N-Benzyladriamycin-14-valerate (AD 198): A Non-Cardiotoxic Anthracycline That Is Cardioprotective through PKC-! Activation

N-Benzyladriamycin-14-valerate (AD 198) is one of several novel anthracycline protein kinase C (PKC)-activating agents developed in our laboratories that demonstrates cytotoxic superiority over doxorubicin (Adriamycin; DOX) through its circumvention of multiple mechanisms of drug resistance. This characteristic is attributed at least partly to the principal cellular action of AD 198: PKC activation through binding to the C1b (diacylglycerol binding) regulatory domain. A significant dose-limiting effect of DOX is chronic, dose-dependent, and often irreversible cardiotoxicity ascribed to the generation of reactive oxygen species (ROS) from the semiquinone ring structure of DOX. Despite the incorporation of the same ring structure in AD 198, we hypothesized that AD 198 might also be cardioprotective through its ability to activate PKC-!, a key component of protective ischemic preconditioning in cardiomyocytes. Chronic administration of fractional LD50 doses of DOX and AD 198 to mice results in histological evidence of dose-dependent ventricular damage by DOX but is largely absent from AD 198-treated mice. The absence of significant cardiotoxicity with AD 198 occurs despite the equal ability of DOX and AD 198 to generate ROS in primary mouse cardiomyocytes. Excised rodent hearts perfused with AD 198 prior to hypoxia induced by vascular occlusion are protected from functional impairment to an extent comparable to preconditioning ischemia. AD 198mediated cardioprotection correlates with increased PKC-! activation and is inhibited in hearts from PKC-! knockout mice. These results suggest that, despite ROS production, the net cardiac effect of AD 198 is protection through activation of PKC-!. The clinical antitumor efficacy of doxorubicin (DOX) is limited by the development of well defined, life-threatening cardiotoxicities, including cardiomyopathy and congestive heart failure (CHF) (Frishman et al., 1996). CHF has been reported to occur in 20% of those patients given 600 mg/m DOX with subsequent 50% mortality after 2 years without cardiac transplantation (Frishman et al., 1996; Jensen et al., 2002). In retrospective analyses of phase III clinical trials using anthracycline-based combination therapies, the frequency of CHF escalates dose-dependently: 5% at 400 mg/m; 26% at 550 mg/m; and 48% at 700 mg/m (Swain et al., 2003). Cardiotoxicity is exacerbated when DOX is administered with the humanized anti-erbB-2 antibody trastuzumab (Herceptin) and cyclophosphamide (Slamon et al., 2001). In addition to overt cardiotoxicity, the association of subclinical cardiac effects with DOX remains largely unquantified but is a contributory factor to subsequent cardiovascular disease in surviving patients (Schultz et al., 2003). Evidence suggests that DOX-related cardiotoxicities are the result of DOX-mediated generation of reactive oxygen species (ROS) and of interference with myocardial Ca homeostasis (Saeki et al., 2002). Numerous therapeutic strategies have been explored to reduce DOX cardiotoxicity, including the design of anthracyThis work was supported in part by National Institutes of Health Grants HL-49939 (to P.A.H), CA37209 (to M.I.), and CA100093 (to L.L.) and The University of Tennessee Vascular Biology Center of Excellence (to L.L.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.126110. ABBREVIATIONS: CHF, congestive heart failure; IPC, ischemic preconditioning; LVDP, left ventricular-developed pressure; PKC, protein kinase C; AD 198, N-benzyladriamycin-14-valerate; MEN 10755, sabarubicin; DAG, diacylglycerol; EDP, end-diastolic pressure; PBS, phosphatebuffered saline; ROS, reactive oxygen species; PAGE, polyacrylamide gel electrophoresis; MAPK, mitogen-activated protein kinase; DMSO, dimethyl sulfoxide; CM-H2DCFDA, 5-(and-6)-chloromethyl-2",7"-dichlorodihydro-fluorescein diacetate, acetyl ester; MTS, mean total score; PMA, phorbol 12-myristate 13-acetate. 0022-3565/07/3232-658–664$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 323, No. 2 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 126110/3265774 JPET 323:658–664, 2007 Printed in U.S.A. 658 by gest on April 9, 2014 jpet.asjournals.org D ow naded rom cline congeners with reduced cardiotoxic potential. Epirubicin (4"-epidoxorubicin) induces cardiotoxicity only after higher cumulative doses than DOX (Frishman et al., 1996). However, clinical studies have revealed that the 850 to 1000 mg/m dose of epirubicin used to treat breast cancer results in a 59% incidence of significantly reduced left ventricular function within 3 years of treatment, with 20% developing severe cardiomyopathy after 5 years (Jensen et al., 2002). Other nuclear-targeted congeners, such as 4-demethoxydaunorubicin (idarubicin), 3"-deamino-3"-hydroxydoxorubicin (hydroxyrubicin), 4"-deoxydoxorubicin (esorubicin), and the disaccharide anthracycline MEN 10755 (sabarubicin), reportedly produce less acute cardiotoxicity than DOX, with long-term effects that are not yet well established (Speyer and Wasserheit, 1998; Binaschi et al., 2001). Of the clinically approved anthracyclines, only valrubicin (Valstar), an Ntrifluoracetamide derivative of adriamycin-14-valerate, is devoid of cardiac toxicity (Sweatman and Israel, 1996). In addition to these anthracyclines, the novel anthracycline N-benzyladriamycin-14-valerate (AD 198) shows promise in demonstrating reduced cardiotoxicity based upon its mechanism of action. AD 198 exhibits no significant organspecific toxicities at therapeutic doses and is less myelosuppressive than DOX at comparable doses (Sweatman and Israel, 1996). Its principal mechanism of cytotoxicity in proliferating cells is the direct, rapid induction of apoptotic signaling through protein kinase C (PKC) activation in a manner that circumvents multiple mechanisms of cellular drug resistance (Lothstein et al., 1998, 2001, 2006; Barrett et al., 2002; Bilyeu et al., 2004). The three-dimensional structure of the 5-carbon alkylester group of AD 198 combined with portions of the chromophore A ring bears a striking similarity to diacylglycerol (DAG), an endogenous ligand for the C1b regulatory domain of PKC (Fig. 1) (Roaten et al., 2001, 2002). AD 198 also competitively inhibits phorbol ester binding to the C1b domain (Roaten et al., 2001, 2002). Within the context of cardiac damage control, PKC signaling plays a critical protective role. Ischemia-induced myocardial damage is attenuated by short duration, transient ischemia (ischemic preconditioning, IPC) (Murry et al., 1986). IPC is associated with receptor-mediated actions of multiple ligands in combination with ROS generated via production of nitric oxide by Ca-dependent nitric oxide reductase (Cohen et al., 2000). This, in turn, activates phospholipase, which generates DAG (Cohen et al., 2000) and activates PKC-!, an essential nexus of signaling for cell surface receptors involved in both the early and late phases of IPC (Liu et al., 1999; Bolli, 2000; Cohen et al., 2000). However, a role for PKC-" activation in promoting preconditioning has also been reported (Kawamura et al., 1998). Multiple events downstream of PKC activation may feed back to enhance cell surface receptor-mediated signaling (Oldenburg et al., 2002) or may signal further downstream events, such as the activation of p38-MAPK# (Yue et al., 2002), p42/p44 ERK, or c-Jun N-terminal kinase (Baines et al., 2002). Recent evidence suggests that PKC-! forms a complex or “signaling module” with MAPKs following translocation of PKC-! to mitochondria and subsequent activation (Baines et al., 2002). This PKC-!/MAPK-active complex results in the inhibition of mitochondrial-dependent apoptosis (Baines et al., 2002). In addition, PKC-! is reported to associate with no less than 36 structural, signaling, and stressactivated proteins in cardiomyocytes (Ping et al., 2001) that ultimately function to maintain homeostasis. Given the ability of AD 198 to target and activate PKC in a variety of cell lines, we determined in the study whether AD 198 activates PKC-! in cardiomyocytes and, subsequently, induces cardioprotective signaling in intact hearts. Materials and Methods Test Agents. DOX was obtained from Sigma-Aldrich (St. Louis, MO), whereas AD 198 hydrochloride salt was prepared in these laboratories according to previously described procedures (Lothstein et al., 1998). For in vitro and ex vivo use, both drugs were dissolved in DMSO and diluted with aqueous media where indicated. For in vivo use, DOX was formulated in sterile saline as usual, whereas AD 198 was formulated in 20% NCI Diluent 12 (polyhydroxylated castor oil/ethyl alcohol, 1:1 by volume), 80% saline. Cardiotoxicity Assessment. The murine chronic anthracycline cardiotoxicity model system developed by Bertazzoli et al. (1979) was used in this study. In this system, dose levels of test drugs for chronic administration are based upon the single dose LD50 (30 days) value of the drug. Preliminary studies in mice of the same strain and sex as used for the cardiotoxicity assessment ultimately gave a tightly titrated i.v. single dose LD50 (30 days) value for DOX (23 mg/kg) and for AD 198 (46 mg/kg). For chronic administration, high, middle, and low dose levels corresponding to 0.2, 0.1, and 0.05 of the i.v. single dose LD50 (30 days) values of each drug were used. Female CD1 mice (PAPIPLUS-virus free; Charles River LaboraCH3O O