Etoposide as a virocidal anticytomegalovirus therapy: intravitreal toxicology and pharmacology in rabbits

Background: Although Cytomegalovirus (CMV) retinitis is now a common intrao cular infection. current therapy is only virostatic so ongoing treatment is required. Etoposide was found to be virocidal for CMV in laboratory experi ments and it might prove to be beneficial clinicaliy. We investigated the t oxicity and intraocular concentration of etoposide (VP16) and its new analo gue etoposide-phosphate (VP16P) following intravitreal injections in rabbit eyes. Methods: First a sequential dose-response was assessed with flash electrore tinogram for both eyes of light- and dark-adapted rabbits (n = 7; one rabbi t for each dose) over a range of light intensities before and after intravi treal injection of VP16 or VPI6P to one eye; the other eye was injected wit h normal saline as a control. A multidose study was then performed on four rabbits. A non-toxic dose of VP16P (50 or 75 g) was injected into the Vitre ous of one eye on four occasions 1 week apart. A photopic electroretinogram was performed before the first injection and 6 weeks after the last inject ion. All the eyes from the electroretinogram studies were fixed in formalin , placed in paraffin, then stained with haematoxylin and eosin and examined under a light microscope.-ro determine the time-course of the intraocular concentrations of VPI6P a sequential pharmacokinetic study was performed us ing a further IZ rabbits. Each rabbit was injected with 50 g VP16P to one e ye and 75 g VP16P to the other eye. Three of these rabbits were killed at 1 ,3, 6 and 9 h after injection. Samples of vitreous were assayed for both VP 16 and VPI6P using HPLC. An in vitro dose-response assay was performed usin g third-passage bovine retinal pigment epithelial (RPE) cells cultured in D ulbecco's modified Eagles medium with fetal calf serum. The effect of a log -dose increment of VPI6P on the RPE cell proliferation was assessed using t ritiated thymidine incorporation. Results: The electroretinogram studies suggested that VP16 was toxic even w ith the 10 g dose. For VP16P a toxic effect was noted following injection o f a single dose greater than 100 g. Multiple injections of 50 or 75 g VP16P did not produce a toxic response. Histological examination demonstrated si gnificant abnormality only with the 500 g dose of VP16 or VP16.VP16P was ra pidly metabolized to VP16 in the eye, producing concentrations of 2.0 g/mL or more for up to 9 h following a 75-mu g dose. This suggests that the elec troretinogram findings following VP16 injections were confounded by a toxic effect of the ethanol solvent (which is absent from the VP16P preparation) . VP16P was quite potent, the ID50 was about 0.1 g/mL for bovine RPE cells in the in vitro assay. Discussion: These results indicate that multiple 75-g VP16P intravitreal in jections were not toxic to the rabbit eye and provide a therapeutic intraoc ular concentration for up to 9 h after the injection.