Journal of Pharmacological and Toxicological Methods xxx (xxxx) xxx

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Journal of Pharmacological and Toxicological Methods

journal homepage: www.elsevier.com/locate/jpharmtox

Characteristics of histamine H4 receptor agonist-induced allergic

conjunctivitis model in Guinea pigs
Hidemi Mochizuki a, *, Susumu Suyama a, So-Young Youm b, Pil-Su Ho b, Akihito Shimoi a
a
Ina Research Inc., 2148-188 Nishiminowa, Ina, Nagano 399-4501, Japan
b
JW Pharmaceutical Corporation, 2477, Nambusunhwan-ro, Seocho-gu, Seoul, 137-864, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: Histamine is strongly associated with the onset of allergic conjunctivitis. The most recent cloned histamine H4
Dug development receptor antagonist is highly expected as a new therapeutic drug candidate. As a model for a therapeutic drug
guinea pig targeting the histamine H4 receptor, a mouse model in which conjunctivitis symptoms are induced by instilling 4-
Allergic conjunctivitis
methylhistamine, a histamine H4 receptor agonist, has been reported. However, the affinity of the H4 receptor for
Animal model
Histamine H4 receptor
histamine varies in species, and it is known that the histamine binding affinity for the guinea pig H4 receptor is
Non-clinical efficacy study closer to that for human receptor than mice receptor. In this paper, we investigated a possibility that a guinea pig
model would become a drug efficacy evaluation model with higher evaluation accuracy than the mouse model.
As a result, hyperemia was observed in the conjunctivae and iris of guinea pigs after instillation of 4-methyl­
histamine and specifically suppressed by the histamine H4 receptor antagonist. Unlikely to the previously re­
ported mouse model, however, none of edema, increased vascular permeability or scratching behavior was
observed, suggesting that there may be differences between mice and guinea pigs not only in the binding affinity
of histamine to the H4 receptor but also in the biological reaction to 4-methylhistamine. Although the symptoms
of allergic conjunctivitis do not appear comprehensively in the guinea pig model, results of this study indicated a
possibility that this model can be used as a simple screening model in the early stages of drug development.

1. Introduction
Histamine is strongly associated with the onset of allergic conjunc­
tivitis. Four types of histamine receptors, H1, H2, H3, and H4, have been
identified by now (Wade, Bielory, & Rudner, 2012), and it is known that
the H1 receptor is mainly involved in allergic conjunctivitis. Histamine
released from mast cells increases vascular dilation and permeability via
H1 receptors and stimulates nerve fibers and proinflammatory cytokine
production by immune cells. This causes symptoms of allergic
conjunctivitis such as itching, conjunctival edema and hyperemia
(Inada, Shoji, Shiraki, Aso, & Yamagami, 2017; Leonardi, 2000; Wade
et al., 2012; Weimer, Gamache, & Yanni, 1998). Accordingly, histamine
H1 receptor antagonists are mainly used as the first-line treatment of
allergic conjunctivitis. On the other hand, mast cells that release hista­
mine are usually not found in the normal epithelium of the eyeballs or
palpebral conjunctivae (Allansmith, Greiner, & Baird, 1978). The
symptoms of allergic conjunctivitis become deteriorated as mast cells
increase in number and infiltrate the epithelium (Morgan, Williams,
Walls, & Holgate, 1991), and the H4 receptor is considered to play an
important role in this exacerbation of inflammatory reaction. The H4
receptor is the most recent cloned receptor that is highly expressed in
immune tissues, such as the thoracic gland and spleen, and is also
expressed in cells from the immune system, such as T-cells, dendritic
cells, monocytes, mast cells, and neutrophils (Zampeli & Tiligada,
2009). Furthermore, the H4 receptor is known to mediate chemotaxis
with histamine as a chemoattractant, and it has been reported that mast
cell chemotaxis is actually mediated by the H4 receptor (de Esch,
Thurmond, Jongejan, & Leurs, 2005; Hofstra, Desai, Thurmond, & Fung-
Leung, 2003). Other similar studies have shown that H4 receptors
modulate the inflammatory cell function and cytokine production
(Gutzmer et al., 2005; Nakayama et al., 2004). Therefore, histamine H4
receptor antagonists are also highly expected as therapeutic drug can­
didates for allergic conjunctivitis.
In the development of therapeutic agents, it is common to evaluate
the drug efficacy using an animal model before administration to
humans. Since there are many candidate compounds in the early stages

* Corresponding author at: Ina Research Inc., 2148-188 Nishiminowa, Ina, Nagano 399-4501, Japan.
E-mail addresses: h-mochizuki@ina-research.co.jp (H. Mochizuki), s-suyama@ina-research.co.jp (S. Suyama), syyoum@jw-holdings.co.kr (S.-Y. Youm), psho@jw-
pharma.co.kr (P.-S. Ho), a-shimoi@ina-research.co.jp (A. Shimoi).

https://doi.org/10.1016/j.vascn.2022.107203
Received 20 January 2022; Received in revised form 5 July 2022; Accepted 11 July 2022
Available online 14 July 2022
1056-8719/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).

Please cite this article as: Hidemi Mochizuki, Journal of Pharmacological and Toxicological Methods, https://doi.org/10.1016/j.vascn.2022.107203
H. Mochizuki et al.
of evaluation, it is preferable to use a species that is easy to handle in the
simple experimental procedures to the extent possible when the effects
of each candidate compound are confirmed. Mice and rats are
commonly used in early efficacy studies, and a mouse model for the
treatment of allergic conjunctivitis targeting the histamine H4 receptor
has been reported (Nakano et al., 2009). On the other hand, a study
investigating the species differences in the histamine affinity for the H4
receptors showed that the H4 receptors in humans and guinea pigs had a
high binding affinity for [3H] histamine, whereas the binding affinity for
[3H] histamine was significantly lower in rat and mouse receptors (Liu,
Wilson, Kuei, & Lovenberg, 2001). In drug development, candidate
compounds are often dropped out due to species differences. Even if the
results of a non-clinical study are good, all the time and resources spent
up to that point will be wasted unless results of the non-clinical study are
reproduced in clinical trials. Therefore, it is necessary to select the
species used for drug efficacy evaluation carefully according to the
characteristics of the target. When the histamine H4 receptor is a target,
guinea pigs are considered to be more suitable species than mice in drug
efficacy evaluation since a binding affinity to histamine in guinea pigs is
closer to humans. In addition, the guinea pig model is considered to be
useful since it is widely used as an animal model for allergic conjunc­
tivitis and is easy to handle. However, there have been no studies on a
guinea pig model for the treatment of allergic conjunctivitis targeting
the histamine H4 receptor.
In this study, we investigated a guinea pig model when conjunctivitis
was induced by a histamine H4 receptor agonist.
2. Materials and methods
This study was conducted in compliance with the Japanese “Law for
the Humane Treatment and Management of Animals” and the “Guidance
for Animal Care and Use” of Ina Research Inc. and in accordance with
the protocol reviewed by the Institutional Animal Care and Use Com­
mittee (IACUC) of Ina Research Inc., a facility which is fully accredited
by AAALAC International.
2.1. Animals and housing conditions
Slc:Hartley guinea pigs (males, 6–10 weeks) were purchased from
Japan SLC, Inc. (Shizuoka, Japan). Up to 3 animals/cage were housed in
stainless steel wire mesh cages (61 W × 22D × 21H cm) with wood
blocks for gnawing as enrichment. The temperature and humidity in the
animal room were maintained between 21.0 ◦ C and 25.0 ◦ C and between
40.0% and 70.0%, respectively. Lighting was set on a 12-h light-dark
cycle (fluorescent lighting from 7:00 to 19:00). The animals were
allowed free access to the pelleted feed (LRC4, Oriental Yeast Co., Ltd.)
and drinking water via an automatic watering system.
2.2. Verification of induction conditions for conjunctivitis
4-Methylhistamine dihydrochloride [(5-(2-Aminoethyl)-4-methyl­
imidazole dihydrochloride)] (R&D Systems) was dissolved in
phosphate-buffered saline (hereafter referred to as “PBS”, pH = 7.4)
before use. 4-Methylhistamine (hereafter referred to as “4MeHA”) is
known as a selective histamine H4 receptor agonist (Lim et al., 2005). In
order to verify the optimal induction conditions for evaluation, the
initial 4MeHA concentration was set at 11 ng/μL with a dose volume of
25 μL/eye. For further verifications, 3 additional models were prepared
at 4MeHA concentrations of 110 ng/μL (25 μL/eye), 550 ng/μL (25 μL/
eye), and 81 mg/mL (12.5 μL/eye, 5000 nmol as 4MeHA). For each
concentration, the 4MeHA solution was instilled once/eye into the right
and left conjunctival sacs of each animal to induce conjunctivitis. The
condition of the conjunctivae and iris was observed macroscopically,
and detailed observations were conducted using a slit lamp to evaluate
the condition of the model. The optimum 4MeHA concentration and
dose level were determined based on the results of observations.
2
Journal of Pharmacological and Toxicological Methods xxx (xxxx) xxx
2.3. Verification of prepared animal model of conjunctivitis
Based on the results of the verification of induction conditions, the
optimum 4MeHA concentration and dose level were set at 81 mg/mL
(5000 nmol/12.5 μL). In order to determine whether the prepared
conjunctivitis model is suitable for use in evaluating the efficacy of a
histamine H4 receptor-targeted therapeutic drug for allergic conjuncti­
vitis, a drug efficacy evaluation test was conducted using a commercially
available reagent that shows H4 receptor antagonistic activity.
JNJ7777120 [(5-chloro-1H-indol-2-yl) (4-methyl-1-piperazinyl)-meth­
anone] (Cayman Chemical) was used as a test article for efficacy eval­
uation, and PBS was used as a control article. JNJ7777120 is an H4
receptor antagonist with high selectivity and binding affinity (Jablo­
nowski et al., 2003). Furthermore, in order to verify that the prepared
model is specific to the histamine H4 receptor, levocabastine hydro­
chloride (hereafter referred to as “levocabastine”, Janssen Pharmaceu­
tical K.K.), which is an H1 blocker eye drop and exhibits histamine H1
antagonistic activity (Noble & McTavish, 1995), was used as a
comparative control article.
The evaluation items commonly used in animal models of conjunc­
tivitis, i.e., scoring of allergic conjunctivitis, observations of scratching
behaviors, and measurements of conjunctival vascular permeability
(Kamei, Izushi, & Tasaka, 1991; Kato et al., 2004; Shii, Oda, Shinomiya,
Katsuta, & Nakamura, 2009), were selected to confirm the inhibitory
effect of JNJ7777120 on allergic conjunctivitis.
2.3.1. Dosing method
4MeHA and JNJ7777120, levocabastine or PBS were instilled into
the right and left conjunctival sacs of each animal. JNJ7777120, levo­
cabastine or PBS was instilled 15 min before 4MeHA instillation. The
lower eyelid was gently pulled away from the eye and the dosing
formulation was instilled into the conjunctival sacs using an autopipette
at 12.5 μL/eye for 4MeHA and at 25 μL/eye for JNJ7777120, levoca­
bastine or PBS. The lower eyelid was held for approximately 15 s after
instillation. The upper and lower eyelids were closed gently for a
moment and then slowly released.
2.3.2. Scoring of allergic conjunctivitis
In the verification experiment using the prepared model, the iris and
conjunctival parts of the Draize scale (Draize, 1959) and McDonald-
Shadduck scoring system (McDonald & Shadduck, 1977) were used
for the conjunctivitis score evaluation (Table 1 and 2), both are
commonly used in drug safety studies to assess the acute toxicity (irri­
tability) of the eyes (Daull, Raymond, Feraille, & Garrigue, 2018;
Kowalski, Romanowski, Yates, & Mah, 2016). The condition of the
conjunctivae and iris was observed macroscopically and scored ac­
cording to the Draize scale and McDonald-Shadduck scoring system
prior to (immediately after instillation of JNJ7777120, levocabastine or
PBS) and at 30, 60, and 90 min after instillation of 4MeHA. Detailed
observations were conducted using a slit lamp. Total evaluation scores of
the iris and conjunctivae were calculated and used as allergic conjunc­
tivitis scores for each eye.
2.3.3. Observations of scratching behaviors
Animals were transferred to observation cages at approximately 60
min prior to induction of allergic conjunctivitis for acclimation to the
observation environment. After dosing with 4MeHA outside the cages,
the animals were promptly returned to their cages and behaviors were
video-recorded for 30 min after induction. The number of eye scratching
behaviors with the hindlimbs was counted from the recorded images.
Continuous scratching behavior was counted as one scratch.
2.3.4. Measurements of conjunctival vascular permeability
Evans blue was dissolved in saline to yield a 1 w/v% Evans blue
solution (hereafter referred to as “Evans blue solution”). At 5 min after
dosing with JNJ7777120, levocabastine or PBS, Evans blue solution was
H. Mochizuki et al.
Table 1
Scoring standards for allergic conjunctivitis (excerption from Draize scoring
system).
Iris
(A) Appearance, values
Normal
Folds above normal, congestion, swelling, circumcorneal injection (any or all of
these or combination of any thereof), iris still reacting to light
(sluggishreaction is positive)
No reaction to light, hemorrhage, gross destruction (any or all of these)
Conjunctivae
(A) Redness (refers to palpebral and bulbar conjunctivae)
Vessels normal
Vessels definitely injected above normal
More diffuse, deeper crimson red, individual vessels not easily discernible
Diffuse beefy red
(B) Chemosis
No swelling
Any swelling above normal
Obvious swelling with partial eversion of lids
Swelling with lids about half closed
Swelling with lids about half closed to completely closed
(C) Discharge
No discharge
Any amount different from normal
Discharge with moistening of the lids and hairs just adjacent to lids
Discharge with moistening of the lids and hairs, and considerable area around
the eye
The iris and conjunctival parts of the Draize scoring system were used for the
conjunctivitis score evaluation. Total evaluation scores of the iris and conjunc­
tivae were calculated and used as allergic conjunctivitis scores for each eye.
administered intravenously at a volume of 0.25 mL/100 g of body
weight, and the 4MeHA solution was administered 10 min later. At 30
min after dosing with the histamine solution, the animals were anes­
thetized by inhalation of isoflurane, JP (Mylan Seiyaku Ltd.) and
euthanized by exsanguination from the posterior vena cava and
abdominal aorta to remove the conjunctival tissues from both eyes. Wet
weights of the removed conjunctival tissues were measured, and the
tissues were cut finely and immersed in a 1 N KOH solution (0.5 mL,
37 ◦ C) overnight (at least 18 h). An H3PO4-acetone solution (4.5 mL, 0.6
N H3PO4:acetone = 5:13) was added to the tissue solution and mixed
well. This extracted solution was centrifuged (approx. 1800 ×g, room
temperature, 20 min) to obtain the supernatant. Absorbance of the su­
pernatant was measured at 620 nm. Extracted dye amounts per 1 g of
conjunctival tissue were calculated based on the calibration curve
generated in advance.
2.4. Statistical analysis
Statistical analysis was performed as follows in the verification
experiment using the prepared model:
For the conjunctivitis scores, the mean group values and standard
errors were calculated from all scores in the left and right eyes at each
observation time point. Data were compared between the control and
JNJ7777120 or levocabastine groups using the Wilcoxon rank sum test.
The mean group values and standard errors were calculated for the
number of scratching behaviors and the amount of conjunctival vascular
permeability. Data were analyzed for homogeneity of variance between
the control and JNJ7777120 or levocabastine groups using the F-test
(5% level of significance). Student t-test was conducted for equality of
variances.
Differences from the control group was evaluated at the two-tailed
5% level of significance and presented as p < 0.05 or p < 0.01 in the
tables.
Journal of Pharmacological and Toxicological Methods xxx (xxxx) xxx
Table 2
Scoring standards for allergic conjunctivitis (excerption from McDonald-
Shadduck scoring system).
Conjunctival congestion
0 = Normal. May appear blanched to reddish pink without perilimbal injection
0 (except at 12:00 and 6:00 o'clock positions) with vessels of the palpebral and bulbar
1 conjunctiva easily observed.
+1 = A flushed, reddish color predominately confined to the palpebral conjunctiva
with some perilimbal injection but primarily confined to the lower and upper parts
2 of the eye from the 4:00 to 7:00 and 11:00 to 1:00 o'clock positions.
+2 = Bright red color of the palpebral conjunctiva with accompanying perilimbal
injection covering at least 75% of the circumference of the perilimbal region.
+3 = Dark, beefy red color with congestion of both the bulbar and the palpebral
conjunctiva along with pronounced perilimbal injection and the presence of
0
petechia on the conjunctiva. The petechia generally predominates along the
1
nictitating membrane.
2
Conjunctival swelling
3
0 = Normal or no swelling of the conjunctival tissue.
+1 = Swelling above normal without eversion of the lids (can be easily ascertained by
0
noting that the upper and lower eyelids are positioned as in the normal eye),
1
swelling generally starts in the lower cul-de-sac near the inner canthus.
2
+2 = Swelling with misalignment of the normal approximation of the lower and upper
3
eyelids; primarily confined to the upper eyelid so that in the initial stages the
4
misapproximation of the eyelids begins by partial eversion of the upper eyelid. In
this stage, swelling is confined generally to the upper eyelid, although it exists in the
0
lower cul-de-sac.
1
+3 = Definite swelling with partial eversion of the upper and lower eyelids essentially
2
equivalent. This can be easily ascertained by looking at the animal head-on and
3
noticing the positioning of the eyelids; if the eye margins do not meet, eversion has
occurred.
+4 = Eversion of the upper eyelid is pronounced with less pronounced eversion of the
lower eyelid. It is difficult to retract the lids and observe the perilimbal region.
Conjunctival discharge
0 = Normal. No discharge.
+1 = Discharge above normal and present on the inner portion of the eye but not on
the lids or hairs of the eyelids. One can ignore the small amount that is in the inner
and outer canthus.
+2 = Discharge is abundant, easily observed, and has collected on the lids and around
the hairs of the eyelids.
+3 = Discharge has been flowing over the eyelids so as to wet the hairs substantially
on the skin around the eye.
Iris
0 = Normal iris without any hyperemia of the iris vessels. Occasionally around the
12:00 to 1:00 o'clock position near the pupillary border and the 6:00 to 7:00 o'clock
position near the pupillary border there is a small area around 1 mm to 3 mm in
diameter in which both the secondary and tertiary vessels are slightly hyperemic.
+1 = Minimal injection of secondary vessels but not tertiary. Generally, it is uniform,
but may be of greater intensity at the 1:00 or 6:00 o'clock position. If it is confined to
the 1:00 or 6:00 o'clock position, the tertiary vessels must be substantially
hyperemic.
+2 = Minimal injection of tertiary vessels and minimal to moderate injection of the
secondary vessels.
+3 = Moderate injection of the secondary and tertiary vessels with slight swelling of
the iris stroma (this gives the iris surface a slightly rugose appearance, which is
usually most prominent near the 3:00 and 9:00 o'clock positions).
+4 = Marked injection of the secondary and tertiary vessels with marked swelling of
the iris stroma. The iris appears rugose; may be accompanied by hemorrhage
(hyperemia) in the anterior chamber.
The iris and conjunctival parts of the McDonald-Shadduck scoring system were
used for the conjunctivitis score evaluation. Total evaluation scores of the iris
and conjunctivae were calculated and used as allergic conjunctivitis scores for
each eye.
3. Results
3.1. Optimal conjunctivitis induction conditions
As a result of instillation of 4MeHA at 11 ng/μL with a dose volume of
25 μL/eye, the score was 0 at all observation points and no change was
observed. When the 4MeHA concentration was increased, no change
was observed at 110 ng/μL (25 μL/eye), and only slight hyperemia was
observed in the bulbar conjunctiva at 550 ng/μL (25 μL/eye). As a result
of further investigation by increasing the concentration of 4MeHA, no
edema was observed, but strong hyperemia of the conjunctivae and iris
was observed at 81 mg/mL (5000 nmol/12.5 μL).
3
H. Mochizuki et al.
3.2. Verification of the prepared conjunctivitis model
3.2.1. Scoring of allergic conjunctivitis
Redness/congestion of the conjunctivae or iris was observed in all
animals in the control group after instillation of 4MeHA, indicating that
allergic conjunctivitis was induced by 4MeHA.
The mean scores of allergic conjunctivitis according to the Draize
scale prior to (immediately after instillation of the dosing substances)
and at 30, 60 and 90 min after instillation of 4MeHA were 0.0, 2.0, 2.0
and 2.0 in the control group, 0.0, 2.0, 1.2 and 1.2 in the JNJ7777120
group and 0.0, 2.0, 2.0 and 2.0 in the levocabastine group, respectively
(Fig. 1). The allergic conjunctivitis scores according to the McDonald-
Shadduck scoring system at these time points were 0.0, 4.0, 3.6 and
3.6 in the control group, 0.0, 2.0, 1.2 and 1.2 in the JNJ7777120 group,
and 0.0, 4.0, 3.5 and 3.0 in the levocabastine group, respectively
(Fig. 2). In comparison between the 2 criteria, Draize scale and
McDonald-Shadduck scoring system, the scores tended to be higher in
the McDonald-Shadduck scoring system.
In the JNJ7777120 group, after instillation of 4MeHA, the mean
scores were statistically significantly lower than those in the control
group after 60 min on the basis of Draize scale and after 30 min on the
basis of McDonald-Shadduck scoring system, and JNJ7777120 showed
inhibitory effects on 4MeHA-induced allergic conjunctivitis (Fig. 3).
No appreciable differences were noted between the levocabastine
and control groups, indicating no inhibitory effects of levocabastine, a
histamine H1 receptor antagonist, on allergic conjunctivitis inducted by
4MeHA, a histamine H4 receptor agonist.
3.2.2. Observations of scratching behaviors
The mean group values for the number of eye scratching behavior
were 3.8 in the control group, 3.0 in the JNJ7777120 group and 2.3 in
the levocabastine group (Table 3). Neither JNJ7777120 nor the levo­
cabastine group showed a significant difference from the control group.
5 JNJ7777120
3
Score
1
0
0 30
Pre
Time (minutes) after induction
Fig. 1. Scoring of allergic conjunctivitis (Draize scoring system).
The condition of the conjunctivae and iris was observed macroscopically and scored according to the Draize scoring system prior to and at 30, 60, and 90 min after
instillation of histamine.
Each value represents Mean ± S.E. (N = 10 or 8) &: P < 0.05, significant difference between the control and JNJ7777120 groups (Wilcoxon's rank-sum test).
4
Journal of Pharmacological and Toxicological Methods xxx (xxxx) xxx
3.2.3. Measurements of conjunctival vascular permeability
The mean group values for extracted dye amounts in the conjunctival
tissue were 22.3 and 21.5 μg/g in the control and JNJ7777120 groups,
respectively (Table 4, levocabastine group was not included). There was
no significant difference between the control group and the
JNJ77777120 group.
4. Discussion and conclusion
Similar to the previously reported mouse model, we examined
whether instillation of 4MeHA, a selective histamine H4 receptor
agonist, could induce allergic conjunctivitis-like symptoms in animals.
As a result, no change was observed under the initial condition of 11 ng/
μL (25 μL/eye). Further investigation by increasing the concentration of
4 MeHA showed no change at 110 ng/μL (25 μL/eye), which was 10
times higher, or at 550 ng/μL (25 μL/eye), which was 55 times higher
than the initial concentration. A slight redness was observed in the
conjunctivae of the eyeballs. When the concentrations of 4MeHA were
further increased, strong hyperemia to the conjunctivae and iris was
confirmed at 81000 ng/μL (5000 nmol/12.5 μL), which was about 7400
times the initial concentration.
However, edema, which has been observed in previously reported
mouse models, was not observed, nor were there any signs of edema. In
the guinea pig model we reported previously, changes were observed in
the conjunctivae but not in the iris when conjunctivitis symptoms were
induced by instillation of histamine (Mochizuki, Suyama, Cha, Ho, &
Shimoi, 2022). In addition, there were no signs of edema even at the
concentrations of 4MeHA higher than those in the previous study with
the mouse model. These results showed that the 4MeHA-induced guinea
pig model is characterized by strong hyperemia in the conjunctivae as
well as the iris. Since there is no study where 4MeHA was instilled in
animals other than mice to observe/compare its effect in detail, the
details of the mechanism of reaction differences between species after
4MeHA instillation are unknown. However, the results suggested that
there may be differences between mice and guinea pigs not only in the
Phosphate-Buffered Saline
Levocabastine
& &
60 90
H. Mochizuki et al. Journal of Pharmacological and Toxicological Methods xxx (xxxx) xxx

Phosphate-Buffered Saline

5 JNJ7777120

Levocabastine

3
Score

&&
2

&& &&
1

0
0 30 60 90
Pre
Time (minutes) after induction

Fig. 2. Scoring of allergic conjunctivitis (McDonald-Shadduck scoring system).

The condition of the conjunctivae and iris was observed macroscopically and scored according to the McDonald-Shadduck scoring system prior to and at 30, 60, and
90 min after instillation of histamine.
Each value represents Mean ± S.E. (N = 10 or 8) &&: P < 0.01, significant difference between the control and JNJ7777120 groups (Wilcoxon's rank-sum test).

30 min 60 min 90 min

PBS

JNJ7777120

Fig. 3. Conjunctivitis-like symptoms in guinea pigs after 4MeHA instillation.

Conjunctival and iris hyperemia in the control group vs the JNJ7777120 group at 30, 60, and 90 min after instillation of 4MeHA.

Table 3 Table 4
Observations of scratching behavior. Measurements of conjunctival vascular permeability.
Group Number of eye scratching behavior (counts) Group Extracted dye amounts (μg/g conjunctival tissues)

Phosphate-Buffered Saline 3.8 ± 1.4 (5) Phosphate-Buffered Saline 22.3 ± 4.1 (3)
JNJ7777120 3.0 ± 0.8 (5) JNJ7777120 21.5 ± 1.8 (3)
Levocabastine 2.3 ± 1.1 (4)
The conjunctival tissues were removed 30 min after instillation of 4MeHA, and
After instillation of 4MeHA, animal behaviors were video-recorded for 30 min the Evans blue pigment was extracted. The absorbance of the extracted solution
after instillation. The number of eye scratching behaviors with the hindlimbs was measured, and the amount of dye per 1 g of conjunctival tissue was calcu­
was counted from the recorded images. Continuous scratching behavior was lated.
counted as one scratch. Each value represents Mean ± S.E. (N = 3).
Each value represents Mean ± S.E. (N = 5 or 4).
receptor antagonism, and levocabastine, which exhibits histamine H1
histamine binding affinity of the H4 receptor but also in the biological antagonism, in guinea pigs for a model created under the conditions of a
response to 4MeHA. 4MeHA concentration of 5000 nmol and an instillation volume of 12.5
As a second step, we used JNJ7777120, which exhibits histamine H4 μL/eye. We examined whether the changes are histamine H4 receptor-

5
H. Mochizuki et al.
specific, and the efficacy of drugs targeting the histamine H4 receptor
can be appropriately evaluated. As the indicators for drug efficacy
evaluation, a commonly used scoring system for allergic conjunctivitis,
observations of scratching behaviors and measurements of conjunctival
vascular permeability, were selected. After instillation of 4MeHA, hy­
peremia was observed in the conjunctivae and iris, and both symptoms
were confirmed to be suppressed by JNJ7777120. Since levocabastine
that exhibits histamine H1 antagonistic activity did not show an inhib­
itory effect, the hyperemia observed after instillation of 4MeHA was
considered to be a histamine H4 receptor-specific change. However, in
the observation of scratching behaviors and the measurement of
conjunctival vascular permeability, there were no differences between
any treated group and the control group, and the drug efficacy could not
be confirmed. Since edema did not occur in guinea pigs, it was consid­
ered difficult to measure the conjunctival vascular permeability since
vascular permeability did not increase and the amount of dye leakage
into the conjunctival tissue was not sufficient for evaluation. For the
observation of scratching behaviors, the scratching behavior was clearly
increased after instillation of 4MeHA in the mouse model, whereas the
values in the control group were clearly lower in guinea pigs than those
in the previously reported animals with conjunctivitis-like symptoms
(Kato et al., 2004; Mochizuki et al., 2022). These results indicated that
itching leading to scratching behavior was not induced only by instil­
lation of 4MeHA, or that there were no physical irritation leading to
scratching behaviors since swelling of the conjunctiva did not occur due
to the absence of edema.
As described above, although we expected that the same reactions as
those in the previously reported mouse model would be obtained in
guinea pigs, only conjunctival and iris hyperemia was observed in
guinea pigs. The indices generally used in the evaluation of the efficacy
of allergic conjunctivitis could not be evaluated comprehensively.
However, the conjunctival and iris hyperemia observed this guinea pig
model was specifically suppressed by histamine H4 receptor antagonist,
and the changes were evaluable by using the conjunctivitis scoring
systems. Although edema does not occur as in the mouse model, it was
confirmed that it is possible to evaluate drugs with the H4 receptor
antagonistic activity by evaluating hyperemia with a conjunctivitis
scoring system. Although the symptoms of allergic conjunctivitis do not
appear comprehensively in the guinea pig model, results of this study
indicated possibility that this model can be used as a simple screening
model in the early stages of drug development in multiple candidate
compounds targeting the histamine H4 receptor. In the evaluation of
conjunctivitis using the two criteria of Draize scale and McDonald-
Shadduck scoring system, both are commonly used in drug develop­
ment, the scores tended to be higher according to the McDonald-
Shadduck scoring system. This discrepancy was considered to be
caused by the total number of severity grades (3 in Draize scale vs. 5 in
McDonald-Shadduck system), and the iris was evaluated more precisely
by the McDonald-Shadduck system than the Draize scale. Since the main
symptom is changes in the iris, changes are considered to be captured
appropriately by using the McDonald-Shadduck system in the 4MeHA-
induced model of guinea pigs.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
6
Journal of Pharmacological and Toxicological Methods xxx (xxxx) xxx
Data availability
No data was used for the research described in the article.
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