Differential toxic effect of dissolved triamcinolone and its crystalline deposits on cultured human retinal pigment epithelium (ARPE19) cells☆
Introduction
Triamcinolone acetonide (TA), a synthetic, lipophilic corticosteroid with low solubility in aqueous solution, has been used as a depot drug for decades. Its advantageous pharmacokinetic profile with rapid bioavailability and sustained release characteristics let Machemer to suggest the intravitreal use of crystalline TA to maintain therapeutic drug levels within the vitreous cavity (Machemer et al., 1979, Tano et al., 1980). Since then, intravitreal administration of crystalline TA suspension has become increasingly popular for the treatment of various intraocular disorders such as chronic uveitis (Antcliff et al., 2001), diabetic macular edema (Jonas et al., 2003, Martidis et al., 2002), and age-related macular degeneration (Danis et al., 2000, Jonas et al., 2005). While the clinical side effects such as cataractogenesis and glaucoma are well known (Jaissle et al., 2004), the underlying mode of action and a potential cytotoxicity have still not been conclusively defined. Although generally considered as safe, the biocompatibility of crystalline TA in direct contact with cells is controversial.
The safety of intravitreal TA administration has been supported by prior animal studies (Kivilcim et al., 2000, McCuen et al., 1981, Schindler et al., 1982) and human trials (Danis et al., 2000, Jonas et al., 2003). In contrast, other studies have found a marked ocular toxicity of some commercial TA preparations (Schlaegel and Wilson, 1974). Hida and co-workers investigated the effect of the preservatives and osmolarity of the vehicle and postulated that adverse effects are probably caused by the vehicles rather than the corticosteroid itself (Hida et al., 1986). Hence, several authors suggested the use of various purification techniques in order to remove the vehicle prior to injection (Hernaez-Ortega and Soto-Pedre, 2004, Jaissle et al., 2005, Jonas et al., 2001, Nishimura et al., 2003).
In a recent laboratory work, however, Yeung and colleagues comprehensively investigated the growth inhibitory effect of TA and its corresponding vehicle on ocular cell lines in clinically relevant concentrations. They found the impact of the vehicle to be less pronounced than formerly assumed. The inhibitory corticosteroid effect on cellular proliferation was shown to be by far more pronounced. They concluded that TA is generally cytotoxic and assumed an apoptotic cell death to be involved (Yeung et al., 2003, Yeung et al., 2004).
These results are highly contradictory to the hitherto good clinical experience and, if confirmed will have a strong impact on the clinical use of intravitreal TA. However, several fundamental ambiguities still remain to be clarified. Firstly, the pharmacological effect of TA within the vitreous cavity must be distinguished from a potential direct toxicity of sedimented crystalline particles. The latter are the equivalent to localized epiretinal deposits that are often seen in the lower periphery, mostly in vitrectomized eyes, and might be more critical in terms of biocompatibility. Secondly, the well-known and beneficial antiproliferative drug effect of corticosteroids is barely discriminable from irreversible cellular toxicity in a proliferating cell culture, if standard assays are used, although this is decisive for a conclusive biocompatibility assessment. Finally, the significance of apoptosis induction has to be further analyzed.
The discrepancy between the good clinical results and recent laboratory evidence for a potential cytotoxicity of TA is currently unsettled. In view of the wide-spread clinical application and the off-label use of intravitreal TA this ambiguity may warrant additional investigations for the safety and efficacy of TA, if to be used with confidence clinically. In this laboratory study, crystalline TA is investigated for its antiproliferative properties, its direct cell toxicity and the significance of epicellular deposits. Finally, the effect of the vehicle and the significance of purification are further evaluated.
Section snippets
Preparation of triamcinolone acetonide and vehicle removal
Volon-A (Dermapharm, Grünwald, Germany) containing triamcinolone acetonide 40 mg was diluted with culture medium to a stock concentration of 1 mg/ml directly from the original vials. To get preservative-free TA, purification was performed prior to dilution according to a standard protocol for human use (Hernaez-Ortega and Soto-Pedre, 2004). Briefly, the content of the vial was transferred to a sterile syringe (1 ml), centrifuged at 2000 rpm for 20 min (Multifuge 3S-R, Heraeus, Osterode, Germany),
Growth inhibitory effect of triamcinolone
In our experimental setting, we used three concentrations of TA suspension resulting in a different amount of crystalline deposits. At the lowest concentration (0.01 mg/ml) TA dissolved almost completely and crystalline particles were rarely seen. In higher concentrations TA particles randomly settled on top of the cells (0.1 mg/ml). At the highest concentration of 1 mg/ml the cells were totally covered by TA particles. TA caused a significant reduction in mitochondrial dehydrogenase activity
Discussion
In regard to a potential toxicity of commercial TA preparations, our results clearly show that the growth inhibitory effect of the corticosteroid itself is by far more important than the toxicity of the vehicle. For both purified and unpurified TA the inhibition of cell proliferation was significant and dose-dependent, although the total number of viable cells still increased even at the highest TA concentration. These results are in accordance with previous studies, which demonstrated a
Acknowledgements
The study was supported by the European Community, Marie Curie Research Grant WLG5-CT-2001-60034 and the Gertrud Kusen Foundation. The authors thank Dr. Barbara Wallenfels-Thilo for excellent editorial assistance.
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Disclosure: P. Szurman, None; R. Kaczmarek, None; M.S. Spitzer, None, G.B. Jaissle, None; P. Decker, None; S. Grisanti, None; S. Henke-Fahle, None; S. Aisenbrey, None; K.U. Bartz-Schmidt, None.