Cathepsin S-cleavable, multi-block HPMA copolymers for improved SPECT/CT imaging of pancreatic cancer

Abstract

This work continues our efforts to improve the diagnostic and radiotherapeutic effectiveness of nanomedicine platforms by developing approaches to reduce the non-target accumulation of these agents. Herein, we developed multi-block HPMA copolymers with backbones that are susceptible to cleavage by cathepsin S, a protease that is abundantly expressed in tissues of the mononuclear phagocyte system (MPS). Specifically, a bis-thiol terminated HPMA telechelic copolymer containing 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was synthesized by reversible addition−fragmentation chain transfer (RAFT) polymerization. Three maleimide modified linkers with different sequences, including cathepsin S degradable oligopeptide, scramble oligopeptide and oligo ethylene glycol, were subsequently synthesized and used for the extension of the HPMA copolymers by thiol–maleimide click chemistry. All multi-block HPMA copolymers could be labeled by ¹⁷⁷Lu with high labeling efficiency and exhibited high serum stability. In vitro cleavage studies demonstrated highly selective and efficient cathepsin S mediated cleavage of the cathepsin S-susceptible multi-block HPMA copolymer. A modified multi-block HPMA copolymer series capable of Förster Resonance Energy Transfer (FRET) was utilized to investigate the rate of cleavage of the multi-block HPMA copolymers in monocyte-derived macrophages. Confocal imaging and flow cytometry studies revealed substantially higher rates of cleavage for the multi-block HPMA copolymers containing the cathepsin S-susceptible linker. The efficacy of the cathepsin S-cleavable multi-block HPMA copolymer was further examined using an in vivo model of pancreatic ductal adenocarcinoma. Based on the biodistribution and SPECT/CT studies, the copolymer extended with the cathepsin S susceptible linker exhibited significantly faster clearance and lower non-target retention without compromising tumor targeting. Overall, these results indicate that exploitation of the cathepsin S activity in MPS tissues can be utilized to substantially lower non-target accumulation, suggesting this is a promising approach for the development of diagnostic and radiotherapeutic nanomedicine platforms.

Introduction

In 2015, pancreatic cancer was estimated to be the 11th and 8th most commonly diagnosed cancer for men and women in the United States, respectively. At the same time, it is estimated to account as the 4th leading cause of cancer related death for both men and women [1]. Pancreatic ductal adenocarcinoma (PDAC), which constitutes over 90% of pancreatic cancers in humans, is a devastating and virtually unexceptionally lethal malignancy [2]. Due to a lack of symptoms in early stages of the disease, PDAC is typically not diagnosed until the disease has progressed to a more advanced state [3], [4]. As a result, despite advances in the field of surgery, chemotherapy, and radiation therapy, the prognosis of PDAC remains extremely poor with a five-year survival rate of less than 5% [5], [6], which is the lowest among common malignancies. Because of the high mortality and rapid progression associated with PDAC, effective detection and accurate staging of pancreatic cancer have become a major challenge in disease management.

Over the years, numerous imaging modalities have been utilized to non-invasively detect cancer. In particular, magnetic resonance imaging [7], positron emission tomography [8], single-photon emission computed tomography (SPECT) [9], computed tomography (CT) [10] and ultrasound [11] have played critical roles in improving the diagnosis and staging of many malignancies. As one of the major imaging techniques used in nuclear medicine today, SPECT is often combined with CT to allow co-registration of the radiotracer and anatomy [12]. Utilizing the dual-modality of SPECT/CT has been shown to yield improved sensitivity and accuracy in cancer imaging [13]. Unfortunately, the potential advantages of SPECT/CT imaging for the detection and staging of PDAC cannot currently be realized due to the lack of a clinically approved imaging agent. As a result, the preclinical development of SPECT imaging agents for pancreatic cancer has been an active area of research [14], [15], [16]. These radiotracers have utilized carriers that range from small molecules to macromolecules. To date, the bulk of the macromolecular work has focused on antibodies due to their excellent in vivo targeting and tumor retention properties. Despite the comparable tumor uptake and retention properties, nanomedcines have received relatively little attention in the development of SPECT imaging agents for PDAC and other cancers. This is largely attributable to the high blood retention and uptake of many of these platforms in non-target tissues associated with the mononuclear phagocyte system (MPS) [17]. In order for nanomedicines to become more attractive as a radionuclide carrier, approaches are needed to lower this non-target retention.

During the last two decades, work by Kopeček and other laboratories have demonstrated the potential of poly[N-(2-hydroxypropyl)methacrylamide] (HPMA), a water-soluble, non-immunogenic, non-toxic polymeric platform, as a drug carrier [18]. For cancer, HPMA copolymers have shown great potential as a drug delivery system to enhance the delivery of chemotherapeutics to a variety of tumors through the enhanced permeability and retention (EPR) effect [19]. Although these copolymers exhibited remarkable advantages for drug delivery, the accumulation of this polymeric platform, as with many nanomedicines, in MPS-associated tissues (e.g., blood, liver and spleen) diminishes its potential as a diagnostic agent. Our laboratory has been focused on developing approaches to substantially improve the clearance of radiolabeled HPMA copolymers from MPS-associated, non-target tissues. Our efforts have focused on cathepsin S, a protease that is abundantly expressed in antigen presenting cells (APCs) such as monocytes and macrophages - the primary components of the MPS [20], [21]. Utilizing known peptidic substrates of cathepsin S, we have used these peptides to substantially enhance the clearance of radiolabeled HPMA copolymers from MPS-associated tissues, such as the blood, liver and spleen [22], [23].

Inspired by the recent work on backbone biodegradable copolymers [24], [25], this manuscript investigates the incorporation of cathepsin S susceptible linkers into the HPMA backbone and its impact on the biological performance of these agents for PDAC imaging. We hypothesized that inclusion of the cathepsin S cleavable, peptidic linker would result in fragmentation of the HPMA copolymers and faster clearance of the copolymer fragments from phagocytic cells (e.g., monocytes and macrophages) and tissues associated with MPS uptake and retention. Furthermore, as a consequence of this fragmentation, the smaller copolymer blocks would be expected to clear more quickly from the body. To investigate, we designed two multi-block HMPA copolymer platforms, depicted in Fig. 1: MPs and FMPs. The MPs, which contains the chelation moiety, were radiolabeled with lutetium-177 (¹⁷⁷Lu) and evaluated using various in vitro and in vivo studies. Whereas, the FMPs, which are capable of Förster Resonance Energy Transfer (FRET), were utilized as a tool to investigate in vitro fragmentation kinetics of the biodegradable, multi-block HPMA copolymers. For both the MPs and FMPs, a known cathepsins S peptide substrate (PMGLP) was utilized as the linker as well as controls containing a scrambled peptide analogue and a non-cleavable polyethylene glycol (PEG). Herein, the syntheses, characterization, in vitro cleavage assays and cell trafficking studies for the MPs and FMPs are described. In addition, the biodistribution studies and SPECT/CT imaging results of the MPs in a PDAC mouse model are reported.