these receptors can exist in multiple active receptor conformations, which are associated with different signaling pathways and couple to multiple downstream effector proteins [
6,
7]. Regarding H
4R, β-arrestin2 recruitment that occurs independently of G protein was shown [
8]. Furthermore, it was presented that certain ligands are able to preferentially activate one pathway with the opposite effect or no impact on the other-biased H
4R ligands [
8,
9]. There is also evidence of constitutive activity of H
4Rs, which means that these receptors are able to undergo agonist-independent isomerization from an inactive state to an active one [
3,
10]. Thereby, the biological response in the absence of a bound ligand can be inhibited by H
4R inverse agonists—the ligands that stabilize the inactive receptor conformation and are therefore able to reduce constitutive activity. It is important that primarily human H
4R receptor possesses a high constitutive activity, whereas canine, murine and rat H
4R orthologs show substantially lower activity in the absence of an agonist [
11,
12].
The H
4Rs are predominantly expressed in a variety of immune cells. The majority of them such as dendritic cells, neutrophils, mast cells, eosinophils, basophils, monocytes and CD4
+ T cells are derived from the hematopoietic stem cells, which also present expression of this receptor [
13,
14]. Furthermore, the H
4Rs were detected in the enteric and central nervous system as well as on dermal fibroblasts and nerves of the human nasal mucosa [
1]. The H
4R is involved in many functional inflammatory responses mediated by histamine, including chemotaxis, cell recruitment, increased expression of adhesion molecules and modulation of cytokine and chemokine release [
2,
15,
16].
Research into the first potent and selective H
4R antagonist (JNJ 7777120) provided a large body of evidence that inhibition of H
4R function (antagonists, inverse agonists) could result in attenuating an inflammatory response. This compound, along with other H
4R antagonists, has shown activity in models of asthma, dermatitis, pain, and pruritus among others [
17‐
20]. It supports the hypothesis that the histamine H
4R offers a great potential for new therapeutic strategies for the treatment of inflammation-based diseases.
The aim of this report is to present the pharmacological profile of two recently synthesized ligands of H
4R with particular reference to their anti-inflammatory and analgesic activity. These compounds (4-(4-Methylpiperazin-1-yl)-6-(4-chloro-phenyl)-1,3,5-triazin-2-amine as 1 and 4-(4-Methylpiperazin-1-yl)-6-(4-bromo-phenyl)-1,3,5-triazin-2-amine as 2) have been chosen from the series of previously described 1,3,5-triazine derivatives. In the previous experiments both compounds showed submicromolar affinities for the H
4R and caused a blockade of the histamine-induced cAMP reduction in CHO-h H
4R-cAMPzen cells co-treated with forskolin. Moreover, tests for their interaction with H
3Rs, which show the highest sequence homology to the H
4R, revealed low affinity for these receptors. Such results entitled to classify the compounds as selective H
4R antagonists [
21].
Discussion
We found that new H4 receptor antagonists attenuated inflammatory and nociceptive response in two in vivo models of inflammation. Both compounds (cAMP dependently) inhibited inflammatory mediators release and ROS production in RAW 264.7 cells.
Inflammation and inflammatory pain are very complex processes associated with the release of numerous inflammatory mediators. Histamine is one of the most important autacoid engaged in the formation of inflammatory response. This mediator, acting also as neurotransmitter, exerts its function through four different types of GPCRs: H
1R, H
2R, H
3R and H
4R [
13]. The identification of H
4Rs, the newest receptors in the group, and their fairly selective expression on cells involved in inflammatory and immune responses suggested their important role in inflammation [
3,
28]. The subsequent discovery of the potent and selective H
4R antagonist-JNJ7777120 [
29]-enabled studies on physiological and the pathophysiological functions of H
4R and provided the first evidence that H
4R blockade could result in anti-inflammatory effect [
30]. In recent years, scientists reported that H
4R antagonists are effective in models of asthma, dermatitis, arthritis, pain, pruritus and colitis [
17‐
20,
31‐
33].
Despite numerous studies on histamine H
4R, the differences between species in response to specific ligands remain ambiguous. Most current data indicate that JNJ7777120 is H
4R antagonist. However, researchers proved that in some species and transfected cell models, the compound acted as H
4R agonist [
8‐
10]. Moreover, it exerted functional selectivity, i.e., β-arrestin activation [
8‐
10]. Thus, in our opinion, research into H
4R-dependent pharmacological effects are valuable.
Our present research investigated pharmacological activity of the two previously synthesized aryl-1,3,5-triazine derivatives (compound 1 and compound 2) with affinity for histamine H
4R. Previous studies demonstrated that both compounds showed submicromolar affinity and antagonist potency at hH
4R (compound 1, Ki-203 nM, IC
50-512 nM; compound 2, Ki-524 nM, IC
50-1630 nM) as well as good selectivity over hH
3R. Additionally, preliminary pharmacological experiments demonstrated their anti-inflammatory properties (carrageenan-induced paw edema in mice) and lack of antiproliferative effect (in HEK-293 and IMR-32 cell lines) [
21].
To evaluate anti-inflammatory and anti-nociceptive properties of compounds, we used two in vivo models of inflammation (carrageenan-induced model of inflammation and zymosan-induced peritonitis). We intentionally performed the experiments on mice and rats, to demonstrate that the compounds show effects regardless of the species and the related variability in expression and activity of the histamine H4R.
In the carrageenan-induced model of inflammation both test compounds reduced edema in all time points. Since Coruzzi and colleagues reported that H
4R antagonists were effective only at the acute inflammatory response in this model of inflammation [
19], we measured the activity of the compounds during the first 3 h of the test. In this period, the compounds showed greater anti-inflammatory properties than the reference compound-JNJ777120 (compared at the same doses). Nevertheless, the investigated compounds at the dose of 50 mg/kg were not as potent as indomethacin at the dose of 10 mg/kg. The obtained results prove that the blockade of H
4R function results in an anti-edematous effect.
We confirmed the anti-inflammatory activity of investigated aryl-1,3,5-triazine derivatives in the model of zymosan-induced peritonitis. Administration of test compounds resulted in attenuated vascular permeability and decreased intraperitoneal influx of inflammatory cells. The pretreatment with the highest doses of compounds (50 mg/kg) reduced vascular permeability, whereas lower doses and JNJ7777120 had no significant effect. Given the lower affinity of triazines for H
4R compared with JNJ7777120, we suggest that decreased vascular permeability was not associated with the direct influence on H
4R. We think that it resulted from other mechanisms e.g., decreased release of inflammatory mediators such as leukotrienes or cytokines, which regulate vascular permeability during inflammation [
23]. The inhibitory effect of H
4R antagonist on cell migration (especially neutrophils) in zymosan-induced inflammation is well known [
30]. However, other cells also express H
4R i.e., hematopoietic progenitor cells [
14] mast cells, eosinophils [
5], Th
2 lymphocytes [
31], monocytes, dendritic cells [
34‐
36], Natural killer cells [
15], monocytes, macrophages and some other cell types [
13]. Thus, we assessed the amount of various cell populations involved in inflammatory response in peritoneal lavage. In fact, we showed significantly decreased granulocytes migration. However, we observed a tendency in decreasing the number of NK cells and dendritic cells (data not published). We think that evaluating cell number in peritoneal cavity in additional time points would clarify the role of H
4R antagonist in other cell type migration.
The test compounds inhibited both, thermal and mechanical hyperalgesia induced by carrageenan injection. As Figs.
3 and
4 present their effect was stronger and lasted longer than that of JNJ7777120. This is particularly visible in case of compound 1. The compounds also reduced body writhnes in the model of zymosan-induced peritonitis (Fig.
5 panel A). In both models, nociceptive reactions are not related to the direct stimulation of nociceptors, but rather result from the secondary release of the inflammatory mediators such as prostanoids or cytokines from immunocompetent cells [
23,
37]. The majority of these cells shows expression of H
4R [
2]. Although the contribution of H
4R in the mechanism of pain still remains controversial, studies confirm that H
4R antagonists elicit analgesic effects in inflammatory and neuropathic pain models [
19,
20]. In contrast, recently published papers revealed that H
4R stimulation exhibited pain-reducing effects [
38‐
40]. Taken together, all these data suggest that H
4R antagonists might possess anti-hyperalgesic properties that are secondary to decreased release of inflammatory mediators, whereas activation of neuronal H
4Rs, especially those localized on sensory dorsal root ganglion neurons, might result in antinociceptive effects in the absence of inflammation [
39].
Thus, we concluded that the anti-inflammatory and anti-hyperalgesic activity of the investigated compounds resulted from their secondary and H
4R-dependent inhibitory influence on the release of inflammatory mediators. To confirm this hypothesis, we assessed the influence of test compounds on LPS-stimulated RAW 264.7 macrophages. The activation of macrophage TLR-4 (Toll-like Receptor) by LPS induces the expression of the histamine-generating enzyme
l-histidine decarboxylase and subsequent histamine synthesis [
16]. Histamine, released form macrophages, activates H
4R leading to a decreased level of intracellular cAMP among others [
13].
Both aryl-1,3,5-triazine derivatives incubated with RAW 264.7 increased intracellular cAMP concentration and significantly decreased TNFα and IL-1β release. Moreover, test compounds decreased reactive oxygen species (ROS) level stronger than JNJ7777120. In the same conditions, JNJ7777120 attenuated NO production, while compound 1 had no effect and compound 2 increased NO formation. Compound 2 increased NO formation similarly to rolipram.
The impact of the tested compounds on NO synthesis was not in line with the result obtained for JNJ7777120, which indicates that they may alter some other pathways involved in NO synthesis. NO is generated by inducible NO synthase (iNOS) in macrophages following exposure to cytokines or microbial products, such as LPS [
41]. Furthermore, a vast amount of NO can cause tissue damage and contribute to the development of a wide spectrum of inflammatory diseases.
Under inflammatory conditions ROS open inter-endothelial junctions and promote the migration of inflammatory cells across the endothelium of postcapillary venules. Therefore, we believe that ROS formation inhibition is an important mechanism of anti-inflammatory activity of the investigated aryl-1,3,5-triazine derivatives. Additionally, ROS play role in edema formation by inducing the paracellular permeability, which is the major route of vascular leakage observed in a variety of inflammatory states and is associated with the extravasation of protein-rich fluid from the luminal to abluminal side of the endothelium [
42]. Decreased level of ROS might also contribute to the antinociceptive effect of the test compounds. ROS evoke nociceptive response in neurogenic inflammation through different targets including TRPA1 receptors [
24].
As described above, the tested compounds increased cAMP level, which was similar to JNJ7777120. The increased concentration of cAMP in LPS-stimulated RAW 264.7 observed after their incubation with JNJ7777120 or the test compounds might result from H
4R blockade. The same mechanism might contribute to the reduced release of pro-inflammatory cytokines such as IL-1β and TNFα [
2,
31,
43,
44]. This effect, in turn, might be involved in the antinociceptive activity of the tested compounds [
45‐
48].
Altogether, our results suggest that the influence of JNJ7777120 on NO production is cAMP-independent and may result from the activation of β-arrestin-dependent pathway. Since, in contrast to JNJ7777120, the compounds increased NO production, we hypothesize that they did not activate β-arrestin-dependent pathway. However, the effect of cellular cAMP on NO release is still not confirmed, since elevation of cAMP level can either stimulate or inhibit NO formation [
41,
49]. The increased level of NO may be also considered as secondary effect of decreased level of ROS, since ROS such as superoxide can rapidly combine with NO to form reactive nitrogen species (RNS) [
42]. The tested triazines are more potent than JNJ7777120 in reducing the ROS formation and consequently they may prevent the ROS-dependent inactivation of NO.
Neurotoxicity or potent sedative effect of the new compounds can limit their future utility, and what is more, result in ambiguous or incorrect interpretation of the results of in vivo tests. Our experiments investigating the influence of the test compounds on spontaneous locomotor activity and their influence on motor coordination in rotarod test revealed that all in vivo effects of compounds were observed at the doses that did not cause neurologic deficits and did not significantly reduce spontaneous locomotor activity.
Although both studied compounds possess significantly lower affinity for H
4R than JNJ7777120, their activity in attenuating inflammatory and nociceptive response was comparable to the reference compound. We propose two main explanations of this phenomenon. The first is species difference in the H
4R structure and function. The affinity of the test compounds for H
4R was assessed in human receptors, whereas all experiments were carried out on mice and rats. In our opinion, it is unlikely that the affinity for mice receptors would be much higher than for human receptors. On the other hand, the species differences might be partially responsible for this effect. Some histamine H
4R ligands act as inverse agonists at the human H
4R, which is constitutively active, whereas as neutral antagonists at the constitutively inactive mouse and rat H
4R [
11]. Moreover, the test compounds and JNJ 7777120 may vary in their impact on β-arrestin-dependent intracellular pathway. JNJ 7777120 appears to be a partial agonist in β-arrestin recruitment [
9]. In case of test compounds, at this stage of research it is difficult to assess their character of interaction with H
4Rs on the molecular level.
The second explanation is that some additional mechanisms are involved in the final effect of the investigated compounds. The experiments on isolated ileum suggested that we cannot definitely exclude the potential involvement of histamine H
1R, since the investigated compounds produced a slight shift of the histamine concentration-response curve. The obtained pA
2 values (see Table
1) indicate that the affinity of compound 1 and compound 2 for H
1R was rather low. Nevertheless, even weak antagonism at H
1R might decrease vascular permeability [
22]. This might be particularly important when high doses are administered.
We showed that the influence on cAMP level in RAW 264.7 cells was similar for the test compounds and JNJ7777120. Taking into account the lower affinity of the compounds for H
4R, we suggest that phosphodiesterase inhibition could be involved in this effect. The fact that the chemical structure of the test compounds is similar to the structure of Irsogladine—the PDE inhibitor—supports this hypothesis [
50,
51]. Phosphodiesterase inhibition could be one of the pharmacological mechanisms of action of 1,3,5-triazine derivatives. PDE inhibitors elicit similar effects to those observed for the investigated compounds. Non-selective inhibition of PDE could explain the impact of these compounds on NO synthesis, since it might increase cGMP level and subsequently activate an important regulator of the activity of NO synthase (NOS)-sGC (soluble guanylyl cyclase). Several studies showed that either PDE4-specific inhibitors such as rolipram [
52‐
54] or non-selective PDE inhibitors such as pentoxifylline or theophylline [
55‐
58] were effective in attenuating pain and inflammation. Moreover, the antinociceptive activity of non-selective [
56] as well as selective [
54] PDE inhibitors was associated with the decreased release of cytokines such as IL-1β and TNFα. It is widely accepted that this effect is due to the increase of intracellular concentration of cAMP as a result of PDE inhibition in inflammatory and immunocompetent cells [
59]. To confirm or rule out the involvement of PDE inhibition in a mechanism of action of the investigated compounds, we investigated the inhibitory potential of studied compounds for the PDE 4B1 enzyme. PDE 4B1 is the phosphodiesterase isozyme, which is expressed in inflammatory cells, such as macrophages [
54]. The obtained results allow to definitely exclude the involvement of PDE inhibition from the pharmacological activity of the investigated aryl-1,3,5-triazines. Thus, we claim that anti-inflammatory and analgesic activities of these compounds are primarily H
4R dependent and the differences between them and JNJ7777123 are due to the different interaction with H
4R on the molecular level.
In conclusion, we demonstrated that two new H4R antagonists attenuated inflammatory and nociceptive response in in vivo models of inflammation. Both compounds (cAMP dependently) inhibited inflammatory mediators release in RAW264.7 cells and ROS production. Although our results provided new insight into the pharmacological profile of H4R ligands, some questions remain open, which encourages further studies.