Micellar Delivery of Cyclopamine and Gefitinib for Treating Pancreatic Cancer Deepak Chitkara, †,§ Saurabh Singh, † Virender Kumar, † Michael Danquah, † Stephen W. Behrman, ‡ Neeraj Kumar, § and Ram I. Mahato* ,† † Department of Pharmaceutical Sciences, and ‡ Department of Surgery, University of Tennessee Health Science Center, Memphis, Tennessee 38163, United States § Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Mohali, India 160062 ABSTRACT: Hedgehog (Hh) and epidermal growth factor receptor (EGFR) signaling are involved in pancreatic cancer progression. Targeting these pathways simultaneously with cyclopamine (Hh inhibitor) and gefitinib (EGFR inhibitor) is a promising approach for treating pancreatic cancer. However, the major limitation for effective clinical translation of these molecules is their low aqueous solubility. We have previously demonstrated that methoxy polyethyleneglycol-b-poly- (carbonate-co-lactic acid) {mPEG-b-P(CB-co-LA)} copolymer solubilizes hydrophobic anticancer drugs and has the potential to deliver to tumors by an enhanced permeability and retention (EPR) effect. In this study, using the nanoprecipitation method, cyclopamine and gefitinib were efficiently loaded into mPEG-b- P(CB-co-LA) micelles with encapsulation efficiencies of 94.4 and 88.6%, respectively. These micelles had a narrow particle size distribution with a mean particle size of 54.3 nm and a PDI of 0.14. Combination therapy showed a synergistic effect against L3.6pl cells but an additive effect against MIA PaCa-2cells. Caspase 3/7 activity was also increased when this combination therapy was used, indicating apoptotic cell death. Gene and protein expression analysis indicated cross-talk between Hh and EGFR signaling. Furthermore, the combination decreased tumor growth rate in L3.6pl-derived xenograft mouse tumors. These data suggest the applicability of our micellar system to effectively load and deliver cyclopamine and gefitinib for combination chemotherapy. KEYWORDS: pancreatic cancer, cyclopamine, gefitinib, combination, micelles, drug delivery 1. INTRODUCTION Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States with less than 5% 5 year survival. 1 The major concern in pancreatic cancer is metastasis to other organs due to late diagnosis. 2,3 At the time of diagnosis, 52% patients have distant disease, and 26% have regional spread, which decreases overall survival. Current treatment strategies for pancreatic cancer employ either surgery and/or chemo- therapy using gemcitabine in combination with other anticancer agents. Chemotherapy has been shown to have only modest results due to the generation of multidrug resistance (MDR), and relapse is significant. Several lines of evidence have indicated that aberrant activation of Hedgehog (Hh) 4,5 and epidermal growth factor receptor (EGFR) 6 cascades plays a major role in pancreatic carcinogenesis, disease progression, metastasis, and their contribution to chemoresistance and subsequent relapse. Binding of Hh ligands to patched (Ptch) receptor initiates a cascade of downstream signaling by relieving inhibition of smoothened (Smo) protein. Activated Smo in turn transduces downstream signals leading to translocation of active forms of glioma-associated oncogene (GLI) transcriptional effectors to the nucleus resulting in the increased expression of target genes. 7,8 In addition, EGFR and its ligands, EGF and transforming growth factor-α (TGF-α), can regulate key cellular events involved in cell proliferation and are normally overexpressed in pancreatic carcinomas. Ligand binding to EGFR induces dimerization of the receptor leading to autophosphorylation of intracellular tyrosine kinase, thereby resulting in activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-k) pathways. 9 Several studies have shown that there is a cross-talk between Hh and EGFR pathways, 10 which can increase chemoresistance and metastasis. Palma et al. have shown that sonic hedgehog (Shh) and EGFR cooperatively stimulated the cell proliferation in neocortical stem cells. 11,12 Furthermore, Shh overexpression in human keratinocyte cell line activates EGFR signaling. 13 Therefore, targeting these pathways simultaneously is a promising approach to improve the therapeutic outcome of pancreatic cancer treatment. Cyclopamine (CYA), a steroidal alkaloid derived from Veratrum californicum, is a selective Hh pathway inhibitor and binds to c-terminal of the Smo receptor leading to blockade of Received: May 18, 2012 Revised: June 28, 2012 Accepted: July 10, 2012 Article pubs.acs.org/molecularpharmaceutics © XXXX American Chemical Society A dx.doi.org/10.1021/mp3002792 | Mol. Pharmaceutics XXXX, XXX, XXX-XXX