Pharmacokinetic Characterization of Amrubicin Cardiac Safety in an Ex Vivo Human Myocardial Strip Model. II. Amrubicin Shows Metabolic Advantages over Doxorubicin and Epirubicin

Abstract

Anthracycline-related cardiotoxicity correlates with cardiac anthracycline accumulation and bioactivation to secondary alcohol metabolites or reactive oxygen species (ROS), such as superoxide anion (O 2 . ) and hydrogen peroxide (H 2 O 2 ). We reported that in an ex vivo human myocardial strip model, 3 or 10 M amrubicin [(7S,9S)-9-acetyl-9-amino-7-[(2-deoxy-␤-D-erythro-pentopyranosyl)oxy]-7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-napthacenedione hydrochloride] accumulated to a lower level compared with equimolar doxorubicin or epirubicin (J Pharmacol Exp Ther 341: 464 -473, 2012). We have characterized how amrubicin converted to ROS or secondary alcohol metabolite in comparison with doxorubicin (that formed both toxic species) or epirubicin (that lacked ROS formation and showed an impaired conversion to alcohol metabolite). Amrubicin and doxorubicin partitioned to mitochondria and caused similar elevations of H 2 O 2 , but the mechanisms of H 2 O 2 formation were different. Amrubicin produced H 2 O 2 by enzymatic reduction-oxidation of its quinone moiety, whereas doxorubicin acted by inducing mitochondrial uncoupling. Moreover, mitochondrial aconitase assays showed that 3 M amrubicin caused an O 2 . -dependent reversible inactivation, whereas doxorubicin always caused an irreversible inactivation. Low concentrations of amrubicin therefore proved similar to epirubicin in sparing mitochondrial aconitase from irreversible inactivation. The soluble fraction of human myocardial strips converted doxorubicin and epirubicin to secondary alcohol metabolites that irreversibly inactivated cytoplasmic aconitase; in contrast, strips exposed to amrubicin failed to generate its secondary alcohol metabolite, amrubicinol, and only occasionally exhibited an irreversible inactivation of cytoplasmic aconitase. This was caused by competing pathways that favored formation and complete or near-to-complete elimination of 9-deaminoamrubicinol. These results characterize amrubicin metabolic advantages over doxorubicin and epirubicin, which may correlate with amrubicin cardiac safety in preclinical or clinical settings.

The Novel Anthracenedione, Pixantrone, Lacks Redox Activity and Inhibits Doxorubicinol Formation in Human Myocardium: Insight to Explain the Cardiac Safety of Pixantrone in Doxorubicin-Treated Patients

“…Quantitative correlations between DCF and H 2 O 2 were obtained by measuring DCF in strips incubated for 15 minutes in 4 ml of 50 mM phosphate buffer/123 mM NaCl/5.6 mM glucose, pH 7.4, to which H 2 O 2 was added at 0.1-10 mM. DCF formation in these strips was plotted against a DCF standard curve, and a DCF:H 2 O 2 stoichiometry of 0.43 could be established (Salvatorelli et al, 2006a(Salvatorelli et al, , 2012a. The lack of a 1:1 stoichiometry could be attributed to H 2 O 2 -independent oxidation of DCFH or side reactions of DCF with redox active-cell constituents (Rota et al, 1999).…”

“…DOX metabolism was characterized in isolated soluble fractions of myocardial strips, as reported previously (Salvatorelli et al, 2012a). Proteins were measured by bicinchoninic acid (Stoscheck, 1990).…”

“…Basal or drugstimulated ROS formation was measured by an HPLC adaptation of the method based on the uptake of membrane-permeable DCFH-DA, its intracellular deacetylation to membrane-impermeable DCFH, and oxidation of DCFH to DCF by H 2 O 2 (Salvatorelli et al, 2006a(Salvatorelli et al, , 2007(Salvatorelli et al, , 2012a.…”

“…We validated an ex vivo human myocardial strip model that eliminated this problem and helped characterize DOX accumulation and bioactivation to ROS or DOXOL in human myocardium (Salvatorelli et al, 2006a(Salvatorelli et al, ,b, 2007(Salvatorelli et al, . 2009(Salvatorelli et al, , 2012a. In the present study, the same model was used to explore the determinants of PIX cardiac safety in anthracyclinepretreated patients.…”

Pharmacokinetic Characterization of Amrubicin Cardiac Safety in an Ex Vivo Human Myocardial Strip Model. I. Amrubicin Accumulates to a Lower Level than Doxorubicin or Epirubicin

“…This pharmacokinetic pattern anticipates an important determinant of the cardiac tolerability of amrubicin in approved or investigational clinical settings. In Salvatorelli et al (2012) we will characterize how amrubicin compared with doxorubicin or epirubicin in converting to metabolites or by-products possibly involved in cardiotoxicity. The box on the left indicates that in the membrane fraction of the strips amrubicin is metabolized by carbon-nitrogen lyase-type enzymes to give 9-deaminoamrubicin (the solid red circle indicates amino group removal and double-bond addition in the A ring).…”