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

Abstract

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.

1. Introduction

Histamine action via distinct receptors (H₁R–H₄R) 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 H₁–H₄ are widespread throughout the body but there is limited knowledge about the H₄R. 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 H₄ 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 H₃R and H₄R 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 H₁R and H₂R antagonists have pro-delirium potential, while H₃R antagonists have proved to be beneficial in combating delirium. The H₄R 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 H₄ has been recognized as a new target for inflammatory and immune diseases, and H₄R ligands reduced inflammation and oxidative stress in lung tissue. It has been shown that poly(ADP-ribose) polymerase (PARP-1) and H₄R are both involved in inflammatory and fibrotic responses. Treatment with H₄R 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].

2. The Histamine Receptors—Localization and Function

Histamine receptors, numbered in the order of their discovery H₁R-H₄R, 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]. H₁R and H₂R are found in the brain and periphery, H₃R is abundant in the CNS, while H₄R 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 H₄R in brain nervous tissue is yet elusive and not fully known but the presence of H₄R in non-neuronal cells in the brain has been confirmed [13,14]. Functional H₄ receptors that increase [³⁵S]-GTPγS binding and/or decrease noradrenaline release have not been identified in human, guinea pig, and mouse cortex [15]. In human mast cells, H₄R 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 H₄R 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 H₄R 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 (1–7) 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 H₄R [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 between the H₄R binding pocket and the structural properties of most H₄R antagonists. They consist of a minimum of one, or preferably two, positively charged groups complementary to two negatively charged residues in the binding pocket, namely D3.32 and E5.46, and such double interaction is crucial for the interaction of high affinity ligands with H₄R [21].

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 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].

3. 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. Sequence similarities of species specific H₄R to the human orthologue.

	Species	Scientific Name	UniProt ID	Similarity to hH4R
1	Human	Homo sapiens	Q9H3N8	-
2	Chimpanzee	Pan troglodytes	H2QED2	99%
3	Gorilla	Gorilla	G3QS38	98%
4	Pygmy chimpanzee	Pan paniscus	A0A2R9BQY6	98%
5	Orangutan	Pongo abelii	H2NW27	98%
6	Crab-eating macaque	Macaca fascicularis	Q3V8G8	94%
7	Pig-tailed macaque	Macaca nemestrina	A0A2K6D1G7	94%
8	Rhesus macaque	Macaca mulatta	G7NKH9	94%
9	Olive baboon	Papio anubis	A0A096NGN9	94%
10	Drill	Mandrillus leucophaeus	A0A2K5YBZ5	94%
11	Angolan colobus	Colobus angolensis palliatus	A0A2K5HHL6	93%
12	Sooty mangabey	Cercocebus atys	A0A2K5LQL7	93%
13	Black snub-based monkey	Rhinopithecus bieti	A0A2K6MXG3	93%
14	Golden snub-based monkey	Rhinopithecus roxellana	A0A2K6RWF0	93%
15	Green monkey	Chlorocebus sabaeus	A0A0D9RYY4	90%
16	Ma’s Night monkey	Aotus nancymaae	A0A2K5CHI5	90%
17	Cebus capucinus imitator	Cebus capucinus imitator	A0A2K5RKQ4	90%
18	White-tufted-ear marmoset	Callithrix jacchus	F7IT43	89%
19	Squirrel monkey	Saimiri boliviensis	A0A2K6TG45	88%
20	Philippine tarsier	Tarsius syrichta	A0A1U7UM57	78%
21	Small-eared galago	Otolemur garnettii	H0WYC8	73%
22	Thirteen-lined ground squirrel	Ictidomys tridecemlineatus	I3MG71	72%
23	Dog	Canis lupus familiaris	J9P1C3	71%
24	Golden hamster	Mesocricetus auratus	A0A1U7Q7T1	71%
25	Grizzly bear	Ursus arctos horribilis	A0A3Q7WBT8	70%
26	Polar bear	Ursus maritimus	A0A384C2G0	70%
27	Pig	Sus scrofa	Q8WNV9 (Pig 1)	70%
28			A0A5G2QV28 (Pig 2)	10%
29	Red fox	Vulpes vulpes	A0A3Q7SYT7	70%
30	Black flying fox	Pteropus alecto	L5K5C7	69%
31	African elephant	Loxodonta africana	G3STF1	69%
32	Giant panda	Ailuropoda melanoleuca	G1M6D3	69%
33	Chinese hamster	Cricetulus griseus	A0A3L7I1V9	69%
34	Horse	Equus caballus	F6Z8L3	69%
35	Sea cow	Trichechus manatus latirostris	A0A2Y9E7N3	69%
36	Rabbit	Oryctolagus cuniculus	G1TKW6	68%
37	Iberian lynx	Lynx pardinus	A0A485N8M7	68%
38	Cat	Felis catus	M3WE71	68%
39	Pacific walrus	Odobenus rosmarus divergens	A0A2U3WW63	68%
40	Rat	Rattus norvegicus	Q91ZY1	68%
41	Kangaroo rat	Dipodomys ordii	A0A1S3F272	68%
42	Hawaiian monk seal	Neomonachus schauinslandi	A0A2Y9GRV4	68%
43	Northern fur seal	Callorhinus ursinus	A0A3Q7Q9W4	67%
44	Sea otter	Enhydra lutris kenyoni	A0A2Y9ITU9	67%
45	Hedgehog	Erinaceus europaeus	A0A1S3A2Y6	67%
46	European domestic ferret	Mustela putorius furo	M3Y4H4	67%
47	Mouse	Mus musculus	Q91ZY2 (Mouse 1)	67%
48			B2ZGH2 (Mouse 2)	66%
49	Goat	Capra hircus	A0A452DKI0	65%
50	Sheep	Ovis aries	W5PBL0	65%
51	Sperm whale	Physeter macrocephalus	A0A2Y9F727	65%
52	Hybrid cattle	Bos indicus*Bos taurus	A0A4W2DVG0	64%
53	Yak	Bos mutus	L8IEJ5	64%
54	Bovine	Bos taurus	E1BBS2	64%
55	Guinea pig	Cavia porcellus	Q91ZY3	63%
56	Black bear	Ursus americanus	A0A452QKW6	62%
57	Yangtze river dolphin	Lipotes vexillifer	A0A340YGS9	61%
58	American mink	Neovison vison	U6CNR7	61%
59	Beluga whale	Delphinapterus leucas	A0A2Y9PB56	59%
60	Yangtze finless porpoise	Neophocaena asiaeorientalis	A0A341CIF8	59%
61	European red deer	Cervus elaphus hippelaphus	A0A212C702	59%
62	Indo-pacific humpbacked dolphin	Sousa chinensis	A0A484GQ08	57%
63	Narwhal	Monodon monoceros	A0A4U1FGC1	56%
64	Wolverine	Gulo gulo	A0A3P4RYS2	55%
65	Atlantic bottle-nosed dolphin	Tursiops truncatus	A0A2U3V3K5	54%
66	Gray short-tailed opossum	Monodelphis domestica	F6QB56	47%
67	North-Pacific minke whale	Balaenoptera acutorostrata scammoni	A0A452C640	46%
68	Tasmanian devil	Sarcophilus harrisii	G3X3P1	45%
69	Weddell seal	Leptonychotes weddellii	A0A2U3YB28	42%
70	White-tailed sea-eagle	Haliaeetus albicilla	A0A091PX74	42%
71	Trogon	Apaloderma vittatum	A0A091NQC4	41%
72	Cuckoo	Cuculus canorus	A0A091G9T7	40%
73	Turbot	Scophthalmus maximus	A0A2U9BJT1 (Turbot 1)	36%
74			A0A2U9C3Q1 (Turbot 2)	36%
75	Channel catfish	Ictalurus punctatus	A0A2D0RQW6	36%
76	Chinese tree shrew	Tupaia chinensis	L8YD15	35%
77	Rifleman	Acanthisitta chloris	A0A091MN56	31%
78	Scallop	Mizuhopecten yessoensis	A0A210PRL2 (Scallop 1)	26%
79			A0A210PS14 (Scallop 2)	22%
80	Oyster	Crassostrea gigas	K1PU39	24%
81	Coral	Stylophora pistillata	A0A2B4RTL0 (Coral 1)	17%
82			A0A2B4RX53 (Coral 2)	14%
83	Sea cucumber	Stichopus japonicus	A0A2G8KHM7 (Sea cucumber 1)	15%
84			A0A2G8L2L5 (Sea cucumber 2)	13%
85			A0A2G8JXR8 (Sea cucumber 3)	20%

). 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 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 H₄R orthologues [45]. Receptor sequence differences have implications even for ligand function as the JNJ7777120 ligand acts as a partial inverse agonist at the human H₄R, but as a partial agonist at the rat and mouse H₄R which possess lower constitutive activity than their human counterpart. Therefore, differences in pharmacological activities of H₄R ligands between different species might hamper preclinical development of future H₄R drugs [46].

4. 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].

4.1. 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 H₄R ligands especially antagonists, (and, to a lesser extent H₃R and even H₂R 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 H₄R 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 H₄R in the calcium influx and cellular chemotaxis [53,54], hence establishing a link between the potential therapeutic application of selectively acting H₄R ligands to inflammatory conditions while also indicating involvement of H₄R 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 H₄R antagonist JNJ7777120, followed by reexamination and synthesis of a plethora of H₄R-targeted compounds [50,51].

Currently, many H₄R ligands are known, synthesized, and evaluated [56,57]. Studies using selective H₄R ligands in animal models of pruritus revealed a role for H₄R in mediating chronic pruritus associated with conditions such as atopic dermatitis [51,58]. Antagonists of H₄R (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 H₁R and H₂R antagonist (in fact, inverse agonist) with weak inverse agonistic activity also towards H₄R [60]. Administration of H₁R/H₄R antagonists or co-administration of H₁R and H₄R antagonists will probably be effective also in humans. Such antagonists are more efficacious as compared to olopatadine (H₁R antagonist without H₄R activity) [61]. Consequently, these studies indicate that H₄R is involved in mediating pruritic responses in humans, and that H₄R 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 H₄R 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 H₄R 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]. H₄R 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 H₄R antagonists important for reducing inflammation and normalizing postinflammatory visceral hypersensitivity [66].

4.2. Asthma

H₄R 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 H₄R 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 H₄R 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 H₄R 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 H₄R agonists (e.g., 4-methylhistamine) [72]. Some selective H₄R antagonists in animal models of asthma proved beneficial by mediating lung function and inflammation [51,73]. In asthma animal models, H₄R 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 H₄R antagonists have been introduced to treat the above disorders.

4.3. Diabetes

The histamine receptor H₄ may also be a therapeutic target in diseases not directly related to inflammation. For instance, H₄R is suggested to be important in the pathogenesis of diabetes. In streptozotocin-induced diabetic rats H₄R is overexpressed in tubular epithelial cells [75], and administration of a H₄R antagonist resulted in a decreased blood sugar [76]. H₄R 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, H₄R antagonism emerges as a possible new multi-mechanism therapeutic approach to counteract development of diabetic nephropathy [77].

4.4. Parkinson’s and Alzheimer’s Diseases

Evidence about the H₄R 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 H₄R 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 H₃R can regulate cognitive and memory functions in the hippocampus so it could be involved in Alzheimer’s pathology [80]. Since H₄R is also present in the brain and its stimulation regulates neuronal functions, then stimulating H₄ receptors may have some beneficial effects in the brain of Alzheimer’s disease patients. Recently, it has been found that clobenpropit, a selective H₃R antagonist with partial H₄R 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 H₃R and H₄R entails considerable similarity in ligand affinities and facilitates simultaneous activation of both receptors. Dual-acting H₃R/H₄R ligands may exhibit therapeutic potential in diverse pathological conditions, such as neuropathic pain, cancer, Parkinson’s, and inflammatory diseases [7,82]. Dual H₃R/H₄R imidazole containing ligands used so far includes compounds such as imetit, immepip, clobenpropit, and thioperamide [7].

4.5. Autoimmune Diseases

The characterization of a histamine receptor H₄R 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 H₄R has been evident in patients with RA [83]. Studies in the animal model of RA have shown that the H₄R 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]. H₄R involvement has been also confirmed in several types of cancers: melanoma [85], breast cancer [86], pancreatic cancer [87], and colorectal cancer [88]. H₄R can regulate the aging and apoptosis of cancer cells and blocking H₄R by antagonists inhibits tumor cell proliferation [86]. Histamine receptors play also an important role in the pathogenesis of multiple sclerosis. It turned out that H₁R and H₂R play a propathogenic role while H₃R and H₄R may reduce the risk of the disease [89].

5. Clinical Trials of Drug Candidates Targeting H₄R

Recently, H₄R 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. Details of compounds which are/have been in clinical trial studies which started/ended/terminated in the 2014–2019 period.

Compound	Clinical Indications	Phase	Status	ClinicalTrials.Gov Database Entry	Ref.
JNJ38518168 (Toreforant)	RA	2	T	NCT01862224	[92]
	RA	2	T	NCT01679951	[93]
	Asthma	2	C	NCT01823016	[94]
	Psoriasis	2	C	NCT02295865	[95]
JNJ39758979	RA	2	W	NCT01480388	[99]
	Asthma	2	W	NCT01493882	[100]
ZPL3893787 (Adriforant/PF3893787/ZPL389)	AD	2	C	NCT02424253	[102]
	Psoriasis	2	C	NCT02618616	[103]
	AD	2	R	NCT03517566	[104]
	AD	2	R	NCT03948334	[105]
SENS-111 (Seliforant)	Unilateral Vestibulopathy	2	R	NCT03110458	[106]

Status: T: terminated; C: completed; R: recruiting; W: withdrawn.

) [91]. Toreforant (JNJ38518168), the first oral H₄R 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 H₄R 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 H₄R 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 H₄R 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 H₄R 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 H₄R 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 H₄R ligands.

6. 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 protein-dependent 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.