Enigmatic Histamine Receptor H4 for Potential Treatment of Multiple Inflammatory, Autoimmune, and Related Diseases.

The histamine H4 receptor, belonging to the family of G-protein coupled receptors, is an increasingly attractive drug target. It plays an indispensable role in many cellular pathways, and numerous H4R ligands are being studied for the treatment of several inflammatory, allergic, and autoimmune disorders, including pulmonary fibrosis. Activation of H4R is involved in cytokine production and mediates mast cell activation and eosinophil chemotaxis. The importance of this receptor has also been shown in inflammatory models: peritonitis, respiratory tract inflammation, colitis, osteoarthritis, and rheumatoid arthritis. Recent studies suggest that H4R acts as a modulator in cancer, neuropathic pain, vestibular disorders, and type-2 diabetes, however, its role is still not fully understood.


Introduction
Histamine action via distinct receptors (H 1 R-H 4 R) modulates diverse physiological as well as pathological processes. Due to their differential receptor pharmacology and signal transduction properties, histamine has characteristic effects dependent upon the histamine receptor subtype it is bound to. Histamine receptors H 1 -H 4 are widespread throughout the body but there is limited knowledge about the H 4 R. The role of H4R in neuropathic pain transmission and other diseases is still controversial after nearly 20 years since its discovery. This may be due to biased signaling of histamine and H 4 receptor agonists and differential effects on multiple signaling pathways in central and peripheral parts of the sensory nervous system. However, in the last two decades, there was a particular increment in evidence supporting participation of H 3 R and H 4 R in neuropathic pain modulation [1]. Histamine has also been identified to be responsible for a vascular type headache, e.g., migraine, hence the antihistamines are regarded as a possible treatment [2]. The proper action of particular subtypes of histamine receptors is of special importance as it has been shown for instance for the delirium syndrome in which H 1 R and H 2 R antagonists have pro-delirium potential, while H 3 R antagonists have proved to be beneficial in combating delirium. The H 4 R may also play an indirect role requiring further intensive exploration [3]. Pulmonary fibrosis is the most frequent form of interstitial lung disease. Unavailability of effective therapies has led to the urge of exploiting novel curative approaches. Histamine receptor H 4 has been recognized as a new target for inflammatory and immune diseases, and H 4 R ligands reduced inflammation and oxidative stress in lung tissue. It has been shown that poly(ADP-ribose) polymerase (PARP-1) and H 4 R are both involved in inflammatory and fibrotic responses. Treatment with H 4 R antagonist JNJ7777120 ((5-chloro-1H-indol-2-yl)(4-methyl-1-piperazinyl)-methanone; CAS Number 459168-41-3; Molecular Weight: 277.8) in a condition of PARP-1 inhibition, provides anti-inflammatory and anti-fibrotic effects, causing reduction in airway remodeling and bronchoconstriction. Its synergistic effect with selective PARP-1 inhibitors could be of potential use for the treatment of pulmonary fibrosis [4]. Viral infections can be important contributors to development of asthma and chronic obstructive pulmonary disease. Pulmonary fibrosis is the main factor leading to pulmonary dysfunction and quality of life decline in SARS survivors. Gaining a deeper understanding of the interaction between Coronaviruses and the innate immune system of the host may shed light on the development and persistence of inflammation in the lungs and can possibly reduce the risk of lung inflammation caused by CoVs [5].

The Histamine Receptors-Localization and Function
Histamine receptors, numbered in the order of their discovery H 1 R-H 4 R, are G protein-coupled receptors (GPCRs) that constitute the largest family of cell surface receptors in humans and play a key role in cellular signaling. In the central nervous system (CNS), the histaminergic system is mainly modulated by histamine, an inflammatory biogenic amine involved in wide range of pathophysiological effects through interaction with histamine GPCRs which belong to class A (rhodopsin-like) GPCRs. These GPCRs differ in localization and cellular signaling mechanisms and they even differ in the level of constitutive activity, i.e., the ability to adopt an active conformation independent of ligand binding [6,7]. H 1 R and H 2 R are found in the brain and periphery, H 3 R is abundant in the CNS, while H 4 R has low expression, if any, in the CNS and is predominantly expressed on a variety of peripheral immune cells such as eosinophils, dendritic cells, mast cells (HMC-1, LAD-2, and primary cord blood derived CD34+ human mast cells), leukocytes, and T-cells (including γδT, T helper 1, 2, Th17, and CD8 cells) [6,[8][9][10][11][12]. The presence and role of H 4 R in brain nervous tissue is yet elusive and not fully known but the presence of H 4 R in non-neuronal cells in the brain has been confirmed [13,14]. Functional H 4 receptors that increase [ 35 S]-GTPγS binding and/or decrease noradrenaline release have not been identified in human, guinea pig, and mouse cortex [15]. In human mast cells, H 4 R mediates release of cytokines, leukotrienes, and chemokines (TGF-β1, TNF-α, TNF-β, PDGF-BB, TIMP-2, M-CSF, IP-10,  IL-16, IL-6, IL-3, IL-10, MIP-1α, IL-1α, ICAM-1, Eotaxin-2, RANTES, IL-8, MCP-1, and IL-6sR) [10].
Being a member of the most populated class A of the GPCR superfamily, human H 4 R also contains seven transmembrane helices and a short amphipathic helix that possibly runs parallel to the cytosolic membrane surface. It consists of 390 amino acid residues possessing all of the highly conserved sequence motifs [16,17] of the class A GPCRs including the most evolutionary conserved residues in each of the transmembrane helices: N1.50, D2.50, R3.50, W4.50, P5.50, P6.50, and P7.50 (Ballesteros-Weinstein numbering [18]) indicating the same activation mechanism of H 4 R as that of the other receptors in class A GPCRs [19]. The Ballesteros-Weinstein numbering scheme of GPCRs provides information about the relative positions of amino acids present in seven transmembrane helices. Each residue of the receptor is recognized by two numbers separated by a dot; the first number (1)(2)(3)(4)(5)(6)(7) indicates the number of the transmembrane helix where the residue is located while the second number indicates its position in relation to the most conserved residue, assigned number 50, of the same helix. The prominent residues such as D3.32 and W7.40, specific for amine-activated GPCRs, are also present in the H 4 R [20]. It has been observed that the two agonists (histamine and OUP-16) exhibit complementary interactions with residues D3.32, E5.46, and T6.55, while the reference antagonist JNJ7777120 exhibits interactions with D3.32 and E5.46 only (Figure 1), implicating a differentiating role of T6.55 in ligand binding and receptor activation [21,22]. There are also striking complementarities Among the histamine receptors, H1R and H4R possess 40% amino acid identity in the transmembrane region and they recognize the same endogenous ligand that is histamine. Due to such similarity the crystal structure of H1R has been used by many researchers for building the homology models of H4R. However, there are substantial differences in histamine receptor binding sites. For instance, N4.57 in H4R is equivalent to W4.56, L5.39 to K5.39, E5.46 to N5.46, and Q7.42 to G7.42 in H1R. Additionally, the mutations of residues N4.57 and E5.46 resulted in significant alteration of inhibition constants of JNJ7777120 which was the first reported H4R antagonist [23] and the homology model of H4R featured two specific hydrogen bonds and ionic interactions of JNJ7777120 to D3.32 and E5.46 [24]. H4R has the highest sequence homology with H3R as it possesses 37% amino acid identity in protein sequence and 58% identity in the transmembrane region. It is evident that a number of ligands of H4R also have a high affinity for H3R due to the identical amino acids within the binding site of both receptors, including E5.46, Y3.33, and Y6.51, involved in ligand binding [25]. These amino acids residues contribute to the similarity between the binding sites of hH3R and hH4R forcing similar conformations of ligands. This explains the number of ligands which are antagonists of both receptors. Additionally, various substituted histamine derivatives such as R-(α)methylhistamine have significant H4R binding in addition to H3R [6]. Istyastono et al. have shown that the E5.46Q mutation impaired the binding strength of clobenpropit and its derivatives in both those receptors [26]. Moreover, the L5.39V and E5.46Q mutations resulted in a decrease of binding of the reported ligands to H4R. This finding emphasized the importance of the E5.46 residue which provides a crucial interaction with antagonists [27].
A plethora of studies have related the heterogenic and complex pharmacology of histamine receptors to various diseases: H1R to the allergic inflammation, anaphylaxis, and motion sickness [28,29], H2R to the stimulation of gastric acid secretion leading to peptic ulcer, GERD and aspiration pneumonitis [30,31], H3R to the neurotransmission controlling sleep, cognitive processes, schizophrenia, epilepsy, and pain [32][33][34][35][36][37], and H4R to the immune responses (cancers, myocarditis) and inflammation [38][39][40][41][42] (Figure 2). The H3 and H4 receptors have relatively high affinity for Among the histamine receptors, H 1 R and H 4 R possess 40% amino acid identity in the transmembrane region and they recognize the same endogenous ligand that is histamine. Due to such similarity the crystal structure of H 1 R has been used by many researchers for building the homology models of H 4 R. However, there are substantial differences in histamine receptor binding sites. For instance, N4.57 in H 4 R is equivalent to W4.56, L5.39 to K5.39, E5.46 to N5.46, and Q7.42 to G7.42 in H 1 R. Additionally, the mutations of residues N4.57 and E5.46 resulted in significant alteration of inhibition constants of JNJ7777120 which was the first reported H 4 R antagonist [23] and the homology model of H 4 R featured two specific hydrogen bonds and ionic interactions of JNJ7777120 to D3.32 and E5.46 [24]. H 4 R has the highest sequence homology with H 3 R as it possesses 37% amino acid identity in protein sequence and 58% identity in the transmembrane region. It is evident that a number of ligands of H 4 R also have a high affinity for H 3 R due to the identical amino acids within the binding site of both receptors, including E5.46, Y3.33, and Y6.51, involved in ligand binding [25]. These amino acids residues contribute to the similarity between the binding sites of hH 3 R and hH 4 R forcing similar conformations of ligands. This explains the number of ligands which are antagonists of both receptors. Additionally, various substituted histamine derivatives such as R-(α)-methylhistamine have significant H 4 R binding in addition to H 3 R [6]. Istyastono et al. have shown that the E5.46Q mutation impaired the binding strength of clobenpropit and its derivatives in both those receptors [26]. Moreover, the L5.39V and E5.46Q mutations resulted in a decrease of binding of the reported ligands to H 4 R. This finding emphasized the importance of the E5.46 residue which provides a crucial interaction with antagonists [27].
A plethora of studies have related the heterogenic and complex pharmacology of histamine receptors to various diseases: H 1 R to the allergic inflammation, anaphylaxis, and motion Life 2020, 10, 50 4 of 17 sickness [28,29], H 2 R to the stimulation of gastric acid secretion leading to peptic ulcer, GERD and aspiration pneumonitis [30,31], H 3 R to the neurotransmission controlling sleep, cognitive processes, schizophrenia, epilepsy, and pain [32][33][34][35][36][37], and H 4 R to the immune responses (cancers, myocarditis) and inflammation [38][39][40][41][42] (Figure 2). The H 3 and H 4 receptors have relatively high affinity for histamine (5-10 nM) compared to the low affinity of H 1 R and H 2 R which is in the µM range [6,43]. Hence, the biological response has been linked directly with the local tissue histamine concentration and functional expression of different receptors [6].
Life 2020, 10, 50 4 of 17 histamine (5-10 nM) compared to the low affinity of H1R and H2R which is in the μM range [6,43]. Hence, the biological response has been linked directly with the local tissue histamine concentration and functional expression of different receptors [6].

Species Differences of H4R
Following the identification of the human H4R (UniProt id: Q9H3N8), various sequences of mouse, rat, guinea pig, pig, dog, and monkey H4R have been reported and functionally expressed [38]. Eighty-five protein sequences of H4R orthologues from different species have been extracted from the UniProt database and aligned to draw the phylogenetic relationship between H4R orthologues (Scheme 1). The H4 receptors of the chimpanzee, gorilla, and orangutan show the highest sequence homology (98-99%) with the human orthologue (hH4R). H4 receptors of some species are highly homologous to hH4R with sequence homology between 78% and 94%, specifically those of macaques, baboon, drill, Angolan colobus, mangabey, Cebus capucinus imitator, marmoset, and Philippine tarsier (Table 1). Orthologues in some species were only moderately homologous to hH4R with sequence homology between 54% and 73% while the least homologous showed homology ranging from 10% to 47%. Pig, mouse, smooth cauliflower coral, Japanese scallop, turbot, and pig have each two H4R orthologues while sea cucumber has three orthologues. However, these orthologues, show only 10-36% homology to hH4R while all others show a substantially higher homology (>50%). As some of the sequences are still incomplete, changes in the phylogenetic tree are to be expected. Within these GPCR sequences, the typical aminergic GPCR features (D3.32 in TM3 and E5.46 in TM5) can often be found. Detailed analysis of most of these species variants is however lacking even though it could provide useful tools to dissect receptor-ligand binding. Using sitedirected mutagenesis Wifling et al. have proved that the F169, located in the second extracellular loop ECL2, is a crucial amino acid for differential interactions, affinities, and potencies of certain agonists with the human and mouse H4R orthologues [45]. Receptor sequence differences have implications

Species Differences of H 4 R
Following the identification of the human H 4 R (UniProt id: Q9H3N8), various sequences of mouse, rat, guinea pig, pig, dog, and monkey H 4 R have been reported and functionally expressed [38]. Eighty-five protein sequences of H 4 R orthologues from different species have been extracted from the UniProt database and aligned to draw the phylogenetic relationship between H 4 R orthologues (Scheme 1). The H 4 receptors of the chimpanzee, gorilla, and orangutan show the highest sequence homology (98-99%) with the human orthologue (hH 4 R). H 4 receptors of some species are highly homologous to hH 4 R with sequence homology between 78% and 94%, specifically those of macaques, baboon, drill, Angolan colobus, mangabey, Cebus capucinus imitator, marmoset, and Philippine tarsier ( Table 1). Orthologues in some species were only moderately homologous to hH 4 R with sequence homology between 54% and 73% while the least homologous showed homology ranging from 10% to 47%. Pig, mouse, smooth cauliflower coral, Japanese scallop, turbot, and pig have each two H 4 R orthologues while sea cucumber has three orthologues. However, these orthologues, show only 10-36% homology to hH 4 R while all others show a substantially higher homology (>50%). As some of the sequences are still incomplete, changes in the phylogenetic tree are to be expected. Within these GPCR sequences, the typical aminergic GPCR features (D3.32 in TM3 and E5.46 in TM5) can often be found. Detailed analysis of most of these species variants is however lacking even though it could provide useful tools to dissect receptor-ligand binding. Using site-directed mutagenesis Wifling et al. have proved that the F169, located in the second extracellular loop ECL2, is a crucial amino acid for differential interactions, affinities, and potencies of certain agonists with the human and mouse H 4 R orthologues [45]. Receptor sequence differences have implications even for ligand function as the JNJ7777120 ligand acts as a partial inverse agonist at the human H 4 R, but as a partial agonist at the rat and mouse H 4 R which possess lower constitutive activity than their human counterpart. Therefore, differences in pharmacological activities of H 4 R ligands between different species might hamper preclinical development of future H 4 R drugs [46].
Life 2020, 10, 50 5 of 17 human counterpart. Therefore, differences in pharmacological activities of H4R ligands between different species might hamper preclinical development of future H4R drugs [46]. Scheme 1. Phylogenetic tree of H4R orthologues. The sequences were obtained from UniProt [47] and the sequences were aligned with ClustalW and the cladogram was created with Clustal Omega service [48].

The Pharmacological Effects of H 4 R Ligands
Although the pharmacology of H 4 R ligands is yet not fully elucidated H 4 R has been widely studied and reviewed since its characterization and cloning in 2000 [25,49]. The vast body of accumulating knowledge on physiological and pathophysiological functions associated with H 4 R modulation can be exploited for therapeutic purposes [11]. The properties of H 4 R make this amine receptor and its ligands of interest to specialists in the field of allergology, neurobiology, gastroenterology, endocrinology, and also to researchers of cardiovascular functions [6,50]. The results of research on the role of H 4 R in various pathophysiological and immunological processes indicate its association with the development and course of many diseases including a crucial role of H 4 R in airway and dermal inflammation (Figure 3), pruritus, ocular inflammation, arthritis, systemic lupus erythematosus, Sjogren's syndrome, multiple sclerosis, gastric ulcer, cancer, and pain [12,51].

The Pharmacological Effects of H4R Ligands
Although the pharmacology of H4R ligands is yet not fully elucidated H4R has been widely studied and reviewed since its characterization and cloning in 2000 [25,49]. The vast body of accumulating knowledge on physiological and pathophysiological functions associated with H4R modulation can be exploited for therapeutic purposes [11]. The properties of H4R make this amine receptor and its ligands of interest to specialists in the field of allergology, neurobiology, gastroenterology, endocrinology, and also to researchers of cardiovascular functions [6,50]. The results of research on the role of H4R in various pathophysiological and immunological processes indicate its association with the development and course of many diseases including a crucial role of H4R in airway and dermal inflammation (Figure 3), pruritus, ocular inflammation, arthritis, systemic lupus erythematosus, Sjogren's syndrome, multiple sclerosis, gastric ulcer, cancer, and pain [12,51]. Allergen may crosslink immunoglobulin E (IgE) on mast cells to release histamine, lipid mediators, and cytokines. Antigen is also processed by dendritic cells and macrophages for presentation to Thelper cells. During this process a local release of histamine and cytokines may occur. Histamine can act on a variety of cells and at different levels. In asthma histamine can facilitate the recruitment of inflammatory cells by regulating the chemotaxis of additional dendritic cells, eosinophils, and mast cells to the airways via the action at H4R. Histamine may additionally affect cytokine release from CD8 + cells via binding to H4R and from eosinophils, neutrophils, and mast cells through multiple histamine receptors. Allergen may crosslink immunoglobulin E (IgE) on mast cells to release histamine, lipid mediators, and cytokines. Antigen is also processed by dendritic cells and macrophages for presentation to T-helper cells. During this process a local release of histamine and cytokines may occur. Histamine can act on a variety of cells and at different levels. In asthma histamine can facilitate the recruitment of inflammatory cells by regulating the chemotaxis of additional dendritic cells, eosinophils, and mast cells to the airways via the action at H 4 R. Histamine may additionally affect cytokine release from CD8 + cells via binding to H 4 R and from eosinophils, neutrophils, and mast cells through multiple histamine receptors.

Allergic Diseases
Inflammatory conditions were for a long time thought to be mediated by activation of the histamine receptor subtype 1. However, the discovery and pharmacological characterization of H 4 R ligands especially antagonists, (and, to a lesser extent H 3 R and even H 2 R ligands) on mast cells, eosinophils, and T cells demonstrates the possibility of its involvement in inflammatory conditions/symptoms such as atopic dermatitis (AD), asthma, allergic rhinitis, rheumatoid arthritis (RA), and pruritus in humans. This is evident from the results obtained in diverse experimental models of inflammation including hepatic ischemia-reperfusion, colitis, atopic dermatitis, in which H 4 R antagonists (JNJ7777120, JNJ10191584, thioperamide) proved to be efficient anti-inflammatory agents with reduced neutrophil recruitment and release of cytokines [51,52]. Preclinical and clinical data strongly suggest the regulatory involvement of H 4 R in the calcium influx and cellular chemotaxis [53,54], hence establishing a link between the potential therapeutic application of selectively acting H 4 R ligands to inflammatory conditions while also indicating involvement of H 4 R in diseases accompanied by itch and pain [55]. The investigations of histamine in the inflammation process have led to a development of the first highly potent and selective non-imidazole H 4 R antagonist JNJ7777120, followed by reexamination and synthesis of a plethora of H 4 R-targeted compounds [50,51].
Currently, many H 4 R ligands are known, synthesized, and evaluated [56,57]. Studies using selective H 4 R ligands in animal models of pruritus revealed a role for H 4 R in mediating chronic pruritus associated with conditions such as atopic dermatitis [51,58]. Antagonists of H 4 R (JNJ7777120, JNJ39758979, INCB38579, and others) reduced pruritus in a number of animal studies [59] as well as itching sensation in different conditions in human patients. Alcaftadine, a topical ophthalmic drug indicated for the prevention of itching associated with allergic conjunctivitis, is a potent H 1 R and H 2 R antagonist (in fact, inverse agonist) with weak inverse agonistic activity also towards H 4 R [60]. Administration of H 1 R/H 4 R antagonists or co-administration of H 1 R and H 4 R antagonists will probably be effective also in humans. Such antagonists are more efficacious as compared to olopatadine (H 1 R antagonist without H 4 R activity) [61]. Consequently, these studies indicate that H 4 R is involved in mediating pruritic responses in humans, and that H 4 R antagonists are ought to be effective in the treatment of pruritic histamine-mediated conditions, such as AD, acute urticaria, allergic rhinitis, or allergic conjunctivitis.
The histamine receptor H 4 R was also found on cartilage cells-chondrocytes [62,63]. As the presence of the histamine triggering protein (HRF) has been identified in the joints of people with RA, it seems very likely that H 4 R antagonists will be used in the future in the treatment of RA [64]. This receptor may also be important in the pathogenesis of Sjörgen's syndrome, erythematous lupus erythematosus, and atopic dermatitis [65]. H 4 R activation not only results in phosphorylation of ERK and PI3K in a time dependent manner but it also leads to enhanced synthesis of inflammatory mediators associated with allergic reactions. It leads to inflammatory conditions as well as contributes to postinflammatory visceral hypersensitivity, thus, making H 4 R antagonists important for reducing inflammation and normalizing postinflammatory visceral hypersensitivity [66].

Asthma
H 4 R seems to be an interesting pharmacological target in the treatment of asthma [6]. Asthma is a condition typically characterized by involvement of eosinophils and mast cells [67][68][69]. Extensive studies have provided evidence detailing the functional profile of H 4 R in eosinophil biology [70] and in the chemotaxis and differentiation of other immune cell types. In experiments carried out on animal models of inflammation of the airways, it was observed that in mice lacking the H 4 R gene, there was a significant reduction in the allergic reaction caused by the administration of a chicken protein-ovalbumin [71]. Chemotaxis of eosinophils was shown to be blocked by H 4 R selective antagonists (JNJ7777120, JNJ39758979, or JNJ10191584) in animal asthma models due to priming and T cell activation [51,72] while induced by histamine and selective H 4 R agonists (e.g., 4-methylhistamine) [72]. Some selective H 4 R antagonists in animal models of asthma proved beneficial Life 2020, 10, 50 9 of 17 by mediating lung function and inflammation [51,73]. In asthma animal models, H 4 R antagonists act either directly by reducing the number of T cells at the site of inflammation [74] or indirectly when it is involved in dendritic cell function driving the response [51]. However, none of the H 4 R antagonists have been introduced to treat the above disorders.

Diabetes
The histamine receptor H 4 may also be a therapeutic target in diseases not directly related to inflammation. For instance, H 4 R is suggested to be important in the pathogenesis of diabetes. In streptozotocin-induced diabetic rats H 4 R is overexpressed in tubular epithelial cells [75], and administration of a H 4 R antagonist resulted in a decreased blood sugar [76]. H 4 R participates in diabetic nephropathy progression through both a direct effect on tubular reabsorption and an indirect action on renal tissue architecture via inflammatory cell recruitment. Therefore, H 4 R antagonism emerges as a possible new multi-mechanism therapeutic approach to counteract development of diabetic nephropathy [77].

Parkinson's and Alzheimer's Diseases
Evidence about the H 4 R antagonist JNJ7777120 inhibiting propagation of microglial inflammation by attenuating the release of M1 microglial cells and largely preventing the pathological progression of Parkinson's disease-like pathology and motor dysfunction has been provided by the latest research [78]. These findings support H 4 R as a promising novel therapeutic target for Parkinson's disease. For Alzheimer's disease the precise mechanism of histamine-induced Alzheimer's pathology is not well known although the increased levels of histamine in plasma and in some areas of the brain are seen in Alzheimer's dementia brain [79]. It is known that H 3 R can regulate cognitive and memory functions in the hippocampus so it could be involved in Alzheimer's pathology [80]. Since H 4 R is also present in the brain and its stimulation regulates neuronal functions, then stimulating H 4 receptors may have some beneficial effects in the brain of Alzheimer's disease patients. Recently, it has been found that clobenpropit, a selective H 3 R antagonist with partial H 4 R agonist property, caused a significant reduction in amyloid-β deposits in a rat model of Alzheimer-like brain pathology. This effect was accompanied by marked reduction in neuronal or glial reactions so such dual-action compounds may have neuroprotective properties [81].
High similarity between H 3 R and H 4 R entails considerable similarity in ligand affinities and facilitates simultaneous activation of both receptors. Dual-acting H 3 R/H 4 R ligands may exhibit therapeutic potential in diverse pathological conditions, such as neuropathic pain, cancer, Parkinson's, and inflammatory diseases [7,82]. Dual H 3 R/H 4 R imidazole containing ligands used so far includes compounds such as imetit, immepip, clobenpropit, and thioperamide [7].

Autoimmune Diseases
The characterization of a histamine receptor H 4 R with putative immunomodulating properties encouraged new hopes for the translational exploitation of this new therapeutic target for the still unmet medical needs, specifically asthma, autoimmune diseases, and a host defense. Rheumatoid arthritis (RA), which is a systemic autoimmune disorder, is characterized by chronic synovitis of peripheral joints, cartilage and bone destruction followed by joint disability. It was found that histamine and Th17 cytokines induced osteoclast differentiation from monocytes and JNJ7777120 decreased the osteoclastogenesis and the osteoclastogenic role of H 4 R has been evident in patients with RA [83]. Studies in the animal model of RA have shown that the H 4 R antagonist JNJ7777120 reduces the degree and severity of joint damage and reduces the number of cells producing IL-17 in the joint, thus, significantly inhibiting the inflammatory process in joints [84]. H 4 R involvement has been also confirmed in several types of cancers: melanoma [85], breast cancer [86], pancreatic cancer [87], and colorectal cancer [88]. H 4 R can regulate the aging and apoptosis of cancer cells and blocking H 4 R by antagonists inhibits tumor cell proliferation [86]. Histamine receptors play also an important role in the pathogenesis of multiple sclerosis. It turned out that H 1 R and H 2 R play a propathogenic role while H 3 R and H 4 R may reduce the risk of the disease [89].

Clinical Trials of Drug Candidates Targeting H 4 R
Recently, H 4 R research has been gaining a lot of importance and the clinical studies were initiated for the putative therapeutic exploitation in inflammatory and allergic disorders [38] such as atopic dermatitis (AD) [59,90], pruritus, asthma, rheumatoid arthritis (RA), as well as in vestibular disease ( Table 2) [91]. Toreforant (JNJ38518168), the first oral H 4 R antagonist, has been explored for the treatment of RA patients with active disease despite concomitant methotrexate therapy (phase 2 trials, ClinicalTrials.gov database entry NCT01862224 and dose range finding study NCT01679951) [92,93]. Both studies were prematurely terminated in 2014 because of the lack of efficacy. The similar phase 2 clinical studies for the same compound evaluating efficacy and safety of toreforant in patients with symptomatic uncontrolled, persistent eosinophilic asthma (NCT01823016) [94], and in patients with moderate to severe plaque-type psoriasis (NCT02295865) [95] were completed in 2015 and 2016. In the former study toreforant (at the dose tested) failed to provide any therapeutic benefit [96]. Preclinical toxicity studies of another H 4 R antagonist, JNJ39758979, provided sufficient evidence of an excellent safe profile encouraging the clinical level testing [72]. JNJ39758979 was observed to mitigate RA in the collagen-induced arthritis models (CIAM) [59]. The completed phase 2 clinical trial demonstrating its safety and effectiveness in human volunteers with persistent asthma (NCT00946569) whereas several phase 1 studies stating its safety and pharmacokinetics, as well as its effect on histamine-induced itch (pruritus) (NCT01068223) in healthy male volunteers have successfully been accomplished [97,98]. Simultaneously, the two phase 2 clinical studies were initiated to find a dose range of JNJ39758979 in patients with RA despite concomitant methotrexate therapy (NCT01480388) and patients with uncontrolled asthma (NCT01493882) but they were withdrawn in 2014 and 2015, respectively, due to the same reasons [99,100]. This adverse effect was predicted to be related with reactive metabolites of JNJ39758979 and not with H 4 R antagonism. Hence, the significant reduction in the pruritus after JNJ39758979 administration can be concluded in the way that drug-induced agranulocytosis can be most likely an off-target effect and other H 4 R antagonists could be beneficial in the treatment of AD, particularly pruritus, without serious adverse effects [101]. In the similar clinical studies, another oral, potent, and selective H 4 R antagonist ZPL3893787 has completed phase 2 clinical trials determining its safety, efficacy, and tolerability on pruritus in adult subjects with moderate to severe AD (NCT02424253) [102] and in patients with plaque psoriasis (NCT02618616) [103] in 2016 but no results for both these studies were posted on ClinicalTrials.gov. Results showed that ZPL3893787 improved inflammatory skin lesions in patients with AD, confirming H 4 R antagonism as a novel therapeutic option [90]. Additionally, in two different phase 2 trials, there is an evaluation safety and efficacy of ZPL3893787 in patients with moderate to severe AD (NCT03517566) [104] and the impact of its concomitant use along with topical corticosteroids (TCS) and/or topical calcineurin inhibitors (TCI) in patients with AD (NCT03948334) [105]. The efficacy of Seliforant (SENS-111) in patients suffering from acute unilateral vestibulopathy is currently under evaluation in Phase 2 trial (NCT03110458) [106]. The above-mentioned observations indicate a wide range of potential clinical applications of H 4 R ligands. corticosteroids (TCS) and/or topical calcineurin inhibitors (TCI) in patients with AD (NCT03948334) [105]. The efficacy of Seliforant (SENS-111) in patients suffering from acute unilateral vestibulopathy is currently under evaluation in Phase 2 trial (NCT03110458) [106]. The above-mentioned observations indicate a wide range of potential clinical applications of H4R ligands. *Status: T: terminated; C: completed; R: recruiting; W: withdrawn.

Challenges and Perspectives
The H4R research triggered serious concern as to the role of histamine in the regulation of immune (patho)physiology. It has been established that JNJ7777120 acts as an antagonist in respect to G protein-dependent signaling, but it also recruits β-arrestin to the receptor in a non-G proteindependent manner [107]. Moreover, JNJ7777120 acts as a partial inverse agonist at the human H4R but as a partial agonist at the rat and mouse H4 receptors [46], which show a lower constitutive activity than their human counterpart [45,46,108,109]. Frequently generated controversies and even in vivo misleading results in a variety of experimental models have been the repercussions of these problems [109]. The clinical development of JNJ7777120 as a prototype experimental tool was hampered due to several setbacks that surfaced over the past two decades including: localized concerns over the receptor subtypes, ligand binding and functional selectivity, constitutive and intrinsic activity and the biased signaling [6,46,50,51,95,110], its short half-life in vivo, and the hypoadrenocorticism toxicity concerns [50]. Therefore, the experimental findings on the role of H4R cannot be relied upon and need thorough investigation with caution.
Although GPCR biased signaling significantly complicates drug discovery attempts, it makes a great promise to design specific ligands with minor side effects [95,111]. The precise drugs have rapidly become the center of research for therapeutic exploitation in immunopharmacology as well as clinical immunology [90,112,113]. However, in addition to H4R, significant evidence attributes some immunomodulatory properties to H2R [90,110], thus, dissection of histamine functions in the immune system becomes indispensable. Although there are many problems in H4R research, a significant number of studies focusing on H4R provide an optimistic research perspective for this new drug target.

Challenges and Perspectives
The H4R research triggered serious concern as to the role of histamine in the regulation of immune (patho)physiology. It has been established that JNJ7777120 acts as an antagonist in respect to G protein-dependent signaling, but it also recruits β-arrestin to the receptor in a non-G proteindependent manner [107]. Moreover, JNJ7777120 acts as a partial inverse agonist at the human H4R but as a partial agonist at the rat and mouse H4 receptors [46], which show a lower constitutive activity than their human counterpart [45,46,108,109]. Frequently generated controversies and even in vivo misleading results in a variety of experimental models have been the repercussions of these problems [109]. The clinical development of JNJ7777120 as a prototype experimental tool was hampered due to several setbacks that surfaced over the past two decades including: localized concerns over the receptor subtypes, ligand binding and functional selectivity, constitutive and intrinsic activity and the biased signaling [6,46,50,51,95,110], its short half-life in vivo, and the hypoadrenocorticism toxicity concerns [50]. Therefore, the experimental findings on the role of H4R cannot be relied upon and need thorough investigation with caution.
Although GPCR biased signaling significantly complicates drug discovery attempts, it makes a great promise to design specific ligands with minor side effects [95,111]. The precise drugs have rapidly become the center of research for therapeutic exploitation in immunopharmacology as well as clinical immunology [90,112,113]. However, in addition to H4R, significant evidence attributes some immunomodulatory properties to H2R [90,110], thus, dissection of histamine functions in the immune system becomes indispensable. Although there are many problems in H4R research, a significant number of studies focusing on H4R provide an optimistic research perspective for this new drug target.

Challenges and Perspectives
The H4R research triggered serious concern as to the role of histamine in the regulation of immune (patho)physiology. It has been established that JNJ7777120 acts as an antagonist in respect to G protein-dependent signaling, but it also recruits β-arrestin to the receptor in a non-G proteindependent manner [107]. Moreover, JNJ7777120 acts as a partial inverse agonist at the human H4R but as a partial agonist at the rat and mouse H4 receptors [46], which show a lower constitutive activity than their human counterpart [45,46,108,109]. Frequently generated controversies and even in vivo misleading results in a variety of experimental models have been the repercussions of these problems [109]. The clinical development of JNJ7777120 as a prototype experimental tool was hampered due to several setbacks that surfaced over the past two decades including: localized concerns over the receptor subtypes, ligand binding and functional selectivity, constitutive and intrinsic activity and the biased signaling [6,46,50,51,95,110], its short half-life in vivo, and the hypoadrenocorticism toxicity concerns [50]. Therefore, the experimental findings on the role of H4R cannot be relied upon and need thorough investigation with caution.
Although GPCR biased signaling significantly complicates drug discovery attempts, it makes a great promise to design specific ligands with minor side effects [95,111]. The precise drugs have rapidly become the center of research for therapeutic exploitation in immunopharmacology as well as clinical immunology [90,112,113]. However, in addition to H4R, significant evidence attributes some immunomodulatory properties to H2R [90,110], thus, dissection of histamine functions in the immune system becomes indispensable. Although there are many problems in H4R research, a significant number of studies focusing on H4R provide an optimistic research perspective for this new drug target.

Challenges and Perspectives
The H 4 R research triggered serious concern as to the role of histamine in the regulation of immune (patho)physiology. It has been established that JNJ7777120 acts as an antagonist in respect to G protein-dependent signaling, but it also recruits β-arrestin to the receptor in a non-G protein-dependent manner [107]. Moreover, JNJ7777120 acts as a partial inverse agonist at the human H 4 R but as a partial agonist at the rat and mouse H 4 receptors [46], which show a lower constitutive activity than their human counterpart [45,46,108,109]. Frequently generated controversies and even in vivo misleading results in a variety of experimental models have been the repercussions of these problems [109]. The clinical development of JNJ7777120 as a prototype experimental tool was hampered due to several setbacks that surfaced over the past two decades including: localized concerns over the receptor subtypes, ligand binding and functional selectivity, constitutive and intrinsic activity and the biased signaling [6,46,50,51,95,110], its short half-life in vivo, and the hypoadrenocorticism toxicity concerns [50]. Therefore, the experimental findings on the role of H 4 R cannot be relied upon and need thorough investigation with caution.
Although GPCR biased signaling significantly complicates drug discovery attempts, it makes a great promise to design specific ligands with minor side effects [95,111]. The precise drugs have rapidly become the center of research for therapeutic exploitation in immunopharmacology as well as clinical immunology [90,112,113]. However, in addition to H 4 R, significant evidence attributes some immunomodulatory properties to H 2 R [90,110], thus, dissection of histamine functions in the immune system becomes indispensable. Although there are many problems in H 4 R research, a significant number of studies focusing on H 4 R provide an optimistic research perspective for this new drug target.