A New Safety Concern for Glaucoma Treatment Demonstrated by Mass Spectrometry Imaging of Benzalkonium Chloride Distribution in the Eye, an Experimental Study in Rabbits

an Experimental Study in Rabbits. PLoS ONE 7(11): e50180. doi:10.1371/ journal.pone.0050180 A New Safety Concern for Glaucoma Treatment Demonstrated by Mass Spectrometry Imaging of Benzalkonium Chloride Distribution in the Eye, an Experimental Study in Rabbits Fran coise Brignole-Baudouin 0 Nicolas Desbenoit 0 Gregory Hamm 0 Hong Liang 0 Jean- 0 Pierre Both 0 Alain Brunelle 0 Isabelle Fournier 0 Vincent Guerineau 0 Raphael Legouffe 0 Jonathan Stauber 0 David Touboul 0 Maxence Wisztorski 0 Michel Salzet 0 Olivier Laprevote 0 Christoph 0 B 0 u 0 ouin 0 Bang V. Bui, Univeristy of Melbourne, Australia 0 1 INSERM, U968, Paris, France , 2 UPMC Univ Paris 06, UMR_S 968 , Institut de la Vision , Paris, France, 3 CNRS, UMR_7210, Paris , France , 4 Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 503, Paris, France, 5 Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Gif-sur-Yvette, France, 6 Laboratoire d'Inte gration des Syste`mes et des Technologies, CEA-LIST, Gif-sur-Yvette, France, 7 Laboratoire de Spectrome trie de Masse Biologique, Fondamentale et Applique e , EA 4550 , Universite Lille Nord de France - Universite Lille 1, Villeneuve d'Ascq, France, 8 Imabiotech Campus Cite Scientifique, Villeneuve d'Ascq, France, 9 Chimie Toxicologie Analytique et Cellulaire , EA 4463 , Faculte des Sciences Pharmaceutiques et Biologiques, Universite Paris Descartes, Paris, France, 10 Universite Versailles Saint-Quentin-en-Yvelines, Versailles, France, 11 Assistance Publique - Ho pitaux de Paris Ho pital Ambroise Pare , Service d'Ophtalmologie , Boulogne-Billancourt , France We investigated in a rabbit model, the eye distribution of topically instilled benzalkonium (BAK) chloride a commonly used preservative in eye drops using mass spectrometry imaging. Three groups of three New Zealand rabbits each were used: a control one without instillation, one receiving 0.01%BAK twice a day for 5 months and one with 0.2%BAK one drop a day for 1 month. After sacrifice, eyes were embedded and frozen in tragacanth gum. Serial cryosections were alternately deposited on glass slides for histological (hematoxylin-eosin staining) and immunohistological controls (CD45, RLA-DR and vimentin for inflammatory cell infiltration as well as vimentin for M uller glial cell activation) and ITO or stainless steel plates for MSI experiments using Matrix-assisted laser desorption ionization time-of-flight. The MSI results were confirmed by a roundrobin study on several adjacent sections conducted in two different laboratories using different sample preparation methods, mass spectrometers and data analysis softwares. BAK was shown to penetrate healthy eyes even after a short duration and was not only detected on the ocular surface structures, but also in deeper tissues, especially in sensitive areas involved in glaucoma pathophysiology, such as the trabecular meshwork and the optic nerve areas, as confirmed by images with histological stainings. CD45-, RLA-DR- and vimentin-positive cells increased in treated eyes. Vimentin was found only in the inner layer of retina in normal eyes and increased in all retinal layers in treated eyes, confirming an activation response to a cell stress. This ocular toxicological study confirms the presence of BAK preservative in ocular surface structures as well as in deeper structures involved in glaucoma disease. The inflammatory cell infiltration and M uller glial cell activation confirmed the deleterious effect of BAK. Although these results were obtained in animals, they highlight the importance of the safety-first principle for the treatment of glaucoma patients. - Funding: This study was supported by the Agence Nationale de la Recherche (grant ANR-09-PIRI-0012 MASDA-EYE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors acknowledge the fact that three authors are employed by Imabiotech Campus Cite Scientifique, a company specializing in mass spectrometry. These authors made independent analyses that confirmed the results obtained by the other members of the consortium in different laboratories. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. Glaucoma is a severe optic neuropathy leading to blindness without treatment and affecting more than 70 million people worldwide. This insidious disease is the main cause of irreversible blindness and is associated with increased intraocular pressure due to a resistance in the trabecular meshwork outflow pathway of aqueous humor [1]. Once diagnosed, treatment must be taken throughout life to prevent or halt retinal ganglion cell loss and visual deterioration. Consequently, patients have to be treated for the rest of their life with intraocular pressure (IOP)-lowering multidose eye drops (1). Most of these eye drops contain a preservative: the most commonly used is benzalkonium chloride (BAK), a quaternary ammonium salt composed of a mixture of benzododecinium C21H38N+ (BAK C12) and myristalkonium C23H42N+ (BAK C14) chlorides. BAK is a cationic surfactant and tensioactive compound, acting as a detergent for the lipid layer of the tear film as well as for the lipids of cell plasma membranes. It is reputed to increase bioavailability or penetration of active compounds and can be used as a penetration enhancer [2,3]. At a concentration ranging from 0.004 to 0.2% in eye drops, this preservative is required by pharmacopeia guidelines to prevent the multidose eye drop containers from bacterial and fungi contamination [4,5]. Although it has the advantage of inducing fewer allergic-type side effects and of being relatively well tolerated, it has been reported to induce ocular surface disorders combining irritation, inflammation and cell death processes, especially in long-term treatment [6]. There is a growing body of evidence that BAK induces apoptosis, oxidative stress and inflammation on the ocular surface epithelia, unlike antiglaucoma active compounds that have been demonstrated to be safe for epithelial cells [6]. While the deleterious effects of BAK could be negligible for a short-term treatment, they need to be considered for long-term or repeated treatment such as in chronic open-angle glaucoma. In this case, patients are most often treated for the rest of their life, often with several BAKcontaining eye drops, since about 40% of patients require multiple therapies to control their IOP and prevent further optic nerve damage [7]. The ocular surface structures, tear film, cornea, conjunctiva as well as the eyelids are the tissues most apparently involved in treatment tolerance. BAK-containing antiglaucoma eye drops have been reported to cause disruption of the blood aqueous barrier inducing cystoid macular edema following cataract surgery [8]. Little is known about BAK penetration and distribution in the eye. A (...truncated)