Contact Lens and Anterior Eye xxx (xxxx) xxx

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Contact Lens and Anterior Eye

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

Safety and efficacy of lotilaner ophthalmic solution, 0.25% for the

treatment of blepharitis due to demodex infestation: A randomized,
controlled, double-masked clinical trial
Roberto Gonzalez-Salinas a, Paul Karpecki b, Elizabeth Yeu c, *, Mark Holdbrook d,
Stephanie N. Baba d, Juan Carlos Ceballos a, Martha Massaro-Corredor a,
Claudia Corredor-Ortega a, Nallely Ramos-Betancourt a, Hugo Quiroz-Mercado a
a
Asociación para Evitar la Ceguera en México I.A.P., Mexico City, Mexico
b
Kentucky Eye Institute, Lexington, KY, USA
c
Virginia Eye Consultants, Norfolk, VA, USA
d
Tarsus Pharmaceuticals Inc, Irvine, CA, USA

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

Keywords: Purpose: To evaluate the safety and efficacy of lotilaner ophthalmic solution, 0.25% for the treatment of ble­
Demodex Blepharitis pharitis due to Demodex infestation compared to vehicle control.
Lotilaner Methods: In this phase II, randomized, controlled, double-masked clinical trial, 60 eligible participants with
Ocular surface disease
Demodex blepharitis were randomly assigned in a 1:1 ratio to receive either topical lotilaner ophthalmic solu­
Collarettes
Cylindrical dandruff
tion, 0.25% (Tarsus Pharmaceuticals, Inc., Irvine, CA) (study group) or the vehicle without lotilaner (control
TP-03 group) bilaterally twice a day for 28 days. Participants were followed at Days 7, 14, 28, 60 and 90. The efficacy
parameters were change in collarette grade and Demodex density at Day 28. Safety parameters were adverse
events, changes in corrected distance visual acuity (CDVA), intraocular pressure (IOP) and slit-lamp
biomicroscopy.
Results: The study group showed a statistically significant decrease in collarette grade compared to the control
group beginning at Day 14 (p = 0.003) in the upper eyelid and at Day 28 (p = 0.003) in the lower eyelid.
Decreases in both lids were maintained through Day 90 (p < 0.001). At Day 28, mite eradication was achieved in
66.7% and 25.9% of eyes in the study and control group (p = 0.005); at Day 90, these proportions were 68.2%
and 18.5% (p = 0.001), respectively. No serious adverse events or clinically significant changes in CDVA and IOP
were observed.
Conclusion: For Demodex blepharitis, treatment with lotilaner ophthalmic solution, 0.25% for 4 weeks is safe and
effective. The improvement in collarette grade and mite density observed during the treatment period persisted
for at least two months following treatment cessation.

1. Introduction
Blepharitis is a commonly encountered disease in eye care practices
that presents with inflammation of the eyelids, particularly the eyelid
margin, causing frequent ocular irritation and discomfort [1–5]. The
pathogenesis of blepharitis is multifactorial, with infectious, allergic,
systemic and environmental components [6]. Demodex mite infestation
has been found to be frequently associated with blepharitis [7]. It has
been reported that 42%–81% of blepharitis patients have concomitant
infestation with Demodex mites [8–11]. Despite its high prevalence
among blepharitis patients, Demodex mite infestation is an often-
overlooked cause of chronic blepharitis [12–14]. When assessed in
community populations, the prevalence of ocular Demodex reportedly
ranges from 16% to 70% [15]. The rate of infestation increases with age,
reaching 84% of the population by the age of 60 and 100% in those older
than 70 [9,16,17].
Demodex mites are microscopic ectoparasites of the phylum
arthropoda with a semi-transparent, elongated body and four pairs of

* Corresponding author at: Virginia Eye Consultants, 241 Corporate Boulevard, Suite 210, Norfolk, VA 23502, USA.
E-mail address: eyeulin@gmail.com (E. Yeu).

https://doi.org/10.1016/j.clae.2021.101492
Received 9 April 2021; Received in revised form 16 July 2021; Accepted 19 July 2021
Available online 28 July 2021
1367-0484/© 2021 Published by Elsevier Ltd on behalf of British Contact Lens Association.

Please cite this article as: Roberto Gonzalez-Salinas, Contact Lens and Anterior Eye, https://doi.org/10.1016/j.clae.2021.101492
R. Gonzalez-Salinas et al.
legs [9,15,18]. Demodex folliculorum and Demodex brevis are known to
inhabit human skin [9,15,19]. These mites tend to gather in the face,
cheeks, forehead, nose, and external ear tract, where they find a favor­
able habitat for breeding and active sebum excretion provides nutrition
[20]. While D. folliculorum is approximately 0.3 to 0.5 mm long and
mostly exists in clusters in the eyelash follicles, D. brevis is typically
smaller (0.2 to 0.3 mm long) and resides deep in the sebaceous glands of
the eyelids and the lobules of meibomian glands [15,20]. D. folliculorum
mites consume epithelial cells at the eyelash follicle, induce epithelial
hyperplasia and hyperkeratinzation, subsequently leading to the for­
mation of collarettes (cylindrical dandruff) and redness, potential
eyelash loss and/or misdirection [3,18,19]. The collarettes appear as
solidified exudative excretions that extrude out of the base of the eyelash
follicle and are considered a pathognomonic sign of Demodex blephar­
itis [3,4,19]. The pathogenic mechanism of Demodex infestation leads to
direct mechanical damage as they burrow into the lash follicle and lay
eggs [18]. Demodex also acts as a vector for bacteria, most notably
Staphylococcus aureus and Propionibacterium acnes [3,18,19]. The
chemicals excreted by both the Demodex and bacteria induce a hyper­
sensitivity reaction, and thus clinical inflammation which manifests as
blepharoconjunctivitis, marginal and phlyctenular keratitis, and ocular
rosacea [19].
Currently, there are no FDA approved treatments for Demodex ble­
pharitis. A number of proposed treatments, including 1% sulfur oint­
ment [21,22], 1% mercury oxide ointment [22–25], pilocarpine gel
[26,27], and iodized solutions [27,28], have been found to be largely
ineffective. Treatment with the oral antiparasitic drugs ivermectin and
metronidazole and lid wipes containing tea tree oil (TTO), a natural oil
distilled from Melaleuca alternifolia, have shown varying levels of success
[3,4,15,18,20,29,30].
Lotilaner is a new oral acaricide from the isoxazoline class approved
for the treatment of flea and tick infestations in pets [31–33]. Isoxazo­
lines are safe for mammals due to their non-competitive antagonism to
gamma-aminobutyric acid (GABA) receptor, with higher selectivity for
GABA receptors in insects or ticks, than for those in mammals, including
humans. Pre-clinical studies did not find any neurobehavioral or loco­
motor effects with the oral or ophthalmic administration of lotilaner in
animals (Data on file with Tarsus Pharmaceuticals).
In a recent pilot study, the safety and efficacy of topical lotilaner
ophthalmic solution, 0.25% was evaluated in humans for the first time
[34]. Participants with Demodex blepharitis were treated for 28 days
and followed for an additional 2 months after treatment cessation.
Statistically significant improvement in collarette grade and mite den­
sity was demonstrated as early as 14 days and was maintained through
the 90-day follow-up.
The purpose of the present study was to evaluate the safety and ef­
ficacy of lotilaner ophthalmic solution, 0.25%, for the treatment of
blepharitis due to Demodex infestation, compared to vehicle control.
2. Methods
This 3-month, phase II, randomized, controlled, double-masked
clinical trial was conducted in participants with blepharitis due to
Demodex infestation. The study was conducted at the Asociación para
Evitar la Ceguera en México I.A.P., Mexico City, Mexico (APEC). The
study adhered to the tenets of the Declaration of Helsinki and was
approved by the APEC Ethics Committee. All enrolled participants
provided written informed consent using the APEC Ethics Committee-
approved informed consent form.
Participants were screened up to 14 days prior to enrollment and
initiation of treatment. Participants aged ≥ 18 years were enrolled if
they met all of the following criteria in at least one eye: More than 10
collarettes present on the upper eyelid, at least mild lid margin erythema
and Demodex density of ≥ 1.5 mites per lash (both eyelids combined).
The eye that met all the inclusion criteria was considered as the analysis
eye. If both eyes met all the inclusion criteria, then the analysis eye was
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Contact Lens and Anterior Eye xxx (xxxx) xxx
the eye with the higher Demodex density at the screening visit; if both
eyes had equal Demodex density, the right eye was the analysis eye.
Participants were excluded if they had used any systemic or topical
antibacterial, antiparasitic or anti-inflammatory steroid treatment,
topical TTO or hypochlorous acid treatment of the ophthalmic area or
any lid hygiene products (lid scrubs) in the last 14 days or were un­
willing to forego the use of lid hygiene products during the study. They
were also excluded if they had used a topical prostaglandin analogue
(PGA) to promote eyelash growth, had initiated PGA treatment for
medical reasons within the past 30 days or planned to change or dis­
continue PGA treatment for medical reasons during the study treatment
phase. The use of contact lenses and artificial eyelashes or eyelash ex­
tensions in the last 7 days prior to enrollment were also exclusion
criteria. Pregnant participants and those with lid structural abnormal­
ities, previous surgery of the eyelid margin, acute ocular infection, or
inflammation other than blepharitis, severe dry eye, hypersensitivity to
lotilaner or any of the formulation components were also excluded.
Since there is limited available data on the change in Demodex
density with lotilaner 0.25% or the vehicle control, the sample size
calculation was based on clinical and practical considerations. A sample
size of 60 participants (30 per treatment group) was considered
adequate.
Sixty participants meeting the recruitment criteria were enrolled and
were randomly assigned in a 1:1 ratio to receive either the TP-03 study
medication, a topical ophthalmic solution containing lotilaner, 0.25%
(Tarsus Pharmaceuticals Inc., Irvine, CA) (study group) or the vehicle
formulation without lotilaner (control group), bilaterally.
Both drops- study medication and the vehicle control were supplied
as a 10 mL fill in a 15 cc low-density polyethylene eye dropper bottle.
The label on the bottle specified the participant number, expiration date
and included the statement “for investigational use only” in Spanish.
Once the participant was assigned a participant number, the site
administered the first dose of the study medication from the bottle with
the same participant number. Throughout the study, both the partici­
pant and site personnel performing study assessments were masked to
the study medication.
On the first treatment day, site staff applied the first dose of study
medication or vehicle control, bilaterally. Subsequent doses were
applied bilaterally by the participants, one drop in each eye twice a day,
in the morning and evening. Treatment was discontinued at Day 28.
Participants were followed at Days 7, 14, 28, 60 and 90.
Efficacy parameters included change from baseline in collarette
grade and Demodex density at Day 28. The collarette grading scale used
in the present study (upper and lower eyelids separately) was: 0 = no
collarettes; 1 = 1–10 collarettes per eyelid; 2 = more than 10 but less
than 1/3 of lashes per eyelid have collarettes; 3 = ≥1/3 of lashes but less
than 2/3 have collarettes; or 4 = 2/3 or more of lid lashes have collarettes.
The findings from Gao et al and Hosseini et al guided the development of
the above described collarette grading scale with grades 0 and 1 (10 or
fewer collarettes) being clinically meaningful [35,36]. Demodex density
was assessed by selecting two or more lashes from each of the upper and
lower eyelids, one lash from each half of each lid and were gently rotated
for approximately 10 s, then epilated using fine forceps. When present,
eyelashes with collarettes were intentionally selected; if there was more
than one lash with collarettes, it was randomly selected. With treatment
(generally in the active group), there may have come a time when there
were no more lashes with collarettes. If no collarettes were present,
investigators were asked to epilate one lash from each half of the lid. The
lashes from each lid were placed in artificial tear drops (Refresh Optive®
Advanced or Refresh Optive Mega 3®) on four separate glass slides.
These artificial tear drops contain castor oil, a surfactant (Tween 80) and
glycerin, ingredients which can penetrate the collarette and allow mites
to move and disperse for easier counting. The number of Demodex mites
observed and the number of lashes epilated were recorded.
Safety parameters included assessment of treatment-related adverse
events, changes in corrected distance visual acuity (CDVA), intraocular
R. Gonzalez-Salinas et al. Contact Lens and Anterior Eye xxx (xxxx) xxx

pressure (IOP) and slit-lamp biomicroscopy (to assess the eyelids,
cornea, conjunctiva, anterior chamber, and lens for any pathology and
corneal fluorescein staining (NEI scale)). An adverse event was defined
as any untoward medical occurrence, unintended disease or injury or
any untoward clinical signs (including an abnormal laboratory finding)
whether related to the investigational drug or not. CDVA was performed
using an Early Treatment Diabetic Retinopathy Study chart and was
recorded as logMAR. IOP using applanation tonometry, such as the
Goldmann or Perkins was assessed at all visits. Drop comfort was eval­
uated at Days 7, 14 and 28. Participants were asked to evaluate the
comfort of the drop using the following scale: (1) The drop was very
comfortable, (2) The drop was slightly comfortable, (3) The drop was
neither comfortable or uncomfortable, (4) The drop was slightly un­
comfortable, (5) The drop was very comfortable.
2.1. Statistical analysis
All analyses were conducted using SAS software, version 9.4 (SAS
Institute, Cary, NC). Continuous data were described using descriptive
statistics (i.e., n, mean, standard deviation, and range) and categorical
data were described using the participant count and percentage in each
category. A two-sample t-test or its non-parametric counterpart Wil­
coxon rank-sum test was used as appropriate to assess the statistical
significance of the difference between treatment groups in the efficacy
analysis. Fisher’s exact test was used to analyze the mite eradication
rate. Study and fellow eyes were analyzed separately. Statistical signif­
icance was set at α = 0.05. All participants randomized to either study
drug or vehicle control were included in the analysis; missing data was
not imputed. No adjustments were made for multiple comparisons.
3. Results
Sixty participants who met the recruitment criteria were enrolled in
the study. Fig. 1 represents the participant disposition from enrollment
to each follow-up visit in the study and control group. The mean age of
participants in the study and control group was 59.6 ± 2.1 years and
61.7 ± 1.9 years respectively (range 36 to 81 years). The study group
was 76.7% (n = 23) female, while the control group was 60.0% (n = 18)
female. All participants were Hispanic.
3.1. Collarettes
Fig. 2 A shows the collarette grade for the upper eyelid of the analysis
eye for both study and control groups; the study group showed a sta­
tistically significant decrease in collarette grade compared to the control
group beginning at Day 14 (p = 0.003) and continuing post-treatment
through Day 90 (p < 0.001 at Day 28, 60 and 90). For the lower
eyelid of the analysis eye, the study group showed a statistically sig­
nificant decrease in collarette grade compared to the control group
beginning at Day 28 (p = 0.003) and continuing through Day 60 and 90
(p < 0.001 at Day 60 and 90) (Fig. 2 B). Clinically meaningful collarette
cure (10 or fewer collarettes on the upper eyelid of the analysis eye) was
achieved in 87.5% (21/24) of subjects in the study group at Day 28,
compared to 22.2% (6/27) in the control group (p < 0.001).
3.2. Mite density
The mean mite density at baseline and subsequent post-treatment
visits for the study and control group are shown in Fig. 3. In the anal­
ysis eyes, there was a statistically significant decrease in mean mite
density in the study group compared to the control group at Day 14 and

Fig. 1. Flow diagram of participant disposition at each time point.

3
R. Gonzalez-Salinas et al.
Fig. 2. Mean collarette score in the (A) upper and (B) lower eyelid of the
analysis eye of the study and control groups.
Fig. 3. Mean mite density in the analysis eye of the study and control groups.
all subsequent follow-up visits.
Mite eradication (mite density of 0) was achieved in 66.7% of eyes in
the study group at Day 28, compared to 25.9% in the control group (p =
0.005). At Day 90, these proportions were 68.2% and 18.5% (p = 0.001)
in the study and control group, respectively (Fig. 4).
3.3. Adverse events
No serious adverse events were observed in this study. In the study
group, 5 adverse events were observed that were not related to treat­
ment: mild diarrhea (n = 2); moderate systemic hypertension (n = 1);
cataract surgery for pre-existing cataract (n = 1); and moderate to
marked pharyngotonsillitis (n = 1). In the control group, 6 adverse
events were observed that were not related to treatment: mild common
cold (n = 3), moderate diarrhea (n = 2) and mild diarrhea (n = 1).
4
Contact Lens and Anterior Eye xxx (xxxx) xxx
Fig. 4. Proportion of eyes that achieved mite eradication in the study and
control groups.
3.4. Additional outcomes
There was little or no change in mean CDVA during the study in
either group. No participants in either group demonstrated a decrease in
CDVA greater than two lines of visual acuity (0.2 logMAR). There was
little or no change in IOP through Day 28 in either group. In the study
group, one eye showed mild increase in corneal staining at Day 7 and
another at Day 14. In the control group, four eyes showed mild to
moderate increase in corneal staining at Day 7 to Day 90. In both groups,
no clinically significant changes in slit lamp biomicroscopy findings
were observed.
In the study group, drop comfort was rated as “very/slightly
comfortable” by 17/24 (70.8%), “neither comfortable or uncomfort­
able” by 5/24 (20.8%), and “slightly uncomfortable” by 2/24 (8.3%) at
Day 28. In the control group, drop comfort was rated as “very/slightly
comfortable” by 19/27 (70.4%), “neither comfortable or uncomfortable
by 5/27 (18.5%), and “slightly uncomfortable by 3/27 (11.1%) at Day
28. No subjects in either the control or study group rated the study
medication as “very uncomfortable” at any visit during the study
treatment phase.
4. Discussion
The presence of collarettes is considered a pathognomonic sign of
Demodex blepharitis; therefore, it may be clinically valuable to monitor
the improvement in collarettes after any treatment [4]. However, there
is paucity of studies that have measured collarettes or cylindrical
dandruff as a study outcome in patients with Demodex blepharitis
[3,34,37]. The present study is the first randomized vehicle-controlled
trial to study change in collarette grade in eyes with Demodex ble­
pharitis. A statistically significant decrease in collarette grade in the
study group compared with the control group was found beginning at
Day 14 in the upper eyelid and at Day 28 in the lower eyelid, which was
maintained through Day 90, or at least 2 months after treatment
cessation. These findings confirm the results of our earlier, single-arm
pilot study, in which there was a statistically significant improvement
in collarette grade from Day 14 onward [34].
In the present study, the 28-day treatment with lotilaner also resul­
ted in statistically significant reduction in mean mite density compared
to vehicle control, similarly confirming results of our earlier pilot. The
significant decrease in mite density was sustained for at least two
months following discontinuation of the treatment. Although the control
group also showed a decrease in mean mite density at Day 14, it
increased at Day 28 and this increase was maintained in subsequent
visits.
Previous publications have used a variety of methods for measuring
mite density. While some studies have recorded the number of mites
present per eye or per eyelid [7,20,30,38], others have recorded the
R. Gonzalez-Salinas et al.
number of mites present per patient [29,39]. Given different method­
ologies used across different study populations in the literature, it is
difficult to compare mean mite density across studies.
In the present study, the mite eradication rate was found to be 66.7%
after 28 days of treatment with lotilaner, which was maintained (68.2%)
2 months after treatment cessation. Previously, lid scrubs with T4O/TTO
have been shown to reduce Demodex mite density [3,40,41]. Koo et al.
showed a mean eradication rate of 24% [39]. In another study, the mite
eradication rate was reported to be 36%, when low-concentration
(7.5%) TTO shampoo was used over a period of 4 weeks to reduce the
risks of possible side-effects of TTO [7]. As such, the efficacy of TTO for
mite eradication remains uncertain [4]. The primary side-effects asso­
ciated with TTO treatment are contact dermatitis, ocular irritation, and
allergic reactions [3,4,39]. Further, a recent in vitro study has found
Terpinen-4-ol, a TTO component to be toxic to human meibomian gland
epithelial cells [42].
Recently, there have been speculations that the anti-oxidative and
anti-inflammatory effects of okra may confer therapeutic effects in pa­
tients with blepharitis and dry eye. Liu and Gong evaluated the
anti‑demodectic and therapeutic effects of okra eyelid patch on Demo­
dex blepharitis and reported 40.7% mite eradication [43].
Besides TTO lid hygiene therapy, antiparasitic drugs have also been
used empirically for the treatment of Demodex blepharitis in the recent
past. Oral treatment with 6 mg ivermectin (twice for 1 day and repeated
after 7/14 days), resulted in improvement in symptoms of chronic ble­
pharitis and a mite eradication rate of 35.3% [20,30]. Further, combined
therapy of oral ivermectin and metronidazole (250 mg three times per
day for 2 weeks) was reported to be better than ivermectin alone in
reducing the mite count (<3 mites/eyelash) [29]. In-office, topical
ivermectin-metronidazole combined gel therapy has also recently been
tested in the management of Demodex-associated blepharitis, with good
results [8]. Although studies investigating oral ivermectin and metro­
nidazole for Demodex blepharitis have not reported any side effects, a
number of systemic side effects, such as Mazzotti reaction (swollen and
tender lymph nodes, tachycardia, hypotension, arthralgias, edema and
abdominal pain), Steven-Johnson and Lyell disease (allergic reaction,
characterized by extensive bullous eruption of the skin and mucous
membranes fever, malaise, conjunctivitis, and diffuse erythema), diar­
rhea, dizziness, nausea, allergic reactions, joint pain, and yellowing of
the eyes or skin, have been described after usage of ivermectin or
metronidazole in other parasitic infections [20,44,45].
In the present study, no treatment-related adverse events were
observed. No participants demonstrated clinically significant changes in
CDVA or IOP. These results indicate good tolerability of lotilaner
treatment. Nevertheless, future studies with larger datasets are neces­
sary to validate the tolerability.
In the present study, the lotilaner treatment was administered for 28
days. Mite density decreased further at Day 60 (1 month after the
cessation of the treatment), then slightly increased thereafter. It is pre­
sumed that the late increases in mite density during the follow-up period
may be due to the presence of mite eggs inside hair follicles. Given that
the life cycle of Demodex mite is estimated to range from 14 to 21 days,
extending the lotilaner treatment for at least two mite life cycles should
be considered to ensure adequate reduction in mite density [15].
The symptoms of blepharitis, as with other ocular surface disorders,
often do not correlate with signs of the disease [46]. Therefore, in the
present study, subjective symptoms were not included as inclusion
criteria or as study parameters. This could be considered as a potential
limitation. Future studies are being planned to understand the effect of
treatment with lotilaner ophthalmic solution, 0.25% on collarette
clearance and symptoms of blepharitis, such as lid margin itchiness etc.
along with additional benefit and impact of longer treatment duration
on reduction in mite density in Demodex blepharitis.
In conclusion, from the results of the present phase 2b study, the first
randomized, controlled trial of this novel therapy, it appears that
treatment with lotilaner ophthalmic solution, 0.25% for 4 weeks is safe
5
Contact Lens and Anterior Eye xxx (xxxx) xxx
and effective for the short term treatment of Demodex blepharitis. The
improvement in collarette grade and mite density observed during the
treatment phase persisted for at least two months following cessation of
treatment.
Funding
This study was funded via a grant from Tarsus Pharmaceuticals to
Asociación para Evitar la Ceguera (APEC) Research Department, Mexico
City. RGS and EY have received consulting fees from Tarsus Pharma­
ceuticals. MH and SNB are employees of Tarsus Pharmaceuticals.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
Jan Beiting (Wordsmith Consulting, Cary, North Carolina) and
Raman Bedi, MD (IrisARC - Analytics, Research & Consulting, Chandi­
garh, India) provided editorial assistance in the preparation of this
manuscript.
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