Alkylphosphocholine Analogs Research Articles

In Silico Docking of Alkylphosphocholine Analogs to Human Serum Albumin Predicts Partitioning and Pharmacokinetics.

Alkylphosphocholine (APC) analogs are a novel class of broad-spectrum tumor-targeting agents that can be used for both diagnosis and treatment of cancer. The potential for clinical translation for APC analogs will strongly depend on their pharmacokinetic (PK) profiles. The aim of this work was to understand how the chemical structures of various APC analogs impact binding and PK. To achieve this aim, we performed in silico docking analysis, in vitro and in vivo partitioning experiments, and in vivo PK studies. Our results have identified 7 potential high-affinity binding sites of these compounds on human serum albumin (HSA) and suggest that the size of the functional group directly influences the albumin binding, partitioning, and PK. Namely, the bulkier the functional groups, the weaker the agent binds to albumin, the more the agent partitions onto lipoproteins, and the less time the agent spends in circulation. The results of these experiments provide novel molecular insights into the binding, partitioning, and PK of this class of compounds and similar molecules as well as suggest pharmacological strategies to alter their PK profiles. Importantly, our methodology may provide a way to design better drugs by better characterizing the PK profile for lead compound optimization.

Diapeutic cancer-targeting alkylphosphocholine analogs may advance management of brain malignancies.

The following is a special report on alkylphosphocholine analogs as targeted imaging and therapy agents for cancer, and their potential role in diagnosis and treatment in glioblastoma and brain metastases. These novel cancer-targeting agents display impressive tumor avidity with low background in the normal brain, and multimodal diagnostic imaging and therapy capabilities. The use of these agents may significantly improve diagnosis, treatment and post-treatment follow-up in patients with brain malignancies.

Analysis of Cancer-Targeting Alkylphosphocholine Analogue Permeability Characteristics Using a Human Induced Pluripotent Stem Cell Blood-Brain Barrier Model.

Cancer-targeting alkylphosphocholine (APC) analogues are being clinically developed for diagnostic imaging, intraoperative visualization, and therapeutic applications. These APC analogues derived from chemically synthesized phospholipid ethers were identified and optimized for cancer-targeting specificity using extensive structure-activity studies. While they strongly label human brain cancers associated with disrupted blood-brain barriers (BBB), APC permeability across intact BBB remains unknown. Three of our APC analogues, CLR1404 (PET radiotracer), CLR1501 (green fluorescence), and CLR1502 (near-infrared fluorescence), were tested for permeability across a BBB model composed of human induced pluripotent stem cell-derived brain microvascular endothelial cells (iPSC-derived BMECs). This in vitro BBB system has reproducibly consistent high barrier integrity marked by high transendothelial electrical resistance (TEER > 1500 Ω-cm(2)) and functional expression of drug efflux transporters. The radioiodinated and fluorescent APC analogues demonstrated fairly low permeability across the iPSC-BMEC (35 ± 5.7 (CLR1404), 54 ± 3.2 (CLR1501), and 26 ± 4.9 (CLR1502) × 10(-5) cm/min) compared with BBB-impermeable sucrose (13 ± 2.5) and BBB-permeable diazepam (170 ± 29). Only the fluorescent APC analogues (CLR1501, CLR1502) underwent BCRP and MRP polarized drug efflux transport in the brain-to-blood direction of the BBB model, and this efflux can be specifically blocked with pharmacological inhibition. None of the tested APC analogues appeared to undergo substantial P-gp transport. Limited permeability of the APC analogues across an intact BBB into normal brain likely contributes to the high tumor to background ratios observed in initial human trials. Moreover, addition of fluorescent moieties to APCs resulted in greater BMEC efflux via MRP and BCRP, and may affect fluorescence-guided applications. Overall, the characterization of APC analogue permeability across human BBB is significant for advancing future brain tumor-targeted applications of these agents.

WE‐H‐207A‐08: Characterization of a Broad‐Spectrum Cancer Targeted MRI Contrast Agent

Purpose:To characterize the relaxation properties and tumor targeting capabilities of a novel alkylphosphocholine (APC) analog MR contrast agent, Gd‐DO3A‐404.Methods:Relaxivities were measured via T1 and T2 mapping of Gd‐DO3A‐404 with inversion recovery and spin echo pulse sequences, respectively. Uptake was characterized in flank xenograft models of non‐small cell lung cancer (A549) and glioma (U87) and compared with uptake of Dotarem. Mice (N=3 per model per agent) were delivered 2.34 moles contrast intravenously. T1‐weighted MRI and T1 maps were acquired pre‐contrast and at multiple time points up to seven days post‐contrast. For Dotarem imaging, T1‐weighted MRI was performed at multiple time points from one minute to one day.Results:Relaxivities of Gd‐DO3A‐404 in plasma were r1=5.74 and r2=20.4 s‐1/mm at 4.7T, comparing favorably to clinical contrast agent Dotarem (r1=3.3, r2=4.7). Specific, sustained uptake of Gd‐DO3A‐404 was observed in U87 and A549. The ratio of tumor:muscle T1‐weighted signal increased from 1.24 pre‐contrast to 2.12 twenty‐four hours post‐contrast in U87 and from 1.14 to 2.16 (same time points) in A549. Significant signal enhancement was maintained until 7 and 4 days post‐contrast in U87 and A549, respectively. In comparison, uptake and washout of Dotarem in U87 occurred over the course of fifteen minutes. The ratio of tumor:muscle T1‐weighted signal increased only 59% as much as Gd‐DO3A‐404, ranging from 1.15 pre‐contrast to a maximum of 1.67 five minutes post‐contrast. Significant signal enhancement from Dotarem was not sustained beyond one hour post‐contrast.Conclusion:These results indicate that with favorable relaxation characteristics and sustained signal‐enhancing uptake in multiple tumor models, Gd‐DO3A‐404 has great potential as a tumor‐targeting MR contrast agent. As part of a library of APC analogs labeled with PET/optical tracers and therapeutic radionuclides, Gd‐DO3A‐404 further expands theranostic capabilities. Future work will investigate applications in orthotopic glioma imaging, simultaneous PET/MR, and neutron capture therapy.

DDEL-05DIFFERENTIAL BLOOD-BRAIN BARRIER (BBB) PERMEABILITY OF ALKYLPHOSPHOCHOLINE (APC) ANALOGS ANALYZED USING AN IN VITRO PLURIPOTENT STEM CELL-DERIVED BRAIN MICROVASCULAR ENDOTHELIAL CELL SYSTEM

BACKGROUND: Agents with cancer specificity against the highly aggressive glioblastoma (GBM) have potential diagnostic, intraoperative visualization, and therapeutic applications. Recently reported alkylphosphocholine (APC) analogs demonstrated tumor selectivity in 55 rodent and human cancer and cancer stem cell models, including GBM. Although these APC analogs strongly label GBM, their permeability across an intact blood-brain barrier (BBB) vasculature remains unknown. METHODS: APC analogs -124I-CLR1404 (PET radiotracer), CLR1501 (green fluorescence), CLR1502 (near infrared fluorescence) - were obtained from Cellectar Biosciences, and tested for permeability across an in vitro BBB composed of human induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (BMECs). This in vitro BBB Transwell system consistently exhibits satisfactory barrier tightness as marked by a high transendothelial electrical resistance (TEER > 1000 Ω-cm2) and functional drug efflux transporters. We investigated the permeability of these different APC analogs by measuring changes in cell diffusion rates using the clearance method and assessed the contribution of drug efflux transporters using pharmacological inhibitors. RESULTS: APC analogs demonstrated limited diffusion across the BBB with permeability values (Pe, x10−5 cm/min) of 40.8 ± 15.1 (124I-CLR1404), 34.4 ± 14.0 (CLR1501), and 35.4 ± 17.5 (CLR1502); compared to impermeable sucrose (12.95 ± 2.49) and highly permeable diazepam (174.0 ± 28.57). Only the fluorescent analogs CLR1501 and CLR1502, and not CLR1404, were substrates for drug efflux transporters, and MRP or BCRP inhibition (using MK571 and Ko143, respectively) increased permeability of these compounds 3-fold. Interestingly, inhibition of P-gp did not affect permeability of any compounds. CONCLUSIONS: Limited BBB diffusion of APC analogs helps explain the excellent GBM to normal brain ratios achievable in initial human trials. Addition of fluorescent moieties to APCs resulted in greater BMEC efflux via MRP and BCRP, and transporter inhibition may help optimize APC analog delivery to GBM during fluorescence-guided neurosurgical resection. Overall, characterizing BBB permeability of APC analogs will help guide future applications of these agents against brain tumors.

Fluorescent cancer-selective alkylphosphocholine analogs for intraoperative glioma detection.

5-Aminolevulinic acid (5-ALA)-induced tumor fluorescence aids brain tumor resections but is not approved for routine use in the United States. We developed and describe testing of 2 novel fluorescent, cancer-selective alkylphosphocholine analogs, CLR1501 (green) and CLR1502 (near infrared), in a proof-of-principle study for fluorescence-guided glioma surgery. To demonstrate that CLR1501 and CLR1502 are cancer cell-selective fluorescence agents in glioblastoma models and to compare tumor-to-normal brain (T:N) fluorescence ratios with 5-ALA. CLR1501, CLR1502, and 5-ALA were administered to mice with magnetic resonance imaging-verified orthotopic U251 glioblastoma multiforme- and glioblastoma stem cell-derived xenografts. Harvested brains were imaged with confocal microscopy (CLR1501), the IVIS Spectrum imaging system (CLR1501, CLR1502, and 5-ALA), or the Fluobeam near-infrared fluorescence imaging system (CLR1502). Imaging and quantitative analysis of T:N fluorescence ratios were performed. Excitation/emission peaks are 500/517 nm for CLR1501 and 760/778 nm for CLR1502. The observed T:N ratio for CLR1502 (9.28±1.08) was significantly higher (P<.01) than for CLR1501 (3.51±0.44 on confocal imaging; 7.23±1.63 on IVIS imaging) and 5-ALA (4.81±0.92). Near-infrared Fluobeam CLR1502 imaging in a mouse xenograft model demonstrated high- contrast tumor visualization compatible with surgical applications. CLR1501 (green) and CLR1502 (near infrared) are novel tumor-selective fluorescent agents for discriminating tumor from normal brain. CLR1501 exhibits a tumor-to-brain fluorescence ratio similar to that of 5-ALA, whereas CLR1502 has a superior tumor-to-brain fluorescence ratio. This study demonstrates the potential use of CLR1501 and CLR1502 in fluorescence-guided tumor surgery.

NOVEL ALKYLPHOSPHOCHOLINE ANALOGS FOR BROAD SPECTRUM CANCER IMAGING AND THERAPY

BACKGROUND: We present in vitro and in vivo imaging and therapeutic studies using a novel alkylphosphocholine (APC)-based molecular scaffold (CLR1404) that combines selective targeting of cancer (including cancer stem cells) with broad-spectrum activity against many different cancer types. CLR1404 APC analogs were created to exploit the finding that phospholipid ethers selectively accumulate in many cancers versus normal cells. METHODS: Depending on the iodine isotope used, radioiodinated CLR1404 is either a PET imaging (124I) or molecular radiotherapeutic agent (131I), with fluorescence analogs created by replacing iodide with various fluorophores. Standard tissue culture, xenograft and molecular biology protocols were used for these studies. RESULTS: CLR1404 displayed preferential uptake and retention in cancer and cancer stem cells compared to normal cells in vitro and in vivo. After 24 hours, selective uptake of fluorescent or radioiodinated CLR1404 analogs by multiple human cancer cell lines in vitro compared to patient-matched normal human fibroblasts was observed (2.3-2.8x). CLR1404 showed highly specific labeling of both human patient-matched glioblastoma stem cells and serum-cultured glioblastoma cells (3x over minimal labeling of control normal human astrocytes and neural stem cells). Lipid raft disruption reduced in vitro CLR1404 analog uptake.In vivo, CLR1404 analogs displayed prolonged tumor-selective retention in 55 different rodent and human cancer models (including triple-negative breast, lung, pancreatic, colorectal, prostate, renal, melanoma, GBM). Tumor accumulation was seen by 24 hours with continued normal tissue clearance between 48-120 hours. In vivo cancer stem cell labeling was also demonstrated. CLR1404 analogs do not visualize inflammatory or premalignant lesions (unlike currently used 18F-fluorodeoxy glucose (FDG)-PET tumor imaging).131I-CLR1404 also displayed therapeutic efficacy (tumor growth suppression, survival extension) especially in renal, colorectal, ovarian, prostate and triple-negative breast cancer, GBM models. CONCLUSIONS: The novel APC CLR1404 analogs potentially offer a powerful multi- modality strategy to detect, treat and follow primary and metastatic cancers in humans. Initial clinical trials strongly suggest CLR1404 has similar broad tumor targeting and retention properties in animals and humans. The pathological correlation between CLR1404 imaging and GBM is being tested in an ongoing multi-institutional Phase II clinical trial. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

Alkylphosphocholine analogs for broad-spectrum cancer imaging and therapy.

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 ((124)I) or molecular radiotherapeutic ((131)I) agent. CLR1404 analogs displayed prolonged tumor-selective retention in 55 in vivo rodent and human cancer and cancer stem cell models. (131)I-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 (124)I-CLR1404 or (131)I-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 (124)I-CLR1404 tumor imaging for planning (131)I-CLR1404 therapy.

Cationic Type I Amphiphiles As Modulators of Membrane Curvature Elastic Stress in Vivo

Recently we proposed that the antineoplastic properties observed in vivo for alkyl-lysophospholipid and alkylphosphocholine analogues are a direct consequence of the reduction of membrane stored elastic stress induced by these amphiphiles. Here we report similar behavior for a wide range of cationic surfactant analogues. Our systematic structure-activity studies show that the cytotoxic properties of cationic surfactants follow the same pattern of activity we observed previously for alkyl-lysophospholipid analogues, indicating a common mechanism of action that is consistent with the theory that these amphiphiles reduce membrane stored elastic stress. We note that several of the cationic surfactant compounds we have evaluated are also potent antibacterial and antifungal agents. The similarity of structure-activity relationships for cationic surfactants against microorganisms and those we have observed in eukaryotic cell lines leads us to suggest the possibility that the antibacterial and antifungal properties of cationic surfactants may also be due to modulation of membrane stored elastic stress.

Alkylphosphocholine Analogues induce Apoptosis in Tumour Cells of the Immune System

Alkylphosphocholine Analogues induce Apoptosis in Tumour Cells of the Immune System

Comparison of selective cytotoxicity of alkyl lysophospholipids

Alkyl lysophospholipids have been shown to be cytooxic to a number of neoplastic tissues. One, ET-18-OCH3, has been used to selectively purge leukemic cells from mixtures with normal marrow progenitor cells, in vitro and in vivo. We have measured the 50% inhibitory (IC50) effect of a series of ether lipids (EL) on leukemic cells (HL60, K562, Daudi, KG-1, KG-1a) and normal marrow progenitor cells. Cells were incubated with varying concentrations of EL for 4 hr and assayed for viability, [3H]thymidine incorporation and clonogenicity in semi-solid media. The effect on protein kinase C (PKC) activity was assayed for each compound. Compounds tested included three glycerophosphocholine analogs--ET-18-OCH3, ET-16-NHCOCH3, and BM 41.440. In addition, a lipoidal amine, CP 46665, an ethyleneglycolphospholipid, AEPL, and four single chain alkylphosphocholine analogs, HePC2, HePC3, HePC4 and HePC6 were also tested. During the period of incubation, the cells remained viable (greater than 70%) as judged by trypan blue dye exclusion. The glycerophosphocholines were the most active and showed the highest therapeutic index. The lipoidal amine was active, but toxic to normal marrow progenitor cells. The ethyleneglycolphospholipid was active against HL60, but not against the other cell lines. The single chain alkylphosphocholine analogs were less active. All of the compounds inhibited PKC activity; however, the glycerophosphocholines were the most inhibitory.