The cataractogenic potential of the thiazolidinedione ciglitazone (CIG) was investigated in vivo and in vitro. In the rat, CIG caused a dose-dependent (30 -300 mg/kg/day) increase in incidence and severity of nuclear cataract formation during a 3-month nonclinical safety assessment study. Potential mechanisms of toxicity were surveyed using whole rat lens explants exposed to CIG with or without various inhibitors of cataract formation. In vitro, CIG caused a concentration-(0.375-30 M) and time-dependent (3-24 h) change in biochemical [ATP content or mitochondrial reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) and reduced glutathione (GSH) content] and morphometric (lens wet weight and clarity) markers of damage. Within 3 h of exposure, 7.5 M CIG decreased lens ATP content 37 Ϯ 7% (percentage of difference from control, p Ͻ 0.05). After 24 h of exposure, lens ATP content, MTT reduction, and GSH content declined 57 Ϯ 5, 30 Ϯ 28, and 42 Ϯ 8%, respectively. Lens wet weight increased 17 Ϯ 4% with a concomitant decrement in lens clarity. Pretreating lenses with the mitochondrial calcium uniport inhibitor ruthenium red (RR) partially or fully protected lenses from toxicity. In contrast, the antioxidant dithiothreitol, aldose reductase inhibitor sorbinil, and selective cell-permeable calpain inhibitors [calpain II inhibitor and (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (E64d)] were ineffective in providing protection under the present testing conditions. Early and selective changes in lenticular ATP content and the partial or full protective effect of RR suggest that alterations in lens bioenergetics may play an important role in CIG-induced cataract formation. Lens explant cultures were successfully used to select two thiazolidinediones that lacked cataractogenic activity when evaluated in 3-month rat safety assessment studies.
These data suggest a novel mechanism for naphthalene-induced cataract, facilitated by the direct effects of 1,2-NQ on lipid peroxidation and cholesterol domain formation.
CJ-12,918, a 5-lipoxygenase (5-LO) inhibitor, caused cataracts during a 1-month safety assessment studies in rats whereas the structurally similar ZD-2138 was without effect. For CJ-12,918 analogs, blocking different sites of metabolic liability reduced (CJ-13,454) and eliminated (CJ-13,610) cataract formation in both rats and dogs. Using this chemical series as a test set, models and mechanisms of toxicity were first explored by testing the utility of ex vivo rat lens explant cultures as a safety screen. This model overpredicted the cataractogenic potential of ZD-2138 due to appreciably high lens drug levels and was abandoned in favor of a mechanismbased screen. Perturbations in lens sterol content, from a decline in lathosterol content, preceded cataract formation suggesting CJ-12,918 inhibited lens cholesterol biosynthesis (LCB). A 2-day bioassay in rats using ex vivo LCB assessments showed that the level of LCB inhibition was correlated with incidence of cataract formation in animal studies by these 5-LO inhibitors. Thereafter, this 2-day bioassay was applied to other pharmaceutical programs (neuronal nitric oxide synthase, sorbitol dehydrogenase inhibitor, squalene synthetase inhibitor and stearoyl-CoA desaturase-1 inhibitors/D 4 antagonists) that demonstrated cataract formation in either rats or dogs. LCB inhibition >40% was associated with a high incidence of cataract formation in both rats and dogs that was species specific. Bioassay sensitivity/specificity were further explored with positive (RGH-6201/ciglitazone/U18666A) and negative (tamoxifen/naphthalene/galactose) mechanistic controls. This body of work over two decades shows that LCB inhibition was a common mechanism of cataract formation by pharmaceutical agents and defined a level of inhibition >40% that was typically associated with causing cataracts in safety assessment studies typically ≥1 month.
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