The best-laid plans

the results of the 52-wk Phase IIb Bardoxolone Methyl Treatment: Renal Function in CKD/Type 2 Diabetes (BEAM) study that were published in the New England Journal of Medicine in 2011 (4) indicating that patients with diabetic nephropathy from type II diabetes had preservation and even increased estimated glomerular filtration rates (eGFR) in response to bardoxolone methyl generated enormous interest, as well as raising a number of questions and concerns. Bardoxolone methyl, an oleanolic acid derivative, is structurally and functionally similar to cyclopentenone prostaglandins, which are endogenous activators of Nrf2, a transcriptional regulator of a multitude of antioxidant and cytoprotective genes. Bardoxolone interacts with cysteine residues on Keap 1, which then dissociates from cytoplasmic Nrf2, allowing Nrf2 to translocate to the nucleus (5). Since oxidant stress is widely recognized as an important factor in the progression of diabetic nephropathy (1), there was biological plausibility for bardoxolone as a potential therapeutic intervention. Although the effects of bardoxolone on eGFR in the diabetic patients were striking, there were some worrisome signals in the clinical trial, including increased albuminuria and increased alanine aminotransferase levels. It was also noteworthy that although there had been animal studies in experimental models of acute kidney injury (6), there were no previously published studies of the effects of bardoxolone in experimental models of diabetic nephropathy, although other putative activators of Nrf2 have been reported to be effective (7). To investigate potential therapeutic mechanisms of bardoxolone, Zoja et al. (8) used a model of type II diabetic nephropathy, the leptin receptor-deficient Zucker diabetic fatty (ZDF) rat, and the results of treatment of this model are published in an issue of the American Journal of Physiology-Renal Physiology. Because rodents may metabolize bardoxolone methyl to toxic metabolites, animals were treated with an analog triterpenoid, RTA 405. The experimental strategy utilized by, these investigators was straightforward, with ZDF rats treated with RTA 405, with the angiotensin-converting enzyme (ACE) inhibitor ramipril or with a combination of RTA 405 and ramipril. The rats were treated from the age of 3 mo, at a time when they already exhibited proteinuria, and treatment was continued for up to 6 mo. The results were, to say the least, not what was expected. ZDF rats given the bardoxolone analog ate less, lost weight, and had lower blood sugars but had higher blood pressures and serum cholesterol levels. Unlike what was expected, they also had more proteinuria and more glomerulosclerosis. These adverse effects were not necessarily dose related, at least at the two concentrations of RTA 405 used in the study and were minimally affected by simultaneous ramipril treatment. Because of the transient elevations in alanine aminotransferase (ALT) levels seen in the BEAM study, Zoja et al. (8) also examined both ALT as well as another hepatic enzyme, aspartate aminotransferase (AST). Nrf2 is known to transcriptionally upregulate both ALT and AST and was associated with hepatoprotection in animal models of liver injury (3), so transient elevations in these enzymes were not necessarily deemed unexpected or seen as harmful. Similar to the human studies, Zoja et al. (8) did observe increases in ALT and AST after 1 mo of RTA 405 treatment, which decreased to levels seen with vehicle by 3 mo of treatment. However, unexpectedly, they also observed significant increases in liver weight, which was associated with marked histological changes, characterized by swelling of hepatocytes, diffuse vacuolization, and cellular degeneration. Post hoc analysis by Reata Pharmaceuticals, who had supplied RTA 405 to the investigators, suggested that there were small concentrations of both identifiable and unknown impurities in the drug that had been supplied to Zoja et al. (8). The identified impurities were intermediates in RTA 405 synthesis, RTA 401, and its 1,2-dihydro derivative. The observed concentrations of these impurities was very low, which suggested they were either extremely toxic or were not the cause of the observed hepatic pathology. Unfortunately, Reata was not able to supply any “pure” RTA 405 for further study. Instead, they provided a compound said to be well tolerated by rodents, dihydro-CDDO-trifluoroethyl amide (dh404) (2). In preliminary studies performed with dh404, there was a trend for GFR to increase at lower concentrations. However, this was accompanied by a corresponding trend for proteinuria to increase, associated with increased glomerulosclerosis and increased interstitial inflammation. In addition, in kidneys of 3 of 20 treated rats, inflammatory granulomatous lesions suggestive of a renal cell pseudotumor were identified. What can be learned from these studies? First and foremost perhaps is that carefully performed studies in experimental animals may provide important insights not only into pathogenic mechanisms but also into unexpected side effects. As this editorial focus is written, the reasons for the premature termination of the phase 3 BEACON trial of bardoxolone methyl in patients with CKD from type 2 diabetes have not been made public besides a statement that their Independent Data Monitoring Committee recommended cancellation due to “excess serious adverse events and mortality in the Bardoxolone methyl arm.” The carefully performed studies in rats by Zoja et al. (8) may provide some insight into negative side effects of bardoxolone and whether these side effects are specific for the drug or will be seen with other Nrf2 inhibitors. The promise and the ultimate disappointment produced by bardoxolone methyl reminds us of the words of the poet Robert Burns to another species of rodent: To a Mouse, on Turning Her Up in Her Nest with the Plough (1785) But Mousie, thou art no thy lane, In proving foresight may be vain; The best-laid schemes o' mice an' men Gang aft agley, An' lea'e us nought but grief an' pain, For promis'd joy!