Marc Longino

Alkylphosphocholine Analogs for Broad-Spectrum Cancer Imaging and Therapy

Tumor-specific alkylphosphocholine analogs were evaluated as imaging and therapy agents in patients and in animal models of human cancer. A Broad View of Cancer Many consider targeted or molecular imaging to be the optimal way to image cancer. Weichert and colleagues feel differently: Uptake of certain small molecules by all cancer cells can give a broad view of cancer, and perhaps also treat it. These small molecules are alkylphosphocholine (APC) analogs, which are taken up preferentially by cancer cells—as compared to, for example, fibroblasts—via plasma membranes and transported into the cells by lipid rafts. The authors tested the uptake of radiolabeled APC analogs in vitro and in vivo in animals in 57 different spontaneous and transgenic tumors, of both human and rodent origin. Because of the well-established efficacy of radiotherapy, the authors demonstrated that the APC analogs could be used to not only visualize tumors but also kill them. Translating this to cancer patients, Weichert et al. showed preliminary preferential uptake of a radiolabeled APC analog in brain tumors. These broadly applicable imaging and therapeutic APC-based agents have been tested in dozens of different human cancers, and preliminarily in people, and are now well poised for further translation to clinical trials. 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 therapy.

Polyiodinated triglyceride lipid emulsions for use as hepatoselective contrast agents in CT: effects of physicochemical properties on biodistribution and imaging profiles.

RATIONALE AND OBJECTIVES A novel lipid emulsion (LE) was developed for hepatoselective delivery of a polyiodinated triglyceride (ITG) with potential for use in CT. This work assessed the effects of mean particle size, total administered dose, and formulation composition on the in vivo biodistribution and imaging profiles of the ITG-LE in rats. METHODS The concentration of radioactivity derived from intravenously administered 125I-ITG-LE was determined as a function of time after injection. CT imaging studies of the abdomen evaluated the extent of hepatic enhancement after administration of ITG-LE. RESULTS Mean emulsion particle diameter and total administered dose exerted the greatest effect on ITG-LE biodistribution profiles. In the optimal delivery scenario, >70% of the administered dose localized to the liver 30 minutes after injection. Liver enhancement profiles in CT imaging studies were consistent with biodistribution profiles. CONCLUSIONS These results suggest that an appropriately formulated and administered dose of ITG-LE provides tissue-selective localization of contrast material for use in CT.

Imaging efficacy of a hepatocyte-selective polyiodinated triglyceride for contrast-enhanced computed tomography.

Rationale and objectivesDHOG-LE is an injectable polyiodinated triglyceride lipid emulsion providing contrast enhancement of the liver in CT. Studies were conducted to characterize the imaging efficacy of various DHOG-LE formulations as a function of both the administered iodine dose and the formulation composition. Materials and methodsFour DHOG-LE preparations consisting of either 10, 20, 25, or 30% (w/v) total lipid were administered to anesthetized female Sprague Dawley rats as single intravenous bolus doses of 50, 100, 150, and/or 300 mg I/kg (n = 3 to 6 rats/formulation and dose). A 25% triolein lipid emulsion prepared without iodine was administered as a vehicle control at the highest dose volume (n = 6). Liver enhancement was evaluated as a function of time (0 to 24 hours) after administration of contrast by analyzing regions of interest from sequential body scans. ResultsAt all dose levels, liver enhancement was observed after injection of each DHOG-LE formulation. Regardless of formulation composition, similar enhancement of the liver was noted when administered at an equivalent iodine dose. Liver enhancement increased proportionately with increasing iodine dose. Mean peak intensities for 50, 100, 150, and 300 mg I/kg doses were 78 HU (42% above baseline), 101 HU (84% above baseline), 125 HU (127% above baseline), and 195 HU (255% above baseline), respectively. Liver time-intensity profiles exhibited rapid uptake, prolonged enhancement up to 3 hours, and complete clearance of the majority of the formulations tested by 24 hours. Time and duration of peak intensities were also directly related to iodine dose. ConclusionsIn the animal model tested, DHOG-LE imaging efficacy was directly related to iodine dose and was independent of formulation composition. Thus, administration of DHOG-LE in highly concentrated lipid preparations minimized administered dose volume and resulted in appreciable liver enhancement, even at the lowest dose of 50 mg I/kg.

Quantification of CLR1401, a novel alkylphosphocholine anticancer agent, in rat plasma by hydrophilic interaction liquid chromatography-tandem mass spectrometric detection

A rapid and specific LC-MS/MS based bioanalytical method was developed and validated for the determination of 18-(p-iodophenyl)octadecyl phosphocholine (CLR1401), a novel phosphocholine drug candidate, in rat plasma. The optimal chromatographic behavior of CLR1401 was achieved on a Kromasil silica column (50 mm x 3 mm, 5 microm) under hydrophilic interaction chromatography. The total LC analysis time per injection was 2.8 min with a flow rate of 1.5 mL/min under gradient elution. Liquid-liquid extraction in a 96-well format using ethyl acetate was developed and applied for method validation and sample analysis. The method validation was conducted over the curve range of 2.00-1000 ng/mL using 0.0500 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed < or = 5.9% relative standard deviation (RSD) and -10.8 to -1.4% relative error (RE). The method was successfully applied to determine the toxicokinetics of CLR1401 in rats from three dose groups of 0.4, 4.0, and 10.0 mg/kg/day via intravenous administration.

Lipid Soluble Contrast Agents for Computed Tomography of the Liver: Results with Cholesteryl Iopanoate

A new class of compounds, polyiodinated sterol esters, has recently been synthesized and found to have characteristics suggesting potential value as site-specific hepatic computed tomographic (CT) contrast agents. A prototype compound, cholesteryl iopanoate, was administered intravenously to rabbits in doses of 65 mg I/kg. The compound was found to significantly enhance hepatic parenchyma to a maximal level of 65 Hounsfield units (HU) above base-line values without observable splenic enhancement. A subsequent study comparing the administered dose with the change in CT attenuation values of liver demonstrated that near-maximum enhancement reaching 22 HU was achieved at a dose of 30 mg I/kg within 24 h after injection. The enhancement characteristics suggest the mechanism of hepatic accumulation may not be mediated solely by the reticuloendothelial system. Administration of cholesteryl iopanoate (30 mg I/kg) to rabbits with hepatic tumors (V x 2 adenocarcinoma) resulted in the CT imaging of tumors as small as 2 mm in diameter. Further investigation of this new class of lipid-soluble contrast agents seems warranted.

Esters of iopanoic acid as liver-specific CT contrast agents: biodistribution and CT evaluation

The synthesis and preliminary biodistribution data for a series of sterol-like esters of iopanoic acid having potential value as liver-specific CT contrast agents are described. Structural modification of the sterol portion of the iopanoate ester afforded a group of compounds that displayed tissue specificity similar to chqlesteryl iopanoate, the prototype ester of this series, but were rapidly cleared from the target tissues after hydrolysis. From the biodistribution data, the most promising of these agents, pregnenolone iopanoate (PI), was evaluated by CT in rabbits receiving a radiologic dose equivalent to 30 mg I/kg. The hepatic parenchyma was enhanced within 2 h of infusion to a maximal level of 31 HU above precontrast values. Hepatic CT attenuation returned to normal within 24 h. However, CT performed after PI infusion into Vx2 tumor-bearing rabbits failed to provide superior images compared with those acquired following bolus administration of urographic contrast.

Abstract 3831: 131I-CLR1404 and CLR1404: Broad spectrum, cancer-targeted molecular radio- and chemotherapeutic phospholipid ether analogs

The effectiveness of current approaches to cancer therapy is often limited by off-target toxicity or, if relatively selective, by lack of drug target expression in tumors. To address these shortcomings, structure-activity relationship studies were undertaken and identified a series of iodophenyl-containing phospholipid ether (PLE) analogs that selectively accumulate in cancer cells/cancer stem cells compared to normal cells/stem cells, in vitro and in vivo. Isosteric iodine substitution in CLR1404 affords either a diagnostic/imaging agent (e.g. using 124 I for cancer-selective PET imaging) or a molecular radiotherapeutic agent (e.g. using 131 I for cancer-selective cytotoxicity), both of which are in clinical development. We suggest the term “diapeutic” to describe such drugs which can be used in one form to identify and characterize patients who will benefit from a specific therapy and, in another form, to effect that therapy. Here we describe the broad-spectrum efficacy of the 131 I-labeled homolog of one such diapeutic compound (CLR1404) in human tumor xenograft models in mice including Caki-2 (renal; clear cell carcinoma), HCT-116 (colorectal carcinoma), Ovcar-3 (ovarian adenocarcinoma), MDA-MB-231 (triple negative mammary gland adenocarcinoma, MES-SA/Dx5 (uterine sarcoma), U87 (glioma), Mia Paca-2 (pancreatic carcinoma) and PC-3 (prostate carcinoma). In these models, a single i.v. injection of 131 I-CLR1404 (100 αCi, 3.8 μg/mouse) resulted in significant tumor growth suppression and extension of survival. A separate radiotoxicology study in normal rats followed for six months or until death after a single dose of 131 I-CLR1404 did not find significant radiation toxicity below 5 mCi suggesting that 131 I-CLR1404 may display an acceptable therapeutic index in man. At 100-fold higher mass dose (380 μg/mouse, i.v.) CLR1404, the non-radioactive, 127 I-containing homolog, was also highly efficacious in human tumor mouse xenograft models including MDA-MB-231 (triple negative breast cancer) and A549 (non-small cell lung cancer). CLR1404 inhibited proliferation of multiple human cancer cell lines in vitro with an IC 50 of ∼5 μM compared to ∼50 μM for growth suppression of normal fibroblasts. Similar concentrations inhibited the PI3K/Akt cell signaling pathway and stimulated pro-apoptotic caspases in human cancer cell lines but not in normal fibroblasts. Thus, 131 I-CLR1404 and CLR1404 each combine wide-ranging, cancer cell-selective targeting with cytotoxicity mechanisms known be broadly effective (intracellular radiation or PI3K/Akt inhibition, respectively). As such, both agents have the potential to provide effective, well-tolerated therapy across numerous cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3831. doi:1538-7445.AM2012-3831

Abstract 5740: 124I-CLR1404 and 131I-CLR1404: Broad spectrum diapeutic agents for cancer cell-targeted PET imaging and molecular radiotherapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Selective delivery of therapeutic and diagnostic agents to malignant tumors versus normal tissues continues to be an important goal in cancer research and clinical practice. We have been developing cancer specific, iodinated phospholipid ether (PLE) analogs for tumor imaging and molecular radiotherapy. These PLEs localize in cancer cells selectively via plasma membrane lipid rafts, more abundant in cancer cells compared to normal cells, using them as portals of entry. Isosteric iodine substitution in CLR1404 affords either a diagnostic/imaging agent (e.g. using 124I for cancer-selective PET imaging) or a molecular radiotherapeutic agent (e.g. using 131I for cancer-selective cytotoxicity), both of which are in clinical development. We suggest the term “diapeutic” to describe such drugs which can be used in one form to identify and characterize patients who will benefit from a specific therapy and, in another form, to effect that therapy. A fluorescent analog of CLR1404, CLR1501, as well as a radioactive homolog displayed increased, lipid raft-dependent selective uptake and prolonged retention in a wide variety of cancer cell lines in comparison to normal human cells. Intracellular accumulation was observed in organellar membranes (ER, Golgi, mitochondria, autophagosomes). Lipid raft involvement in uptake was supported by co-localization of CLR1501 and lipid raft molecular markers. Lipid raft-dependence of uptake into cancer cells was confirmed by the finding that it was significantly decreased after disruption of lipid rafts with Filipin III. In vivo, nuclear imaging with radioiodinated CLR1404 revealed tumor-selective uptake and retention in 52 of 54 rodent cancer models tested, including human tumor xenograft, transgenic and spontaneous tumor models. In these models, 124I-CLR1404 clearly imaged both primary tumors and metastases. In vivo tumor conspicuity was apparent by 24 hours after i.v. administration and appeared undiminished at 120 hours. In contrast, the extra-tumoral 124I-CLR1404 PET signal decreased dramatically over the same time period as the agent was excreted. By 48 hours, no other tissues or organs showed significant labeling above the background of the blood compartment. Tumor uptake was not seen in models of benign hyperplasia. SPECT/CT imaging after administration of 131I-CLR1404 to cancer patients in the context of a Phase 1a dosimetric clinical trial also demonstrated selective uptake into malignant tumors, including metastases. Used in tandem, these chemically identical isosteres offer a personalized, diapeutic approach to cancer therapy, with 124I-CLR1404 imaging serving as an ideal qualitative and quantitative biomarker for subsequent treatment with 131I-CLR1404. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5740. doi:1538-7445.AM2012-5740

Abstract 3495: The novel phospholipid ether analog CLR1404 decreases glioblastoma stem cell proliferation, suppresses GBM growth, and improves survival

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Most patients treated for glioblastoma multiforme (GBM), the most common primary brain malignancy, have less than 2 years survival due to rapid recurrence. The GBM stem cell (GSC) sub-population exhibits therapeutic resistance and is hypothesized to drive tumor recurrence. Therefore, GSC-specific targeting is likely critical for improving outcome. Phospholipid ether analog, CLR1404 and its radioiodinated isosteres, exhibit cancer cell-specific uptake and prolonged retention in 57/61 cancer cell lines and xenografts (including GBM) due to affinity for cancer cell lipid rafts. In this study, we investigated the therapeutic potential of CLR1404 against GBM and its GSC subpopulation. Multiple sphere-forming GSC lines were isolated from patient specimens with IRB approval, and rigorously validated for self-renewal, multi-lineage potential, and high efficiency orthotopic tumor initiation in NOD-SCID mice. Proliferation assays were performed by addition of CLR1404 in serum-free medium for 24 hours. To test effects on stem cell properties, GSCs were treated with CLR1404, dissociated to single cells, plated at 500 cells in a 96-well plate, and allowed to form spheres (≥2-4 weeks). CLR1404 inhibition of the AKT oncogenic signaling pathway in GSCs was assayed with immunoblot analysis. In vivo, GSCs were pre-treated for 24 hours with CLR1404 prior to orthotopic injection of 200,000 live cells into immunodeficient mice. Subsequent survival curves were then constructed. CLR1404 anti-proliferative effects were seen on all 7 different GSC and GBM lines tested with IC50 values ranging between 5-10 µM using MTS assay; control normal differentiated neural cells exhibited an IC50 of approximately 40 µM. CLR1404 treatment also decreased sphere-forming ability of multiple GSC lines with IC50 values between 5-10 µM. CLR1404 inhibition of AKT activation was observed using immunoblot analysis. CLR1404 pre-treatment of GSCs significantly increased survival time in an orthotopic mouse model (Control: 59±6.1 days; CLR1404: 94±4.4 days), suggesting CLR1404 treatment of the GSC tumor initiating cells improves outcome. This data of CLR1404s therapeutic potential against GBM and its GSCs, combined with previously demonstrated tumor cell targeting specificity of CLR1404 and its radioactive isosteres, provides strong evidence for the potential of novel CLR1404-based therapies to improve GBM outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3495. doi:1538-7445.AM2012-3495