Prospective randomized study to evaluate the efficacy and tolerability of Ectoin® containing Eye Spray (EES09) and comparison to the liposomal Eye Spray Tears Again® (TA) in the treatment of dry eye disease

Open AccessPublished:April 27, 2020DOI:https://doi.org/10.1016/j.clae.2020.04.003

      Abstract

      Purpose

      To subjectively and objectively evaluate the efficacy and tolerability of preservative-free Ectoin ® Eye Spray – Colloidal (EES09) and Tears Again ® Eye Spray (TA) in subjects with mild-moderate dry eye disease (DED), and to compare efficacy of these two eye sprays with each other.

      Methods

      Thirty-six volunteers (average age 32.3 ± 16.1 years; 26 females) were successfully recruited for this prospective double-blind study with between-subject design and randomly divided into two groups (gender and age balanced): Group A received EES09 and Group B received TA during the treatment phase. Inclusioncriteria were a minimum age of 18 years, a score of 18.0 or higher on the OSDI questionnaire, and a non-invasive tear break up time (NIKBUT, Oculus Keratograph M5, Oculus, Germany) of no more than 10s in at least one eye. The objective variables NIKBUT, conjunctival redness, lipid layer and osmolarity (TearLab Cooperation, USA) were assessed at baseline, 10 min. after spray application and after a treatment phase of 10±3 days (3x applications daily). Symptoms, tolerance and handling were evaluated with the OSDI and VAS questionnaires.

      Results

      A statistically significant increase in NIKBUT and improvement in DED symptoms were obtained for the overall group (mean NIKBUT 7.7 ± 1.7s vs. 11.6 ± 4.6s, p<0.001; mean OSDI score: 36.1 ± 12.7 vs. 20. 7± 12.7, p<0.001) during course of treatment. No statistically significant effect was established for the variables lipid layer (p = 0.406), conjunctival redness (p = 0.766) and osmolarity (p = 0.378). No statistically significant differences were observed between the groups, for any variable. The noninferioritycriterion for EES09 towards TA could be shown for the scores of the dry eye symptoms via VAS questionnaire and the variable NIKBUT.

      Conclusions

      A beneficial treatment effect was confirmed for both, symptoms of DED and the objective variable NIKBUT. Both eye sprays were rated favourably in view of perceived tolerability and handling of the spray bottle.

      Keywords

      1. Introduction

      Dry eye disease (DED) affects hundreds of millions of people throughout the world and is one of the most frequent causes of patient visits to eye care practitioners. It is a symptomatic disease, characterized by a vicious cycle of tear film instability and hyperosmolarity, which leads to increased ocular surface inflammation, damage and neurosensory abnormalities. [
      • Craig J.P.
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      • et al.
      TFOS DEWS II report executive summary.
      ] In the classification of DED, the latest evidence supports a scheme based on its pathophysiology in which aqueous deficient dry eye and evaporative dry eye exist as a continuum [
      • Craig J.P.
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      • Azar D.T.
      • Belmonte C.
      • Bron A.J.
      • Chauhan S.K.
      • et al.
      TFOS DEWS II report executive summary.
      ].
      The lipid layer of the tear film has long been recognised to play an important role as it lowers the surface tension at the air interface of the preocular tear film, hence allowing the tear film to spread over the ocular surface, and consequently avoiding evaporation. [
      • Mishima S.
      • Maurice D.M.
      The oily layer of the tear film and evaporation from the corneal surface.
      ,
      • Bron A.J.
      • Tiffany J.M.
      • Gouveia S.M.
      • Yokoi N.
      • Voon L.W.
      Functional aspects of the tear film lipid layer.
      ] Interferometry has shown a fourfold higher rate of tear film evaporation in subjects with no visible or compromised lipid layer quality [
      • Craig J.P.
      • Tomlinson A.
      Importance of the lipid layer in human tear film stability and evaporation.
      ]. The lipid layer is almost entirely derived from meibomian gland secretions and its complete nature is still unknown, but it is likely to have a thinner polar layer of surfactant molecules at the muco-aqueous interface and a thicker non-polar outer layer of lipophilic molecules at the air interface [
      • Bron A.J.
      • Tiffany J.M.
      • Gouveia S.M.
      • Yokoi N.
      • Voon L.W.
      Functional aspects of the tear film lipid layer.
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      • Willcox M.D.P.
      • Argüeso P.
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      • et al.
      TFOS DEWS II tear film report.
      ]. One of the major causes for ocular discomfort and ocular surface abnormality represents meibomian gland dysfunction resulting in abnormal lipid production [
      • Foulks G.N.
      The correlation between the tear film lipid layer and dry eye disease.
      ,
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      Eyelid disorders: the meibomian gland, blepharitis, and contact lenses.
      ,
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      Ocular surface changes and discomfort in patients with meibomian gland dysfunction.
      ].
      Liposomal eye sprays represent an effective therapy option. [
      • Dausch D.
      • Lee S.
      • Dausch S.
      • Kim J.
      • Schwert G.
      • Michelson W.
      [Comparative study of treatment of the dry eye syndrome due to disturbances of the tear film lipid layer with lipid-containing tear substitutes].
      ] These sprays contain phospholipid liposomes, which are spherical vesicles consisting of an aqueous core surrounded by at least one phospholipid bilayer. This liposomal form is used to bring phospholipids to the ocular surface, with the aim to stabilize the lipid layer of the tear film, which is affected in approximately 80 % of the patients with DED [
      • Dausch D.
      • Lee S.
      • Dausch S.
      • Kim J.
      • Schwert G.
      • Michelson W.
      [Comparative study of treatment of the dry eye syndrome due to disturbances of the tear film lipid layer with lipid-containing tear substitutes].
      ]. Several studies have shown significant improvements in tear film quality and dry eye symptoms with their use [
      • Dausch D.
      • Lee S.
      • Dausch S.
      • Kim J.
      • Schwert G.
      • Michelson W.
      [Comparative study of treatment of the dry eye syndrome due to disturbances of the tear film lipid layer with lipid-containing tear substitutes].
      ,
      • Craig J.P.
      • Purslow C.
      • Murphy P.J.
      • Wolffsohn J.S.W.
      Effect of a liposomal spray on the pre-ocular tear film.
      ,
      • Pult H.
      • Gill F.
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      Effect of three different liposomal eye sprays on ocular comfort and tear film.
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      • Hueck A.
      • Wehrmann R.
      Comparison of the clinical efficacy of four different liposomal sprays for the treatment of dry eye.
      ,
      • Lee S.
      • Dausch S.
      • Maierhofer G.
      • Dausch D.
      [A new therapy concept for the treatment of dry eye - the usefulness of phospholipid liposomes].
      ,
      • Khaireddin R.
      • Schmidt K.-G.-G.
      [Comparative investigation of treatments for evaporative dry eye].
      ]. The eye spray is applied to the closed eyelid, and the liposomes reach the tear film during blinking. The liposomal eye spray Tears Again® (TA, Optima Pharmazeutische GmbH) represents such an option to treat mild to moderate evaporative DED.
      Furthermore, a novel preservative-free eye spray, Ectoin® Eye Spray – Colloidal (EES09; bitop AG), was developed, in which phospholipid based colloids serve as a carrier for ectoine. These colloids are droplet like structures with a lipid / triglyceride core surrounded by a monolayer of ambiphilic molecules. Typically, the monolayer consists of phospholipids and optionally, co-surfactants. A unique characteristic of colloids is their small droplet size and homogenous droplet size distribution. Hence, they can be sterile filtered and do not need a preservative.
      Ectoine, ((S)-2-Methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) belonging to the substance class of extremolytes, is known as a natural cell-protective and inflammatory-reducing molecule: [
      • Galinski E.
      • Pfeiffer H.
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      1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid. A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira.
      ,
      • Sydlik U.
      • Gallitz I.
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      • Unfried K.
      The compatible solute ectoine protects against nanoparticle-induced neutrophilic lung inflammation.
      ,
      • Sydlik U.
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      • Keymel S.
      • Krämer U.
      • Weissenberg A.
      • et al.
      Recovery of neutrophil apoptosis by ectoine: a new strategy against lung inflammation.
      ,
      • Unfried K.
      • Kroker M.
      • Autengruber A.
      • Gotić M.
      • Sydlik U.
      The compatible solute ectoine reduces the exacerbating effect of environmental model particles on the immune response of the airways.
      ] It is a compatible water molecule-binding solute (osmoprotectant) produced by microorganisms living in an extremely saline environment [
      • Bownik A.
      • Stępniewska Z.
      Ectoine as a promising protective agent in humans and animals.
      ]. Using ectoine, the microorganisms are able to maintain their physiologic metabolism in the presence of exogenous cell stress like osmotic water loss, UV light or temperature stress, and to protect themselves against chemical and physical noxa [
      • Galinski E.A.
      Compatible solutes of halophilic eubacteria: molecular principles, water-solute interaction, stress protection.
      ]. Ectoine binds strongly to water molecules and forms a hydrate shield that prevents cell dehydration and proteins’ irreversible denaturation [
      • Zaccai G.
      • Bagyan I.
      • Combet J.
      • Cuello G.J.
      • Demé B.
      • Fichou Y.
      • et al.
      Neutrons describe ectoine effects on water H-bonding and hydration around a soluble protein and a cell membrane.
      ,
      • Lentzen G.
      • Schwarz T.
      Extremolytes: natural compounds from extremophiles for versatile applications.
      ]. This mechanism of action grants Ectoin® the ability to stabilize protein and biomembrane structures such as cell membranes and monolayered lipid layer [
      • Harishchandra R.K.
      • Wulff S.
      • Lentzen G.
      • Neuhaus T.
      • Galla H.-J.-J.
      The effect of compatible solute ectoines on the structural organization of lipid monolayer and bilayer membranes.
      ]. The evidence of a protecting and inflammatory-reducing effect of ectoine from preclinical studies has heralded its medical application in the treatment of dermatitis, dry eye syndrome, allergic rhinoconjunctivitis, sinusitis, mucositis, pharyngitis, and lung inflammation [
      • Casale M.
      • Moffa A.
      • Carbone S.
      • Fraccaroli F.
      • Costantino A.
      • Sabatino L.
      • et al.
      Topical ectoine: a promising molecule in the upper airways inflammation—a systematic review.
      ,
      • Eichel A.
      • Bilstein A.
      • Werkhäuser N.
      • Mösges R.
      Meta-analysis of the efficacy of ectoine nasal spray in patients with allergic rhinoconjunctivitis.
      ,
      • Veselovska Z.F.
      • Veselovska N.M.
      • Zherebko I.B.
      • Kukhar N.V.
      Improvement in the functional state of the eye surface in patients with dry eye syndrome under the influence of eye drops based on natural substances.
      ,
      • Drozhzhyna G.I.
      • Troychenko L.F.
      New possibilities for substitution of the lipid layer of the tear film.
      ].
      The aim of this study was to subjectively and objectively evaluate the efficacy and tolerability of EES09 and TA in patients with mild-moderate dry eye disease, and to compare efficacy of these two eye sprays with each other. Subjective variables include the Ocular Surface Disease Index (OSDI) questionnaire [
      • Schiffman R.M.
      Reliability and validity of the ocular surface disease index.
      ] and VAS questionnaires regarding dry eye symptoms as well as tolerability and handling of the eye sprays. Objective variables were non-invasive tear break up time (NIKBUT), tear lipid layer assessment, bulbar conjunctival redness and tear film osmolarity.
      The primary endpoint was to test the difference in subjective and objective variables 10 min after application and after a treatment period of 10 ± 3 days with the eye sprays, in comparison to baseline measurements.
      The secondary endpoint was to compare subjective and objective variables after the treatment period between the two eye sprays.

      2. Methods

      This was a prospective randomized double-blind study with between-subject design. The study followed the tenets of the Declaration of Helsinki, GCP and ISO 14155 and ethical approval was obtained from the Swiss Ethics Committees on research involving humans (study number 2018-00462). The study was listed on clinicaltrials.gov under NCT03519815.
      One hundred and eight subjects were recruited from the patient pool of the optometry clinic at the Institute of Optometry (Olten, Switzerland). All subjects invited to take part in the study were given a subject information sheet explaining the study prior to giving signed consent. Inclusion criteria were a minimum age of 18 years, a score of 18.0 or higher on the OSDI questionnaire [
      • Schiffman R.M.
      Reliability and validity of the ocular surface disease index.
      ], and a NIKBUT of no more than 10 s in at least one eye. Exclusion criteria were post-operative ocular trauma or injury, ocular disease with exception of DED, hypersensitivity to any of the ingredients in the products used for this study, contact lens wear less than 24 h before or on the day of study visits, and use of eye drops / other eye sprays on either visit day or during the duration of the study period.
      Eligible subjects were divided randomly into two groups (group A and group B), whereby it was aimed to balance OSDI for subject age: Group A received EES09 and Group B received TA during the treatment phase. All subjects filled in the OSDI questionnaire before visit 1. Every subject attended the clinic for two appointments, with an interval of 10 ± 3 days between visit 1 and visit 2 (Fig. 1).

      2.1 Visit 1

      All subjects filled in the VAS questionnaire (symptoms) for evaluation of dryness, foreign body and burning / stinging / itching sensations on the day. Subsequently, NIKBUT, lipid layer thickness and conjunctival bulbar redness were evaluated on both eyes (Oculus 5 M Keratograph, Oculus, D-Wetzlar). Upon fulfilment of all inclusion and exclusion criteria, the osmolarity measurement of the tear film was carried out (Tear Lab, TearLab Cooperation, USA) on the eye with the lower NIKBUT value.
      Subjects were demonstrated how to apply the eye spray onto the closed eyelids. Subsequently, a new eye spray bottle for the treatment phase was handed over to the subjects in an opaque, sealed envelope. Any manufacturer’s labelling on the bottle was removed beforehand. Hence, subjects and examiner were blinded to the type of eye spray being dispensed.
      The examiner then left the examination room whilst the subject applied the eye spray onto both closed eyelids, at a distance of 10 cm. Precisely 10 min after the application the subject filled in the VAS (symptoms) questionnaire again, plus one additional question regarding the tolerability of the eye spray. Subsequently, the following objective measurements were repeated, again on both eyes: NIKBUT, lipid layer thickness and conjunctival bulbar redness. Subjects who did not fulfil the NIKBUT inclusion criteria at baseline during visit 1, were excluded from the study.

      2.2 Treatment phase: 10 ± 3 days

      All subjects applied two spray puffs per eye 3x daily (morning / mid-day / evening), for a duration of 10 ± 3 days. They recorded online their daily number and times of spray applications and filled in the VAS questionnaire for evaluation of dry eye symptoms (same as above) every evening at a predetermined time frame.

      2.3 Visit 2

      Appointments were scheduled at the same time of day as visit 1 (max. ± 1 h) and approximately 2 h after the last application of the eye spray. Subjects filled in again the VAS (symptoms) and OSDI questionnaire, as well as one additional VAS questionnaire considering performance and acceptance of the spray. Subsequently, the same measurements were carried out on both eyes analogous to visit 1.

      2.4 NIKBUT, lipid layer thickness and conjunctival bulbar redness measurements

      These measurements were carried out with use of the Oculus 5 M Keratograph (Oculus, D-Wetzlar). NIKBUT represents the measured time in seconds between the full opening of the eyelids after a complete blink and the first break in the tear film, with use of the Oculus 5 M Keratograph (Oculus, D-Wetzlar). Each NIKBUT measurement was carried out three times on each eye, with an interval of a minimum of two min. between each measurement, in order to ensure that the tear film stabilized again after each measurement. Interference patterns of the tear film (lipid layer thickness) were evaluated with white light projected onto the ocular surface, using the Oculus 5 M Keratograph: open meshwork (grade 1), closed meshwork (grade 2), wave (grade 3), amorphous (grade 4), colour fringes (grade 5), globular appearance (grade 6). Conjunctival bulbar redness was graded on the nasal and temporal conjunctiva in 0.1 steps, with use of the Oculus 5 M Keratograph software (grade 0–4; 0 for no redness and 4 for a maximum level of redness).

      2.5 Osmolarity measurement

      The tip of a disposable TearLab chip (TearLab Cooperation, USA) was placed on the lower tear meniscus and the instrument carried out the recording in mOsm/L. Each measurement was carried out three times and the mean value was noted.

      2.6 Ocular Surface Disease questionnaire (OSDI)

      The OSDI questionnaire (12-item scale) was developed to grade the severity of DED. It is used worldwide to discriminate between people with varying levels of ocular surface disease, [
      • Schiffman R.M.
      Reliability and validity of the ocular surface disease index.
      ] and it is accepted by the U.S. Food and Drug Administration (FDA) for use in clinical trials. For this study, a validated German translation of the OSDI questionnaire was applied to fulfil the inclusion criterion that participants were symptomatic of DED, showing an OSDI score of at least 18.0 [
      • Michel M.
      • Sickenberger W.
      • Pult H.
      The effectiveness of questionnaires in the determination of contact lens induced dry eye.
      ]. The official score used to assess dry eye symptoms was: OSDI = (sum of scores) x 25 / (number of questions answered).

      2.7 VAS_questionnaire (symptoms)

      Visual analogue scale with four questions for evaluation of dry eye symptoms: dryness, foreign body and burning, stinging, itching sensations on the day: for each on a scale of 0–10; 0 for no symptoms and 10 for the highest possible level of symptoms. After spray application during visit 1, one additional question No 5 regarding the tolerability of the eye spray was added (with a score of 0–10; 0 for no discomfort and 10 for highest level of discomfort).

      2.8 VAS_ questionnaire (spray evaluation: performance and acceptance of the spray)

      Visual analogue scale for evaluation of the eye sprays (on a scale of 0–10): Questions 1−3 for burning sensation, ocular comfort and tolerability of the spray application (0 for ‘no symptoms’, 10 for the ‘highest possible level of symptoms’); Question 4 for tolerability, if contact lenses were worn during the treatment phase; Questions 5 and 6 for sensation / tolerability of smell of eye spray (0 for ‘smell not noticeable’ / ‘extremely unpleasant’, 10 for ‘extremely strong’ / ‘extremely pleasant’); Question 7 for handling of the eye spray (0 for ‘extremely difficult’, 10 for ‘extremely easy’); Question 8 for improvement of dry eye symptoms with spray application (0 for ‘no improvement’, 10 for ‘extremely easy’); Question 9 for intention to carry on with the use of the eye spray after completion of the study (0 for ‘never’, 10 for ‘always’).

      2.9 Investigational products

      • “Ectoin® Eye Spray – Colloidal” (EES09; bitop AG) – CE marked medical device; Ingredients: Ectoin®, Soy-Lecithin, Vitamin A, Vitamin E, water, physiological buffer system
      • Liposomal eye spray: Tears Again® (TA, Optima Pharmazeutische GmbH) – CE marked medical device; Ingredients: Soy-Lecithin, Sodium Chloride, Ethanol, Phenoxyethanol, Vitamin A-Palmitate, Vitamin E, Aqua purificata

      2.10 Statistical analysis

      No standard tool for the power calculation such as G-Power could be applied for the sample size calculation of this study. This can be explained by the repeated measurements on the subjects’ eyes, resulting in a complicated dependence (correlation vs. covariance). Hence, a Monte-Carlo procedure was applied (alpha = 0.05, power = 0.80), which resulted in 10 subjects for the primary endpoint and 16 subjects for the secondary endpoint per group, for the variable NIKBUT, assuming that each measurement was carried out 3 times. For better balancing, the sample size was increased to 18 per group.
      An important part of this study is dedicated to demonstrate non-inferiority of the product compared to the established product. In order to do so, a non-inferiority threshold of 33 % was estimated to be adequate.
      The statistical analysis was carried out with R (v. 3.5.3, R Foundation). Package “nlme” was applied for repeated measures ANOVA, i.e. the statistical analysis was primarily carried out with mixed-effects linear models. Time (baseline, 10 min. after treatment and after treatment phase) was treated as an ordered factor. The study arm was treated as a factor. Both, TIME and ARM were independent factors. Variables to be tested, such as NIKBUT, were dependent variables.
      For NIKBUT, the data of the eye with the lower mean NIKBUT value at baseline (visit 1) were statistically analysed. Osmolarity measurements were only carried out and analysed on the eye with the lower NIKBUT value at baseline. For the other two variables (conjunctival redness and lipid layer thickness), the mean values of the two eyes of each subject were included in the analysis.

      3. Results

      Of the 106 recruited subjects, only 36 fulfilled the inclusion criteria and consequently completed this clinical study. This considerable dropout can be explained by the NIKBUT criterion of a maximum value of 10 s in at least one eye, in order to participate in this study. Twenty six subjects were female (13 females in each treatment group and the overall average age was 32.3 ± 16.1 years. For Group A (receiving “Ectoin® Eye Spray – Colloidal”), average age was 34.1 ± 18.6 years, and for Group B (receiving Tears Again®), average age was 30.1 ± 12.5 years.
      Descriptive data are summarised in Table 1, Table 2.
      Table 1Descriptive data for the overall group: mean values ± standard deviation and Median / interquartile ranges.
      BaselineVisit 1: after treatmentVisit 2
      OSDIMean ± SD36.1 ± 12.7N/A20.7 ± 12.7
      Median / IR33.7 /

      26.6−40.6
      N/A19.7 /

      11.4–27.3
      VAS (symptoms)

      questions 1−4
      Mean ± SD2.9 ± 1.91.7 ± 1.51.1 ± 1.3
      Median / IR2.5 / 1.5 - 4.21.4 / 0.8 - 2.00.9 /

      0.4–1.6
      VAS (symptoms) question 5Mean ± SDN/A0.4 ± 1.10.6 ± 1.1
      Median / IRN/A0.0 / 0.0 - 0.00.0 / 0.0–1.0
      NIKBUTMean ± SD7.7 ± 1.79.3 ± 2.811.6 ± 4.6
      Median / IR8.1 / 6.7−8.88.9 / 7.6−10.211.0 / 9.1−13.1
      Lipid layerMean ± SD3.2 ± 1.003.2 ± 1.02.9 ± 1.0
      Median / IR3.0 / 3.0−3.23.0 / 3.0−4.03.0 / 2.0−3.0
      Conjunctival bulbar rednessMean ± SD1.00 ± 0.431.00 ± 0.411.00 ± 0.45
      Median / IR0.90 / 0.70−1.220.95 / 0.70−1.200.95 / 0.67−1.12
      Osmolarity [mOsm/L]Mean ± SD311 ± 8.02N/A309 ± 10.88
      Median / IR309 / 306−316M/A310 / 302−315
      Table 2Descriptive data for group A and B: mean values ± standard deviation and Median / interquartile ranges.
      BaselineVisit 1: after treatmentVisit 2
      GROUP A: EES09GROUP B:

      Tears Again
      GROUP A

      EES09
      GROUP B

      Tears Again
      GROUP A

      EES09
      GROUP B

      Tears Again
      OSDIMean ± SD33.2 ± 10.639.0 ± 14.1N/AN/A17.7 ± 9.823.6 ± 14.8
      Median / IR30.6 / 25.6 - 36.337.0 /

      27.7 - 49.4
      N/AN/A14.2 /

      11.4 - 24.4
      21.8 /

      12.3–32.4
      VAS (symptoms)

      questions 1−4
      Mean ± SD2.9 ± 1.72.9 ± 2.01.7 ± 1.71.7 ± 1.21.1 ± 1.61.1 ± 0.8
      Median / IR2.9 / 2.0 - 3.82.5 / 1.5 - 4.51.1 / 0.6–1.91.6 / 0.8 - 2.00.8 / 0.1 - 1.20.9 / 0.5–1.7
      VAS (symptoms)

      question 5
      Mean ± SDN/AN/A0.6 ± 1.40.2 ± 0.50.6 ± 1.30.5 ± 0.9
      Median / IRN/AN/A0.0 / 0.0 - 0.80.0 / 0.0 - 0.00.0 / 0.0 - 0.00.0 / 0.0–1.0
      NIKBUT [s]Mean ± SD7.9 ± 1.57.4 ± 1.99.5 ± 2.69.0 ± 2.912.0 ± 4.611.3 ± 4.7
      Median / IR7.9 /

      7.0 - 9.0
      8.2 /

      5.9 - 8.6
      9.2 /

      8.0 - 10.6
      8.6 /

      7.5 - 10.0
      11.3 /

      10.1 - 12.5
      10.9 /

      9.0–13.8
      Lipid layerMean ± SD2.9 ± 0.93.4 ± 1.03.0 ± 1.03.5 ± 0.92.7 ± 1.23.1 ± 0.7
      Median / IR3.0 / 2.6 - 3.03.0 / 3.0 - 4.03.0 / 3.0−3.03.0 / 3.0 - 4.03.0 / 2.0 - 3.03.0 / 3.0−3.0
      Conjunctival bulbar rednessMean ± SD1.0 ± 0.51.0 ± 0.41.1 ± 0.40.9 ± 0.41.1 ± 0.40.9 ± 0.5
      Median / IR1.0 /

      0.7−1.2
      0.9 /0.7−1.31.1 / 0.8–1.20.8 / 0.6–1.11.1 / 0.8–1.10.7 / 0.6–1.2
      Osmolarity [mOsm/L]Mean ± SD310.3 ± 7.9311.3 ± 8.4N/AN/A307.7 ± 10.5310.5 ± 11.3
      Median / IR308.0 /

      304.0–316.1
      309.2 /

      306.4 - 316.6
      N/ANA/304.8 /

      300.2 - 311.9
      310.5 /

      302.8 - 317.9

      3.1 Non-invasive Tear-Breakup-Time (NIKBUT)

      The measurements show that the averaged NIKBUT increased during the course of the study (baseline, 10 min. after spray application during visit 1, and at visit 2) for the overall group to a statistically significant degree (mean difference NIKBUT = 3.98 s; p < 0.001, 95 % CI [2.92 s; 5.04 s], Table 1, Fig. 2). This means that the model estimates 7.53 s for baseline, 9.52 s for 10 min after treatment at visit 1, and finally 11.5 s for after the treatment at visit 2. Based on the specific statistical analysis carried out and described above, the treatment effect observed 10 min. after spray application (step one) was about as large as the effect recorded during the rest of treatment phase (step two).
      Fig. 2
      Fig. 2Boxplot of NIKBUT values for the overall subject group before treatment, 10 min. after spray.
      From the analysis of the statistical model it was concluded that only the linear term of TIME was statistically significant. Hence the results show the effects per TIME step, where one TIME step means the step from either “baseline” to “10 min. after application at visit 1” or “10 min. after application visit 1” to “at visit 2”. As the quadratic term of TIME was not significant, it could be concluded that the estimate for the effects for the two time steps were equal.
      Boxplots illustrate NIKBUT measurements for the individual Groups A and B (Fig. 3): The increase in NIKBUT was statistically significant for each individual group (Group A: difference NIKBUT = 4.12 s; p < 0.001, 95 % CI [2.66 s;5.56 s]; Group B: difference NIKBUT = 3.84 s p < 0.001, 95 % CI [2.28 s;5.40 s]. However, no significant differences between the two Groups A and B with regards to effect of eye spray application could be observed (p = 0.411). Instead, the non-inferiority criterion of Group A treatment defined by a treatment effect that is not smaller than 67 % for Group B treatment, could be confirmed: The effect of treatment for the arm ‘Group B’ with respect to the variable ‘NIKBUT’ was estimated to be 3.84 s, with a 33 % limit of 2.57 s.
      Fig. 3
      Fig. 3Boxplot of NIKBUT values for the individual groups A and B before treatment, 10 min. after spray.
      It may be noteworthy to mention that the increase in NIKBUT is statistically significant for the step from “before treatment” to “10 min. after application”. This holds true for both individual groups (group A: p = 0.0045; group B: p = 0.0077), as well as for the overall group (p = 0.0001). Effect sizes are half the amounts mentioned in the preceding paragraph and can be considered to be clinically relevant improvements of NIKBUT.

      3.2 OSDI and VAS symptoms questionnaires

      The OSDI score reduced significantly during the course of the study for the overall group of subjects participating in this study (p < 0.001; with an estimated effect of 95 % confidence interval (CI) for difference in OSDI score [-19.7; -11.2]), whereby no significant difference could be observed between Group A and Group B (p = 0.988). The reduction in OSDI score was also statistically significant for the individual groups A and B (Group A: mean difference of -15.5, p < 0.001, with a 95 % CI for difference in OSDI score [-20.1; -10.8]; Group B: mean difference of -15.4, p < 0.001, with a 95 % CI for difference in OSDI score [-22.9; -7.86]). The mean and median scores (Fig. 4 and Table 2) as well as variability, were higher for Group B at all measurement time points.
      Fig. 4
      Fig. 4Boxplots for OSDI scores for Group A and Group B at baseline (before treatment) and during visit.
      The scores for VAS symptoms (evaluation of dry eye symptoms: dryness, foreign body and burning / stinging / itching sensations on the day) also reduced significantly during the course of the study for the overall group of subjects participating in this study (p < 0.001, with an estimated effect of 95 % confidence interval (CI) for difference in VAS score [-2.20; -1.36], according to the linear mixed-effect model using polynomial contrasts, reflecting the nature of the ordered factor ‘time’). The treatment effect was estimated at -0.891 score points for each treatment (during visit 1 and treatment phase), resulting in a total of -1.78 score points from baseline to visit 2. No significant differences could be observed between Group A and Group B (p = 0.987), however the difference in VAS score was statistically significant for the individual Groups A and B. The mean and median scores (Fig. 5 and Table 2) as well as variability, were higher for Group B during all measurement points.
      Fig. 5
      Fig. 5Scores for VAS symptoms (including dryness, foreign body and burning, stinging, itching.
      Question No 5 of the VAS symptoms questionnaire rated subjectively perceived tolerability of the spray 10 min. after application during visit 1 and at the end of the study during visit 2. No statistically significant changes were observed between the two timepoints for the overall group (p = 0.838). No significant differences could be observed between the two groups at either visit (p = 0.370 and p = 0.564, respectively).
      A statistically significant trend towards lower daily scores for VAS symptoms (questions 1−4) during the treatment phase could be observed during the course of the treatment phase (p < 0.001, Fig. 5, whereby no significant difference could be observed between Group A and Group B (p = 0.922).
      The subjectively perceived tolerability of the spray was recorded 10 min. after application during visit 1 and at the end of the study during visit 2. No statistically significant changes were observed between the two timepoints for the overall group (p = 0.838). No significant differences could be observed between the two groups at either visit (p = 0.370 and p = 0.564, respectively), both sprays were tolerated well by the subjects.
      Fig. 6 summarises the pooled online scores and those during visits for questions 1−4. An additional statistical analysis showed that the treatment effect for Group A was not inferior to the Group B treatment with a 20 % effect threshold: The 80 % effect of the VAS symptoms questionnaire for Group B was -1.062 and the 95 % CI for the VAS symptoms in Group A was [–1.918−1.064]. Thus, the non-inferior limit of Group B is above the upper limit of the 95 % confidence interval of Group A.
      Fig. 6
      Fig. 6Pooled scores for online data and those during visits for VAS I (average scores for questions.

      3.3 VAS (spray performance and acceptance evaluation)

      Table 3 summarises the score results for the VAS (spray performance and acceptance evaluation). For questions 1−3, the summarised score indicates the comfort perceived by application of the eye spray (0 for extremely uncomfortable, 10 for extremely comfortable).
      Table 3Result scores for VAS on spray performance and acceptance evaluation (statistically significant p-value in bold).
      Overall groupGroup AGroup Bp-value

      Group A vs B
      Questions 1−3

      Comfort application
      Mean ± SD3.2 ± 1.73.4 ± 1.82.9 ± 1.60.329
      Median / IR3.5 /

      2.4−4.5
      3.8 / 3.1−4.93.5 / 2.1−4.0
      Question 4 (n = 8)

      Comfort application
      Mean ± SD2.62 ± 1.602.25 ± 1.503.0 ± 1.80.552
      Median / IR2.5 /

      1.0−4.0
      2.0 /

      1.0 – 3.3
      3.0 /

      1.8–4.3
      Question 5s

      Smell of spray
      Mean ± SD4.1 ± 3.52.78 ± 3.325.50 ± 3.130.019
      Median / IR2.5 / 1.0−7.01.5 / 0−5.86.0 / 2.3−7.8
      Question 6

      Smell pleasant?
      Mean ± SD6.9 ± 3.37.9 ± 3.55.9 ± 2.70.0576
      Median / IR7.5 / 4.8−9.39.0 / 5.8−11.06.0 / 3.5−7.8
      Question 7

      Handling
      Mean ± SD9.2 ± 1.09.4 ± 0.99.0 ± 1.10.275
      Median / IR10.0 / 8.0−10.010.0 / 9.0−10.09.0 / 8.0−10.0
      Question 8

      Improvement symptoms
      Mean ± SD6.4 ± 2.56.6 ± 2.76.2 ± 2.30.543
      Median / IR7.0 / 5.0−8.07.0 / 5.3−8.87.0/5.0−8.0
      Question 9

      Continue spray use?
      Mean ± SD6.3 ± 2.96.6 ± 3.35.9 ± 2.50.201
      The ratings only differed to a statistically significant degree between Group A and B for question 5, indicating that the spray ‘TA’ was perceived to have a stronger smell. There was a tendency towards the smell perceived as more pleasant for Group A, however this difference was not statistically significant (p = 0.0576).
      When combining the scores for questions 1−3, 5–7 and 9 (a higher score indicating better performance / acceptance), Group A exhibited a better performance (p = 0.019, Fig. 7).
      Fig. 7
      Fig. 7Combined VAS score for evaluation for subjective performance and acceptance.

      3.4 Lipid Layer

      No statistically significant change in lipid layer interference patterns was observed, neither for the overall group (p = 0.406, Table 1), nor between Group A and B (p = 0.082; Table 2).

      3.5 Conjunctival bulbar redness

      No statistically significant change in conjunctival bulbar redness was observed, neither for the overall group (p = 0.766, Table 1), nor between Group A and B (p = 0.343; Table 2).
      Osmolarity
      No significant dependency on treatment could be observed for osmolarity, neither for the overall group (p = 0.378), nor between the individual Groups A and Groups B (p = 0.458).

      4. Discussion

      Various clinical trials have shown a significant beneficial effect of ectoine as the key ingredient in different eye drops for the treatment of dry eyes: Veselovska et al. carried out a prospective clinical study with use of 0.5 % Ectoin® eye drops on 60 subjects, in comparison to no treatment and treatment with synthetic artificial tears. [
      • Veselovska Z.F.
      • Veselovska N.M.
      • Zherebko I.B.
      • Kukhar N.V.
      Improvement in the functional state of the eye surface in patients with dry eye syndrome under the influence of eye drops based on natural substances.
      ] They observed significant improvements in respect to conjunctival redness and ocular irritation in the group that used the Ectoin® eye drops. Drozhzhyna and Troychenko also reported an improvement in dry eye symptoms (OSDI) and with respect to TBUT with use of 0.5 % Ectoin® eye drops on 21 subjects with moderately dry eyes. [
      • Drozhzhyna G.I.
      • Troychenko L.F.
      New possibilities for substitution of the lipid layer of the tear film.
      ] Dwivedi and colleagues showed that ectoine is able to stabilize the meibomian lipid film against shear stress as occurs with blinking of the eye lids [
      • Dwivedi M.
      • Backers H.
      • Harishchandra R.K.
      • Galla H.-J.-J.
      Biophysical investigations of the structure and function of the tear fluid lipid layer and the effect of ectoine. Part A: natural meibomian lipid films. Biochimica et Biophysica Acta (BBA) -.
      ]. Based on these literature findings, a general improvement of the DED symptoms after use of an Ectoin® eye spray was expected.
      A considerable number of published studies have also shown a significant beneficial effect of regular eye spray application on the quality of the lipid layer of the tear film: Lee et al. carried out a prospective double-blind study with use of the eye spray TA and physiological saline as comparative product (placebo) on 382 subjects. [
      • Lee S.
      • Dausch S.
      • Maierhofer G.
      • Dausch D.
      [A new therapy concept for the treatment of dry eye - the usefulness of phospholipid liposomes].
      ] They found significant improvements in lid-parallel conjunctival folds (LIPCOF), tear film break up time (TBUT), with Schirmer I test and in level of inflammation, as well as subjective improvements in symptoms. In their prospective double-blind study with use of the eye spray TA and physiological saline as comparative product (placebo), Craig et al. obtained a statistically significant improvement in respect to NIBUT, lipid layer thickness and tear meniscus height, with a study sample size of 22 subjects [
      • Craig J.P.
      • Purslow C.
      • Murphy P.J.
      • Wolffsohn J.S.W.
      Effect of a liposomal spray on the pre-ocular tear film.
      ]. Dausch et al. investigated the use of TA in comparison to Carbomerum 980 eye gel (Liposic) on 74 subjects (Prospective randomised multi-center cross-over study) [
      • Dausch D.
      • Lee S.
      • Dausch S.
      • Kim J.
      • Schwert G.
      • Michelson W.
      [Comparative study of treatment of the dry eye syndrome due to disturbances of the tear film lipid layer with lipid-containing tear substitutes].
      ]. They observed greater significant improvements with TA with respect to LIPCOF, TBUT, Schirmer and level of inflammation in comparison to Liposic eye gel. Khaireddin and Schmidt published a prospective randomized bi-center study where they used TA and an artificial tear product based on hyaluronic acid (Vismed light, eye drops) on 216 subjects [
      • Khaireddin R.
      • Schmidt K.-G.-G.
      [Comparative investigation of treatments for evaporative dry eye].
      ]. They noted greater significant improvements with TA with respect to grade of inflammation, NIBUT and LIPCOF. Pult et al. confirmed these findings of lipid based eye sprays and designed a prospective double-blind multi-center study, comparing three different eye sprays (TA, DryEyesMist und TearMist) on 80 subjects. Significant improvements in respect to subjective comfort and NIBUT was observed only for TA [
      • Pult H.
      • Gill F.
      • Riede-Pult B.
      Effect of three different liposomal eye sprays on ocular comfort and tear film.
      ]. Hueck and Wehrmann also published a prospective randomised clinical study with use of four different eye sprays (Ocuvers Hyaluron, Ocuvers Lipostamin, TA and TA Sensitive) on 166 subjects [
      • Hueck A.
      • Wehrmann R.
      Comparison of the clinical efficacy of four different liposomal sprays for the treatment of dry eye.
      ]: Compared to results of the other clinical investigations, they observed significantly more short-term burning with use of TA than with the other eyelid sprays. Subjective comfort and TBUT were found to be significantly better with Ocuvers Hyaluron and Ocuvers Lipostamin than with TA and TA Sensitive. Conjunctival bulbar redness was significantly better with Ocuvers Lipostamin, compared to when treated with the other eyelid sprays.
      This study discussed here evaluated the efficacy and tolerability of two eye sprays, the preservative-free Ectoin® Eye Spray (EES09) and TA in patients with mild-moderate DED, and subsequently compared the efficacy of these two eye sprays with each other. The results confirm a beneficial treatment effect of these two eye sprays for both symptoms of DED and the objective variable NIKBUT. As expected from previous clinical data, DED symptoms improved in both subject groups progressively during the course of this study, to similar degrees in both study groups, i.e. the improvement effect was cumulative.
      NIKBUT measurement represents an indirect measure of tear film stability. Hence, the increase in NIKBUT recorded in this study indicated an improvement in tear film stability, which was similar in the two study groups. This improvement in an objective variable rules out a positive subjective evaluation purely based on a potential placebo effect. Similarly to symptoms, the NIKBUT improvement effect was cumulative, increasing during the course of the study. This suggests that for optimal treatment effect, the eye sprays should be recommended for use on a regular basis (ideally daily), rather than sporadically.
      Whilst the findings tended towards being more favourable for the treatment with EES09 than for TA, most of the differences were not statistically significant. However, the non-inferiority criterion for EES09 towards TA could be shown for both the scores of the dry eye symptoms via VAS questionnaire and the objective variable NIKBUT.
      The lipid layer of the tear film plays an important role, allowing the tear film to spread over the ocular surface, and consequently avoiding evaporation. Craig et al. observed the greatest increase in tear lipid layer thickness 30−60 min. after spray application, and after 135 min. it had returned to baseline level. [
      • Craig J.P.
      • Purslow C.
      • Murphy P.J.
      • Wolffsohn J.S.W.
      Effect of a liposomal spray on the pre-ocular tear film.
      ] This may explain, why no treatment effect with respect to the lipid layer could be observed in this study: Ten minutes after spray application being possibly too early (visit 1), and two hours after application being possibly too late (visit 2). Some researchers have questioned a significant correspondence between DED and lipid layer thickness and suggest that the lipid is a poor barrier to evaporation [
      • King-Smith P.E.
      • Reuter K.S.
      • Braun R.J.
      • Nichols J.J.
      • Nichols K.K.
      Tear film breakup and structure studied by simultaneous video recording of fluorescence and tear film lipid layer images.
      ], which may explain the discrepancy found between NIKBUT and lipid measurement changes obtained during the course of this study.
      Conjunctival redness represents the most common clinical sign, suggestive of ocular surface inflammation. [
      • Papas E.B.
      Key factors in the subjective and objective assessment of conjunctival erythema.
      ,
      • Fieguth P.
      • Simpson T.
      Automated measurement of bulbar redness.
      ,
      • Amparo F.
      • Wang H.
      • Emami-Naeini P.
      • Karimian P.
      • Dana R.
      The Ocular Redness Index: a novel automated method for measuring ocular injection.
      ] Indicative of treatment success, an improvement was expected in this subject group. However, conjunctival redness levels were already low at baseline in this subject group, possibly explaining the lack of difference.
      Tear hyperosmolarity, along with tear instability, was confirmed by the TFOS DEWS I and II workshops as one of the core drivers of DED. [
      • Craig J.P.
      • Nelson J.D.
      • Azar D.T.
      • Belmonte C.
      • Bron A.J.
      • Chauhan S.K.
      • et al.
      TFOS DEWS II report executive summary.
      ,
      • Lemp M.A.
      • Baudouin M.C.C.
      • Baum P.J.
      The definition and classification of dry eye disease: report of the definition and classification subcommittee of the International Dry Eye Workshop.
      ] Osmolarity has been shown to generally increase with disease severity [
      • Willcox M.D.P.
      • Argüeso P.
      • Georgiev G.A.
      • Holopainen J.M.
      • et al.
      TFOS DEWS II tear film report.
      ]: It is considered as normal up to 302 ± 8.3 mOsm/L, 315.0 ± 11.4 mOsm/L has been found to indicate mild-to-moderate DED, whereas 336.4 ± 22.3 mOsm/L classifies severe DED. Furthermore, variability in measurements between eyes and visits has been observed to increase as the disease process progresses [
      • Tomlinson A.
      • Khanal S.
      • Ramaesh K.
      • Diaper C.
      • McFadyen A.
      Tear film osmolarity: determination of a referent for dry eye diagnosis invest.
      ,
      • Keech A.
      • Senchyna M.
      • Jones L.
      Impact of time between collection and collection method on human tear fluid osmolarity.
      ]. In view of the subjective and objective improvements obtained in this study, it seems counterintuitive that osmolarity levels did not improve during the course of the treatment phase, for either subject group. The baseline osmolarity values at 311 ± 8.02mOsm/L for the overall group confirmed the presence of mild-to-moderate DED in the subject group recruited. One possible reason may represent the timepoint of measurement two hours after last application as not ideal, as the momentary effect of the spray may have already worn off. Interestingly, Larmo et al. did not report any osmolarity change one hour after application of two lid sprays (TA and sea buckthorn oil spray emulsion, respectively) either [
      • Larmo P.
      • Järvinen R.
      • Laihia J.
      • Löyttyniemi E.
      • Maavirta L.
      • Yang B.
      • et al.
      Effects of a sea buckthorn oil spray emulsion on dry eye.
      ].
      Both eye sprays were rated favourably in view of tolerability and handling. The smell of TA was perceived as stronger. However, the majority of subjects expressed an interest to continue the use of the individual eye spray after completion of the study.

      5. Conclusions

      A beneficial treatment effect for both eye sprays tested in this study was confirmed for both symptoms of DED and NIKBUT. Whilst the findings tended towards being more favourable for the treatment with EES09 than for TA, most of them were not statistically significant. However, the non-inferiority criterion for EES09 towards TA could be shown for both, the scores of the DED symptoms via VAS questionnaire and the objective variable NIKBUT. The subjects stated a favourable handling and tolerability for both eye sprays and reported a stronger smell of TA. Both eye sprays, the established TA as well as the ectoine based colloid eye spray may represent a valid alternative in the treatment regimen of DED.

      Declaration of Competing Interest

      This study was sponsored by bitop AG covering all study costs including any necessary study materials and monitoring. The authors have no financial interest in any of the products used for this study.

      Acknowledgements

      We would like to acknowledge A. Heinrich, A. Bilstein, A. Supersaxo and N. Kalsberger for their significant contribution to the study planning and review of this manuscript.

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