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.

Antitumor anthracyclines such as doxorubicin and epirubicin are known to cause cardiotoxicity that correlates with anthracycline accumulation in the heart. The anthracycline amrubicin [(7S,9S)-9-acetyl-9-amino-7-[(2-deoxy-ß-d-erythro-pentopyranosyl)oxy]-7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-napthacenedione hydrochloride] has not shown cardiotoxicity in laboratory animals or patients in approved or investigational settings; therefore, we conducted preclinical work to characterize whether amrubicin attained lower levels than doxorubicin or epirubicin in the heart. Anthracyclines were evaluated in ex vivo human myocardial strips incubated in plasma to which anthracycline concentrations of 3 or 10 µM were added. Four-hour incubations were performed to characterize myocardial anthracycline accumulation derived from anthracycline uptake in equilibrium with anthracycline clearance. Short-term incubations followed by multiple washouts were performed to obtain independent measurements of anthracycline uptake or clearance. In comparison with doxorubicin or epirubicin, amrubicin attained very low levels in the soluble and membrane fractions of human myocardial strips. This occurred at both 3 and 10 µM anthracycline concentrations and was caused primarily by a highly favorable clearance of amrubicin. Amrubicin clearance was facilitated by formation and elimination of sizeable levels of 9-deaminoamrubicin and 9-deaminoamrubicinol. Amrubicin clearance was not mediated by P glycoprotein or other drug efflux pumps, as judged from the lack of effect of verapamil on the partitioning of amrubicin and its deaminated metabolites across myocardial strips and plasma. Limited accumulation of amrubicin in an ex vivo human myocardial strip model may therefore correlate with the improved cardiac tolerability observed with the use of amrubicin in preclinical or clinical settings.

Pharmacokinetic characterization of amrubicin cardiac safety in an ex vivo human myocardial strip model. II. Amrubicin shows metabolic advantages over doxorubicin and epirubicin.

Anthracycline-related cardiotoxicity correlates with cardiac anthracycline accumulation and bioactivation to secondary alcohol metabolites or reactive oxygen species (ROS), such as superoxide anion (O2·â») and hydrogen peroxide H2O2). We reported that in an ex vivo human myocardial strip model, 3 or 10 µM amrubicin [(7S,9S)-9-acetyl-9-amino-7-[(2-deoxy-ß-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 H2O2, but the mechanisms of H2O2 formation were different. Amrubicin produced H2O2 by enzymatic reduction-oxidation of its quinone moiety, whereas doxorubicin acted by inducing mitochondrial uncoupling. Moreover, mitochondrial aconitase assays showed that 3 µM amrubicin caused an O2·â»-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.

Increased cellular accumulation and distribution of amrubicin contribute to its activity in anthracycline-resistant cancer cells.

PURPOSE: Multi-drug resistance and cumulative cardiotoxicity are major limitations for the clinical use of anthracyclines. Here, we evaluated and compared the cross-resistance of amrubicin, a third-generation synthetic anthracycline and potent topoisomerase (topo)-II inhibitor with little or no observed cardiotoxicity to other anthracyclines and the topo-II inhibitor etoposide in drug-resistant tumor models in order to elucidate its potential mechanisms of action. METHODS: Amrubicin activity was assessed in multi-drug-resistant cell lines and human tumor explants using cytotoxicity assays, confocal microscopy, fluorescence time-lapse imaging, flow cytometry, immunoblotting, and gene expression profiling techniques. RESULTS: We demonstrate that both doxorubicin-resistant tumor cell lines and several drug-resistant human ovarian and breast tumor explants retain sensitivity to amrubicin. In addition, we observed similar levels of amrubicin uptake and accumulation in doxorubicin-sensitive versus doxorubicin-resistant cell lines. Although amrubicin is a weak P-glycoprotein substrate, transport and retention of amrubicin were not solely modulated by P-glycoprotein in the resistant cell lines overexpressing drug efflux pumps. The cellular retention of amrubicin is likely to be a result of rapid influx due to its high intrinsic permeability and lipophilic properties, and this may explain why amrubicin overcomes pleiotropic drug resistance. Consistent with drug accumulation studies, amrubicin induced DNA damage, G(2)-M cell cycle arrest, and apoptosis in both doxorubicin-sensitive and doxorubicin-resistant lines. Using gene expression profiling studies, several classes of genes were significantly and uniquely regulated following amrubicin, but not doxorubicin or etoposide, treatment. CONCLUSIONS: Amrubicin appears to have a distinct mode of action that overcomes typical anthracycline resistance mechanisms. Therefore, amrubicin may be useful in the treatment of anthracycline-refractory or anthracycline-resistant tumors.

A comparison of azacitidine and decitabine activities in acute myeloid leukemia cell lines.

BACKGROUND: The cytidine nucleoside analogs azacitidine (AZA) and decitabine (DAC) are used for the treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Few non-clinical studies have directly compared the mechanisms of action of these agents in a head-to-head fashion, and the agents are often viewed as mechanistically similar DNA hypomethylating agents. To better understand the similarities and differences in mechanisms of these drugs, we compared their in vitro effects on several end points in human AML cell lines. METHODOLOGY/PRINCIPAL FINDINGS: Both drugs effected DNA methyltransferase 1 depletion, DNA hypomethylation, and DNA damage induction, with DAC showing equivalent activity at concentrations 2- to 10-fold lower than AZA. At concentrations above 1 microM, AZA had a greater effect than DAC on reducing cell viability. Both drugs increased the sub-G1 fraction and apoptosis markers, with AZA decreasing all cell cycle phases and DAC causing an increase in G2-M. Total protein synthesis was reduced only by AZA, and drug-modulated gene expression profiles were largely non-overlapping. CONCLUSIONS/SIGNIFICANCE: These data demonstrate shared mechanisms of action of AZA and DAC on DNA-mediated markers of activity, but distinctly different effects in their actions on cell viability, protein synthesis, cell cycle, and gene expression. The differential effects of AZA may be mediated by RNA incorporation, as the distribution of AZA in nucleic acid of KG-1a cells was 65:35, RNA:DNA.

Recombinant interleukin-2 significantly augments activity of rituximab in human tumor xenograft models of B-cell non-Hodgkin lymphoma.

Recombinant interleukin-2 (rIL-2) is a pleiotropic cytokine that activates select immune effector cell responses associated with antitumor activity, including antibody-dependent cellular cytotoxicity (ADCC). Rituximab is an anti-CD20 monoclonal antibody that activates ADCC in non-Hodgkin lymphoma (NHL). The ability of rIL-2 to augment rituximab-dependent tumor responses was investigated. The efficacy of rIL-2 in combination with rituximab was evaluated in 2 NHL tumor xenograft models: the CD20hi, rituximab-sensitive, low-grade Daudi model and the CD20lo, aggressive, rituximab-resistant Namalwa model. Combination of rIL-2 plus rituximab was synergistic in a rituximab-sensitive Daudi tumor model, as evidenced by significant tumor regressions and increased time to tumor progression, compared with rIL-2 and rituximab single agents. In contrast, rituximab-resistant Namalwa tumors were responsive to single-agent rIL-2 and showed an increased response when combined with rituximab. Using in vitro killing assays, rIL-2 was shown to enhance activity of rituximab by activating ADCC and lymphokine-activated killer activity. Additionally, the activity of rIL-2 plus rituximab F(ab')2 was similar to that of rIL-2 alone, indicating a critical role for immunoglobulin G1 Fc-FcgammaR-effector responses in mediating ADCC. Antiproliferative and apoptotic tumor responses, along with an influx of immune effector cells, were observed by immunohistochemistry. Collectively, the data suggest that rIL-2 mediates potent tumoricidal activity against NHL tumors, in part, through activation and trafficking of monocytes and natural killer cells to tumors. These data support the mechanistic and therapeutic rationale for combination of rIL-2 with rituximab in NHL clinical trials and for single-agent rIL-2 in rituximab-resistant NHL patients.

Novel robust hepatitis C virus mouse efficacy model.

The lack of a robust small-animal model for hepatitis C virus (HCV) has hindered the discovery and development of novel drug treatments for HCV infections. We developed a reproducible and easily accessible xenograft mouse efficacy model in which HCV RNA replication is accurately monitored in vivo by real-time, noninvasive whole-body imaging of gamma-irradiated SCID mice implanted with a mouse-adapted luciferase replicon-containing Huh-7 cell line (T7-11). The model was validated by demonstrating that both a small-molecule NS3/4A protease inhibitor (BILN 2061) and human alpha interferon (IFN-alpha) decreased HCV RNA replication and that treatment withdrawal resulted in a rebound in replication, which paralleled clinical outcomes in humans. We further showed that protease inhibitor and IFN-alpha combination therapy was more effective in reducing HCV RNA replication than treatment with each compound alone and supports testing in humans. This robust mouse efficacy model provides a powerful tool for rapid evaluation of potential anti-HCV compounds in vivo as part of aggressive drug discovery efforts.

CHIR-258: a potent inhibitor of FLT3 kinase in experimental tumor xenograft models of human acute myelogenous leukemia.

PURPOSE: Fms-like tyrosine kinase 3 (FLT3) encodes a receptor tyrosine kinase (RTK) for which activating mutations have been identified in a proportion of acute myelogenous leukemia (AML) patients and associated with poor clinical prognosis. Given the relevance of FLT3 mutations in AML, we investigated the activity of CHIR-258, an orally active, multitargeted small molecule, with potent activity against FLT3 kinase and class III, IV, and V RTKs involved in endothelial and tumor cell proliferation in AML models. EXPERIMENTAL DESIGN: CHIR-258 was tested on two human leukemic cell lines in vitro and in vivo with differing FLT3 mutational status [MV4;11 cells express FLT3 internal tandem duplications (ITD) versus RS4;11 cells with wild-type (WT) FLT3]. RESULTS: Antiproliferative activity of CHIR-258 against MV4;11 was approximately 24-fold greater compared with RS4;11, indicating more potent inhibition against cells with constitutively activated FLT3 ITD. Dose-dependent down modulation of receptor phosphorylation and downstream signaling [signal transducer and activator of transcription 5 (STAT5) and extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase] in MV4;11 cells with CHIR-258 confirmed the molecular mechanism of action. Target modulation of phospho-FLT3, phospho-STAT5, and phospho-ERK in MV4;11 tumors was achieved at biologically active doses of CHIR-258. Tumor regressions and eradication of AML cells from the bone marrow were shown in s.c. and bone marrow engraftment leukemic xenograft models. Tumor responses were characterized by decreased cellular proliferation and positive immunohistochemical staining for active caspase-3 and cleaved poly(ADP-ribose) polymerase, suggesting cell death was mediated in part via apoptosis. CONCLUSIONS: Our data indicate that CHIR-258 may be an effective therapy in FLT3-associated AML and warrants clinical trials.

beta-Catenin regulates vascular endothelial growth factor expression in colon cancer.

To evaluate whether beta-catenin signaling has a role in the regulation of angiogenesis in colon cancer, a series of angiogenesis-related gene promoters was analyzed for beta-catenin/TCF binding sites. Strikingly, the gene promoter of human vascular endothelial growth factor (VEGF, or VEGF-A) contains seven consensus binding sites for beta-catenin/TCF. Analysis of laser capture microdissected human colon cancer tissue indicated a direct correlation between up-regulation of VEGF-A expression and adenomatous polyposis coli (APC) mutational status (activation of beta-catenin signaling) in primary tumors. In metastases, this correlation was not observed. Analysis by immunohistochemistry of intestinal polyps in mice heterozygous for the multiple intestinal neoplasia gene (Min/+) at 5 months revealed an increase and redistribution of VEGF-A in proximity to those cells expressing nuclear beta-catenin with a corresponding increase in vessel density. Transfection of normal colon epithelial cells with activated beta-catenin up-regulated levels of VEGF-A mRNA and protein by 250-300%. When colon cancer cells with elevated beta-catenin levels were treated with beta-catenin antisense oligodeoxynucleotides, VEGF-A expression was reduced by more than 50%. Taken together, our observations indicate a close link between beta-catenin signaling and the regulation of VEGF-A expression in colon cancer.