Anti-Myeloma and Anti-Angiogenic Effects of the Novel Anthracycline Derivative INNO-206

Abstract 4065

Doxorubicin and pegylated liposomal doxorubicin have shown efficacy for the treatment of multiple myeloma (MM). Most recently, anthracyclines including doxorubicin have inhibited vascular development in tumor tissues by reducing intracellular hypoxia inhibitory factor (HIF)-1α levels. However, the efficacy of these drugs as single agents and in combination therapies is limited by their myelosuppressive, cardiac and dermatological side effects. INNO-206 (CytRx Corporation, Los Angeles, CA) is an albumin-binding prodrug of doxorubicin that is released from albumin under acidic conditions. Extracellularly, tumor tissues including MM are often acidic which allows free doxorubicin to be released from INNO-206. Following cellular uptake, it is released in the acidic endosomal or lysosomal compartments. Thus, this drug offers the possibility to achieve higher levels of active doxorubicin within tumor cells than with conventional doxorubicin. INNO-206 has not been previously evaluated using animal models in any hematologic malignancies including MM. First, we determined the effects of INNO-206 at varying pH levels on MM tumor cell proliferation and apoptosis in vitro. Second, we have developed a novel approach to test the anti-angiogenic activity of new agents in a rapid, accurate and inexpensive way using a combined chorioallantoic membrane (CAM) and feather bud (FB) assay (Chen et al. Int J Oncol 2010) and used this model to evaluate the anti-angiogenic effects of INNO-206 in a concentration- and pH-dependent fashion. Third, we determined the anti-MM effects and toxicity of INNO-206 and conventional doxorubicin administered weekly in vivo using our severe combined immunodeficient (SCID) murine model of human MM LAGκ-1A. We used the MTS cell proliferation and Annexin V assays for determining the number of viable cells and apoptosis, respectively following exposure to INNO-206 at pH 5, 6 and 7. Our CAM/FB model was used to assess the anti-angiogenic effects of this drug at these same pH levels. For the in vivo studies, each SCID mouse received a 20 – 40 mm³ MM tumor piece surgically implanted into the left hind limb superficial gluteal muscle. Seven days post-implantation mice were bled, human IgG levels were measured by ELISA and mice randomized into treatment groups. INNO-206 was diluted in sodium phosphate and administered to SCID mice at 10.8 mg/kg (free doxorubicin equivalent of 7 mg/kg) once weekly via intravenous injection (i.v.). Conventional doxorubicin (Sigma, St Louis, MO) was also diluted in sodium phosphate and mice were treated with the drug at 4 or 8 mg/kg i.v. once weekly. Mice were bled for hIgG levels and the intramuscular tumors were measured using standard calipers on a weekly basis. Data was analyzed as the mean ± SEM. INNO-206 increased MM cell apoptosis and reduced cell proliferation much more markedly at pH 5 compared to pH 7. Using our CAM/FB model to evaluate angiogenesis, INNO-206 inhibited blood vessel and feather formation as well as endothelial gene and protein expression within FB in a concentration- and pH-dependent fashion. Moreover, SCID mice bearing LAGκ-1A treated with INNO-206 at 10.8 mg/kg showed markedly smaller tumor volumes and IgG levels on days 28 (tumor volumes: P=0.0152; hIgG: P=0.0019), 35 (tumor volumes: P=0.0051; hIgG: P=0.0006) and 42 (tumor volumes: P=0.0036; hIgG: P=0.0113) compared to vehicle-treated mice. Overall, 90% of mice were alive for the duration of the study (day 42). In contrast, treatment of tumor-bearing mice with doxorubicin at 4 and 8 mg/kg resulted in significant toxicity and death (4 mg/kg resulted in 3/10 and 9/10 deaths on days 28 and 35, respectively; 8 mg/kg, 5/10, 8/10 and 10/10 deaths occurred on days 21 28 and 35, respectively). We have shown that the novel anthracycline INNO-206 shows marked anti-MM effects both in vitro and in vivo using our SCID-hu MM model LAGκ-1A. Moreover, this albumin-bound drug which is released only under acidic conditions which is commonly present within tumor tissue is able to be administered safely at higher doses than conventional doxorubicin. The drug also shows marked anti-angiogenic effects in a pH-dependent fashion. The results from these studies suggest that this doxorubicin conjugate may provide MM patients with a new anthracycline that may be able to be administered at higher doses safely resulting in superior efficacy compared to the currently available anthracyclines to treat this B-cell malignancy.