Efficacy and Safety of a Multiple-Action Tear Substitute (TriMix) in Dry Eye Disease: A Randomized, Double-blinded, HA-controlled Study
Phase: PHASE2
Conditions: Dry Eye
Sponsor: University of Seville

Study protocol 
 
 
 
 
 
 
 
Efficacy and Safety of a Multiple-Action Tear Substitute (TriMix) in Dry Eye Disease: A 
Randomized, Double-Blinded, HA-controlled Study 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Date: June 14, 2024. 

1. Study design 
This was a randomized, double-masked, HA-controlled clinical trial conducted in the 
ophthalmology departments of Tedesco Eye Center (Girifalco, CZ, Italy) and Russo multi-
specialty medical practice (Corso Garibaldi, CZ, Italy) between July, 2023 and May, 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 1.5:1 ratio to receive either TriMix 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 TriMix tear substitute or 0.15% HA tear substitute into each eye 3 times per day for 
6 months. HA-based tear substitutes can reduce tear film hyperosmolarity and are effective in 
treating DED. Therefore, 0.15% HA tear substitute was selected as a suitable comparator. Both 
tear substitutes are transparent, with no special smell and the bottles were identical in appearance 
such that patients and investigators were masked to treatment assignment.  
4. Clinical outcomes 
The study design is shown in Figure 1. Patients were assessed at screening, baseline (day 1), and 
2 follow-up visits: 3 months (12 ± 1.5 weeks) and 6 months (24 weeks ± 1). All clinical outcomes 
were conducted in the order suggested by Ballesteros et al. 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.  
4.1. 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 [1,2]. This questionnaire was provided during consultations at each follow-up 
visit. 
4.2. 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. 
 

 
4.3. 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). The sample size was estimated using the GRANMO calculator, version 7.12 
(Municipal Institute of Medical Research, Barcelona, Spain). It was calculated based on a 
statistically significant paired difference of 1.24 ± 2.56 s at 2 months after the TriMix tear 
substitute treatment onset. This assumed difference was based on the findings of a pilot study 
with 25 eyes [3]. With these assumptions, a sample size of 40 eyes per group would yield a power 
> 80% and a statistically significant paired difference of 95% confidence. A 20% discontinuation 
rate was considered when calculating sample size. 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 ∆ TriMix 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] 
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. 
[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] 
Vigo L, Senni C, Pellegrini M, Vagge A, Ferro Desideri L, Carones F, et al. Effects of a 
New Formulation of Multiple-Action Tear Substitute on Objective Ocular Surface 
Parameters and Ocular Discomfort Symptoms in Patients with Dry Eye Disease. 
Ophthalmol Ther 2022;11:1441–7. https://doi.org/10.1007/S40123-022-00518-7.