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.

Full text

life
Review
Enigmatic Histamine Receptor H4for Potential
Treatment of Multiple Inflammatory, Autoimmune,
and Related Diseases
Pakhuri Mehta 1, Przemysław Miszta 1, Przemysław Rzodkiewicz 2, Olga Michalak 3,
Piotr Krzeczy ´nski 3and Sławomir Filipek 1, *
1Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, 02-093 Warsaw,
Poland; pmehta@chem.uw.edu.pl or pakhurimehta@gmail.com (P.M.); pmiszta@chem.uw.edu.pl (P.M.)
2
Department of General and Experimental Pathology, Medical University of Warsaw, 02-091 Warsaw, Poland;
przemyslaw.rzodkiewicz@wum.edu.pl
3Łukasiewicz Research Network-Pharmaceutical Research Institute, 01-793 Warsaw, Poland;
o.michalak@ifarm.eu (O.M.); p.krzeczynski@ifarm.eu (P.K.)
*Correspondence: sfilipek@chem.uw.edu.pl
Received: 31 March 2020; Accepted: 20 April 2020; Published: 24 April 2020


Abstract:
The histamine H
4
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 H
4
R ligands are being studied for the treatment of several inflammatory, allergic, and
autoimmune disorders, including pulmonary fibrosis. Activation of H
4
R 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 H
4
R acts as a modulator
in cancer, neuropathic pain, vestibular disorders, and type-2 diabetes, however, its role is still not
fully understood.
Keywords:
histamine H
4
receptor; G protein-coupled receptors; allergic diseases; inflammatory
diseases; autoimmune disorders; neuropathic pain; cancer
1. 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].
Life 2020,10, 50; doi:10.3390/life10040050 www.mdpi.com/journal/life

Life 2020,10, 50 2 of 17
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].
2. 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
γδ
T, T helper 1, 2, Th17, and CD8
cells) [
6
,
8
–
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]-GTP
γ
S 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-
β
1, 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
(1–7) 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

Life 2020,10, 50 3 of 17
between the H
4
R binding pocket and the structural properties of most H
4
R 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].
Life 2020, 10, 50 3 of 17
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].
Figure 1. The homology model of H4R with docked JNJ7777120 antagonist. The specific ligand–
receptor interactions are shown on the right panel. D3.32 forms both a hydrogen bond and an ionic
interaction with the charged amine group of the ligand.
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 [32–37], and H4R to the immune responses (cancers, myocarditis)
and inflammation [38–42] (Figure 2). The H3 and H4 receptors have relatively high affinity for
Figure 1.
The homology model of H
4
R with docked JNJ7777120 antagonist. The specific ligand–receptor
interactions are shown on the right panel. D3.32 forms both a hydrogen bond and an ionic interaction
with the charged amine group of the ligand.
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
–
37
], and H
4
R to the immune responses (cancers, myocarditis) and
inflammation [
38
–
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
µ
M 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].
Figure 2. Classification of histamine receptors (H1R–H4R) in relation to their functions. H1R–H3R
transduce extracellular signals via Gαq/11, Gαis, and Gαi/o, respectively, while H4R acts through Gαi/o
and β-arrestin. H1R and H2R are low-affinity receptors while H3R and H4R are high-affinity receptors
towards histamine. Ligands of H1R–H4R have therapeutic applications in allergic inflammation,
gastric acid secretion, neurotransmission, and immunomodulation, respectively. The information in
the figure is partially based on [44].
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). 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
Figure 2.
Classification of histamine receptors (H
1
R–H
4
R) in relation to their functions. H
1
R–H
3
R
transduce extracellular signals via G
αq/11
, G
αis
, and G
αi/o
, respectively, while H
4
R acts through G
αi/o
and
β
-arrestin. H
1
R and H
2
R are low-affinity receptors while H
3
R and H
4
R are high-affinity receptors
towards histamine. Ligands of H
1
R–H
4
R have therapeutic applications in allergic inflammation, gastric
acid secretion, neurotransmission, and immunomodulation, respectively. The information in the figure
is partially based on [44].
3. Species Differences of H4R
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

Life 2020,10, 50 5 of 17
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 H4R 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].
Table 1. Sequence similarities of species specific H4R to the human orthologue.
Species
Scientific Name
UniProt ID
Similarity to hH
4
R
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%
Scheme 1.
Phylogenetic tree of H
4
R 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].

Life 2020,10, 50 6 of 17
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%

Life 2020,10, 50 7 of 17
Table 1. Cont.
Species Scientific Name UniProt ID Similarity to hH4R
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%
4. The Pharmacological Effects of H4R 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].
Life 2020, 10, 50 7 of 17
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%
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].
Figure 3. Potential role of histamine and histamine H4R-induced recruitment of eosinophils and mast
cells in chronic allergic inflammation. Histamine has been known to be a major mediator of
inflammation. Histamine H4 receptors are expressed on the surface of both eosinophils and mast cells.
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 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.
Figure 3.
Potential role of histamine and histamine H
4
R-induced recruitment of eosinophils and
mast cells in chronic allergic inflammation. Histamine has been known to be a major mediator of
inflammation. Histamine H
4
receptors are expressed on the surface of both eosinophils and mast cells.
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.

Life 2020,10, 50 8 of 17
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 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 H4R-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].
4.2. 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
–
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.
4.3. 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].
4.4. 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].
4.5. 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

Life 2020,10, 50 10 of 17
the pathogenesis of multiple sclerosis. It turned out that H
1
R and H
2
R 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, 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.

Life 2020,10, 50 11 of 17
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)
Life 2020, 10, 50 10 of 17
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) [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.
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]
RA 2 T NCT01862224 [92]
RA 2 T NCT01679951 [93]
Asthma 2 C NCT01823016 [94]
Psoriasis 2 C NCT02295865 [95]
JNJ39758979
Life 2020, 10, 50 11 of 17
ZPL3893787
(Adriforant/PF38937
87/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.
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.
Author Contributions: Conceptualization, P.M. (Pakhuri Mehta) and S.F.; writing—original draft preparation,
P.M. (Pakhuri Mehta); writing—review and editing, P.M. (Pakhuri Mehta), P.M. (Przemysław Miszta), P.R.,
O.M., P.K. and S.F.; visualization, P.M. (Pakhuri Mehta); supervision, S.F.; funding acquisition, S.F., P.R. and
O.M. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by NATIONAL SCIENCE CENTRE, POLAND, grant OPUS
2017/25/B/NZ7/02788.
RA 2 W NCT01480388 [99]
Asthma 2 W NCT01493882 [100]
ZPL3893787
(Adriforant/PF3893787/ZPL389)
Life 2020, 10, 50 11 of 17
ZPL3893787
(Adriforant/PF38937
87/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.
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.
Author Contributions: Conceptualization, P.M. (Pakhuri Mehta) and S.F.; writing—original draft preparation,
P.M. (Pakhuri Mehta); writing—review and editing, P.M. (Pakhuri Mehta), P.M. (Przemysław Miszta), P.R.,
O.M., P.K. and S.F.; visualization, P.M. (Pakhuri Mehta); supervision, S.F.; funding acquisition, S.F., P.R. and
O.M. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by NATIONAL SCIENCE CENTRE, POLAND, grant OPUS
2017/25/B/NZ7/02788.
AD 2 C NCT02424253 [102]
Psoriasis 2 C NCT02618616 [103]
AD 2 R NCT03517566 [104]
AD 2 R NCT03948334 [105]
SENS-111 (Seliforant)
Life 2020, 10, 50 11 of 17
ZPL3893787
(Adriforant/PF38937
87/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.
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.
Author Contributions: Conceptualization, P.M. (Pakhuri Mehta) and S.F.; writing—original draft preparation,
P.M. (Pakhuri Mehta); writing—review and editing, P.M. (Pakhuri Mehta), P.M. (Przemysław Miszta), P.R.,
O.M., P.K. and S.F.; visualization, P.M. (Pakhuri Mehta); supervision, S.F.; funding acquisition, S.F., P.R. and
O.M. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by NATIONAL SCIENCE CENTRE, POLAND, grant OPUS
2017/25/B/NZ7/02788.
Unilateral
Vestibulopathy 2 R NCT03110458 [106]
Status: T: terminated; C: completed; R: recruiting; W: withdrawn.
6. 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.
Author Contributions:
Conceptualization, P.M. (Pakhuri Mehta) and S.F.; writing—original draft preparation,
P.M. (Pakhuri Mehta); writing—review and editing, P.M. (Pakhuri Mehta), P.M. (Przemysław Miszta), P.R., O.M.,
P.K. and S.F.; visualization, P.M. (Pakhuri Mehta); supervision, S.F.; funding acquisition, S.F., P.R. and O.M.
All authors have read and agreed to the published version of the manuscript.

Life 2020,10, 50 12 of 17
Funding:
This research was funded by NATIONAL SCIENCE CENTRE, POLAND, grant OPUS 2017/25/
B/NZ7/02788.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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