Gilaise® X Eye Drops – New Possibilities with Cross-Linked, Urea-Modified, High-Molecular-Weight 0.4% Hyaluronic Acid

Dry eye syndrome (DES), or dry keratoconjunctivitis (DKC), is a multifactorial disease of the tear film and ocular surface that leads to symptoms of discomfort and visual disturbances associated with tear film instability and potential damage to the corneal epithelium (Lemp M.A. et al., 2007). DSE is accompanied by increased tear film osmolarity and inflammation of the ocular surface. If left untreated, this condition can cause pain, ulcers, corneal scarring, and even reduced visual acuity (Lemp M.A. et al., 2007).

In 2017, the Tear Film and Ocular Surface Society (TFOS) published the results of the second Dry Eye Workshop (DEWS II) in a series of consensus articles on the definition, diagnosis, pathophysiology, and treatment of DFS (Nelson J.D. et al., 2017; Craig J.P. et al., 2017; Willcox M.D. et al., 2017; Bron A.J. et al., 2017; Belmonte C. et al., 2017; Wolffsohn J.S. et al., 2017; Jones L. et al., 2017; Gomes J.A. et al., 2017).
DEWS II defined dry eye as “a multifactorial disease of the ocular surface characterized by a loss of tear film homeostasis and accompanied by ocular symptoms in which tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory dysfunction play an etiological role.” The concept of tear film homeostasis disruption is considered a unifying characteristic of dry eye disease (DEO), which is then classified based on the presence or absence of symptoms (symptomatic or asymptomatic), as well as by etiology (aqueous deficiency or evaporative dry eye) (Wolffsohn J.S. et al., 2017). DEWS II proposes a stepwise approach to treating DEO, in which ocular lubricants (artificial tears) play a central role and are considered to replace or supplement the natural tear film (Jones L. et al., 2017) . Corticosteroid eye drops are recommended as an option to increase tear viscosity and improve lubrication due to their non-Newtonian thinning properties (Jones L. et al., 2017).
Contrary to the DEWS II consensus, the Asian Dry Eye Society (ADES) limits the definition of dry eye to the presence of symptoms of discomfort or visual impairment (Tsubota K. et al., 2017). ADES acknowledges that hyperosmolarity is not necessarily a prerequisite for dry eye but is often a consequence of reduced blink rate, for example, in video terminal workers, resulting in the development of ocular surface inflammation in these patients (Niederkorn J.Y., 2006). Accordingly, ADES defines dry eye as “a multifactorial disease characterized by an unstable tear film, causing a variety of symptoms and visual disturbances, potentially accompanied by damage to the ocular surface” (Tsubota K. et al., 2017) . ADES classifies dry eye into three categories: aqueous deficiency, increased evaporation, and reduced wettability, recognizing that disruption of the glycocalyx of the apical epithelial cells of the cornea leads to a reduction in the water-binding and lubricating properties of the ocular surface (Tsubota K. et al., 2020). Secretory and membrane-bound mucins are believed to play an important role in tear film instability, and there is clinical evidence that this underlying mechanism can be improved by topical secretagogues such as the eye drops diclofenac and rebamipide. With this in mind, ADES has developed a tear film-oriented treatment (TFOT) strategy, in which hyaluronan eye drops are indicated for the treatment of aqueous-deficient dry eye, while eye drops containing secretagogues may be prescribed for all three categories of dry eye (Tsubota K. et al., 2020).
There are over 140 million contact lens wearers worldwide, and many of them suffer from signs and symptoms of dry eye syndrome, including discomfort, dryness, and redness of the eyes (Stapleton F. et al., 2017). Clinical studies have shown a prevalence of discomfort ranging from 10% to 50% among contact lens wearers. Among the reported symptoms, a sensation of dry eyes is the most common (Markoulli M. et al., 2017). In addition, contact lens wearers complain of various types of discomfort associated with contact lenses. Eye drops are the first-line treatment for many causes of eye irritation, reducing friction between the eyelids and the cornea (Kathuria A. et al., 2021).
Most published clinical results indicate that hyaluronic acid (HA)-based eye drops, particularly those containing very high molecular weight HA, not only act as water-binding lubricants but can also actively influence the underlying pathophysiological mechanisms of ocular surface diseases (Beck R. et al., 2021; Kojima T. et al., 2020). To discuss the issue of OCS and the use of HA preparations, it is necessary to focus on understanding ocular surface homeostasis.

Ocular Surface Homeostasis
The healthy ocular surface epithelium is topographically smooth. The lipid bilayer plasma membrane of the apical epithelial cells of the cornea is textured with microvilli lined with an anti-adhesive, water-binding protective glycocalyx. The glycocalyx consists primarily of membrane-bound mucins. It is covered by a muco-aqueous tear film with lubricating properties, primarily due to the dissolved gel-forming mucin MUC5AC, which is secreted by the goblet cells of the conjunctiva (Nichols B. et al. , 1983; Argüeso P. et al., 2001; Gipson I.K. et al., 2003; Argüeso P.; Hodges R.R. et al., 2013; Corfield A. Mucins, 2015; Portal C. et al.; Baudouin C. Fini M.E et al., 2019). The glycoprotein MUC5AC consists of a long protein molecule with oligosaccharide side chains linked via O-glycosylation to the protein backbone (Gipson I. K. et al., 2003; Moniaux N., 2001), which consists of monomeric units with an average molecular weight of 2.2 MDa (Sheehan J.K. et al., 2000). The monomeric units are disulfide bonds, resulting in the formation of MUC5AC macromolecules with an average molecular weight exceeding 40 MDa, which form very long, linear, flexible filaments over 10 μm in length (Gipson I.K. et al., 2003; Sheehan J.K. et al., 2000; Perez-Vilar J. et al., 1999). It has been found that MUC5AC in the precorneal tear fluid has a lower average molecular weight (Spurr-Michaud S. et al., 2007). However, this conclusion may have been influenced by the sampling technique and the fact that the chain length of long linear MUC5AC molecules (polymers) may be non-uniform and determined by their tendency to entangle (Graessley W.W., 2006).
Such polymer solutions exhibit viscoelastic shear thinning characteristics for the tear film (Kaura R., Tiffany J.M., 1986; Tiffany J.M., 1991, 1994; Tiffany J.M. et al., 1998). For stability reasons, the tear film requires high viscosity at rest and low viscosity during blinking to prevent excessive shear stress on the ocular surface epithelium.
DSC alters the rheology of the tear fluid (Tiffany J.M., 1991). Every form of dry eye, as well as atopic keratoconjunctivitis, is associated with ocular inflammation, loss of goblet cells, and reduced levels of MUC5AC (Gipson I.K. et al., 2003; Baudouin C., Fini M.E. et al., 2019; Ralph R.A., 1975; Argüeso P. et al., 2002; Dogru M. et al., 2006, 2008; Mantelli F. et al., 2008; Garcia-Posadas L. et al., 2018). A decrease in the concentration, glycosylation, or molecular weight of MUC5AC is associated with a reduction in the lubricating efficiency of the tear film. Increased friction between the cellular structures of the ocular surface is recognized as the driving force not only of SCO but also of all forms of ocular surface disease (Van Setten G.B. et al., 2019). Increased friction between ocular surface tissues may arise not only due to insufficient amounts of MUC5AC dissolved in the muco-aqueous layer of tears, but also due to disruption of the glycocalyx of apical epithelial cells or increased eyelid pressure (Yamaguchi M. et al., 2018). The glycocalyx contains mucins bound to the plasma membrane of the apical epithelial cells of the cornea and conjunctiva conjunctiva. The largest of these mucins, MUC16, extends 200–500 nm from the tips of microvilli into the muco-aqueous layer of the tear film and prevents cell adhesion, as well as bacterial adhesion and invasion (Hilkens J. et al., 1992; Blalock T.D. et al., 2007; Guzmán-Aránguez A. et al., 2010; Gipson I.K. et al., 2014). SSO alters the degree of O-glycosylation, particularly of MUC16, which leads to impaired wettability, water-retention capacity, anti-adhesive, and lubricating properties of the cellular surface barrier and, ultimately, even to the appearance of dry patches on the epithelial surface (Gipson I.K. et al., 2003; Argüeso P.; Hodges R.R. et al., 2013; Gipson I.K. et al., 2014; Danjo Y. et al., 1998; Gipson I.K., Hori Y., Argüeso P., 2014; Sumiyoshi M. et al., 2008; ShimazakiDen S. et al., 2013; Uchino Y., 2018) . It is worth noting that MUC16 plays an important role not only in the function of the cellular epithelial barrier but also promotes tight junctions between epithelial cells and, consequently, the function of the paracellular barrier (Shimazaki-Den S. et al., 2013). Topographical irregularities of the ocular surface have recently attracted attention as an additional source of ocular surface friction (Van Setten G., 2017). The levels of MUC5AC concentration and chain length in the mucinous layer may be either too high, leading to the formation of mucin strands, blurring, and high friction during blinking, or too low, leading to insufficient lubrication and tear film instability (Garcia-Posadas L. et al., 2018). Any sustained increased friction during blinking creates mechanical stress, leading to epithelial damage. This manifests as staining of the cornea and conjunctiva and conjunctival epitheliopathy of the eyelids, inflammation of the eyelid margins associated with obstruction of the meibomian gland orifices, and possible changes in the topography of the ocular surface, such as eyelid-parallel conjunctival folds (LIPCOF) (Höh H. et al., 1995; Korb D.R. et al., 2010; Van Setten G., 2017). In fact, just 60 seconds of eye friction is sufficient to increase levels of inflammatory markers, such as MMP-13, IL-6, and TNF, in healthy eyes (Balasubramanian S.A. et al., 2013). Therefore, it can be assumed that constant stress on the ocular epithelial tissues due to increased friction triggers an acute inflammatory response and may ultimately lead to chronic inflammation. Al-Akaba and colleagues described the potential role of corneal nerve abrasion in neurotrophic keratopathy, post-penetrating keratoplasty, laser refractive surgery, and chronic corneal edema, and concluded that abrasion may also explain the observed lack of correlation between corneal epithelial nerve density and corneal sensitivity (Al Aqaba M.A. et al., 2019). Thus, the osmolarity of the aqueous phase of the tear fluid secreted by the main lacrimal glands is well regulated. The peak increase in osmolarity between two blinks depends largely on the thickness of the tear film, the time interval between blinks, and the water-retaining properties of the glycocalyx and tears (Willcox M.D. et al., 2017). Hyperosmolarity of the aqueous-mucous layer of tears is considered one of the main etiological factors of ocular surface inflammation (Farris R.L., 1994; Baudouin C. et al., 2013, 2016). Innervation of the corneal epithelium includes cold thermoreceptors sensitive to changes in temperature and osmolarity via TRPM8 channels. An increase in extracellular osmolarity or a decrease in the temperature of the tear film due to evaporation triggers blinking (Parra A. et al., 2014). Blinking, in turn, leads to the redistribution of the tear film from the tear meniscus reservoir.
Surface irregularities of the eye cause the formation of localized areas of a thin tear film. Intensive screen viewing causes prolonged intervals between blinks and, consequently, greater fluctuations in osmolarity between blinks (Sheppard A.L. et al., 2018; Van Setten G.-B., 2019). The lipid layer, which covers the aqueous-mucous phase, has been considered for decades to be the most important barrier against tear evaporation, and therefore meibomian gland (MG) disease is the most common cause of DSE (Craig J.P. et al., 1997, 2017; Knop E. et al., 2011). However, recent data suggest that the thickness of the tear lipid layer has only a minor effect on the rate of tear evaporation (King-Smith P.E. et al., 2009, 2013; Georgiev G. et al., 2014). Therefore, the widespread belief that the tear film is disrupted in DKS due to increased evaporation resulting from a defective lipid layer has been called into question (Millar T.J. et al., 2015). On the other hand, gel-forming mucins, such as MUC5AC in the mucous layer of the tear film and the glycocalyx mucins of apical epithelial cells on the ocular surface, have the ability to retain water through their carbohydrate side chains (Willcox M.D. et al., 2017; Bron A.J. et al., 2017; Mantelli F. et al., 2008; Uchino Y., 2018; Carlstedt I. et al., 1985) . Dry eye is associated with reduced MUC5AC concentration, as well as with reduced glycosylation of MUC5AC and MUC16 molecules (Gipson I.K. et al., 2003; Argüeso P., 2002, 2013; Ralph R.A., 1975; Danjo Y. et al., 1998; Versura P. et al., 2009). It is quite likely that this is one of the main causes of increased tear evaporation rate, and consequently, hyperosmolarity or osmolarity fluctuations in patients with ocular surface diseases.

Hyaluronic acid and its properties
The medical community owes the emergence of highly purified HA to Endre A. Balas, who in the late 1960s discovered that HA does not cause an inflammatory reaction in the eyes of marmosets and can be used to replace pathological synovial fluid in arthritic joints, as well as the vitreous body and aqueous humor of the human eye (Nelson J.D. et al., 2017). In 1976, the Swedish pharmaceutical company Pharmacia took over the production and global marketing of highly purified high-molecular-weight hyaluronan (HMW HA) under the trade name Healon (Pharmacia, Uppsala, Sweden) for use as a therapeutic agent for pain relief in arthritis, and later for ophthalmic surgery. The first ophthalmic surgeon was Robert K. Stegman, who in 1978 successfully injected Healon into the anterior chamber of the human eye to prevent damage to the corneal endothelium during cataract surgery (Craig J.P. et al., 2017). The first report of the clinical use of GC eye drops in patients with severe dry eye dates back to 1982. Polak and McNeice used a 0.1% solution of GC from leftover Healon syringes used for cataract surgery to treat patients with severe dry keratoconjunctivitis (Willcox M.D. et al., 2017). By the mid-1990s, it was believed that GCs achieved their physiological functions exclusively through nonspecific interactions, such as lubrication, mechanical buffering, water homeostasis, and macromolecular filtration (Bron A.J. et al., 2017).
In 1994, the European Commission’s Working Group on the Use of Medicinal Products/Devices decided during its meeting that medical products containing HA are regulated as medical devices unless the manufacturer intends to claim pharmacological, immunological, or metabolic activity as the primary intended mechanism of action. Since 1998, numerous brands of eye drops containing HA of various molecular weights in different concentrations have been approved as medical devices in Europe.
In 1995, Hyalein hyaluronan eye drops from the Japanese ophthalmic drug manufacturer Santen (Osaka, Japan) were approved by Japanese authorities as a prescription drug for the treatment of dry eye. These eye drops have proven their effectiveness as a highly biocompatible substitute for the aqueous phase of human tears. While eye drops obtained by diluting Healon contain high-molecular-weight hyaluronic acid (HMW HA), Hyalein eye drops contain low-molecular-weight hyaluronic acid (LMW HA).
In the U.S., tear substitutes are regulated as “ophthalmic emollients,” which, if they contain certain active ingredients at established concentrations, can be registered as over-the-counter drugs without the need for a rigorous approval process (Belmonte C. et al., 2017). Currently, ophthalmic demulcents (water-soluble polymers), particularly HA, are vital components of both over-the-counter and prescription products used for the treatment of dry eye syndrome and contact lens care. They lubricate the epithelium and reduce surface irritation.
HA is a biocompatible and biodegradable polymer. It is a vital component of human eye physiology and is present in the vitreous body, lacrimal gland, corneal epithelium, and conjunctiva (Berriaud N. et al., 2005; Lapcik L. et al., 1998; Stuart J.C. et al., 1985; Yoshida K. et al., 1996), and has also been detected in tear fluid (Berry M. et al., 1998; Frescura M. et al., 1994; Fukuda M. et al., 1996).
HC has a unique viscoelastic profile. During blinking, shear stress causes HC molecules to align with one another. As a result, the solution momentarily loses its viscosity and spreads easily across the corneal surface. Between blinks, the HC chains form a tangled network and the solution becomes more viscous. This stabilizes the precorneal tear film and maximizes the solution’s residence time on the eye’s surface, where HC is able to improve eye hydration due to its hygroscopic and mucoadhesive -adhesive properties (Nakamura M. et al., 1993).
HA is a high-molecular-weight glycosaminoglycan composed of units of D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc), linked by alternating β-(1 → 4)- and β(1 → 3)-glycosidic bonds. HA is one of the most important and well-studied biomolecules of the extracellular matrix, as well as one of the most commonly used ingredients in artificial tears and multipurpose solutions. HA is used as a moisturizer or soothing agent.
Based on molecular weight, HA can be classified into five groups: very high molecular weight HA (vHMM-HA >5000 kDa), high molecular weight HA (HMW-HA, 3000–1000 kDa), medium-molecular-weight HA (MMW-HA, 1000–250 kDa), low-molecular-weight HA (LMW-HA, 250–10 kDa), and oligosaccharides (<10 kDa) (Tavianatou A.G. et al., 2019). The size of a molecule determines its physicochemical properties as well as its biological activity (Müller-Lierheim W.G., 2020; Huerta-Ángeles G. et al., 2021). In fact, the viscoelastic properties of any formulation (composition) containing native and/or modified HA will be significantly influenced by its concentration, molecular weight, and chemical or physical modification. In the case of eye drops, manufacturers typically do not report the molecular weight of HA (Aragona P. et al., 2019). Some studies have used vHMM-HA (Müller-Lierheim W.G., 2020). However, eye drops are typically autoclaved prior to use, so the average molecular weight of HA decreases due to hydrolysis. Ideal HA-based eye drops should likely include HMW-HA, which ensures increased viscosity at low shear rates without exceeding the blurring threshold (Aragona P. et al., 2019). The primary effects of HA are mediated through its mucoadhesive properties.
By definition, mucoadhesion is the adhesion of two surfaces, one of which is a mucous membrane (Smart J.D., 2005) . Mucoadhesion has become a subject of interest due to its potential to optimize local drug delivery by retaining and releasing the active pharmaceutical ingredient near the site of action (Andrews G.P. et al., 2008; Smart J.D., 2005) . Mucoadhesion occurs in two stages: interpenetration and binding of a mucoadhesive polymer containing functional groups such as hydroxyl (OH), carboxyl (COOH), amide (NH2), or sulfate (SO4H), with mucin chains, followed by the formation of hydrogen bonds between the mucoadhesive polymer and the mucin chains (Andrews G.P. et al., 2008). When incorporated into a mucous gel or layer, mucoadhesive polymers increase the gel’s resistance to deformation (known as rheological synergy) and strengthen the layer (Mortazavi S. et al., 1992; Madsen F. et al., 1998; Dodou D. et al., 2005). The flexibility of polymer chains is important for interpenetration and entanglement with mucin chains, allowing the formation of hydrogen bonds (Andrews G.P. et al., 2008; Smart J.D., 2005). Mucoadhesive polymers can serve as drug carriers, but they can also be used independently to coat and protect, for example, damaged tissues or act as lubricants (Smart J.D., 2005) . On the surface of the eye, mucoadhesion involves interaction with the mucins of the muco-aqueous tear film, as well as with the membrane-bound mucins of the glycocalyx of apical epithelial cells. GC is a highly flexible polyanion capable of tightly binding to and adhering to mucin molecules on the ocular surface. The interaction between mucin molecules and mucoadhesive molecules causes changes in the shape or arrangement of macromolecules, which is reflected in a change in viscosity (Hassan E.E. et al., 1990). Based on the results of their experiments, Madsen and his colleagues concluded that rheological synergy, as verified by rheological methods, does not fully explain the mucoadhesive phenomenon and should not be considered an independent method for characterizing the interaction between mucus and polymers (Madsen F., 1998). At the same time, the rheological method is best suited for determining the interaction between a mucus gel, such as MUC5AC, and mucoadhesive polymers. Hansen et al. reported the binding of high-molecular-weight HA to cell-membrane-associated mucins, which consequently strengthens the cellular barrier against pathogen invasion and prolongs the residence time for local drug delivery (Khutoryanskiy V.V. et al., 2010).
The study of the advantages of high-molecular-weight HA in eye drops for the treatment of SCO deserves special attention. Thus, HA preparations with very high molecular weight are capable of replacing MUC5AC in the tear film and are completely non-allergenic. Moreover, very high-molecular-weight GC has the ability to counteract inflammation (Petrey A.C. et al., 2014).
Let us focus on the physiological role of high-molecular-weight GC in ocular surface homeostasis and the clinical efficacy of eye drops containing high-molecular-weight GC. Until the mid-1990s, it was believed that HA performed its physiological functions exclusively through nonspecific interactions, such as lubrication, mechanical buffering, water homeostasis, and macromolecular filtration (Laurent T.C. et al., 1996). The cloning of three cell surface HA receptors—CD44, RHAMM (receptor for HA-mediated motility)—sparked intensive research on HA (Goldstein L.A. et al., 1989; Aruffo A. et al., 1990; Hardwick C. et al., 1992; Borowsky M.L. et al., 1998; Bono P. et al., 2001) . This ultimately led to our current comprehensive understanding of the active role of GCM in tissue homeostasis, wound healing, and inflammation.
R. Tammi and colleagues found that cultured rat epidermal keratinocytes contain GCM, which is catabolized via intracellular lysosomal degradation (Tammi R. et al., 2001). These authors established that the CD44 receptor is involved in the endocytosis of HA by epidermal keratinocytes, but did not detect CD44 in vesicles containing HA. Extracellular and intracellular HA had molecular weights of up to ≤6000 kDa and < 400 kDa, respectively, indicating that keratinocytes selectively remove low-molecular-weight HA from the extracellular matrix (Tammi R. et al., 2001). The long chain of HA molecules is highly susceptible to degradation by reactive oxygen species generated by inflammatory processes as well as radiation (Ågren U.M. et al., 1997). It is quite likely that epidermal keratinocytes are capable of limiting the proportion of low-molecular-weight HA in the extracellular matrix under homeostatic conditions.
In its high-molecular-weight form, HA exerts an immunosuppressive effect (Delmage J.M. et al., 1986; Jiang D. et al., 2011). Large HA molecules protect against lymphocyte-mediated cytolysis, suppress septic reactions to lipopolysaccharides, maintain immune tolerance, induce the production of immunosuppressive macrophages, reduce the expression of inflammatory cytokines, modulate the immune system, and are anti-angiogenic; furthermore, high-molecular-weight hyaluronan has an intrinsic rejuvenating effect (Petrey A.C. et al., 2014; Aya K.L. et al., 2014; Feinberg R. et al., 1983; Meyer L.J. et al., 1994; Mummert M.E. et al., 2002; Stern R. et al., 2008; Papakonstantinou E. et al., 2012; Tian X. et al., 2013). It has also been postulated that particularly high-molecular-weight HA reduces oxidative stress (Litwiniuk M. et al., 2016). On the other hand, it has been reported that reactive oxygen species formed during inflammatory processes depolymerize HA and that HA fragments trigger hypersecretion of MUC5AC mucin (Yu H. et al., 2011). Furthermore, low-molecular-weight degradation products of hyaluronan may induce inflammation and angiogenesis (Aya K.L. et al., 2014; Jiang D. et al., 2011; Jiang D. et al., 2007). In addition to its mucoadhesive effect, HA can bind to a number of cell surface receptors (hyaladherins), including CD44, LYVE-1, HARE, lailin, TLR4, and RHAMM, thereby promoting the detachment of transient cells and influencing cell proliferation, survival, motility, and migration (Tammi R. et al., 2001; Solis M.A. et al., 2012) . It has also been reported that HA is involved in neurogenesis (Preston M., 2011).
HA is present at all stages of the wound healing process, not only as an integral component of the wound environment but also as a factor that actively modulates tissue regeneration (Litwiniuk M. et al., 2016; Price R.D. et al., 2005; Stiebel-Kalish H. et al., 1998). Low-molecular-weight HA is a product of the hydrolysis of high-molecular-weight HA during inflammation, but, more importantly, it also acts as a stimulator of inflammation and the entire wound healing process (Aya K.L. et al., 2014; Kavasi R.-M. et al., 2017). Therefore, K.L. Aya et al. suggested paying attention to the often underestimated role of HA in wound healing (Aya K.L. et al., 2014).
GC and the cell membrane-bound CD44 receptor are localized in basal cells, as well as on the apical surface of surface epithelial cells of the cornea and conjunctiva (Asari A. et al., 1992; Zhu S.-N. et al., 1997; Lerner L.E. et al., 1998; Asari A. et al., 2004). The expression of GC on basal cells is consistent with its role in promoting the migration and proliferation of epithelial cells (Lerner L.E. et al., 1998; Gomes J.A. et al., 2004; Evanko S.P. et al., 2007). Low concentrations of high-molecular-weight GK molecules support cross-linking between GK receptor sites on adjacent cells and may contribute to the mechanical stabilization of the apical layers of the corneal epithelium (Evanko S.P. et al. , 2007; Toole B., 1990; Knudson C.B. et al., 1993; 1999). Elevated levels of CD44 and HC on the apical surface of the epithelium and the confirmed presence of HC in the tear film support the hypothesis that membrane-bound membrane-bound HC can significantly contribute to the hydration, lubrication, and barrier function of the corneal epithelium and ultimately even replace damaged membrane-bound mucins in the glycocalyx (Frescura M. et al., 1994; Dreyfuss J.L. et al., 2015) . The molecular weight of membrane-associated HA determines the activity of immune cells. Thus, low-molecular-weight HA inhibits the chemotaxis and phagocytosis of macrophages, whereas high-molecular-weight HA has almost no effect on chemotaxis (Forrester J.V. et al., 1980; Tamoto K. et al., 1994). High-molecular-weight HA is a potent activator of macrophages and induces IL-1β, TNF, and four members of the neutrophil family, as well as the binding of chemotactic factors to the surface of neutrophils, and the aggregation, adhesion, and binding of neutrophils to surfaces (Forrester J.V. et al. , 1981). Fragments of HA are potent activators of dendritic cells (Termeer C. et al., 2004; Powell J.D. et al., 2005). The ability of HA to function as a pro- or anti-inflammatory molecule depends on its size, microenvironment, localization, and the presence of specific binding partners; HCA acts as a key regulator of inflammation (Petrey A.C. et al., 2014; Lee-Sayer S.S.M. et al., 2015). Thus, in the amniotic membrane stroma, HA plays an important role in trapping inflammatory cells, including lymphocytes (Higa K. et al., 2005). On the other hand, reactive oxygen species formed during inflammatory processes effectively degrade high-molecular-weight HA, which, in turn, sustains the inflammatory process (Martínez-Cayuela M., 1995; Šoltés L. et al., 2006). Therefore, GC metabolism deserves attention when considering the vicious cycle of chronic inflammation in ocular surface diseases (Baudouin C. et al., 2017).
GC plays a key role in the healing of corneal epithelial wounds (Litwiniuk M. et al., 2016; Šoltés L. et al. , 2006; Dua H.S. et al., 1994; Ruppert S.M. et al., 2014) . In this context, it is important that the HARE receptor, responsible for GC binding and endocytosis, is highly expressed on corneal epithelial cells (Falkowski M. et al., 2003; Harris E.N. et al., 2020). It has been shown that high-molecular-weight GCs inhibit activity in nociceptive afferent nerves by modulating the opening rate of polymodal transient receptor potential vanilloid subtype 1 (TRPV1) (Gomis A. et al., 2004; Caires R. et al., 2015). In addition, high-molecular-weight HA appears to be important for the proliferation, differentiation, and maturation of nerve cells (Preston M., 2011). All these properties of high-molecular-weight HA likely contribute to symptom improvement in ocular surface diseases and warrant further investigation.
The results of numerous clinical studies conducted over the past 20–30 years demonstrate the high efficacy of eye drops based on high-molecular-weight corticosteroids in patients with Sjögren’s syndrome, Stevens-Johnson syndrome, dry keratitis, corneal dystrophy, recurrent erosion, contact lens-induced irritation, pemphigoid, filamentary keratitis, and neurotrophic keratitis (Deluise V.P. et al., 1984; Stuart J.C. et al., 1985).
In recent years, dysregulation of the epithelial barrier has been recognized as a key defect in the pathogenesis of atopy and allergic reactions (Yokoi K. et al., 1984; Mantelli F. et al., 2013). It can be expected that eye drops containing high-molecular-weight corticosteroids will also prove effective in the prevention and treatment of allergic eye diseases due to their supportive role in the mechanical removal of allergens from the tear film, their stabilizing effect on epithelial barrier function, and their ability to inhibit the activation of inflammatory cells. It has been demonstrated that high-molecular-weight GC-based eye drops counteract inflammation in patients with atopy and allergies, unlike low-molecular-weight GC preparations, which may even promote inflammation of the ocular surface (Gipson I.K. et al., 2003; Hansen I.M. et al., 2017; Petrey A.C. et al., 2014; Lerner L.E. et al., 1998 ; Lee-Sayer S.S. et al., 2015; Mantelli F. et al., 2013). The presence of HARE receptors on the surface of ocular epithelial cells allows them to internalize GCs via endocytosis. This is a novel method for transporting active pharmaceutical ingredients using high-molecular-weight GC molecules as a carrier across the cell membrane of epithelial cells without damaging the cell membrane. It has been demonstrated that GC mitigates the adverse effects of corneal toxic substances (Wysenbeek Y.S. et al., 1988; Pauloin T. et al., 2008, 2009; Liu X. et al., 2015); therefore, it is expected that patients requiring long-term topical treatment for conditions such as glaucoma, who currently suffer from the adverse side effects of treatment, will benefit from this new technology.
To date, there are numerous publications on eye drops containing high-molecular-weight HA, which have demonstrated the clinical efficacy of these agents. Research findings confirm the role of high-molecular-weight HA in regulating ocular surface inflammation, corneal wound healing, regeneration of damaged nerves, immunoregulation, alleviation of symptoms of allergic keratoconjunctivitis and atopy, as well as in improving pain-related symptoms through interaction with superficial ocular nerves. High-molecular-weight HA facilitates the transport of drug nanoparticles across the ocular epithelial barrier. Given this, high-molecular-weight HA is a candidate to replace current penetration enhancers and will significantly reduce side effects during long-term topical treatment of eye diseases such as glaucoma, allergies/atopy, and chronic inflammation.
A number of studies have demonstrated that HA stimulates the migration of corneal epithelial cells and possesses anti-inflammatory and antioxidant properties; therefore, it may play a role in wound healing (Gomes J.A. et al., 2004; Inoue M. et al., 1993; Nishida T. et al., 1991; Presti D. et al., 1994; Scott J.E., 1995). Numerous studies have been conducted to evaluate the safety and efficacy of GC-based eye drops. All of them demonstrate a marked improvement in the symptoms and signs of SSO, associated with the concentration of GC and the molecular weight of GC (typically 0.1–0.4% solutions and 0.8–1.4 MDa GC, respectively) (Stuart J.C. et al., 1995; Aragona P. et al., 2002; Dumbleton K. et al., 2009; Hamano T. et al., 1996; Johnson M.E. et al., 2006; Prabhasawat P. et al., 2007; Sand B.B. et al., 1989). All of this explains the vast variety of commercially available eye drops containing HA. However, most of these products available on the market contain the linear form of this polymer. Only a few eye drops containing cross-linked HA are available on the Ukrainian market.

Cross-linked hyaluronic acid
Cross-linking is a chemical strategy aimed at increasing the stiffness of the polymer network (i.e., the viscoelasticity of the gel), prolonging its stability at the site of application, and reducing susceptibility to enzymatic degradation to decrease the daily frequency of application (Fallacara A. et al., 2017). Among the most popular strategies are crosslinking via condensation reactions, enzymatic crosslinking, disulfide crosslinking, click chemistry, and polymerization to form double bonds between different chains. Crosslinked HA-based injectable hydrogels have demonstrated excellent photothermal antibacterial properties against E. coli and Staphylococcus aureus in vitro. In vivo studies also showed that one of these hydrogels significantly reduced E. coli infection, decreased inflammation, and promoted angiogenesis and wound healing in patients with E. coli (Ren Y. et al., 2022). Among the various approaches used for HA crosslinking, one of the most studied involves modifying available hydroxyl groups using homo-bifunctional crosslinking agents such as 1,4-butanediol diglycidyl ether (BDDE), glutaraldehyde, ethylene sulfide, methacrylic anhydride, and divinylsulfone (DVS). These derivatives have been used for intra-articular and dermal injections.
The use of the aforementioned compounds as crosslinking agents simplified the synthesis of crosslinked HA and ensured good mechanical properties. However, after release for HA hydrolysis, these compounds became potential sources of side reactions, many of which are recognized as mutagenic and toxic molecules (Schanté, C.E. et al., 2011). A few scientific studies confirm the absence of toxicity to lung cells and resistance to enzymatic degradation of cross-linked HA, where arginine was used as the cross-linking agent (Sciabica S. et al., 2023). The antibacterial properties of arginine-crosslinked HA against S. aureus and P. acnes and its effect on S. pneumoniae are also noted, which may be useful for use in cosmetic preparations for topical application and for bronchopulmonary administration (Sciabica S. et al., 2023). Relatively few scientific publications address the effects of cross-linked HA in ophthalmic preparations (Calles J.A. et al., 2013; Calles J.A. et al., 2016; Postorino E.I. et al., 2017; Cagini C. et al., 2017). In light of this, we would like to focus on a review of the use of urea-crosslinked HA in ophthalmic products.
Urea is well known as a humectant due to its ability to retain water, which promotes cell regeneration and repair (Fallacara A. et al., 2017). J.F. Charlton et al. (1996) found that topical urea is capable of stimulating corneal re-epithelialization and limiting damage to the corneal epithelium following injury. Thus, all the aforementioned studies on the ophthalmic use of HA and urea indicate that HA cross-linked with urea (Citernesi U.R. et al., 2015) , may be an innovative and promising ingredient for eye drops aimed at inducing corneal re-epithelialization. Urea is not only a cross-linking agent that increases the viscosity of native HA by binding its chains, which may account for its longer retention on the corneal epithelium. Urea is also a non-toxic molecule with its own health-promoting activity (Citernesi U.R. et al., 2015; Fallacara A. et al., 2017). Thus, HA cross-linked with urea is a promising polymer, as it was designed to improve not only the molecule’s mechanical properties but also its biological activity (Citernesi U.R. et al., 2015; Fallacara A. et al., 2017).
The study by A. Fallacara et al. (2017) aimed to investigate the safety and efficacy of eye drops based on a new high-molecular-weight HA cross-linked with urea in improving corneal re-epithelialization. The study was conducted on 2D human corneal cells (HCEpiC) and on 3D reconstructed human corneal epithelial tissues (HCE). To investigate the ranges of efficacy, concentrations of urea-crosslinked HA at 0.02% (solution S1) and 0.4% (solution S2) were analyzed to assess the range of efficacy. The release of pro-inflammatory cytokines (IL-8) was evaluated using ELISA, and corneal morphology was assessed using hematoxylin and eosin staining. In addition, to understand the molecular basis of the re-epithelialization properties, cyclin D1 levels were assessed using Western blotting. The results showed no cellular toxicity, a slight decrease in IL-8 release, and restoration of epithelial integrity after treatment of the 3D wound model with solutions S1 and S2. Concurrently, cyclin D1 levels increased in cells treated with both S1 and S2. The corneal epithelium, treated with the two solutions S1 and S2, demonstrated a noticeable improvement in wound closure compared to the positive control, as indicated by corneal morphology. These data suggest that solutions S1 and S2 may accelerate epithelial wound closure by promoting cell proliferation induced by cyclin D1.
The clearly demonstrated promising results for the use of artificial tears containing urea-crosslinked GC for the treatment of CSC and corneal injuries. Although neither S1 nor S2 was able to significantly reduce IL-8 levels, they demonstrated interesting wound-healing properties and re-epithelialization efficacy in the analyzed cell models: clear wound healing was observed in both the 2D and 3D models. This was also confirmed by histological analysis, which showed restoration of the microscopic epithelial structure following treatment with S1 and S2 solutions. The findings of previous in vivo and in vitro studies (Gomes J.A. et al., 2004; Inoue M. et al., 1993; Nishida T. et al., 1991; Condon P.I. et al., 1999; Papa V. et al., 2001; Williams D. et al., 2012) also confirmed that native HA and other types of cross-linked HA promote corneal epithelial wound healing (Williams D.L. et al., 2013, 2014; Yang G. et al., 2010; Wirostko B. et al., 2014; Williams D.L. et al., 2017; Calles J.A. et al., 2013, 2016; Postorino E.I. et al., 2017; Cagini C. et al., 2017) . Furthermore, Western blot analysis showed that after treatment with S1 and S2 eye drops, the level of the proliferative marker cyclin D1 was elevated compared to the control. Thus, the two urea-conjugated GC solutions accelerated the tissue proliferation process associated with re-epithelialization.
This study offers promising prospects, as urea-crosslinked GC can rapidly alleviate both the signs and symptoms of SCO, even when used at a concentration (0.02%) lower than that typically used for GC-based artificial tears (usually 0.1–0.4%) (Stuart J.C. et al., 1985; Aragona P. et al., 2002; Dumbleton K. et al., 2009; Hamano T. et al., 1996; Johnson M.E. et al., 2006; Prabhasawat P. et al., 2007; Sand B.B. et al., 1989; Condon P.I. et al., 1999). Thus, eye drops based on high-molecular-weight HA cross-linked with urea can be effectively used to treat dry eye syndrome and traumatic and postoperative corneal injuries. These studies show promising results and open up interesting prospects for the use of urea-crosslinked HA in ophthalmology.
Innovative Gilays® X eye drops are entering the Ukrainian pharmaceutical market; they contain high-molecular-weight 0.4% HA, produced using patented urea cross-linking technology.

Thanks to its latest formula, Gilays® X offers the following advantages over conventional HA:

• longer resistance to hyaluronidase degradation;
• a more pronounced effect on epithelialization and regeneration of the cornea and conjunctiva;
• better and longer-lasting lubricating and moisturizing action;
• retention of a greater amount of water molecules;
• provides maximum comfort within the first few minutes after application;
• no anti-inflammatory or vasoconstrictive effects;
• no preservatives in the formulation;
• compatibility with contact lenses.

Kyiv Vitamin Plant JSC traditionally offers and guarantees the high quality and effectiveness of Gilaise® X while keeping it affordable.

Gilaise® X can be widely used for the following ophthalmic conditions:

• traumatic and postoperative corneal damage;
• SSO (dry eye disease);
• computer vision syndrome;
• adverse environmental effects (chlorinated water, air conditioning, dust, smoke, UV radiation);
• chronic blepharitis;
• age-related decrease in tear production;
• refractive surgery;
• cataract surgery;
• cosmetic eyelid surgery; wearing contact lenses.

Eye drops Gilaise® X from Kyiv Vitamin Plant JSC – an innovative solution to improve your quality of life amid the “epidemic” of dry eye! Treat your eyes to comfort in any situation!