Efficacy and Safety of Different Hyaluronic Acid Tear Substitutes Formulations in Evaporative Dry Eye: A Randomized, Single-Blind, Comparative Study
Phase: PHASE2
Conditions: Dry Eye
Sponsor: University of Seville

Study protocol 
 
 
 
 
 
 
 
Efficacy and Safety of Different Hyaluronic Acid Tear Substitutes Formulations in Evaporative Dry 
Eye: A Randomized, Single-Blind, Comparative Study 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Date: July 18, 2024. 

1. Study design 
 
This was a randomized, double-masked, comparative clinical trial conducted in the 
ophthalmology departments of Tedesco Eye Center (Girifalco, CZ, Italy) between November, 
2023 and June, 2024. The study protocol fulfilled all the requirements of the Declaration of 
Helsinki. Before initiating the study, informed and written consent was obtained from each 
patient. 
2. Participants 
 
Eligible patients were adults ≥ 18 years old with a self-reported history DED while working with 
computer screens ≥ 6 hours per day. Patients had to meet the following inclusion criteria in ≥ 1 
eye at screening and randomization to be enrolled in the study: (1) ocular surface disease index 
(OSDI) > 13 points; (2) non-invasive tear film break-up time (NIBUT) < 10 s; (3) Schirmer test 
(ST) without anesthesia ≥ 5 mm and (4) MGD grade > 1. For MGD, the Sirius device (CSO, 
Florence, Italy) was used, which determines MGD grade based on loss area of meibomian glands 
(LAMG). MGD grade was scored from 0 to 4 (MGD grade 1 = LAMG < 25%; MGD grade 2 = 
LAMG ≥ 25% and < 50%; MGD grade 3 = LAMG ≥ 50% and < 75%; MGD grade 4 = LAMG ≥ 
75%). 
Patients were excluded from the study if they met any of the following criteria: (1) abnormal lid 
anatomy, including active blepharitis, and active lid margin; (2) all corneal disorders that affect 
diagnostic test, such as active corneal infection and corneal dystrophies; (3) active ocular 
allergies; (4) vectored thermal pulsation (VTP) intense pulse light (IPL), quantum molecular 
resonance (QMR), or other procedure to treat DED within the previous 6 months; (5) intraocular 
surgery or laser ocular surgery within the previous 6 months; (6) use of topical antibiotics and 
anti-inflammatory treatments, including steroids and non-steroidal anti-inflammatory drugs; (7) 
systemic autoimmune diseases; (8) contact lens wearers; (9) pregnant or lactating women; (10) 
patients who did not understand or comprehend the informed consent. Prior use of tear substitutes 
was allowed, and there was a one-month washout period after enrollment. 

3. Randomization, intervention and masking 
 
Eligible participants were randomized in a 1:1 ratio to receive either 0.20% cross-linked HA tear 
substitute (Off Health S.p.a., Firenze, Italy) or 0.15% HA tear substitute (Thea Pharma S.p.a., 
Milan, Italy). Randomization tables were generated by an unmasked statistician not otherwise 
involved in the trial using the software “randomiser” (Appier inc., Taiwan, China). Patients 
were instructed to instill 1 drop of 0.20% cross-linked HA tear substitute or 0.15% HA tear 
substitute into each eye 3 times per day for 3 months. Both tear substitutes are transparent, with 
no special smell and the bottles were identical in appearance such that patients were masked to 
treatment assignment. 
4. Clinical outcomes 
 
Patients were assessed at screening, baseline (day 1), and 2 follow-up visits: 1 month (12 ± 1.5 
weeks) and 3 months (24 weeks ± 1). All clinical outcomes were conducted in the order 
suggested by Ballesteros et al. [1] to maintain the integrity of the tear film. Furthermore, these 
measurements were taken under consistent environmental conditions in the same room by a 
trained optometrist. 
Primary Outcomes 
4.1. Lipid Layer thickness 
 
The Lipiview® (Johnson & JohsonVision Care, San Francisco, CA, USA) II ocular surface 
interferometer evaluates LLT automatically assesses LLT with nanometer precision by recording 
a 20 s video of the tear film interference pattern and then displays the data in interferometric 
color units (ICU), where1 ICU reflects approximately 1 nm of LLT. 
Secondary Outcomes 
4.2. Dry eye symptoms 
 
The OSDI questionnaire were employed to assess the severity of DED symptoms, with scores 
ranging from 0 (indicating no ocular surface disease) to 100 (indicating severe ocular surface 
disease) points [2,3]. This questionnaire was provided during consultations at each follow-up 
visit. 
 
 

4.3. Tear film stability 
 
Tear film stability was automatically assessed using NIBUT by projecting Placido rings from the 
Sirius device (CSO, Florence, Italy) onto the corneal surface. The time interval between the last 
blink and the initial distortion of the ring pattern was defined as first NIBUT. This variable was 
always measured at least 12 hours after administration of the study medication and the average of 
3 consecutive measurements was calculated for statistical analysis. 
4.4. Tear film volume 
 
Tear film volume was evaluated with unanesthetized ST. During the test, the patient is instructed 
to look upward while the test strip is carefully positioned between the palpebral conjunctiva of 
the lower eyelid and the bulbar conjunctiva. Subsequently, the patient is asked to keep their eyes 
gently closed for five minutes. After this period, the test strip is removed, and the Schirmer test 
score is determined by measuring the length of the moistened area on the strip. 
5. Statistical analysis 
 
Statistical analyses were performed with SPSS statistics software, version 25.0 (IBM Corporation, 
NY, USA).Continuous variables were displayed as the mean ± standard deviation (SD), while 
ordinal categorical variables were expressed as frequencies (n) and percentages (%). When both 
eyes were eligible, the eye with a lower baseline NIBUT was selected for the analyses. If the 
baseline NIBUT was the same in both eyes, the right eye was selected. Since the Kolmogorov-
Smirnov test showed that the clinical endpoints did not meet the normality criteria, 
nonparametric tests were performed. The Friedman test (non-parametric) was performed to 
compare intra-group clinical outcomes. Within each group, the increment (∆) was calculated. It 
was defined as the change from the last visit (LV) to baseline (B): D = LV – B. Inter-group 
clinical outcomes were analyzed with the Mann–Whitney U test. Between each group, the 
differences were calculated as ∆ cross-linked HA group - ∆ HA group. Categorical variables were compared 
using the χ2 test. A P value of less than 0.05 is considered to be statistically significant. 

References 
 
[1] 
Ballesteros-Sánchez A, Gargallo-Martínez B, Gutiérrez-Ortega R, Sánchez-González JM. 
Intraobserver Repeatability Assessment of the S390L Firefly WDR Slitlamp in Patients 
With Dry Eye Disease: Objective, Automated, and Noninvasive Measures. Eye Contact 
Lens 2023;49:283–91. https://doi.org/10.1097/ICL.0000000000001001. 
[2] 
Dougherty BE, Nichols JJ, Nichols KK. Rasch analysis of the Ocular Surface Disease 
Index (OSDI). Invest Ophthalmol Vis Sci 2011;52:8630–5. 
https://doi.org/10.1167/IOVS.11-8027. 
[3] 
Schiffman RM, Christianson MD, Jacobsen G, Hirsch JD, Reis BL. Reliability and validity 
of the Ocular Surface Disease Index. Arch Ophthalmol 2000;118:615–21. 
https://doi.org/10.1001/ARCHOPHT.118.5.615.