**1. Introduction**

Dry eye disease (DED) is a multifactorial disorder a ffecting 5% to 35% of the world population [1]. In cases of severe DED, patients experience symptoms of ocular discomfort and visual instability, resulting in a considerable loss of quality of life. Ocular burning or stinging, ocular discomfort, and ocular pain are rated as the most important symptoms by patients [2]. The loss of visual stability additionally causes a negative impact on quality of life and is attributed to an instable tear film [3]. DED has a significant socio-economic e ffect due to considerable direct treatment costs as well as indirect costs due to a loss of work productivity [4–6].

The Tear Film and Ocular Surface Society (TFOS) summarizes the current concepts as a staged managemen<sup>t</sup> and treatment for DED. Lubricating, hydrating eye drops not targeting the underlying pathophysiology of DED are the standard long-term treatment for DED [7]. Secretagogues may have an initial e ffect, but this may fade over time. If ocular lubricants and secretagogues are not providing acceptable relief from symptoms, immunomodulatory eye drops such as cyclosporine may be tried [7]. This treatment approach reflects the current model of pathophysiology of DED. Other models can either reflect a di fferent understanding or the existence of regional di fferences. The Asia Dry Eye Society, e.g., proposes a tear film-oriented therapy distinguishing between lipid layer, aqueous/secretory mucin deficiency, and corneal epithelial surface/membrane bound mucin deficiency [8,9]. It is assumed that the instability of the tear film increases the friction between the eyelids and the eye, which will result in ocular inflammation and epithelial damage [9–11]. Persistent systemic conditions of the patient or environmental adverse conditions may lead to a self-maintaining vicious circle of inflammation, which may result in chronic, eventually irreversible forms of severe dry eye [12]. Hence, a prerequisite for successful therapy is personalized clinical assessment and treatment [13]. Inflammation caused by autoimmune diseases or elevated osmolarity of the tear film due to excess evaporation have been pointed out as driving forces of the vicious circle of inflammation. Here, the treatment of inflammation as a driving force within the vicious circle plays a key role [14–16]. Although hyperosmolarity measured in the tear meniscus has been proposed for diagnosing DED, the level of osmolarity and its absolute value apparently plays a minor role as a local stress factor compared to the extent of diurnal variation [17–19]. Severe discomfort as a symptom of DED may transit to changes in neuroception, reflecting nerve damage. Nerve damage itself may be another underlying pathophysiological mechanism in severe chronic DED [20]. Not only does nerve damage result in a reduction of trophic support for the corneal epithelium, it could also initiate and maintain inflammation, and thus the interplay between nerve damage and inflammation needs to be taken into consideration [21,22]. This might contribute to the well-known discordance between dry eye signs and symptoms [23–26]. Although some substances have recently demonstrated certain potential in the treatment of neuropathic keratopathy, there is currently no therapy that directly addresses the underlying nerve damage [27,28]. Additionally, patients su ffering from neuropathic ocular pain frequently respond poorly to treatment with lubricant eye drops [29,30].

High molecular weight hyaluronan (HMWHA) has in contrast to the majority of lubricating eye drops anti-inflammatory activity and the capability to reduce the activity of the pain transducing channel TRPV1 in nociceptive nerves, thus reducing neuropathic pain [31–35]. HMWHA in eye drops provides excellent lubrication due to shear-thinning properties such as the natural tear film, good hydrating and water-binding properties resulting in reduced evaporation, and stabilization of the ocular surface barrier function to recover the protection against infection [36]. A recent study demonstrated in an environmental dry eye stress model in mice that HMWHA eye drops protect the ocular surface from mechanical damage and inflammation better than low molecular weight hyaluronan (LMWHA) [37].

The aim of the presented study (HYLAN M study) was to investigate the e ffect of HMWHA eye drops in comparison with other tear substitutes in an international multicenter prospective open label clinical investigation.

#### **2. Experimental Section**

#### *2.1. Study Design*

The HYLAN M study, a multicenter prospective randomized open label study, was performed in 11 centers in eight countries. Details of the study centers, administrative structure, planning, and conduct are provided in Appendix A. The study adhered to the Declaration of Helsinki, was approved by ethics committees of all eight countries involved, and registered as outlined in Appendix B.

Patients identified as having severe DED were randomized in two parallel arms. The control group continued with the currently used therapy as by the time of inclusion. In the verum group, the individual lubricant eye drops used by each patient by the time of inclusion were replaced by preservative-free eye drops containing 0.15% HMWHA dissolved in isotonic saline solution bu ffered with 120 mmol/L phosphate (Comfort Shield ® eye drops; i.com medical GmbH, Munich, Germany; see Appendix C). Concomitant treatment for dry eye such as cyclosporine eye drops remained unchanged in both arms.

Demographic data and medical history were recorded during the baseline visits. Symptoms and signs associated with DED were assessed at the baseline visit and at the week 4 and week 8 follow-up visits, respectively (see Table 1).


**Table 1.** Diagnostic testing schedule with optional tests in round brackets.
