Cancer-Targeted Alkylphosphocholine Analogs for Intraoperative Visualization

13 Cancer-Targeted Alkylphosphocholine Analogs for Intraoperative Visualization

Ray R. Zhang, Paul A. Clark, Jamey P. Weichert, and John S. Kuo

Abstract
Within the last decade, several clinical trials pairing targeted fluorophores for cancer with their detection instrumentation have emerged in an effort to improve resection outcomes and survival. This rapidly expanding field of fluorescence-guided surgery (FGS) is ready to revolutionize the way oncological surgeries are performed in the operating room. Alkylphosphocholine (APC) analogs are a group of versatile small molecules that can be attached to different “diapeutic” moieties for targeted diagnostic imaging and therapy of cancer. They work by a unique mechanism in multiple kinds of cancers, through selective uptake through lipid rafts that are overexpressed in cancer cells, and prolonged retention by decreased catabolism in cancer cells. Several APC agents are in clinical trial testing, and the fluorescent APC analogs 1501 and 1502 have successfully demonstrated selective uptake and retention in several orthotopic and xenograft rodent models of human cancer, including orthotopic human-derived cancer stem cell models of glioblastoma multiforme. 1501 carries a green fluorescent BODIPY (boron-dipyrromethene) tag for subcellular localization studies, and 1502 (with a near-infrared fluorophore IR-775) was validated for FGS in multiple preclinical rodent models of cancer with high tumor-to-normal tissue contrast. Dual-labeled PET/fluorescent APC analogs have also been synthesized and validated in order to better characterize and understand the new modality of fluorescence. With an armamentarium of several APC analogs of different diagnostic and therapeutic moieties at our disposal that target cancer by the same purported mechanism, we are able to preoperatively image, intraoperatively resect, and postoperatively treat and follow multiple types of cancers, offering a level of synergy that may improve cancer care and outcomes.

Keywords: alkylphosphocholine analogs, fluorescence-guided surgery, targeted fluorophores, near-infrared fluorophores, glioblastoma multiforme, surgical oncology

13.1 Introduction

Surgical resection remains a primary treatment of cancer, and the extent of tumor resection has prognostic impact on patient survival.1,2,3 In most cancer types, patients who have microscopic residual and positive tumor margins experience cancer recurrence and significantly shorter long-term survival compared to patients with complete resections and negative tumor margins.^4,5,6,7 In the case of brain malignancies, gross total resection (GTR) is associated with improved long-term survival compared to patients with subtotal resections (13 vs. 8 months for high-grade gliomas and 15 vs. 9.9 years for low-grade gliomas).^4,5,8 Furthermore, microscopically positive margins have been reported in up to 65% high-grade glioma resections, highlighting the need for better intraoperative distinction between malignant and nonmalignant tissues.⁸ At the same time, while positive margins are associated with increased rates of recurrence and worse survival compared with GTR, overzealous resection can compromise adjacent vital tissue, resulting in poor neurological functional outcomes.^2,3 Fluorescence-guided surgery (FGS) could achieve better resection margins and also improve functional outcomes of patients undergoing surgeries by providing a superimposable and real-time field of visualization (FOV) that distinguishes malignant tissue from benign tissue.

Traditionally, surgeons rely on subjective assessments during resection and ablative procedures including subtle tactile and visual tissue differences to intraoperatively distinguish cancer from adjacent tissues. Improvements in cancer imaging technology are now aiding surgeons in visualizing cancer in the operating room. Several intraoperative modalities including ultrasound, fluoroscopy, CT, and MRI have all been harnessed to improve and facilitate cancer removal. These intraoperative imaging modalities can be used to provide additional information about anatomical localization of abnormal lesions, adjacent vital structures, and tissue shift during an operation. However, these intraoperative imaging modalities rely on general characteristics such as abnormal perfusion or tissue consistency, lack cancer specificity, are limited in their contrast detection sensitivities, and offer distinctly different and nonsuperimposable FOV from the surgical cavity. Fluorescence imaging has several favorable characteristics that are conducive for clinical implementation. These include real-time detection capabilities with a superimposable FOV, an abundance of cancer-targeted fluorophores, high detection sensitivity compared to other imaging modalities, an excellent safety record with clinically used probes,^9,10,11,12 lack of ionizing radiation exposure, lower costs, and markedly less cumbersome detection instrumentation compared with other intraoperative imaging modalities.^13,14,15,16 Importantly, fluorophores can be attached to a variety of available targeting molecules, enabling the application of this modality to many cancer types.

Fluorescence-guided imaging usually involves the administration of a cancer-selective fluorophore, followed by excitation and detection of its fluorescence at an optimal time point to achieve contrast. Near-infrared (NIR) fluorophores with excitation and emission spectra in the NIR wavelength range (700–900 nm) exhibit improved depth penetration and lower in vivo background compared to fluorophores that excite and emit fluorescence at shorter wavelengths. NIR fluorophores have attracted the most attention because of their favorable characteristics and improved contrast.^17,18

13.2 Fluorescent Alkylphosphocholine Analogs 1501 and 1502

Weichert et al has recently expanded the repertoire of cancer-targeting alkylphosphocholines (APCs; small molecule platform agents) for cancer imaging by creating fluorescent APC analogs for intraoperative surgical illumination of cancer margins. In 2014, Weichert et al reported APC analogs, a new class of small synthetic phospholipid ether molecules for PET imaging and targeted radiotherapy, that demonstrate broad-spectrum tumor-targeting potential in over 60 preclinical cancer models and in early human clinical studies.19 Through extensive structure activity relationship studies, NM404 was selected from 30 related phospholipid ether and APC compounds as the optimal tumor-imaging agent.²⁰ The PET compound ¹²⁴I-NM404 can be used for noninvasive localization and staging of cancer, and ¹³¹I-NM404 (a companion radioisotope) can be used for cancer-targeted radiotherapy ( Fig. 13.1).^19,21,22 Due to the broad tumor-targeting potential of NM404, two fluorescent APC analogs (1501 and 1502) were created by labeling the same APC-targeting backbone with different fluorophores; these fluorescent APC agents were synthesized for subcellular localization studies and FGS, respectively ( Fig. 13.1). 1501 carries a green fluorescent boron-dipyrromethene (BODIPY) tag for subcellular localization studies and 1502 (with an NIR fluorophore [NIRF] IR-775) was validated for FGS in multiple preclinical rodent models of cancer. Other APC analogs are also being synthesized and tested for multimodality cancer imaging and therapy ( Fig. 13.1).

Fig. 13.1 Alkylphosphocholine (APC) analogs for multimodality cancer imaging and therapy. APC analogs are composed of a phosphocholine head, a C18 alkyl chain, and an aromatic ring, which together comprise the targeting moiety and an imaging or therapy moiety. ¹²⁴I is used for PET imaging, ¹³¹I is used for targeted radiotherapy, the fluorophore BODIPY (boron-dipyrromethene) is used for subcellular localization and mechanistic studies, and the fluorophore IR-775 for near-infrared imaging. Other imaging and therapy moieties can also be added to the APC targeting backbone for multimodality cancer imaging and therapy.

Fig. 13.2 Fluorescence imaging of an orthotopic U87 glioblastoma in a mouse brain with the near-infrared alkylphosphocholine analog 1502.

Subcellular localization studies with 1501 offered insight into the mechanism of APC’s cancer specificity.19 In co-culture with normal cells, cancer cells demonstrated more uptake of 1501 compared to normal fibroblasts. As found with other APC analogs, methyl-beta-cyclodextrin disruption of cell membrane lipid rafts prior to addition of 1501 to tumor cells markedly decreased 1501 uptake and fluorescence by over 60%. These key studies suggest an APC-selective uptake in cancer cells through cell membrane lipid rafts, and many groups have also reported that lipid rafts are overexpressed on cancer cells.^23,24,25 These results are consistent with mechanistic studies using multiple other APC analogs.^23,24,25

In a study that compared the properties of fluorescent analogs 1501, 1502, and 5-ALA in mice with stereotactically implanted glioblastoma (GBM) xenografts, both 1501 and 1502 demonstrated high tumor-to-normal brain contrast ratios of 7.23 and 9.28, respectively. The observed tumor-to-normal brain contrast ratio of 9.28 for 1502 is significantly higher than that observed for 5-ALA at 4.81 ( Fig. 13.2).²⁶ This illustrates that 1502 offers superior contrast than the clinically approved FGS fluorophore, 5-ALA, recently approved for FGS of GBM in the United States and already clinically used worldwide. In the mice with orthotopic GBM xenografts treated with 1501, flow cytometry analysis of normal brain cells and tumor cells demonstrated that tumor-associated 1501 fluorescence was 14.8 times higher than the background signal in normal brain cells.26 Histological analysis of the tumor margins illustrated that 1501 fluorescence was localized to the tumor, and not beyond the tumor margins ( Fig. 13.3). These studies illustrate the cancer selectivity of 1501 and 1502 in GBMs, and the potential of 1502 as a viable and sensitive intraoperative fluorescent agent.