Ectoine in the Treatment of Irritations and Inflammations of the Eye Surface

The ocular surface is facing various unspecific stress factors resulting in irritation and inflammation of the epithelia, causing discomfort to the patients. Ectoine is a bacteria-derived extremolyte with the ability to protect proteins and biological membranes from damage caused by extreme environmental conditions like heat, UV-light, high osmolarity, or dryness. Evidence from preclinical and clinical studies attest its effectiveness in treating several epithelium-associated inflammatory diseases, including the eye surface. In this review, we analysed 16 recent clinical trials investigating ectoine eye drops in patients with allergic conjunctivitis or with other unspecific ocular inflammations caused by e.g. ophthalmic surgery. Findings from these studies were reviewed in context with other published work on ectoine. In summary, patients with irritations and unspecific inflammations of the ocular surface have been treated successfully with ectoine-containing eye drops. In these patients, significant improvement was observed in ocular symptoms of allergic rhinoconjunctivitis, postoperative secondary dry eye syndrome, or ocular reepithelisation after surgery. Using ectoine as an add-on therapy to antihistamines, in allergy patients accelerated symptom relief by days, and its use as an add-on to antibiotics resulted in faster wound closure. Ectoine is a natural substance with an excellent tolerability and safety profile thus representing a helpful alternative for patients with inflammatory irritation of the ocular surface, who wish to avoid local reactions and side effects associated with pharmacological therapies or wish to increase the efficacy of standard treatment regimen.


Introduction
Ectoine, an extremolyte, is a natural protection molecule found in bacteria which survive under extreme conditions of salinity, drought, irradiation, pH, and temperature [1,2]. Ectoine forms a protective hydration shield around proteins and other biomolecules [3] that is based on its strong binding capacities to water molecules [4]. This mode of action is known as "preferential exclusion" [5]; i.e., ectoine is preferen-tially excluded from the hydrate shield, leading to the alteration of the aqueous solvent structure [6,7]. That effect protects proteins from damage and irreversible denaturation and stabilizes biological membranes [4,[8][9][10]. In preclinical studies, ectoine was shown to protect lung and skin cells against the damage induced by toxic pollution particles and to prevent the subsequent activation of inflammatory cascades [11][12][13][14][15][16]. A similar effect was observed in model systems for inflammatory bowel disease [17]. Interestingly, ectoine can stabilize lipid layers in pulmonary surfactants, as well as the tear film of the eyes against physical stress [18][19][20][21][22].
Promising findings from clinical trials were reviewed by Casale and colleagues [23] who attributed topical applied ectoine effectiveness in upper airway inflammations such as acute pharyngitis/laryngitis [24,25], rhinosinusitis, rhinitis sicca, and acute bronchitis [26]. In addition, several trials showed efficacy of ectoine in various diseases with barrier dysfunctions such as rhinitis sicca [27], chemotherapyinduced mucositis [28], lung inflammation caused by environmental pollutants [29], prevention of upper respiratory infections [30], and atopic dermatitis [31]. Moreover, studies on allergic rhinoconjunctivitis [27,32,33] and dry eye syndrome [34,35] have been published. Among these published studies, the application of ectoine to treat ophthalmic indications prompted us to perform a detailed analysis of the use of ectoine in this field. The ocular surface (cornea, conjunctivitis, and tear film) is a sensitive part of the human body exposed to various environmental challenges, such as heat, dry air, pollutants, or allergens. Besides these environmental exposures, individuals are sometimes genetically predisposed to-or develop-secondary inflammatory processes.
Allergic diseases, including allergic rhinoconjunctivitis, are a global health burden. The global prevalence of all allergic diseases is reported to be 20%-30% [36], resulting in a high pressure on the socioeconomic systems. The Global Allergy and Asthma European Network report indicated that cost savings of over EUR 100 billion could be realistically expected through better treatment of allergic diseases [37]. The 2008 and 2016 Allergic Rhinitis and its Impact on Asthma (ARIA) guideline supports physicians with a treatment algorithm for allergic rhinitis and conjunctivitis, depending on the severity and duration of the symptoms [38,39]. Pharmacological therapies using oral/topical antihistamines, intranasal glucocorticosteroids (INGS), oral glucocorticosteroids, decongestants, and chromones are-beside avoidance-considered the keystones of allergic rhinitis and conjunctivitis treatment. Nevertheless, a relevant proportion of patients with symptoms are still not sufficiently treated [40][41][42][43][44]. A study reported that about 60% of allergic rhinitis sufferers in the U.S. are "very interested" in trying out new medications [45]. Furthermore, many patients are reluctant to use pharmacological therapies for fear of local irritations and side effects associated with sedative antihistamines, which in turn can lead to poor medication compliance [46,47]. Therefore, nonpharmacological therapies with an advantageous tolerability and safety profile are of interest to many patients with allergic rhinoconjunctivitis.
Beside the allergic irritation of the eye surface, other noxious influences such as injuries, burn, or physical trauma also lead to inflammation and irritation of the air-facing epithelia, the conjunctiva, and cornea. Following the initial damage by noxae, inflammatory irritation leads to symptoms similar to the ones described for dry eye syndrome (DES). In most cases, DES symptoms develop as a consequence of a broad range of different causes and are not only limited to a preceding surgical intervention (cataract, strabismus correction, and laser in situ keratomileusis (LASIK) [48][49][50][51][52]), but DES can also occur in consequence of environmental influences, previous inflammatory diseases (chronic blepharitis, traumatic erosion of the cornea, keratitis of various etiologies, etc.), wearing contact lenses, and taking certain medications (anticholinergic drugs and antihistamines, alpha and beta blockers, antipsychotics, etc. [53]). In all mentioned cases, the initial disturbance of the eye surface is followed by a period of irritation and eye surface discomfort which contributes to a reduced quality of life. Together with the application of needed medication such as antibiotics (in case of surgery), steroids (in case of inflammation), or lubricants and wound healing promoting agents, the eye surface discomfort must be treated and a faster restoration period must be promoted.
Following the initial controlled trials on ectoine treatment of allergic rhinoconjunctivitis reviewed by Eichel [54] and first documented ectoine treatments of DES [35], several real-life, interventional, or noninterventional trials with ectoine-based eye drops have been conducted. In this article, we reviewed the literature regarding the treatment of irritations of the eye surface in the context of various indications. We focused on the ectoine treatment of allergic rhinoconjunctivitis and postsurgery treatment of ocular irritation and discomfort. The systemic review presented here is aimed at investigating the evidence on the use of this interesting substance for topical treatment of ocular surface irritations.

Objectives and Search
Strategy. For this narrative review, the literature search was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement [55]. Primary databases were PubMed, Google Scholar, and Ovid. Initial search language was English. After the search in Google Scholar and PubMed that reported different articles on Ukraine language, we extended the search to Elibrary.ru and the National library of Ukrainian and Russian/Ukrainian language. The country of origin and languages were not limited. The time period was set to the beginning of 2010 until 22 July 2020.
The following search terms/medical subject headings were: "ectoine" and "eye drops," "ectoine" and "allergic conjunctivitis," "ectoine" and "eye irritation," "ectoine" and "allergic rhinoconjunctivitis," "ectoine" and "eye burn," "ectoine" and "wound healing," "ectoine" and "eye," "ectoine" and "LASIK," "ectoine" and "Glaucoma," "ectoine" and "cataract," "ectoine" and "eye surgery," and "ectoine" and "postoperative." Studies published in peer-reviewed journals or presented on scientific congresses, reporting data on the role of the topical administration of ectoine eye drops to treat various irritations of the eye surface, were included. Studies related to other applications were not considered. Additional literature was found by reviewing the reference lists of the selected articles. The authors then independently assessed each publication and excluded those whose content was judged not to be strictly related to the subject of this review. They included only clinical trials where 2% ectoine eye drops were applied, controlled or uncontrolled, and interventional or noninterventional into eyes which were 2 BioMed Research International irritated by various reasons. Reviews, systematic reviews, meta-analyses, retrospective medical record reviews, case series, preclinical or observational studies, letters, editorials, technical notes, errata, and reports of pooled data were excluded ( Figure 1).

Study Design and Study
Population. Except one trial [65] all were real-life studies investigating the application of 2% ectoine eye drops in different settings over a period of up to 6 months, either as monotherapy or in combination with other interventions. Patient-reported outcome (diary) was used in all studies. The scores for patient reported outcome differed greatly in the method of reporting: from combined scores for all symptoms to individual scales for up to 8 symptoms. The summary scores were also calculated differently in the analysed studies. In addition to patient-reported outcomes, several trial and indication-specific parameters were measured and collected, such as wound closing time, reepithelisation time, or tear production (Schirmer test).
A total of 1795 subjects were studied in the 16 different trials. Of those, 1225 applied ectoine eye drops during their respective observation period. Four clinical trials specifically studied the effect of ectoine in children and adolescents (524 subjects in total with 492 using ectoine eye drops); the youngest child included was 2 years of age. Two other trials included children and adults but did not publish the age of the participants. Fourteen of the 16 studies included both, males and females. Two studies included only male patients. Most of the studies were performed in the Ukraine but also in Germany, Poland, Canada, Spain, and Italy. Details on the studies can be found in Tables 1 and 2.

Results from Included Clinical Trials
3.1. Safety of Ectoine Eye Drops. All 16 studies evaluated the safety of ectoine eye drops. This is of particular interest since very sensitive patient groups, like children from the age of 2 years and patients with a very recent eye surgery, were investigated in some studies. In detail, 4 studies especially treated children and adolescents with ectoine eye drops, and 2 studies evaluated the eye drops in children, adolescents, and adults. None of the studies reported a serious adverse event (SAE). Only a small number of adverse effects/adverse events (AEs) were reported. All authors attributed an excellent safety profile towards the ectoine eye drops (Tables 1 and 2).  (Table 2) 4. Discussion

Study Design and Available
Information. This review showed that ectoine eye drops (EED) have been successfully applied in a range of clinical studies covering different indications. Many of these studies are not yet internationally published (especially the Ukrainian studies), and detailed information on some of the studies (8 out of 16) was not available, as only presentations from scientific congresses could be obtained and reviewed. Therefore, an in-depth analysis of the presented data was not possible for these studies.
For most of the studies, information regarding randomization or blinding is missing, nor do entries in international study databases exist for 14 of the 16 studies. However, the combined data from fully published studies together with the data from conference presentations allowed a review of the application of EED in nonspecific irritation or inflammation of the eye surface including allergic conjunctivitis. All 16 studies reviewed here applied an eye drop formulation with 2% ectoine as key ingredient. As 15 studies Refined search strategy to combine "ectoine" with "ocular irritation", "post-surgery", "eye drops", "allergic conjunctivitis", "woundhealing" "eye burn" "conjunctivitis", "LASIK", cataract"         10 BioMed Research International applied the same formulation and the remaining one differed mainly in the type of viscosity enhancer in the formulation, this study could still be included as a confirmatory study, demonstrating that the efficacy of 2% ectoine is not dependent on the lubricant used in the formulation. We found two main areas of ocular irritation and inflammation where EED have been studied: (1) allergic conjunctivitis and (2) nonspecific eye irritation and inflammation caused by physical damage to the eye (e.g., surgery or burn).
Within the scope of allergic conjunctivitis, 5 studies investigated the effect of EED in seasonal allergic rhinitis [33,59,62,65,68], and 2 studies included patients with vernal conjunctivitis [56,57]. Primary outcome parameters of all studies were patient-reported symptoms together with study-specific measurements related to the respective study endpoints. The studies differed in terms of the studied populations (children, adults), EED application period (1 week up to 6 months), and design (comparative, crossover, add-on, noncomparative, with parallel treatments of eyes and nose, retrospective case series) with only one trial being placebo controlled. The overall number of participants for the 7 studies analysing allergic conjunctivitis was 444, including 254 children. Interestingly, one study enrolled 192 patients whereas the other 6 trials included 42 patients on average.
All studies investigating allergic conjunctivitis showed a significant improvement of the patient-reported outcome following application of EED, which was significantly better than placebo [65] and at least comparable to pharmacological standard treatments such as ketotifen or azelastine [33,68]. Usage together with standard therapy resulted in a faster decrease of symptoms than standard therapy alone (e.g., reduction of itching in 2.2 days versus 4.0 days, complete resolution in 5.3 days versus 12.8 days) [68]. Interestingly, one long-term application in patients with vernal conjunctivitis delayed the use of corticosteroids in 75% of the patients [56]. All 7 studies reported a very good tolerability and safety of the EED, both in children and in adults, which was even significantly better than an established over-the-counter drug such as ketotifen or azelastine [33,57].
As shown above, the overall picture described by the reviewed studies is a good efficacy of EED in treating the symptoms of allergic conjunctivitis either as monotherapy or in combination with other interventions, together with a very good tolerability and excellent safety profile. Especially, the results of an add-on effect of ectoine when used together with pharmacotherapies are of interest, as combination therapies are suggested by different studies in allergic rhinitis. The revision of the ARIA guideline in 2016 recommends the combination of intranasal/oral antihistamine and INGS; the combination of INGS and intranasal antihistamines acts faster than INGS alone and thus might be preferred by patients [39]. The combination of oxymetazoline and mometasone furoate nasal spray showed greater reductions in allergic rhinitis symptoms than mometasone furoate nasal spray alone [75]. Greiwe and Bernstein [76] concluded that two combinations-intranasal antihistamine with INGS and INGS with nasal decongestants-are advantageous for patients with complex rhinitis symptoms in terms of symp-tom control. Similar results are to be expected for a combination treatment of allergic rhinoconjunctivitis.
The remaining 9 studies investigated nonspecific eye irritation and inflammation of the ocular surface after a harming impact such as surgery, eye burn, or unclassified disturbance. In 7 studies, treatment with EED was conducted in the postoperative phase for different reasons (strabismus, traumatic injuries, and advanced keratoconus) [58,60,63,64,67]. One study investigated the effects on functional epiphora of unknown origin [61], another on healing after eye burn [66] and one study on the effects during an aseptic uveitis after penetrating injury [67]. A subgroup analysis on irritation due to long-term use of contact lenses was also done in one study [70]. Again, with a total of 268 children and adolescents in 2 studies exclusively conducted in this population [64,67], the EED was applied to a very sensitive group of patients.
One hallmark result from all studies was the positive effect of EED on wound healing and reepithelization in the respective studies: Pastukh et al. [63], Gorokhovskaya et al. [60], and Sarzhevska and Tabakovа [66] reported a faster healing when EED was applied concomitantly to the conventional regime after eye damage, and Rykov et al. reported positive effects on postoperative scar reduction [64]. All 8 studies conducted during the post damage or post-operative phase reported positive effects of the EED compared with standard treatment only or even to sodium hyaluronate instillation [66]. These irritation/inflammation symptoms of the ocular epithelium like conjunctival hyperaemia or foreign body sensation are often referred to as secondary dry eye syndrome. These results are supported by the study of Martinez et al., in which a comparable efficacy of EED with fluorometholone in treating functional epiphora symptoms was shown without having the typical negative side effects like interocular pressure, which is associated with corticosteroid treatment [61].

Mode of Action of Ectoine as Ideal Qualification for Its
Ophthalmic Application. The results from the clinical trials presented here are in line with the mode of the action model of ectoine, which was reported by different researchers. Based on the "preferential exclusion" model presented by Arakawa and colleagues [5] and reviewed by Lentzen and Schwarz [2], ectoine exerts its protective function by its cosmotropic effect on water molecules and-when applied topically to epithelia-results in the stabilisation of the respective tissue ( Figure 2). This stabilisation results in a reduction of inflammation, as seen in the reduction of particulate matter-induced inflammation of lung epithelia [15] and in UV-induced inflammation of the skin [77]. The effective treatment of upper respiratory tract infections has recently been reviewed [23], and effects on inflammatory diseases of the lung were also published [26,29]. Applied on the ocular surface, ectoine stabilizes not only the tissue but also the meibum layer as presented recently [18][19][20]22]. This effect, both on epithelium and on the surrounding tear film, may explain the broad and unspecific positive effect of ectoine on irritations and inflammation of the ocular surface. Moreover, it allows an   BioMed Research International add-on treatment to pharmacological treatments, thus making use of different pathways, resulting in additional effects. Interestingly, different studies were identified during this review, which reported on positive outcomes of application on ectoine-based formulations on dry eye syndrome [34,35]. Although these trials are not subject of this work and were excluded, it is worth to mention that additional evidence is available proving the successful treatment of the ocular surface with ectoine.

Conclusions
In this review, we provide evidence based on the review of 16 independent studies from 6 countries that irritations and inflammations of the ocular surface can be treated with ectoine-based eye drops, either alone or in combination with other (pharmacological) therapies. Although many of the studies showed limitations regarding their study design or reporting and data is not fully available, the following readout can be supported: ectoine is a natural substance with a unique mode of action on the eye surface and with an excellent tolerability and safety profile. This conclusion is supported by the work of other colleagues, who reviewed the efficacy of ectoine in upper respiratory inflammation [23] or allergic rhinitis (systematic review submitted) and other indications. Especially, the studies on dry eye syndrome, which have not been subject of this review, should be analysed in detail to further strengthen the evidence base for ocular application of ectoine.
This systematic review of the literature extends the previously existing knowledge about the substance in two ways.
On the one hand, some of the articles cited in the review report on patients with allergic conjunctivitis or rhinoconjunctivitis treated in real-world scenarios. This means that many of the patients had concomitant diseases and were taking concomitant medications for the underlying pathology or for other coexisting diseases. From this real-world evidence, we can conclude that the effects demonstrated in these situations are generally the same as those observed before in the more selected populations of the controlled trials.
On the other hand, these recently published articles extend our knowledge into the area of new indications beyond allergic conjunctivitis. There are reports about the use of ectoine in traumatic uveitis following a penetrating injury of the eye and similar traumatic situations. Also, the substance was studied in the vulnerable postoperative state of the eye following surgical interventions of the cornea or in corrective strabismus operations, as well as postburn treatment. From these observations we learn that, in the surgical field of ophthalmology, traumatology, nonsurgical damages, or irritations of the eye surface, the very special properties of ectoine regarding the restoration of barrier functions may open a new perspective for this treatment modality.
Therefore, ectoine-based eye drops represent a viable alternative or add-on treatment option for nonspecific eye irritation and ocular inflammation acting through stabilisation of the epithelial barrier of the organ.

AE:
Adverse event ARIA: Allergic rhinitis and its impact on asthma CRP: C-reactive protein DES: Dry eye syndrome EEC: Environmental exposure chamber EED: Ectoine eye drops ENS: Ectoine nasal spray FEC: Phacoemulsification of cataract FML: Fluorometholone INGS: Intranasal glucocorticosteroids LASIK: Laser in situ keratomileusis PRISMA: Preferred Reporting Items for Systematic Review and Meta-Analysis OTC: Over-the-counter SAE: Serious adverse event TNSS: Total Nasal Symptom Score TOSS: Total Ocular Symptom Score VKC: Vernal Keratoconjunctivitis.

Data Availability
Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Conflicts of Interest
AB reports personal fees from bitop AG. AH is employee of bitop AG. RM reports personal fees from ALK; grants from ASIT biotech; personal fees from Allergopharma; personal fees from Allergy Therapeutics; grants and personal fees from Bencard; grants from Leti, grants, personal fees, and nonfinancial support from Lofarma; nonfinancial support from Roxall; grants and personal fees from Stallergenes; grants from Optima; personal fees from Friulchem; personal fees from Hexal; personal fees from Servier; personal fees from Klosterfrau; nonfinancial support from Atmos; personal fees from Bayer; nonfinancial support from Bionorica; personal fees from FAES; personal fees from GSK; personal fees from MSD; personal fees from Johnson & Johnson; personal fees from Meda; personal fees and nonfinancial support from Novartis; nonfinancial support from Otonomy; personal fees from Stada; personal fees from UCB; nonfinancial support from Ferrero; grants from bitop AG; grants from Hulka; personal fees from Nuvo; and grants from Ursapharm, outside the submitted work.

Authors' Contributions
AB conceptualised the review, evaluated the study data, and wrote the manuscript together with AH and RM. AR helped with evaluating the study data and translated the papers and presentations, where applicable. All authors approved the final version of the manuscript before submission.