Purpose We investigated the potential short and long-term effects in cultured human trabecular meshwork (TM) cells of various topical glaucoma formulations containing different preservatives. any long-term effects we assayed release of matrix metalloproteinase 9 (MMP-9) and apoptosis 24 h after treatments. Results BAK exhibited a dose-dependent reduction in TM cell viability ranging from 71±5% live cells at 0.001% BAK (diluted 1:10) to 33±3% live cells Gadd45a at 0.020% BAK (diluted 1:10). Travoprost (0.004%) plus 0.5% timolol preserved with 0.015% BAK had statistically fewer live TM cells (79±7%) than the same preparation preserved Dehydroepiandrosterone with 0.001% polyquad? (PQ; 93±1%; p<0.001). Latanoprost plus timolol preserved with 0.020% BAK (29±9% live cells) was similar to the 0.020% BAK (33±3%) treatment. However travoprost plus timolol preserved in 0.015% BAK had significantly more live cells (83±12%) than the 1:10 dilution of 0.015% BAK (49±10%). We also found 0.020% BAK (diluted 1:100) resulted in elevated levels of extracellular MMP-9 at 24 h. Conclusions These results demonstrate that this substitution of the preservative BAK from topical ophthalmic drugs results in greater in vitro viability of TM cells. Travoprost with timolol but not latanoprost with timolol countered some of the toxic BAK effects. BAK treatment appeared to cause elevated levels of MMP-9 a matrix metalloproteinase implicated in the pathogenesis of glaucoma. Introduction One of the commonly prescribed classes of intraocular pressure (IOP) lowering agents are the prostaglandin analogs (PGAs). PGAs act primarily by enhancing uveoscleral outflow of aqueous humor however PGAs also appear Dehydroepiandrosterone to act around the trabecular meshwork (TM) to facilitate aqueous humor (AH) outflow through the conventional outflow pathway [1-3]. The beta adrenergic receptor antagonist (β-blocker; BB) timolol which reduces IOP by Dehydroepiandrosterone reducing AH production Dehydroepiandrosterone is often combined with PGAs as a second-line treatment after initial PGA monotherapy has failed (PGA+BB). Use of topical ophthalmic formulations with two hypotensive brokers in a single bottle (fixed combination drug therapy) is a cost effective way to treat glaucoma simplifies the treatment regimen and has the added benefit of decreasing the number of daily exposures to the medications and preservatives contained in most topical ophthalmic preparations. Human and animal studies have shown that chronic topical glaucoma therapy associated with daily use can lead to alterations in tear film damage and remodeling of the corneal surface an increase in inflammatory cytokines as well as other deleterious effects [4-9]. Acute exposure models using animals or cell culture systems demonstrate significant damage/death to cornea and conjunctival cells either immediately after exposure or within 24 h [10-14]. Some toxicity can be attributed to either of the active ingredients of the PGA+BB fixed combination therapy [14 15 However much of the ocular surface changes seen with chronic daily topical glaucoma therapy are associated with the commonly used preservative benzalkonium chloride (BAK) [7 8 16 It has long been known that BAK at antiseptic concentrations increases corneal permeability to hydrophilic brokers [17]. While this can potentially increase delivery of topically applied drugs to the aqueous humor (AH) and ultimately the sites of AH production and AH outflow it would also increase delivery of BAK itself. Little is known about the long-term effects of BAK around the TM endothelial cells that populate this conventional outflow pathway. The aim of this study was to compare the in vitro effects on cultured Dehydroepiandrosterone TM cells of three formulations of PGA+BB fixed combination therapies preserved with either BAK (varying concentrations) or the related cationic polymer polyquad (PQ: 0.001%). We assayed the effects of these brokers diluted 1:10 and 1:100 to mimic the concentrations that reach the anterior chamber. Methods Test and control solutions for cell treatment Topical ophthalmic preparations included 0.004% travoprost plus 0.5% timolol with 0.015% BAK (DuoTrav?; Alcon Laboratories Inc. Fort Worth TX) 0.004% travoprost plus 0.5% timolol with 0.001% PQ (DuoTrav? BAK-free; Alcon) and 0.005% latanoprost plus 0.5% timolol with 0.020% BAK (Xalacom?; Pfizer Inc. New York NY). Not all of these therapies are commercially.