Advertisement

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

Open AccessPublished:July 28, 2021DOI:https://doi.org/10.1016/j.clae.2021.101492

      Abstract

      Purpose

      To evaluate the safety and efficacy of lotilaner ophthalmic solution, 0.25% for the treatment of blepharitis due to Demodex infestation compared to vehicle control.

      Methods

      In this phase II, randomized, controlled, double-masked clinical trial, 60 eligible participants with Demodex blepharitis were randomly assigned in a 1:1 ratio to receive either topical lotilaner ophthalmic solution, 0.25% (Tarsus Pharmaceuticals, Inc., Irvine, CA) (study group) or the vehicle without lotilaner (control 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.

      Keywords

      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 [
      • Amescua G.
      • Akpek E.K.
      • Farid M.
      • Garcia-Ferrer F.J.
      • Lin A.
      • Rhee M.K.
      • et al.
      Blepharitis Preferred Practice Pattern(R).
      ,
      • Dias M.R.
      • Guaresch B.L.V.
      • Borges C.R.
      • Biazim D.F.
      • Casagrande D.
      • Luz R.A.
      Blefarite: epidemiologia, etiologia, apresentações clínicas, tratamento e evolução de nossos pacientes.
      ,
      • Messaoud R.
      • El Fekih L.
      • Mahmoud A.
      • Ben Amor H.
      • Bannour R.
      • Doan S.
      • et al.
      Improvement in ocular symptoms and signs in patients with Demodex anterior blepharitis using a novel terpinen-4-ol (2.5%) and hyaluronic acid (0.2%) cleansing wipe.
      ,

      Savla K, Le JT, Pucker AD. Tea tree oil for Demodex blepharitis. Cochrane Database Syst Rev 2020;6:CD013333. 10.1002/14651858.CD013333.pub2.

      ,
      • Wolffsohn J.S.
      • Arita R.
      • Chalmers R.
      • Djalilian A.
      • Dogru M.
      • Dumbleton K.
      • et al.
      TFOS DEWS II Diagnostic Methodology report.
      ]. The pathogenesis of blepharitis is multifactorial, with infectious, allergic, systemic and environmental components [
      • Putnam C.M.
      Diagnosis and management of blepharitis: an optometrist's perspective.
      ]. Demodex mite infestation has been found to be frequently associated with blepharitis [
      • Karakurt Y.
      • Zeytun E.
      Evaluation of the efficacy of tea tree oil on the density of demodex mites (acari: demodicidae) and ocular symptoms in patients with demodectic blepharitis.
      ]. It has been reported that 42%–81% of blepharitis patients have concomitant infestation with Demodex mites [
      • Avila M.Y.
      • Martinez-Pulgarin D.F.
      • Rizo M.C.
      Topical ivermectin-metronidazole gel therapy in the treatment of blepharitis caused by Demodex spp.: A randomized clinical trial.
      ,

      Suresha A, Sadhwini M. Role of demodex infestation in blepharitis and coconut oil as a treatment option. Indian J Clin Exp Ophthalmol 2020;6:270–5. 10.18231/j.ijceo.2020.058.

      ,
      • Biernat M.M.
      • Rusiecka-Ziolkowska J.
      • Piatkowska E.
      • Helemejko I.
      • Biernat P.
      • Gosciniak G.
      Occurrence of Demodex species in patients with blepharitis and in healthy individuals: a 10-year observational study.
      ,
      • Kabatas N.
      • Dogan A.S.
      • Kabatas E.U.
      • Acar M.
      • Bicer T.
      • Gurdal C.
      The effect of demodex infestation on blepharitis and the ocular symptoms.
      ]. Despite its high prevalence among blepharitis patients, Demodex mite infestation is an often-overlooked cause of chronic blepharitis [
      • Bhandari V.
      • Reddy J.K.
      Blepharitis: always remember demodex.
      ,
      • Zhao Y.E.
      • Wu L.P.
      • Hu L.
      • Xu J.R.
      Association of blepharitis with Demodex: a meta-analysis.
      ,
      • Patel N.V.
      • Mathur U.
      • Gandhi A.
      • Singh M.
      Demodex blepharokeratoconjunctivitis affecting young patients: A case series.
      ]. When assessed in community populations, the prevalence of ocular Demodex reportedly ranges from 16% to 70% [
      • Zhang A.C.
      • Muntz A.
      • Wang M.T.M.
      • Craig J.P.
      • Downie L.E.
      Ocular Demodex: a systematic review of the clinical literature.
      ]. The rate of infestation increases with age, reaching 84% of the population by the age of 60 and 100% in those older than 70 [

      Suresha A, Sadhwini M. Role of demodex infestation in blepharitis and coconut oil as a treatment option. Indian J Clin Exp Ophthalmol 2020;6:270–5. 10.18231/j.ijceo.2020.058.

      ,
      • Liu J.
      • Sheha H.
      • Tseng S.C.
      Pathogenic role of Demodex mites in blepharitis.
      ,
      • Sedzikowska A.
      • Oseka M.
      • Skopinski P.
      The impact of age, sex, blepharitis, rosacea and rheumatoid arthritis on Demodex mite infection.
      ].
      Demodex mites are microscopic ectoparasites of the phylum arthropoda with a semi-transparent, elongated body and four pairs of legs [

      Suresha A, Sadhwini M. Role of demodex infestation in blepharitis and coconut oil as a treatment option. Indian J Clin Exp Ophthalmol 2020;6:270–5. 10.18231/j.ijceo.2020.058.

      ,
      • Zhang A.C.
      • Muntz A.
      • Wang M.T.M.
      • Craig J.P.
      • Downie L.E.
      Ocular Demodex: a systematic review of the clinical literature.
      ,
      • Fromstein S.R.
      • Harthan J.S.
      • Patel J.
      • Opitz D.L.
      Demodex blepharitis: clinical perspectives.
      ]. Demodex folliculorum and Demodex brevis are known to inhabit human skin [

      Suresha A, Sadhwini M. Role of demodex infestation in blepharitis and coconut oil as a treatment option. Indian J Clin Exp Ophthalmol 2020;6:270–5. 10.18231/j.ijceo.2020.058.

      ,
      • Zhang A.C.
      • Muntz A.
      • Wang M.T.M.
      • Craig J.P.
      • Downie L.E.
      Ocular Demodex: a systematic review of the clinical literature.
      ,
      • Luo X.
      • Li J.
      • Chen C.
      • Tseng S.
      • Liang L.
      Ocular demodicosis as a potential cause of ocular surface inflammation.
      ]. These mites tend to gather in the face, cheeks, forehead, nose, and external ear tract, where they find a favorable habitat for breeding and active sebum excretion provides nutrition [
      • Holzchuh F.G.
      • Hida R.Y.
      • Moscovici B.K.
      • Villa Albers M.B.
      • Santo R.M.
      • Kara-Jose N.
      • et al.
      Clinical treatment of ocular Demodex folliculorum by systemic ivermectin.
      ]. 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 [
      • Zhang A.C.
      • Muntz A.
      • Wang M.T.M.
      • Craig J.P.
      • Downie L.E.
      Ocular Demodex: a systematic review of the clinical literature.
      ,
      • Holzchuh F.G.
      • Hida R.Y.
      • Moscovici B.K.
      • Villa Albers M.B.
      • Santo R.M.
      • Kara-Jose N.
      • et al.
      Clinical treatment of ocular Demodex folliculorum by systemic ivermectin.
      ]. D. folliculorum mites consume epithelial cells at the eyelash follicle, induce epithelial hyperplasia and hyperkeratinzation, subsequently leading to the formation of collarettes (cylindrical dandruff) and redness, potential eyelash loss and/or misdirection [
      • Messaoud R.
      • El Fekih L.
      • Mahmoud A.
      • Ben Amor H.
      • Bannour R.
      • Doan S.
      • et al.
      Improvement in ocular symptoms and signs in patients with Demodex anterior blepharitis using a novel terpinen-4-ol (2.5%) and hyaluronic acid (0.2%) cleansing wipe.
      ,
      • Fromstein S.R.
      • Harthan J.S.
      • Patel J.
      • Opitz D.L.
      Demodex blepharitis: clinical perspectives.
      ,
      • Luo X.
      • Li J.
      • Chen C.
      • Tseng S.
      • Liang L.
      Ocular demodicosis as a potential cause of ocular surface inflammation.
      ]. 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 blepharitis [
      • Messaoud R.
      • El Fekih L.
      • Mahmoud A.
      • Ben Amor H.
      • Bannour R.
      • Doan S.
      • et al.
      Improvement in ocular symptoms and signs in patients with Demodex anterior blepharitis using a novel terpinen-4-ol (2.5%) and hyaluronic acid (0.2%) cleansing wipe.
      ,

      Savla K, Le JT, Pucker AD. Tea tree oil for Demodex blepharitis. Cochrane Database Syst Rev 2020;6:CD013333. 10.1002/14651858.CD013333.pub2.

      ,
      • Luo X.
      • Li J.
      • Chen C.
      • Tseng S.
      • Liang L.
      Ocular demodicosis as a potential cause of ocular surface inflammation.
      ]. The pathogenic mechanism of Demodex infestation leads to direct mechanical damage as they burrow into the lash follicle and lay eggs [
      • Fromstein S.R.
      • Harthan J.S.
      • Patel J.
      • Opitz D.L.
      Demodex blepharitis: clinical perspectives.
      ]. Demodex also acts as a vector for bacteria, most notably Staphylococcus aureus and Propionibacterium acnes [
      • Messaoud R.
      • El Fekih L.
      • Mahmoud A.
      • Ben Amor H.
      • Bannour R.
      • Doan S.
      • et al.
      Improvement in ocular symptoms and signs in patients with Demodex anterior blepharitis using a novel terpinen-4-ol (2.5%) and hyaluronic acid (0.2%) cleansing wipe.
      ,
      • Fromstein S.R.
      • Harthan J.S.
      • Patel J.
      • Opitz D.L.
      Demodex blepharitis: clinical perspectives.
      ,
      • Luo X.
      • Li J.
      • Chen C.
      • Tseng S.
      • Liang L.
      Ocular demodicosis as a potential cause of ocular surface inflammation.
      ]. The chemicals excreted by both the Demodex and bacteria induce a hypersensitivity reaction, and thus clinical inflammation which manifests as blepharoconjunctivitis, marginal and phlyctenular keratitis, and ocular rosacea [
      • Luo X.
      • Li J.
      • Chen C.
      • Tseng S.
      • Liang L.
      Ocular demodicosis as a potential cause of ocular surface inflammation.
      ].
      Currently, there are no FDA approved treatments for Demodex blepharitis. A number of proposed treatments, including 1% sulfur ointment [

      Norn MS. Demodex folliculorum. Incidence and possible pathogenic role in the human eyelid. Acta Ophthalmol Suppl 1970;108:7-85. https://www.ncbi.nlm.nih.gov/pubmed/4322592.

      ,
      • Rusiecka-Ziółkowska J.
      • Nokiel M.
      • Fleischer M.
      Demodex-an old pathogen or a new one?.
      ], 1% mercury oxide ointment [
      • Rusiecka-Ziółkowska J.
      • Nokiel M.
      • Fleischer M.
      Demodex-an old pathogen or a new one?.
      ,
      • Bourée P.
      • Bisaro F.
      Le Demodex: un ectoparasite commensal et/ou pathogène.
      ,
      • Fulk G.W.
      • Clifford C.
      A case report of demodicosis.
      ,
      • Rodriguez A.E.
      • Ferrer C.
      • Alio J.L.
      Chronic blepharitis and Demodex.
      ], pilocarpine gel [
      • Fulk G.W.
      • Murphy B.
      • Robins M.D.
      Pilocarpine gel for the treatment of demodicosis–a case series.
      ,
      • Gao Y.Y.
      • Di Pascuale M.A.
      • Li W.
      • Baradaran-Rafii A.
      • Elizondo A.
      • Kuo C.L.
      • et al.
      In vitro and in vivo killing of ocular Demodex by tea tree oil.
      ], and iodized solutions [
      • Gao Y.Y.
      • Di Pascuale M.A.
      • Li W.
      • Baradaran-Rafii A.
      • Elizondo A.
      • Kuo C.L.
      • et al.
      In vitro and in vivo killing of ocular Demodex by tea tree oil.
      ,
      • Gao Y.Y.
      • Di Pascuale M.A.
      • Elizondo A.
      • Tseng S.C.
      Clinical treatment of ocular demodecosis by lid scrub with tea tree oil.
      ], 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 [
      • Messaoud R.
      • El Fekih L.
      • Mahmoud A.
      • Ben Amor H.
      • Bannour R.
      • Doan S.
      • et al.
      Improvement in ocular symptoms and signs in patients with Demodex anterior blepharitis using a novel terpinen-4-ol (2.5%) and hyaluronic acid (0.2%) cleansing wipe.
      ,

      Savla K, Le JT, Pucker AD. Tea tree oil for Demodex blepharitis. Cochrane Database Syst Rev 2020;6:CD013333. 10.1002/14651858.CD013333.pub2.

      ,
      • Zhang A.C.
      • Muntz A.
      • Wang M.T.M.
      • Craig J.P.
      • Downie L.E.
      Ocular Demodex: a systematic review of the clinical literature.
      ,
      • Fromstein S.R.
      • Harthan J.S.
      • Patel J.
      • Opitz D.L.
      Demodex blepharitis: clinical perspectives.
      ,
      • Holzchuh F.G.
      • Hida R.Y.
      • Moscovici B.K.
      • Villa Albers M.B.
      • Santo R.M.
      • Kara-Jose N.
      • et al.
      Clinical treatment of ocular Demodex folliculorum by systemic ivermectin.
      ,
      • Salem D.A.
      • El-Shazly A.
      • Nabih N.
      • El-Bayoumy Y.
      • Saleh S.
      Evaluation of the efficacy of oral ivermectin in comparison with ivermectin-metronidazole combined therapy in the treatment of ocular and skin lesions of Demodex folliculorum.
      ,
      • Filho P.A.
      • Hazarbassanov R.M.
      • Grisolia A.B.
      • Pazos H.B.
      • Kaiserman I.
      • Gomes J.A.
      The efficacy of oral ivermectin for the treatment of chronic blepharitis in patients tested positive for Demodex spp.
      ].
      Lotilaner is a new oral acaricide from the isoxazoline class approved for the treatment of flea and tick infestations in pets [
      • Toutain C.E.
      • Seewald W.
      • Jung M.
      The intravenous and oral pharmacokinetics of lotilaner in dogs.
      ,
      • Young L.
      • Karadzovska D.
      • Wiseman S.
      • Helbig R.
      Efficacy of lotilaner (Credelio) against the adult cat flea, Ctenocephalides felis and flea eggs following oral administration to dogs.
      ,
      • Kuntz E.A.
      • Kammanadiminti S.
      Safety of lotilaner flavoured chewable tablets (Credelio(TM)) after oral administration in cats.
      ]. Isoxazolines 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 locomotor 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 [
      • Quiroz-Mercado H.
      • Ramos-Betancourt N.
      • Corredor-Ortega C.
      • Ceballos J.C.
      • Massaro-Corredor M.G.
      • Baba S.N.
      • et al.
      Pilot Study to Evaluate the Safety and Efficacy of TP-03 for the Treatment of Blepharitis Due to Demodex Infestation (Mars Study).
      ]. 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 density 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 efficacy 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 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 unwilling 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 discontinue PGA treatment for medical reasons during the study treatment phase. The use of contact lenses and artificial eyelashes or eyelash extensions in the last 7 days prior to enrollment were also exclusion criteria. Pregnant participants and those with lid structural abnormalities, 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 participant 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 ⅓ of lashes per eyelid have collarettes; 3 = ≥⅓ of lashes but less than ⅔ have collarettes; or 4 = ⅔ 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 [
      • Gao Y.Y.
      • Di Pascuale M.A.
      • Li W.
      • Liu D.T.
      • Baradaran-Rafii A.
      • Elizondo A.
      • et al.
      High prevalence of Demodex in eyelashes with cylindrical dandruff.
      ,
      • Hosseini K.
      • Bourque L.B.
      • Hays R.D.
      Development and evaluation of a measure of patient-reported symptoms of Blepharitis.
      ]. 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 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 evaluated 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 uncomfortable, (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 Wilcoxon 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 significance 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.
      Figure thumbnail gr1
      Fig. 1Flow diagram of participant disposition at each time point.

      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 statistically 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 significant 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).
      Figure thumbnail gr2
      Fig. 2Mean collarette score in the (A) upper and (B) lower eyelid of the analysis eye of the study and control groups.

      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 analysis eyes, there was a statistically significant decrease in mean mite density in the study group compared to the control group at Day 14 and all subsequent follow-up visits.
      Figure thumbnail gr3
      Fig. 3Mean mite density in the analysis eye of the study and control groups.
      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).
      Figure thumbnail gr4
      Fig. 4Proportion of eyes that achieved mite eradication in the study and control groups.

      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 treatment: 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).

      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 uncomfortable” 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 [

      Savla K, Le JT, Pucker AD. Tea tree oil for Demodex blepharitis. Cochrane Database Syst Rev 2020;6:CD013333. 10.1002/14651858.CD013333.pub2.

      ]. However, there is paucity of studies that have measured collarettes or cylindrical dandruff as a study outcome in patients with Demodex blepharitis [
      • Messaoud R.
      • El Fekih L.
      • Mahmoud A.
      • Ben Amor H.
      • Bannour R.
      • Doan S.
      • et al.
      Improvement in ocular symptoms and signs in patients with Demodex anterior blepharitis using a novel terpinen-4-ol (2.5%) and hyaluronic acid (0.2%) cleansing wipe.
      ,
      • Quiroz-Mercado H.
      • Ramos-Betancourt N.
      • Corredor-Ortega C.
      • Ceballos J.C.
      • Massaro-Corredor M.G.
      • Baba S.N.
      • et al.
      Pilot Study to Evaluate the Safety and Efficacy of TP-03 for the Treatment of Blepharitis Due to Demodex Infestation (Mars Study).
      ,
      • Muntz A.
      • Sandford E.
      • Claassen M.
      • Curd L.
      • Jackson A.K.
      • Watters G.
      • et al.
      Randomized trial of topical periocular castor oil treatment for blepharitis.
      ]. The present study is the first randomized vehicle-controlled trial to study change in collarette grade in eyes with Demodex blepharitis. 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 [
      • Quiroz-Mercado H.
      • Ramos-Betancourt N.
      • Corredor-Ortega C.
      • Ceballos J.C.
      • Massaro-Corredor M.G.
      • Baba S.N.
      • et al.
      Pilot Study to Evaluate the Safety and Efficacy of TP-03 for the Treatment of Blepharitis Due to Demodex Infestation (Mars Study).
      ].
      In the present study, the 28-day treatment with lotilaner also resulted 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 [
      • Karakurt Y.
      • Zeytun E.
      Evaluation of the efficacy of tea tree oil on the density of demodex mites (acari: demodicidae) and ocular symptoms in patients with demodectic blepharitis.
      ,
      • Holzchuh F.G.
      • Hida R.Y.
      • Moscovici B.K.
      • Villa Albers M.B.
      • Santo R.M.
      • Kara-Jose N.
      • et al.
      Clinical treatment of ocular Demodex folliculorum by systemic ivermectin.
      ,
      • Filho P.A.
      • Hazarbassanov R.M.
      • Grisolia A.B.
      • Pazos H.B.
      • Kaiserman I.
      • Gomes J.A.
      The efficacy of oral ivermectin for the treatment of chronic blepharitis in patients tested positive for Demodex spp.
      ,
      • Kim J.H.
      • Chun Y.S.
      • Kim J.C.
      Clinical and immunological responses in ocular demodecosis.
      ], others have recorded the number of mites present per patient [
      • Salem D.A.
      • El-Shazly A.
      • Nabih N.
      • El-Bayoumy Y.
      • Saleh S.
      Evaluation of the efficacy of oral ivermectin in comparison with ivermectin-metronidazole combined therapy in the treatment of ocular and skin lesions of Demodex folliculorum.
      ,
      • Koo H.
      • Kim T.H.
      • Kim K.W.
      • Wee S.W.
      • Chun Y.S.
      • Kim J.C.
      Ocular surface discomfort and Demodex: effect of tea tree oil eyelid scrub in Demodex blepharitis.
      ]. Given different methodologies 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  [
      • Messaoud R.
      • El Fekih L.
      • Mahmoud A.
      • Ben Amor H.
      • Bannour R.
      • Doan S.
      • et al.
      Improvement in ocular symptoms and signs in patients with Demodex anterior blepharitis using a novel terpinen-4-ol (2.5%) and hyaluronic acid (0.2%) cleansing wipe.
      ,

      Evren Kemer Ö, Esra Karaca E, Özek D. Efficacy of cyclic therapy with terpinen-4-ol in Demodex blepharitis: Is treatment possible by considering Demodex’s life cycle? Eur J Ophthalmol 2020:1120672120919085. https://pubmed.ncbi.nlm.nih.gov/32329361/.

      ,
      • Tighe S.
      • Gao Y.-Y.
      • Tseng S.C.
      Terpinen-4-ol is the most active ingredient of tea tree oil to kill Demodex mites.
      ]. Koo et al. showed a mean eradication rate of 24% [
      • Koo H.
      • Kim T.H.
      • Kim K.W.
      • Wee S.W.
      • Chun Y.S.
      • Kim J.C.
      Ocular surface discomfort and Demodex: effect of tea tree oil eyelid scrub in Demodex blepharitis.
      ]. 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 [
      • Karakurt Y.
      • Zeytun E.
      Evaluation of the efficacy of tea tree oil on the density of demodex mites (acari: demodicidae) and ocular symptoms in patients with demodectic blepharitis.
      ]. As such, the efficacy of TTO for mite eradication remains uncertain [

      Savla K, Le JT, Pucker AD. Tea tree oil for Demodex blepharitis. Cochrane Database Syst Rev 2020;6:CD013333. 10.1002/14651858.CD013333.pub2.

      ]. The primary side-effects associated with TTO treatment are contact dermatitis, ocular irritation, and allergic reactions [
      • Messaoud R.
      • El Fekih L.
      • Mahmoud A.
      • Ben Amor H.
      • Bannour R.
      • Doan S.
      • et al.
      Improvement in ocular symptoms and signs in patients with Demodex anterior blepharitis using a novel terpinen-4-ol (2.5%) and hyaluronic acid (0.2%) cleansing wipe.
      ,

      Savla K, Le JT, Pucker AD. Tea tree oil for Demodex blepharitis. Cochrane Database Syst Rev 2020;6:CD013333. 10.1002/14651858.CD013333.pub2.

      ,
      • Koo H.
      • Kim T.H.
      • Kim K.W.
      • Wee S.W.
      • Chun Y.S.
      • Kim J.C.
      Ocular surface discomfort and Demodex: effect of tea tree oil eyelid scrub in Demodex blepharitis.
      ]. Further, a recent in vitro study has found Terpinen-4-ol, a TTO component to be toxic to human meibomian gland epithelial cells [
      • Chen D.
      • Wang J.
      • Sullivan D.A.
      • Kam W.R.
      • Liu Y.
      Effects of Terpinen-4-ol on Meibomian Gland Epithelial Cells In Vitro.
      ].
      Recently, there have been speculations that the anti-oxidative and anti-inflammatory effects of okra may confer therapeutic effects in patients with blepharitis and dry eye. Liu and Gong evaluated the anti‑demodectic and therapeutic effects of okra eyelid patch on Demodex blepharitis and reported 40.7% mite eradication [
      • Liu W.
      • Gong L.
      Anti-demodectic effects of okra eyelid patch in Demodex blepharitis compared with tea tree oil.
      ].
      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 blepharitis and a mite eradication rate of 35.3% [
      • Holzchuh F.G.
      • Hida R.Y.
      • Moscovici B.K.
      • Villa Albers M.B.
      • Santo R.M.
      • Kara-Jose N.
      • et al.
      Clinical treatment of ocular Demodex folliculorum by systemic ivermectin.
      ,
      • Filho P.A.
      • Hazarbassanov R.M.
      • Grisolia A.B.
      • Pazos H.B.
      • Kaiserman I.
      • Gomes J.A.
      The efficacy of oral ivermectin for the treatment of chronic blepharitis in patients tested positive for Demodex spp.
      ]. 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) [
      • Salem D.A.
      • El-Shazly A.
      • Nabih N.
      • El-Bayoumy Y.
      • Saleh S.
      Evaluation of the efficacy of oral ivermectin in comparison with ivermectin-metronidazole combined therapy in the treatment of ocular and skin lesions of Demodex folliculorum.
      ]. In-office, topical ivermectin-metronidazole combined gel therapy has also recently been tested in the management of Demodex-associated blepharitis, with good results [
      • Avila M.Y.
      • Martinez-Pulgarin D.F.
      • Rizo M.C.
      Topical ivermectin-metronidazole gel therapy in the treatment of blepharitis caused by Demodex spp.: A randomized clinical trial.
      ]. Although studies investigating oral ivermectin and metronidazole 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), diarrhea, 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 [
      • Holzchuh F.G.
      • Hida R.Y.
      • Moscovici B.K.
      • Villa Albers M.B.
      • Santo R.M.
      • Kara-Jose N.
      • et al.
      Clinical treatment of ocular Demodex folliculorum by systemic ivermectin.
      ,
      • Pacque M.
      • Munoz B.
      • Greene B.M.
      • White A.T.
      • Dukuly Z.
      • Taylor H.R.
      Safety of and compliance with community-based ivermectin therapy.
      ,
      • Navel V.
      • Mulliez A.
      • Benoist d'Azy C.
      • Baker J.S.
      • Malecaze J.
      • Chiambaretta F.
      • et al.
      Efficacy of treatments for Demodex blepharitis: A systematic review and meta-analysis.
      ].
      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 necessary 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 presumed 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 [
      • Zhang A.C.
      • Muntz A.
      • Wang M.T.M.
      • Craig J.P.
      • Downie L.E.
      Ocular Demodex: a systematic review of the clinical literature.
      ].
      The symptoms of blepharitis, as with other ocular surface disorders, often do not correlate with signs of the disease [
      • Blackie C.A.
      • Korb D.R.
      • Knop E.
      • Bedi R.
      • Knop N.
      • Holland E.J.
      Nonobvious obstructive meibomian gland dysfunction.
      ]. 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 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 Pharmaceuticals. 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, Chandigarh, India) provided editorial assistance in the preparation of this manuscript.

      References

        • Amescua G.
        • Akpek E.K.
        • Farid M.
        • Garcia-Ferrer F.J.
        • Lin A.
        • Rhee M.K.
        • et al.
        Blepharitis Preferred Practice Pattern(R).
        Ophthalmology. 2019; 126: P56-P93https://doi.org/10.1016/j.ophtha.2018.10.019
        • Dias M.R.
        • Guaresch B.L.V.
        • Borges C.R.
        • Biazim D.F.
        • Casagrande D.
        • Luz R.A.
        Blefarite: epidemiologia, etiologia, apresentações clínicas, tratamento e evolução de nossos pacientes.
        Revista Brasileira de Oftalmologia. 2019; 78: 300-303
        • Messaoud R.
        • El Fekih L.
        • Mahmoud A.
        • Ben Amor H.
        • Bannour R.
        • Doan S.
        • et al.
        Improvement in ocular symptoms and signs in patients with Demodex anterior blepharitis using a novel terpinen-4-ol (2.5%) and hyaluronic acid (0.2%) cleansing wipe.
        Clin Ophthalmol. 2019; 13: 1043-1054https://doi.org/10.2147/OPTH.S198585
      1. Savla K, Le JT, Pucker AD. Tea tree oil for Demodex blepharitis. Cochrane Database Syst Rev 2020;6:CD013333. 10.1002/14651858.CD013333.pub2.

        • Wolffsohn J.S.
        • Arita R.
        • Chalmers R.
        • Djalilian A.
        • Dogru M.
        • Dumbleton K.
        • et al.
        TFOS DEWS II Diagnostic Methodology report.
        Ocul Surf. 2017; 15: 539-574https://doi.org/10.1016/j.jtos.2017.05.001
        • Putnam C.M.
        Diagnosis and management of blepharitis: an optometrist's perspective.
        Clin Optom (Auckl). 2016; 8: 71-78https://doi.org/10.2147/OPTO.S84795
        • Karakurt Y.
        • Zeytun E.
        Evaluation of the efficacy of tea tree oil on the density of demodex mites (acari: demodicidae) and ocular symptoms in patients with demodectic blepharitis.
        J Parasitol. 2018; 104: 473-478https://doi.org/10.1645/18-46
        • Avila M.Y.
        • Martinez-Pulgarin D.F.
        • Rizo M.C.
        Topical ivermectin-metronidazole gel therapy in the treatment of blepharitis caused by Demodex spp.: A randomized clinical trial.
        Cont Lens Anterior Eye. 2020; 44: 30084-30089https://doi.org/10.1016/j.clae.2020.04.011
      2. Suresha A, Sadhwini M. Role of demodex infestation in blepharitis and coconut oil as a treatment option. Indian J Clin Exp Ophthalmol 2020;6:270–5. 10.18231/j.ijceo.2020.058.

        • Biernat M.M.
        • Rusiecka-Ziolkowska J.
        • Piatkowska E.
        • Helemejko I.
        • Biernat P.
        • Gosciniak G.
        Occurrence of Demodex species in patients with blepharitis and in healthy individuals: a 10-year observational study.
        Jpn J Ophthalmol. 2018; 62: 628-633https://doi.org/10.1007/s10384-018-0624-3
        • Kabatas N.
        • Dogan A.S.
        • Kabatas E.U.
        • Acar M.
        • Bicer T.
        • Gurdal C.
        The effect of demodex infestation on blepharitis and the ocular symptoms.
        Eye Contact Lens. 2017; 43: 64-67https://doi.org/10.1097/ICL.0000000000000234
        • Bhandari V.
        • Reddy J.K.
        Blepharitis: always remember demodex.
        Middle East Afr J Ophthalmol. 2014; 21: 317-320https://doi.org/10.4103/0974-9233.142268
        • Zhao Y.E.
        • Wu L.P.
        • Hu L.
        • Xu J.R.
        Association of blepharitis with Demodex: a meta-analysis.
        Ophthalmic Epidemiol. 2012; 19: 95-102https://doi.org/10.3109/09286586.2011.642052
        • Patel N.V.
        • Mathur U.
        • Gandhi A.
        • Singh M.
        Demodex blepharokeratoconjunctivitis affecting young patients: A case series.
        Indian J Ophthalmol. 2020; 68: 745-749https://doi.org/10.4103/ijo.IJO_1402_19
        • Zhang A.C.
        • Muntz A.
        • Wang M.T.M.
        • Craig J.P.
        • Downie L.E.
        Ocular Demodex: a systematic review of the clinical literature.
        Ophthalmic Physiol Opt. 2020; 40: 389-432https://doi.org/10.1111/opo.12691
        • Liu J.
        • Sheha H.
        • Tseng S.C.
        Pathogenic role of Demodex mites in blepharitis.
        Curr Opin Allergy Clin Immunol. 2010; 10: 505-510https://doi.org/10.1097/ACI.0b013e32833df9f4
        • Sedzikowska A.
        • Oseka M.
        • Skopinski P.
        The impact of age, sex, blepharitis, rosacea and rheumatoid arthritis on Demodex mite infection.
        Arch Med Sci. 2018; 14: 353-356https://doi.org/10.5114/aoms.2016.60663
        • Fromstein S.R.
        • Harthan J.S.
        • Patel J.
        • Opitz D.L.
        Demodex blepharitis: clinical perspectives.
        Clin Optom (Auckl). 2018; 10: 57-63https://doi.org/10.2147/OPTO.S142708
        • Luo X.
        • Li J.
        • Chen C.
        • Tseng S.
        • Liang L.
        Ocular demodicosis as a potential cause of ocular surface inflammation.
        Cornea. 2017; 36: S9-S14https://doi.org/10.1097/ICO.0000000000001361
        • Holzchuh F.G.
        • Hida R.Y.
        • Moscovici B.K.
        • Villa Albers M.B.
        • Santo R.M.
        • Kara-Jose N.
        • et al.
        Clinical treatment of ocular Demodex folliculorum by systemic ivermectin.
        Am J Ophthalmol. 2011; 151e1https://doi.org/10.1016/j.ajo.2010.11.024
      3. Norn MS. Demodex folliculorum. Incidence and possible pathogenic role in the human eyelid. Acta Ophthalmol Suppl 1970;108:7-85. https://www.ncbi.nlm.nih.gov/pubmed/4322592.

        • Rusiecka-Ziółkowska J.
        • Nokiel M.
        • Fleischer M.
        Demodex-an old pathogen or a new one?.
        Adv Clin Exp Med. 2014; 23: 295-298
        • Bourée P.
        • Bisaro F.
        Le Demodex: un ectoparasite commensal et/ou pathogène.
        Antibiotiques. 2008; 10: 176-182https://doi.org/10.1016/j.antib.2008.08.005
        • Fulk G.W.
        • Clifford C.
        A case report of demodicosis.
        J Am Optom Assoc. 1990; 61: 637-639
        • Rodriguez A.E.
        • Ferrer C.
        • Alio J.L.
        Chronic blepharitis and Demodex.
        Arch Soc Esp Oftalmol. 2005; 80: 635-642https://doi.org/10.4321/s0365-66912005001100004
        • Fulk G.W.
        • Murphy B.
        • Robins M.D.
        Pilocarpine gel for the treatment of demodicosis–a case series.
        Optom Vis Sci. 1996; 73: 742-745https://doi.org/10.1097/00006324-199612000-00004
        • Gao Y.Y.
        • Di Pascuale M.A.
        • Li W.
        • Baradaran-Rafii A.
        • Elizondo A.
        • Kuo C.L.
        • et al.
        In vitro and in vivo killing of ocular Demodex by tea tree oil.
        Br J Ophthalmol. 2005; 89: 1468-1473https://doi.org/10.1136/bjo.2005.072363
        • Gao Y.Y.
        • Di Pascuale M.A.
        • Elizondo A.
        • Tseng S.C.
        Clinical treatment of ocular demodecosis by lid scrub with tea tree oil.
        Cornea. 2007; 26: 136-143https://doi.org/10.1097/01.ico.0000244870.62384.79
        • Salem D.A.
        • El-Shazly A.
        • Nabih N.
        • El-Bayoumy Y.
        • Saleh S.
        Evaluation of the efficacy of oral ivermectin in comparison with ivermectin-metronidazole combined therapy in the treatment of ocular and skin lesions of Demodex folliculorum.
        Int J Infect Dis. 2013; 17: e343-e347https://doi.org/10.1016/j.ijid.2012.11.022
        • Filho P.A.
        • Hazarbassanov R.M.
        • Grisolia A.B.
        • Pazos H.B.
        • Kaiserman I.
        • Gomes J.A.
        The efficacy of oral ivermectin for the treatment of chronic blepharitis in patients tested positive for Demodex spp.
        Br J Ophthalmol. 2011; 95: 893-895https://doi.org/10.1136/bjo.2010.201194
        • Toutain C.E.
        • Seewald W.
        • Jung M.
        The intravenous and oral pharmacokinetics of lotilaner in dogs.
        Parasit Vectors. 2017; 10: 522https://doi.org/10.1186/s13071-017-2475-z
        • Young L.
        • Karadzovska D.
        • Wiseman S.
        • Helbig R.
        Efficacy of lotilaner (Credelio) against the adult cat flea, Ctenocephalides felis and flea eggs following oral administration to dogs.
        Parasit Vectors. 2020; 13: 25https://doi.org/10.1186/s13071-019-3873-1
        • Kuntz E.A.
        • Kammanadiminti S.
        Safety of lotilaner flavoured chewable tablets (Credelio(TM)) after oral administration in cats.
        Parasit Vectors. 2018; 11: 1-8https://doi.org/10.1186/s13071-018-2969-3
        • Quiroz-Mercado H.
        • Ramos-Betancourt N.
        • Corredor-Ortega C.
        • Ceballos J.C.
        • Massaro-Corredor M.G.
        • Baba S.N.
        • et al.
        Pilot Study to Evaluate the Safety and Efficacy of TP-03 for the Treatment of Blepharitis Due to Demodex Infestation (Mars Study).
        Invest Ophthalmol Vis Sci. 2020; 61: 2984
        • Gao Y.Y.
        • Di Pascuale M.A.
        • Li W.
        • Liu D.T.
        • Baradaran-Rafii A.
        • Elizondo A.
        • et al.
        High prevalence of Demodex in eyelashes with cylindrical dandruff.
        Invest Ophthalmol Vis Sci. 2005; 46: 3089-3094https://doi.org/10.1167/iovs.05-0275
        • Hosseini K.
        • Bourque L.B.
        • Hays R.D.
        Development and evaluation of a measure of patient-reported symptoms of Blepharitis.
        Health Qual Life Outcomes. 2018; 16: 11https://doi.org/10.1186/s12955-018-0839-5
        • Muntz A.
        • Sandford E.
        • Claassen M.
        • Curd L.
        • Jackson A.K.
        • Watters G.
        • et al.
        Randomized trial of topical periocular castor oil treatment for blepharitis.
        Ocul Surf. 2020; 19: 145-150https://doi.org/10.1016/j.jtos.2020.05.007
        • Kim J.H.
        • Chun Y.S.
        • Kim J.C.
        Clinical and immunological responses in ocular demodecosis.
        J Korean Med Sci. 2011; 26: 1231-1237https://doi.org/10.3346/jkms.2011.26.9.1231
        • Koo H.
        • Kim T.H.
        • Kim K.W.
        • Wee S.W.
        • Chun Y.S.
        • Kim J.C.
        Ocular surface discomfort and Demodex: effect of tea tree oil eyelid scrub in Demodex blepharitis.
        J Korean Med Sci. 2012; 27: 1574-1579https://doi.org/10.3346/jkms.2012.27.12.1574
      4. Evren Kemer Ö, Esra Karaca E, Özek D. Efficacy of cyclic therapy with terpinen-4-ol in Demodex blepharitis: Is treatment possible by considering Demodex’s life cycle? Eur J Ophthalmol 2020:1120672120919085. https://pubmed.ncbi.nlm.nih.gov/32329361/.

        • Tighe S.
        • Gao Y.-Y.
        • Tseng S.C.
        Terpinen-4-ol is the most active ingredient of tea tree oil to kill Demodex mites.
        Transl Vis Sci Technol. 2013; 2: 2
        • Chen D.
        • Wang J.
        • Sullivan D.A.
        • Kam W.R.
        • Liu Y.
        Effects of Terpinen-4-ol on Meibomian Gland Epithelial Cells In Vitro.
        Cornea. 2020; 39: 1541-1546https://doi.org/10.1097/ICO.0000000000002506
        • Liu W.
        • Gong L.
        Anti-demodectic effects of okra eyelid patch in Demodex blepharitis compared with tea tree oil.
        Exp Ther Med. 2021; 21: 338https://doi.org/10.3892/etm.2021.9769
        • Pacque M.
        • Munoz B.
        • Greene B.M.
        • White A.T.
        • Dukuly Z.
        • Taylor H.R.
        Safety of and compliance with community-based ivermectin therapy.
        Lancet. 1990; 335: 1377-1380https://doi.org/10.1016/0140-6736(90)91253-7
        • Navel V.
        • Mulliez A.
        • Benoist d'Azy C.
        • Baker J.S.
        • Malecaze J.
        • Chiambaretta F.
        • et al.
        Efficacy of treatments for Demodex blepharitis: A systematic review and meta-analysis.
        Ocul Surf. 2019; 17: 655-669https://doi.org/10.1016/j.jtos.2019.06.004
        • Blackie C.A.
        • Korb D.R.
        • Knop E.
        • Bedi R.
        • Knop N.
        • Holland E.J.
        Nonobvious obstructive meibomian gland dysfunction.
        Cornea. 2010; 29: 1333-1345https://doi.org/10.1097/ICO.0b013e3181d4f366