Kim et al. BMC Ophthalmology          (2019) 19:217 
https://doi.org/10.1186/s12886-019-1228-5
RESEARCH ARTICLE Open Access
Comparison of cytotoxicities and anti-
allergic effects of topical ocular dual-action
anti-allergic agents
Sung Il Kim1,2, Choul Yong Park3, Gladys Fordjuor4, Jong Heon Lee5, Jong Soo Lee1 and Ji Eun Lee1,2*
Abstract
Background: To investigate the cytotoxicities of the topical ocular dual-action anti-allergic agents (alcaftadine
0.25%, bepotastine besilate 1.5%, and olopatadine HCL 0.1%) on human corneal epithelial cells (HCECs) and their
anti-allergic effects on cultured conjunctival epithelial cells.
Methods: A Methylthiazolyltetrazolium(MTT)-based calorimetric assay was used to assess cytotoxicities using HCECs
at concentrations of 10, 20 or 30% for exposure durations of 30 min, 1 h, 2 h, 12 h or 24 h. Cellular morphologies
were evaluated by inverted phase-contrast and electron microscopy. Wound widths were measured 2 h, 18 h, or 24
h after confluent HCECs monolayers were scratched. Realtime PCR was used to quantify anti-allergic effects on
cultured human conjunctival cells, in which allergic reactions were induced by treating them with Aspergillus
antigen.
Results: Cell viabilities decreased in time- and concentration-dependent manners. Cells were detached from dishes
and showed microvilli loss, cytoplasmic vacuoles, and nuclear condensation when exposed to antiallergic agents;
alcaftadine was found to be least cytotoxic. Alcaftadine treated HCECs monolayers showed the best wound healing
followed by bepotastine and olopatadine (p < 0.0001). All agents significantly reduced the gene expressions of
allergic cytokines (IL-5, IL-25, eotaxin, thymus and activation-regulated chemokine, and thymic stromal
lymphopoietin) and alcaftadine had the greatest effect (p < 0.0001 in all cases).
Conclusions: Alcaftadine seems to have less side effects and better therapeutic effects than the other two anti-
allergic agents tested. It may be more beneficial to use less toxic agents for patients with ocular surface risk factors
or presumed symptoms of toxicity.
Keywords: Allergic conjunctivitis, Cytotoxicity, Topical anti-allergic agents
Background disease, its chronic, recurrent tendencies influence the
The prevalence rates of allergic diseases have been in- quality of patient’s life considerably [5].
creasing due to hereditary factors, environmental pollu- The fundamental treatment for AC is to avoid aller-
tion, increased allergen levels, and changes in life gens that cause hypersensitive reactions as other allergic
patterns including dietary [1, 2]. Approximately 6–30% diseases. However, it is difficult not only to identify the
of individuals are suffering from allergic conjunctivitis causative allergens accurately but also to avoid a known
(AC) and 30~70% of them accompany other allergic dis- allergen completely if they are easily encountered in
eases [3, 4]. Even though AC is not a life-threatening daily life. For these reasons, pharmacotherapy has been
used to provide symptom relief and treatment in AC.
* Correspondence: jiel75@hanmail.net The clinical manifestations of AC such as itching,
1Department of Ophthalmology, School of Medicine, Pusan National hyperemia, chemosis, and eyelid swelling are the result
University, Mulgumup, Yangsan 50612, Gyeongnam Province, Republic of of mast cell degranulation and the release of inflamma-
South Korea
2Research Institute for Convergence of Biomedical Science and Technology, tory chemical mediators (especially histamine), which
Pusan National University Yangsan Hospital, Yangsan, South Korea are initiated by crosslinking between permeated allergen
Full list of author information is available at the end of the article
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Kim et al. BMC Ophthalmology          (2019) 19:217 Page 2 of 9
and sensitized IgE on mast cell surface [6]. Therefore, were subconfluent. Alcaftadine, bepotastine, or olopata-
these pathologic immune reactions have been considered dine (100 μl diluted 10, 20%, or 30%) were added and in-
as main targets for pharmacotherapy, and can be con- cubated for 0.5, 1, 2, 12 or 24 h. DMEM (100 μl) was
trolled by antihistamine agents and mast cell stabilizers. added to controls. After drug exposure, plates were
Olopatadine was the first approved dual-action topical washed three times with PBS to remove the drugs. Cell
agent and other two dual-action agents have been devel- viabilities were evaluated after incubating for 24 h, and
oped and become general trend to treat AC. Although MTT was then added to each well. Samples were incu-
dual-action agents reduce dosage and frequency due to bated in the dark for 4 h at 37 °C, and media were then
its rapid onset and long lasting therapeutic effect, long removed. The formazan reaction product was dissolved
period of use can damage ocular surface cells [7–9]. An by adding 150 μl dimethyl sulfoxide (Sigma, St. Louis,
impaired epithelial barrier may allow allergens to infil- MO, USA), and absorbances were measured on an auto-
trate easily and exacerbate the disease. Therefore, reli- matic plate reader (Molecular Devices, Sunnyvale, CA,
able safety as well as therapeutic effects are required for USA) at 570 nm. The experiment was repeated 5 times.
anti-allergic agents.
We chose three topical ocular dual-action anti-allergic Morphologic assay
agents for this study; alcaftadine 0.25% (Lastacaft®, Aller- HCECs were exposed to three anti-allergic agents at a
gan, Inc., Irvine, CA, USA) and bepotastine besilate 1.5% concentration of 10% for 24 h and photographed under
(Talion®, Dong-A ST, Seoul, Korea), which were intro- an inverted phase-contrast light microscope. For trans-
duced recently, and olopatadine HCL 0.1% (Pataday®, mission electron microscopy, cells that had been grown
Alcon, Fribourg, Switzerland), a traditionally and widely to confluence in 24-well plates were incubated in
used agent. The aim of this study was to investigate the DMEM containing 10% concentrations of the three anti-
cytotoxicities of these agents on cultured human corneal allergic agents or phosphate buffer (control) for 4 or 8 h
epithelial cells and their anti-allergic effects on cultured under 5% CO2 at 37 °C. After rinsing with PBS, cells
human conjunctival epithelial cells in vitro. were incubated at 37 °C for 24 h, fixed with 2.5% glutar-
aldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 12 h
Methods and postfixed with 0.1% osmium tetroxide for 2 h. After
Cell lines rinsing with 0.1 mol/L of a phosphate buffer and dehy-
This study was performed according to the tenets of the drating in a graded ethanol series, specimens were em-
Declaration of Helsinki. The SV-40-transfected human bedded in an Epon 812 mixture. Ultrathin sections
corneal epithelial cell line (HCE-T) was obtained from (60~80 nm) were then stained with uranyl acetate and
the American Type Culture Collection (ATCC-CRL- lead citrate, and examined under a transmission electron
11515; Manassas, VA, USA), and was grown to 80% con- microscope (JEOL1200EX: Jeol Ltd., Tokyo, Japan.)
fluency in keratinocyte serum-free medium (KSFM) con-
taining 0.05 mg/ml bovine pituitary extract and 5 ng/mL Scratch wound healing assay
epidermal growth factor in collagen-coated plates. Before A scratch-wound assay was used to compare the effects
treatment, the cells underwent epidermal growth factor of alcaftadine, bepotastine and olopatadine on corneal
starvation overnight, as previously described [10]. epithelial wound healing. HCECs were cultured to con-
The Wong Kilbourne derivative of the Chang conjunc- fluent monolayers on eight well chamber slides coated
tival epithelial cell line (WKD; clone 1–5c-4, ATCC, Ma- with collagen I (10 mg/cm2; Auspep, Parkville, VIC,
nassas, VA) was cultured under standard conditions Australia) and then scratched with a 100 μl pipette tip.
(moist atmosphere, 5% CO2, 37 °C) in Dulbecco’s mini- Cells were then washed with fresh medium to remove
mum essential medium (DMEM, 340 ± 20mOsM) sup- detached cells and incubated in medium in the presence
plemented with 10% fetal bovine serum (FBS), 1% 10% concentrations of the three anti-allergic agents for
glutamine (200 mM stock solution), 1% penicillin (10, 2, 18, or 24 h. To ensure that wounds in similar areas
000 units/ml), and 1% streptomycin (10,000μg/ml) for were compared, multiple positioning marks were made
24 h to reach confluence before challenges [11]. at the center of denuded surfaces with a needle, and
mean distances between wound edges were measured.
Methylthiazolyltetrazolium (MTT) assay Twenty-four hours after wounding, monolayers were
The viabilities of human corneal epithelial cells (HCECs) fixed, and wound areas in marked fields of view were
were measured using a MTT assay (3-[4,5-dimethyl- imaged. Mean distances between original and migrated
thiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Sigma, wound edges of three separate samples per treatment
St. Louis, MO, USA). Cultured cells (100 μl; 5 × 104 cell/ were determined using an image analysis system (Image
ml) were seeded in 96-well tissue-culture plates and in- J 1.33o;available by ftp at zippy.nimh.nih.gov/ or at
cubated at 37 °C in 5% CO2 for 24 to 48 h until cultures http://rsb.info.nih.gov/nih-imageJ; developed by Wayne
Kim et al. BMC Ophthalmology          (2019) 19:217 Page 3 of 9
Rasband, National Institute of Health, Bethesda, MD, Statistical analysis
USA), and percentage wound closures in response to the Statistical analysis significance was determined by
three anti-allergic agents were compared. The experi- ANOVA followed by Tukey’s post hoc analysis (Prism;
ment was repeated 5 times. GraphPad Software, La Jolla, CA, USA). Statistical sig-
nificance was accepted for p values < 0.05.
Analysis of electrolyte compositions, pH values, and
osmolarities of the eye solutions
The electrolyte compositions of the three anti- Results
allergic agents were assessed using a LX-20 (Beck- HCECs viabilities after exposure to the three anti-
man Coulter, Fullerton, CA, USA). pH and osmolar- allergic agents at different dilutions and exposure
ity were measured using a Metrohm 780 (Metrohm, times are shown in Fig. 1. At a concentration of 30%,
Zofingen, Switzerland) and a Micro-Sample Osmom- viability decline in alcaftadine was the smallest com-
eter (Fiske Associate, Norwood, MA, USA), pared with the control and alcaftdine showed signifi-
respectively. cantly higher viability than bepotastine or olopatadine
at exposure times up to 2 h. At a concentration of
Conjunctival provocation test (CPT) 20%, viabilities in bepotastine and olopatadine were
Cultured conjunctival epithelial cells were seeded significantly lower than in the control after 30 min,
with or without 10% concentrations of anti-allergic whereas alcaftadine had no significant effect at expos-
agents and incubated 2 h at 37 °C under 5% CO2. ure times up to 2 h. At exposure times of ≥12 h, the
Conjunctival cells were subsequently treated with or comparisons between agents were meaningless be-
without 1 mg/ml Aspergillus fumigatus allergen ex- cause viabilities were extremely low.
tract (Jubilant Hollister-Stier, Kirkland, Quebec, Phase-contrast microscopy revealed many epithelial
Canada) for 1 h. Cells were then collected, lysed, and cells were densely arrayed in control culture media
treated with Tri-RNA reagent (Favorgen, Taiwan) to (Fig. 2a), but in the presence of either of the three agents
extract mRNA, according to the manufacturer’s in- HCECs progressively detached from dishes (Fig. 2b-d),
structions. Total extracted RNA was used to gener- although alcaftadine exposed cells were less detached
ate cDNA using oligo-dT, dNTP, RNasin® and more densely arrayed than cells exposed to bepotas-
ribonuclease Inhibitor, and M-MLV reverse Tran- tine or olopatadine (Fig. 2b).
scriptase (Promega, Madison, Wisconsin, USA). To Electron microscopy showed that HCECs exposed
quantify cytokine gene expression, cDNA samples to alcaftadine, bepotastine, or olopatadine demon-
were amplified in AMPOGENE® qPCR Green Mix strated more cytoplasmic bleb formation and loss of
Lo-ROX (Enzo Life Sciences, Farmingdale, NY, microvilli (Fig. 3b-d) than control cells (Fig. 3a).
USA). The primer pairs used for RT-PCR are shown Whereas cells exposed to alcaftadine showed minimal
in Table 1. The experiment was repeated 5 times. changes, cells exposed to bepotastine or olopatadine
showed more and larger cytoplasmic vacuoles and nu-
clear chromatin condensation along nuclear peripher-
Table 1 List of PCR primer sequences ies (Fig. 3c, d).
Cytokines Primer sequence (5′-3′) Eighteen and 24 h after scratching HCECs monolayers,
GAPDH F: AAT CCC ATC ACC ATC TTC CA alcaftadine exposed HCECs exhibited significantly better
R: TGG ACT CCA CGA CGT ACT CA wound healing than bepotastine or olopatadine exposed
Eotaxin F: TCT GTG GTC ATC CCC TCT CC cells (p < 0.0001). Olopatadine treated wounds showed
R: TTG GCG TCC AGG TTC TTC AT almost no change (Fig. 4).
The measured values of electrolytes, pH, and osmo-
IL-5 F: TAC GTG TAT GCC ATC CCC AC
larity, and preservative are shown in Table 2. The
R: CCC CCT TGC ACA GTT TGA CT concentrations of Na+ and Cl− in bepotastine were
IL-25 F: GCT GCT CTA CCA CAA CCA GA lower and in olopatadine were higher than ideal
R: GTG GTT GTA CAC CTG GCT CC ranges, whereas the concentration of K+ in all three
TSLP F: TGG GTG TCC ACG TAT GTT CC agents was lower than the ideal range; bepotastine
R: ACT CGG TAC TTT TGG TCC CAC had the lowest value. Alcaftadine had the highest Cl
−
level and was more acidic than the other agents. The
TARC F: TGT TCG GAC CCC AAC AAC AA
osmolarities of all agents were within normal limits.
R: TCA CTG TGG CTC TTC TTC GTC Benzalkonium chloride (BAC) was the preservative
F Forward, GAPDH Glyceraldehyde-3-phosphate dehydrogenase, IL interleukin, used in all agents and its concentration in olopatadine
R Reverse, TARC thymus and activation-regulated chemokine, TSLP thymic
stromal lymphopoietin was twice as high as in alcaftadine or bepotastine.
Kim et al. BMC Ophthalmology          (2019) 19:217 Page 4 of 9
Fig. 1 The viabilities of human corneal epithelial cells (HCECs) evaluated by MTT assay. Cell viability was found to be time and concentration
dependent and to be significantly reduced after 12 h exposure to all antiallergic agents. At a concentration of 30%, bepotastine and olopatadine
treated HCECs were significantly less viable than alcaftadine treated HCECs at exposure times up to 2 h. Survival rates are provided as means ± SDs
All three anti-allergic agents significantly reduced the Discussion
gene expressions of allergic cytokines induced by Asper- Dual-action anti-allergic agents are widely and com-
gillus allergen provocation, except eotaxin induction by monly used for AC which is not severe as demanding
olopatadine in conjunctival cells. Alcaftadine had the steroid or immune modulators. The aim of the present
greatest effect on the reduction of all cytokine gene ex- study was to compare the cytotoxicities and anti-allergic
pressions examined (Fig. 5). effects of the commercially available topical dual-action
Fig. 2 Inverted phase contrast micrographs of human corneal epithelial cells (HCECs) exposed to 10% antiallergic agents (bar length 50 μm,
original magnification × 200). Many epithelial cells were visible in control culture media (a). HCECs were less detached from dishes after treatment
with alcaftadine (b) than after treatment with bepotastine (c) or olopatadine (d)
Kim et al. BMC Ophthalmology          (2019) 19:217 Page 5 of 9
Fig. 3 Transmission electron micrograph of human corneal epithelial cells (HCEC) (bar length 2 μm, original magnification × 3000–4000). HCECs
were exposed to culture media (a), and 10% diluted solutions of antiallergic agents; alcaftadine (b), bepotastine (c), and olopatadine (d). Normal
corneal epithelial cells (a) showed microvilli, homogenous cytoplasm and intact cells and nuclear membranes. Antiallergic agents exposed cells
(b, c and d) exhibited damage to plasma membranes, loss of microvilli (black arrowheads), increased and enlarged vacuoles (white arrows), and
nuclear chromatin condensation (white arrowheads). Olopatadine treated cells showed more and larger vacuoles and condensed nuclear
remnants along nuclear peripheries
anti-allergic agents; alcaftadine 0.25%, bepotastine besi- damage caused by chemical, mechanical or hypoxic in-
late 1.5%, and olopatadine HCL 0.1%. jury [18]. Alcaftadine exposure resulted in less severe
MTT assay revealed that cell viabilities decreased with cellular changes than bepotastine or olopatadine.
exposure time and agent concentration. Bepotastine and Abnormal electrolyte composition, pH, and osmolarity
olopatadine induced significantly lower viabilities than can damage cellular functions [19]. In the present study,
the control even after 30 min at concentrations of 20 most measured electrolyte values were beyond ideal
and 30%, whereas alcaftadine induced significantly lower ranges. Abnormal electrolyte composition not only aug-
viabilities after 2 h at a concentration of 20% and 1 h at a ments agent toxicity, due to changes in cell membrane
concentration of 30%. In addition, cell viability in the permeabilities, but can also cause other types of cell
presence of alcaftadine was significantly higher than in damage. On the other hand, osmolarity and pH of all
the presence of bepostatine or olopatadine at a concen- agents may not affect the toxicity because they were
tration of 30%. However, after treatment for more than similar and in normal ranges. Preservatives are necessary
12 h including 24, 48 and 72 h (data not shown for 48 to prevent ocular infection by prohibiting microorgan-
and 72 h), all agents proved toxic to HCECs, which ism proliferation, but they can damage the ocular surface
agrees with other studies [15, 16]. [20–22]. All three agents examined contained BAC, the
A comparative study of olopatadine and alcaftadine on most commonly used ophthalmic preservative. BAC
murine conjunctival epithelial cells concluded that alcaf- causes surface-active molecules to bind to cellular epi-
tadine had a protective effect on epithelial tight junction thelium and rapidly intercalate into the bilaminar mem-
protein expression [17]. This property could explain why brane, and thus, BAC can disrupt the precorneal tear
in our study alcaftadine treated cells detached to a lesser film and damage the ocular surface [16, 23–25]. BAC
extent than bepotastine or olopatadine treated cells. concentrations in topical ocular solutions typically range
Cytoplasmic blebbing, chromatin clumping and margin- between 0.004 and 0.025% [20]. Recently, it was reported
ation, and loss of microvilli are the evidences of cellular that even at the lowest concentration tested, 0.001%,
Kim et al. BMC Ophthalmology          (2019) 19:217 Page 6 of 9
Fig. 4 The closure of human corneal epithelial cells (HCECs) wounds in response to 10% antiallergic agents (bar length 200 μm, original
magnification × 4). Migration was assessed after 2, 18 or 24 h after scratching confluent HCECs in the presence or absence of antiallergic agents.
Micrographs show wound widths immediately after and 24 h after wounding in the absence of any agent (a, e) or in the presence of 10%
alcaftadine (b, f), bepotastine (c, g) or olopatadine (d, h). The effects of antiallergic agents are expressed as percentage reductions in average
wound widths. Results are expressed as means ± SDs of percentage wound widths (defined as average widths at 10 positions). Wound widths
were significantly narrower for cells exposed to alcaftadine than for cells exposed to bepotastine or olopatadine
BAC caused significant loss of cellular metabolic activity drugs used to treat other chronic ocular pathologies, like
at exposure times as short as 1 min [26]. Although we glaucoma, have been reported to be more cytotoxic than
found all three agents had BAC levels in the recom- preservative free preparations [24, 25].
mended concentration range, olopatadine had twice as Damage, abrasions, or wounding of epithelial cells are
much BAC than alcaftadine or bepotastine. This higher caused by various insults including ophthalmic agents
level of BAC could partly explain the lower viability of which can impair healing [27]. Healing involves a series
olopatadine treated HCECs. These results are consistent of events that includes the proliferation and migration of
with those of previous studies, in which cell viabilities cells to seal wounds [28, 29]. In the present study, alcaf-
were found to be affected more by anti-allergic drugs tadine treated cell layers showed the best wound healing
containing BAC [16, 21, 23]. Similarly, BAC-containing followed by bepotastine treated cell layers. Olopatadine
Table 2 Electrolyte compositions, pH values, osmolalities, and preservative contents of the three anti-allergic agents
Parameters Anti-allergic agents Ideal range
Alcaftadine 0.25% Bepostatine besilate 1.5% Olopatadine HCL 0.1% [12–14]
Na + (mEq/L) 151.7 135.9 177 142.0–152.7
K+ (mEq/L) 0.93 0.75 1.46 4.3–4.6
Cl− (mEq/L) 145.0 99.3 118.1 104.0–117.4
Osmolarity (mOsm/kg) 292 275–350 296 260–320
Preservative (BAC, %) 0.005 0.005 0.01 < 0.025
pH 7.2 6.5–7.1 7.3 7.0–7.7
BAC benzalkonium chloride
Kim et al. BMC Ophthalmology          (2019) 19:217 Page 7 of 9
Fig. 5 Quantitative real time PCR analyses of the mRNA expression of allergic cytokines in conjunctival cells. Levels of interleukin(IL)-5 (a), IL-25 (b),
eotaxin (c), TARC (thymus and activation-regulated chemokine) (d), and TSLP (thymic stromal lymphopoietin) (e) are shown. Expression levels of all
5 cytokines were lowest for alcaftadine. Results are presented as means ± SDs
treated cell layers showed least wound healing, in fact, exposure of cultured conjunctival cells to agents before
wound gaps did not almost change. Interestingly olopa- they are sensitized, which thus, differs from real life situ-
tadine has been reported to inhibit monocyte migration ations because anti-allergic agents are usually used to
by binding to S100A12 protein, which is involved in in- already sensitized patients in clinic.
flammation [30]. It is generally considered well- The most obvious limitation of the present study is its
maintained healing capacity helps to minimize harmful in vitro design. However, although in vitro results do
effects resulting from a damaged ocular surface barrier. not always reflect in vivo effects, we believe that our
To the best of our knowledge, this is the first study to findings provide a valuable guide with respect to optimal
use a CPT based on an Aspergillus allergen stimulus to clinical usage. In particular, in patients with decreased
investigate the anti-allergic effect of agents on cultured tear clearance or lack of sufficient tear amounts, such as
conjunctival epithelial cells in vitro. We measured the the elderly, nasolacrimal duct obstruction, and dry eye
gene expression of 5 cytokines related to the allergic re- syndrome, anti-allergic agents administered to ocular
action cascade, that is, interleukin (IL)-5 for eosinophil surfaces may cause cytotoxic effects [42–45]. Therefore,
activation, IL-25 for Th2 response maintenance, eotaxin if these risk factors are anticipated or symptom suggest-
for eosinophil recruitment, thymus and activation- ive of toxicity is encountered, it might be more beneficial
regulated chemokine (TARC) for Th2 cell migration, to use a less toxic agent.
and thymic stromal lymphopoietin (TSLP) for dendritic
cell differentiation to prime Th2 cells [31–35]. The re- Conclusion
sults obtained showed all three agents reduced gene ex- In summary, our in vitro results indicate alcaftadine has
pressions. In particular, alcaftadine was superior in less side effects and better therapeutic effects than bepo-
terms of attenuating the gene expressions of all five cy- tastine or olopatadine. Although these effects may not
tokines, the levels of which were similar to those in un- correspond with actual response to eye-drops in pa-
treated cells. Furthermore, alcaftadine has a 10 times tients, we believe our results provide an advanced guide-
stronger effect on H1 and H2 receptors than olopatadine line to clinicians and better treatment to patients. Well
and affinity for H4 receptor, which olopatadine does not designed in vivo studies should be followed in the
possess [36–38]. Because Th2 cell-driven allergic re- future.
sponse is caused by H1 and H4 receptor activations,
Abbreviations
these results are reasonable and consistent with those of AC: Allergic conjunctivitis; ATCC: American type culture collection;
previous studies [39–41]. The lower cytokine gene ex- BAC: Benzalkonium chloride; CPT: Conjunctival provocation test;
pression observed indicated that alcaftadine, bepotastine, DMEM: Dulbecco’s minimum essential medium; FBS: Fetal bovine serum;
HCECs: Human corneal epithelial cells; IL: Interleukin; KSFM: Keratinocyte
and olopatadine have strong anti-allergic effects. How- serum-free medium; MTT: Methylthiazolyltetrazolium; TARC: Thymus and
ever, the CPT has a limitation because it involves the activation-regulated chemokine; TSLP: Thymic stromal lymphopoietin
Kim et al. BMC Ophthalmology          (2019) 19:217 Page 8 of 9
Acknowledgements 11. Clouzeau C, Godefroy D, Riancho L, Rostène W, Baudouin C, Brignole-
Not applicable. Baudouin F. Hyperosmolarity potentiates toxic effects of benzalkonium
chloride on conjunctival epithelial cells in vitro. Mol Vis. 2012;18:851–63.
Authors’ contributions 12. Park YS. Physiology of body fluid. In: Kang DH, editor. Physiology. Seoul: Sin-
JSL and JEL were primarily responsible for experimental concept and design. Kwang; 2000. p. 77–98.
SIK and CYP performed data acquisition and analysis, as well as drafting of 13. Dutescu RM, Panfil C, Schrage N. Osmolarity of prevalent eye drops, side
the manuscript. GF and JHL were involved in data analysis. All authors effects, and therapeutic approaches. Cornea. 2015;34(5):560–6.
reviewed and approved the final manuscript. 14. Khoh-Reiter S, Jessen BA. Evaluation of the cytotoxic effects of ophthalmic
solutions containing benzalkonium chloride on corneal epithelium using an
Funding organotypic 3-D model. BMC Ophthalmol. 2009;9:5.
This study was supported by Research Institute for Convergence of 15. Lee JS, Lee JE, Kim N, Oum BS. Comparison of the conjunctival toxicity of
biomedical science and technology Grant (30–2016-017), Pusan National topical ocular antiallergic agents. J Ocul Pharmacol Ther. 2008;24(6):557–62.
University Yangsan Hospital. The funding organization had no role in the 16. Ayaki M, Iwasawa A, Yaguchi S, Koide R. In vitro assessment of the
design of the study and collection, analysis, and interpretation of data, and cytotoxicity of anti-allergic eye drops using 5 cultured corneal and
in writing the manuscript. conjunctival cell lines. J Oleo Sci. 2011;60(3):139–44.
17. Ono SJ, Lane K. Comparison of effects of alcaftadine and olopatadine on
Availability of data and materials conjunctival epithelium and eosinophil recruitment in a murine model of
The datasets during and/or analyzed during the current study are available allergic conjunctivitis. Drug Des Devel Ther. 2011;5:77–84.
from the corresponding author on reasonable request. 18. Ziegler U, Groscurth P. Morphological features of cell death. News Physiol
Sci. 2004;19:124–8.
Ethics approval and consent to participate 19. Merrill DL, Fleming TC, Girard LJ. The effects of physiologic balanced salt
Not applicable. solutions and normal saline on intraocular and extraocular tissue. Am J
Ophthalmol. 1960;49:895–8.
Consent for publication 20. Baudouin C, Labbé A, Liang H, Pauly A, Brignole-Baudouin F. Preservatives in
Not applicable. eyedrops: the good, the bad and the ugly. Prog Retin Eye Res. 2010;29(4):312–34.
21. Guzman-Aranguez A, Calvo P, Ropero I, Pintor J. In vitro effects of preserved
Competing interests and unpreserved anti-allergic drugs on human corneal epithelial cells. J
Choul Yong Park is an section editor and Ji Eun Lee is an associate editor. Ocul Pharmacol Ther. 2014;30(9):790–8.
22. Ayaki M, Iwasawa A, Yaguchi S, Koide R. Preserved and unpreserved 12 anti-
Author details allergic ophthalmic solutions and ocular surface toxicity: in vitro assessment
1Department of Ophthalmology, School of Medicine, Pusan National in four cultured corneal and conjunctival epithelial cell lines. Biocontrol Sci.
University, Mulgumup, Yangsan 50612, Gyeongnam Province, Republic of 2010;15(4):143–8.
South Korea. 2Research Institute for Convergence of Biomedical Science and 23. Pauly A, Brasnu E, Riancho L, Brignole-Baudouin F, Baudouin C. Multiple
Technology, Pusan National University Yangsan Hospital, Yangsan, South endpoint analysis of BAC-preserved and unpreserved antiallergic eye drops
Korea. 3Department of Ophthalmology, Dongguk University Ilsan Hospital, on a 3D-reconstituted corneal epithelial model. Mol Vis. 2011;17:745–55.
Dongguk University College of Medicine, Goyang, South Korea. 24. Brasnu E, Brignole-Baudouin F, Riancho L, Guenoun JM, Warnet JM,
4Ophthalmology Unit, Department of Surgery, Korle-Bu Teaching Hospital, Baudouin C. In vitro effects of preservative-free tafluprost and preserved
Accra, Ghana. 5Busan Medical Center, Busan, South Korea. latanoprost, travoprost, and bimatoprost in a conjunctival epithelial cell line.
Curr Eye Res. 2008;33(4):303–12.
Received: 6 August 2018 Accepted: 28 October 2019 25. De Saint JM, Debbasch C, Brignole F, Rat P, Warnet JM, Baudouin C. Toxicity
of preserved and unpreserved antiglaucoma topical drugs in an in vitro
model of conjunctival cells. Curr Eye Res. 2000;20:85–94.
References 26. Feng MM, Baryla J, Liu H, Laurie GW, McKown RL, Ashki N, et al.
1. Bloomfield SF, Stanwell-Smith R, Crevel RW, Pickup J. Too clean, or not too Cytoprotective effect of lacritin on human corneal epithelial cells exposed
clean: the hygiene hypothesis and home hygiene. Clin Exp Allergy. 2006; to benzalkonium chloride in vitro. Curr Eye Res. 2014;39:604–10.
36(4):402–25. 27. Pinheiro R, Panfil C, Schrage N, Dutescu RM. The impact of Glaucoma
2. Platts-Mills TA, Erwin E, Heymann P, Woodfolk J. Is the hygiene hypothesis medications on corneal wound healing. J Glaucoma. 2016;25(1):122–7.
still a viable explanation for the increased prevalence of asthma? Allergy. 28. Lee JS, Lee SU, Che CY, Lee JE. Comparison of cytotoxicity and wound
2005;60(Suppl 79):25–31. healing effect of carboxymethylcellulose and hyaluronic acid on human
3. Thong BY. Allergic conjunctivitis in Asia. Asia Pac Allergy. 2017;7(2):57–64. corneal epithelial cells. Int J Ophthalmol. 2015;8(2):215–21.
4. ISAAC Phase Three Study Group. Global map of the prevalence of 29. Ljubimov AV, Saghizadeh M. Progress in corneal wound healing. Prog Retin
symptoms of rhinoconjunctivitis in children: the international study of Eye Res. 2015;49:17–45.
asthma and allergies in childhood (ISAAC) phase three. Allergy. 2009;64(1): 30. Kishimoto K, Kaneko S, Ohmori K, Tamura T, Hasegawa K. Olopatadine
123–48. suppresses the migration of THP-1 monocytes induced by S100A12 protein.
5. Fukagawa K, Fujishima H, Fukushima A, Sumi T, Okamoto S, Shoji J, et al. A Mediat Inflamm. 2006;2006(1):42726.
quality of life questionnaire for Japanese allergic conjunctival disease. 31. Wong AH, Barg SS, Leung AK. Seasonal and perennial allergic conjunctivitis.
Nippon Ganka Gakkai Zasshi. 2012;116(5):494–502. Recent Patents Inflamm Allergy Drug Discov. 2014;8(2):139–53.
6. Solomon A, Pe'er J, Levi-Schaffer F. Advances in ocular allergy: basic 32. Wang YH, Liu YJ. Thymic stromal lymphopoietin, OX40-ligand, and
mechanisms, clinical patterns and new therapies. Curr Opin Allergy Clin interleukin-25 in allergic responses. Clin Exp Allergy. 2009;39(6):798–806.
Immunol. 2001;1(5):477–82. 33. Menzies-Gow A, Ying S, Sabroe I, Stubbs VL, Soler D, Williams TJ, Kay AB.
7. Leonardi A, Quintieri L. Olopatadine: a drug for allergic conjunctivitis Eotaxin (CCL11) and eotaxin-2 (CCL24) induce recruitment of eosinophils,
targeting the mast cell. Expert Opin Pharmacother. 2010;11(6):969–81. basophils, neutrophils, and macrophages as well as features of early- and
8. Bergmann MT, Williams JI, Gomes PJ. Treatment of allergic conjunctivitis late-phase allergic reactions following cutaneous injection in human atopic
with bepotastine besilate ophthalmic solution 1.5%. Clin Ophthalmol. 2014; and nonatopic volunteers. J Immunol. 2002;169(5):2712–8.
8:1495–505. 34. Imai T, Baba M, Nishimura M, Kakizaki M, Takagi S, Yoshie O. The T cell-
9. Greiner JV, Edwards-Swanson K, Ingerman A. Evaluation of alcaftadine 0.25% directed CC chemokine TARC is a highly specific biological ligand for CC
ophthalmic solution in acute allergic conjunctivitis at 15 minutes and 16 chemokine receptor 4. J Biol Chem. 1997;272(23):15036–42.
hours after instillation versus placebo and olopatadine 0.1%. Clin 35. Liu YJ, Soumelis V, Watanabe N, Ito T, Wang YH, Malefyt Rde W, et al.
Ophthalmol. 2011;5:87–93. TSLP: an epithelial cell cytokine that regulates T cell differentiation by
10. Lee JE, Sun Y, Gjorstrup P, Pearlman E. Inhibition of corneal inflammation by conditioning dendritic cell maturation. Annu Rev Immunol. 2007;25:
the Resolvin E1. Invest Ophthalmol Vis Sci. 2015;56(4):2728–36. 193–219.
Kim et al. BMC Ophthalmology          (2019) 19:217 Page 9 of 9
36. Gallois-Bernos A, Thurmond R. Alcaftadine, a new antihistamine with
combined antagonist activity at histamine H1, H2, and H4 receptors. J
Recept Ligand Channel Res. 2012;5:9–20.
37. Bohets H, McGowan C, Mannens G, Schroeder N, Edwards-Swanson K,
Shapiro A. Clinical pharmacology of alcaftadine, a novel antihistamine for
the prevention of allergic conjunctivitis. J Ocul Pharmacol Ther. 2011;27(2):
187–95.
38. Sharif NA, Xu SX, Yanni JM. Olopatadine (AL-4943A): ligand binding and
functional studies on a novel, long acting H1-selective histamine antagonist
and anti-allergic agent for use in allergic conjunctivitis. J Ocul Pharmacol
Ther. 1996;12(4):401–7.
39. Chigbu DI, Coyne AM. Update and clinical utility of alcaftadine ophthalmic
solution 0.25% in the treatment of allergic conjunctivitis. Clin Ophthalmol.
2015;9:1215–25.
40. Ackerman S, D'Ambrosio F Jr, Greiner JV, Villanueva L, Ciolino JB, Hollander
DA. A multicenter evaluation of the efficacy and duration of action of
alcaftadine 0.25% and olopatadine 0.2% in the conjunctival allergen
challenge model. J Asthma Allergy. 2013;6:43–52.
41. McLaurin EB, Marsico NP, Ackerman SL, Ciolino JB, Williams JM, Villanueva L,
et al. Ocular itch relief with alcaftadine 0.25% versus olopatadine 0.2% in
allergic conjunctivitis: pooled analysis of two multicenter randomized
clinical trials. Adv Ther. 2014;31(10):1059–71.
42. Zheng X, Yamaguchi M, Kamao T, Sakane Y, Goto T, Shiraishi A, et al.
Visualization of tear clearance using anterior segment optical coherence
tomography and Polymethylmethacrylate particles. Cornea. 2016;35(Suppl
1):S78–82.
43. Prabhasawat P, Tseng SC. Frequent association of delayed tear clearance in
ocular irritation. Br J Ophthalmol. 1998;82(6):666–75.
44. Yen MT, Pflugfelder SC, Feuer WJ. The effect of punctal occlusion on tear
production, tear clearance, and ocular surface sensation in normal subjects.
Am J Ophthalmol. 2001;131(3):314–23.
45. Yuan Y, Wang J, Chen Q, Tao A, Shen M, Shousha MA. Reduced tear
meniscus dynamics in dry eye patients with aqueous tear deficiency. Am J
Ophthalmol. 2010;149(6):932–8.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.