Dmd064295 922..927

The anthracyclines doxorubicin and daunorubicin are used in the treatment of various human and canine cancers, but anthracyclinerelated cardiotoxicity limits their clinical utility. The formation of anthracycline C-13 alcohol metabolites (e.g., doxorubicinol and daunorubicinol) contributes to the development of anthracyclinerelated cardiotoxicity. The enzymes responsible for the synthesis of anthracycline C-13 alcohol metabolites in canines remain to be elucidated. We hypothesized that canine carbonyl reductase 1 (cbr1), the homolog of the prominent anthracycline reductase human CBR1, would have anthracycline reductase activity. Recombinant canine cbr1 (molecular weight: 32.8 kDa) was purified from Escherichia coli. The enzyme kinetics of “wild-type” canine cbr1 (cbr1 D218) and a variant isoform (cbr1 V218) were characterized with the substrates daunorubicin and menadione, as well as the flavonoid inhibitor rutin. Canine cbr1 catalyzes the reduction of daunorubicin to daunorubicinol, with cbr1 D218 and cbr1 V218 displaying different kinetic parameters (cbr1 D218 Km: 1886 144 mM versus cbr1 V218 Km: 5276 136 mM, P < 0.05, and cbr1 D218 Vmax: 6446 6 3615 nmol/min per milligram versus cbr1 V218 Vmax: 15539 6 2623 nmol/min per milligram, P < 0.01). Canine cbr1 also metabolized menadione (cbr1 D218 Km: 104 6 50 mM, Vmax: 2034 6 307 nmol/min per milligram). Rutin acted as a competitive inhibitor for the reduction of daunorubicin (cbr1 D218 Ki: 1.84 6 1.02 mM, cbr1 V218 Ki: 1.38 6 0.47 mM). These studies show that canine cbr1 metabolizes daunorubicin and provide the necessary foundation to characterize the role of cbr1 in the variable pharmacodynamics of anthracyclines in canine cancer patients.