**4. The role of Benzalkonium chloride (BAK)**

Benzalkonium chloride (BAK) has been used in ophthalmology since the 1940s. It is by far most common preservative, found in approximately 70% of eye drops while only 10% use other preservatives. It is used in different concentrations varied from 0.005% to 0.02% [16]. It is a quaternary ammonium compound that acts as a detergent, lysing cell membranes and thus killing microorganisms [17], in this way prevents bacterial and fungal contamination in multidose eye drop containers. It is highly effective as a preservative. It was also initially thought that the detergent effect of BAK might be necessary for the penetration of the active ingredient.

BAK toxicity for eye structures has been reported in many studies, in experimental or cell models have consistently and reliably shown its toxic effects. One major argument that is proposed to maintain the use of quaternary ammoniums in eyedrop formulations is that BAK reportedly enhances the penetration of the

drug into the anterior chamber, through disruption of the hydrophobic barrier of the corneal epitheliumm [18], however there are a large number of non-inferiority studies for bak free drugs that demonstrate the effectiveness of these medications.

#### **4.1 Bak toxicity in globet cells**

Low doses of BAK can induced proapoptotic efect on conjutival cells lines involving, as shown in vitro, reactive oxygen species and, therefore, oxidative stress reducing the number of goblet cells [19]. This cytotoxicity was proportional to the BAK concentration. In an interesting study by Guenoun, it was shown that these effects can be neutralized by topical prostaglandins analogs [20].

#### **4.2 Corneal toxicity**

Like the conjunctiva, the corneal cells have shown similar apoptotic response to BAK in superficial and deeper layers. Liang et al. confirmed the cytotoxicity of BAK in a model of a 3D culture of corneal epithelium. This study confirmed that BAK, depending on the time and concentration used, acts as a pro-inflammatory and pro-apoptotic agent able to impair the normal epithelium turnover irreversibly, even after BAK withdrawal [21].

Other study has been show topical application of BAK to the eye causes neurotoxicity, this secondarily produces reduction in aqueous tear production. Cessation of BAK treatment leads to resolution of inflammation, normalization of tear production, and recovery of stromal nerve density [22].

The damage to the ocular surface (conjuntiva, globet cells, Meibomian gland dysfunction and cornea) by topical treatment manifests itself with dry eye symptoms that have been described more frequently with the use of BAK. Jaenen et al. show a very high Prevalence of symptoms in patients using preserved drops, with 43% discomfort upon instillation, 40% burning or stinging sensation, 31% foreign body sensation, 23% dry eyes, 21% tearing, and 18% itchy eyelids [11]. Clinical impairment of the tear film and a rapid decrease of goblet cell density were also demonstrated after starting treatment with preserved timolol [22]. Schirmer's test and break-up time were altered compared to the basal control already significantly in the first month of treatment (P < 0.01 and P < 0.001, respectively). Similarly, impression cytology showed a progressive decrease in goblet cell density also significant at the first month.

#### **5. Tear production and surface staining**

The alteration of glaucoma medications affect the globet cells and meibomian glands affecting the tear film, both in quantity and quality [23]. This is accompanied by increased osmolarity.

Hyperosmolarity stimulates a cascade of inflammatory events in the epithelial surface cells, involving MAP kinases and NFkB signaling pathways and the generation of inflammatory cytokines (IL-1A; -1B; TNF-A) and MMPs (MMP9), which arise from or activate inflammatory cells at the ocular surface, these inflammatory events lead to apoptotic death of surface epithelial cells, including goblet cells, as already explained. In this way, a vicious circle is created that began with the topical treatment and is perpetuated over time. Clearly there are other factors such as the existence of previous ocular surface disease and individual variability that can give a wide range of symptoms [24].
