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    Purite May be Gentler Preservative for Lubrication Solutions

    Stabilized oxychloro complex shows no cytotoxic effects to the corneal surface

    Almost all ophthalmic medications contain preservatives, which inhibit bacterial growth. The use of preservatives allows for the use of multidose bottles, which are the most cost-effective and user-friendly packaging for ophthalmic medications. However, some of the preservatives present in commonly used eye drops today have been shown to be disruptive to the cornea surface.1-5

    Prolonged or repeated exposure to these agents may disrupt an already compromised corneal epithelial layer. When medications are used for short durations or sporadically in normal eyes, any adverse effects of the preservatives on the ocular surface are probably negligible. However, some patients with chronic conditions such as glaucoma or keratitis sicca may require dosing with considerable numbers of drops daily for long periods. While medications such as artificial tears provide relief from dry eye and physical irritants, intensive or long-term use of drops may compromise the ocular surface and exacerbate underlying conditions.

    The preservatives that are present in ophthalmic preparations vary from product to product. These preservatives are generally well tolerated, but certain preservatives are more toxic and may affect the degree of overall ocular comfort and have a disruptive effect on the ocular surface at the cellular level. This is why we evaluated the effect of various preservatives on in vitro cellular integrity, as well as on rabbit ocular corneal surface.

    Our results suggest that there is a measurable difference in cytotoxicity among common preservatives used in lubricating solutions.

    MethodsWe ran two sets of assays, one with American-marketed products and one with European-marketed products (Table 1, Page 75). Preservative components of lubricating eye drops included a stabilized oxychloro complex (Purite, 0.005%); a quaternary ammonium compound, benzalkonium chloride (BAK, 0.005% and 0.01%); cetrimide; phenylmercury borate (<0.003%); sodium perborate; and polyquaternium-1 (Polyquad, 0.001%).

    Our study used these parameters:

    • Cultured epithelial monolayers in 96 well plates were treated with neutral red for 3 hours.
    • Cells were exposed to the artificial tear solutions or control agents for 15 minutes up to 3 hours.
    • Remaining cellular neutral red was extracted, absorbance was read by a Molecular Devices microplate spectrophotometer, and the data were expressed as a percentage of retention relative to the negative control.

    In vitro cytotoxicity was assessed by a neutral red retention assay using Madin-Darby canine kidney (MDCK) epithelial cells, which were exposed for 15 minutes to 3 hours.

    We know that viable epithelial cells incorporate neutral red into cellular lysosomes. Neutral red is released upon damage to the membrane.6,7 Assay measures loss of neutral red exposure time.

    In addition, corneal surface evaluation was done via scanning electron microscopy (SEM) following a 7-day topical ocular treatment administered four times a day with each lubricating eye drop solution.

    ResultsThe American-marketed artificial tears showed the following results with regard to cytotoxicity from epithelial membrane disruption:

    • oxychloro complex: no membrane disruption;
    • polyquaternium-1: minimal membrane disruption; and
    • BAK (0.01%) and sodium perborate: disruption of the epithelial membrane with 58% +/- 2% and 31% +/- 1% neutral red retention, respectively.

    The European-marketed artificial tears showed the following results with regard to cytotoxicity from epithelial membrane disruption:

    • oxychloro complex: no membrane disruption;
    • BAK (0.01%): 5% +/- 1% retention of neutral red;
    • cetrimide: 34% +/- 1% retention;
    • phenylmercury borate: 27% +/- 2% retention; and
    • BAK (0.005%): 56% +/- 4% retention.

    As measured by neutral red release, the oxychloro complex showed no cytotoxicity. By this measure, BAK showed the highest cytotoxicity, with a 95%, 69%, and 56% release at 100, 100, and 50 ppm, respectively, after a 2-hour exposure. Phenylmercury borate and cetrimide showed 73% and 66% neutral red release, respectively, after a 2-hour exposure. Sodium perborate and polyquaternium-1 showed 20% and 13% release, respectively, after a 2-hour exposure.

    SEM studies (Table 2, Page 75) of the rabbit cornea showed no defect or superficial erosion in the eyes treated with the oxychloro complex and sodium perborate, compared with the control. BAK, and to a lesser extent polyquaternium-1, presented extensive superficial erosion of the epithelium and a lack of protruding microvilli.

    We found that lubricating eye drops containing the oxychloro complex had no adverse effect on epithelial cells in vitro or on rabbit corneas in vivo. Polyquaternium-1, cetrimide, phenylmercury borate, sodium perborate, and BAK all showed cytotoxic effects, either in vivo or in vitro. Our results indicate that the oxychloro complex was less disruptive to cellular integrity than other preservatives currently used.

    In general, the cytotoxicity observed for each preservative, from least to most, is as follows: the oxychloro complex; polyquaternium-1; sodium perborate; BAK 0.005%; cetrimide, phenylmercury borate; and BAK 0.01%.

    These results suggest that there is a measurable difference in cytotoxicity among preservatives used in lubricating solutions. The oxychloro complex Purite had no cytotoxic effects and was the least disruptive, compared with the other preserved solutions tested. The oxychloro complex Purite may be a gentler and more acceptable preservative for ophthalmic solutions than other currently used preservatives. Clinically, ophthalmic medications containing the oxychloro complex Purite preservative may be better tolerated and more comfortable than those using more toxic preservatives.

    References

    1. Debbasch C, Brignole F, Pisell PJ, et al. Quaternary ammoniums and other preservatives contribution in oxidative stress and apoptosis on Chang conjunctival cells. Invest Ophth Vis Sci 2001;42:642-652.
    2. Vaughan JS, Porter DA. A new in vitro method for assessing the potential toxicity of soft contact lens care solutions. Contact Lens 1993;19:54-57.
    3. Debbasch C, de Saint Jean M, Rat P, et al. Quaternary ammonium cytotoxicity in a human conjunctival cell line. J Fr Ophthalmol 1999;9:950-958.
    4. Ichijima II, Peroll WM, Jester JV, et al. Confocal microscopic studies of living rabbit cornea treated with benzalkonium chloride. Cornea 1992;11:368.
    5. Pfister RR, Burstein N. The effects of ophthalmic drugs, vehicles, and preservatives on corneal epithelium: a scanning electron microscope study. Invest Ophthalmol 1976;15:246-259.
    6. Bornefreund E, Puerner JA. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol Lott 1985;24:119-124.
    7. Shaw AJ, Clothier RH, Balls M, et al. Loss of transepithelial impermeability of a confluent monolayer of Madin-Darby canine kidney (MDCK) cells as a determinant of ocular irritancy potential. ATLA 1990;18:145-151.

    Robert J. Noecker, MD
    Dr. Noecker, vice chair, University of Pittsburgh Medical Center Eye Center, and director, Glaucoma Service and associate professor of ...

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