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 Omega-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) X 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.