An analysis of the effects of CARP-1/CCAR1 functional mimetics on drug efflux transporters

Abstract

Background: The most aggressive malignant phenotypes of neuroblastoma result in poor prognoses. Treatment of central nervous system (CNS) neuroblastoma metastases represents an unmet need, largely attributable to poor penetration of many chemotherapeutics across the blood-brain barrier (BBB). BBB endothelial cells express high levels of ATP-binding cassette (ABC) efflux transporters for which, importantly, several chemotherapeutic agents used to treat neuroblastoma are substrates. Therefore, novel compounds which inhibit or downregulate ABC transporter activity may increase their own CNS bioavailability and that of concomitant chemotherapies. The cytotoxic effects of CARP-1/CCAR1 functional mimetics (CFMs) are largely mediated by upregulating CARP-1, whose downstream effectors include NF-kappaB, p53 and glucocorticoid receptors, all of which can modulate ABC transporter expression and activity. Aims and Objectives: Initially, the effects of CFM-4.16 and CFM-4.17 on the viability of SH-SY5Y neuroblastoma cells and porcine brain endothelial cells (PBECs) were determined. Since reduced activity of BBB-associated efflux transporters may (i) increase intracellular drug accumulation in malignant cells and (ii) increase CNS delivery, subsequent studies investigated the effect of CFM-4.16 and CFM-4.17 on the activities of ABCB1 and ABCC in SH-SY5Y cells and ABCB1 and ABCG2 in PBECs. The ability of CFM-4.16 and CFM-4.17 to penetrate an in vitro model of the BBB was also explored. Methods: Effects of CFM compounds on cell viability were quantified using the MTT assay in SH-SY5Y cells and the neutral red assay in PBECs. Activities of ABCB1, ABCG2 and ABCC transporters were determined by measuring intracellular accumulation of the respective fluorescent probes calcein, Hoescht 33342 or GS-MF. Penetration of CFM-4.16 and CFM-4.17 was assessed using an in vitro BBB model comprising PBECs co-cultured with the CTX-TNA2 rat astrocyte cell line in the Transwell apparatus. Initial studies investigated the ability of CFM-4.16 and CFM-4.17 to penetrate PBEC monolayers in Transwell inserts by measuring the cytotoxicity towards SH-SY5Y cells of media from apical and basolateral chambers. Results: Short-term (30 min) incubation with CFM-4.17 but not CFM-4.16 inhibited ABCB1 transporter activity in SH-SY5Y cells. Short-term incubation with CFM-4.16 or CFM-4.17 lacked inhibitory effects on ABCC transporter activity in SH-SY5Y cells or on ABCB1 or ABCG2 transporter activity in PBECs. Long-term (24 h or 48 h) incubation with CFM-4.16 or CFM-4.17 did not modulate either ABCB1 transporter activity in SH-SY5Y cells or ABCB1 and ABCG2 activities in PBECs. Initial studies in PBEC monolayers housed in Transwell inserts suggested a lack of detectable penetration of CFM-4.17 and CFM-4.16 across the barrier. Penetration of CFM-4.16 across BBB endothelial cells was not significantly affected by inhibition of ABCB1 or ABCG2, suggesting that these efflux transporters do not influence this process. Conclusions: The greater potency of CFM-4.16 and CFM-4.17 in SH-SY5Y cells than in PBECs is consistent with a favourable therapeutic index. In addition, inhibition of ABCB1 transporters in SH-SY5Y cells by CFM-4.17 may circumvent ABCB1-mediated drug resistance to CFM-4.17 or other ABCB1 substrates. Lack of effects of CFM-4.16 or CFM-4.17 on ABCB1 or ABCG2 transporters in PBECs suggests that they are unlikely to modulate their own transport or that of concomitant therapies across the BBB.

Date of Award	31 Dec 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jeffrey Penny (Supervisor) & Costas Demonacos (Supervisor)