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

Abstract

Cancer-targeting alkylphosphocholine (APC) analogs are being clinically developed for diagnostic imaging, intraoperative visualization, and therapeutic applications. These APC analogs 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 analogs, 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-cm2) and functional expression of drug efflux transporters. Our radioiodinated and fluorescent APC analogs 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 our fluorescent APC analogs (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 our tested APC analogs appeared to undergo substantial P-gp transport. Limited permeability of our APC analogs 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 analog permeability across human BBB is significant for advancing future brain tumor-targeted applications of these agents.

Paving the Way Toward Complex Blood-Brain Barrier Models Using Pluripotent Stem Cells

“…The functional tightness can be validated by the flux of molecules with different molecular weights such as dextran, lucifer yellow, sodium fluorescein, glucose, insulin, or mannitol. Another useful control is sucrose because it is BBB impermeable, hydrophilic and there is no brain-specific uptake mechanism [41]. To test for the expression of specific receptors or transporter proteins, these can also be selectively inhibited and the flux can be measured [42,43].…”

New experimental models of the blood-brain barrier for CNS drug discovery

“…Importantly, the TEER responses in stem cell based models are substantially higher as compared to primary human BMEC or hCMEC/D3 cultures [96]. Given a large dynamic range of permeability values (diazepam to sucrose), hPSC derived BBB models could have potential applications as reliable drug screening tools [123, 125], although more functional assays with a diverse array of compounds are required to test the predictive strength of the model [122]. Importantly, stem cells can be integrated with microfluidic approaches towards development of robust BBB-on-chip platforms for high throughput studies [123].…”

Commentary on human pluripotent stem cell-based blood–brain barrier models

“…We defined this cell type as hPSC-derived BMECs. The resultant cells have proven useful for examining interactions between cells of the neurovascular unit [1][2][3][4][5][6][7][8][9][10], evaluating experimental drug permeability at the BBB [1,[11][12][13][14][15][16], and modeling human genetic disease using hiPSCs derived from patient sources [9,10,13], among other applications.…”