Therapeutic efficacy of a lipid-based prodrug of mitomycin C in pegylated liposomes: Studies with human gastro-entero-pancreatic ectopic tumor models Alberto Gabizon a, b, ⁎, Yasmine Amitay c , Dina Tzemach a , Jenny Gorin a , Hilary Shmeeda a , Samuel Zalipsky d a Shaare Zedek Medical Center, Jerusalem, Israel b Hebrew University—School of Medicine, Jerusalem, Israel c Lipomedix Pharmaceuticals Ltd., Ramat Hasharon, Israel d Independent Consultant, Redwood City, CA, USA abstract article info Article history: Received 23 September 2011 Accepted 15 November 2011 Available online 26 November 2011 Keywords: Liposome Mitomycin C Cancer Chemotherapy Prodrug Background: A mitomycin-C lipid-based prodrug (MLP) formulated in pegylated liposomes (PL-MLP) was previously reported to have significant antitumor activity and reduced toxicity in mouse tumor models (Clin Cancer Res 12:1913–20, 2006). MLP is activated by thiolysis releasing mitomycin-C (MMC) which rap- idly dissociates from liposomes. The purpose of this study was to examine the plasma stability, pharmacoki- netics, and antitumor activity of PL-MLP in mouse models of human gastroentero-pancreatic tumors. Methods: MLP was incorporated with almost 100% efficiency in pegylated liposomes composed of hydroge- nated phosphatidylcholine, with or without cholesterol (Chol). Mean vesicle size was 45–65 nm for liposome preparations downsized by homogenization, and 80–100 nm when downsized by extrusion, the latter dis- playing narrower polydispersity. MLP to phospholipid mole ratio was 5% (~20 μg MMC-equivalents/μmol). Therapeutic studies were carried out in the N87 gastric carcinoma (Ca), HCT15 colon Ca, and Panc-1 pancre- atic Ca models implanted s.c. in CD1 nude mice. Treatment was administered i.v. in mice with established tu- mors. Results: PL-MLP was very stable when incubated in plasma, and whole blood with a maximum of 5% release and activation to free MMC after 24 h. In the presence of a strong reducing agent (dithiotreitol), MLP was al- most entirely activated to free MMC. Pharmacokinetic studies revealed major differences in plasma clearance between free MMC and PL-MLP. The longest half-lives were observed for extruded and Chol-containing prep- arations. Using a liposome radiolabel, it was found that the plasma levels of liposomes and prodrug were nearly superimposable confirming the absence of drug leakage in circulation. In vivo prodrug activation was significantly increased by co-injection of a large dose of a biocompatible reducing agent, N- acetylcysteine. PL-MLP was significantly more effective in delaying tumor growth and resulted in more tumor regressions than irinotecan in the N87 and HCT15 models, and than gemcitabine in the Panc-1 model. PL-MLP was ~3-fold less toxic than free MMC at MMC-equivalent doses, and displayed mild myelo- suppression at therapeutic doses. Conclusions: Delivery of MLP in pegylated liposomes is more effective than conventional chemotherapy in the treatment of gastroentero-pancreatic ectopic tumor models, and may represent an effective tool for treat- ment of these malignancies in the clinical setting with improved safety over free MMC. Reducing agents offer a tool for controlling in vivo prodrug release. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Mitomycin C (MMC) is a powerful anti-bacterial and anti-tumor fungal antibiotic isolated from cultures of Streptomyces caespitosus. Following enzymatic or chemical reduction, MMC becomes a bifunc- tional alkylator, cross-linking complementary strands of the DNA double helix. Mitomycin C has an extraordinary ability to crosslink DNA with high efficiency and absolute specificity for the CpG sequence [1]. The molecular mechanism of activity has been reviewed by Tomasz [1]. MMC activity appears to be enhanced in hypoxic tu- mors [2]. The pharmacokinetics and metabolic studies of mitomycin C demonstrate a rapid elimination pattern for the drug (alpha half- life of 8 min and beta, or terminal, half-life of 48 min), and an abso- lute requirement for reductive enzymatic activation of the drug to mono- and bifunctional alkylating species [3]. MMC often retains ac- tivity against P-glycoprotein-mediated multidrug resistant (MDR) tumor cells, with resistance more often related to the tumor levels of DT-diaphorase (known also as NADPH quinone oxidoreductase or NQO1) [4]. Despite a broad spectrum of antitumor activity, MMC clin- ical use is relatively limited due to dose-limiting toxicity, particularly Journal of Controlled Release 160 (2012) 245–253 ⁎ Corresponding author at: Oncology Institute, Shaare Zedek MC, POB 3235, Jerusalem 91031, Israel. Tel.: +972 2 6555036; fax: +972 2 6555080. E-mail address: albertog@ekmd.huji.ac.il (A. Gabizon). 0168-3659/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2011.11.019 Contents lists available at SciVerse ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel NANOMEDICINE