Abstract 5538: Enhanced tumor cytotoxicity of a combinational therapy using liposomal copper-doxorubicin, rapamycin, and ultrasound
Authors: Kheirolomoom Azadeh , Mahakian Lisa , Lai Chun-Yen , Lindfors Heather , Seo Jai Woong , Watson Katherine E. , Haynam Eric , Ingham Elizabeth , Xing Li , Borowsky Alexander D. , Cardiff Robert D. , Ferrara Katherine W. 
Published: February 2011

Abstract
We introduce an effective local therapeutic strategy using a combined treatment with: a) increased and stable loading of doxorubicin (Dox) in liposomes using a complex of Dox and copper (II), b) suppression of tumor proliferation using rapamycin, c) insonation of the entire tumor to increase microvascular permeability, extravasation and accumulation of liposomes.
Liposomes composed of HSPC: Cholesterol: DSPE-PEG2k, 56: 39: 5 were prepared in the presence of 100 mM copper(II) gluconate and 270 mM triethanolamine (TEA), pH 7.4 and extruded with 100 nm membrane filters. Cu-liposomes were loaded with Dox up to a maximum concentration of 0.6 mg-drug/mg-lipid with 100% loading. We studied the efficacy of Cu-Dox liposomes and optimized the treatment strategy using the highly invasive and metastatic Met-1 tumor, a syngeneic model of human breast carcinoma which was transplanted bilaterally into the mouse mammary fat pad. Over the 28 days of the study, Cu-Dox liposomes were administrated intravenously (iv) two times per week at a therapeutic level of 6 mg-drug/kg-body weight and rapamycin was injected intraperitoneally (ip) three times per week at 0.9 mg-drug/kg-body weight. Ultrasound was applied using a peak negative pressure of 1.23 MPa (MI=0.99), cycle length of 100 and pulse repetition rate maintained between 0.1 and 5 kHz, resulting in a temperature of 42°C for a two-minute duration.
A dotted and diffuse structure of the Cu- Dox complex within the liposomes was verified by cryo-electron microscopy. For the Met-1 tumor line, the IC50 for liposomal doxorubicin with an identical lipid formulation decreased from 2.2±0.91 to 0.412±0.16 µM in the presence of copper gluconate during loading. The toxicity of copper-loaded liposomes (without doxorubicin) was not increased over the control (no copper, no doxorubicin) liposome formulation. Insonifying one tumor per mouse after systemic injection of Cu-Dox liposomes resulted in an increase in local accumulation of liposomes from 7.7±8 to 14±9 %ID/g-tumor as detected using ICP-MS. Although single therapy using Cu-Dox liposomes suppressed tumor growth, significant tumor regression was accomplished only when Cu-Dox liposomes were combined with rapamycin and tumor insonation. At 15 days following the onset of therapy, the percent change in tumor diameter was −20±42, 114±250, 234±213, 833±247, 5533±4200 for treatments of Cu-Dox liposomes/rapamycin/ultrasound, Cu-Dox liposomes/rapamycin, Cu-Dox liposomes, rapamycin, rapamycin diluent, respectively. Diluent-treated animals survived 18 days after the onset of treatment; all animals receiving the full combined therapy survived throughout the 28-day course of treatment, demonstrating a lower fraction of angiogenic vessels and decreased tumor proliferation upon immunohistochemistry.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5538.