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Safety and Tolerability of Overdosed Artificial Tears by Abraded Rabbit Corneas


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Purpose: Preservative-free cationic emulsion-based artificial tear (AT) is an innovative eye drop based on the Novasorb® technology with cetalkonium chloride (CKC) as the cationic agent. The cationic emulsion Cationorm is designed for the management of mild-to-moderate dry eye disease (DED) patients that present cornea epithelium alterations. The aim of the present study was to evaluate the safety and tolerability of overdosed ATs by altered corneal epithelium in vivo and assess the usefulness of the ex vivo eye irritation test (EVEIT) as a predictive alternate toxicity test method. Methods: The experimental procedure, treatment duration, and instillation frequency closely mimic in vivo the ex vivo protocol described by Pinheiro et al. and discussed in the Discussion and Conclusion section of this article. Two to 3-month-old female New Zealand white rabbits, n = 6 per group, were treated with ATs (21 instillations/day over 3 days) following corneal abrasion. Corneal fluorescein staining, in vivo confocal microscopy (IVCM), and slit lamp examinations were performed to assess corneal epithelium recovery and the ocular tolerability of the overdosed ATs. Results: All abraded eyes experienced almost complete epithelium recovery within 3 days following treatments with Cationorm, Optive, Vismed, and Saline. Benzalkonium chloride (BAK, 0.02%) treatment resulted in 82.4% reepithelialization. IVCM data illustrated corneal epithelium normal recovery. Acute local tolerability of the overdosed ATs was confirmed using Draize and McDonald-Shadduck's test scales. Conclusions: The different ATs were demonstrated to be well tolerated by abraded corneas in vivo, and the extreme overdosing regimen did not hamper the wound healing process of the rabbit eye in comparison to saline. These data did not confirm the ones obtained with the nonvalidated ex vivo eye irritation test.
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Safety and Tolerability of Overdosed Artificial
Tears by Abraded Rabbit Corneas
Philippe Daull,
Elisabeth Raymond,
Laurence Feraille,
and Jean-Se´ bastien Garrigue
Purpose: Preservative-free cationic emulsion-based artificial tear (AT) is an innovative eye drop based on the
technology with cetalkonium chloride (CKC) as the cationic agent. The cationic emulsion Catio-
norm is designed for the management of mild-to-moderate dry eye disease (DED) patients that present cornea
epithelium alterations. The aim of the present study was to evaluate the safety and tolerability of overdosed ATs
by altered corneal epithelium in vivo and assess the usefulness of the ex vivo eye irritation test (EVEIT) as a
predictive alternate toxicity test method.
Methods: The experimental procedure, treatment duration, and instillation frequency closely mimic in vivo the
ex vivo protocol described by Pinheiro et al. and discussed in the Discussion and Conclusion section of this
article. Two to 3-month-old female New Zealand white rabbits, n=6 per group, were treated with ATs (21
instillations/day over 3 days) following corneal abrasion. Corneal fluorescein staining, in vivo confocal mi-
croscopy (IVCM), and slit lamp examinations were performed to assess corneal epithelium recovery and the
ocular tolerability of the overdosed ATs.
Results: All abraded eyes experienced almost complete epithelium recovery within 3 days following treatments
with Cationorm, Optive, Vismed, and Saline. Benzalkonium chloride (BAK, 0.02%) treatment resulted in
82.4% reepithelialization. IVCM data illustrated corneal epithelium normal recovery. Acute local tolerability of
the overdosed ATs was confirmed using Draize and McDonald–Shadduck’s test scales.
Conclusions: The different ATs were demonstrated to be well tolerated by abraded corneas in vivo, and the
extreme overdosing regimen did not hamper the wound healing process of the rabbit eye in comparison to
saline. These data did not confirm the ones obtained with the nonvalidated ex vivo eye irritation test.
Keywords: cationic emulsions, cornea abrasion, in vivo model, wound healing, safety evaluation
Dry eye disease (DED) is a complex multifactorial
disease of the ocular surface that manifests by struc-
tural alterations of the cornea (keratitis) and the presence of
symptoms of discomfort of variable severity (scratchy and
dryness sensation, stinging or burning, pain, and eye red-
Patients with DED may also experience a blurred
vision, eye fatigue, and heavy eyelid sensations. The tear
film (TF), whose normal function is to protect, hydrate, and
nurture the corneal cells and thus preserve vision’s quality,
is often altered and instable in DED patients, thus not able to
play its roles.
Artificial tears (ATs) represent the first line
therapy for restoring the TF and for the management of the
symptoms and signs of DED patients.
Many different
ATs exist with different formulation strategies used to re-
store a healthy TF.
Aqueous-based AT formulations, with
or without gel-forming polymers (eg, carboxymethyl cellu-
lose, hydroxypropyl guar, and hyaluronic acid), are classi-
cally used in aqueous-deficient DED,
while lipid-containing
ATs are used for evaporative DED.
For the temporary
relief of the less severe (ie, mild to moderate) symptoms,
patients are advised to instill one drop in the affected eye
when needed and up to 4 drops per day.
The demonstra-
tion of the efficacy and safety of most of the ATs was
performed in 1–3 month clinical trials in mild-to-moderate
DED patients treated with up to 4 instillations per eye and
per day.
SANTEN SAS, Novagali Innovation Center, Evry Cedex, France.
Iris Pharma, La Gaude, France.
ªPhilippe Daull et al. 2018; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative
Commons License (, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Volume 34, Number 10, 2018
Mary Ann Liebert, Inc.
DOI: 10.1089/jop.2018.0040
The management of the more severe symptoms generally
requests the need of prescription eye drop containing potent
anti-inflammatoryactives like cyclosporine,
or glucocorticoids.
However, depending on the severity of the
discomfort some patients might feel the need to instill ATs in
their eyes above the recommended dosing regimen of 4 eye
drops per day, with the risk of developing acute toxicity events.
The safety of the ATs is generally demonstrated during the
preclinical development stage of the ATs in in vitro and in vivo
relevant animal models with dosing regimens exceeding the
expected human posology. This approach usually allows for the
definition of a margin of safety (fordrugs under development),
which can be defined as the range between the minimal thera-
peutic dose and the minimal toxic dose of a drug. However, it is
often not possible to precisely determine a margin of safety for
ATs, as for the latter the maximal dose assayed in the animal
ocular tolerability/toxicology tests is not high/extreme enough
for the development of adverse events to appear (and thus de-
fine the minimal toxic dose) due to the inherent verygood safety
of unpreserved ATs.
With the idea of reducing the use of animal models for
toxicology and safety evaluations, alternative in vitro and ex
vivo models are being developed. Unfortunately, most of
them do not generate sufficiently reliable data to be validated
and used as good alternative test methods.
Recently, an ex
vivo eye irritation test (EVEIT) was developed and used as an
alternate assay model to evaluate the toxic effects of ATs on a
wounded corneal epithelium subjected to hourly (24 instil-
lations per day) instillations over a 3–6 day period. This
highly overdosed dosage regimen in the nonphysiological
experimental setting of the EVEIT assay suggests dramatic
ocular toxicity of the preservative-free cationic emulsion
Cationorm and the Purite
preserved Optive eye drops. The
goal of the present study was to evaluate the effects of these 2
eye drops in a rabbit model with similarly wounded corneas
and receiving 21 instillations (one eye drop every 45 min) per
day over a 3-day experimental period and, thus, assess the
reliability of the EVEIT assay data.
Thirty female New Zealand white rabbits (Hypham,
Roussay, France) of approximately 2–3 months of age were
used in this study. Iris Pharma Internal Ethics Committee
approved the protocol. All animals were treated according
to the Directive 2010/63/UE European Convention for the
Protection of Vertebrate Animals used for Experimental and
Other Scientific Purposes and to the Association for Re-
search in Vision and Ophthalmology (ARVO) statement for
the Use of Animals in Ophthalmic and Vision Research. All
animals were housed individually in standard cages under
identical temperature (18C3C), humidity (45%–80%
relative humidity), and controlled enrichment conditions
and exposed to a 12-h light/12-h dark cycle in continuously
ventilated rooms (15 air volumes per hour). They received a
standard dry pellet diet and water ad libitum.
Experimental procedure and treatment groups
The animals were randomly assigned to 5 groups (n=6per
group) defined as follows: a negative control group receiving
saline (0.9% NaCl), a toxic control group treated with a 0.02%
benzalkonium chloride (BAK) solution (in saline), and 3
treatment groups: Cationorm (Santen SAS, Evry, France),
Vismed (Horus Pharma, Saint-Laurent du Var, France), and
Optive (Allergan, Inc., Dublin, Ireland). Twenty microliters of
each eye drop was applied for up to 21 instillations (except on
the day of induction with 16instillations) per day (45 min apart)
in the right eye and started 1h after the completion of the
corneal abrasion procedure. The treatment period lasted 3 days.
The study design used in this experiment is presented in Fig. 1.
Corneal abrasion procedure
Rabbits were deeply anesthetized by an intramuscular in-
jection of a mixture of ketamine (35 mg/kg)/xylazine (5 mg/
kg), and local cornea anesthesia was performed by instillation
of a drop of 0.4% oxybuprocaine (Cebesine
, Laboratoire
Chauvin, Montpellier, France) in the study eye. Standardized
superficial corneal abrasions (4 per right eye) were performed
with a classic biopsy punch (2mm in diameter) to a depth of
approximately 35 mm (Fig. 2). An analgesic (buprenorphine,
20 mg/kg, SC) was administered 30min before corneal abra-
sion and once a day during the following 2 days.
Corneal fluorescein staining
and healing measurement
One drop of a 0.5% fluorescein sodium solution (Fluor-
esceine Faure, 0.4-mL unit-dose vials, Novartis Pharma SAS,
FIG. 1. Schematic repre-
sentation of the experimental
design of the in vivo study.
France) was instilled into the inferior conjunctival sac using a
micropipette. The cornea was examined through a biomi-
croscope by light passing through a cobalt blue filter. Pictures
of cornea lesions (area stained by fluorescein) were taken
using a CCD camera and analyzed using ImageJ software to
measure the size of the 4 abrasions on each cornea.
Local tolerability evaluation with Draize,
McDonald–Shadduck, and IVCM scales
Ocular examinations for clinical evaluations. Both eyes
were examined under a light source for cornea, conjunctiva,
and iris adverse reaction scoring according to Draize’s scale.
Slit lamp examinations of both eyes were performed to as-
sess cornea, conjunctiva, iris, and the inner parts of the
eye adverse reactions according to McDonald–Shadduck’s
scale. Figure 1 presents the schedule of the different ob-
servations performed during the experiment; slit lamp ob-
servations were performed at baseline and at the end of the
experiment, while ocular observations were also performed
each day before the first and after the 10th and 21st instil-
lation of the day.
In vivo confocal microscopy
A laser scanning in vivo confocal microscope Heidelberg
Retina Tomograph (HRT II)/Rostock Cornea Module
(RCM, Heidelberg Engineering GmbH, Heidelberg, Ger-
many) was used to examine the cellular structure of the
cornea. Pictures (400 ·400 mm) of the superficial and basal
epithelium, of the anterior and posterior stroma, and the
endothelium were taken in the center of the cornea with at
least one wounded area in the field of the microscope.
Statistical analysis
Statistical analysis was performed with GraphPad Prism
software, and intergroup comparisons were performed by 1-
way or 2-way ANOVA followed by Tukey’s multiple com-
parison test. The significance threshold was set at P<0.05.
Effects of overdosed ATs on the healing process
Four standardized lesions were performed in the central
part of the cornea (Fig. 2). The total abrasion area at day 1
(immediately after the debridement procedure) was approxi-
mately 15%, ranging from 14.8% 1.4% to 16.2% 0.5% for
the different groups (Fig. 3A). Twenty one eye drops per day
of various ATs were instilled for 3 days. At the end of the
treatment period (day 3), the size of the remaining lesion was
measured by fluorescein staining (Figs. 2 and 3B). The re-
maining lesion size varied from 0.3% 0.5% to 0.9% 2.1%
for the 3 commercially available ATs and saline, with no
statistically significant difference between them. Only the
0.02% BAK solution had a lesion at day 3 that remains vis-
ible with the naked eye (Fig. 2) following fluorescein stain-
ing. The area of the remaining lesion represents 2.8%1.3%
of the total cornea area. Corneal reepithelialization at day 3
was almost complete (from 94.4% 12.3% to 97.9% 3.5%)
FIG. 2. Illustration of the cornea defect during the ex-
periment. Corneal fluorescein staining of abraded rabbit
eyes at the beginning of the treatment period (day 1) and
after 3 days of treatment (day 3). Color images available
online at
FIG. 3. (A) Percentage of cornea abrasion (SD) at day 1
and day 3 for the different treatment groups. There is no
statistically significant difference between the groups (2-
way ANOVA, Tukey’s multiple comparison test). (B) Per-
centage of reepithelialization (SD) at day 3. *P<0.05 (1-
way ANOVA, Tukey’s multiple comparison test) versus
0.02% BAK.
following the treatment with saline and the 3 commercially
available ATs (Fig. 3B). Surprisingly, the 21 instillations per
day of a 0.02% BAK solution were not accompanied by a
worsening of the lesion, that is, an increase in the size of the
abraded area; the healing process remained effective, even
though it appeared to be slower.
Ocular tolerability of overdosed ATs
by diseased corneas
Examination of the ocular surface (cornea and conjunc-
tiva), as well as the inner parts of the eye (iris) and the
aqueous flare, was performed, with a slit lamp and by IVCM
at baseline, during the treatment period, and at the end of the
experiment to characterize the potential side effects that may
result from the high number of daily instillations. Ocular ex-
amination of the cornea, conjunctiva, and iris before the first
and after the 10th and 21st instillations of each day did not
reveal any major findings throughout the experiment. The
resulting mean score per group on the conjunctiva and cornea
using the Draize’s scale (Fig. 4) remains extremely low for all
treatments, including the 0.02% BAK solution. The only
findings (minor in intensity and duration) that affected the
conjunctiva: slight conjunctival discharge and redness present
in most of the eyes of each group. They were seen only on the
first day of instillation just after the corneal abrasion. No
findings were observed on days 2 and 3 (Fig. 4). Slit lamp
biomicroscopic observations on day 3 revealed a slight con-
junctiva congestion and a slight iris hyperemia for one eye in
the Vismed group. The other findings affected the cornea.
Some opacities were observed for one eye in the saline group,
2 eyes in the Vismed and Optive groups, 4 eyes in the Ca-
tionorm group, and for all the eyes in the 0.02% BAK group
(Fig. 5). However, the overall mean score per group on the
McDonald–Shadduck’s scale remains low, even for the 0.02%
BAK group. There was no statistically significant difference
between saline and the 3 commercially available ATs (Ca-
tionorm, Vismed, and Optive); however, saline, Vismed, and
Optive were significantly different from the 0.02% BAK.
IVCM pictures of the superficial and basal epithelium, the
anterior and posterior stroma, and the endothelium (Fig. 6) did
not reveal significant differences between the AT groups and
the saline group. Superficial epithelial cells appeared to be
relatively large with an irregular size, with some smaller cells
visible in the 0.02% BAK and saline groups. In a basal epi-
thelium that looked overall homogeneous within the different
groups, areas with irregular intercellular spaces were visible,
with hyper-reflective cells being more present in the 0.02%
BAK and Optivegroups compared to the other treatments. The
anterior stroma in the 0.02% BAK group presented numerous
hyper-reflective elongated spindle-shaped cells with a linear
cell body suggesting apoptosis. Fewer presumably apoptotic
cells can also be seen in the anterior stroma from the other
treatment groups. The posterior stroma and endothelium ap-
peared to be normal for all treatment groups.
Discussion and Conclusion
DED is a very common eye disease that affects millions
of people worldwide.
It manifests by symptoms of dis-
comfort associated with clinical signs of corneal and con-
junctival alterations. The vast majority of DED patients
experience mild-to-moderate signs and symptoms that are
generally treated by repeated instillations of ATs.
the patients are advised to instill ATs when necessary, that
is, upon feeling sensations of discomfort, the dosage regi-
men of ATs for the alleviation of DED symptoms rarely
exceeds 4 instillations per eye and per day.
for the management of very severe symptoms, more fre-
quent instillations of ATs might be needed on an acute basis
(eg, one drop per hour) before switching to prescription (Rx)
eye drops designed for the treatment of severe DED con-
An hourly dosing regimen in a 12-h awaken
FIG. 4. Mean Draize test score (SEM) for the different
treatment groups before the first and after the 10th and 21st
instillations at days 1, 2, and 3.
FIG. 5. Mean McDonald–Shadduck’s test score (SEM) for
the different treatment groups at day 3 for corneal opacity degree
(A) and corneal opacity area (B).*P<0.05 (1-way ANOVA,
Tukey’s multiple comparison test) versus 0.02% BAK.
period per day would represent 12 eye drops per day, quite a
high burden for the patient. Indeed, an hourly dosing regi-
men is not sustainable for the patients in the long term,
especially when Rx medicine containing potent (and safe)
anti-inflammatory compounds, such as cyclosporine, de-
signed for the treatment of severe DED conditions in dry eye
patients that did not improve with ATs, are available and
were demonstrated to be effective following the instillation
of just one drop per day and per eye.
Recently, an ex vivo eye irritation assay was used to
compare commercially available ATs with the goal of ex-
ploring the toxicity of ATs.
To make sure that some toxic
events arise following the instillations of the ATs an hourly
dosage regimen (ie, 24 eye drops a day) was applied over
the 3-day experiment. The cultured half eye balls received
72 eye drops over a 3-day period. With such overdosing
toxicity side effects were indeed observed in this EVEIT
assay for Cationorm and Optive.
The aim of the present work was to explore in vivo the
potential side effects that may be induced on abraded rabbit
cornea epithelium by Cationorm and other commercially
available ATs following a high dosing regimen. Four small
and standardized abrasions were performed on the corneas of
rabbits’ right eye before receiving one eye drop every 45min,
that is, 21 instillations per day over a 3-day period (except on
the day of induction with 16 instillations). This design was
chosen to mimic in vivo the extreme instillation protocol (24
eye drops per day) used in the ex vivo irritation test, EVEIT,
and confirm, or not, the relevance of the EVEIT assay as an
alternate toxicity test method for the evaluation of eye drops.
The data presented on Figs. 2 and 3 demonstrate that the
healing process in the rabbit is normal and that even one in-
stillation every 45 min (ie, 58 instillations over 3 days) of Ca-
tionorm for 3 days has no negative impact on abraded rabbit
eyes compared to saline instillations. Indeed, BID instillations
of cationic emulsions
were demonstrated to contribute to
the promotion of the healing process of abraded cornea in a rat
scrapping assay. The IVCM pictures (Fig. 6) confirmed struc-
turally that the corneal epithelium did heal properly. In addi-
tion, in an in vivo rabbit acute toxicity assay, the animals
received 15 instillations over a 90-min period, that is, one in-
stillationevery 5 min for 90 min;no toxic reactions were found
following the applications of cetalkonium chloride (CKC)-
containing cationic emulsions.
During the development pro-
cess of the cationic emulsions (Cationorm, Ikervis), the 28-day
local tolerability studies (6 daily instillations over 28 days)
confirmed that the cationic emulsions, with CKC as the cationic
agent, were very well tolerated by rabbit eyes.
This good
safety profile in animal models was also observed in patients
during the different clinical trials evaluating the safety and effi-
cacy of the cationic emulsions.
Nosika trial, Cationorm vs. Vismed, 4 instillations per day over 3
months in patients with mild-to-moderate DED signs and
symptoms; 22.7% of the patients treated with Cationorm have a
complete cornea clearing (ie, a completely healed cornea) as soon
as 7 days after treatment initiation.
By comparison, Vismed
treatment reached this effectiveness (22.0%) only after 3 months
of treatment.
In addition, the annual update safety reports of
Cationorm also confirmed that Cationorm is very well tolerated
by the altered and inflamed ocular surface of DED patients.
The improved wound healing and anti-inflammatory prop-
erties of the cationic emulsions demonstrated during clinical
trials (Nosika and Sansika trials) in patients were suggested
previously by in vitro studies in human corneal epithelial cells
and in vivo.
Indeed, in these early experiments, it was
noticed that the cationic emulsions were able to improve the
wound healing process (both in pace and ‘‘quality’’). The
reason for the wound healing clinical efficacy of the cationic
emulsions was furtherexplored at the mechanistic level. Recent
studies demonstrated that CKC was a specific inhibitor of
protein kinase C (PKC) alpha
(Daull, 2018, in press). Re-
cently, Pflugfelder and collaborators (2010) demonstrated that
FIG. 6. IVCM representa-
tive pictures of the superficial
and basal epithelium, the an-
terior and posterior stroma,
and the endothelium of the
cornea for the different treat-
ment groups. IVCM, in vivo
confocal microscopy.
PKCa‘inhibition’’ improved wound healing by reducing the
level of inflammatory cell infiltration in the wounded area with
PKCaknockout mice which have a ‘more rapid corneal epi-
thelial wound healing, perhaps due to a decreased neutrophil
Hence, the inhibition of PKCaby CKC might
explain why cationic emulsions like Cationorm and the Ikervis
vehicle did perform so well in the improvement of corneal
epithelium healing in the clinical trials.
Following the rationale for reducing the use of live animals
in toxicology studies, alternate toxicity test methods are being
developed. These new in vitro (and ex vivo) test methods are
particularly valuable for the scientific community and the so-
ciety in general. However, these alternate test methods used for
toxicity evaluation need an extended validation phase to make
sure that they produce reliable data that can confidently be used
to adequately assess the toxicity of the tested eye drops or
Unfortunately, most of these alternate irritation
assays failed in their validation phase. Indeed, the interagency
coordinating committee on the validation of alternative meth-
ods (ICCVAM) and ICCVAM Ocular Toxicology Working
Group did not recommend the use of the bovine corneal opacity
and permeability, Cytosensor
Microphysiometer (CM), hen’s
egg test-chorioallantoic membrane, isolated chicken eye, and
isolated rabbit eye test methods to distinguish substances not
labeled as irritants.
In most of the cases the false positive rates
were high. Recently, Frentz et al. developed an EVEIT as an
alternative to the in vivo evaluation of toxicity or ocular toler-
So far, the EVEIT assay has not yet undergone
formal validation
irritation test method by Abdelkader et al. in 2015
and Lotz
et al. in 2016
in their reviews.
In a direct comparison of the EVEIT assay and an in vivo
assay in rabbits (this study) based on the Draize test, the most
reliable eye irritation test, used for more than 70 years
drug development to address regulatory requirements for
safety testing, it appears that the EVEIT assay failed to ad-
equately predict the toxicity of compounds not classified as
irritant. In conclusion, the EVEIT as it is presently designed
and used appears not to be an adequate alternate test method
for safety testing of eye drops. Modifying its design so that
the EVEIT test method more closely mimics the eye’s normal
lubrication and physiology would probably improve its pre-
dictability and decrease its overprediction rate and position
this test method, once it has passed formal validation, as a
potential alternate toxicity assay for eye drops.
Author Disclosure Statement
No competing financial interests exist.
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Received: March 22, 2018
Accepted: August 15, 2018
Address correspondence to:
Dr. Jean-Se
´bastien Garrigue
Novagali Innovation Center
1, rue Pierre Fontaine
ˆtiment Genavenir IV
Evry Cedex F-91458
... Only 34% of these systematic reviews reported that acupuncture was effective, 50% indicated that it was either ineffective or equivalent to the placebo group, and 16% were uncertain. Currently, the level of clinical evidence regarding acupuncture is not strong, with 58% being low-level evidence [34][35][36][37][38] , 8% being moderate [39][40][41][42][43] , and 34% being insufficient [44][45][46] . painkillers, side effects of treatments like surgery, radiotherapy, and chemotherapy, as well as the need for lifelong treatment and rehabilitation of multi-factorial chronic diseases, have prompted the international search for non-toxic, safe, and effective treatment methods. ...
... Many researchers have used this method to study TrPs and acupoints. The evaluation of TrPs by this pressure instrument has demonstrated that it is a highly sensitive and reliable tool [45][46] . ...
... The method is simple, environmentally friendly, efficient, low-cost, and has a high quantum yield. [7,17,[33][34][35][36][37][38][39][40][41][42][43][44][45] Hydrothermal method A carbon precursor is placed in a hightemperature and high-pressure sealed container for a long period of time for the reaction to synthesize CQDs. ...
... Both test formulations were used in quintuple for each treatment group. In accordance with the in vivo experimental study design of Daull et al., 17 19 -1 mL of the respective test substance (BAC or Cationorm) was applied 45 min apart, for up to 21 installations per day. The exception is day 1, which started 1 h after the completion of the corneal abrasion procedure and had 16 installations (Fig. 1). ...
... As a negative control for this study, we refer to several of our own previous experiments that presented perfect corneal healing within 2 days for hyaluronic acid-based unpreserved lubricant eye drops. 13,18,19 The main discussion of this article is that the challenging work of Daull et al. 17 was reproduced exactly here, giving insight into the differences between living animal and ex vivo corneas. Daull's group is questioning the results of EVEIT compared with patients, showing that BAC and CAC are well tolerated by living rabbits, and thus that the EVEIT is over-predictive and gives false positive results in pharmaceutical evaluation. ...
... To understand the differences between the EVEIT results of our study presented here and the living animals result of Daull et al., 17 we must focus on the differences in the circumstances of living animals and isolated exposed corneas. In living animals, we have continuous production of tears, especially with a higher tear clearance in wounded eyes. ...
Purpose: High-frequency applied cetalkonium chloride (CAC) and benzalkonium chloride (BAC) 0.02% did not hamper corneal healing in a living rabbit model of induced corneal erosion. In contrast, the ex vivo eye irritation test (EVEIT) shows inhibition of healing for these substances. In a systematic ex vivo reproduction of the in vivo experiments, we discuss the background of these differences. Methods: Excised rabbit corneas (n = 5 per group) were cultured in artificial anterior chambers (EVEIT). Four erosions were induced for each cornea before starting regular 21 installations/day over 3 days of (1) CAC containing eye drops (Cationorm®), (2) 0.02% BAC. Corneal fluorescein staining, quantification of glucose-/lactate consumption, and histology were performed. Results: BAC 0.02% treated corneas showed increased epithelial lesions from 10.13 ± 0.65 mm2 to 10 ± 0.8 mm2 on day 0, to 86.82 ± 5.18 mm2 (P < 0.0001) by day 3. After a trend toward smaller lesions for CAC on day 1, erosion sizes increased significantly by day 3 from 9.82 ± 0.30 mm2 to 29.51 ± 16.87 mm2 (P < 0.05). For 1 cornea, corneal erosions nearly disappeared on day 3 (0.89 mm2). Corneal lactate increased significantly for BAC and CAC, whereas glucose concentrations were unchanged. Histology revealed disintegration of the corneal structures for both compounds. Conclusions: The data underline the EVEIT as a predictive toxicity test to show side effects in a time-compressed manner. The consistency of these predictions was previously demonstrated by the EVEIT for BAC, phosphate buffer, and others. The EVEIT is suited for a chronic application prediction of tolerability and toxic side effects of eye drops in particular, and other chemicals in general.
... The reported effects, both in in vitro and in vivo models, include dose-dependent and time-dependent toxicity to the corneal epithelium, the conjunctival epithelium, the stroma, and tear film constituents. BAK may also reduce epithelial cell integrity, impair healing, induce cytokine secretion, cause elevated production of conjunctival inflammatory cells, and reduce goblet cell number [5,6,[9][10][11][12][13]. Furthermore, BAK may impair tear function and reduce tear film integrity through its effects on the integrity of the meibomian layer and disruption of the lipid film continuous multilayer structure, thus decreasing tear film break-up time [6,[13][14][15]. ...
... BAK may also reduce epithelial cell integrity, impair healing, induce cytokine secretion, cause elevated production of conjunctival inflammatory cells, and reduce goblet cell number [5,6,[9][10][11][12][13]. Furthermore, BAK may impair tear function and reduce tear film integrity through its effects on the integrity of the meibomian layer and disruption of the lipid film continuous multilayer structure, thus decreasing tear film break-up time [6,[13][14][15]. At the cellular level, BAK not only induces growth arrest, but also increases epithelial cell apoptosis and cytotoxicity [5,[9][10][11]. ...
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Background Benzalkonium chloride (BAK), the most commonly used preservative in anti-glaucoma eye drops, inflicts damage to the ocular surface. A novel anti-glaucoma formulation that avoids the use of BAK has been developed. The aim of this study was to evaluate the cytotoxicity of this formulation and to compare it with an ophthalmic solution containing BAK. Methods Two different latanoprost eye drops were used: one ophthalmic solution (LSc) containing BAK 0.02% and one ophthalmic nanoemulsion (LNe) with a soft preservative (potassium sorbate 0.18%). Human epithelial conjunctival cells were incubated for 15, 30, and 60 min with either LSc or LNe. The cytotoxicity was determined by MTT assay. Cell death was measured by flow cytometry using annexin V–FITC and propidium iodide. Results The values of cell viability and proliferation obtained from cells exposed to LNe were between 80 and 90% relative to the control group, whereas values obtained from cells exposed to LSc were around 30% at all study times (p < 0.05 at 15 and 30 min; p < 0.01 at 60 min). The percentage of viable cells decreased significantly when cells were incubated with LSc compared with cells incubated with LNe at all the study times, while the percentage of cells in late apoptosis/necrosis increased significantly in cells exposed to LSc compared to LNe. Conclusions The new latanoprost nanoemulsion is significantly less cytotoxic on human conjunctival cells than LSc. These results suggest that the new formulation might be gentler on the eye surface than currently available BAK-preserved latanoprost solutions.
... The argument that the ex vivo system is too susceptible to toxicity, especially in the area where clinical, non-preserved, high-frequency artificial tear formulations are used, was discussed by Daull et al. [22]. This argumentation has been followed up by our group with a 1:1 experiment of animals versus EVEIT copying the Daull approach. ...
Full-text available
Introduction: Corneal epithelial toxicity and delayed healing process have already been attributed to preservatives or some excipients. We study the effects of galenic components in antiglaucoma drugs such as benzalkonium chloride (BAC) or surfactants like macrogolglycerol hydroxystearate 40 (MGHS 40) on corneal toxicity in an ex vivo system mimicking chronic use. Methods: Latanoprost-containing eyedrops are available with and without preservatives on the market. Unpreserved, they are available in different formulations with various excipients like MGHS at different concentrations (0%, 2.5%, and 5%). We studied these in the ex vivo bioreactor (EVEIT) on initially injured rabbit corneas. The drugs were applied six times daily for observation periods of 3 or 5 days. BAC, 5% MGHS 40 solution, and 0.18% hyaluronic acid served as controls. Macroscopic photographic, biochemical methods and corneal integrity quantification were used for evaluation. Toxicity was assessed by measuring wound healing and corneal fluorescein sodium permeability and was confirmed by histology studies. Results: The BAC-preserved formulation resulted in high corneal toxicity, which was expected. Interestingly, the preservative-free (PF) formulation containing 5% MGHS 40, carbomer, macrogol 4000, and sorbitol showed the highest corneal toxicity, followed by the control formulation with equal MGHS 40 concentration, which presented significantly less damage. No toxicity was shown by eyedrops containing 2.5% MGHS 40 or salts only. Conclusion: Our study demonstrates a significant corneal toxicity of certain formulations of PF antiglaucoma ophthalmic drugs containing 5% MGHS 40 with other excipients compared to other formulations with lower MGHS 40 concentrations (2.5% or 0%), or even compared to the solution containing 5% MGHS alone. This suggests a concentration-dependent toxicity of MGHS 40, especially in interaction with other excipients, which may increase its epithelial toxicity, and that has to be considered in clinical glaucoma therapy. Further single-component formulation trials are needed to support this interpretation.
... Artificial tears increase tear volume, minimize eye dehydration, reduce tear osmolarity, and lubricate the ocular surface. In addition, they provide temporary relief for various mild symptoms of DED, such as temporary improvement in eye irritation, tear breakup time, and corneal surface regularity [11]. According to Business Wire ( ...
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Citation: Mehdaoui, H.; Ait Abderrahmane, H.; de Loubens, C.; Nait Bouda, F.; Hamani, S. Dynamics of a Gel-Based Artificial Tear Film with an Emphasis on Dry Disease Treatment Applications. Gels 2021, 7, 215. gels7040215 Academic Editors: Abstract: This paper discusses the spreading of gel-based ophthalmic formulation on the cornea surface assumed to be flat. We show that gel-based formulations exhibit rheological behaviors that the Herschel-Bulkley model can describe. The continuity and momentum equations are solved numerically using the monofluid formulation and the volume-of-fluid (VOF) method. We investigated the influence of the rheological properties, namely the consistency, the yield stress, and the flow behavior index, on the spreading of a gel-based artificial tear over the cornea surface. We propose optimal values of these properties for efficient gel-based artificial tears.
... 57 CKC is a lipophilic surfactant from the benzyl alkyl quaternary ammonium chloride family. Although it is the most hydrophobic subcomponent of the benzalkonium chloride (BAK) mixture-which explains its improved safety profile when formulated in emulsion 8,9,[58][59][60][61] versus the benzyl alkyl quaternary ammoniums chloride with shorter aliphatic chains (12 or 14 carbons) 8,9 -it was not anticipated that CKC, through its modulation of PKCa, could be even partly responsible for the good clinical performance of the CEs. 10,11,35 Indeed, Pflugfelder and collaborators demonstrated in a PKCa knock-out mouse the benefits that PKCa ''inhibition'' has on corneal inflammation and its healing process. ...
Full-text available
Dry eye disease (DED) is a complex multifactorial disease that affects an increasing number of patients worldwide. Close to 30% of the population has experienced dry eye (DE) symptoms and presented with some signs of the disease during their lifetime. The significant heterogeneity in the medical background of patients with DEs and in their sensitivity to symptoms renders a clear understanding of DED complicated. It has become evident over the past few years that DED results from an impairment of the ocular surface homeostasis. Hence, a holistic treatment approach that concomitantly addresses the different mechanisms that result in the destabilization of the tear film (TF) and the ocular surface would be appropriate. The goal of the present review is to compile the different types of scientific evidence (from in silico modeling to clinical trials) that help explain the mechanism of action of cationic emulsion (CE)-based eye drop technology for the treatment of both the signs and the symptoms of DED. These CE-based artificial tear (AT) eye drops designed to mimic, from a functional point of view, a healthy TF contribute to the restoration of a healthy ocular surface environment and TF that leads to a better management of DE patients. The CE-based AT eye drops help restore the ocular surface homeostasis in patients who have unstable TF or no tears.
... Corneal epithelium was removed by gentle scraping with a dulled scalpel or by using a classic biopsy punch (2 mm in diameter), as previously described. [6][7][8] Each cornea was then examined using the iOS application on an iPhone 8 Plus (Apple Inc.) after applying a fluorescein sodium ophthalmic strip (Bio Glo; HUB Pharmaceuticals, LLC, Rancho Cucamonga, CA). The smartphone's liquid crystal display was held approximately 4 inches away from the ex vivo specimen. ...
Purpose: To describe the feasibility of an attachment-free iOS-based cobalt blue light application to accurately detect corneal injury. Methods: Corneal epithelium from New Zealand White rabbits was removed by gentle scraping with a dulled scalpel or by using a classic biopsy punch (2 mm in diameter). Each cornea was then examined using a smartphone-enabled blue light after applying a fluorescein sodium ophthalmic strip. Photographs of each cornea before and after using the smartphone-based light were captured with a second smartphone device. Results: The iOS-based (Apple Inc., Cuptertino, CA) cobalt blue light effectively illuminated fluorescein dye in all ex vivo samples. Both circular punch biopsy and linear scalpel-induced corneal injuries were readily identified. Conclusions: The iOS-based cobalt blue light is a portable, low-cost, and effective alternative to commercially available cobalt blue lights.
Histamine is strongly associated with the onset of allergic conjunctivitis. The most recent cloned histamine H4 receptor antagonist is highly expected as a new therapeutic drug candidate. As a model for a therapeutic drug targeting the histamine H4 receptor, a mouse model in which conjunctivitis symptoms are induced by instilling 4-methylhistamine, a histamine H4 receptor agonist, has been reported. However, the affinity of the H4 receptor for histamine varies in species, and it is known that the histamine binding affinity for the guinea pig H4 receptor is 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-methylhistamine and specifically suppressed by the histamine H4 receptor antagonist. Unlikely to the previously reported 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.
Objective This study aimed to lay the foundation for the research on Panax notoginseng saponins (PNS) in pH-sensitive in situ gel and the development and improvement of related preparations. Methods We used Carbopol ® 940, a commonly used pH-sensitive polymer, and the thickener hydroxypropyl methylcellulose (HPMC E4M) as an ophthalmic gel matrix to prepare an ophthalmic in situ gel of PNS. In addition, formula optimization was performed by assessing gelling capability with the results of in vitro release studies. In vitro (corneal permeation, rheological, and stability) and in vivo (ocular irritation and preliminary pharmacokinetics in the vitreous) studies were also performed. Results The results demonstrated that the in situ gelling systems containing PNS showed a sustained release of the drug, making it an ideal ocular delivery system for improving posterior ocular bioavailability. Conclusions This study lays the foundation for the research of PNS contained in an in situ pH-triggered gel as well as the development and improvement of related preparations. It concurrently traditional Chinese medicine with a contemporary in situ gelling approach to provide new directions for the treatment of posterior ocular diseases such as diabetic retinopathy.
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Components of the ocular surface synergistically contribute to maintaining and protecting a smooth refractive layer to facilitate the optimal transmission of light. At the air-water interface, the tear film lipid layer (TFLL), a mixture of lipids and proteins, plays a key role in tear surface tension and is important for the physiological hydration of the ocular surface and for ocular homeostasis. Alterations in tear fluid rheology, differences in lipid composition, or downregulation of specific tear proteins are found in most types of ocular surface disease, including dry eye disease (DED). Artificial tears have long been a first line of treatment in DED and aim to replace or supplement tears. More recently, lipid-containing eye drops have been developed to more closely mimic the combination of aqueous and lipid layers of the TFLL. Over the last 2 decades, our understanding of the nature and importance of lipids in the tear film in health and disease has increased substantially. The aim of this article is to provide a brief overview of our current understanding of tear film properties and review the effectiveness of lipid-based products in the treatment of DED. Liposome lid sprays, emulsion eye drops, and other lipid-containing formulations are discussed.
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Introduction: Dry eye disease (DED) is a common ocular disorder that can have a substantial burden on quality of life and daily activities. Lifitegrast ophthalmic solution 5.0% is the first medication approved in the US for the treatment of the signs and symptoms of DED. The aim of this article is to summarize the preclinical and clinical data on lifitegrast and discuss how lifitegrast may fit into the current treatment landscape for DED. Areas covered: A literature search of published preclinical and clinical data was conducted to review the chemistry, pharmacodynamics, pharmacokinetics, and clinical efficacy/safety of lifitegrast. The impact that lifitegrast may have on DED treatment practices is also discussed. Expert opinion: The introduction of lifitegrast provides a potentially important additional option for eye care professionals treating DED. In clinical trials conducted in adults with DED, lifitegrast ophthalmic solution 5.0% improved both signs and symptoms of DED. Of note, in 2 phase 3 trials, symptom improvements were observed as early as 2 weeks, which may be explained by lifitegrast’s unique mechanism of action of blocking a specific signaling pathway in inflammation. Future research should include evaluation of whether lifitegrast can be used in combination with other DED treatments.
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Purpose The SANSIKA study evaluated the efficacy/safety of 0.1% (1 mg/mL) cyclosporine A cationic emulsion (CsA CE) for treating dry eye disease (DED) with severe keratitis. The double-masked phase demonstrated that CsA CE was effective in reducing corneal damage and ocular surface inflammation, and was well-tolerated over 6 months. Here we report efficacy and safety findings of SANSIKA’s open-label extension (OLE). Methods In this multicenter, double-masked, phase III study, patients with severe DED (corneal fluorescein staining [CFS] grade 4, modified Oxford scale) were randomized to once-daily CsA CE (Ikervis®) or its vehicle for 6 months, followed by 6-month open-label, once-daily CsA CE (CsA CE/CsA CE and vehicle/CsA CE groups). Results A total of 177 patients completed the OLE. Efficacy results reiterated the double-masked phase: CsA CE reduced CFS score and human leukocyte antigen-antigen D related expression, improved corneal clearing, and produced continuous improvements in global symptom scores (ocular surface disease index [OSDI], visual analogue scale). The CFS-OSDI response rates (≥2 CFS points, ≥30% OSDI improvement vs baseline) at 12 vs 6 months were 39.1% vs 28.6%, respectively, for CsA CE/CsA CE and 38.0% vs 23.1% for vehicle/CsA CE. Cyclosporine A CE’s safety profile was similar to the initial 6 months. The most common treatment-related treatment-emergent adverse event was instillation site pain (7.8%, CsA CE/CsA CE group; 19.0%, vehicle/CsA CE group). No unexpected safety signals were observed; systemic CsA levels were undetectable/negligible in all patients except 2 previously treated with systemic CsA. Conclusions In this 12-month study, once-daily CsA CE was well-tolerated and showed reductions in ocular surface inflammation and improvements in signs/symptoms in DED patients with severe keratitis.
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Dry eye can damage the ocular surface and result in mild corneal epithelial defect to blinding corneal pannus formation and squamous metaplasia. Significant progress in the treatment of dry eye has been made in the last two decades; progressing from lubricating and hydrating the ocular surface with artificial tear to stimulating tear secretion; anti-inflammation and immune regulation. With the increase in knowledge regarding the pathophysiology of dry eye, we propose in this review the concept of ocular surface microenvironment. Various components of the microenvironment contribute to the homeostasis of ocular surface. Compromise in one or more components can result in homeostasis disruption of ocular surface leading to dry eye disease. Complete evaluation of the microenvironment component changes in dry eye patients will not only lead to appropriate diagnosis, but also guide in timely and effective clinical management. Successful treatment of dry eye should be aimed to restore the homeostasis of the ocular surface microenvironment.
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Purpose: The SICCANOVE study aimed to compare the efficacy and safety of 0.1% cyclosporine A cationic emulsion (CsA CE) versus vehicle in patients with moderate to severe dry eye disease (DED). Methods: In this multicenter, double-masked, parallel-group, controlled study, patients were randomized (1:1) to receive CsA CE (Ikervis?) or vehicle for 6 months. The co-primary efficacy endpoints at month 6 were mean change from baseline in corneal fluorescein staining (CFS; modified Oxford scale) and in global ocular discomfort (visual analogue scale [VAS]). Results: The mean change in CFS from baseline to month 6 (CsA CE: n = 241; vehicle: n = 248) was significantly greater with CsA CE than with vehicle (-1.05 ? 0.98 and -0.82 ? 0.94, respectively; p = 0.009). Ocular discomfort improved similarly in both groups; however, the percentage of patients with ?25% improvement in VAS was significantly higher with CsA CE (50.2%) than with vehicle (41.9%; p = 0.048). In a post hoc analysis of patients with severe ocular surface damage (CFS score 4) at baseline (CsA CE: n = 43; vehicle: n = 42), the percentage of patients with improvements of ?2 grades in CFS score and ?30% in Ocular Surface Disease Index score was significantly greater with CsA CE (p = 0.003). Treatment compliance and ocular tolerability were satisfactory and as expected for CsA use. Conclusion: Cyclosporine A CE was well-tolerated and effectively improved signs and symptoms in patients with moderate to severe DED over 6 months, especially in patients with severe disease, who are at risk of irreversible corneal damage.
Dry eye disease (DED) represents a heterogeneous group of conditions with tear film insufficiency and signs and/or symptoms of ocular surface irritation. The clinical manifestations of DED can be highly variable; hence the diagnosis is often based on a combination of symptoms, signs, and clinical tests, given that any one of these alone would miss a significant number of patients. Similarly, the treatment must often be tailored to each patient by targeting the specific mechanisms involved in his or her disease. The purpose of this review is to summarize recent advances that have allowed us to better recognize, categorize, and treat patients with DED. The most notable new diagnostic tests in DED are tear film osmolarity, inflammatory biomarkers, and meibomian gland imaging. Therapeutically, anti-inflammatory therapy, meibomian gland heating and expression, and scleral contact lenses are some of the latest options available for treating DED.
The members of the Management and Therapy Subcommittee undertook an evidence-based review of current dry eye therapies and management options. Management options reviewed in detail included treatments for tear insufficiency and lid abnormalities, as well as anti-inflammatory medications, surgical approaches, dietary modifications, environmental considerations and complementary therapies. Following this extensive review it became clear that many of the treatments available for the management of dry eye disease lack the necessary Level 1 evidence to support their recommendation, often due to a lack of appropriate masking, randomization or controls and in some cases due to issues with selection bias or inadequate sample size.
The TFOS DEWS II Pathophysiology Subcommittee reviewed the mechanisms involved in the initiation and perpetuation of dry eye disease. Its central mechanism is evaporative water loss leading to hyperosmolar tissue damage. Research in human disease and in animal models has shown that this, either directly or by inducing inflammation, causes a loss of both epithelial and goblet cells. The consequent decrease in surface wettability leads to early tear film breakup and amplifies hyperosmolarity via the Vicious Circle. Pain in dry eye is caused by tear hyperosmolarity, loss of lubrication, inflammatory mediators and neurosensory factors, while visual symptoms arise from tear and ocular surface irregularity. Increased friction targets damage to the lids and ocular surface, resulting in characteristic punctate epithelial keratitis, superior limbic keratoconjunctivitis, filamentary keratitis, lid parallel conjunctival folds, and lid wiper epitheliopathy. Hybrid dry eye disease, with features of both aqueous deficiency and increased evaporation, is common and efforts should be made to determine the relative contribution of each form to the total picture. To this end, practical methods are needed to measure tear evaporation in the clinic, and similarly, methods are needed to measure osmolarity at the tissue level, to better determine the severity of dry eye. Areas for future research include the role of genetic mechanisms in non-Sjögren syndrome dry eye, the targeting of the terminal duct in meibomian gland disease and the influence of gaze dynamics and the closed eye state on tear stability and ocular surface inflammation.
The subcommittee reviewed the prevalence, incidence, risk factors, natural history, morbidity and questionnaires reported in epidemiological studies of dry eye disease (DED). A meta-analysis of published prevalence data estimated the impact of age and sex. Global mapping of prevalence was undertaken. The prevalence of DED ranged from 5 to 50%. The prevalence of signs was higher and more variable than symptoms. There were limited prevalence studies in youth and in populations south of the equator. The meta-analysis confirmed that prevalence increases with age, however signs showed a greater increase per decade than symptoms. Women have a higher prevalence of DED than men, although differences become significant only with age. Risk factors were categorized as modifiable/non-modifiable, and as consistent, probable or inconclusive. Asian ethnicity was a mostly consistent risk factor. The economic burden and impact of DED on vision, quality of life, work productivity, psychological and physical impact of pain, are considerable, particularly costs due to reduced work productivity. Questionnaires used to evaluate DED vary in their utility. Future research should establish the prevalence of disease of varying severity, the incidence in different populations and potential risk factors such as youth and digital device usage. Geospatial mapping might elucidate the impact of climate, environment and socioeconomic factors. Given the limited study of the natural history of treated and untreated DED, this remains an important area for future research.
The members of the Tear Film Subcommittee reviewed the role of the tear film in dry eye disease (DED). The Subcommittee reviewed biophysical and biochemical aspects of tears and how these change in DED. Clinically, DED is characterized by loss of tear volume, more rapid breakup of the tear film and increased evaporation of tears from the ocular surface. The tear film is composed of many substances including lipids, proteins, mucins and electrolytes. All of these contribute to the integrity of the tear film but exactly how they interact is still an area of active research. Tear film osmolarity increases in DED. Changes to other components such as proteins and mucins can be used as biomarkers for DED. The Subcommittee recommended areas for future research to advance our understanding of the tear film and how this changes with DED. The final report was written after review by all Subcommittee members and the entire TFOS DEWS II membership.