Introduction
The composition, integrity, and stability of the tear film can be adversely affected by multiple patient-related and environmental factors, resulting in the development of dry eye disease (DED), one of the most common ocular disorders. Both its incidence and prevalence are continuously growing [1,2]. Aging is one of the main contributors to tear film changes, with an incidence ranging from 8 % in subjects under 60 to 20 % in those over 80 [3]. However, the huge spread of digital devices, surgical refractive treatments, and contact lenses use are causing an increasing prevalence of DED among both young people and adults [4]. According to the revised definition provided by the second Tear Film and Ocular Surface Society Dry Eye Workshop (TFOS DEWS II) report [5,6], DED is an ocular surface multifactorial disease, characterized by a disruption of tear film homeostasis. Etiology involves tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities. DED impacts patients' quality of life heavily, limiting daily activities (such as reading, using digital devices, driving, and watching television) [5,7] to such an extent  that the patient may develop mood disorders like anxiety, depression, and frustration [8]. DED can be classified as aqueous deficient or evaporative. Evaporative DED is most common, accounting for about 50 % of cases [5,9]. Mixed forms are also quite common as they account for about one-third of cases [9]. Depending on symptoms and signs, DED can be further classified as mild, moderate, or severe [10]. Biomicroscopic examination of the ocular surface, of the tear film, and the use of validated questionnaires (such as the Ocular Surface Disease Index - OSDI – questionnaire [11]) are the principal diagnostic tools for evaluating DED and its symptoms. Local inflammation markers, such as matrix metalloproteinases (MMPs), may also be quantified by adjunctive tests [12]. Metalloproteinase- 9 (MMP-9), an MMP produced by corneal epithelial cells, fibroblasts, leukocytes, and lacrimal gland cells, plays a role in the remodeling process of the extracellular matrix, acting in both normal corneal healing processes and pathological states (i.e., ocular allergy, keratitis, blepharitis, and DED). Tear hyperosmolarity, a core mechanism of DED pathogenesis, stimulates the over-expression of MMP-9, as well as that of other pro-inflammatory cytokines, which in turn promote inflammation and epithelial cell loss [13-15]. Tear substitutes are the current mainstay of DED therapy, regardless of the disease severity. Lipid-based substitutes are emulsions containing mineral oils, castor oil, olive oil, glycerin carbopol, lecithin, phospholipids, and saturated and unsaturated fatty acids. They are particularly suitable for treating evaporative DED since their composition is closer to that of the deficient tear lipid layer [16]. Liposomal eye drops formulations–made of bilayered spherical lipid vesicles encapsulating the aqueous-soluble material – have also been tested, showing favorable outcomes in managing DED, improving both subjective and objective endpoints [17-21]. To improve the dwell time and lubricating effect, carboxymethylcellulose and other agents can be used to increase viscosity, protecting the ocular surface, and increasing the density of goblet cells [22]. Tear substitutes may also incorporate free amino acids, given their role in protecting the tear film, such as proline and glycine, the main components of type I collagen - the most abundant protein in the corneal stroma - and lysine and leucine, which contribute to the spatial arrangement and assembly of the fibrils, and the transparency of the corneal stroma [23]. Tear substitutes may also be added with antioxidant agents, such as vitamin A (retinyl palmitate) and vitamin E (α-tocopherol) to contrast oxidative stress and the consequent inflammation that contributes to the establishment, maintenance, and progression of DED. In DED patients, vitamin A-based tear substitutes have been shown to increase the quality of vision and contribute to improving objective parameters, such as Tear Film Break-Up Time (TF-BUT), Schirmer's test score, rose bengal and fluorescein staining scores, as well as subjective DED symptoms [24,25]. Tear substitutes containing vitamin E have also been shown to be effective in managing DED as measured by the variation in the TF-BUT time as well as the OSDI and Schirmer test scores [26]. A new liposome-based ophthalmic solution containing L-proline, L- glycine, L-lysine hydrochloride, and L-leucine as well as vitamin A and vitamin E, (Lacrisek Ofta Plus, Sooft Italia S.p.A., Italy) has been recently placed on the market. Its formulation has been conceived to exploit both the benefits of liposome-mediated solution delivery and the protecting and anti-oxidative action of the other constituents, possibly enhancing its overall effectiveness in managing DED symptoms. At present, this tear substitute – to the authors’ knowledge – has still not been investigated in the clinical setting. This pilot study, therefore, aims to gather preliminary evidence concerning its efficacy and safety.

30 days of treatment. The study also primarily aimed to gather preliminary data concerning device safety, by assessing the frequency and nature of adverse events, if any would occur. TF-BUT was evaluated after instilling 2 µl fluorescein, as the mean of three consecutive measures per eye. The fluorescein staining pattern of the corneal and conjunctival epithelium was scored from 0 to 5 according to the modified Oxford scale [27]. Corneal and conjunctival staining was repeated using Lissamine green and scoring the corresponding pattern according to the van Bijsterveld 0-9 scale [28]. The OSDI is a 12-item questionnaire, whose questions are grouped into three sections, namely ocular symptoms, vision-related function, and environmental factors making eyes feeling uncomfortable.

It allows patients to subjectively assess ocular symptoms of DED and how they impact their quality of life. Each item is assigned a score from 0 (the discomfort being present none of the time) to 4  (the discomfort being present all the time); the sum of scores allows to detect normal eyes or to stratify DED as mild, moderate, or severe [11]. The InflammaDry test (Rapid Pathogen Screening, Inc, Sarasota, FL, USA) is a rapid, disposable immunological test for semi-quantitative measurement of MMP-9, a nonspecific inflammatory marker that has consistently been shown to beelevated in the tears of patients suffering from DED. Normal MMP-9 levelsin human tears range from 3 ng/ml to 40 ng/ml [13-15]. The InflammaDry test returns a positive result if MMP-9 level is > 40 ng/ml, thus adjuvating in confirming a diagnosis of DED and pathologic ocular surface inflammation [29,30]. Adjunctive endpoints included: measuring Meibomian gland obstruction through a 0 to 3 score (0 = no obstructed glands; 2 = 1-2 obstructed glands; 2 = 3-4 obstructed glands; 3 = all glands obstructed); the doctor assessing conjunctival hyperemia and the patient self- reporting burning, photophobia, foreign body sensation, itchy eyes, and tearing by using a 0-4 scoring system (0 = absent, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe). All eyes were also routinely scoped by biomicroscopic evaluation of the whole ocular surface.

At the 30-day visit, they underwent routinely biomicroscopic examination and were subjected to the InflammaDry test. TF-BUT was measured, and patients answered the OSDI questionnaire. Meibomian gland obstruction assessment and signs and symptoms evaluation were carried out as on the first visit. Even if performing any comparison between the investigational device and the alternative substitute was explicitly out of the scope of the study, all evaluations at baseline and the 30-day control visit were carried out at both eyes for sake of completeness.

No patient experienced any adverse effects. For sake of completeness, results at the contralateral eyes treated with the alternative tear substitute were as follows: the difference in TF- BUT was not statistically significant (4.82±3.10 seconds after 30 days vs. 4.73±3.20 seconds at baseline, p=0.80); the difference in the Oxford score (2.00±1.00 at 30 days vs. 2.09±1.04 at baseline) as well as that in the van Bijsterveld score for Lissamine green staining score (1.00±1.00 at 30 days vs. 1.90±1.00 at baseline) and that in the OSDI score (27.90±13.40, range 19-52 at 30 days vs. 26.60±8.90, range 16- 38; mean variation, +1.30±8.30 units) were not statistically significant (p>0.05 in all cases). At 30 days, the eyesthat were positive at the InflammaDry test were 14 (87.5 %) versus 16 at baseline (100%, as per inclusion criteria), the difference being not statistically significant. The Meibomian gland score  was  not  significantly different  (3.60±0.50 vs. 3.40 ±0.80, p>0.05). Conjunctival hyperemia at 30 days was not significantly different from that at baseline (1.73±1.19 vs. 1.64±0.67, p>0.05). Even the scores concerning the other symptoms of interest did not change significantly (burning,1.36±0.67 vs 1.82±1.08; photophobia, 2.91±1.30 vs. 3.45±0.93; foreign body sensation, 1.55±0.69 vs. 1.91±1.04; itchy eyes, 1.45±0.82 vs. 1.27±1.27; tearing, 2.27±0.79 vs 2.55±1.04; p>0.05 in all cases). No patient complained concerning any adverse effects.

Figure 1. Plots showing the variation of the main study endpoints after DED-affected eyes were treated using the investigational device. *marks significance; NS, not significant. A) the TF-BUT showed a significant decrease; B) the fraction of patients positive for a pathologic MMP-9 level also significantly decreased; C) the OSDI score increased, but the difference was not statistically significant; D) the Oxford fluoresceine staining score decreased significantly; the E) van Bijsterveld Lissamine staining score decreased, but the difference was not statistically significant. The F) Meibomian gland score did not change.

Table 1. Signs and symptoms of DED measured on the day of enrollment (baseline) and 30 days after treatment using the investigational device.

* marks significance. Conjunctival hyperemia was assessed by the clinician; all other symptoms were self-assessed by patients.

Discussion
Results of the present study suggest that Lacrisek Ofta Plus is effective in managing DED symptoms when applied daily over 30 days as per its manufacturer instructions. Most primary study endpoints, namely the mean TF-BUT time, the number of eyes positive to the InflammaDry test, and the fluoresceine Oxford score concerning ocular surface staining pattern were, in fact, significantly improved after the treatment period compared to baseline. The OSDI score increased, even if the difference was not statistically significant (possibly because of the small sample size). These positive outcomes may be ascribed to the tear substitute composition. Lipid-based and liposome-based tear substitutes are known to be beneficial because they mimic – better than water-based eye drops – the aqueous and lipid layers of the tear film [16]. Results of the present study are indeed consistent with previously published evidence concerning using lipid- based and liposome-based substitutes to manage DED [17- 20,31-36]. Results of the present study are also consistent with clinical observations concerning lipid-based tear substitutes containing amino acids. Indeed, similar beneficial effects were reported in a prospective, double-masked, controlled, clinical study comparing the effects of two tear substitutes based on sodium hyaluronate (SH) alone or SH enriched with amino acids (L-glycine, L-proline, L-lysine hydrochloride, L-leucine) in patients with tear film disorders. After 1 month of treatment, a statistically significant improvement of TF- BUT was observed in the aminoacid-enriched- SH treated group compared to baseline value [37]; eyes treated with eye drops containing amino acids had better corneal staining, and observation at the confocal microscope showed less hyper-reflecting cells (a sign of metabolic damage) in the epithelial corneal layer compared to those receiving hyaluronic acid alone. Results of the present study, as well as those by Aragona et al. [37], are consistent with the supposed beneficial effect ascribed to exogenous supplementation of amino acids (including L-proline, L-glycine, L- lysine, and L-leucine) through tear substitutes [23], given the fact that their concentration changes in patients affected by DED [38] as well as in other ocular pathologies [38,39]. Such consistency between the beneficial effect of exogenous amino acid supplementation through tear substitutes in DED patients and the variation of the amino acid level in the pathologic eye represents one topic at the forefront of current research [23,38] and should be the subject of further investigations. As in the present study, positive clinical results have also been reported in using eye drops containing vitamin A or E to manage DED [24-26]. As far as the investigational device that was preliminary assessed in the present study is concerned, as well as for the different tear substitutes currently marketed to manage DED, it is not possible to tell which of the formulation components may have been more relevant in determining efficacy, let alone to identify if they have had any synergistic effect. Indeed, despite the widespread of DED, only a few randomized clinical trials (RCTs) have compared the efficacy of different tear substitutes [40]. In 2016, Pucker et al. reviewed  43 RCTs assessing the efficacy of several tear substitutes in DED and performed a meta-analysis of those comparing head-to-head different tear substitutes; the analysis showed most of the tear substitutes tested were equally effective, concluding that at present the literature indicates uncertainty as to which tear substitute works best [41]. Indeed, studies aimed to collect evidence-based guidance for managing DED through artificial tear supplements are only very  recent [42]. Incidentally, data of the present study seem to suggest the alternative tear substitute was not as effective as the investigational one. Yet, data were not subjected to statistical analysis, which would have been out of the scope of this investigation and meaningless given its small sample size; the small sample size itself might have hidden significant results that could have instead been observed by assessing a greater number of subjects. This again confirms that thorough, well- designed appropriate clinical studies should be performed to better distinguish if any currently marketed tear substitute performs better than others. Limitations of the present study arise from it being altogether preliminary in nature and include the reduced sample size, the lack of a control group, and the limited follow-up. Studies with a greater number of subjects would allow choosing appropriate control groups, either including healthy subjects, or subjects to be treated with a comparator. Longer follow-up times would be required to assess longer-term efficacy and safety, including the development of habituation. Appropriate comparative studies would also allow to fine- tune the composition of the eyedrop formulations to specific subgroups of patients, suffering from DED with varying severity.

Author Disclosure Statement: No competing financial interests exist.

Funding sources: No funding was received for this study.