**1. Introduction**

Glaucoma is one of the leading causes of blindness worldwide, with an overall global prevalence of 3.54% (95%CI 2.09–5.82) in patients aged 40–80 years [1]. Elevated intraocular pressure (IOP) is a recognized risk factor in the development of glaucoma, and lifelong treatment is essential to prevent the progression of the disease [2–4]. Prostaglandin analog (PGA) eye drops are often the first choice when prescribing anti-glaucomatous eye drops because of their well-documented IOP-lowering effect and high tolerability [4].

**Citation:** Freiberg, J.C.; Hedengran, A.; Heegaard, S.; Petrovski, G.; Jacobsen, J.; Cvenkel, B.; Kolko, M. An Evaluation of the

Physicochemical Properties of Preservative-Free 0.005% (*w*/*v*) Latanoprost Ophthalmic Solutions, and the Impact on In Vitro Human Conjunctival Goblet Cell Survival. *J. Clin. Med.* **2022**, *11*, 3137. https:// doi.org/10.3390/jcm11113137

Academic Editors: Margaret M. DeAngelis and Michele Lanza

Received: 17 February 2022 Accepted: 25 May 2022 Published: 31 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Several studies have shown that long-term treatment with preservative-containing eye drops induces ocular surface inflammation, instability of the tear film, and damage to the corneal surface [5–7].

The tear film consists of a mucin/aqueous layer and an external lipid layer [8]. Mucin is produced by the conjunctival goblet cells. Goblet cells are essential for obtaining a stable tear film, as well as protecting and lubricating the ocular surface [8–10].

Various clinical, animal, and cellular studies document the finding that preservative agents, such as benzalkonium chloride (BAK), harm the ocular tissue and induce an inflammatory response [11–14].

Prostaglandins are lipophilic molecules and, thus, demand a solubilizing agent that is compatible with the tear film [15]. Besides the solution's antibacterial properties, preservative agents such as BAK solubilize the ophthalmic solution. If preservative agents are removed from an ophthalmic solution, they should be replaced with other solubilizing or stabilizing agents. In 2012, a preservative-free (PF) 0.005% latanoprost ophthalmic solution with Macrogol glycerol hydroxy stearate 40 (MGH40) as a solubilizing agent was developed, patented, and marketed by Laboratoires Théa (Clermont-Ferrand, France) (Monoprost®) [16]. PF formulations can be dispensed as single-dose units or multidose bottles. To prevent contamination of the formulations with a water content above 20% (*w*/*w*), multidose containers have incorporated systems, such as filters or two-layer bottles, to protect against contamination. Pharmaceutical companies have patented multi-dose bottles, e.g., ABAK® and EASYGRIP®, developed by Laboratoires Théa, Novelia®, developed by Nemerand (La Verpillière, France), and the 3K® pump/COMOD® device developed by Ursapharm (Saarbrücken, Germany) [17–19].

The European Medicines Agency (EMA, Amsterdam, The Netherlands) requires the active drug used in generics to be identical to the original preparation, with the same concentration, indication, route of administration, and bioavailability. However, the excipients may differ from the original preparation, and generics are not tested with the same efficacy and safety studies as the original products [20,21]. Differences in the physicochemical properties of preserved PGA eye drops have been identified [22,23], but little is known about the differences among PF PGA eye drops and their effect on the ocular surface, cells, and tissue.

In this pre-clinical study, five PF 0.005% latanoprost eye drops were investigated in terms of their chemical and physical properties: the pH value, osmolality, and surface tension. As a proxy for the eye drops' effect on the viability of human conjunctival goblet cells, lactate dehydrogenase (LDH) and tetrazolium dye (MTT) colorimetric assays were conducted on cultured human conjunctival goblet cells. The presence of mucin in the goblet cells was evaluated with immunohistochemical staining.

#### **2. Materials and Methods**
