Review of clinical outcomes of a cationic emulsion tear substitute in patients with dry eye disease

First‐line options for the treatment of dry eye disease (DED) rely on artificial tears (ATs), among which cationic emulsion (CE)‐based ATs have been developed in order to mimic the healthy tear film for an improved restoration of the ocular surface homeostasis. In this review, we describe the outcomes reported in several studies, assessing the mode of action, ocular tolerance and clinical performance of a CE‐based AT. Pilot studies have revealed that CE‐based ATs can increase the volume and stability of the tear film while limiting its evaporation rate. Larger studies have demonstrated that CE‐based ATs play a significant role in the improvement of both objective and subjective DED parameters, including superior efficacy on DED symptoms compared to several other available AT formulation types. Concomitantly, CE‐based ATs have been shown to help patients to prevent or recover from corneal defects associated with refractive surgery. These positive outcomes on ocular surface epithelia are likely due to the combination of unique rheological behaviour and intrinsic anti‐inflammatory properties. Based on all clinical findings, CE‐based ATs represent a valuable treatment option for patients with various etiologies of DED including evaporative forms and would deserve evaluation of benefits in other surgical intervention types triggering DED.


| I N T RODUC T ION
Dry eye disease (DED) is a multifactorial and chronic disorder identified in 5%-50% of the population worldwide, depending on age, sex and diagnostic criteria used.Ageing of population, increasing use of electronic screens, air conditioning use, global warming and pollution make DED a growing health issue worldwide, due to the significant decrease in the quality of life of patients (Nelson et al., 2017), and direct and indirect costs for health insurances and civil society (Yu et al., 2011).
DED is often clinically classified into two categories based on the predominant pathogeny (Craig et al., 2017).The first, is aqueous-deficient dry eye (ADDE), resulting from reduced secretion by lacrimal glands, and the second, is evaporative dry eye (EDE), secondary to either Meibomian gland dysfunction (MGD), blink abnormalities, mucin deficiency and/or contact lens wear (Craig et al., 2017).Pure ADDE accounts for approximately 10% of cases, and the remaining 90% of DED due to pure EDE or combined ADDE/EDE in equal proportions (Lemp et al., 2012;Teo et al., 2020).
Aiming at restoring homeostasis of the ocular surface when taking in charge of DED patients, artificial tears (ATs) are the keystone among all other treatment options.Initially composed of water and electrolytes, the composition of commercially available ATs has become more and more sophisticated over time, targeting the multiple etiologies and mechanisms associated with DED, and to better mimicking the composition and rheologic properties of natural tears (Barabino et al., 2020;Kathuria et al., 2021).AT ingredients have been recently classified into three groups, based upon composition and mode of action: wetting agents, multiple-action tear substitutes and ocular surface modulators (Barabino et al., 2020;Kathuria et al., 2021).While wetting agents and multiple-action tear substitutes aim to improve the quantity and eventually the quality of the tear film, ocular surface modulators contain ingredients able to influence the ocular surface and counteract DED signs (Barabino et al., 2020).ATs based on cationic emulsions (CE-based ATs) represent a new generation of ocular surface modulator, acting on all main DED signs including tear film instability and hyperosmolarity, ocular surface cell apoptosis and ocular inflammation.
The aim of this review is to discuss the clinical studies that have been conducted over the past decade in order to optimize the formulation, define the mode of action and to compare the safety and performance of CE-based ATs with other commercially available ATs.

| FOR M U L AT ION A N D BIOC H E M ICA L PROPE RT I E S OF C E -BA SED AT S
The composition of one CE-based AT is detailed in Table 1 below as example.

| Properties
The human tear film is composed of three layers (Figure 1a).The tear film lipid layer (TFLL) helps to prevent the ocular surface from desiccation and contains

Contribution to the ocular surface homeostasis and mimetic properties compared to natural tears
Mineral oils 1 (50% heavy and 50% light chain) Oily core of the emulsion (oily phase) Apolar lipid mixture for restoration of the TFLL (Daull et al., 2020;Georgiev et al., 2017) Emollient Cetalkonium chloride 0.002 Cationic surfactant Polar lipid, stabilization of the TFLL (Cwiklik et al., 2017) Anti-inflammatory properties (Daull et al., 2018(Daull et al., , 2020) ) Tylopaxol 0.2 Polymeric surfactant Stabilization TFLL in replacement of surface-active glycoproteins Anti-inflammatory properties (Daull et al., 2018(Daull et al., , 2020) ) Poloxamer non-polar and polar lipids which allow its stability on the aqueous layer.The aqueous layer is predominantly composed of water and together with the TFLL maintains the proper moisture of the ocular surface.It also contains surface active proteins providing the first line defence against chemical and biological injuries, as well as the stability of the outer layer.The mucin layer represents the inner layer of the tear film and the interface between the aqueous part of the tear film and the corneal surface.Secreted by the corneal epithelial cells (CEC) and goblets cells, the negatively charged mucins form gel-glycoproteins at the top of the CEC and solubilized in the mucin layer (Willcox et al., 2017).At the ocular surface tissue level, DED eventually results in loss of ocular surface homeostasis, tear film instability, hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities (Figure 1a) (Craig et al., 2017).Cationic emulsions (CEs) (Cationorm®/Retaine® MGD, Cationorm® Pro/ Ocutears® Pro+ from Santen Pharmaceutical) are ATs aimed at mimicking, as much as possible, the composition of normal tear film (Figure 1b and Table 1).Various preclinical studies have demonstrated that the oil-inwater emulsions can replenish both the lipid and aqueous layers (Daull et al., 2020;Lallemand et al., 2012) due to the characteristics and properties including: (1) The ratio between the oily phase and the aqueous phase found in the CE-based ATs is similar to the ratio in healthy tear film; (2) the polar (i.e.cetalkonium chloride (CKC)) and non-polar lipids (i.e.mineral oil) present in CE-based ATs allow the restoration of the outer TFLL, increase its thickness and help stabilize the interface with the underlying aqueous layer (Daull et al., 2020); (3) the electrostatic attraction naturally occurring between positively charged oily nanodroplets and negatively charged ocular mucins increase residence time (Lallemand et al., 2012;Rabinovich-Guilatt et al., 2004;Royle et al., 2008); (4) surface-active polymeric excipients in the emulsion (e.g.poloxamer 188 and tyloxapol) may help compensate for the deficiency of polar lipids and stabilize the lipid film like natural surface-active proteins such as lipocalin (Eftimov et al., 2020;Willcox et al., 2017); (5) intrinsic anti-inflammatory and corneal healing properties of CKC have been demonstrated and attributed to specific protein kinase C alpha (PKCα) inhibition, providing an additional pharmacological activity to the mechanical action of CEs (Daull et al., 2018;Daull, Feraille, Barabino, et al., 2016).
Due to the combination of mechanical and antiinflammatory properties, CE has also been shown to promote corneal re-epithelization in an in vitro cell culture model, in an in vivo rat model of corneal scraping (Daull, Feraille, Elena, et al., 2016;Daull, Guenin, et al., 2016), as well as in humans, CE-based ATs have also demonstrated their ability to help prevent or recover from corneal defects (Kochergin et al., 2015;Nubile, 2010).
The selection and optimization of each ingredient used in CE-based ATs have been assessed during preclinical studies (Patents EP1655021, EP1809237, EP1891939 and EP2049079) and early-stage clinical studies described in the next subsections.

| Cationic agent
Compared to anionic emulsions, the use of a cationic agent can improve the tear film stability and residence by interacting with the negatively charged mucin layer (Daull et al., 2020;Georgiev et al., 2017;Lallemand et al., 2012).The choice of cationic agent was a key part of the formulation development with screening of various cationic surfactants (e.g.primary amines, cationic phospholipids and polymeric cationic agents), that can provide high cationic charges without toxicity, and conform to regulatory standards (GRAS, pharmacopoeias) (Lallemand et al., 2012).Commonly used as a preservative of eye drops, benzalkonium chloride (BAK) could theoretically be used as the cationic agent in this type of emulsion, but the adverse effects of BAK, such as dry eye exacerbation and cell damage, are well known (Baudouin et al., 2010;Burstein, 1980;Kuppens et al., 1995).In comparison, the composition of CKC, with a 16 carbon backbone vs. a mix of 12 or 14 for BAK, allows a much higher hydrophobicity, with highly positive zeta potential (+20-40 mV) at low concentrations (e.g.0.002% w/w) (Lallemand et al., 2012), and thus an increased stability of the oil droplets.The reduced number of free molecules of CKC in the water phase explains the very low toxicity, if any, on epithelial cells.The resulting polar lipid structure is thus suitable for stabilizing the tear film, effectively replacing natural polar lipids of the TFLL and helping with the spreading of meibum (Cwiklik et al., 2017;Georgiev et al., 2017).

| Oily phase
Some studies have been also performed to optimize the oil-in-water composition in terms of both choice and concentration of lipidic agents.Mineral oils (heavy and light alkane chains 50:50 mixture) are apolar lipids with good tolerability and lubrication properties, used for decades in DED ointments (21 CFR 349.14 -Ophthalmic emollients, 2023; Garrigue et al., 2017).Ex vivo studies on human meibum showed that CE increased film elasticity and thickness and can compensate for moderate meibum deficiency (Georgiev et al., 2017).
A first phase II, multicenter, randomized clinical study, referred to as the 'Catiodry' trial, was performed to assess and compare the tolerance of CEs prepared with two concentrations of oily agents.For 28 days, 75 patients with bilateral and moderate DED symptoms used eye drops containing 1% or 2% CEs four times daily (Binlich et al., 2006).Both 1% and 2% CEs were well tolerated by the patients, but 1% CEs showed greater improvement in the DED symptoms compared with 2% CEs, and therefore, were chosen as the concentration for the development of these ATs.
The safety and performance of 1% CEs-based ATs were confirmed in another clinical trial which enrolled 42 patients diagnosed with DED (Ousler et al., 2015).After one to two drops of CEs-based eye drops given twice daily for 14 days, a significant reduction of breakup area (i.e.mean corneal surface exposure) and corneal fluorescein staining was observed, compared to baseline.
Moreover, patients reported an enhanced feeling of comfort and quality of life (Ousler et al., 2015).

| CLINICAL STUDIES ASSESSING THE DIFFERENT MODES OF ACTION
After formulation optimization, other exploratory clinical studies have been performed to better understand the mode of action of CEs on the ocular surface.In these studies, the effect of a single instillation of CE-based AT on the ocular surface was evaluated.

| Influence on the thickness of the tear-film lipid layer
The thickness of the TFLL after the instillation of CEs has been measured using non-invasive methods.An ultrahigh-resolution optical coherence tomography (OCT) system was used for imaging of the TFLL.After baseline measurement, each of the 20 healthy subjects in the control group received one drop of 0.9% NaCl solution (Hydrabak®, Thea).Each of the 10 healthy subjects in the study group received one drop of CE-based AT.TFLL quantity increased on average by 34% after administration of CE-based AT with a maximum increase of 60.8% ±28% 30 min post-instillation.In the control group, an average increase of 7.2% of TFLL quantity was recorded overall measurement time points after the instillation of saline solution (Aranha dos Santos et al., 2018).
For the measurement of TFLL thickness, a commercially available non-invasive interferometer system (LipiView®, Tear Science) was also used.TFLL thickness measurements were performed every 10 min for 1 h in 10 subjects with no symptoms of DED to determine the reproducibility of the technique.In a second cohort of 7 healthy subjects receiving one drop of CE-based AT, TFLL thickness measurements were performed before administration and up to 60 min post administration in 10-min intervals.TFLL thickness increased up to 20%, reaching a maximum lipid layer thickness 50 min after administration of CE-based AT (Garhofer et al., 2017).A recent study using LipiView® interferometer in 84 patients also revealed an increase in the TFLL thickness over 15 min after a single use of CE-based AT and another lipid-containing AT (Artelac® Lipids, Bausch & Lomb) (Lim et al., 2020).Participants with baseline TFLL thickness <60 nm had greater changes in TFLL thickness after CE-based instillation, compared with those with a TFLL thickness of >60 nm.

| Residence time on the ocular surface
A pilot study assessed the residence time after one instillation of CE-based AT in 9 patients diagnosed with ADDE (n = 4) or MGD (n = 5) with a mean Ocular Surface Disease Index (OSDI) symptom score of 40.4 (range 15.9-77.3)(Study OTG #14-30, conducted by Optometric Technology Group Ltd, see Appendix S1).The primary endpoints were the mineral oil concentrations in the tear samples collected 30, 60 and 120 min post-instillation.Results showed that mineral oils (i.e.oily agents) were found to be still present after 120 min in both ADDE and MGD patients with a concentration of 0.065 ± 0.103 μg/μL of tears (range 0.053-0.099μg/μL).This remaining concentration of oily agents was estimated to be on average 0.13% of the oil quantity initially instilled, which corresponds to a 3-times higher concentration of lipids compared to the amount of triglycerides naturally present in MGD patients at baseline.Despite the short follow-up period of this study, the outcomes revealed that the CE-based AT can successfully replenish lipid components of the tear film.This can be particularly useful for patients with DED affecting the TFLL such as MGD.Two recent studies confirmed that a single drop of CE-based AT resulted in an increase in the tear film volume in healthy individuals and in males subjects with DED (Abusharha, 2021;Makri et al., 2021).The outcomes also revealed that tear film stability is enhanced after a single instillation of CE-based AT, which can effectively control the evaporation rate (Abusharha, 2021).

| Influence on tear film stability
The effects on tear film stability of single instillation of CE-based AT were compared with hyaluronic acid (HA)-based eye drops (Vismed®, Horus Pharma) over 4 h in 25 patients with DED symptoms (mean OSDI score = 42.0 ± 15.4) looking at both evaporation rate and non-invasive tear break-up time (NIBUT) (Study OTG #08-03, conducted by Optometric Technology Group Ltd, see Appendix S1).After instillation, the evaporation rate measured with evaporimeter system and sealed goggles at 30% room humidity was statistically lower for CE-based vs. HA-based AT at a 3-h time point (p = 0.033).The tear evaporation rate measured at 30% RH and 40% RH was lower than the baseline value at all time points post-instillation from 1 h for CE-based AT.Similarly, greater improvement in the Minimum NIBUT at 60 min (p = 0.01) and the Median NIBUT at 180 min post-installation for (p = 0.05) was observed with CE vs. HA.These differences were associated with mean differences of 1.7 and 2 s respectively.Moreover, post instillation visual disturbances were significantly lower for the CE-based AT compared to HA (p = 0.008).However, it is reminded that the original results shown in this chapter have not been peerreviewed (Appendix S1).

| RANDOMIZED STUDIES COMPARING CE-BASED ATS TO OTHER ATS FOR THE TREATMENT OF DED
Over the last decade, several randomized comparative clinical trials have been performed to assess the safety and performance of CE-based ATs and to compare them with other available ATs (Table 2).

| Clinical studies in patients with primary DE
The first phase II study compared the ocular tolerability of a CE-based AT in 79 patients with mild to moderate DED compared with polyvinyl alcohol and povidone eye drops (PVA-P; Refresh®, Allergan) for 1-month follow-up (Amrane et al., 2014).This study reported a significant improvement in favour of CEbased ATs across multiple parameters: TBUT, ocular surface staining (OSS), subjective scores of symptoms not related to eye drops installation, overall efficacy assessed by investigators and palpebral erythema score.Interestingly, the improvement of TBUT was greater in patients with MGD (+2 s vs. +0.5 s; p = 0.035) than in the non-MGD group (+1.4 s vs. 0.6 s) for CE and PVA-P, respectively.The significantly greater efficacy of CE in MGD patients on TBUT and symptoms, compared with PVA-P, confirms that the CE may act on the lipid layer of the tear film (Amrane et al., 2014).Another randomized study was performed in 85 patients with bilateral moderate to severe DED with keratitis or keratoconjunctivitis (Robert et al., 2016).For this study, CE-based AT was compared with HAbased AT for a 3-month follow-up.Improvements in clinical signs of DED (TBUT, Schirmer test, OSS) were clinically relevant and similar in both groups as well as the incidence of adverse events.However, global symptom scores of ocular discomfort such as itching and dryness were found significantly improved for CEbased AT (Figure 2a,b) (Robert et al., 2016).
CE-based AT was also compared with 0.5% carboxymethyl cellulose (CMC) (Optive® UD, Allergan) and natural phospholipids (Emustil®, SIFI) eye drops for a 3-month follow-up in 71 patients with moderate DED (Spinella, 2010).CE-based AT were found F I G U R E 2 Change from baseline in symptoms of ocular discomfort (Visual Analogic Scale) for Itching (a) and Dryness (b) sensations respectively, in patients with bilateral moderate to severe DED with keratitis or keratoconjunctivitis (Robert et al., 2016).
similar to CMC eye drops and superior to natural phospholipids in terms of subjective symptom scores, TBUT and OSS.In addition, the tear film osmolarity significantly decreased only for the group using CEbased AT (Figure 3d) which highlights the benefits of the CE-based AT formulation.A similar comparative study, conducted in 2009 in Indonesia at the Dr Cipto Mangunkusumo National Public Hospital (Study CMNPH), with 0.5% CMC eye drops vs. CE-based AT investigated the DED signs and symptoms in 46 postmenopausal women (Appendix S1).In this study, the ocular protection index (OPI) was used, which is calculated by the ratio of TBUT and the inter-blink interval (IBI), when TBUT is shorter than the blink interval (IBI), the eyes are exposed to the risk of ocular surface damage (Ousler et al., 2002).Results showed that CEbased AT significantly improved TBUT compared to 0.5% CMC eye drops, providing effective ocular protection in the management of DED in menopausal women, a population known at risk for DED (Figure 3a,b).
Regarding the safety outcomes, CE-based ATs showed no increase in the incidence of adverse events compared to other ATs in the different clinical studies performed (Nubile, 2010;Spinella, 2010) Only very rare cases of transient discomfort (eye irritation, eye pain, foreign body sensation in the eye, eye redness, eye itching, watery eyes, burning sensation in the eye, temporarily blurred vision, eyelids inflammation or eyelids oedema) were observed, showing that CE-based ATs represent a safe option for patients with DED.
All of these results suggest that the formulation of CEbased ATs, particularly the presence of oily agents, can significantly reduce DED signs and symptoms.These data highlight the importance of having lipid-based ATs to help stabilize the tear film as also suggested in the literature (Garrigue et al., 2017).

| Clinical studies with patients with secondary DED
In addition to patients with DED, CE-based ATs have been tested in patients with other ocular conditions such as photorefractive keratectomy (PRK) and for the prevention of ocular complications in the intensive care unit (ICU) (Kochergin et al., 2015;Nubile, 2010).
The effect of CE-based AT was evaluated in 40 myopic patients after bilateral PRK for 3 months and compared with 0.2% HA eye drops (Hyalistil, SIFI) (Nubile, 2010).After 7 days of treatment, subjective symptoms of ocular discomfort were improved by similar amounts in both groups, except for foreign body sensation and ocular dryness, which was significantly improved in the CEs group.TBUT was also significantly increased at day 7 and day 30 in patients receiving CE-based AT compared with HA-based AT.While the corneal re-epithelialization time was similar in both groups (~3 days), the quality of the corneal epithelium was significantly improved at day 7 in the CE group vs. HA (p = 0.007) (Figure 4a) (Nubile, 2010).It took 1 month to observe similar corneal epithequality or symptom level with HA-based AT.All these outcomes suggest that CE-based ATs represent a good candidate for faster recovery from the corneal damage induced by PRK surgery, echoing preclinical data showing a good corneal healing process after corneal alkali burn, scraping or de-epithelization (Klang et al., 1999;Lallemand et al., 2012;Liang et al., 2012).
Another study assessed the effects of a CE-based AT for the prevention of ophthalmic complications in 50 patients with lagophthalmos in the ICU (Kochergin et al., 2015).While 28% of the patients in the non-treated group showed initial signs of corneal xerosis and exposure keratitis, the use of CE-based AT increased the tear film stability and prevented corneal epithelial defects for all patients in the group (Kochergin et al., 2015).These results suggest that CE-based ATs can be beneficial in patients with lagophthalmos.
These clinical outcomes showed that the CE-based ATs can also be used in patients to prevent secondary dry eye symptoms caused by different types of injuries and disorders (Klang et al., 1999) and would deserve evaluation in other surgical intervention types associated with DED such as Laser-Assisted In-Situ Keratomileusis (LASIK) or cataract surgery.

| CONC LUSION
A large range of ATs are available on the market, and they are made of various components, which can be very challenging for physicians to determine the most suitable one based on the aetiology of each patient (Labetoulle et al., 2022).The clinical outcomes presented in this review show how the instillation of CEbased AT can significantly increase the volume and stability of the lipid layer of the tear film.Compared to various AT formulation types (saline solution, anionic emulsions, polymers and hyaluronic acid), CE-based ATs were found to be at least non-inferior and even superior in the improvement of some DED signs and symptoms.
Despite the limited number of patients in the comparative studies, the level of evidence indicates that CE-based AT, due to its composition, improved impacts on DED symptoms compared to other marketed ATs.Together with the preclinical compilation of data from CE-based AT in DED, (Daull et al., 2020) this clinical review is shedding light on the interest of ophthalmic emulsions, including cationic ones, for the management of DED in various cases including MGD.Knowledge on the role of lipids in tears is advancing, (Garrigue et al., 2017) additional translational research may help to better understand the role of other tear constituents in tears so that improved tear substitutes can be developed to help manage DED.

F
I G U R E 3 Clinical outcomes of comparative studies assessing the safety and performance of CE-based AT.(a) TBUT and (b) OPI (defined as TBUT/IBI) after a single instillation of CE-based or 0.5%-CMC eye drops or after 4 instillations a day on both eyes for 14 days in postmenopausal women with DED signs and symptoms (c) OSDI score after 4 instillations a day of CE-based or 0.5%-CMC eye drops for 14 days (Study CMNPH, see Appendix S1) and (d) Ocular osmolarity after instillation 4 times a day of CE-based, 0.5%-CMC or NP eye drops for 3 months in patients with moderate level of DED (Spinella, 2010).*p < 0.05 compared with baseline, # p < 0.05 compared with CMC-based AT.CE, cationic emulsion; CMC, carboxymethyl cellulose; NP, natural phospholipids.

F
Effect of 3-months treatment of CE-based and HA-based ATs on ocular surface staining in patients who underwent photorefractive keratectomy (Nubile, 2010).(a) Corneal epithelium quality based on Ocular Staining Score and (b) Ocular Dryness symptom score on Visual Analogic Scale (VAS)