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 u003e 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.