Article entitled "Alkylphosphocholine Analogs for Broad-Spectrum Cancer Imaging and Therapy" from Science Translational Medicine

Article entitled "Alkylphosphocholine

Analogs for Broad-Spectrum Cancer Imaging and Therapy" from Science Translational Medicine, June 11, 2014, vol. 6 issue

240. Reprinted with permission from AAAS.

Sci. Transl. Med. DOI: 10.1126/scitranslmed.3007646

Alkylphosphocholine Analogs for Broad-Spectrum Cancer Imaging

Jamey P. Weichert1,2,3,4,*,

Irawati K. Kandela4,

Anatoly N. Pinchuk1,4,

Joseph Grudzinski2,4,

Christopher J. Pazoles4,

Perry J. Pickhardt1 and

+ Author Affiliations

1Department of Radiology, University of Wisconsin

School of Medicine and Public Health, Madison, WI 53792, USA.

2Department of Medical Physics, University of

Wisconsin School of Medicine and Public Health, Madison, WI 53792, USA.

3Carbone Cancer Center, University of Wisconsin

School of Medicine and Public Health, Madison, WI 53792, USA.

4Cellectar Biosciences Inc., 3301 Agriculture

Drive, Madison, WI 53716, USA.

5Department of Neurological Surgery, University

of Wisconsin School of Medicine and Public Health, Madison, WI 53792, USA.

6Department of Medicine, University of Wisconsin

School of Medicine and Public Health, Madison, WI 53792, USA.

*Corresponding author. E-mail: jweichert@uwhealth.org

(J.P.W.); j.kuo@neurosurgery.wisc.edu (J.S.K.)

Many solid tumors contain an overabundance of phospholipid ethers

relative to normal cells. Capitalizing on this difference, we created cancer-targeted alkylphosphocholine (APC) analogs through

structure-activity analyses. Depending on the iodine isotope used, radioiodinated APC analog CLR1404 was used as either a positron

emission tomography (PET) imaging (124I) or molecular radiotherapeutic (131I) agent. CLR1404 analogs displayed

prolonged tumor-selective retention in 55 in vivo rodent and human cancer and cancer stem cell models. 131I-CLR1404

also displayed efficacy (tumor growth suppression and survival extension) in a wide range of human tumor xenograft models. Human

PET/CT (computed tomography) and SPECT (single-photon emission computed tomography)/CT imaging in advanced-cancer patients with

124I-CLR1404 or 131I-CLR1404, respectively, demonstrated selective uptake and prolonged retention in both

primary and metastatic malignant tumors. Combined application of these chemically identical APC-based radioisosteres will enable

personalized dual modality cancer therapy of using molecular 124I-CLR1404 tumor imaging for planning 131I-CLR1404

Selective delivery of diagnostic imaging and therapy agents

to malignant tumors while sparing healthy tissues continues to be the major goal in cancer research and in clinical practice. Approaches

to achieve this goal are reflected in therapeutic agents that are already approved or in development. Tumor-targeting vehicles

based on antibodies, viruses, peptides, and nanoparticles have been used to deliver a wide range of "payloads" to cancer

cells, including radioactive isotopes, imaging agents, oncolytic viruses, metals, and chemotherapeutic drugs (1-8).

Currently available cancer therapy modalities offer either broad applicability across cancer types (for example, external beam

radiation, surgery, cytotoxic drugs) or cancer selectivity based on higher expression of drug targets (genes) in cancer cell subpopulations

compared to normal cells (for example, PML-RAR , BCR-ABL, c-kit, EGFR, ERBB2/Her2/neu).

Still, many cancers recur despite the initial disease control and therapeutic efficacy of many available cancer therapies. Recent

evidence suggests that cancer recurrence likely involves failure to eradicate cancer cells that are relatively resistant to current

therapies, such as cancer stem cells (or cancer stem-like cells) (9-11).

A new therapeutic modality that minimizes damage to normal cells

and combines cancer selectivity (including cancer stem cells) with a broad anticancer mechanism would represent a paradigm shift

in cancer treatment. Naturally occurring phospholipid ethers (PLE) selectively accumulate in human cancer cells compared to normal

cells (12-14). Structure-activity relationship studies were previously performed at the University of Michigan

with radioiodinated aryl PLE and a subset of alkylphosphocholine (APC) derivatives to see how alterations in molecular structure

affected tumor retention of these compounds (15-18). These studies indicated that, for glycerol-derived PLE

analogs, the stereochemistry at the sn-2 position of glycerol did not affect tumor uptake and retention in a rat sarcoma

model. The length of the hydrophobic alkyl chain was discovered as another key characteristic that affected tumor retention of

these compounds. Decreasing the alkyl chain length from 12 carbons (C12) to C7 resulted in little or no tumor

accumulation, whereas increasing the chain length had the opposite effect, with C15 and C18 analogs showing

delayed plasma clearance and enhanced tumor uptake. Furthermore, in the C12 APC series, substitution of the choline

part of the molecule with ethanol, ethanolamine, or 3,3-dimethyl-1-butanol reduced tumor uptake. From these studies, we found that

the glycerol backbone was not required for tumor avidity, the alkyl chain must contain >11 methylene groups, and the position

of iodine on the phenyl ring did not influence tumor uptake or specificity.

Ultimately, CLR1404 [18-(p-iodophenyl)octadecyl phosphocholine]

was identified among nine PLE and APC analogs as the best tumor-imaging agent in rodent models (18). In follow-up pharmacologic

toxicology studies, CLR1404 additionally exhibited low rodent toxicity and lower liver, kidney, bladder, and abdominal exposure

compared to other derivatives. Moreover, replacement of iodine with optically active moieties yielded similar tumor selectivity

and retention, suggesting bulk structural tolerance in that location. Therefore, we hypothesized that the CLR1404 chemical scaffold

(Fig. 1) is uniquely well suited to deliver radioiodine isotopes and other reporters that are useful for either diagnostic imaging

or therapy for a broad spectrum of solid tumors.

Fig. 1. APC analog chemical structures.

CLR1404 could be synthesized as a radioactive compound (125I,

124I, or 131I), as a fluorescent analog (CLR1501), or as a near-infrared analog (CLR1502).

Unlike classical DNA-targeted cytotoxic agents, alkylphospholipids,

including APC, target cellular and intracellular membranes (19). When administered at therapeutic doses, alkylphospholipids

inhibit phosphatidylcholine biosynthesis, interfere with lipid transduction pathways, and block the endoplasmic reticular transport

of cholesterol, thereby ultimately disrupting cholesterol homeostasis and membrane lipid raft function (20). Lipid rafts

are specialized plasma membrane microdomains rich in cholesterol and sphingomyelin (21), which spatially organize signaling

pathways and regulate cell proliferation and survival (apoptosis) (22). Lipid rafts, which are more abundant in cancer cells

relative to normal cells (23), serve as cellular portals of entry for CLR1404 and other alkylphospholipids into tumors.

Here, we report results of in vitro and in vivo testing with

radioisotope-labeled CLR1404 for diagnostic imaging (124I-CLR1404) and for cancer therapy (131I-CLR1404),

as well as imaging data with the fluorescent analog CLR1501, in a wide variety of tumor models. We also present first-in-human

imaging results showing that CLR1404 has tumor-targeting and retention properties in cancer patients. CLR1404 represents a new

class of synthetic APC analogs to be used for broad-spectrum, tumor-selective molecular imaging and therapy in human cancers.

Preferential uptake and retention of APC analogs in cancer

CLR1501, a fluorescently labeled CLR1404 analog (Fig.

1), was administered to seven different cancer cell lines (renal, colorectal, glioma, ovarian, pancreatic, melanoma,

and prostate) and a normal human skin fibroblast line in vitro. Twenty-four hours later, CLR1501 exhibited five- to ninefold preferential

uptake in these cancer cell lines in vitro compared to normal fibroblasts (Fig. 2A and

fig. S1). Retained CLR1501 was associated with plasma and organelle membranes. Cancer-selective localization of APC analogs was

also observed in a coculture of human prostate carcinoma cells (PC-3) and a primary normal human skin fibroblast cell line treated

with CLR1501 for 24 hours and imaged with confocal microscopy (Fig. 2B).

Fig. 2. Uptake of CLR1404 analogs in human cancer cell lines.

(A) Fluorescence confocal microscopy shows CLR1501 (5

M) uptake in multiple cancer types, and less uptake in normal human fibroblasts at 24 hours. Scale bar, 10 m. (B)

Coculture of human prostate cancer (PC-3) cells and normal human fibroblasts. Cells were treated with the fluorescent analog CLR1501

(5 M) for 24 hours and counterstained with the nuclear stain, Hoechst 33342. Scale bar, 10 m. (C) Quantitative uptake

and retention of 125I-CLR1404 in three cancer cell lines derived from patients and the corresponding patient-matched

normal cell lines. Data are means SEM (n = 3 independent experiments with three biological replicates each). *P

< 0.001, analysis of variance (ANOVA) with post hoc Tukey test.

To further examine cancer cell-selective targeting, three