An Analysis of the Effects of CARP-1/CCAR1 Functional Mimetics on Drug Efflux Transporters

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

A thesis submitted to The University of Manchester

for the degree of Master of Philosophy in the

Faculty of Biology, Medicine and Health

2019

Shelley R Morris

School of Health Sciences

Division of Pharmacy and Optometry

List of Contents

List of Contents ...... 2 List of Tables ...... 7 List of Figures ...... 8 List of Abbreviations ...... 10 Abstract ...... 13 Declaration ...... 14 Copyright Statement ...... 15 Acknowledgements ...... 16 The Author ...... 17 1 Introduction ...... 18 1.1 Definition and hallmarks of cancer ...... 18 1.2 Neuroblastoma and current treatments ...... 19 1.3 CNS metastases – the requirement of novel treatments for neuroblastoma to cross the blood-brain barrier ...... 23 1.4 Characteristics of the blood-brain barrier ...... 23 1.5 In vitro models of the BBB ...... 27 1.6 SH-SY5Y cell line as an in vitro model of neuroblastoma...... 29 1.7 Multidrug resistance and prognostic implications for ABCB1, ABCG2 and ABCC in neuroblastoma ...... 30 1.7.1 ABCB1 ...... 30 1.7.2 ABCG2 ...... 31 1.7.3 ABCC ...... 32 1.8 Limitations of current approaches to circumvent chemoresistance and increase BBB penetration ...... 33 1.8.1 ABCB1 Inhibitors ...... 33 1.8.2 ABCG2 Inhibitors ...... 35 1.8.3 ABCC Inhibitors ...... 36 1.8.4 Chemotherapeutics with dual ABC transporter inhibitory properties ...... 37 1.8.5 Modulators of ABC transporter expression ...... 38 1.9 Novel approach to the cytotoxic treatment of neuroblastoma-derived CNS metastases and modulation of ABC transporters based on CARP-1/CCAR1 ...... 38 1.9.1 Validation of CARP-1 as a drug target ...... 38 1.9.2 Discovery of CARP-1 functional mimetics (CFMs) based on disruption of the CARP-1/Anaphase-promoting complex (APC)-2 interaction ...... 39 1.9.3 CFM-mediated upregulation of CARP-1 signalling in a range of cancer cell types 40 2

1.9.4 CFM-mediated cytotoxicity ...... 41 1.9.5 Additional mechanistic evidence supporting the proposed use of CFM-4.16 and CFM-4.17 in neuroblastoma ...... 41 1.9.6 Effects of CFMs on signalling molecules implicated in the regulation of ABCB1 and ABCG2 activity and expression ...... 42 1.10 Aims and objectives ...... 43 1.10.1 Aims ...... 43 1.10.2 Objectives ...... 45 2 Methods ...... 46 2.1 Reagents and materials ...... 46 2.2 Isolation of porcine brain endothelial cells ...... 47 2.3 Coating of PBEC culture surfaces ...... 48 2.4 Culture of PBECs ...... 48 2.5 Subculture of PBECs from 6-well plates onto 96-well plates ...... 48 2.6 Subculture of PBECs from 6-well plates onto Transwell® inserts ...... 49 2.7 Trypan blue assay ...... 49 2.8 Preparation of CFM-4.16 and CFM-4.17 ...... 49 2.9 Culture of CTX-TNA2 astrocytes and production of astrocyte-conditioned medium . 50 2.10 Cryopreservation of CTX-TNA2 astrocytes ...... 50 2.11 Subculture of CTX-TNA2 astrocytes onto 12-well plates ...... 50 2.12 Culture, cryopreservation and subculture of SH-SY5Y neuroblastoma cells ...... 51 2.13 Measurement of the effect of CFM-4.16 and CFM-4.17 on SH-SY5Y viability using the MTT assay...... 51 2.14 Measurement of the effect of CFM-4.16 and CFM-4.17 on PBEC viability using the neutral red assay ...... 52 2.15 Measurement of the effect of ABCB1 and ABCG2 inhibitors on CFM-4.16 and CFM- 4.17–mediated toxicity in PBECs...... 53 2.16 Measurement of ABC transporter functional activity in SH-SY5Y cells and in porcine brain endothelial cells ...... 53 2.17 Measurement of ABCB1 transporter functional activity ...... 53 2.18 Measurement of ABCG2 transporter functional activity ...... 55 2.19 Measurement of ABCC transporter functional activity ...... 56 2.20 Measurement of cellular protein content ...... 57 2.21 Generating the in vitro blood-brain barrier model ...... 57 2.22 Measurement of transendothelial electrical resistance ...... 58 2.23 Measurement of the apparent permeability of PBEC monolayers to Lucifer yellow . 58 2.24 Determination of absorption characteristics of CFM-4.16 and CFM-4.17 ...... 59 2.25 Measurement of the penetration of CFM-4.16 and CFM-4.17 across an in vitro model of the blood-brain barrier ...... 60 3

2.26 Indirect measurement of the permeability of CFM-4.16 and CFM-4.17 across an in vitro model of the blood-brain barrier...... 60 2.27 Statistical analysis ...... 61 3 Results 1 (Neuroblastoma cell viability and functional assays) ...... 62 3.1 Visualisation of the morphology of SH-SY5Y cells ...... 62 3.2 Evaluation of the effect of CFM-4.16 and CFM-4.17 on the viability of SH-SY5Y cells 62 3.3 Determination of ABCB1 functional activity in SH-SY5Y cells ...... 65 3.4 Determination of the specificity of Hoechst 33342 as a transporter substrate in SH- SY5Y cells ...... 65 3.5 Determination of ABCG2 functional activity in SH-SY5Y cells ...... 66 3.6 Determination of ABCC functional activity in SH-SY5Y cells ...... 67 3.7 Determination of the effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCB1 activity in SH-SY5Y cells ...... 68 3.8 Determination of the effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCC activity in SH-SY5Y cells ...... 69 3.9 Determination of the effect of CFM-4.16 and CFM-4.17 (24 h incubation) on ABCB1 activity in SH-SY5Y cells ...... 70 3.10 Determination of the effect of CFM-4.16 and CFM-4.17 (48 h incubation) on ABCB1 activity in SH-SY5Y cells ...... 72 4 Results 2 (Porcine brain endothelial cell viability and functional assays)...... 74 4.1 Visualisation of the morphology of porcine brain endothelial cells ...... 74 4.2 Evaluation of the effect of CFM-4.16 and CFM-4.17 on the viability of porcine brain endothelial cells ...... 74 4.3 Determination of the effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCB1 activity in PBECs ...... 76 4.4 Determination of the effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCG2 activity in PBECs ...... 77 4.5 Determination of the effect of CFM-4.16 and CFM-4.17 (24 h incubation) on ABCB1 activity in PBECs ...... 78 4.6 Determination of the effect of CFM-4.16 and CFM-4.17 (48 h incubation) on ABCB1 activity in PBECs ...... 81 4.7 Determination of the effect of CFM-4.16 and CFM-4.17 (24 h incubation) on ABCG2 activity in PBECs ...... 83 4.8 Determination of the effect of CFM-4.16 and CFM-4.17 (48 h incubation) on ABCG2 activity in PBECs ...... 84 4.9 Assessment of whether a washout period modifies the effects of CFM-4.16 or CFM- 4.17 on ABCB1 or ABCG2 functional activities in PBECs ...... 87 5 Results 3 (Transwell® studies) ...... 88 5.1 Measurement of transendothelial electrical resistance of PBEC monolayers ...... 88 5.2 Measurement of the apparent permeability of PBEC monolayers to Lucifer yellow . 89

5.3 Determination of the absorption characteristics of CFM-4.16 and CFM-4.17 ...... 89 5.4 Indirect measurement of the penetration of CFM-4.16 across an in vitro model of the blood-brain-barrier ...... 90 5.5 Indirect measurement of the penetration of CFM-4.17 across an in vitro model of the blood-brain-barrier ...... 93 5.6 Indirect measurement of the effect of ABCB1 inhibition on the penetration of CFM- 4.16 across an in vitro model of the blood-brain barrier ...... 95 5.7 Indirect measurement of the effect of ABCG2 inhibition on the penetration of CFM- 4.16 across an in vitro model of the blood-brain barrier ...... 97 5.8 Measurement of the effect of ABCB1 or ABCG2 inhibition on CFM-4.16 or CFM-4.17- mediated toxicity in PBECs ...... 99 6 Discussion ...... 101 6.1 Overview ...... 101 6.2 Effects of CFM-4.16 and CFM-4.17 on the viability of SH-SY5Y cells and PBECs ...... 101 6.3 Characterisation of SH-SY5Y cell morphology and ABC transporter functional activity 104 6.4 Determination of ABCB1 functional activity in SH-SY5Y cells ...... 105 6.5 Determination of ABCG2 functional activity in SH-SY5Y cells ...... 105 6.6 Determination of ABCC functional activity in SH-SY5Y cells ...... 106 6.7 Characterisation and validity of the PBEC model ...... 107 6.8 Determination of the effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCB1 and ABCC activities in SH-SY5Y cells ...... 108 6.8.1 Chemical perspective ...... 108 6.8.2 Clinical implications of ABCB1 inhibition ...... 109 6.8.3 Determination of the effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCC activity in SH-SY5Y cells ...... 112 6.9 Determination of the effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCB1 or ABCG2 activity in PBECs ...... 112 6.10 Determination of the effect of CFM-4.16 and CFM-4.17 (24 h or 48 h incubation) on ABCB1 activity in SH-SY5Y cells and on ABCB1 and ABCG2 activity in PBECs ...... 113 6.10.1 Potential mediation of chronic regulatory effects of CFMs on ABCB1 and ABCG2 activity by modulation of CARP-1 and the glucocorticoid receptor signalling pathway .... 114 6.10.2 Potential mediation of chronic regulatory effects of CFMs on ABCB1 and ABCG2 activity by modulation of CARP-1 and the NF-κB signalling pathway ...... 116 6.10.3 Potential mediation of chronic regulatory effects of CFMs on ABCB1 and ABCG2 activity by modulation of CARP-1 and the p53 signalling pathway...... 118 6.11 Characterisation of the integrity of the in vitro blood-brain barrier model ...... 121 6.12 Toxicity-based indirect measurement of CFM penetration of the in vitro blood-brain barrier model ...... 123 6.13 Assessing penetration of CFM-4.16 and CFM-4.17 across the in vitro blood-brain barrier model ...... 123 5

6.14 Evaluation of the substrate-like properties of CFM-4.16 and CFM-4.17 ...... 125 6.15 Clinical implications of limited penetration ...... 125 6.16 Limitations and clinical extrapolation of the Transwell® model ...... 127 6.17 Potential experimental refinements ...... 128 7 Conclusion ...... 129 7.1 Implications for the treatment of neuroblastoma CNS metastases ...... 129 7.2 Future work ...... 129 Appendix: Assessment of the effects of CFM-4.16 or CFM-4.17 on ABCB1 or ABCG2 functional activity in PBECs following a washout period ...... 132 8 References ...... 135

Word Count 34,372

List of Tables

Chapter 1

Table 1.1. International Neuroblastoma Staging System (INSS) 20 Table 1.2. Risk Stratification Scheme of the Children’s Oncology Group 20 based on the INSS Table 1.3. International Neuroblastoma Risk Group (INRG) Staging System 21 Table 1.4. International Neuroblastoma Risk Group (INRG) pre-treatment 21 classification

Chapter 5

Table 5.1. Apparent permeability of PBEC monolayers to Lucifer yellow 89

List of Figures

Chapter 1

Figure 1.1. The Hallmarks of Cancer 18 Figure 1.2. Cellular associations at the blood-brain barrier 25 Figure 1.3. Potential routes for penetration of blood-brain barrier 27 endothelial cells Figure 1.4. Schematic representation of a Transwell® model of the blood- 29 brain barrier Figure 1.5. Structure of CFM-4, CFM-4.16 and CFM-4.17 40 Figure 1.6. Predicted net effects of CFM-4.16 or CFM-4.17 on signalling 43 molecules which regulate the activity and expression of ABCB1 and ABCG2 transporters

Chapter 3

Figure 3.1. Morphology of SH-SY5Y cells 62 Figure 3.2. The effect of CFM-4.16 and CFM-4.17 on SH-SY5Y cell viability 64 Figure 3.3. Determination of ABCB1 functional activity in SH-SY5Y cells 65 Figure 3.4. Determination of the specificity of Hoechst 33342 as a 66 transporter substrate in SH-SY5Y cells Figure 3.5. Determination of ABCG2 functional activity in SH-SY5Y cells 67 Figure 3.6. Determination of ABCC functional activity in SH-SY5Y cells 68 Figure 3.7. Effect of short-term treatment with CFM-4.16 or CFM-4.17 69 on ABCB1 activity in SH-SY5Y cells Figure 3.8. Effect of short-term treatment with CFM-4.16 or CFM-4.17 70 on ABCC activity in SH-SY5Y cells Figure 3.9. Effect of long-term (24 h) treatment with CFM-4.16 or CFM- 71 4.17 on ABCB1 activity in SH-SY5Y cells Figure 3.10. Effect of long-term (48 h) treatment with CFM-4.16 or CFM- 73 4.17 on ABCB1 activity in SH-SY5Y cells

Chapter 4

Figure 4.1. Morphology of porcine brain endothelial cells 74

Figure 4.2. The effect of CFM-4.16 and CFM-4.17 on the viability of PBECs 76 Figure 4.3. Effect of short-term treatment with CFM-4.16 or CFM-4.17 77 on ABCB1 activity in PBECs Figure 4.4. Effect of short-term treatment with CFM-4.16 or CFM-4.17 78 on ABCG2 activity in PBECs Figure 4.5. Effect of long-term (24 h) treatment with CFM-4.16 or CFM- 80 4.17 on ABCB1 activity in PBECs Figure 4.6. Effect of long-term (48 h) treatment with CFM-4.16 or CFM- 82 4.17 on ABCB1 activity in PBECs Figure 4.7. Effect of long-term (24 h) treatment with CFM-4.16 or CFM- 84 4.17 on ABCG2 activity in PBECs Figure 4.8. Effect of long-term (48 h) treatment with CFM-4.16 or CFM- 86 4.17 on ABCG2 activity in PBECs

Chapter 5

Figure 5.1. Transendothelial Electrical Resistance of PBEC monolayers 88 Figure 5.2. Absorption Spectra of CFM-4.16 and CFM-4.17 90 Figure 5.3. Indirect measurement of the penetration of CFM-4.16 across 92 an in vitro model of the blood-brain barrier Figure 5.4. Indirect measurement of the penetration of CFM-4.17 across 94 an in vitro model of the blood-brain barrier Figure 5.5. Indirect measurement of the effect of ABCB1 inhibition on 96 the penetration of CFM-4.16 across an in vitro model of the blood-brain barrier Figure 5.6. Indirect measurement of the effect of ABCG2 inhibition on 98 the penetration of CFM-4.16 across an in vitro model of the blood-brain barrier Figure 5.7. The effect of ABCB1 or ABCG2 inhibition on CFM-4.16 or CFM- 100 4.17-mediated toxicity in PBECs

Appendix

Figure A1. Effect of long-term treatment with CFM-4.16 or CFM-4.17 133 followed by a washout period on ABCB1 activity in PBECs Figure A2. Effect of long-term treatment with CFM-4.16 or CFM-4.17 134 followed by a washout period on ABCG2 activity in PBECs

List of Abbreviations

ABC ATP-binding cassette ABIN A20 binding inhibitor of nuclear factor κB ACM astrocyte-conditioned medium ANOVA analysis of variance APC/C anaphase promoting complex/cyclosome APC-2 anaphase-promoting complex 2 ATCC American Type Culture Collection ATP adenosine 5-triphosphate AUC area under the curve BBB blood-brain barrier BCR-ABL breakpoint cluster region-abelson BHK baby hamster kidney BSA bovine serum albumin Calcein-AM calcein acetoxymethyl ester cAMP cyclic adenosine monophosphate CARP-1 cell cycle and apoptosis regulatory protein-1 CCAR1 cell division cycle and apoptosis regulator protein 1 Cdc20 cell division cycle protein 20 Cdh1 cadherin 1 CFM CARP-1/CCAR1 functional mimetic cm centimetre Cmax maximum concentration CMFDA 5-Chloromethylfluorescein diacetate CNS central nervous system COX-2 cyclooxygenase 2 CYP cytochrome P450 Da Daltons DMEM Dulbecco’s modified eagle medium DMSO dimethylsulfoxide DNA deoxyribonucleic acid EDTA ethylenediaminetetraacetic acid EFS event-free survival EGFR epidermal growth factor receptor FBS foetal bovine serum FLT3 fms-like tyrosine kinase 3 FTC fumitremorgin C GI gastrointestinal GN ganglioneuroma GNB ganglioneuroblastoma GR glucocorticoid receptor GS-MF glutathione methylfluorescein h hour HBSS Hank’s balanced salt solution HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC high performance liquid chromatography IAP inhibitor of apoptosis protein 10

IC50 half maximal inhibitory concentration IKKγ inhibitor of nuclear factor κB kinase subunit γ INRG International Neuroblastoma Risk Group INSS International Neuroblastoma Staging System IκB inhibitor of nuclear factor κB JNK c-Jun N-terminal kinase L litre logD distribution coefficient logP partition coefficient logS aqueous solubility coefficient MAPK mitogen activated protein kinase mdr multidrug resistance protein mg milligram min minute miR513a-5p microRNA 513a-5p mL millilitre mM millimolar mol mole mRNA messenger ribonucleic acid MSDAC metastatic site-derived aggressive cell MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H- tetrazolium bromide n.s. non-significant NEMO NF-kappa B essential modulator NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells nm nanometre nM nanomolar NMDA N-methyl-D-aspartate NSCLC non-small cell lung cancer oC degree celsius Papp apparent permeability PARP1 poly(ADP-ribose) polymerase 1 PBEC porcine brain endothelial cell PBS phosphate buffered saline PDGFR platelet-derived growth factor receptor PDS plasma derived serum PPARγ peroxisome proliferator-activated receptor gamma PpIX protoporphyrin IX Rac1 ras-related C3 botulinum toxin substrate 1 RCC renal cell carcinoma RFU relative fluorescence units RT-PCR reverse transcriptase polymerase chain reaction s second siRNA small interfering ribonucleic acid TAZ transcriptional co-activator with PDZ-binding motif TEER transendothelial electrical resistance TKI tyrosine kinase inhibitor TNBC triple negative breast cancer 11

TNF tumour necrosis factor TUNEL terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labelling U international units UK United Kingdom v/v volume/volume VEGFR vascular endothelial growth factor receptor x g gravity ZO zonula occludens μg microgram μL microlitre μm micrometre μM micromolar Ω ohms

Abstract

The University of Manchester. Shelley Morris. Degree Title: Master of Philosophy, August 2019. MPhil Thesis Title: An analysis of the effects of CARP-1/CCAR1 functional mimetics on drug efflux transporters. 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-κB, 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. 13

Declaration

No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.

Shelley Morris

Copyright Statement i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trademarks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=24420), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.library.manchester.ac.uk/about/regulations/) and in The University’s policy on Presentation of Theses.

Acknowledgements

First and foremost I would like to thank my primary supervisor Dr Jeff Penny for challenging me to meet his exceptionally high standards and expectations and for nearly two years of encouragement and support. I also want to thank my second supervisor Dr Costas Demonacos for readily giving his advice and feedback whenever it was requested. I am particularly grateful for the training, technical advice and companionship from everyone that I worked alongside in the lab throughout the long weekdays and, as the project progressed, increasingly frequent evenings and weekends. Finally, I would like to thank my friends and family for putting up with my retreat into academia while I strived to attain a postgraduate research qualification.

The Author

Shelley Morris gained a 2.1 BSc(Hons) in pharmacology from the University of Leeds during which she attained several academic prizes, including the Pfizer prize for the highest mark in the final year research project. Additional research experience includes a funded vacation studentship at the same university and a summer project at the University of Manchester. Shelley also holds an MSc in pharmacology from the University of Oxford, completing a four-month research project in molecular neuropharmacology and attaining an overall degree grade of 69%. Further qualifications include a PgCert in cancer therapeutics from Queen Mary, University of London and a PgCert in neuroscience from King’s College London, both with distinction. Shelley has worked at a senior level within the pharmacovigilance and medical information sectors of the pharmaceutical industry and has gained recent experience as a medical writer across a range of therapy areas, including oncology.

1 Introduction

1.1 Definition and hallmarks of cancer The term ‘cancer’ refers to a group of diseases involving rapid uncontrolled cell division, which are characterised by several common hallmarks (Figure 1.1) (Hanahan and Weinberg, 2000, 2011). These hallmarks include evading apoptosis, limitless replicative potential, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion and metastasis, sustained angiogenesis, reprogramming of energy metabolism and evasion of immune destruction (Hanahan and Weinberg, 2000, 2011). Cancer represents a major health burden, with an estimated 3.91 million new cases (excluding non-melanoma skin cancer) and 1.93 million associated deaths in Europe projected to occur in 2018 alone (Ferlay et al., 2018). Within the UK, approximately 363,000 new cancer cases were reported annually between 2014 and 2016 with 166,135 cancer- related deaths occurring in 2016 (CRUK, 2019).

Figure 1.1. The Hallmarks of Cancer.

(Hanahan and Weinberg, 2011)

1.2 Neuroblastoma and current treatments Neuroblastoma is a form of cancer arising from neural crest cells in the developing sympathetic nervous system (Matthay et al., 2016; Tolbert and Matthay, 2018). Primary tumours typically originate in the adrenal medulla or sympathetic ganglia, presenting in the neck, chest, abdomen or pelvis (Louis and Shohet, 2015; Maris, 2010). Common metastatic sites include the cortical bone, bone marrow, liver and lymph nodes (Newman et al., 2019; Whittle et al., 2017). The number and location of the primary tumours and metastases determine the symptoms experienced (Tolbert and Matthay, 2018; Whittle et al., 2017). Whilst some tumours may present asymptomatically, localised symptoms may arise from compression of the airway, blood vessels, spinal cord or other proximal organs (Newman et al., 2019). In addition, systemic symptoms such as weight loss, fever and fatigue may indicate more advanced disease (Newman et al., 2019).

Consistent with pathogenesis in developing tissues, neuroblastoma is typically a paediatric malignancy with 90% of patients presenting under the age of 10 years and a median age at diagnosis of 18 months (Matthay et al., 2016). Neuroblastoma can develop in adolescents and young adults however this accounts for less than 5% of cases and tumours tend to be more indolent and chemoresistant than in younger children (Matthay et al., 2016). The estimated incidence of neuroblastoma within North America and Europe is 10.5 cases per million children under 15 (Whittle et al., 2017). While accounting for 10% of paediatric malignancies, neuroblastoma disproportionately results in up to 15% of paediatric deaths from cancer (Whittle et al., 2017).

Neuroblastoma is characterised by marked heterogeneity in its prognosis and response to therapy, ranging from tumours which spontaneously regress or differentiate without intervention, to very aggressive malignant phenotypes unresponsive to intensive multimodal treatment (Johnsen et al., 2018; Louis and Shohet, 2015). Likewise, survival rates vary widely according to tumour classification (Johnsen et al., 2018).

The two primary methods of categorising neuroblastoma are the long-established International Neuroblastoma Staging System (INSS) and its associated risk stratification system (Tables 1.1, 1.2) and the more recent International Neuroblastoma Risk Group (INRG) staging system and associated classification system (Tables 1.3, 1.4) which have

19 largely superseded INSS approaches (Brodeur et al., 1993; Cohn et al., 2009; Ganeshan and Schor, 2011).

Table 1.1. International Neuroblastoma Staging System (INSS)

Stage Definition 1 Localised tumour with complete gross excision, with or without microscopic residual disease; representative ipsilateral lymph nodes negative for tumour microscopically (nodes attached to and removed with primary tumour may be positive) 2A Localised tumour with incomplete gross excision, with ipsilateral nonadherent lymph nodes positive for tumour. Enlarged contralateral lymph nodes must be negative microscopically 2B Localised tumour with or without complete gross excision with ipsilateral nonadherent lymph nodes positive for tumour. Enlarged contralateral lymph nodes must be negative microscopically 3 Unresectable unilateral tumour infiltrating across the midline, with or without regional lymph node involvement; or localised unilateral tumour with contralateral regional lymph node involvement; or midline tumour with bilateral extension by infiltration (unresectable) or by lymph node involvement 4 Any primary tumour with dissemination to distant lymph nodes, bone marrow, bone, liver, skin and/or other organs (except as defined for stage 4S). 4S Localised primary tumour (as defined for stage 1 and 2A or 2B) with dissemination limited to skin, liver and/or bone marrow (bone marrow involvement <10%), limited to infants <1 year of age. Adapted from (Davidoff, 2012) and (Brodeur et al., 1993)

Table 1.2. Risk Stratification Scheme of the Children’s Oncology Group based on the INSS

Risk group INSS Age at MYCN Ploidy Histology Survival Rate stage diagnosis amplification (%) status Low 1 Any Any Any Any >98% 2A/2B Any Not amplified Any Any 4S <12 Not amplified DNA index Favourable >1 Intermediate 3 <18 Not amplified Any Any 90-95% ≥18 Not amplified Any Favourable 4 <12 Not amplified Any Any 12-18 Not amplified DNA index Favourable >1 4S <12 Not amplified DNA index = Any 1 <12 Not amplified Any Unfavourable High 2A/2B Any Amplified Any Any 40-50% 3 Any Amplified Any Any ≥18 Not amplified Any Unfavourable 4 <12 Amplified Any Any 12-18 Amplified Any Any 12-18 Any DNA index = Any 1 12-18 Any Any Unfavourable ≥18 Any Any Any Tumour 4S <12 Amplified Any Any >90% Stage 4S Adapted from (Ganeshan and Schor, 2011) and (Maris, 2010)

Table 1.3. International Neuroblastoma Risk Group (INRG) Staging System

Stage Definition L1 Localised tumour not involving vital structures as defined by the list of image-defined risk factors and limited to one body compartment L2 Locoregional tumour with one or more image-defined risk factors present M Metastatic disease with distant metastases (except stage MS) MS Metastatic disease in children aged <18 months with metastases confined to skin, liver, and/or bone marrow Adapted from (Louis and Shohet, 2015) and (Cohn et al., 2009)

Table 1.4. International Neuroblastoma Risk Group (INRG) pre-treatment classification

Risk Group INRG Age Histological Grade of Tumour MYCN 11q Ploidy 5-year Stage (months) Category Differentiation aberration EFS Very low L1/L2 Any GN maturing or Any Any Any Any >85% GNB intermixed

L1 Any Any except GN Any NA Any Any maturing or GNB intermixed

MS <18 Any Any NA No Any Low L2 <18 Any except GN Any NA No Any >75% maturing or to GNB intermixed ≤85%

L2 ≥18 GNB nodular, Differentiating NA No Any neuroblastoma M <18 Any Any NA Any Hyper- diploid Intermediate L2 <18 Any except GN Any NA Yes Any ≥50% maturing or to GNB intermixed ≤75%

L2 ≥18 GNB nodular, Differentiating NA Yes Any neuroblastoma

L2 ≥18 GNB nodular, Poorly NA Any Any neuroblastoma differentiated or undifferentiated M <12 Any Any NA Any Diploid M 12-<18 Any Any NA Any Diploid High L1 Any Any except GN Any Amp Any Any <50% maturing or GNB intermixed

L2 ≥18 GNB nodular, Any Amp Any Any neuroblastoma M <18 Any Any Amp Any Any M ≥18 Any Any Any Any Any MS <18 Any Any NA Yes Any MS <18 Any Any Amp Any Any

Amp, amplified; diploid, DNA index ≤1.0; EFS, event-free survival; GN, ganglioneuroma; GNB, ganglioneuroblastoma; hyperdiploid, DNA index >1.0 and includes near-triploid and near-tetraploid tumours; NA, nonamplified. Adapted from (Cohn et al., 2009; Louis and Shohet, 2015)

In patients stratified in the very low or low risk groups either minimal or no treatment or surgery alone may be required to achieve excellent survival, while those in the intermediate group typically receive low intensity chemotherapy followed by surgical resection of the residual tumour (Johnsen et al., 2018; Matthay et al., 2016). The current standard of care for high risk neuroblastoma incorporates induction chemotherapy and surgery, consolidation therapy with intensive myeloablative chemotherapy, autologous stem cell rescue and radiotherapy and postconsolidation treatment to target minimal residual disease (Newman et al., 2019; Pinto et al., 2015). Induction chemotherapy regimens may typically include cisplatin, cyclophosphamide, doxorubicin, etoposide, topotecan and vincristine, while subsequent myeloablative chemotherapy regimens include combinations of agents such as cisplatin, etoposide, melphalan, busulfan, thiotepa, and cyclophosphamide (Pinto et al., 2015). Other chemotherapeutic agents employed to treat high risk neuroblastoma may include carboplatin, ifosfamide, vindesine, dacarbazine, temozolomide and irinotecan (Matthay et al., 2016; Morgenstern et al., 2013).

Postconsolidation therapy involves anti-ganglioside 2 monoclonal antibody and cytokine immunotherapy together with isotretinoin-mediated differentiating treatment (Matthay et al., 2016; Pinto et al., 2015). It is the high risk treatment group that is associated with the greatest clinical unmet need with overall survival rates remaining below 50% (Newman et al., 2019). Furthermore, for those with refractory or relapsed high risk neuroblastoma, there are at present no curative salvage treatments available (Newman and Nuchtern, 2016). Adverse events associated with the aggressive treatment strategy are also a concern and may include severe transient myelosuppression, renal toxicity, growth failure and poor weight gain (Matthay et al., 2016). Even when high risk patients are successfully treated with initial therapy, additional chronic complications may include hearing loss, cardiac dysfunction, infertility and secondary malignancies (Wagner and Danks, 2009). It is therefore evident that novel treatments with improved efficacy and a wider therapeutic index are urgently required (Applebaum et al., 2017).

1.3 CNS metastases – the requirement of novel treatments for neuroblastoma to cross the blood-brain barrier Although primary neuroblastoma tumours are typically located extracranially, the CNS represents a relatively rare but increasingly important site of metastasis (Kramer et al., 2001; Louis and Shohet, 2015; Matthay et al., 2003). Estimated frequencies of post- therapeutic CNS involvement, including either leptomeningeal or CNS parenchymal disease, range from 1% to 16% (Kramer et al., 2001). Possible risk factors for CNS metastasis include MYCN amplification, age between 2-3 years, disease in the head and neck area and peridiagnostic lumbar punctures although causality of the latter is disputed (Kramer et al., 2001; Matthay et al., 2003; Sidi-Fragandrea et al., 2010). However longer survival associated with more intensive treatment of high risk patients may be contributing to increased emergence of CNS relapse (Kramer et al., 2001; Matthay et al., 2016).

Critically, the CNS has been described as a potential inaccessible ‘sanctuary site’, protected from systemic therapy, given that it is sometimes the sole site of recurrence and that the blood-brain barrier (BBB) impedes permeation of many of the standard neuroblastoma treatments, including monoclonal antibodies which cannot penetrate the CNS at all (Kramer et al., 2001; Matthay et al., 2016; Morgenstern et al., 2013). Some therapeutic agents, including topotecan, thiotepa, carboplatin, vincristine, temozolomide and irinotecan, which cross the BBB, have been employed and additionally some intrathecal approaches have been attempted with isolated individual successes (Gains et al., 2012; Morgenstern et al., 2013; Yamada et al., 2013; Zhu et al., 2015). However, there is a lack of consistently effective treatments and CNS metastasis in neuroblastoma remains almost uniformly fatal with very short median survival (Kramer et al., 2001; Matthay et al., 2003; Sidi-Fragandrea et al., 2010).

Consequently, there is an unmet need for novel chemotherapeutic agents to treat neuroblastoma which can also cross the BBB.

1.4 Characteristics of the blood-brain barrier The BBB has been described as a ‘bottleneck’ in the development of centrally acting drugs, preventing penetration of 100% of large molecule compounds and over 98% of

23 small molecules (Pardridge, 2005). The BBB was first conceptualised by Ehrlich after demonstrating restricted molecular exchange between the blood and the brain whereby intravenous injection of dyes into rats stained peripheral organs but not the brain and cerebrospinal fluid (Ehrlich, 1885). Conversely, following administration of trypan blue into the cerebrospinal fluid, Goldmann found that staining was restricted to brain tissue (Goldmann, 1913). The primary function of the BBB is to preserve homeostasis, facilitate carrier uptake of nutrients and protect the brain from potentially harmful metabolites and xenobiotics (Wilhelm and Krizbai, 2014). The predominant brain capillary endothelial cell basis of the barrier is supported by electron microscopy revealing tight intercellular junctions which prevented the passage of protein (i.e. peroxidase) (Brightman and Reese, 1969; Reese and Karnovsky, 1967). Additionally the endothelium is characterised by a lack of fenestrations (Fenstermacher et al., 1988), relatively sparse pinocytic vesicular transport (Sedlakova et al., 1999) and high density of mitochondria (Oldendorf et al., 1977). Correspondingly high transendothelial electrical resistance (TEER) has been measured in vivo, in the range of 1000-2000 Ω.cm2, and consequently the BBB confers low permeability to polar and ionic substances (Butt et al., 1990).

However intact cerebrovascular endothelium is increasingly recognised to act as part of a neurovascular unit in concert with astrocytes, pericytes, neurons and the extracellular matrix of the basal lamina, all of which contribute to development and maintenance of the BBB phenotype (Figure 1.2) (Harder et al., 2002; Ramsauer et al., 2002).

Figure 1.2. Cellular associations at the blood-brain barrier.

Tight junctions are formed between cerebral endothelial cells which restrict aqueous paracellular diffusion. Pericytes are in discontinuous association with cerebral capillaries and partially surround the endothelium. The basement membrane forms an extracellular matrix associated with endothelial cells and pericytes (basal lamina 1, BL1) or with glial endfeet surrounding the brain parenchyma (BL2). Astrocytes induce and maintain barrier properties. Neuronal projections release vasoactive neurotransmitters and peptides, and microglia possess immune functions. Based on (Abbott et al., 2010).

The basal lamina provides an anchoring function and astrocytes may upregulate expression of BBB properties including tight junction proteins, ATP-binding cassette (ABC) transporters and increased enzymatic activity (Dehouck et al., 1990; Hayashi et al., 1997; Rubin et al., 1991; Yang and Rosenberg, 2011). Furthermore, pericytes may indirectly modulate the BBB by reducing permeability and inducing BBB differentiation (Berezowski et al., 2004; Hayashi et al., 2004; Hori et al., 2004).

Drug metabolising enzymes within BBB endothelial cells confer a metabolic barrier, with cytochrome P450 enzymes mediating Phase I functionalisation reactions and glutathione-s-transferases, sulphotransferases and methyltransferases potentially mediating Phase II conjugation reactions (Shawahna et al., 2011). A transport barrier is further comprised of ABC transporters which mediate efflux of metabolites and xenobiotics (Dauchy et al., 2008). In particular ABCB1, ABCG2 and ABCC are extensively studied efflux transporters in the apical membrane of brain endothelial cells. Expression of ABCB1 and ABCG2 transporters was detected in human cerebrovascular endothelium in 1989 and 2002 respectively (Cooray et al., 2002; Cordon-Cardo et al., 1989). ABCC efflux transporters are also important, although there are mixed reports about

25 expression of individual ABCC family members (Gutmann et al., 1999; Perriere et al., 2007; Regina et al., 1998; Seetharaman et al., 1998).

The net result of the physical barrier presented particularly by endothelial tight junctions is to restrict paracellular permeation of hydrophilic molecules (Wolburg et al., 1994). However, even lipophilic molecules capable of penetrating via passive transcellular diffusion across endothelial cell membranes may be intercepted and effluxed into the systemic circulation by ABC transporters for which they are substrates (Figure 1.3) (Levin, 1980; Mahar Doan et al., 2002).

Several of the current drug treatments for high risk neuroblastoma are substrates for ABC transporters. For example doxorubicin, etoposide, irinotecan, temozolomide, thiotepa, topotecan and vincristine are confirmed substrates and melphalan is a possible substrate of ABCB1 (de Vries et al., 2007; Goldwirt et al., 2014; Landini et al., 2017; Loschmann et al., 2016; Veringa et al., 2013). Furthermore doxorubicin, etoposide, irinotecan, temozolomide, and topotecan are substrates of ABCG2 (de Gooijer et al., 2018b; de Vries et al., 2007; Kopecka et al., 2015; Maliepaard et al., 2001; Yuan et al., 2009). Finally carboplatin, doxorubicin, etoposide, irinotecan, topotecan, and potentially dacarbazine are substrates of one or more ABCC transporters (Chartrain et al., 2012; Chen et al., 1999; Helms et al., 2014; Kopecka et al., 2015; Li et al., 2008). Therefore ABC transporter-mediated efflux may be a limiting factor in the penetration of several established neuroblastoma therapies across the BBB and influence that of any future treatments in the development pipeline.

Figure 1.3. Potential routes for penetration of blood-brain barrier endothelial cells.

Potential routes across the BBB endothelial cells vary according to the properties of the penetrating substance. Cells may cross endothelial cells either through or adjacent to tight junctions. Small lipid soluble molecules may permeate the membrane via passive diffusion, some of which may pumped out the endothelial cells by active efflux transporters. Essential polar molecules can undergo carrier mediated influx while macromolecules such as peptides and proteins are transported by receptor-mediated transcytosis. Positively charged macromolecules can induce adsorptive-mediated transcytosis. Additionally tight junction modulation can increase penetration across the paracellular aqueous diffusional route. Based on (Serlin et al., 2015).

1.5 In vitro models of the BBB Primary brain endothelial cell cultures provide the closest morphological and biochemical phenotypic resemblance in vitro to BBB cells in vivo (Smith et al., 2007). Such monolayers typically generate high TEER values and display restrictive paracellular permeability to solutes (Cantrill et al., 2012). Ideally human cells would be employed over cells from other sources to avoid any species differences, however most human samples necessarily originate from diseased tissue acquired from surgical biopsies or post-mortem, affected for example by epilepsy or multiple sclerosis (Bernas et al., 2010; Prat et al., 2000; Subileau et al., 2009). Immortalised human brain endothelial cell lines have been created, however their TEER values and paracellular restrictiveness are typically insufficient for permeability studies (Kannan et al., 2000; Stins et al., 2001; Weksler et al., 2005).

Rat primary cultures offer a readily available alternative source of brain endothelial cells (Bowman et al., 1981). Rat models also permit establishment of syngeneic co-cultures 27 with astrocytes of the same species, and may also enable in vitro/in vivo correlations given that rodents are usually used for preclinical pharmacokinetic studies (Perriere et al., 2007). Typical TEER values vary according to whether monoculture or astrocyte/pericyte-based co/triple culture are employed, however optimal conditions can produce TEER exceeding 600 Ω.cm2 (Abbott et al., 2012; Nakagawa et al., 2009).

While it is helpful that the rat genome and transcriptome are particularly well characterised, available comparative data highlight sequence differences in ABCB1 between human and rodent species which influence substrate specificity (Booth-Genthe et al., 2006). Sequence comparability of ABC transporters might be expected to be greater in more closely related sources such as bovine, and particularly porcine cells which in turn could theoretically affect affinity and transport rate. Relative expression of different transporter isoforms and hence their contribution to efflux may also show interspecies differences (Warren et al., 2009).

The primary limitation of use of rat brains is that the low yield per animal introduces ethical and financial constraints (Demeuse et al., 2002). In contrast, the larger brain size of bovine and porcine species permits isolation of high numbers of endothelial cells per brain (Thomsen et al., 2015). Early bovine endothelial cell models have been refined using approaches such as cAMP potentiation and co-culture with resultant increase in typical TEER values from initial values of ~600 Ω.cm2 to those frequently exceeding 1000 Ω.cm2 (Bowman et al., 1983; Cecchelli et al., 1999; Helms et al., 2014; Rubin et al., 1991). Functional expression of ABCB1, ABCG2 and ABCC isoforms has also been demonstrated at the level of both efflux activity and transporter protein in bovine experiments (Helms et al., 2014).

BBB models which employ primary porcine brain endothelial cells (PBECs) similarly form restrictive monolayers with high TEER which, in the host laboratory, range from almost 400 Ω.cm2 to over 2000 Ω.cm2 depending on the culture conditions (Cantrill et al., 2012; Skinner et al., 2009). PBECs display further phenotypic features which help to validate their resemblance to the in vitro BBB. For example, expression of the tight junction proteins ZO-1, claudin-1, claudin-5 and occludin is consistent with the ability to form a well-differentiated highly restrictive permeability barrier (Cantrill et al., 2012; Smith et al., 2007). PBECs also express functionally active marker enzymes including gamma- glutamyl transpeptidase and alkaline phosphatase, levels of which are enhanced in the 28 presence of astrocyte-conditioned medium (Cantrill et al., 2012). Quantitative proteomic analysis of PBECs demonstrates expression of transporters including ABCB1, ABCG2 and ABCC5 with an ABCG2:ABCB1 ratio more closely approximating that of human or monkey than rodent brain capillaries (Kubo et al., 2015). Expression and functional activity of the same transporters is also detectable in PBECs via Western blot and spectrofluorometric efflux assays (Shubbar and Penny, 2018). PBECs therefore enable evaluation of the effects of experimental compounds on transporter activity. However, when seeded onto a Transwell® insert (Figure 1.4) they permit quantification of compound penetration across the PBEC monolayer. PBECs may be maintained on the Transwell® insert in monoculture (Lelu et al., 2017; Thomsen et al., 2015). Alternatively contact or non-contact co-culture with astrocytes and/or pericytes can be employed to mimic elements of the neurovascular unit and minimise downregulation of BBB properties over time (Cantrill et al., 2012; Cohen-Kashi Malina et al., 2009; Skinner et al., 2009; Smith et al., 2007).

Figure 1.4. Schematic representation of a Transwell® model of the blood-brain barrier.

In this model of the BBB the Transwell® insert, seeded with porcine brain endothelial cells, rests in the well of a multiwell plate and is not in direct contact with an astrocyte cell line. In a contact co-culture model the astrocytes would be located directly on the underside of the insert. The upper apical chamber represents the blood side of the BBB while the lower basolateral chamber represents the brain compartment. Adapted from (Bicker et al., 2014).

1.6 SH-SY5Y cell line as an in vitro model of neuroblastoma SH-SY5Y represents a well-characterised cell line derived from a bone marrow biopsy in a patient with metastatic neuroblastoma (Biedler et al., 1973; Biedler et al., 1978). The tumour originated in the chest cavity and spread to the femur, bone marrow, liver and epidural space (Biedler et al., 1973). The original cell line, SK-N-SH, was tumorigenic when introduced into Syrian hamster cheek pouches and displayed rapid growth in vitro

29 and in vivo (Biedler et al., 1973). Morphological features included long cell processes, while high levels of dopamine-β-hydroxylase characteristic of sympathetic neurons were also consistent with cells of neuronal origin (Biedler et al., 1973). SK-N-SH was subcloned into the neuroblast-like clonal subline SH-SY, from which the SH-SY5 line and in turn SH- SY5Y were generated (Biedler et al., 1978). Capacity for transmitter synthesis evidencing neuronal properties, specifically generation of the noradrenaline precursor dopamine from tyrosine, has also been demonstrated in the SH-SY line (Biedler et al., 1978). SH- SY5Y consists of a homogenous neuroblast-like cell population (lacking the epithelial- like cells additionally present in SK-N-SH and SH-SY lines) with moderate dopamine-β- hydroxylase activity (Biedler et al., 1978). Proliferative capacity and maintenance of neuroblastic characteristics make the SH-SH5Y cell line an ideal in vitro model of neuroblastoma.

1.7 Multidrug resistance and prognostic implications for ABCB1, ABCG2 and ABCC in neuroblastoma ABC transporters including ABCB1, ABCG2 and ABCC, in addition to effluxing chemotherapeutic substrates in brain endothelial cells and thereby contributing to the BBB, can also confer multidrug resistance when overexpressed in cancer cells (Bates et al., 2004; Durmus et al., 2015; Gana et al., 2019; Haber et al., 1999; Helms et al., 2014; Li et al., 2018; Loschmann et al., 2016). In turn, reduced intracellular accumulation and efficacy of cytotoxic agents may follow, impairing clinical outcomes and prognosis (Chan et al., 1991; Chen et al., 2016b; Eadie et al., 2017; Goldstein et al., 1990; Porro et al., 2010). The prognostic significance of ABC transporter expression levels has been studied in neuroblastoma and is outlined below.

1.7.1 ABCB1 There is a lack of consensus regarding the prognostic implications of ABCB1 overexpression in neuroblastoma, however increased ABCB1 levels have been identified in metastatic and chemoresistant cell sublines (Blanc et al., 2003) and inversely correlated with sensitivity to chemotherapy in tumour samples (Bourhis et al., 1989; Chan et al., 1991). Evidence exists both for and against higher expression of ABCB1 in tumour from patients with treated, chemoresistant or advanced neuroblastoma

(Bourhis et al., 1989; Chan et al., 1991; Corrias et al., 1990; Dhooge et al., 1997; Goldstein et al., 1990; Nakagawara et al., 1990). Absence of ABCB1 expression in clinical tumour biopsies has also been linked to improved survival outcomes (Benard et al., 1994; Chan et al., 1991). However, others have found no such association with survival, disease progression or recurrence (Dhooge et al., 1997; Haber et al., 2006; Lu et al., 2004; Norris et al., 1996).

1.7.2 ABCG2 Expression of ABCG2 in neuroblastoma appears heterogenous and is potentially determined by the presence of stem cell subpopulations referred to as side populations (Hirschmann-Jax et al., 2004). For example, ABCG2 expression has been confirmed by moderate diffuse cytoplasmic immunostaining in a range of neuroblastoma cell lines, including SH-SY5Y, whilst others have failed to detect ABCG2 expression in SH-SY5Y using similar techniques (Acosta et al., 2009; Barron et al., 2013). In SH-SY5Y cells, the proportion of side population cells has been quantified as 0.4% which in turn expressed high levels of ABCG2 mRNA and protein (Xing et al., 2015).

The ABCG2-expressing side population, identified in one study within primary neuroblastoma cells from 15 of 23 patients, and also in five neuroblastoma cell lines (JF, SK-N-SH, IMR32, LAN-1 and LAN-5) may also determine responsiveness to treatment (Hirschmann-Jax et al., 2004). For example, ABCG2 expression was associated with enhanced efflux of cytotoxic drugs such as mitoxantrone, conferring a cellular survival advantage (Hirschmann-Jax et al., 2004). Given that neuroblastoma samples were obtained from patients in relapse, and in vitro exposure to mitoxantrone increased the proportion of side population cells, treatment may select for ABCG2 expressing resistant cells (Hirschmann-Jax et al., 2004). The side-population in LAN-5 cells is also relatively resistant to doxorubicin, consistent with ABCG2-mediated efflux, and was enriched following doxorubicin exposure (Mahller et al., 2009). However there was insufficient evidence in either study to conclude whether presence of such a population is a prognostic marker (Hirschmann-Jax et al., 2004; Mahller et al., 2009). Related to the concept of stem cell subpopulations, metastatic site-derived aggressive cell (MSDAC) clones have been generated from parental SH-SY5Y cells (Pandian et al., 2015). MSDACs exhibit stem cell like properties, uncontrolled growth and activated cell survival signalling along with increased expression of ABCG2 (Pandian et al., 2015). However the

31 role of ABCG2 in maintaining the aggressive phenotype was not determined. In a more direct clinical evaluation employing a large set of 251 neuroblastoma tumour samples, the oncogene MYCN directly regulated ABCG2 expression (Porro et al., 2010). However, low levels of ABCG2 expression correlated with reduced patient survival contrary to the expected overexpression that would be consistent with a multidrug resistance phenotype (Porro et al., 2010). Consequently, the prognostic significance of ABCG2 may be difficult to predict and dependent on the proportion of side population cells and on exposure to chemotherapeutic agents.

1.7.3 ABCC The main ABCC isoforms with prognostic significance in neuroblastoma are ABCC1, ABCC3 and ABCC4 (Porro et al., 2010). High tumour expression levels of ABCC1 and ABCC4 and, in contrast, low levels of ABCC3 are correlated with reduced survival (Henderson et al., 2011; Porro et al., 2010). Furthermore, expression of ABCC1 and ABCC4 positively correlates with expression of the oncogene MYCN in neuroblastoma specimens, while that of ABCC3 correlates negatively (Porro et al., 2010). Consistent with this, MYCN stimulates transcription of ABCC1 and ABCC4, while repressing ABCC3 in a TET21/N human neuroblastoma-derived cell line (Porro et al., 2010). Further evidence supports the relevance of these transporters in neuroblastoma cell phenotype and tumour progression (Henderson et al., 2011). For example, in the BE(2)-C human neuroblastoma cell line ABCC1 knockdown inhibits cell migration and proliferation, while ABCC4 knockdown enhances morphological differentiation and reduces proliferation (Henderson et al., 2011). Conversely, ABCC3 overexpression in the same cell line inhibits cell migration and proliferation (Henderson et al., 2011).

Nevertheless, ABCC1 has been implicated in multidrug resistance in neuroblastoma cell lines, in particular to the chemotherapeutic substrates etoposide, doxorubicin and vincristine (Haber et al., 1999). Low expression levels of ABCC3 do not necessarily fit with expectations of a transporter-mediated multidrug resistance (Yu et al., 2015). However relevant chemotherapy substrates to which ABCC3 confers varying degrees of resistance in non-neuroblastoma systems include etoposide and teniposide, while mediation of resistance to doxorubicin and vincristine in such systems is controversial (Kool et al., 1999; Zelcer et al., 2001; Zeng et al., 1999). There are limited examples of ABCC4 overexpression in HEK293 or Saos-2 cells, conferring resistance to substrates

32 employed in neuroblastoma chemotherapy regimens including irinotecan and topotecan respectively (Leggas et al., 2004; Norris et al., 2005). However, given that ABCC4 expression acts as a predictive marker of clinical outcome in patients with neuroblastoma treated with chemotherapeutic agents that are not substrates, this may also implicate a non-drug transport role (Henderson et al., 2011).

1.8 Limitations of current approaches to circumvent chemoresistance and increase BBB penetration Coadministration of a small molecule inhibitor of specific ABC transporters could theoretically overcome chemoresistance and increase penetration of chemotherapeutic compounds that are transporter substrates, across the BBB to reach CNS metastases. However, inhibitors in development are associated with toxicity, pharmacokinetic interactions, or lack of specificity, potency or efficacy. Alternatively, they require more detailed clinical evaluation. Many inhibitors of ABC transporters have yet to be investigated at the BBB, in neuroblastoma cells, or in patients with neuroblastoma necessitating use of data from non-neuroblastic cancers as an indicator of whether inhibitors may overcome an ABC transporter efflux barrier in an alternative cellular or clinical setting.

1.8.1 ABCB1 Inhibitors Most investigation has been carried out into the use of ABCB1 inhibitors. First generation compounds include repurposed drugs such as verapamil and cyclosporine A, both of which reverse multidrug resistance preclinically, restoring sensitivity to chemotherapeutic agents in neuroblastoma (Cowie et al., 1998; Tebbi et al., 1991). However, when verapamil was administered clinically, in patients with a range of non- neuroblastic cancers, cardiotoxicity was dose-limiting (Benson et al., 1985; Pennock et al., 1991). Furthermore, a lack of benefit in response rate or survival has been reported in refractory myeloma (Dalton et al., 1995). Cyclosporine A is additionally immunosuppressive and interacts with drugs such as etoposide and epidoxorubicin (Lum and Gosland, 1995; Verweij et al., 1991; Yahanda et al., 1992). Critically, cyclosporine A augmentation of chemotherapy is associated with only limited clinical efficacy in colorectal cancer and acute myeloid leukaemia (Daenen et al., 2004; List et

33 al., 2001; Verweij et al., 1991). Similarly disappointing results were reported in a pilot study involving coadministration of verapamil and cyclosporine A with chemotherapy in patients with neuroblastoma (Miniero et al., 1994). Second generation compounds include PSC-833, a non-immunosuppressive analogue of cyclosporine A (Twentyman and Bleehen, 1991). Whilst reversing preclinical multidrug resistance with approximately 10 times greater potency than cyclosporine A in a range of drug-resistant non-neuroblastoma cell lines (Boesch et al., 1991a; Boesch et al., 1991b; Twentyman and Bleehen, 1991) and effecting analogous reversal of multidrug resistance in neuroblastoma cells (Helson et al., 1994), PSC-833 has been linked to increased incidence of severe toxicities and cytochrome P450 pharmacokinetic interactions (Fischer et al., 1998b; Friedenberg et al., 2006; Kang et al., 2001). However clinical trials identified only limited therapeutic benefit of PSC-833 in acute myeloid leukaemia or ovarian or peritoneal cancers, whilst its efficacy in neuroblastoma has yet to be evaluated (Baer et al., 2002; Kolitz et al., 2004; Kolitz et al., 2010; Lhomme et al., 2008). VX-710 is a further second generation compound which increased uptake, retention and cytotoxicity of chemotherapeutic compounds in vitro in ABCB1 expressing neuroblastoma cell lines, however it only increased clinical efficacy in a limited subset of patients with soft tissue sarcoma with as yet unknown effects in neuroblastoma patients (Bramwell et al., 2002; Yanagisawa et al., 1999). Third generation compounds are typified by zosuquidar and tariquidar, both exhibiting potent nanomolar reversal of preclinical multidrug resistance and relative selectivity versus other ABC transporters and CYP3A4 enzymes however neither appears to have been evaluated in neuroblastoma (Dantzig et al., 1996; Dantzig et al., 1999; Mistry et al., 2001). While zosuquidar demonstrated acceptable clinical toxicity in patients with a range of non- neuroblastic malignancies it has not achieved clear improvements in clinical outcomes (Cripe et al., 2010; Fracasso et al., 2004). Furthermore, reports of toxicity with tariquidar are mixed, some Phase 1 trials identifying a lack of significant adverse events, while two Phase 3 trials were terminated prematurely due to toxicity in the tariquidar arm (Abraham et al., 2009; Fox and Bates, 2007; Kelly et al., 2011; Stewart et al., 2000). Again none of the above studies with zosuquidar or tariquidar involved patients with neuroblastoma.

In the absence of studies specifically investigating the effects of ABC transporter inhibitors at the BBB in the treatment of CNS neuroblastoma metastases, the potential for several of the above ABCB1 inhibitors to increase BBB penetration of chemotherapeutics in other contexts is described. In one study neither verapamil nor cyclosporine A enhanced distribution of vinblastine across the BBB in mice, confirmed in a second study for verapamil (Arboix et al., 1997; Cisternino et al., 2001). PSC-833 has been associated with increased penetration of vinblastine but not for example of vincristine or doxorubicin (Cisternino et al., 2001). More positively, cyclosporine A and PSC-833 increase BBB penetration of paclitaxel and docetaxel in mice (Kemper et al., 2003; Kemper et al., 2004b). Furthermore PSC-833 and zosuquidar can increase the distribution of imatinib across the BBB in mice with zosuquidar additionally increasing penetration of paclitaxel across the BBB in mice (Bihorel et al., 2007; Kemper et al., 2004a). Cyclosporine A also potentiates penetration of INNO-406, a tyrosine kinase inhibitor, with augmented growth suppressive effects on CNS leukaemia cells (Yokota et al., 2007). These findings illustrate the potential for this therapeutic approach but suggest that new candidate inhibitors and further investigation are required.

1.8.2 ABCG2 Inhibitors ABCG2 inhibitors are at a much earlier stage in the development process. Elacridar inhibits both ABCB1 and ABCG2 transporters and restores chemotherapy sensitivity in ABCB1 or ABCG2 overexpressing cancer non-neuroblastic cell lines (de Bruin et al., 1999; Hyafil et al., 1993). Elacridar, when administered to mice in vivo increases BBB penetration of diverse chemotherapeutics such as gefitinib, imatinib, dasatinib, temozolomide, topotecan, vemurafenib, lapatinib, lorlatinib and crizotinib (Agarwal et al., 2010; Bihorel et al., 2007; Breedveld et al., 2005; Chen et al., 2009; de Gooijer et al., 2018b; de Vries et al., 2007; Durmus et al., 2012; Karbownik et al., 2019; Lagas et al., 2009; Li et al., 2018; Tang et al., 2014). When administered clinically elacridar increases oral bioavailability of topotecan, paclitaxel and doxorubicin consistent with inhibition of transporters within the GI tract, with a favourable safety profile (Kruijtzer et al., 2002; Malingre et al., 2001; Planting et al., 2005), however elacridar appears to have yet to demonstrate potentiation of BBB penetration of chemotherapeutics, or to have improved outcomes in patients with brain cancers or metastases.

Fumitremorgin C (FTC) inhibits ABCG2 and reverses multidrug resistance with greater specificity than elacridar (Rabindran et al., 1998; Rabindran et al., 2000). Although examples exist of FTC administration to mice without any resulting associated major toxicities, contrasting reports of neurotoxicity preclude clinical use (Allen et al., 2002; Garimella et al., 2005). Ko143 is a more potent ABCG2 inhibitor than elacridar and FTC, increasing intracellular uptake, retention and cytotoxicity of dasitinib in patient-derived leukaemia cells, and lacking toxic effects in mice (Allen et al., 2002; Hiwase et al., 2008). Pantoprazole additionally inhibits ABCG2-mediated efflux of chemotherapeutics in cell lines and enhances brain penetration of imatinib in mice (Breedveld et al., 2005; Breedveld et al., 2004). However a role of pantoprazole in inhibiting metabolic degradation of imatinib rather than solely efflux, has been advanced (Oostendorp et al., 2009). Although, as an established proton pump inhibitor, it already has demonstrable clinical safety and minimal CYP3A4 interaction (Huber et al., 1996). Use of flavonoids has also been explored, quercetin, chrysin and biochanin amongst those demonstrating reversal of multidrug resistance in ABCG2-overexpressing cell lines including, for quercetin, neuroblastoma although in this instance the effects of quercetin were likely attributable to heat shock protein modulation and notably quercetin has the disadvantage of being a CYP3A4 inhibitor (Hsiu et al., 2002; Yoshikawa et al., 2004; Zanini et al., 2007; Zhang et al., 2004) . Both quercetin and chrysin have been demonstrated to enhance brain penetration of vincristine in mice, although biphasicity is observed with activation of efflux by quercetin at low concentrations (Mitsunaga et al., 2000). However, to date, clinical efficacy of these ABCG2 inhibitors in overcoming multidrug resistance or increasing penetration of the BBB has yet to be established in patients.

1.8.3 ABCC Inhibitors ABCC inhibitors similarly lack clinical validation. Furthermore, available compounds typically exhibit low specificity with respect to individual ABCC isoforms or other unintended targets, and low binding affinity. For example, MK571 lacks ABCC subtype specificity, inhibiting ABCC1, ABCC2, ABCC3, ABCC4 and ABCC5 and additionally acting as a potent leukotriene D4 receptor antagonist (Jones et al., 1989; Leier et al., 2000; Morrow et al., 2006; Reid et al., 2003; Videmann et al., 2009). MK571 sensitises neuroblastoma cells to paclitaxel at the level of enhancing drug accumulation (Sietsma et al., 2000). Probenecid and sulfinpyrazone inhibit at least the ABCC1 isoform in

36 addition to inhibiting non-ABC organic anion transporters (Bakos et al., 2000; Gollapudi et al., 1997). Probenecid sensitises neuroblastoma cells and in vivo xenograft models to the cytotoxic effects of cisplatin although sulfinpyrazone lacks similar data (Campos- Arroyo et al., 2016). Several compounds are dual ABCC1 and ABCB1 inhibitors including cyclosporine A, VX-710, S9788, MS-209 and PAK-104P (Bichat et al., 1998; Germann et al., 1997; Marbeuf-Gueye et al., 2000). However, S9788 has been found to disrupt BBB integrity so this may confound evaluation of BBB penetration (Monnaert et al., 2004). Preliminary evidence suggests MS-209 may increase efficacy of vincristine in treating transplanted brain gliomas in rats (Sasajima et al., 2007). Repurposed molecules include glibenclamide which inhibits ATP-sensitive K+ channels alongside ABCC1 and ABCC2 transporters, and the dual ABCC1 and reverse transcriptase inhibitors delavirdine and efavirenz (Payen et al., 2001; Weiss et al., 2007). Similarly the antifolate methotrexate and topoisomerase inhibitor etoposide both inhibit ABCC3 (Zelcer et al., 2001). There is therefore an acute need for development of potent inhibitors of ABCC transporters with greater selectivity and for their evaluation in neuroblastoma and at the BBB.

1.8.4 Chemotherapeutics with dual ABC transporter inhibitory properties Use of chemotherapeutic agents with dual ABC transporter inhibitory properties represents an underdeveloped therapeutic means of overcoming resistance.

Compounds which have been investigated primarily include tyrosine kinase inhibitors (TKIs). For example, nilotinib, a breakpoint cluster region-abelson (BCR-ABL) TKI, and sunitinib, which targets multiple kinases (vascular endothelial growth factor receptor (VEGFR), c-KIT, platelet-derived growth factor receptor (PDGFR) and fms-like tyrosine kinase 3 (FLT3)), both inhibit efflux activity of ABCB1 and ABCG2 transporters (Dai et al., 2009; Shukla et al., 2009; Tiwari et al., 2009). Similarly, the EGFR TKIs erlotinib and gefitinib both inhibit ABCB1 and ABCG2 transporters with erlotinib additionally inhibiting ABCC10 (Kitazaki et al., 2005; Kuang et al., 2010; Ozvegy-Laczka et al., 2004; Shi et al., 2007). In addition, the novel taxanes SB-T-1213 and SB-T-1250 inhibit ABCB1 transporters (Ferlini et al., 2000). However, the availability of compounds which are cytotoxic to neuroblastoma, penetrate the BBB to treat CNS metastases, inhibit ABC transporter activity and are safe and clinically effective has yet to be described.

1.8.5 Modulators of ABC transporter expression Given the limitations of some direct inhibitors of ABC transporters, of which toxicity is a frequent concern, identification of compounds which indirectly affect transporter activity by regulating transporter expression is a promising alternative. More subtle alterations in activity and expression may preserve partial basal transporter function and associated protective efflux capacity. For example, glutamate and NMDA both increase ABCB1 expression and activity in isolated rodent brain capillaries, with cyclooxygenase 2 (COX-2) acting downstream of NMDA receptor signalling (Bauer et al., 2008). However COX-2 inhibition using celecoxib abolishes ABCB1 induction by glutamate whilst preserving basal expression and ABCB1 activity (Bauer et al., 2008). Therefore modulation of ABC transporter expression warrants further investigation either within separate compounds or within a dual chemotherapeutic and ABC transporter regulator. The potential to increase BBB penetration of the dual molecule itself or of concomitant chemotherapeutics may therefore increase CNS bioavailability of treatments for CNS neuroblastoma metastases.

1.9 Novel approach to the cytotoxic treatment of neuroblastoma-derived CNS metastases and modulation of ABC transporters based on CARP-1/CCAR1 CARP (cell cycle and apoptosis regulatory protein)-1 is an oncological signalling target (Rishi et al., 2003) whose downstream effectors, as well as affecting growth, apoptosis and cytotoxicity, include known regulators of ABC transporter expression as discussed below. Hence compounds which influence CARP-1 signalling such as CARP-1 functional mimetics (CFMs) could conceivably act as dual modulators of cancer cell viability and ABC transporter function, in turn determining CNS bioavailability of both CFMs and other concomitant chemotherapeutic agents.

1.9.1 Validation of CARP-1 as a drug target The effects of CARP-1 on apoptosis and cell growth contribute towards its validation as a drug target in the treatment of cancer, potentially including neuroblastoma, given that this represents a mechanism of cytotoxicity. CARP-1 was initially identified as a mediator of CD437, adriamycin or etoposide-dependent growth arrest and/or apoptosis in breast cancer cells (Rishi et al., 2003). CARP-1 also mediates apoptosis induced by epidermal growth factor receptor (EGFR) blockade, for example using gefitinib, in breast cancer 38 cells (Rishi et al., 2006). CARP-1 signalling further results in upregulation of phosphorylated p38 MAPK and activation of caspases 3 and 9 with CARP-1 depletion attenuating cell death, thus implicating CARP-1 as a pro-apoptotic mediator (Jiang et al., 2010; Rishi et al., 2006). CARP-1 further coactivates the proapoptotic tumour suppressor p53 in breast cancer cells (Kim et al., 2008). Similar tumour suppressor or proapoptotic activity of CARP-1 has also been demonstrated in lymphoma and leukaemia cells (Levi et al., 2011; Lu et al., 2013).

Interference with unregulated cell cycle progression and proliferation represents an additional potential mechanism of disrupting the neuroblastoma disease course, therefore it is relevant that CARP-1 regulates expression of genes involved in these processes including c-myc, cyclin B1, DNA topoisomerase IIα, p21rac1, p21WAF1/CIP1 and histone deacetylase 3C (Rishi et al., 2003). CARP-1 further interacts with the differentiation, cell cycle and apoptosis-regulating 14-3-3 protein and the associated TAZ ligand (Jiang et al., 2010; Rishi et al., 2003). These mechanistic insights prompted further research resulting in the discovery of CARP-1 functional mimetics (CFMs).

1.9.2 Discovery of CARP-1 functional mimetics (CFMs) based on disruption of the CARP-1/Anaphase-promoting complex (APC)-2 interaction The specific interaction between CARP-1 and the anaphase-promoting complex 2 (APC- 2) protein, a subunit of the APC/C E3 ubiquitin ligase, was predicted to mediate effects of CARP-1 on cell growth and apoptosis (Puliyappadamba et al., 2011). APC/C is involved in cell cycle checkpoints which are frequently disrupted in cancer; its substrates or its regulators also correlate with malignancy (Lehman et al., 2007; Puliyappadamba et al., 2011). Given respective roles in apoptosis and cell cycle progression of CARP-1 and APC/C and the capacity for CARP-1 to bind APC-2 and coactivate APC/C (alongside regulators Cdc20 and Cdh1), disruption of the CARP-1/APC-2 interaction was advanced as an oncological therapeutic target (Puliyappadamba et al., 2011). Small molecule inhibitors of this interaction, CFMs, were identified through high throughput screening of a chemical library (Puliyappadamba et al., 2011). Compounds CFM-1, -4, and -5 were the most potent inhibitors of the interaction, with IC50 values of 4.1, 0.75, and 1.4 µM respectively (Puliyappadamba et al., 2011). Additional structure-activity relationship studies initially led to the development of six analogues of CFM-4, CFM-4.1 to CFM-4.6, of which CFM-4.6 displayed an IC50 of 3.96 µM of the CARP-1/APC-2 interaction and was

39 also the most potent cytotoxic (Muthu et al., 2015). Further medicinal chemistry led to the development of 12 additional structurally similar compounds, of which CFM-4.16 and CFM-4.17 had the most favourable activity profile, superior to the lead compound CFM-4 (Cheriyan et al., 2016) (Figure 1.5).

Figure 1.5. Structure of CFM-4, CFM-4.16 and CFM-4.17.

(Cheriyan et al., 2017)

1.9.3 CFM-mediated upregulation of CARP-1 signalling in a range of cancer cell types Although upregulation of CARP-1 was first demonstrated in CFM-4-treated breast cancer cells, CFM-mediated disruption of CARP-1/APC-2 appears to consistently elevate levels of CARP-1 in a range of cancer cell types and with several CFM compounds demonstrating its broad applicability, including to neuroblastoma (Puliyappadamba et al., 2011). For example, CFM-4 upregulates CARP-1 in neuroblastoma, alongside medulloblastoma, non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), and renal cell carcinoma (RCC) cells (Ashour et al., 2013; Cheriyan et al., 2018; Cheriyan et al., 2017; Jamal et al., 2014; Muthu et al., 2014; Muthu et al., 2015). Of the compounds employed in the present work, the effects of CFM-4.17 on CARP-1 have yet to be reported, and CFM-4.16 is yet to be studied in neuroblastoma. However, CFM-4.16 upregulates CARP-1 in cisplatin or Adriamycin-resistant TNBC cells, parental or everolimus-resistant RCC cells, and parental or tyrosine kinase inhibitor or gemcitabine resistant NSCLC cells, in addition to TNBC, cervical cancer and kidney cancer cells (Cheriyan et al., 2018; Cheriyan et al., 2017; Sekhar et al., 2019). It was therefore 40 considered likely that CFM-4.16 and CFM-4.17 might similarly upregulate CARP-1 in neuroblastoma, and possibly also PBECs.

1.9.4 CFM-mediated cytotoxicity CFM-4 has been reported to inhibit cell viability in a dose- and time-dependent manner in four different neuroblastoma cell lines (Muthu et al., 2014). CFM-4 further elicits cytotoxic effects in breast, colon, prostate, pancreatic, pleural malignant mesothelioma, lymphoma, medulloblastoma, RCC and NSCLC cancer cell lines (Ashour et al., 2013; Cheriyan et al., 2018; Cheriyan et al., 2017; Jamal et al., 2014; Muthu et al., 2015; Puliyappadamba et al., 2011). Of the subsequently developed CFM-4 analogues, CFM- 4.16 and CFM-4.17 have reported cytotoxic effects in TNBC, cervical cancer, RCC, NSCLC, and mesothelioma cells (Ali, 2017; Alsaab et al., 2018; Cheriyan et al., 2018; Cheriyan et al., 2017; Cheriyan et al., 2016; Sekhar et al., 2019). Given the range of cell types in which CFM-4.16 and CFM-4.17 are cytotoxic, it was expected that these compounds would also, like CFM-4, be effective in neuroblastoma, although the SH-SY5Y cell line had yet to be evaluated with this class of compounds prior to the present study.

1.9.5 Additional mechanistic evidence supporting the proposed use of CFM-4.16 and CFM-4.17 in neuroblastoma While CFM-4.16 and CFM-4.17 have yet to be evaluated in neuroblastoma cell lines, insights regarding their likely effectiveness can be derived from the parent compound CFM-4. Consistent with the aforementioned effects of the downstream mediator CARP- 1 in breast cancer cells, CFM-4 induces cell cycle arrest with accumulation of cells in the

G2M phase in neuroblastoma cells (Muthu et al., 2014). CFM-4 also results in loss of cyclin B1 and of oncogenic MYCN and c-myc in the same cells (Muthu et al., 2014). In addition, CFM-4 increases TUNEL staining, PARP1 cleavage, p38 kinase and c-Jun N- terminal kinase (JNK) phosphorylation and miR513a-5p levels, suggesting potentiation of pro-apoptotic signalling (Muthu et al., 2014). CFM-4 also inhibits the migratory, colony forming and invasive properties of neuroblastoma and downregulates matrix metalloproteinase activity which are intrinsic metastatic processes (Muthu et al., 2014).

Similar observations regarding cell cycle arrest, apoptosis and metastasis have been reported with CFM-4.16 in other cell lines, although CFM-4.17 is less well studied (Cheriyan et al., 2018; Cheriyan et al., 2017; Cheriyan et al., 2016). CFM-4 and CFM-4.16 both display in vitro anti-angiogenic properties (Cheriyan et al., 2016; Muthu et al., 41

2015). CFM-4.16 also possesses other anticancer properties that could conceivably be extrapolated to the treatment of neuroblastoma including suppression of three dimensional growth (evaluated in mammospheres and spheroids derived from RCC and NSCLC cells) induction of DNA damage and fragmentation (in TNBC and cervical cancer cells), and immune modulation (Alsaab et al., 2018; Cheriyan et al., 2018; Cheriyan et al., 2017; Cheriyan et al., 2016). There is therefore the potential for CFM-4.16 and CFM- 4.17 to exert multimodal effects in neuroblastoma treatment.

1.9.6 Effects of CFMs on signalling molecules implicated in the regulation of ABCB1 and ABCG2 activity and expression In addition to exerting diverse effects on cancer cell growth and signalling, both in vitro and in vivo within murine tumour xenografts, CARP-1 functional mimetics elevate CARP- 1 expression in a range of cancer cell types (Alsaab et al., 2018; Ashour et al., 2013; Cheriyan et al., 2018; Cheriyan et al., 2017; Cheriyan et al., 2016; Jamal et al., 2014; Muthu et al., 2014; Muthu et al., 2015; Sekhar et al., 2019). In turn, CARP-1 can modulate glucocorticoid receptor signalling, typically in a stimulatory direction (Kim et al., 2008; Ou et al., 2014; Wu and S, 2014). CFMs and CARP-1 also produce complex time- and cell type-specific effects on NF-κB signalling, however overall, derepression and hence stimulation of NF-κB pathways appears to predominate (Ashour et al., 2013; Beg et al., 1993; Bouwmeester et al., 2004; DiDonato et al., 1997; Jamal et al., 2014; Mercurio et al., 1997; Muthu et al., 2014; Muthu et al., 2015; Woronicz et al., 1997; Yamaoka et al., 1998). CARP-1 also coactivates p53, and both wild-type and various mutant p53 isoforms have been studied (Kim et al., 2008). The overall consensus in the literature is that signalling by glucocorticoid receptors, NF-κB and most p53 mutants potentiates ABCB1 and ABCG2 transporter activity and expression whilst wild-type p53 elicits opposite effects (Alam et al., 2016; Alms et al., 2014; Bauer et al., 2004; Bauer et al., 2007; Chan et al., 2013; Chin et al., 1992; de Kant et al., 1996; Hirose and Kuroda, 1998; Jiang et al., 2018; Johnson et al., 2001; Marroni et al., 2003; Mei et al., 2004; Narang et al., 2008; Nwaozuzu et al., 2003; Oka et al., 1997; Ou et al., 2014; Sampath et al., 2001; Strauss et al., 1995; Thottassery et al., 1997; Tonigold et al., 2014; Torres-Vergara and Penny, 2018; Wang et al., 2013; Wang et al., 2014; Wang et al., 2010; Wu and S, 2014; Yu et al., 2008).

The effects of CFM-4.16 and CFM-4.17 on CARP-1 and therefore glucocorticoid receptor, NF-κB, and p53 may therefore be expected to produce net upregulation of ABCB1 and

ABCG2 activity and expression in brain endothelial cells and in neuroblastoma cell lines (Fig 1.6), however to date, this hypothesis has yet to be tested.

Figure 1.6. Predicted net effects of CFM-4.16 or CFM-4.17 on signalling molecules which regulate the activity and expression of ABCB1 and ABCG2 transporters.

GR: glucocorticoid receptor

1.10 Aims and objectives

1.10.1 Aims Current treatments for CNS neuroblastoma metastases are characterised by limited efficacy and very poor survival outcomes (Kramer et al., 2001; Matthay et al., 2003; Sidi- Fragandrea et al., 2010). Furthermore, most small molecules are unable to penetrate the BBB due to tight junctions and expression of ABC efflux transporters in BBB endothelial cells (Pardridge, 2005). ABC transporter efflux activity in neuroblastoma cells may additionally confer resistance to chemotherapeutic agents by limiting intracellular accumulation of transporter substrates. ABCB1, ABCG2 and ABCC transporter expression can also influence neuroblastoma prognosis, further illustrating their importance. Chemotherapeutic agents which downregulate or inhibit transporter activity may therefore potentiate both CNS penetration and intracellular accumulation within neuroblastoma cells increasing the likelihood of attaining therapeutic concentrations. The bioavailability of concomitant treatments may also increase if the chemotherapeutic is a transporter substrate. However, many inhibitors of ABC

43 transporters evaluated to date have been limited by safety concerns, drug interactions, lack of specificity, inadequate potency or lack of efficacy, and others remain clinically unproven (see section 1.8).

The CARP-1 functional mimetics were originally identified based on their disruption of the interaction between CARP-1 and APC-2 and were subsequently discovered to upregulate CARP-1 expression (Ashour et al., 2013; Cheriyan et al., 2018; Cheriyan et al., 2017; Cheriyan et al., 2016; Jamal et al., 2014; Muthu et al., 2014; Muthu et al., 2015; Puliyappadamba et al., 2011; Sekhar et al., 2019). CARP-1 regulates the downstream effectors NF-κB, p53 and glucocorticoid receptors, which are known to modulate ABC transporter activity and expression (see Figure 1.6). It was therefore hypothesised that CFM compounds such as CFM-4.16 and CFM-4.17 may, via CARP-1, affect transporter function in brain endothelial cells and in neuroblastoma cells. CFMs also have demonstrable cytotoxic efficacy in other cell types (see section 1.9.4) therefore it was envisaged that these compounds may have dual cytotoxic and transporter modulating properties useful in the treatment of CNS neuroblastoma metastases subject to penetration of the BBB.

In the present work, primary porcine brain endothelial cells were employed as an in vitro model of the BBB based on their phenotypic resemblance to human cells and established use in the host laboratory (Cantrill et al., 2012; Shubbar and Penny, 2018; Skinner et al., 2009; Torres-Vergara and Penny, 2018). SH-SY5Y cells were also selected as a well-characterised and representative neuroblastoma cell line (Biedler et al., 1973; Biedler et al., 1978). Several in vitro properties were evaluated in order to predict whether CFM-4.16 and CFM-4.17 may be candidates for further in vivo and clinical investigation.

1.10.2 Objectives • Evaluate the effects of CFM-4.16 and CFM-4.17 on the viability of SH-SY5Y cells and PBECs and compare their susceptibilities • Determine the functional activities of ABCB1, ABCG2 and ABCC transporters in SH-SY5Y cells • Investigate the effects of short-term (30 min) treatment with CFM-4.16 and CFM- 4.17 on both ABCB1 and ABCC activities in SH-SY5Y cells • Investigate the effects of long-term (24 h and 48 h) incubation with CFM-4.16 and CFM-4.17 on ABCB1 activity in SH-SY5Y cells • Investigate the effects of short-term treatment of PBECs with CFM-4.16 and CFM-4.17 on both ABCB1 and ABCG2 activities • Investigate the effects of long-term incubation with CFM-4.16 and CFM-4.17 on ABCB1 and ABCG2 activities, both with and without a 5 h washout period • Establish an in vitro Transwell®-based BBB model and validate its barrier

properties by measuring both the apparent permeability (Papp) to Lucifer yellow and the TEER of PBEC monolayers • Determine a means of detecting and assess the extent of penetration of CFM- 4.16 and CFM-4.17 across the BBB model • Measure the effect of ABCB1 or ABCG2 inhibition on the penetration of CFM- 4.16 across the BBB model to detect transporter substrate-like behaviour • Quantify the effect of ABCB1 or ABCG2 inhibition on CFM-4.16 or CFM-4.17- mediated toxicity in PBECs as an alternative means of detecting substrate-like properties

2 Methods

2.1 Reagents and materials

CFM-4.16 and CFM-4.17 were gifts from Professor Arun Rishi, Wayne State University, USA. 8-4-chlorophenylthio-cAMP, acetic acid, bovine serum albumin (BSA), Dulbecco’s Modified Eagle Medium (DMEM) (high glucose, with phenol red), Dulbecco’s phosphate buffered saline (calcium and magnesium free) (PBS), DMSO, ethanol (molecular biology grade), foetal bovine serum (FBS), Hank’s balanced salt solution (HBSS), HEPES, heparin sodium salt from porcine intestinal mucosa, hydrocortisone, Lucifer yellow, MK571, 3- (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), neutral red powder, penicillin/streptomycin, puromycin, trypsin-EDTA solution, verapamil, Hoeschst 33342, L-glutamine, and M199 medium were purchased from Sigma-Aldrich (Poole, UK). CMFDA was purchased from Abcam (Cambridge, UK) and telmisartan and nefazodone were supplied by Alfa Aesar (Heysham, England and Ward Hill, USA respectively). Bradford protein assay dye reagent concentrate and trypan blue solution were purchased from Bio-Rad (Hemel Hempstead, UK) while rat tail collagen and isopropanol were obtained from SLS Ltd (Nottingham, UK). Absolute ethanol and human fibronectin were purchased from Fisher Scientific (Loughborough, UK), RO-20-1724 was supplied by EMD Millipore Corp (Billerica, USA) and plasma derived serum (PDS) was obtained from First Link (Birmingham, UK). Calcein-AM and HEPES buffer solution were purchased from Invitrogen (Paisley, UK) while DMEM (both high glucose, phenol red- free and low glucose, phenol red-free) were obtained from Life Technologies (Paisley, UK). Ko143 was supplied by Tocris Bioscience (Bristol, UK), and collagenase I, DNAase I and trypsin powders were purchased from Worthington Lorne Laboratories (Twyford, UK). Cell culture plastic materials were supplied by Greiner Bio-one (Stonehouse, UK), sterile syringe filters were purchased from Merck Millipore Ltd (Cork, Ireland) and nylon membranes originated from Plastok Associates Ltd (Wirral, UK). The SH-SY5Y cell line was a gift from Professor Nigel Hooper, University of Manchester, while the CTX-TNA2 astrocyte cell line was purchased from the American Type Culture Collection (ATCC) repository.

2.2 Isolation of porcine brain endothelial cells

Primary cultures of PBECs were prepared essentially as described by (Skinner et al., 2009) using methodology adapted from (Rubin et al., 1991). Fresh porcine brain hemispheres were collected from the abattoir and transported on ice in high glucose DMEM supplemented with 1% penicillin/streptomycin (penicillin (100 U/mL) and streptomycin (100 μg/mL)). Brain hemispheres were washed in phosphate-buffered saline (PBS) containing 1% penicillin/streptomycin. Meninges, including those within sulci, were then removed using fine forceps, and the hemispheres placed in fresh ice- cold PBS containing 1% penicillin/streptomycin. White matter was subsequently removed with curved forceps and discarded and the remaining cortical grey matter placed into ice-cold isolation medium (high glucose DMEM supplemented with 10% (v/v) FBS, 1% penicillin/streptomycin and 20 mM HEPES). The grey matter was cut into smaller pieces with scissors, passed through a 50 mL syringe, and mixed with isolation medium at a ratio of approximately 10 mL brain tissue: 35 mL medium. The tissue was then gently homogenised in a Dounce tissue grinder with loose-fitting (89-127 μm clearance, 15 strokes) followed by tight-fitting pestles (25-76 μm clearance, 15 strokes). The homogenate was sequentially filtered under vacuum through a 150 μm nylon mesh to capture residual white matter and meninges which were discarded, and then through a 60 μm nylon mesh to retain cerebromicrovessel fragments. The fragments attached to the 60 μm mesh were incubated in M199 medium supplemented with 10% (v/v) FBS, 1% penicillin/streptomycin, 210 U/mL collagenase, 114 U/mL DNase I, and 91 U/mL trypsin for 1 h at 37 oC with continuous shaking. Digested material was washed off the mesh with the digest mixture, and centrifuged for 5 min at 330 x g. The supernatant was aspirated and the pellet resuspended in isolation medium via trituration. The centrifugation step was repeated on a further two occasions and the final microvessel pellet resuspended in freezing medium containing a 9:1 ratio of FBS:dimethylsulfoxide (DMSO). The microvessel suspension was aliquoted into cryovials, maintained in an isopropanol-containing freezing container at a temperature of -80 oC overnight, and then transferred to liquid nitrogen for storage until use.

2.3 Coating of PBEC culture surfaces

Six-well and 96-well plates were treated with 1 mL or 32 μL respectively of 0.1 mg/mL rat tail collagen type I solution in 20 mM acetic acid. Following a 1.5 h incubation period at room temperature, excess solution was removed by aspiration and wells gently washed with PBS. Individual wells were then treated with 1 mL or 32 μL respectively of 7.5 μg/mL fibronectin solution in sterile distilled water and the plates incubated overnight at 4 oC. Immediately prior to seeding, fibronectin was aspirated and the wells washed with PBS. Transwell® membranes were similarly treated with 400 μL volumes of collagen and fibronectin appropriate to their surface area, with an intermediate PBS wash step and a further PBS wash prior to seeding.

2.4 Culture of PBECs

Cryopreserved aliquots of microvessel suspension were rapidly thawed in a 37 oC water bath, and added dropwise to PBEC growth medium (low glucose, phenol red-free DMEM with 1% penicillin/streptomycin, 2 mM L-glutamine, 10% (v/v) bovine PDS and 125 μg/mL heparin). Following centrifugation (5 min, 330 x g) the pellet was resuspended in 6 mL growth medium, seeded onto a pre-coated 6-well plate and plates maintained at

o 37 C in humidified air with 5% CO2 for 48 h. Subsequently, PBECs originating from the microvessels were treated with fresh growth medium supplemented with 4 μg/mL puromycin (1 mL/well) for a further 48 h. Medium was then replaced with a 1:1 (v/v) mixture of growth medium:astrocyte-conditioned medium (ACM) (2 mL/well) and the cells incubated for a further 72 h to attain confluence. At this point PBECs were subcultured onto 96-well plates or onto Transwell® inserts.

2.5 Subculture of PBECs from 6-well plates onto 96-well plates

Following removal of medium, cells were washed with warm PBS and detached by incubation with trypsin (1 mL/well) for 5 min at 37 oC followed by gentle tapping. Trypsin was inactivated by adding an equal volume of PBEC growth medium and remaining cells were loosened by repeatedly flushing the wells with the cell suspension. The suspension was then centrifuged (5 min, 330 x g), the supernatant aspirated, and the pellet homogenously resuspended in 1 mL 1:1 (v/v) growth medium:ACM. After cell counting

48 using the trypan blue assay (see section 2.7), the cell suspension was diluted further with sufficient 1:1 (v/v) growth medium:ACM to achieve a cell concentration of 125,000 cells/mL and mixed thoroughly. A 200 µL volume of suspension containing 25,000 cells was seeded per well. PBS was added to empty peripheral wells to maintain humidity.

2.6 Subculture of PBECs from 6-well plates onto Transwell® inserts

PBECs were detached from 6-well plates and counted as described above. The 1 mL cell suspension was then mixed with 1:1 (v/v) growth medium:ACM to attain a cell concentration of 240,000 cells/mL. Medium was aspirated from each individual well of a 12-well plate, pre-seeded with CTX-TNA2 astrocytes 48 h earlier to attain confluency, and a Transwell® polycarbonate insert (pore size 0.4 μm; surface area 1.2 cm2) transferred to the same well. A 500 μL volume of PBEC suspension (containing 120,000 cells) was pipetted into the apical chamber, and 1.5 mL 1:1 (v/v) growth medium:ACM was applied to the basolateral compartment. A blank was generated by adding 500 μL 1:1 ACM:growth medium instead of cell suspension to the apical chamber of the final insert.

2.7 Trypan blue assay

Based on the principle that viable cells possess intact cell membranes which exclude the trypan blue dye whereas non-viable cells are stained blue, this assay was used to facilitate counting of viable unstained cells (Strober, 2001). A 10 µL sample of 50% (v/v) cell suspension and 50% (v/v) trypan blue solution was applied to a haemocytometer chamber and the average viable unstained cell count per square determined and converted to cells/mL via a multiplication factor of 2 x 104.

2.8 Preparation of CFM-4.16 and CFM-4.17

Stock solutions of 10 mM CFM-4.16 and CFM-4.17, dissolved in DMSO, were stored at - 20 oC until required. Prior to treatment, the stock was thawed and diluted 10-fold in molecular biology grade ethanol. Media or transport buffer HBSS supplemented with

HEPES (10 mM)) were used for further dilutions. In cell studies, final solvent concentrations did not exceed 0.05 % (v/v) DMSO or 0.45 % (v/v) ethanol.

2.9 Culture of CTX-TNA2 astrocytes and production of astrocyte-conditioned medium

The rat astrocyte cell line CTX-TNA2 (passage 18-21), originally derived from primary cultures of type 1 astrocytes transfected with an oncogenic sequence of SV40, was maintained in astrocyte growth medium (high glucose DMEM supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin). Cryovials containing CTX-TNA2 aliquots were thawed rapidly at 37 oC and the contents were then added dropwise to growth medium and the cell suspension centrifuged (5 min, 330 x g). The pellet was resuspended in a small volume of growth medium and used to seed three to five 75 cm2 culture flasks. When CTX-TNA2 cells reached 40% confluency, medium was collected every 48 h, and passed through a 0.22 µm sterile syringe filter. Filtered medium (thereafter termed astrocyte-conditioned medium) was frozen at -20 oC and thawed as required. Cells were passaged at 70% confluency. Following aspiration of medium, cells were washed with PBS, each flask was incubated with trypsin for 5 min at 37 oC, and the enzymatic reaction terminated with addition of growth medium. The cell suspension was centrifuged, the pellet resuspended and cells subcultured.

2.10 Cryopreservation of CTX-TNA2 astrocytes

Frozen stocks of the CTX-TNA2 cell line were generated by resuspending the cell pellet, obtained after trypsinisation and centrifugation, in freezing medium composed of 90% (v/v) growth medium and 10% (v/v) DMSO. Typically cells from four confluent 75 cm2 flasks were resuspended in 10 mL and frozen at -80 oC in 1 mL aliquots.

2.11 Subculture of CTX-TNA2 astrocytes onto 12-well plates

CTX-TNA2 cells were detached and centrifuged as described in section 2.9 and the pellet resuspended in 1 mL of growth medium. Cells were counted using the trypan blue assay

(section 2.7) and diluted further with sufficient growth medium to permit seeding of 80,000 astrocytes per 1.5 mL in each well. Cells were not seeded onto one well for subsequent use as a blank Transwell® insert.

2.12 Culture, cryopreservation and subculture of SH-SY5Y neuroblastoma cells

SH-SY5Y cells (passage 2-19) were cultured in SH-SY5Y growth medium (high glucose, phenol-red free DMEM containing L-glutamine and 25 mM HEPES, supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin) at 37 oC. Passaging of SH-SY5Y cells was carried out as for CTX-TNA2 cells as described in section 2.9 and cryopreservation of SH- SY5Y cells was as described in section 2.10. Following trypsin treatment, SH-SY5Y cells were seeded into 96-well plates, after resuspension of the cell pellet in growth medium to a cell density of 9 x 104 cells/mL. A 200 µL volume of cell suspension was pipetted into each experimental well (18,000 cells/well) and 200 µL of PBS was added to each peripheral well.

2.13 Measurement of the effect of CFM-4.16 and CFM-4.17 on SH-SY5Y viability using the MTT assay The MTT assay, developed and optimised by (Mosmann, 1983) and (Sylvester, 2011) respectively, was employed to measure the effect of CFM-4.16 and CFM-4.17 on SH- SY5Y viability. The assay is based on catalytic conversion of the yellow tetrazolium salt MTT to a purple formazan product by mitochondrial enzymes, the amount of which is directly proportional to the number of viable cells exposed to MTT (Sylvester, 2011). SH- SY5Y cells were plated at a density of 18,000 cells/well in 96-well plates and maintained in culture for 48 h. Growth medium was aspirated and the cells equilibrated with SH- SY5Y treatment medium (high glucose, phenol red-free DMEM supplemented with 1% v/v FBS) (200 μL/well) for 30 min at 37 oC. Medium was then replaced with vehicle, CFM- 4.16 or CFM-4.17 (at concentrations 0.0025–20 µM), each prepared with treatment medium as the final diluent, and the cells incubated for 24 h at 37 oC. A 5 mg/mL stock solution of MTT in PBS was prepared and filtered through a 0.22 μm filter. The stock was then diluted 1:10 in SH-SY5Y assay medium (high glucose, phenol red-free DMEM) immediately prior to use. Following treatment, the medium was aspirated and replaced 51 with MTT-containing medium (100 µL/well). Following a 4 h incubation at 37 oC the medium was aspirated and the purple formazan reaction product solubilised by addition of DMSO (100 µL/well). The plate was then agitated on a shaker for 15 min. Absorbance at 570 nm was measured using a Safire multiplate reader in turn reflecting the concentration of the purple product (Tecan, Germany).

2.14 Measurement of the effect of CFM-4.16 and CFM-4.17 on PBEC viability using the neutral red assay

The neutral red cell viability assay, adapted by (Repetto et al., 2008) from methodology originally developed by (Borenfreund and Puerner, 1985), was employed to measure the effect of CFM-4.16 and CFM-4.17 on PBEC viability. The assay is based on the intracellular concentration of the neutral red dye within lysosomes of treated cells (Repetto et al., 2008). Given the requirement for a lysosomal proton gradient for retention to occur, the amount of retained dye, quantified by measuring absorbance, reflects the number of viable cells (Repetto et al., 2008). PBECs were plated at a density of 25,000 cells/well in 96-well plates and maintained in culture for 48 h. Cell monolayers were then pre-equilibrated with PBEC treatment medium (low glucose, phenol red-free DMEM supplemented with 1% (v/v) FBS and 1% (v/v) glutamine) (200 μL/well) for 30 min, the medium aspirated, CFM-4.16 or CFM-4.17 (at 1.25-20 µM) added and cells incubated for 24 or 48 h. Neutral red was dissolved in PBS to produce a 4 mg/mL stock solution which was diluted 1:100 in PBEC assay medium (low glucose, phenol red-free DMEM with 1% (v/v) glutamine) and filtered through a 0.22 μm filter immediately prior to each experiment. Following treatment, cell monolayers were washed with warm PBS and neutral red-containing assay medium (100 µL/well) added. The cells were then incubated at 37 oC for 2 h, the neutral red-containing medium was removed and excess neutral red was washed with cold PBS (150 µL/well). PBS was aspirated and the intracellular neutral red dye extracted by addition of destain solution (50% (v/v) absolute ethanol, 49% (v/v) deionised water, 1% (v/v) acetic acid) (150 µL/well). Destain solution alone was used as a blank. The plate was then placed on a shaker for 15 min at room temperature. Fluorescence was quantified using a Safire multiplate reader (Tecan, Germany) with an excitation wavelength of 530 nm and an emission wavelength of 645 nm. 52

2.15 Measurement of the effect of ABCB1 and ABCG2 inhibitors on CFM-4.16 and CFM-4.17–mediated toxicity in PBECs

The neutral red assay was carried out as in section 2.14, with modifications, in order to determine whether inhibitors of ABCB1 and ABCG2 modulated the effects of CFM-4.16 and CFM-4.17 on PBEC viability. Forty-eight hours after plating PBECs into 96-well plates (25,000 cells/well), growth medium was aspirated and PBECs washed with PBS (200 μL/well). PBECs were pre-incubated for 30 min at 37 oC with 100 μL/well of PBEC treatment medium or PBEC treatment medium containing either 10 μM verapamil or 0.5 μM Ko143. Stock solutions of CFM-4.16, CFM-4.17 and vehicle were prepared in each of the three media types. PBECs were then treated with 100 μL/well of CFM-4.16, CFM- 4.17 or vehicle (within the same media type employed for pre-incubation) to attain a final concentration of 1.25 μM CFM-4.16, 5 μM CFM-4.17 or solvent equivalent to that present in the 5 μM CFM-4.17. Incubation was then carried out for a further 6 h, followed by measurement of cell viability. All other steps of the neutral red assay were identical to section 2.14.

2.16 Measurement of ABC transporter functional activity in SH-SY5Y cells and in porcine brain endothelial cells

Measurement of ABCB1, ABCG2 and ABCC transporter functional activity was based on the methodology of (Cantrill et al., 2012) and (Dalzell et al., 2015). The technique is a dye retention assay which involves incubation of cells with a fluorescent substrate for the transporter type under investigation followed by wash steps to remove non-retained extracellular substrate. The intracellular fluorescent signal, quantified spectrophotometrically, is inversely related to the extent of transporter-mediated efflux.

2.17 Measurement of ABCB1 transporter functional activity

SH-SY5Y cells were subcultured into 96-well plates at a density of 18,000 cells/well and maintained in SH-SY5Y growth medium. PBECs were subcultured into 96-well plates at a density of 25,000 cells/well and maintained in PBEC growth medium.

In experiments to measure ABCB1 transporter activity, SH-SY5Y cells were used 72 h after subculture. Cells were first washed with warm PBS (200 μL/well) and pre- equilibrated for 30 min at 37 oC with SH-SY5Y assay medium (high glucose, phenol red- free DMEM) at a volume of 100 μL/well or 150 μL/well respectively for those wells subsequently treated or untreated with inhibitor. Where appropriate, 50 μL/well of the ABCB1 inhibitor verapamil in assay medium (final concentration 10 μM) was added. Cells were then incubated for a further 30 min, following which the ABCB1 substrate calcein- AM in assay medium was added to a final concentration of 0.5 μM and cells incubated for 30 min at 37 oC. The plate was then placed on ice, the media aspirated and the cells washed with ice cold PBS (200 µL/well). A 160 µL volume of PBS was then added to each well and fluorescence was measured immediately (excitation 484 nm; emission 530 nm) using a Safire multiplate reader (Tecan, Germany). Measurements were corrected by subtracting fluorescence associated with blank wells (i.e. wells containing no cells). The Bradford assay was then conducted (see section 2.20) to determine the protein content of each individual well. Relative fluorescence units (RFU) were normalised to μg of protein and expressed as a percentage of non-treated control cells.

In experiments to measure the effect of short-term (30 min) treatment with CFM-4.16 or CFM-4.17 on ABCB1 transporter activity, SH-SY5Y cells and PBECs were used 72 h or 48 h respectively after subculture. For SH-SY5Y cells, assays were carried out as for determination of activity except that cells were incubated with assay medium containing vehicle, CFM-4.16 or CFM-4.17 (final concentration 0.04 μM) in lieu of medium containing verapamil. In addition, for PBECs, PBEC assay medium (low glucose, phenol red-free DMEM with 1% (v/v) glutamine) and a 1.25 μM final concentration of CFM-4.16 and CFM-4.17 were employed.

In order to measure the effect of long-term (24 h or 48 h) treatment with CFM-4.16 or CFM-4.17 on ABCB1 transporter activity in SH-SY5Y cells and PBECs, cells were subcultured as above and both cell types maintained in growth medium for 48 h. Cells were initially pre-equilibrated with 200 μL/well of SH-SY5Y treatment medium (high glucose, phenol red-free, DMEM supplemented with 1% (v/v) FBS) or PBEC treatment medium (low glucose, phenol red-free, DMEM with 1% (v/v) FBS and 1% (v/v) glutamine) for 30 min at 37 oC. The medium was then aspirated, and vehicle, CFM-4.16 or CFM-4.17 prepared in the respective treatment medium added (0.04 μM for SH-SY5Y cells or 1.25

μM for PBECs). The cells were then incubated for 24 h or 48 h, washed with 200 μL/well of PBS and all subsequent steps carried out as for determination of ABCB1 transporter activity. The equilibration period with assay medium was extended to 5 h for those experiments in PBECs in which the effect of long-term (24 h or 48 h) incubation followed by a 5 h washout was evaluated. The rationale behind use of the washout period was that dissociation of CFM compounds from the ABCB1 transporter prior to measuring ABCB1 activity may allow chronic regulatory effects to be distinguished from any acute reversible inhibitory effects of these compounds.

2.18 Measurement of ABCG2 transporter functional activity

SH-SY5Y cells and PBECs were maintained in 96-well plates as detailed in section 2.17. In experiments to measure ABCG2 transporter activity, SH-SY5Y cells were used 72 h after subculture. Cells were washed with PBS and pre-equilibrated with assay medium as described above (section 2.17). Where appropriate, 50 μL/well of the ABCG2 inhibitors Ko143 (0.5 μM or 5 μM), telmisartan or nefazodone (1.5 μM or 5 μM), all in assay medium and reported as final concentrations were added. Cells were then incubated for a further 30 min, following which the ABCG2 substrate Hoechst 33342 in assay medium was added to a final concentration of 1 μM and cells incubated for 30 min at 37 oC. The plate was then placed on ice, the media aspirated and the cells washed with ice cold PBS (200 µL/well). A 160 µL volume of PBS was then added to each well and fluorescence was measured immediately (excitation 370 nm; emission 450 nm) using a Safire multiplate reader (Tecan, Germany). Measurements were corrected by subtracting fluorescence associated with blank wells (i.e. wells containing no cells). The Bradford assay was then conducted (see section 2.20) to determine the protein content of each individual well. RFU were normalised to μg of protein and expressed as a percentage of non-treated control cells.

In order to measure the effect of short-term (30 min) treatment with CFM-4.16 or CFM- 4.17 on ABCG2 transporter activity in PBECs, cells were used 48 hours following subculture and assays were carried out as for determination of activity except that PBEC rather than SH-SY5Y assay medium was employed. In addition, PBECs were incubated

55 with assay medium containing vehicle, CFM-4.16 or CFM-4.17 (final concentration 1.25 μM) in lieu of medium containing Ko143, telmisartan or nefazodone.

In order to measure the effect of long-term (24 h or 48 h) treatment with CFM-4.16 or CFM-4.17 on ABCG2 transporter activity, PBECs were initially pre-equilibrated with 200 μL/well of treatment medium for 30 min at 37 oC. The medium was then aspirated, and vehicle, CFM-4.16 or CFM-4.17 (1.25 μM prepared in treatment medium) added. The cells were then incubated for 24 h or 48 h, washed with 200 μL/well of PBS and all subsequent steps carried out as for determination of ABCG2 transporter activity, using PBEC assay medium and Ko143 at a concentration of 0.5 μM. The equilibration period with assay medium was extended to 5 h for those experiments in PBECs in which the effect of long-term (24 h or 48 h) incubation followed by a 5 h washout was evaluated.

2.19 Measurement of ABCC transporter functional activity

SH-SY5Y cells were maintained in 96-well plates as detailed in section 2.17. In experiments to measure ABCC transporter activity, SH-SY5Y cells were used 72 h after subculture. Cells were washed with PBS and pre-equilibrated with assay medium as described above (section 2.17). Where appropriate, 50 μL/well of the ABCC inhibitor MK571 (final concentration 25 μM in assay medium) was added. Cells were then incubated for a further 30 min, following which the ABCC substrate CMFDA in assay medium was added to a final concentration of 4 μM and cells incubated for 30 min at 37 oC. The plate was then placed on ice, the media aspirated and the cells washed with ice cold PBS (200 µL/well). A 160 µL volume of PBS was then added to each well and fluorescence was measured immediately (excitation 492 nm; emission 516 nm) using a Safire multiplate reader (Tecan, Germany). Measurements were corrected by subtracting fluorescence associated with blank wells (i.e. wells containing no cells). The Bradford assay was then conducted (see section 2.20) to determine the protein content of each individual well. RFU were normalised to μg of protein and expressed as a percentage of non-treated control cells.

In experiments to measure the effect of short-term (30 min) treatment with CFM-4.16 or CFM-4.17 on ABCC transporter activity in SH-SY5Y cells, assays were carried out as for determination of activity except that cells were incubated with assay medium containing 56 vehicle, CFM-4.16 or CFM-4.17 (final concentration 0.04 μM) in lieu of medium containing MK571.

In order to measure the effect of long-term (24 h or 48 h) treatment with CFM-4.16 or CFM-4.17 on ABCB1 transporter activity in the SH-SY5Y cell line, cells were subcultured as above and maintained in growth medium for 48 h. Cells were initially pre-equilibrated with 200 μL/well of treatment medium (high glucose, phenol red-free, DMEM supplemented with 1% (v/v) FBS) for 30 min at 37 oC. The medium was then aspirated, and vehicle, CFM-4.16 or CFM-4.17 (0.04 μM prepared in treatment medium) added. The cells were then incubated for 24 h or 48 h, washed with 200 μL/well of PBS and all subsequent steps carried out as for determination of ABCC transporter activity.

2.20 Measurement of cellular protein content

Protein quantification of cell monolayers in 96-well plates was based on the technique developed by (Bradford, 1976). A 40 μL volume of concentrated Bradford reagent was added to each well containing 160 μL of PBS. The contents of each individual well were mixed by pipetting and the plate incubated for 5 min to permit colour development. A standard protein calibration curve was generated from BSA (3.125–100 μg/mL) dissolved in PBS and treated in the same manner as the samples. Absorbance was measured at 595 nm with a Safire multiplate reader (Tecan, Germany). The linear regression equation generated from the standard curve was used to interpolate sample protein concentrations.

2.21 Generating the in vitro blood-brain barrier model

On day 0, PBECs were seeded onto collagen and fibronectin-pretreated 6-well plates and maintained as described in section 2.4. CTX-TNA2 cells were seeded onto 12-well plates on day 5 and maintained in ACM/PBEC growth medium. Following trypsinisation, PBECs were subcultured onto collagen and fibronectin-pretreated Transwell® inserts on day 7 and placed directly into wells containing confluent astrocytes to produce a non-contact coculture system which was maintained for 3 days. ACM/PBEC growth medium was replaced on days 8 and 9, and TEER was measured daily from day 9 to day 11. On day

10, 24 h prior to measuring the permeability of CFM-4.16 or CFM-4.17 across PBEC monolayers, individual Transwell® inserts were transferred to a fresh 12-well plate lacking astrocytes and maintained in switch medium (low glucose, phenol red-free DMEM with 312.5 µM 8-4-chlorophenylthio-cAMP, 17.5 µM RO-20-1724 and 55 nM hydrocortisone and 1% v/v penicillin/streptomycin). On day 11, the apparent permeability of Lucifer yellow across PBEC monolayers was measured (a Transwell® insert lacking cells was used a blank). Only monolayers with TEER values of >500 Ω.cm2 were employed. On day 11, CFM-4.16 or CFM-4.17 were added to the apical compartment of PBEC monolayers and the contents of apical and basolateral compartments were collected after 6 h (see section 2.25).

2.22 Measurement of transendothelial electrical resistance

The TEER of PBEC monolayers grown on Transwell® inserts was determined two days after seeding and daily thereafter using an EVOM voltohmmeter and chopstick electrodes (World Precision Instruments, Aston, Stevenage, UK) positioned in the respective apical and basolateral compartments. TEER was calculated by subtracting resistance measurements obtained for a blank Transwell® insert (collagen/fibronectin- coated but without cells) from those obtained for inserts with cells and multiplying final resistance by 1.2 cm2 to correct for the insert surface area.

2.23 Measurement of the apparent permeability of PBEC monolayers to Lucifer yellow

The apparent permeability coefficient (Papp) of Lucifer yellow in PBEC monolayers in the apical to basolateral direction was determined. The Papp was also measured across a blank Transwell® insert. Switch medium was carefully removed from the PBEC monolayers in inserts, cells were washed three times with transport buffer (HBSS supplemented with HEPES (10 mM)) and 1.5 mL of fresh transport buffer was added to the basolateral compartment. 500 μL transport buffer containing 100 μM Lucifer yellow was added to the apical chamber and the Transwell® inserts were incubated at 37 oC for 2 h. A 50 μL and 100 μL sample was taken from the apical (donor) and basolateral

(receiver) compartments respectively at 0 h and at 2 h and 50 μL of transport buffer was added to apical samples. Samples were transferred to a 96-well plate and Lucifer yellow fluorescence quantified spectrophotometrically (excitation 485 nm, emission 530 nm) using a Safire multiplate reader (Tecan, Germany). The concentrations of Lucifer yellow in samples were determined via interpolation from a Lucifer yellow standard curve (0– 60 μM). Calculated concentrations of apical samples were doubled to correct for dilution. Papp was calculated using Equation 1, as described by (Artursson, 1990).

-1 Papp (cm s ) = (dc/dt) x (V/AC0) Equation 1

dc/dt = the change in receiver (basolateral) compartment concentration of Lucifer yellow over time (mol.L-1.s-1)

V = the volume in the receiver compartment reservoir (cm3)

A = the surface area of the Transwell® insert membrane (1.2 cm2)

- C0 = the initial concentration of Lucifer yellow in the donor (apical) compartment (mol.L 1)

Permeability coefficients specific to the PBEC monolayer were corrected for blank filters using Equation 2, as described by (Mark and Miller, 1999):

1/Pendothelial = 1/Ptotal – 1/Pfilter Equation 2

2.24 Determination of absorption characteristics of CFM-4.16 and CFM-4.17

In an attempt to quantify levels of CFM-4.16 and CFM-4.17 in the basolateral (receiver) compartment in studies investigating permeation of the compounds across the in vitro BBB model, the absorption characteristics of CFM-4.16 and CFM-4.17 were evaluated. Absorbance spectra of CFM-4.16 and CFM-4.17 were obtained. Compounds were initially evaluated as 10 mM stock dissolved in DMSO. The 10 mM stock, prepared in 59

DMSO, was diluted 10-fold in ethanol and further diluted in transport buffer to final concentrations of 1 or 100 µM. Transport buffer containing DMSO and ethanol was employed as a vehicle control. Samples (100 µL/well) were added to 96-well plates and absorbance spectra determined between 230 nm and a maximum of 900 nm using a Safire multiplate reader (Tecan, Germany). The spectra were examined for the presence of a unique absorbance peak.

2.25 Measurement of the penetration of CFM-4.16 and CFM-4.17 across an in vitro model of the blood-brain barrier

To measure the penetration of CFM-4.16 and CFM-4.17 across an in vitro model of the BBB, PBEC monolayers grown on Transwell® inserts were washed with PBS and 1.5 mL transport buffer added to the basolateral compartment. Five hundred microlitres of transport buffer containing either CFM-4.16 (1.25 μM), CFM-4.17 (1.25 μM or 5 μM) or solvent was added to the apical chamber of each Transwell® insert. Following a 6 h incubation period at 37 oC, the full volume of transport buffer from the apical and basolateral chambers of each individual Transwell® was collected and stored at -20 oC until use.

In order to assess whether the ABCB1 and ABCG2 efflux transporters influenced the permeation of CFM-4.16 and CFM-4.17 across the in vitro model of the BBB, PBEC monolayers grown on Transwell® inserts were preincubated in 250 μL transport buffer with or without either verapamil (10 μM) or Ko143 (0.5 μM) for 30 min at 37 oC. Subsequently 250 μL of transport buffer containing 1.25 μM CFM-4.16, or 5 μM CFM- 4.17 or solvent, with or without verapamil (10 μM), or Ko143 (0.5 μM) was added to the apical compartment. Following a 6 h incubation at 37 oC, transport buffer from apical and basolateral chambers was collected and stored at -20 oC.

2.26 Indirect measurement of the permeability of CFM-4.16 and CFM-4.17 across an in vitro model of the blood-brain barrier

In order to indirectly assess the permeability of CFM-4.16 and CFM-4.17 across the in vitro model of the BBB, SH-SY5Y cells were treated with samples from the apical and 60 basolateral compartments following permeation studies (section 2.25), and SH-SY5Y cell viability measured. SH-SY5Y cells in 96-well plates were pre-equilibrated with 200 μL/well of transport buffer for 30 min at 37 oC. The transport buffer was then aspirated and replaced with either 200 μL/well of transport buffer obtained from the apical or basolateral compartments or replaced with 200 μL/well of CFM-4.16 (1.25 µM) and CFM-4.17 (1.25 µM or 5 µM) or with vehicle. SH-SY5Y cells were incubated for 24 h at 37 oC and cell viability measured using the MTT assay (section 2.13).

2.27 Statistical analysis

Data are expressed as mean ± standard deviation. Initial data analysis was carried out using Excel 2013 (Microsoft, USA) and subsequent statistical analysis and graphical representation were performed in Prism version 7 (GraphPad, USA). Statistical tests included the unpaired Student’s t test for groups of two, and one way analysis of variance (ANOVA) with Tukey or Dunnett’s post hoc analyses for groups of three or more. A probability of observed differences arising due to chance alone of less than 5% (P<0.05) was taken as the threshold for statistical significance.

3 Results 1 (Neuroblastoma cell viability and functional assays)

3.1 Visualisation of the morphology of SH-SY5Y cells The morphology of SH-SY5Y cells in the exponential growth phase was visualised using Incucyte live cell imaging (Figure 3.1). SH-SY5Y cells exhibited rounded pyramidal shaped cell bodies with multiple short, fine neuritic processes. The neuroblastic cells initially grew as diffuse clusters followed by progressively denser aggregations and subsequently attained confluence. Figure 3.1. Morphology of SH-SY5Y cells.

The SH-SY5Y neuroblastoma cell line was maintained in growth medium (phenol red- free high glucose DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin). One millilitre of cell suspension (9 x 104 cells) was seeded into each well of a 6-well plate. Images were acquired 48 h after seeding using the Incucyte ZOOM Live Cell System (model SXL 9R) in conjunction with Incucyte ZOOM (version 2016A) software. Scale bar 300 μm.

3.2 Evaluation of the effect of CFM-4.16 and CFM-4.17 on the viability of SH- SY5Y cells In order to identify a non-toxic concentration of CFM-4.16 and CFM-4.17 for use in subsequent assays of transporter activity in SH-SY5Y cells, the effects of 24 h exposure to CFM-4.16 and CFM-4.17 on the viability of the SH-SY5Y cell line were determined.

CFM-4.16 had no significant effect on the viability of SH-SY5Y cells between 0.0025 μM and 0.04 μM however, at concentrations between 0.08 μM and 20 μM, exposure of SH- SY5Y cells to CFM-4.16 significantly (p<0.001 for 0.08 µM, p<0.0001 for concentrations ≥0.1 µM) reduced viability (Figure 3.2A). CFM-4.17 had no significant effect on the

62 viability of SH-SY5Y cells between 0.0025 μM and 0.2 μM except for a small but significant (p<0.05) increase at 0.1 μM (Figure 3.2B). However, at concentrations between 0.4 μM and 20 μM, except for 10 μM, exposure of SH-SY5Y cells to CFM-4.17 significantly (p<0.0001) reduced viability (Figure 3.2B).

A concentration of 0.04 μM of both CFM-4.16 and CFM-4.17 was selected for use in transporter functional assays in SH-SY5Y cells.

Figure 3.2. The effect of CFM-4.16 and CFM-4.17 on SH-SY5Y cell viability.

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C F M -4 .1 7 C o n c e n tra tio n ( M )

SH-SY5Y cells were seeded (18,000 cells/well) in 96-well plates and incubated for 48 h. Cells were then treated with treatment medium containing either vehicle or the indicated concentration (0.0025-20 μM) of (A) CFM-4.16 or (B) CFM-4.17. Following 24 h exposure, the MTT cell viability assay was carried out and absorbance quantified at 570 nm. Data were analysed using ANOVA and Dunnett’s post hoc test and are expressed as mean ± standard deviation of at least 3 individual wells from 1 or 2 independent experiments. * p<0.05, *** p<0.001, **** p<0.0001 relative to the vehicle control. All other comparisons with the control lacked statistical significance.

3.3 Determination of ABCB1 functional activity in SH-SY5Y cells In order to assess the functional activity of ABCB1 in SH-SY5Y cells, intracellular accumulation of the fluorescent probe calcein was determined in the presence and absence of the ABCB1 inhibitor verapamil. In SH-SY5Y cells treated with verapamil there was a significant, p<0.01, 67.8% increase in intracellular accumulation of calcein relative to control cells (Figure 3.3) indicating the presence of verapamil-sensitive ABCB1 functional activity in this cell line.

Figure 3.3. Determination of ABCB1 functional activity in SH-SY5Y cells.

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SH-SY5Y cells were seeded (18,000 cells/well) into 96-well plates and incubated for 72 h. Cells were subsequently washed with PBS, equilibrated for 30 min with assay medium (high-glucose, phenol red-free DMEM), and pre-incubated for a further 30 min in medium with or without verapamil (final concentration 10 μM). Cell monolayers were then incubated for 30 min in assay medium containing calcein-AM (final concentration 0.5 μM) with or without verapamil as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of calcein was measured at an excitation wavelength of 484 nm and an emission wavelength of 530 nm. Data were analysed using an unpaired Student’s t test and are expressed as mean ± standard deviation of six replicate wells in a single experiment. RFU: relative fluorescence units. ** p<0.01.

3.4 Determination of the specificity of Hoechst 33342 as a transporter substrate in SH-SY5Y cells In order to ascertain whether Hoechst 33342 is a substrate for ABCB1 in SH-SY5Y cells, in addition to its more established action as a substrate for ABCG2 in other cell types,

65 intracellular accumulation of the fluorescent probe was determined in the presence and absence of verapamil. In SH-SY5Y cells treated with the ABCB1 inhibitor verapamil there was no significant increase in intracellular accumulation of Hoechst 33342 (Figure 3.4) indicating that Hoechst 33342 did not behave as a substrate for ABCB1 transporters.

Figure 3.4. Determination of the specificity of Hoechst 33342 as a transporter substrate in SH-SY5Y cells.

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SH-SY5Y cells were seeded (18,000 cells/well) into 96-well plates and incubated for 72 h. Cells were subsequently washed with PBS, equilibrated for 30 min with assay medium (high-glucose, phenol red-free DMEM), and pre-incubated for a further 30 min in medium with or without verapamil (final concentration 10 μM). Cell monolayers were then incubated for 30 min in assay medium containing Hoechst 33342 (final concentration 1 μM) with or without verapamil as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of Hoechst 33342 was measured at an excitation wavelength of 370 nm and an emission wavelength of 450 nm. Data were analysed using an unpaired Student’s t test and are expressed as mean ± standard deviation of at least five replicate wells in a single experiment. RFU: relative fluorescence units. n.s. non-significant.

3.5 Determination of ABCG2 functional activity in SH-SY5Y cells In order to assess the functional activity of ABCG2 in SH-SY5Y cells, the effects of the ABCG2 inhibitors Ko143, telmisartan and nefazodone on the intracellular accumulation of the fluorescent probe Hoechst 33342 were determined.

In SH-SY5Y cells treated with Ko143 (0.5 or 5 μM), telmisartan (1.5 μM) or nefazodone (4 μM) there was no significant increase in intracellular accumulation of the ABCG2

66 substrate Hoechst 33342 (Figure 3.5). These findings indicate a lack of demonstrable ABCG2 functional activity in the SH-SY5Y cell line.

Figure 3.5. Determination of ABCG2 functional activity in SH-SY5Y cells.

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SH-SY5Y cells were seeded (18,000 cells/well) into 96-well plates and incubated for 72 h. Cells were subsequently washed with PBS, equilibrated for 30 min with assay medium (high-glucose, phenol red-free DMEM), and pre-incubated for a further 30 min in medium with or without additional Ko143 (0.5 μM or 5 μM), telmisartan (1.5 μM) or nefazodone (4 μM). Cell monolayers were then incubated for 30 min in assay medium containing Hoechst 33342 (final concentration 1 μM) with or without inhibitor as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of Hoechst 33342 was measured at an excitation wavelength of 370 nm and an emission wavelength of 450 nm. Data were analysed using ANOVA and Dunnett’s post hoc test and are expressed as mean ± standard deviation of six replicate wells in a single experiment. RFU: relative fluorescence units. n.s. non-significant.

3.6 Determination of ABCC functional activity in SH-SY5Y cells In order to assess the functional activity of ABCC in SH-SY5Y cells, intracellular accumulation of the fluorescent probe GS-MF was determined in the presence and absence of the ABCC inhibitor MK571. In SH-SY5Y cells treated with MK571 there was a significant, p<0.01, 2.4-fold increase in intracellular accumulation of the ABCC substrate (Figure 3.6), indicating the presence of MK571-sensitive ABCC functional activity in this cell line.

Figure 3.6. Determination of ABCC functional activity in SH-SY5Y cells.

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1 0 0 %

0 l o 1 tr 7 n 5 o K C M

SH-SY5Y cells were seeded (18,000 cells/well) into 96-well plates and incubated for 72 h. Cells were subsequently washed with PBS, equilibrated for 30 min with assay medium (high-glucose, phenol red-free DMEM), and pre-incubated for a further 30 min in medium with or without MK571 (final concentration 25 μM). Cell monolayers were then incubated for 30 min in assay medium containing CMFDA (final concentration 4 μM) with or without MK571 as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of GS-MF was measured at an excitation wavelength of 492 nm and an emission wavelength of 516 nm. Data were analysed using an unpaired Student’s t test and are expressed as mean ± standard deviation of three independent experiments each including at least seven replicate wells. RFU: relative fluorescence units. ** p<0.01.

3.7 Determination of the effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCB1 activity in SH-SY5Y cells Studies were carried out in order to determine whether short-term exposure (30 min) to either CFM-4.16 or CFM-4.17 inhibits ABCB1 activity in the SH-SY5Y cell line. Changes in ABCB1 expression would not be expected in this timeframe. In SH-SY5Y cells treated with 0.04 μM CFM-4.17 for 30 min, there was a significant, p<0.01, 18.2% increase in intracellular accumulation of calcein relative to cells treated with vehicle, indicating that short-term treatment with CFM-4.17 significantly reduced ABCB1 activity (Figure 3.7). Treatment with 0.04 μM CFM-4.16 for the same duration did not result in a significant increase in calcein accumulation (Figure 3.7) indicating that short-term treatment with CFM-4.16 had no significant inhibitory effect on ABCB1 activity.

Figure 3.7. Effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCB1 activity in SH-SY5Y cells.

1 5 0 * *

n .s .

i e

t 1 0 0



. U

F 5 0

e 6 7 l 1 1 ic . . h -4 -4 e V M M F F C C

SH-SY5Y cells were seeded (18,000 cells/well) into 96-well plates and incubated for 72 h. Cells were subsequently washed with PBS, equilibrated for 30 min with assay medium (high-glucose, phenol red-free DMEM), and pre-incubated for a further 30 min with medium containing vehicle, CFM-4.16 or CFM-4.17 (final concentration 0.04 μM). Cell monolayers were then incubated for 30 min in assay medium containing calcein-AM (final concentration 0.5 μM), and vehicle or CFM compound as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of calcein was measured at an excitation wavelength of 484 nm and an emission wavelength of 530 nm. Data were analysed using ANOVA and Dunnett’s post hoc test and are expressed as mean ± standard deviation of four independent experiments, each including at least four replicate wells. RFU: relative fluorescence units. ** p<0.01, n.s. non-significant.

3.8 Determination of the effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCC activity in SH-SY5Y cells Studies were carried out in order to determine whether short-term exposure to either CFM-4.16 or CFM-4.17 inhibits ABCC activity in the SH-SY5Y cell line. In SH-SY5Y cells treated with 0.04 μM CFM-4.16 or CFM-4.17 for 30 min, there was no significant increase in intracellular accumulation of the fluorescent probe GS-MF relative to control cells treated with vehicle (Figure 3.8), indicating that short-treatment with CFM-4.16 or CFM-4.17 had no significant inhibitory effect on ABCC activity in this cell line.

Figure 3.8. Effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCC activity in SH-SY5Y cells.

2 0 0

n .s .

1 -

n n .s .

i 1 5 0

g 1 0 0



F R

5 0 %

e 6 7 l 1 1 ic . . h -4 -4 e V M M F F C C

SH-SY5Y cells were seeded (18,000 cells/well) into 96-well plates and incubated for 72 h. Cells were subsequently washed with PBS, equilibrated for 30 min with assay medium (high-glucose, phenol red-free DMEM), and pre-incubated for a further 30 min with medium containing vehicle, CFM-4.16 or CFM-4.17 (final concentration 0.04 μM). Cell monolayers were then incubated for 30 min in assay medium containing CMFDA (final concentration 4 μM), and vehicle, or CFM-4.16 or CFM-4.17 as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of GS-MF was measured at an excitation wavelength of 492 nm and an emission wavelength of 516 nm. Data were analysed using ANOVA and Dunnett’s post hoc test and are expressed as mean ± standard deviation of four independent experiments, each including at least four replicate wells. RFU: relative fluorescence units. n.s. non-significant.

3.9 Determination of the effect of CFM-4.16 and CFM-4.17 (24 h incubation) on ABCB1 activity in SH-SY5Y cells In order to determine the effects of long-term (24 h) treatment with CFM-4.16 or CFM- 4.17 on ABCB1 activity in the SH-SY5Y cell line, intracellular accumulation of the fluorescent probe calcein was determined in the presence or absence of verapamil and with pre-incubation with either CFM-4.16, CFM-4.17 or vehicle. In SH-SY5Y cells treated with 0.04 μM CFM-4.16 or CFM-4.17 for 24 h there was no significant difference in intracellular accumulation of calcein relative to control cells treated with vehicle (Figure 3.9). Treatment with verapamil (10 µM) was associated with significant 44.4–118.6% increases in calcein accumulation demonstrating the presence of verapamil-sensitive

ABCB1 functional activity (Figure 3.9). There was no significant difference in calcein accumulation in SH-SY5Y cells treated with vehicle and verapamil compared to SH-SY5Y cells treated with CFM-4.16 and verapamil or CFM-4.17 and verapamil (Figure 3.9). These findings indicate that long-term (24 h) treatment with CFM-4.16 or CFM-4.17 had no significant effect on ABCB1 activity, both in the presence and absence of verapamil.

Figure 3.9. Effect of long-term (24 h) treatment with CFM-4.16 or CFM-4.17 on ABCB1 activity in SH-SY5Y cells.

n .s . n .s . A B n .s . n .s . 3 0 0 4 0 0 n .s . n .s .

n .s .

1 -

- n .s .

n i

i 3 0 0 e

e * * * * t

t 2 0 0 * * * * * * * * * * * * *

g 2 0 0



 .

U F

F 1 0 0

1 0 0 % %

0 0 l l l l l l e i 6 i 7 i e i 6 i 7 i l m 1 m 1 m l m 1 m 1 m ic a . a . a ic a . a . a h p -4 p -4 p h p -4 p -4 p e a a a e a a a V r M r M r V r M r M r e F e F e e F e F e V C V C V V C V C V & & & & & & e 6 7 e 6 7 l 1 1 l 1 1 ic . . ic . . h 4 4 h 4 4 e - - e - - V M M V M M F F F F C C C C

SH-SY5Y cells were seeded (18,000 cells/well) into 96-well plates and incubated for 48 h. Cells were then pre-equilibrated with treatment medium (high-glucose, phenol red- free DMEM supplemented with 1% FBS (v/v) for 30 min. The medium was subsequently aspirated, and medium containing vehicle, CFM-4.16 or CFM-4.17 (0.04 µM) added. SH- SY5Y cells were incubated for 24 h, washed with PBS, equilibrated for 30 min with assay medium (high-glucose, phenol red-free DMEM), and pre-incubated for a further 30 min in medium with or without verapamil (final concentration 10 μM). Cell monolayers were then incubated for 30 min in assay medium containing calcein-AM (final concentration 0.5 μM), and vehicle, CFM-4.16, CFM-4.17 or verapamil as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of calcein was measured at an excitation wavelength of 484 nm and an emission wavelength of 530 nm. Data were analysed using ANOVA and Tukey’s post hoc test and are expressed as mean ± standard deviation of six replicate wells in each experiment. A) and B) each represent data from individual independent experiments. RFU: relative fluorescence units. * p<0.05, ** p<0.01, **** p<0.0001, n.s. non-significant.

3.10 Determination of the effect of CFM-4.16 and CFM-4.17 (48 h incubation) on ABCB1 activity in SH-SY5Y cells In order to determine the effects of long-term (48 h) treatment with CFM-4.16 or CFM- 4.17 on ABCB1 activity in the SH-SY5Y cell line, intracellular accumulation of the fluorescent probe calcein was determined in the presence or absence of verapamil and with pre-incubation with either CFM-4.16, CFM-4.17 or vehicle.

In SH-SY5Y cells treated with 0.04 μM CFM-4.16 or CFM-4.17 for 48 h there was no significant difference in intracellular accumulation of calcein relative to cells treated with vehicle (Figure 3.10). Treatment with verapamil (10 µM) was associated with significant 83.3–289.6% increases in calcein accumulation (Figure 3.10), again demonstrating the presence of verapamil-sensitive ABCB1 functional activity. In the presence of verapamil, CFM-4.16 elicited variable effects on calcein accumulation, however there was no significant difference in calcein accumulation in SH-SY5Y cells treated with vehicle and verapamil compared to SH-SY5Y cells treated with CFM-4.17 and verapamil (Figure 3.10). These findings indicate that long-term (48 h) treatment with CFM-4.16 or CFM-4.17 had no significant consistent effect on ABCB1 activity, both in the presence and absence of verapamil.

Figure 3.10. Effect of long-term (48 h) treatment with CFM-4.16 or CFM-4.17 on ABCB1 activity in SH-SY5Y cells.

n .s . A n .s . B 1 0 0 0 4 0 0 * * * * n .s . n .s .

n .s . 1

1 n .s . -

8 0 0 - n

n .s . n i

i 3 0 0 e

e * * * * t

* * t o

n .s . o * * * * r

6 0 0 r * * * *

* g

g 2 0 0



 .

4 0 0 .

U F

1 0 0

2 0 0

% %

SH-SY5Y cells were seeded (18,000 cells/well) into 96-well plates and incubated for 48 h. Cells were then pre-equilibrated with treatment medium (high-glucose, phenol red- free DMEM supplemented with 1% FBS (v/v) for 30 min. The medium was subsequently aspirated, and medium containing vehicle, CFM-4.16 or CFM-4.17 (0.04 µM) added. SH- SY5Y cells were incubated for 48 h, washed with PBS, equilibrated for 30 min with assay medium (high-glucose, phenol red-free DMEM), and pre-incubated for a further 30 min in medium with or without verapamil (final concentration 10 μM). Cell monolayers were then incubated for 30 min in assay medium containing calcein-AM (final concentration 0.5 μM), and vehicle, CFM-4.16, CFM-4.17, or verapamil as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of calcein was measured at an excitation wavelength of 484 nm and an emission wavelength of 530 nm. Data were analysed using ANOVA and Tukey’s post hoc test and are expressed as mean ± standard deviation of six replicate wells in each experiment. A) and B) each represent data from individual independent experiments. RFU: relative fluorescence units. * p<0.05, ** p<0.01, **** p<0.0001, n.s. non-significant.

4 Results 2 (Porcine brain endothelial cell viability and functional assays)

4.1 Visualisation of the morphology of porcine brain endothelial cells The appearance of PBECs on day 7 of culture was determined using phase contrast microscopy (Figure 4.1). By this stage, PBECs had differentiated to exhibit characteristic elongated fusiform spindle-like morphology and formed a near-confluent cell monolayer. These findings were consistent in samples derived from multiple isolations.

Figure 4.1. Morphology of porcine brain endothelial cells.

A B

Phase contrast photograph of a PBEC monolayer at 7 days post-seeding. A 1 mL aliquot of microvessel suspension isolated from porcine brain was centrifuged (5 min, 330 xg) and the pellet resuspended in 6 mL PBEC growth medium (phenol red-free low glucose DMEM supplemented with 10% (v/v) PDS, 1% (v/v) penicillin/streptomycin, 2 mM L- glutamine, 125 μg/mL heparin). The resultant suspension was seeded into a collagen/fibronectin-treated 6-well plate (1 mL/well) and cultured for 48 h. PBECs were subsequently treated with medium supplemented with puromycin 4 μg/mL for 48 h, the medium was then replaced with a 1:1 (v/v) mixture of growth medium:astrocyte- conditioned medium (ACM) (2 mL/well) and cells were incubated for a further 72 h prior to imaging with a Nikon Eclipse TE2000-U inverted microscope. Figure 4.1A, 4x magnification; Figure 4.1B, 20x magnification. Scale bar 100 μm.

4.2 Evaluation of the effect of CFM-4.16 and CFM-4.17 on the viability of porcine brain endothelial cells In order to identify a non-toxic concentration of CFM-4.16 and CFM-4.17 for use in subsequent assays of transporter activity in PBECs, the effects of 24 h and 48 h exposure to CFM-4.16 and CFM-4.17 on the viability of PBECs were determined.

Following a 24 h treatment, CFM-4.16 and CFM-4.17 both had no significant effect on the viability of PBECs between 1.25 μM and 20 μM despite non-significant trends towards reduced viability at 10 μM and 20 μM (Figure 4.2A,B). Following a 48 h treatment, CFM-4.16 had no significant effect on the viability of PBECs between 1.25 μM and 10 μM but produced a significant (p<0.01) 31.0% reduction in viability at 20 μM (Figure 4.2C). A 48 h treatment with CFM-4.17 had no significant effect on the viability of PBECs between 1.25 μM and 20 μM (Figure 4.2D).

A concentration of 5 μM of both CFM-4.16 and CFM-4.17 was initially selected for PBEC transporter assays. However, following initial results indicating less protein with the Bradford assay in cells treated with either compound for 24 h or 48 h versus cells treated with vehicle, which may have reflected toxicity, a lower concentration of 1.25 μM was employed going forward.

Figure 4.2. The effect of CFM-4.16 and CFM-4.17 on the viability of PBECs.

A 2 0 0 B 2 0 0

1 5 0 1 5 0

y y

t t

i i

l l

i i b

b a

a 1 0 0 1 0 0

i i

V V

% % 5 0 5 0

0 0

0 5 .5 5 0 0 0 5 .5 5 0 0 .2 2 1 2 .2 2 1 2 1 1 C F M -4 .1 6 C o n c e n tra tio n ( M ) C F M -4 .1 7 C o n c e n tra tio n ( M )

C 2 0 0 D 2 0 0

1 5 0 1 5 0

y y

t t

i i

l l

i i

b b a

a 1 0 0 1 0 0 i

V V

* *

% % 5 0 5 0

0 0

0 5 .5 5 0 0 0 5 .5 5 0 0 .2 2 1 2 .2 2 1 2 1 1

C F M -4 .1 6 C o n c e n tra tio n ( M ) C F M -4 .1 7 C o n c e n tra tio n ( M )

PBECs were seeded (25,000 cells/well) in 96-well plates and incubated for 48 h. Cells were then treated with treatment medium containing either vehicle or the indicated concentration (1.25-20 μM) of (A, C) CFM-4.16 or (B, D) CFM-4.17. Following 24 h (A, B) or 48 h (C, D) exposure, the neutral red assay was carried out and fluorescence quantified using an excitation wavelength of 530 nm and an emission wavelength of 645 nm. Data were analysed using ANOVA and Dunnett’s post hoc test and are expressed as mean ± standard deviation of at least two independent experiments, each with at least three replicate wells. ** p<0.01 relative to the vehicle control. All other comparisons with the control lacked statistical significance.

4.3 Determination of the effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCB1 activity in PBECs In order to assess the effect of short-term treatment with CFM-4.16 or CFM-4.17 on functional activity of ABCB1 in PBECs, intracellular accumulation of the fluorescent probe calcein was determined in the presence of vehicle, CFM-4.16 or CFM-4.17. In PBECs treated with 1.25 μM CFM-4.16 or CFM-4.17 for 30 min there was no significant increase in intracellular accumulation of calcein relative to control cells treated with 76 vehicle (Figure 4.3) indicating that short-term treatment with CFM-4.16 or CFM-4.17 had no significant inhibitory effect on ABCB1 activity in this cell type.

Figure 4.3. Effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCB1 activity in PBECs.

2 0 0

n .s .

- n

i 1 5 0 n .s .

g 1 0 0



F R

5 0 %

e 6 7 l 1 1 ic . . h -4 -4 e V M M F F C C

PBECs were seeded (25,000 cells/well) into 96-well plates and incubated for 48 h. Cells were subsequently washed with PBS, equilibrated for 30 min with assay medium (low- glucose, phenol red-free DMEM supplemented with 1% (v/v) glutamine), and pre- incubated for a further 30 min with medium containing vehicle, CFM-4.16 or CFM-4.17 (final concentration 1.25 μM). Cell monolayers were then incubated for 30 min in assay medium containing calcein-AM (final concentration 0.5 μM) and vehicle, CFM-4.16, or CFM-4.17 as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of calcein was measured at an excitation wavelength of 484 nm and an emission wavelength of 530 nm. Data were analysed using ANOVA and Dunnett’s post hoc test and are expressed as mean ± standard deviation of five independent experiments, each including at least three replicate wells. RFU: relative fluorescence units. n.s. non-significant.

4.4 Determination of the effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCG2 activity in PBECs In order to assess the effect of short-term treatment with CFM-4.16 or CFM-4.17 on functional activity of ABCG2 in PBECs, intracellular accumulation of the fluorescent probe Hoechst 33342 was determined in the presence of vehicle, CFM-4.16 or CFM-4.17. In PBECs treated with 1.25 μM CFM-4.16 or CFM-4.17 for 30 min there was no significant increase in intracellular accumulation of Hoechst 33342 relative to control cells treated

77 with vehicle (Figure 4.4) indicating that short-term treatment with CFM-4.16 or CFM- 4.17 had no significant inhibitory effect on ABCG2 activity in this cell type.

Figure 4.4. Effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCG2 activity in PBECs.

2 0 0 n .s .

n .s .

- n

i 1 5 0

g 1 0 0



F R

5 0 %

e 6 7 l 1 1 ic . . h -4 -4 e V M M F F C C

PBECs were seeded (25,000 cells/well) into 96-well plates and incubated for 48 h. Cells were subsequently washed with PBS, equilibrated for 30 min with assay medium (low- glucose, phenol red-free DMEM supplemented with 1% (v/v) glutamine), and pre- incubated for a further 30 min with medium containing vehicle, CFM-4.16 or CFM-4.17 (final concentration 1.25 μM). Cell monolayers were then incubated for 30 min in assay medium containing Hoechst 33342 (final concentration 1 μM) and vehicle, CFM-4.16 or CFM-4.17 as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of Hoechst 33342 was measured at an excitation wavelength of 370 nm and an emission wavelength of 450 nm. Data were analysed using ANOVA and Dunnett’s post hoc test and are expressed as mean ± standard deviation of four independent experiments, each including at least three replicate wells. RFU: relative fluorescence units. n.s. non- significant.

4.5 Determination of the effect of CFM-4.16 and CFM-4.17 (24 h incubation) on ABCB1 activity in PBECs In order to determine the effects of long-term (24 h) treatment with CFM-4.16 or CFM- 4.17 on ABCB1 activity in PBECs, intracellular accumulation of the fluorescent probe calcein was determined in the presence or absence of verapamil and with pre- incubation with either CFM-4.16, CFM-4.17 or vehicle. In PBECs treated with 1.25 μM CFM-4.16 or CFM-4.17 for 24 h there was no significant difference in intracellular

78 accumulation of calcein relative to control cells treated with vehicle (Figure 4.5). Treatment with verapamil (10 μM) was associated with significant 7.9–13.5 fold increases in calcein accumulation (Figure 4.5), demonstrating the presence of verapamil- sensitive ABCB1 functional activity. Furthermore, there was no significant difference in calcein accumulation in PBECs treated with vehicle and verapamil compared to PBECs treated with CFM-4.16 and verapamil or CFM-4.17 and verapamil (Figure 4.5). These findings indicate that long-term (24 h) treatment with CFM-4.16 or CFM-4.17 had no significant effect on ABCB1 activity, both in the presence and absence of verapamil.

Figure 4.5. Effect of long-term (24 h) treatment with CFM-4.16 or CFM-4.17 on ABCB1 activity in PBECs.

n .s . n .s . n .s . n .s . A n .s . B n .s . 2 0 0 0 n .s . 2 0 0 0 * * *

n .s . * * * * 1

1 * * * * -

- * * * * * * *

n i

i 1 5 0 0 1 5 0 0

r p

p * * *

g 1 0 0 0 1 0 0 0



5 0 0 5 0 0

% %

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n .s .

- n i 1 5 0 0

n .s .

e t

r p

* * * *

g 1 0 0 0 * * * * 

. * * * *

F R

5 0 0 % 0 l l l e i 6 i 7 i l m 1 m 1 m ic a . a . a h p -4 p -4 p e a a a V r M r M r e F e F e V C V C V & & & e 6 7 l 1 1 ic . . h 4 4 e - - V M M F F C C

PBECs were seeded (25,000 cells/well) into 96-well plates and incubated for 48 h. Cells were then pre-equilibrated with treatment medium (low-glucose, phenol red-free DMEM supplemented with 1% (v/v) FBS and 1% (v/v) glutamine) for 30 min. The medium was subsequently aspirated, and medium containing vehicle, CFM-4.16 or CFM-4.17 (1.25 µM) added. PBECs were incubated for 24 h, washed with PBS, equilibrated for 30 min with assay medium (low-glucose, phenol red-free DMEM supplemented with 1% (v/v) glutamine), and pre-incubated for a further 30 min in medium with or without verapamil (final concentration 10 μM). Cell monolayers were then incubated for 30 min in assay medium containing calcein-AM (final concentration 0.5 μM) and vehicle, CFM- 4.16, CFM-4.17 or verapamil as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of calcein was measured at an excitation wavelength of 484 nm and an emission wavelength of 530 nm. Three independent experiments were carried out (A-C). Data were analysed using ANOVA and Tukey’s post hoc test and are expressed as mean ± standard deviation of at least three replicate wells within each experiment. RFU: relative fluorescence units. *** p<0.001, **** p<0.0001, n.s. non-significant.

4.6 Determination of the effect of CFM-4.16 and CFM-4.17 (48 h incubation) on ABCB1 activity in PBECs In order to determine the effects of long-term (48 h) treatment with CFM-4.16 or CFM- 4.17 on ABCB1 activity in PBECs, intracellular accumulation of the fluorescent probe calcein was determined in the presence or absence of verapamil and with pre- incubation with either CFM-4.16, CFM-4.17 or vehicle.

In PBECs treated with 1.25 μM CFM-4.16 or CFM-4.17 for 48 h there was no significant difference in intracellular accumulation of calcein relative to control cells treated with vehicle (Figure 4.6). Treatment with verapamil (10 μM) was associated with significant 6.8–16.4 fold increases in calcein accumulation (Figure 4.6), further evidencing the presence of verapamil-sensitive ABCB1 functional activity. Furthermore, there was no significant difference in calcein accumulation in PBECs treated with vehicle and verapamil compared to PBECs treated with CFM-4.16 and verapamil or CFM-4.17 and verapamil (Figure 4.6). These findings indicate that long-term (48 h) treatment with CFM-4.16 or CFM-4.17 had no significant effect on ABCB1 activity, both in the presence and absence of verapamil.

Figure 4.6. Effect of long-term (48 h) treatment with CFM-4.16 or CFM-4.17 on ABCB1 activity in PBECs.

n .s . n .s . n .s . A n .s . B 3 0 0 0 * * 3 0 0 0 n .s . n .s .

n .s .

1 -

* * -

n i * i

n .s .

e t

2 0 0 0 t 2 0 0 0

* * * *

* * * * * * * *



 .

U F

1 0 0 0 F 1 0 0 0

% %

3 0 0 0 C n .s .

n .s . 1

- n

i n .s . e

t 2 0 0 0

r p

n .s .

 .

U * * * * * * * * * * * *

F 1 0 0 0

0 l l l e i 6 i 7 i l m 1 m 1 m ic a . a . a h p -4 p -4 p e a a a V r M r M r e F e F e V C V C V & & & e 6 7 l 1 1 ic . . h 4 4 e - - V M M F F C C PBECs were seeded (25,000 cells/well) into 96-well plates and incubated for 48 h. Cells were then pre-equilibrated with treatment medium (low-glucose, phenol red-free DMEM supplemented with 1% (v/v) FBS and 1% (v/v) glutamine) for 30 min. The medium was subsequently aspirated, and medium containing vehicle, CFM-4.16 or CFM-4.17 (1.25 µM) added. PBECs were subsequently incubated for 48 h, washed with PBS, equilibrated for 30 min with assay medium (low-glucose, phenol red-free DMEM supplemented with 1% (v/v) glutamine), and pre-incubated for a further 30 min in medium with or without verapamil (final concentration 10 μM). Cell monolayers were then incubated for 30 min in assay medium containing calcein-AM (final concentration 0.5 μM) and vehicle, CFM-4.16, CFM-4.17 or verapamil as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of calcein was measured at an excitation wavelength of 484 nm and an emission wavelength of 530 nm. Three independent experiments were carried out (A-C). Data were analysed using ANOVA and Tukey’s post hoc test are expressed as mean ± standard deviation of at least three replicate wells in each experiment. RFU: relative fluorescence units. * p<0.05, ** p<0.01, **** p<0.0001, n.s. non-significant.

4.7 Determination of the effect of CFM-4.16 and CFM-4.17 (24 h incubation) on ABCG2 activity in PBECs In order to determine the effects of long-term (24 h) treatment with CFM-4.16 or CFM- 4.17 on ABCG2 activity in PBECs, intracellular accumulation of the fluorescent probe Hoechst 33342 was determined in the presence or absence of Ko143 and with pre- incubation with either CFM-4.16, CFM-4.17 or vehicle. In PBECs treated with 1.25 μM CFM-4.16 or CFM-4.17 for 24 h there was no significant difference in intracellular accumulation of Hoechst 33342 relative to control cells treated with vehicle (Figure 4.7). Treatment with Ko143 (0.5 μM) was associated with significant 63.0–103.4% increases in Hoechst 33342 accumulation (Figure 4.7), demonstrating the presence of Ko143- sensitive ABCG2 functional activity. There was no significant difference in Hoechst 33342 accumulation in PBECs treated with vehicle and Ko143 compared to PBECs treated with CFM-4.16 and Ko143 or CFM-4.17 and Ko143 (Figure 4.7). These findings indicate that long-term (24 h) treatment with CFM-4.16 or CFM-4.17 had no significant effect on ABCG2 activity, both in the presence and absence of Ko143.

Figure 4.7. Effect of long-term (24 h) treatment with CFM-4.16 or CFM-4.17 on ABCG2 activity in PBECs.

6 0 0 A 6 0 0 B n .s . n .s .

n .s .

1 1 - - n .s .

n .s .

n n

i i

e e t t n .s .

4 0 0 4 0 0 n .s .

o o

r r p

p n .s .

g * * * * 

 * *

. . U

U * * * * * F

F 2 0 0 * 2 0 0

R R

% %

0 0

e 3 6 3 7 3 e 3 6 3 7 3 l 4 1 4 1 4 l 4 1 4 1 4 ic 1 . 1 . 1 ic 1 . 1 . 1 h o -4 o -4 o h o -4 o -4 o e K K K e K K K V M M V M M & F & F & & F & F & e C 6 C 7 e C 6 C 7 l 1 1 l 1 1 ic . . ic . . h -4 -4 h -4 -4 e e V M M V M M F F F F C C C C

PBECs were seeded (25,000 cells/well) into 96-well plates and incubated for 48 h. Cells were then pre-equilibrated with treatment medium (low-glucose, phenol red-free DMEM supplemented with 1% (v/v) FBS and 1% (v/v) glutamine) for 30 min. The medium was subsequently aspirated, and medium containing vehicle, CFM-4.16 or CFM-4.17 (1.25 µM) added. PBECs were incubated for 24 h, washed with PBS, equilibrated for 30 min with assay medium (low-glucose, phenol red-free DMEM supplemented with 1% (v/v) glutamine), and pre-incubated for a further 30 min in medium with or without Ko143 (final concentration 0.5 μM). Cell monolayers were then incubated for 30 min in assay medium containing Hoechst 33342 (final concentration 1 μM) and vehicle, CFM- 4.16, CFM-4.17, or Ko143 as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of Hoechst 33342 was measured at an excitation wavelength of 370 nm and an emission wavelength of 450 nm. Two independent experiments were carried out (A-B). Data were analysed using ANOVA and Tukey’s post hoc test and are expressed as mean ± standard deviation of at least three replicate wells within each experiment. RFU: relative fluorescence units. * p<0.05, ** p<0.01, *** p<0.001, n.s. non-significant.

4.8 Determination of the effect of CFM-4.16 and CFM-4.17 (48 h incubation) on ABCG2 activity in PBECs In order to determine the effects of long-term (48 h) treatment with CFM-4.16 or CFM- 4.17 on ABCG2 activity in PBECs, intracellular accumulation of the fluorescent probe Hoechst 33342 was determined in the presence or absence of Ko143 and with pre- incubation with either CFM-4.16, CFM-4.17 or vehicle. In PBECs treated with 1.25 μM CFM-4.16 or CFM-4.17 for 48 h there was no significant difference in intracellular accumulation of Hoechst 33342 relative to control cells treated with vehicle (Figure 4.8).

Treatment with Ko143 (0.5 μM) was associated with significant 2.2 – 3.2 fold increases in Hoechst 33342 accumulation (Figure 4.8) again evidencing the presence of Ko143- sensitive ABCG2 functional activity. In the presence of Ko143, there was no consistent increase in Hoeschst 33342 accumulation in PBECs treated with either CFM-4.16 or CFM- 4.17 compared with vehicle (Figure 4.8).

Figure 4.8. Effect of long-term (48 h) treatment with CFM-4.16 or CFM-4.17 on ABCG2 activity in PBECs.

n .s . * A B n .s . 5 0 0 n .s . 5 0 0 n .s .

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PBECs were seeded (25,000 cells/well) into 96-well plates and incubated for 48 h. Cells were then pre-equilibrated with treatment medium (low-glucose, phenol red-free DMEM supplemented with 1% (v/v) FBS and 1% (v/v) glutamine) for 30 min. The medium was subsequently aspirated, and medium containing vehicle, CFM-4.16 or CFM-4.17 (1.25 µM) added. PBECs were incubated for 48 h, washed with PBS, equilibrated for 30 min with assay medium (low-glucose, phenol red-free DMEM supplemented with 1% (v/v) glutamine), and pre-incubated for a further 30 min in medium with or without Ko143 (final concentration 0.5 μM) and vehicle, CFM-4.16, CFM-4.17, or Ko143 as appropriate. Cell monolayers were then incubated for 30 min in assay medium containing Hoechst 33342 (final concentration 1 μM). The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of Hoechst 33342 was measured at an excitation wavelength of 370 nm and an emission wavelength of 450 nm. Four independent experiments were carried out (A-D). Data were analysed using ANOVA and Tukey’s post hoc test and are expressed as mean ± standard deviation of at least three replicate wells in each experiment. RFU: relative fluorescence units. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, n.s. non- significant.

4.9 Assessment of whether a washout period modifies the effects of CFM- 4.16 or CFM-4.17 on ABCB1 or ABCG2 functional activities in PBECs A washout period may permit dissociation of CFM-4.16 and CFM-4.17 from the ABCB1 or ABCG2 transporter prior to the evaluation of ABCB1 or ABCG2 activity, potentially allowing chronic regulatory effects of CFM-4.16 and CFM-4.17 to be distinguished from any acute inhibitory effects of these compounds. Therefore, preliminary experiments were carried out to determine whether a 5 h washout period modifies the effects of long-term (24 h or 48 h) treatment with CFM-4.16 or CFM-4.17 on ABCB1 or ABCG2 activity in PBECs.

Following inclusion of a washout period in the experimental protocol, long-term treatment with either compound remained without significant effect on the activity of ABCB1 or ABCG2 transporters (Appendix A).

5 Results 3 (Transwell® studies)

5.1 Measurement of transendothelial electrical resistance of PBEC monolayers In order to determine the integrity of PBEC monolayers seeded onto Transwell® inserts, the TEER was measured. There was no significant difference between TEER values of cell monolayers on days 2 and 3 of culture on Transwell® inserts (Figure 5.1). Mean TEER values were recorded as 460 ± 164 Ω.cm2 and 534 ± 145 Ω.cm2 respectively (Figure 5.1). Following 24 h incubation of the monolayers in switch medium, mean TEER increased to 866 ± 122 Ω.cm2 on day 4 of culture (p<0.001 vs day 3; p<0.0001 vs day 2) indicating significantly increased monolayer integrity (Figure 5.1).

Figure 5.1. Transendothelial Electrical Resistance of PBEC monolayers.

c n

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i 1 0 0 0

)

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5.2 Measurement of the apparent permeability of PBEC monolayers to Lucifer yellow In order to further characterise the restrictiveness of PBEC monolayers seeded onto

Transwell® inserts, the apparent permeability coefficient (Papp) of the paracellular pathway marker Lucifer yellow was measured in the apical to basolateral direction. The mean Papp for the blank collagen/fibronectin-coated Transwell® inserts (Pfilter) was 4.6- fold higher than for Transwell® inserts containing cell monolayers (Ptotal), indicating reduced permeability in the presence of the PBEC monolayer (Table 5.1). The net

-7 apparent permeability coefficient of the PBEC monolayer (Pendothelial) was 10.5 x 10 ± 5.4 x 10-7 cm.s-1 (Table 5.1).

Table 5.1. Apparent permeability of PBEC monolayers to Lucifer yellow*.

-1 -1 -1 Ptotal (cm.s ) Pfilter (cm.s ) Fold Difference† Pendothelial‡ (cm.s ) 6.6 x 10-7 3.0 x 10-6 4.58 10.5 x 10-7 ± 1.7 x 10-7 ± 2.7 x 10-6 ± 5.4 x 10-7

† Ratio of Pfilter and Ptotal

‡ Calculated using the equation: 1/Pendothelial = 1/Ptotal – 1/Pfilter *Apparent permeability coefficients are expressed as mean ± standard deviation of seven independent experiments each employing one individual blank Transwell® insert and one individual cell-covered Transwell® insert.

5.3 Determination of the absorption characteristics of CFM-4.16 and CFM- 4.17 In order to determine whether CFM-4.16 and CFM-4.17 exhibit detectable absorbance at a specific wavelength, the absorption characteristics of both compounds and corresponding vehicle controls were obtained (Figure 5.2).

The absorbance spectra of 1 or 100 μM CFM-4.16 and CFM-4.17 dissolved in vehicle showed absorbance at approximately ≤290 nm (Figure 5.2). However spectra of CFM- 4.16 and CFM-4.17 were indistinguishable from the spectrum of the vehicle control, suggesting no significant absorbance of CFM-4.16 and CFM-4.17 at this wavelength 89

(Figure 5.2). The wavelength of maximal absorbance is also in the range expected for DMSO, a constituent of the vehicle.

Since no unique peak in absorbance was identifiable which would have allowed quantification of CFM-4.16 and CFM-4.17 in Transwell®-based studies to investigate penetration of the compounds across PBEC monolayers, indirect measurements of the penetration of CFM-4.16 and CFM-4.17 were employed.

Figure 5.2. Absorption Spectra of CFM-4.16 and CFM-4.17.

The absorption characteristics of CFM-4.16 and CFM-4.17 were evaluated at two concentrations A) 100 µM and B) 1 µM. Stock solutions were prepared in DMSO and diluted 10-fold in ethanol and further diluted in transport buffer to final concentrations of 1 or 100 µM. Samples (100 µL/well) were added to 96-well plates and absorbance spectra determined. Data presented are from one experiment.

5.4 Indirect measurement of the penetration of CFM-4.16 across an in vitro model of the blood-brain-barrier In order to determine whether CFM-4.16 penetrates PBEC monolayers housed on Transwell® inserts, CFM-4.16 was applied to the apical chamber at a concentration of 1.25 µM, established in the present work to be cytotoxic to SH-SY5Y cells but non-toxic 90 to PBECs. Following incubation, the effects of apical and basolateral samples on SH-SY5Y viability were quantified as an indirect measure of CFM-4.16 concentrations (Figure 5.3).

There was a significant 44.2 ± 17.0% reduction in cell viability in SH-SY5Y cells exposed to apical samples from CFM-4.16-treated Transwell® inserts versus vehicle-treated Transwell® inserts (p<0.001) (Figure 5.3). However the 15.6 ± 9.2% reduction in viability with basolateral samples from CFM-4.16-treated Transwell® inserts versus vehicle- treated Transwell® inserts was not statistically significant (Figure 5.3), consistent with no significant penetration of CFM-4.16 across the PBEC monolayer (or penetration to such a low extent that did not significantly affect cell viability). CFM-4.16 (1.25 µM), when applied directly to SH-SY5Y cells as a control, resulted in a significant 47.7 ± 10.0% reduction in viability relative to the vehicle equivalent (p<0.01) (Figure 5.3).

Figure 5.3. Indirect measurement of the penetration of CFM-4.16 across an in vitro model of the blood-brain barrier.

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1 5 0

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t i

l * *

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0 ) l) l) l) l) l a a a a ro M c c r r t µ i i e e n p p t t 5 a a o 2 A (A l l C . ( o o 1 e 6 s s e ( l a a l l c .1 ic i B B ro h -4 ( ( h t e e 6 e n V M l 1 V o F ic . C C h -4 e 6 V M .1 F 4 C - M F C

PBECs were seeded onto Transwell® inserts and maintained in co-culture with the CTX- TNA2 rat astrocyte cell line in 1:1 (v/v) growth medium:astrocyte-conditioned medium for three days. On day 3 of culture, astrocytes were removed and PBECs maintained in switch medium (low glucose, phenol red-free DMEM supplemented with 312.5 µM 8-4- chlorophenylthio-cAMP, 17.5 µM RO-20-1724, 55 nM hydrocortisone and 1% v/v penicillin/streptomycin) for 24 h. On day 4 of culture, Transwell® inserts were washed with PBS and 1.5 mL transport buffer added to the basolateral compartment. 500 µL of transport buffer containing either CFM-4.16 (1.25 μM) or solvent was added to the apical compartment. Following 6 h incubation at 37 oC, the transport buffer was collected and added (200 µL/well) to monolayers of SH-SY5Y cells pre-seeded into 96- well plates. Following 24 h exposure, the MTT cell viability assay was carried out and absorbance quantified at 570 nm. Percentage viabilities were normalised to a transport buffer control. Data were analysed using ANOVA and Tukey’s post hoc test. Data from apical and basolateral samples are expressed as mean ± standard deviation of four independent experiments, each of which included at least three Transwell® inserts per condition. Data from directly applied CFM-4.16 and vehicle controls are expressed as mean ± standard deviation of three independent experiments, each of which included at least seven wells of a 96 well plate per condition. ** p<0.01, *** p<0.001, n.s. non- significant.

5.5 Indirect measurement of the penetration of CFM-4.17 across an in vitro model of the blood-brain-barrier In order to determine whether CFM-4.17 penetrates PBEC monolayers housed on Transwell® inserts, CFM-4.17 was applied to the apical chamber at a concentration of 5 µM. Following incubation, the effects of apical and basolateral samples on SH-SY5Y viability were quantified as an indirect measure of CFM-4.17 concentrations (Figure 5.4). Apical samples from CFM-4.17-treated Transwell® inserts resulted in a 17.8 ± 16.4% non- significant reduction in SH-SY5Y viability relative to vehicle-treated Transwell® inserts (Figure 5.4). Basolateral samples from CFM-4.17-treated Transwell® inserts also resulted in a 7.9 ± 5.5% non-significant reduction in SH-SY5Y viability relative to vehicle-treated Transwell® inserts (Figure 5.4), consistent with no significant penetration of CFM-4.17 across the PBEC monolayer (or penetration to such a low extent that did not significantly affect cell viability). When applied directly to SH-SY5Y cells as a control, CFM-4.17 (5 μM) resulted in a significant 29.5 ± 6.5% reduction in viability relative to the vehicle equivalent (p<0.01) (Figure 5.4).

Figure 5.4. Indirect measurement of the penetration of CFM-4.17 across an in vitro model of the blood-brain barrier.

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n .s .

1 5 0 n .s .

t i

l * *

i b

a 1 0 0

% 5 0

0 ) l) l) l) l) l a a a a ro M c c r r t µ i i e e n p p t t o 5 a a ( (A (A l l C l o o o le 7 s s le tr c 1 a a c n i . B B i h 4 ( ( h o e - 7 e C M le 1 V V c . 7 F i 4 1 C h - . e M -4 V F M C F C

PBECs were seeded onto Transwell® inserts and maintained in co-culture with the CTX- TNA2 rat astrocyte cell line in 1:1 (v/v) growth medium:astrocyte-conditioned medium for three days. On day 3 of culture, astrocytes were removed and PBECs maintained in switch medium (low glucose, phenol red-free DMEM supplemented with 312.5 µM 8-4- chlorophenylthio-cAMP, 17.5 µM RO-20-1724, 55 nM hydrocortisone and 1% v/v penicillin/streptomycin) for 24 h. On day 4 of culture, Transwell® inserts were washed with PBS and 1.5 mL transport buffer added to the basolateral compartment. 500 µL of transport buffer containing CFM-4.17 (A) 1.25 μM or B) 5 μM) or solvent was added to the apical compartment. Following 6 h incubation at 37 oC, the transport buffer was collected and added (200 μL/well) to monolayers of SH-SY5Y cells pre-seeded into 96 well plates. Following 24 h exposure, the MTT cell viability assay was carried out and absorbance quantified at 570 nm. Percentage viabilities were normalised to a transport buffer control. Data were analysed using ANOVA and Tukey’s post hoc test. Data from apical and basolateral samples are expressed as mean ± standard deviation of three Transwell® inserts per condition from a single experiment. Data from directly applied CFM-4.17 and vehicle controls are expressed as mean ± standard deviation of nine individual wells of a 96 well plate per condition from a single experiment. ** p<0.01, n.s. non-significant.

5.6 Indirect measurement of the effect of ABCB1 inhibition on the penetration of CFM-4.16 across an in vitro model of the blood-brain barrier

In order to determine whether efflux of CFM-4.16 by ABCB1 transporters affects penetration of CFM-4.16 across a Transwell®-based in vitro model of the BBB, CFM-4.16 (1.25 µM) was applied to the apical chamber, in the presence or absence of the ABCB1 inhibitor verapamil.

Apical samples from CFM-4.16-treated cell monolayers in Transwell® inserts produced a non-significant 30.7 ± 14.0% reduction in SH-SY5Y viability relative to the corresponding vehicle-treated control (Figure 5.5). Apical samples from cell monolayers treated with both CFM-4.16 and verapamil produced a 24.7 ± 9.7% reduction in SH-SY5Y viability relative to the corresponding vehicle-treated control (Figure 5.5), which was not significantly different from treatment with CFM-4.16 alone.

Once again, in the absence of verapamil, samples from the basolateral compartments of CFM-4.16-treated Transwell® inserts did not significantly reduce SH-SY5Y viability relative to samples from vehicle-treated Transwell® inserts (Figure 5.5), consistent with no significant penetration of CFM-4.16 across PBEC monolayers or penetration to such a low extent that did not significantly affect cell viability.

Analysis of samples from the basolateral compartments revealed there was no significant difference in the viability of SH-SY5Y cells when PBECs monolayers were treated with CFM-4.16 or both CFM-4.16 and verapamil, indicating that inhibition of ABCB1 had no significant effect on penetration of CFM-4.16 across PBEC monolayers (Figure 5.5).

CFM-4.16 (1.25 µM), when applied directly to SH-SY5Y cells as a control, resulted in a non-significant 41.2 ± 20.7% reduction in viability relative to the vehicle equivalent, of comparable magnitude to the statistically significant 47.7 ± 10.0% reduction observed in previous experiments. The lack of statistical significance could be accounted for by experimental variability.

Figure 5.5. Indirect measurement of the effect of ABCB1 inhibition on the penetration of CFM-4.16 across an in vitro model of the blood-brain barrier.

2 0 0 n .s . n .s . n .s . n .s .

1 5 0 n .s . n .s . n .s .

y t

i n .s .

i b

a 1 0 0

i n .s .

% 5 0

0 ) ) ) ) ) l) l l) l l) l l) l l a a a a a o M a c c ra r ra r tr ic i ic i e e µ p p te t te t n p p a a o 5 A A A A la l la l 2 ( ( ( ( o o C . ) ) o o 1 e il 6 il s s s s e ( l a a a a l l c m .1 m ic i B (B B (B ro h a -4 a ( ( h t e p p l) l) e n a M a le i 6 i V V r r c 1 o e F e i m . m C C h a -4 a V V e p p 6 + + V a M a .1 ( ( r F r e e -4 le 6 V C V c .1 M i + + F h -4 ( ( e e 6 C V M l 1 F ic . C h 4 e - V M F C

PBECs were seeded onto Transwell® inserts and maintained in co-culture with the CTX- TNA2 rat astrocyte cell line in 1:1 (v/v) growth medium:astrocyte-conditioned medium for three days. On day 3 of culture, astrocytes were removed and PBECs maintained in switch medium (low glucose, phenol red-free DMEM supplemented with 312.5 µM 8-4- chlorophenylthio-cAMP, 17.5 µM RO-20-1724, 55 nM hydrocortisone and 1% v/v penicillin/streptomycin) for 24 h. On day 4 of culture, Transwell® inserts were washed with PBS and 1.5 mL transport buffer added to the basolateral compartment. A 30 min pre-incubation was then carried out at 37oC after adding 250 µL of transport buffer with or without verapamil (10 µM) to the apical compartment. A further 250 µL of transport buffer containing either CFM-4.16 (1.25 μM) or solvent (and verapamil (10 µM) in those Transwell®s pre-incubated with this inhibitor) was added to the apical compartment. Following 6 h incubation at 37 oC, the transport buffer was collected and added (200 µL/well) to monolayers of SH-SY5Y cells pre-seeded into 96 well plates. Following 24 h exposure, the MTT cell viability assay was carried out and absorbance quantified at 570 nm. Percentage viabilities were normalised to a transport buffer control. Data were analysed using ANOVA and Tukey’s post hoc test. Data from apical and basolateral samples are expressed as mean ± standard deviation of two independent experiments, each of which included at least two Transwell® inserts per condition. Data from directly applied CFM-4.16 and vehicle controls are expressed as mean ± standard deviation of two independent experiments, each of which included at least five wells of a 96 well plate per condition. n.s. non-significant.

5.7 Indirect measurement of the effect of ABCG2 inhibition on the penetration of CFM-4.16 across an in vitro model of the blood-brain barrier

In order to determine whether efflux of CFM-4.16 by ABCG2 transporters affects penetration of CFM-4.16 across a Transwell®-based in vitro model of the BBB, CFM-4.16 (1.25 µM) was applied to the apical chamber, in the presence or absence of the ABCG2 inhibitor Ko143.

Apical samples from CFM-4.16-treated cell monolayers in Transwell® inserts produced a non-significant 26.3 ± 4.7% reduction in SH-SY5Y viability relative to the corresponding vehicle-treated control (Figure 5.6).

Apical samples from cell monolayers treated with both CFM-4.16 and Ko143 did not significantly reduce SH-SY5Y viability relative to the corresponding vehicle-treated control (Figure 5.6). This effect was not significantly different from treatment with CFM- 4.16 alone, accounted for by experimental variability (Figure 5.6).

As before, in the absence of Ko143, samples from the basolateral compartments of CFM- 4.16-treated Transwell® inserts did not significantly reduce SH-SY5Y viability relative to samples from vehicle-treated Transwell® inserts (Figure 5.6), consistent with lack of CFM-4.16 penetration across PBEC monolayers.

Analysis of samples from the basolateral compartments revealed there was no significant difference in the viability of SH-SY5Y cells when PBECs monolayers were treated with CFM-4.16 or both CFM-4.16 and Ko143 (Figure 5.6), indicating that inhibition of ABCG2 had no significant effect on penetration of CFM-4.16 across PBEC monolayers.

CFM-4.16 (1.25 µM), when applied directly to SH-SY5Y cells as a control, resulted in a non-significant 37.8 ± 15.4% reduction in viability relative to the vehicle equivalent (Figure 5.6) comparable in magnitude to the statistically significant 47.7 ± 10.0% reduction observed in initial experiments. The lack of statistical significance could be accounted for by experimental variability.

Figure 5.6. Indirect measurement of the effect of ABCG2 inhibition on the penetration of CFM-4.16 across an in vitro model of the blood-brain barrier.

2 0 0 n .s . n .s . n .s . n .s . 1 5 0 n .s .

n .s . n .s . y

t n .s . n .s .

i b

a 1 0 0

% 5 0

0 ) l) l) l) l) l) l) l) l) l a a a a a a a a ro M c c c c r r r r t µ i i i i e e e e n p p p p t t t t 5 a a a o A A A A la l l l .2 ( ( ( ( o o o C ) ) o s 1 e 6 s s s le ( l 3 1 3 a a a a l ic 4 . 4 ic o 1 1 B (B B (B r h -4 ( ( h t e o o ) 6 ) e n K M K le 3 3 V o V c 1 4 + F + i 4 . C ( C ( h 1 -4 1 e o o 6 e 6 1 l 1 V K M K . ic . F + + -4 h -4 ( C ( e M M le 6 V c .1 F F i C C h 4 e - V M F C

PBECs were seeded onto Transwell® inserts and maintained in co-culture with the CTX- TNA2 rat astrocyte cell line in 1:1 (v/v) growth medium:astrocyte-conditioned medium for three days. On day 3 of culture, astrocytes were removed and PBECs maintained in switch medium (low glucose, phenol red-free DMEM supplemented with 312.5 µM 8-4- chlorophenylthio-cAMP, 17.5 µM RO-20-1724, 55 nM hydrocortisone and 1% v/v penicillin/streptomycin) for 24 h. On day 4 of culture, Transwell® inserts were washed with PBS and 1.5 mL transport buffer added to the basolateral compartment. A 30 min pre-incubation was then carried out at 37oC after adding 250 µL of transport buffer with or without Ko143 (0.5 µM) to the apical compartment. A further 250 µL of transport buffer containing either CFM-4.16 (1.25 μM) or solvent (and Ko143 (0.5 µM) in those Transwell®s pre-incubated with this inhibitor) was added to the apical compartment. Following 6 h incubation at 37 oC, the transport buffer was collected and added (200 µL/well) to monolayers of SH-SY5Y cells pre-seeded into 96 well plates. Following 24 h exposure, the MTT cell viability assay was carried out and absorbance quantified at 570 nm. Percentage viabilities were normalised to a transport buffer control. Data were analysed using ANOVA and Tukey’s post hoc test. Data from apical and basolateral samples are expressed as mean ± standard deviation of two independent experiments, each of which included at least two Transwell® inserts per condition. Data from directly applied CFM-4.16 and vehicle controls are expressed as mean ± standard deviation of two independent experiments, each of which included at least five wells of a 96 well plate per condition. n.s. non-significant.

5.8 Measurement of the effect of ABCB1 or ABCG2 inhibition on CFM-4.16 or CFM-4.17-mediated toxicity in PBECs In order to determine whether CFM-4.16 and CFM-4.17 are substrates of ABCB1 and ABCG2, an alternative experimental approach was employed. This involved measuring the effect of CFM-4.16 or CFM-4.17 on PBEC viability in the presence or absence of the ABCB1 inhibitor verapamil or the ABCG2 inhibitor Ko143. The underlying rationale was that if CFM-4.16 or CFM-4.17 are substrates of ABCB1 or ABCG2, inhibition of the transporters may reduce transporter-mediated efflux of CFM-4.16 or CFM-4.17, leading to increased intracellular accumulation and decreased PBEC viability.

Furthermore, as the concentrations and duration of incubation were identical to those employed in the Transwell® experiments, the experiment was also designed to investigate whether CFM-4.16 may have compromised PBEC viability when co- incubated with either verapamil or Ko143 (section 5.6 and 5.7).

Neither CFM-4.16 (1.25 µM) nor CFM-4.17 (5 µM) elicited a statistically significant effect on PBEC viability relative to vehicle control whether applied in the presence or absence of verapamil or Ko143 (Figure 5.7).

Figure 5.7. The effect of ABCB1 or ABCG2 inhibition on CFM-4.16 or CFM-4.17- mediated toxicity in PBECs.

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% 5 0

0 ) ) ) ) 6 il il le 1 3 3 c . m m 4 4 i 4 a a 1 1 h - p p o o e M a a K K V F r r e e + (+ C V V ( e 6 + + l 1 ( ( ic . 4 le 6 h - c .1 e i V M h -4 F e C V M F C

B 2 0 0

1 5 0

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PBECs were seeded (25,000 cells/well) in 96 well plates and incubated for 48 h. Growth medium was then aspirated and PBECs washed with PBS. PBECs were pre-incubated for 30 min at 37 oC with 100 μL/well of either PBEC treatment medium, PBEC treatment medium containing 10 μM verapamil or PBEC treatment medium containing 0.5 μM Ko143. PBECs were then treated with 100 μL/well of A) CFM-4.16, final concentration 1.25 μM, B) CFM-4.17, final concentration 5 μM, or vehicle. The cells were incubated for a further 6 h, following which the neutral red assay was carried out and fluorescence quantified using an excitation wavelength of 530 nm and an emission wavelength of 645 nm. Percentage viability values were normalised to results from PBECs which were incubated with media alone. Data were analysed using ANOVA and Tukey’s post hoc test and are expressed as mean ± standard deviation of six wells of a 96 well plate within a single experiment. n.s. non-significant.

100

6 Discussion

6.1 Overview Treatment of CNS metastases arising from neuroblastoma represents an unmet clinical need (Matthay et al., 2003). Novel therapeutic agents must not only exhibit cytotoxic effects towards this cell type, but are also required to penetrate the BBB. Furthermore their effects on ABC transporter activity and their transporter substrate-like behaviour may influence their own capacity to access the site of the tumour and retention by tumour cells together with that of concomitantly administered established treatments. The present work evaluated the cytotoxicity of novel compounds CFM-4.16 and CFM- 4.17 in the SH-SY5Y neuroblastoma cell line and in PBECs. Experiments also detected ABCB1 and ABCC but not ABCG2 transporter activity in SH-SY5Y cells and reaffirmed the presence of ABCB1 and ABCG2 transporter activity in PBECs. The effects of short and long-term acute and chronic incubation with CFM-4.16 and CFM-4.17 on transporter activity were subsequently evaluated in both cell types. An in vitro model of the BBB was generated, its barrier properties validated and the penetration of CFM-4.16 and CFM- 4.17 evaluated. Finally the potential for CFM-4.16 and CFM-4.17 to act as substrates for ABCB1 and ABCG2 transporters was explored.

6.2 Effects of CFM-4.16 and CFM-4.17 on the viability of SH-SY5Y cells and PBECs In the present work, CFM-4.16 and CFM-4.17 exhibited potent inhibitory effects on SH- SY5Y cell viability, as determined by the MTT assay. This is the first report of the effects of CFM-4.16 and CFM-4.17 on cell viability in neuroblastoma and also represents the first report of the effects of any of the CARP-1 functional mimetics on the viability of the SH-SY5Y cell line or of PBECs.

The findings were consistent with effects of the CFM-4 lead compound on the SK-N-BE and SK-N-SH neuroblastoma cell lines (Muthu et al., 2014), however the potency was markedly higher in the present work. Muthu et al (2014) reported IC50 values of ≤5 μM following 24 h treatment and a concentration of 20 μM was required to elicit a 70-80% loss of viability (Muthu et al., 2014). However in the present work, concentrations as low

101 as 0.4 µM CFM-4.16 and 0.8 µM CFM-4.17 elicited a 72 ± 1.2 % and 84 ± 7.1 % loss of viability respectively in SH-SY5Y cells.

The effects of CFM-4.16 and CFM-4.17 in SH-SY5Y cells were also consistent with those reported in other cancer cell types. In the A498 and ACHN RCC cell lines the IC50 for CFM-

4.16 was approximately 1 μM, and in the CAKI-1 and CAKI-2 RCC cell lines the IC50 for CFM-4.16 was between 2 μM and 5 μM (CFM-4.17 not reported) (Alsaab et al., 2018;

Cheriyan et al., 2017). The respective IC50 values for CFM-4.16 and CFM-4.17 in the UOK 262 and UOK 268 RCC cell lines are reported to be between 2 μM and 5 μM (Cheriyan et al., 2017). In the A549 and H1299 NSCLC cell lines IC50 values for CFM-4.16 and CFM- 4.17 were approximately 2 μM and 5 μM respectively (Cheriyan et al., 2018), with similar

IC50 values reported in the MDA-MB-468 breast cancer cell line (Cheriyan et al., 2016).

The current studies indicate that CFM-4.16 and CFM-4.17 were more potent than CFM- 4 in attenuating cell growth in neuroblastoma cells, which is likely explained by the fact that chemical modification of the lead compound is associated with enhanced cytotoxicity, particularly of CFM-4.16, in the UOK 262 and UOK 268 RCC cell lines (Cheriyan et al., 2017), the A549 and H1299 NSCLC cell lines (Cheriyan et al., 2018) and the MDA-MB-468 breast cancer cell line (Cheriyan et al., 2016). The data in the current study suggest that SH-SY5Y cells are more susceptible to CFM-4.16 than CFM-4.17, consistent with previous studies reporting CFM-4.16 to be more potent than CFM-4.17 in UOK 262 and UOK 268 RCC cell lines (Cheriyan et al., 2017) and A549 and H1299 NSCLC cell lines (Cheriyan et al., 2018) suggesting that this differential sensitivity to the effects of CFM-4.16 and CFM-4.17 is common to a variety of cancer cells. The presence of a chloride atom in the R1 group of CFM-4.16 versus a methoxy group in CFM-4.17 (Figure 1.5) may underlie these differences.

The effects of CFM-4.16 or CFM-4.17 on the viability of PBECs were quantified using the neutral red assay. The MTT assay was not employed due to the potential confounding issue of the MTT compound acting as a substrate of ABC transporters which are expressed at high density in PBECs. In contrast to studies in SH-SY5Y cells, much higher concentrations of CFM-4.16 or CFM-4.17 were required to elicit equivalent cytotoxic effects. For example, in PBECs, concentrations of 10-20 μM resulted in an approximate 50% reduction in viability whereas similar effects were observed with 0.1-0.2 μM CFM- 4.16 and 0.2-0.4 μM CFM-4.17 in SH-SY5Y cells. 102

Furthermore, CFM-4.16 exhibited only marginally higher potency relative to CFM-4.17 in PBECs whereas in SH-SY5Y cells CFM-4.16 consistently displayed higher potency than CFM-4.17.

The differential sensitivity of PBECs and SH-SY5Y cells to CFM-4.16 or CFM-4.17 is consistent with the potential for a favourable therapeutic index in clinical settings, reflected by the margin between effective and toxic doses. Therapeutic indices of cancer chemotherapeutics, including those employed to treat neuroblastoma, are typically narrow, underscoring the need for more targeted agents with wider indices which CFM- 4.16 and CFM-4.17 may exemplify (Applebaum et al., 2017; Ganeshan and Schor, 2011; Patel and Papachristos, 2015).

The differential sensitivity of PBECs and SH-SY5Y cells to CFM-4.16 or CFM-4.17 is consistent with the differential sensitivity of MDA-MB-231, MDA-MB-468 and SKBR-3 breast cancer cells versus MCF-10A immortalised non-tumorigenic mammary epithelial cells to CFM-4 (Puliyappadamba et al., 2011). Growth of the latter was not inhibited at concentrations up to 20 μM whilst growth of the former was attenuated by >90% at this concentration (Puliyappadamba et al., 2011). The data are also consistent with the 10- fold higher IC50 for CFM-4.16 in renal epithelial cells compared with that for renal carcinoma cells (Cheriyan et al., 2017).

The differential sensitivity of SH-SY5Y cells and PBECs to CFM-4.16 and CFM-4.17 may be attributed to susceptibility of rapidly dividing cancer cells versus primary endothelial cell cultures to the cell cycle-disrupting and pro-apoptotic effects of the CFM compounds.

The CFM compounds disrupt CARP1-mediated co-activation of cell cycle regulatory anaphase promoting complex/cyclosome (APC/C) E3 ligase, which is in turn implicated in cell cycle transitions (Harper et al., 2002; Puliyappadamba et al., 2011; Zachariae and Nasmyth, 1999). This is reflected in previous reports of G2M phase accumulation with the CFM-4 parent compound in neuroblastoma cell lines (Muthu et al., 2014). CFM-4 also causes loss of the mitotic cell cycle regulator cyclin B1 in neuroblastoma cell lines (Jamal et al., 2014). CFM-4.16 similarly effects depletion of cyclin B1 in parental and drug resistant TNBC, NSCLC, and RCC cell lines although similar data for CFM-4.17 have not been published (Cheriyan et al., 2018; Cheriyan et al., 2017; Cheriyan et al., 2016). Rapid

103 proliferation and hence progression through the cell cycle of SH-SY5Y would be expected to increase susceptibility to cell cycle arrest relative to comparatively more quiescent PBECs.

CFM-4 has also been reported to increase DNA fragmentation and expression of cleaved PARP-1 (poly(ADP-ribose) polymerase-1) in neuroblastoma cell lines, both markers of apoptosis (Muthu et al., 2014). Mechanistically, CFM-4 activates pro-apoptotic p38 MAP kinase and JNK and induces expression of apoptosis-promoting CARP-1 (Muthu et al., 2014). CFM-4 also upregulates microRNAs responsible for downregulating inhibitor of apoptosis proteins (IAPs) including XIAP, cIAP1, cIAP2, and survivin proteins (Muthu et al., 2014). IAPs inhibit the activity of caspases 3, 7 and 9 thereby preventing apoptosis (Muthu et al., 2014). CFM-4.16 also augments PARP-1 cleavage, p38 MAP kinase and JNK activation, CARP1 expression and additionally caspase 8 cleavage, in parental and drug resistant TNBC, NSCLC, and RCC cell lines (Cheriyan et al., 2018; Cheriyan et al., 2017; Cheriyan et al., 2016). The effects of CFM-4.17 might be expected to be similar.

Cancer cells may be predicted to be inherently resistant to chemotherapeutic strategies which rely on pro-apoptotic mechanisms given their characteristic evasion of apoptosis and dysregulation of apoptotic or survival signalling cascades (Hanahan and Weinberg, 2011; Taylor et al., 2006). However, scope exists for targeted therapies to re-engage apoptosis (Makin, 2018). Furthermore, despite having a high threshold for apoptosis, cancer cells may be considered primed for cellular death having proliferated without protective survival signals, and being exposed to proapoptotic stress originating from oncogenes and the microenvironment (Makin, 2018; Makin and Dive, 2003).

6.3 Characterisation of SH-SY5Y cell morphology and ABC transporter functional activity The present study reports SH-SY5Y morphology consistent with that previously described, including rounded cell bodies with neuritic processes and formation of dense cell aggregates (Ross et al., 1983). The potential functional activities of ABCB1, ABCG2 and ABCC transporters were also evaluated in this cell line.

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6.4 Determination of ABCB1 functional activity in SH-SY5Y cells Evidence for functionally active ABCB1 transporters in SH-SY5Y cells in this study derives from a 67.8 ± 17.0% increase in calcein accumulation in the presence of verapamil, reflecting inhibition of ABCB1 activity. These findings are consistent with the previously published functional expression of this transporter in the SH-SY5Y cell line. For example ABCB1 protein and mRNA have been detected by Western blot and RT-PCR (Atil et al., 2016; Campos-Arroyo et al., 2016; Chen et al., 2014; Dalzell et al., 2015; Radic-Sarikas et al., 2017). Cell surface ABCB1 protein expression has also been quantified by flow cytometry (Atil et al., 2016; Campos-Arroyo et al., 2016).

ABCB1 functional activity has additionally been demonstrated via verapamil-dependent increases in calcein accumulation although the measurements of calcein flux over time do not permit numerical comparisons with the present work (Sieczkowski et al., 2010). ABCB1 activity has been further determined by measuring rhodamine 123 efflux, initially without verification using an established ABCB1 inhibitor (Atil et al., 2016). A more recent publication using the rhodamine 123 efflux assay reported very similar ~70% increases in intracellular fluorescence in the presence of verapamil to those reported in the present work using the calcein assay (Burger et al., 2018).

6.5 Determination of ABCG2 functional activity in SH-SY5Y cells In the present work, several known inhibitors of ABCG2 failed to increase Hoechst 33342 accumulation, consistent with a lack of functional ABCG2 activity in the SH-SY5Y cell line. These findings are in keeping with a previously reported lack of detectable ABCG2 protein in cell lysates and lack of Ko143-sensitive ABCG2 activity quantified via PpIX accumulation (Barron et al., 2013). The present findings also align with a previously reported lack of effect of Ko143 on Hoechst 33342 accumulation at 0.5 μM Ko143 (Dalzell et al., 2015). Notably these authors did report increased Hoechst 33342 accumulation at concentrations (2-5 μM), 200-500 fold greater than the IC50 for Ko143, which was not replicated in the present study with 5 μM Ko143 (Dalzell et al., 2015). However, in view of the high Ko143 concentrations, these increases were attributed to non-specific effects (Dalzell et al., 2015).

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Some conflicting findings in the literature positively identify ABCG2 in SH-SY5Y. Examples include moderate ABCG2 immunostaining (Acosta et al., 2009) and demonstration of ABCG2 mRNA in SH-SY5Y cells, although normalisation precluded comparisons of absolute levels of different transporters (Atil et al., 2016). Others have similarly detected ABCG2 mRNA and protein expression (Campos-Arroyo et al., 2016; Pandian et al., 2015; Xing et al., 2015). Surface expression of ABCG2 has additionally been identified by flow cytometric analysis, although, at a lower level than for ABCB1 and ABCC transporters where quantified (Campos-Arroyo et al., 2016; Xing et al., 2015). One explanation to reconcile these positive reports of ABCG2 functional expression with the present findings of lack of ABCG2 functional activity is the presence of cancer stem cell subpopulations, within the neuroblastoma cell line, which are characterised by ABCG2 expression (Campos-Arroyo et al., 2016; Pandian et al., 2015; Xing et al., 2015).

6.6 Determination of ABCC functional activity in SH-SY5Y cells The MK571-sensitive ABCC activity demonstrated in the present study, characterised by a 2.4-fold increase in GS-MF accumulation in the presence of MK571, provides additional evidence for the functional expression of ABCC transporters in SH-SY5Y cells. However, given that the substrate CMFDA and inhibitor MK571 lack ABCC subtype specificity, it is not possible to confirm which ABCC isoform(s) are responsible. Expression of ABCC1 in SH-SY5Y cells has been detected at the mRNA and protein levels (Atil et al., 2016; Chen et al., 2014). However in another report, ABCC1 was not detectable via Western blot (Henderson et al., 2011). The presence of ABCC2 protein and mRNA in SH-SY5Y cells has been verified alongside ABCC4 protein (Campos-Arroyo et al., 2016; Henderson et al., 2011). This suggests that several isoforms could potentially contribute to transport of the substrate. CMFDA and MK571 have been employed previously in the SH-SY5Y cell line (Dalzell et al., 2015). Although these data are not directly comparable to the present work as they derive from a range of CFMDA concentrations rather than a single value, increased steepness of the relationship between extracellular CMFDA concentration and intracellular fluorescence in the presence of MK571 was consistent with inhibition of functionally active ABCC transporters (Dalzell et al., 2015).

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6.7 Characterisation and validity of the PBEC model The present study reports that PBECs demonstrate a spindle-like morphology, consistent with previous findings within the host laboratory and reports of others (Cantrill et al., 2012; Smith et al., 2007). The absence of contaminating cells reflects successful isolation procedures designed to enhance cell purity, including dissection of white matter and filtration steps followed by pericyte-eliminating puromycin treatment (Perriere et al., 2005).

Previous studies have validated PBECs as a model of the BBB by demonstrating the functional expression of ABCB1 and ABCG2 transporters. For example, expression in PBECs of both ABCB1 and ABCG2 has been verified at the protein level in the host laboratory (Cantrill et al., 2012; Shubbar and Penny, 2018). Further evidence for ABCB1 and ABCG2 expression at the protein and mRNA levels within the same cell type has been reported by other groups (Eisenblatter et al., 2003; Kido et al., 2002; Mahringer et al., 2009; Patabendige et al., 2013; Smith et al., 2007; von Wedel-Parlow et al., 2009).

The present study found 6.8 to 16.4-fold increases in calcein accumulation in the presence of verapamil, reflecting inhibition of ABCB1 activity, in turn consistent with 5.7- fold and 7.8-fold increases previously determined in the host laboratory (Cantrill et al., 2012; Shubbar and Penny, 2018). Other groups have also similarly reported 5.5 to 6.0- fold increases in calcein accumulation and 7.5-fold increases in rhodamine 123 retention with verapamil pre-treatment (Smith et al., 2007; Steglich et al., 2012).

The present study also found 2.2 to 3.2-fold increases in Hoechst 33342 accumulation in the presence of Ko143, reflecting inhibition of ABCG2 activity, in turn consistent with 2.0-fold and 2.1-fold increases previously observed in the host laboratory (Cantrill et al., 2012; Shubbar and Penny, 2018). An approximately 1.6-fold Ko143-evoked increase in accumulation of mitoxantrone, an ABCG2 substrate, has also been reported (Mahringer et al., 2009). ABCG2 functional activity has also been evidenced previously by the elevation of intracellular Hoechst 33342 accumulation in the presence of other ABCG2 inhibitors such as FTC (von Wedel-Parlow et al., 2009).

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6.8 Determination of the effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCB1 and ABCC activities in SH-SY5Y cells In this study the effects of short-term incubation with CFM-4.16 and CFM-4.17 on ABCB1 and ABCC transporter activities were evaluated in SH-SY5Y cells. This study demonstrated an inhibitory effect of CFM-4.17, but not CFM-4.16, on the activity of ABCB1 in SH-SY5Y cells treated for 30 min. In contrast, this study demonstrated a lack of inhibitory effect of either CFM-4.16 or CFM-4.17 on the activity of ABCC transporters in SH-SY5Y cells treated for the same duration.

These findings represent the first reported account of the effects of any of the CFM compounds on ABC transporter function.

6.8.1 Chemical perspective Given the absence of reports of similar investigations of the effects this class of compounds on ABC transporter activity, it is not possible to draw direct comparisons with published work. Medicinal chemistry offers an alternative perspective to explore whether inhibition is consistent with expectations. Predictions regarding the properties of putative ABCB1 inhibitors have been hindered by technical difficulties in structural characterisation of ABCB1, partly stemming from the transporter’s intrinsic flexibility (Darby et al., 2011; Wen et al., 2013).

However in silico modelling approaches suggest that certain physicochemical characteristics may be associated with ABCB1 inhibition, of which hydrophobicity appears to be the most consistent. For example a logP ≥ 2.92 has been positively correlated with effective inhibition (Wang et al., 2003), and others employ this coefficient or related measures of hydrophobicity (such as logD, logS or hydrophobic surface area) to draw similar conclusions (Broccatelli et al., 2011; Chen et al., 2011; Tan et al., 2013). While corresponding values of these parameters for CFM-4.16 and CFM- 4.17 are not published in the literature, the CFM compounds are consistently described as having poor aqueous solubility, that of CFM-4.16 being <1 mg/mL (Cheriyan et al., 2017), in turn consistent with having a high logP. Furthermore, the electronegative chloride atom of CFM-4.16 would be expected to be slightly more polar than the methoxy group of CFM-4.17 implying that CFM-4.17 would be at least as, if not slightly more, hydrophobic than CFM-4.16.

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Aromaticity (quantified by the total harmonic oscillator model of aromaticity index) of compounds has been implicated in inhibitory activity (Tan et al., 2013). Both CFM-4.16 and CFM-4.17 contain three non-polar aromatic ring structures which would confer a level of aromaticity, allowing interaction with the large hydrophobic binding region composed of multiple aromatic residues of ABCB1 (Tan et al., 2013). However, it should be noted other structural features would influence a compound’s ability to inhibit ABCB1.

A central positive ionisable feature such as a N-based cationic group has also been deemed important for complex formation with electron rich ABCB1 aromatic residues (AlQudah et al., 2016). Others similarly report a requirement for at least one tertiary protonatable nitrogen capable of acting as a hydrogen bond acceptor or donor (Pajeva et al., 2009; Wang et al., 2003). One of the N atoms near the R4 group of either compound may be protonatable in which case it could be cationic (Figure 1.5).

ABCB1 inhibition has been associated with features involved in molecular recognition such as the number of hydrogen-bonding groups of the inhibitory compound particularly hydrogen bond acceptors, although hydrogen bond donors serve as additional interaction mechanisms (AlQudah et al., 2016; Pajeva et al., 2009; Pajeva and Wiese, 2002).

The difference between CFM-4.16 and CFM-4.17 concerns the R1 functional group in which the chloride atom of CFM-4.16 is replaced with a methoxy group in CFM-4.17 (Figure 1.5). The methoxy group possess two hydrogen bond acceptors, whereas the chloride atom does not. The different functional groups in turn may influence how the compounds interact with ABCB1.

6.8.2 Clinical implications of ABCB1 inhibition Potentially, inhibition of ABCB1 functional activity by CFM-4.17 in SH-SY5Y cells could have several clinical implications for treatment of neuroblastoma. If CFM-4.17 were also a substrate for this transporter, a question which remains untested in the Transwell® model, and which was inconclusive in neutral red studies of the effect of verapamil on CFM-4.17-mediated toxicity in PBECs, inhibition would increase cellular retention of the compound. Increased intracellular accumulation could, in turn, increase cytotoxic efficacy and/or reduce the therapeutic dose of chemotherapy required to overcome any

109 intrinsic or acquired ABCB1-mediated resistance. Dual properties of ABCB1 inhibition and cytotoxicity within a single molecule may offer convenience and regimen simplicity, and potentially aversion of additional side effects associated with a separate ABCB1 transporter inhibitor. Use of a molecule with dual properties may compromise dosing flexibility given that inhibition emerges at lower concentrations than cytotoxicity. However, the observed degree of inhibition, 18.2%, whilst statistically significant, may be greater and more clinically important at the higher cytotoxic concentrations that would be required for chemotherapy. When evaluating inhibitory effects in the present work, non-toxic concentrations were selected to ensure that any observed inhibition was independent of a loss of viability. In drug resistant neuroblastoma cells exposed to ordinarily cytotoxic concentrations of CFM-4.17, ABCB1 inhibition may result in restored sensitivity. Should CFM-4.17 be administered in combination with other cytotoxic agents which are ABCB1 substrates, cellular retention of these too may be increased, counteracting ABCB1-mediated resistance. Examples of ABCB1 substrates employed to treat neuroblastoma include doxorubicin, etoposide, irinotecan, thiotepa, topotecan, and vincristine (de Vries et al., 2007; Goldwirt et al., 2014; Loschmann et al., 2016; Veringa et al., 2013). However, when considering potential effects of CFM-4.17- mediated ABCB1 inhibition on clinical outcomes, both the role of ABCB1 overexpression in multidrug resistance and its prognostic significance are especially controversial in neuroblastoma.

Evidence implicating ABCB1 in multidrug resistance and tumour progression in neuroblastoma includes increased ABCB1 expression in metastatic and chemoresistant sublines of an untreated human neuroblastoma xenograft model (Blanc et al., 2003). Lack of ABCB1 expression in tumour samples has also been associated with responsiveness to chemotherapy (Bourhis et al., 1989; Chan et al., 1991). In addition, significantly higher expression of ABCB1 in tumour samples from treated than untreated patients, and in those with more advanced stage disease, have been reported (Bourhis et al., 1989; Chan et al., 1991; Goldstein et al., 1990). However others have failed to identify differences in ABCB1 expression in tumour samples collected before and after either chemotherapy or loss of sensitivity to chemotherapy (Dhooge et al., 1997; Nakagawara et al., 1990) or failed to identify a correlation between ABCB1 expression and tumour progression (Corrias et al., 1990). One confounding factor in positive reports

110 may be contamination of malignant cell specimens with normal infiltrating cells or residual adrenal gland, which may express ABCB1 at high levels (Favrot et al., 1991).

Some groups report overexpressed ABCB1 to be functionally active in neuroblastoma, consistent with a role in drug resistance (Blanc et al., 2003; Porro et al., 2010). Alternatively, suggestions that ABCB1 may have therapeutically relevant regulatory functions independent of drug efflux, and potentially reflect differentiation rather than resistance, stem from induction of increased ABCB1 protein levels in several human neuroblastoma cell lines, including SH-SY5Y, by the chemotherapeutic and differentiating agent retinoic acid without an expected decrease in accumulation of ABCB1 substrates (Bates et al., 1989). However, both presence and absence of correlation between ABCB1 expression and neuroblastoma differentiation have been reported (Dhooge et al., 1997; Lu et al., 2004).

One approach to determining the likely prognostic significance of ABCB1 inhibition could involve correlation of ABCB1 overexpression with established adverse prognostic factors such as MYCN amplification, age at diagnosis or clinical stage (Brodeur, 2003; Pinto et al., 2015). Positive correlation between ABCB1 and MYCN expression has been observed in a human neuroblastoma xenograft model and in patient tumour samples, with mixed evidence regarding positive regulation of ABCB1 by MYCN via interaction with the ABCB1 promoter as an underlying mechanism (Blanc et al., 2003; Porro et al., 2010). However, a lack of correlation between ABCB1 and MYCN was documented in several studies (Benard et al., 1994; Bourhis et al., 1989; Corrias et al., 1990; Dhooge et al., 1997; Goldstein et al., 1990; Haber et al., 1999; Haber et al., 2006; Norris et al., 1996) with inverse correlation in a further study (Nakagawara et al., 1990). ABCB1 expression was higher in tumour samples from patients aged >1 year at diagnosis in one study (Lu et al., 2004) but was not associated with age at diagnosis or clinical stage in others (Benard et al., 1994; Haber et al., 2006).

A few studies have evaluated the association between ABCB1 expression and clinical outcomes more directly. Lack of ABCB1 expression in patient tumour samples has been associated with responsiveness to chemotherapy (Bourhis et al., 1989; Chan et al., 1991), prolonged relapse free and overall survival (Chan et al., 1991) and reduced risk of mortality (Benard et al., 1994; Dhooge et al., 1997). However, ABCB1 expression levels

111 have failed to predict event free or overall survival or progressive disease or recurrence (Dhooge et al., 1997; Haber et al., 2006; Lu et al., 2004; Norris et al., 1996).

Based on the divergent literature, the clinical impact of treatment of neuroblastoma with an ABCB1 inhibitor such as CFM-4.17 is difficult to predict. One study found that ABCB1 overexpression was prognostic in a subset of patients lacking MYCN amplification or in children aged >1 year at diagnosis but not in the overall population, therefore it is possible that the role and prognostic significance of ABCB1 may be restricted to specific clinical subgroups (Haber et al., 1997). In turn this may justify patient stratification by risk factors to identify those most likely to benefit.

6.8.3 Determination of the effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCC activity in SH-SY5Y cells Although CFM-4.17 inhibited ABCB1 transporter activity in SH-SY5Y cells, neither CFM- 4.16 nor CFM-4.17 inhibited ABCC transporters in the present work. Discrimination between ABCB1 and ABCC transporters may stem from structural differences between the two subtypes. For example, ABCC1 only shares 19% sequence identity at the amino acid level with ABCB1, with the most notable global structural difference in ABCC1 being an additional transmembrane domain with five segments (Fletcher et al., 2016; Pajeva et al., 2009). Sequence differences may also affect the geometry and physicochemical characteristics of the CFM-4.17 binding site, for example reducing binding affinity, or interfering with any allosteric modulation that CFM-4.17 may elicit.

6.9 Determination of the effect of short-term treatment with CFM-4.16 or CFM-4.17 on ABCB1 or ABCG2 activity in PBECs In the present study, a lack of ABCB1 (or ABCG2) inhibition was reported following 30 min treatment with CFM-4.16 or CFM-4.17 in PBECs, despite the application of a 31.25- fold higher concentration than that producing ABCB1 inhibitory effects with CFM-4.17 in SH-SY5Y cells (1.25 versus 0.04 μM).

Potential explanations include subtle species differences in amino acid sequences between human and porcine ABCB1 transporters, or structural differences between ABCB1 and ABCG2 transporters, both affecting CFM binding sites. Furthermore PBECs typically exhibit denser expression of ABCB1 and ABCG2 transporters than cancer cells 112 which may facilitate more efficient extrusion of weak substrates (although substrate- like properties of CFM-4.16 and CFM-4.17 were not confirmed in this study) (de Gooijer et al., 2018a). Denser transporter expression in PBECs may additionally necessitate administration of higher CFM concentrations than in SH-SY5Y to detect an inhibitory effect due to a functional reserve of ‘spare’ transporters. Finally, in the event of CFM- 4.16 and CFM-4.17 being substrates for enzymes such as cytochrome P450s which are particularly abundant in PBECs, more extensive metabolism may result in degradation (Shawahna et al., 2013).

6.10 Determination of the effect of CFM-4.16 and CFM-4.17 (24 h or 48 h incubation) on ABCB1 activity in SH-SY5Y cells and on ABCB1 and ABCG2 activity in PBECs CFM-4.16 and CFM-4.17 (0.04 μM) applied for 24 or 48 h to SH-SY5Y cells were both without significant effect on calcein accumulation, indicative of a lack of effect of chronic incubation with either compound on ABCB1 transporter activity.

It is perhaps surprising that the inhibitory effects of CFM-4.17, apparent with a 30 min incubation, were not observed over an extended 24 h or 48 h timeframe. This may be attributable to or metabolism or degradation of CFM-4.17 over the extended time period.

Similarly CFM-4.16 and CFM-4.17 (1.25 μM) applied for the same duration to PBECs were without significant effect on calcein or Hoechst 33342 accumulation, indicative of a lack of effect on ABCB1 or ABCG2 transporter activity.

Whilst this absence of effect may be attributable to these compounds failing to modulate ABCB1 or ABCG2 expression, alternative explanations include the possibility that CFM-4.16 and CFM-4.17 induced transporter expression, but transporters were not functional; Western blot with a specific anti-ABCB1 or anti-ABCG2 antibody would be required to test this hypothesis.

It is also feasible that CFM-induced modulation of transcription factors known to regulate ABCB1 or ABCG2 expression may have occurred, however the net result of alterations in stimulatory and/or inhibitory factors may have cancelled each other out.

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The glucocorticoid receptor, NF-κB and p53 signalling pathways in particular are candidates for involvement. Most published evidence is consistent with upregulation of transporter expression via potentiation of glucocorticoid receptor, NF-κB, and some mutant p53 pathways and opposing downregulation of transporter expression via wild- type p53 (section 6.10.1-6.10.3) each of which merits further discussion.

6.10.1 Potential mediation of chronic regulatory effects of CFMs on ABCB1 and ABCG2 activity by modulation of CARP-1 and the glucocorticoid receptor signalling pathway Chronic exposure to CFM-4.16 or CFM-4.17 was without net effect on the activity of ABCB1 transporters in SH-SY5Y cells and ABCB1 and ABCG2 transporters in PBECs. One molecular pathway which may have contributed to the net result, is that of a CARP-1 mediated increase in glucocorticoid signalling and resultant glucocorticoid receptor- mediated regulation of ABCB1 or ABCG2 expression, most likely in a stimulatory direction.

Although equivalent data for CFM-4.17 are lacking, CFM-4.16 and the parent compound CFM-4 have been reported to increase CARP-1 expression in several cancer cell types (Ashour et al., 2013; Cheriyan et al., 2018; Cheriyan et al., 2017; Cheriyan et al., 2016; Jamal et al., 2014; Muthu et al., 2014; Muthu et al., 2015). This phenomenon has also been demonstrated, at least for CFM-4, in other neuroblastoma lines (Muthu et al., 2014).

There is no direct published evidence to confirm whether CFM compounds exert effects on glucocorticoid receptor signalling, however at least three examples of glucocorticoid receptor signalling regulation by CARP-1 have been reported (Kim et al., 2008; Ou et al., 2014; Wu and S, 2014). Firstly recombinant CARP-1 has been shown to bind to glucocorticoid receptors and additionally potentiate uninduced basal and dexamethasone-evoked transcription of a glucocorticoid receptor-driven reporter construct (Kim et al., 2008). CARP-1 coactivation may involve facilitating recruitment of RNA polymerase II and mediator 1 to the promoter (Kim et al., 2008). Secondly genome- wide analysis of the effects of CARP-1 siRNA knockdown in lung adenocarcinoma demonstrated that 254 of 1208 glucocorticoid-regulated genes were modulated by CARP-1, 80% of these in the same direction as dexamethasone (Wu and S, 2014). Thirdly, CARP-1 positively regulates PPARγ transcriptional activity by facilitating chromatin 114 remodelling, increased accessibility and glucocorticoid receptor binding at a specific PPARγ glucocorticoid receptor binding region (Ou et al., 2014).

These data are consistent with compounds such as CFM-4.16 and CFM-4.17 having the potential to potentiate glucocorticoid receptor signalling.

In turn, glucocorticoid receptor signalling has been demonstrated to regulate activity and expression of both ABCB1 and ABCG2 transporters. The most recent evidence, derived from the same PBEC model system and employing similar experimental protocols to the present work, reported hydrocortisone and dexamethasone-mediated upregulation of ABCB1 expression and activity (Torres-Vergara and Penny, 2018). This is consistent with findings in rat brain capillaries where dexamethasone treatment similarly increased ABCB1 protein levels and functional activity (Bauer et al., 2004; Narang et al., 2008). It is also consistent with mouse in vivo studies in which dexamethasone-induced ABCB1 upregulation in brain capillary fractions was observed (Chan et al., 2013).

However contrasting findings include lack of effect of dexamethasone on ABCB1 expression in rat microvessels in vivo and on ABCB1 activity in PBECs (Alms et al., 2014; Mei et al., 2004). Hydrocortisone-induced downregulation of ABCB1 expression in PBECs has also been reported (von Wedel-Parlow et al., 2009). Differences may be accounted for by low doses of dexamethasone or confounds of an extended 72 h puromycin treatment which itself may have inductive properties (Alms et al., 2014; Mei et al., 2004; Torres-Vergara and Penny, 2018).

In a similar manner to ABCB1, the consensus in the literature is that glucocorticoid receptor signalling induces ABCG2 expression and activity. For example dexamethasone increases levels of ABCG2 mRNA, protein and transporter activity (Narang et al., 2008). However the caveat exists that, CARP-1 exhibits differential specificity for different subsets of glucocorticoid-regulated physiological pathways, for example affecting adipogenic but not anti-inflammatory genes (Ou et al., 2014; Wu and S, 2014). This opens the possibility that, even if the compounds affected glucocorticoid receptors, modulation of ABCB1 and ABCG2 transporters may not follow.

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6.10.2 Potential mediation of chronic regulatory effects of CFMs on ABCB1 and ABCG2 activity by modulation of CARP-1 and the NF-κB signalling pathway A second major pathway that may have contributed to the net absence of effects of chronic exposure to CFM-4.16 and CFM-4.17 on ABCB1 and ABCG2 transporter activity in the present work is that of a CARP-1-mediated modulation of NF-κB signalling and associated regulation of ABCB1 or ABCG2 expression, most likely in a stimulatory direction.

As discussed above, although data are lacking for CFM-4.17, both CFM-4.16 and CFM-4 increase CARP-1 expression in a range of cancer cell lines (Ashour et al., 2013; Cheriyan et al., 2018; Cheriyan et al., 2017; Cheriyan et al., 2016; Jamal et al., 2014; Muthu et al., 2014; Muthu et al., 2015). CARP-1 is known to modulate NF-κB signalling by interacting with IκB kinase-γ (IKKγ/NEMO), a regulatory subunit of the IKK complex, in turn an upstream kinase of the IκB inhibitory proteins which sequester NF-κB (Beg et al., 1993; Bouwmeester et al., 2004; DiDonato et al., 1997; Mercurio et al., 1997; Woronicz et al., 1997; Yamaoka et al., 1998). Phosphorylation of IκB results in its ubiquitination and targeted degradation, derepressing the NF-κB pathway (Beg et al., 1993; DiDonato et al., 1997; Mercurio et al., 1997). Hence CARP-1 has a net effect of stimulating the NF- κB pathway.

Therefore CFM-4.16 and CFM-4.17 might in turn be expected to promote NF-κB signalling, further supported by a reported net depletion of NF-κB inhibitors, and hence NF-κB derepression, in the presence of CFMs, particularly CFM-4, in neuroblastoma, medulloblastoma, breast cancer, and malignant pleural mesothelioma (Ashour et al., 2013; Muthu et al., 2014; Muthu et al., 2015).

Prediction of the direction of CFM-4.16 and CFM-4.17-evoked regulation of NF-κB in the present work, whilst expected to be predominantly stimulatory, is complicated by association of CFM compounds with cell type-specific and biphasic treatment duration- dependent effects on individual mediators of the NF-κB pathway, often with initial inhibition and subsequent activation. For example, in the SK-N-SH and SK-N-BE(2) neuroblastoma cell lines, CFM-4 primarily activated NF-κB signalling via disinhibition (Muthu et al., 2014). CFM-4 consistently reduced IκBα and IκBβ expression, effected an initial reduction in ABIN2 levels within SK-N-BE(2) cells and produced biphasic effects on ABIN1 varying according to the cell line (Muthu et al., 2014). ABIN1 and ABIN2 bind the 116

NF-κB inhibitor protein A20 and therefore also mediate disinhibition (Verstrepen et al., 2009).

In view of the absence of published data relating to the effect of CFM compounds on NF-κB signalling in PBECs, it is instructive to explore the other cell types in which these CFM-modulated pathways have been studied and in which complexity is also exhibited. In medulloblastoma cell lines, CFM-4 induced regulation is similarly biphasic, and consistent with initial inhibition and subsequent activation of NF-κB signalling (Ashour et al., 2013). In malignant pleural mesothelioma and p53 mutation positive, estrogen receptor negative, breast cancer cell lines, CFM-4 evoked activation of NF-κB signalling, while inhibition was reported in TNBC cell lines (Jamal et al., 2014; Muthu et al., 2014; Muthu et al., 2015).

However, despite the time-dependency of effects of CFM-4 on NF-κB, the observed nuances and biphasic characteristics have only been described up to 12 h in all cells except for mesothelioma (Ashour et al., 2013; Jamal et al., 2014; Muthu et al., 2014; Muthu et al., 2015). In the present work, incubation with CFM-4.16 or CFM-4.17 for 24 and 48 h was without effect on transporter activity at 24 and 48 h in both SH-SY5Y cells and PBECs. The net effects on NF-κB signalling mediators at 12 h in the literature are consistent with activation of the pathway in most evaluated cell types including neuroblastoma, and this is reinforced by similar findings in mesothelioma at 24 h. However evidence would be required to confirm that these NF-κB signalling mediator level trends are maintained at 24 h or 48 h in neuroblastoma and to determine whether similar events occur in PBECs.

The reported relationship between NF-κB and expression and activity of ABCB1 and ABCG2 transporters can in turn be employed to help predict the downstream NF-κB- mediated effects of CFM-4.16 or CFM-4.17 on transporter function. NF-κB binding to the rat ABCB1 promotor and associated transcriptional activation have been implicated in basal and TNF-induced expression of ABCB1 in a cerebral microvessel endothelial cell line and also in TNF, endothelin, and sulforaphane-induced expression and activity of ABCB1 in isolated rat brain capillaries (Bauer et al., 2007; Wang et al., 2014; Yu et al., 2008). Positive modulatory effects of NF-κB on ABCB1 expression have been replicated in other cell types including liver, kidney proximal tubule, hepatocellular carcinoma and colon cancer (Bentires-Alj et al., 2003; Ros et al., 2001; Thevenod et al., 2000; Yang et 117 al., 2012). However evidence also exists for negative regulatory effects of NF-κB in basal ABCB1 expression in rat endothelial cells (Nwaozuzu et al., 2003).

ABCG2 appears to be differentially regulated given the respective absence of involvement or negative regulatory effects of NF-κB in TNF and endothelin-mediated expression of this transporter in rat brain capillaries (Bauer et al., 2007). However NF-κB can also positively regulate sulforaphane-induced ABCG2 expression in rat brain capillaries and arsenic trioxide-induced ABCG2 expression in lung adenocarcinoma cells (Jiang et al., 2018; Wang et al., 2014).

Whilst, on balance, the effects of CFMs on NF-κB mediated regulation of transporters are more likely to be stimulatory, prediction of the net direction of effect may be complicated by the state of co-activation of the signalling pathways transduced by NF- κB such as TNF, and the extent of cross-talk with for example with glucocorticocoid receptor signalling (Auphan et al., 1995; Ray and Prefontaine, 1994; Scheinman et al., 1995) both of which could also conceivably be modified by malignancy and chemotherapeutics, including CFM compounds. Therefore there are molecular mechanisms through which CFM-4.16 or CFM-4.17 via a pathway involving NF-κB, could conceivably upregulate or downregulate transporter expression.

6.10.3 Potential mediation of chronic regulatory effects of CFMs on ABCB1 and ABCG2 activity by modulation of CARP-1 and the p53 signalling pathway A third major pathway that may have contributed to the net lack of effect of chronic CFM-4.16 or CFM-4.17 exposure on ABCB1 and ABCG2 transporter activity in the present work is that of CFM-evoked, CARP-1-mediated regulation of p53, given that CARP-1 coactivates this proapoptotic tumour suppressor (Kim et al., 2008). The direction of transcriptional regulation of ABCB1 and ABCG2 expression by p53 may vary with cell type, the drug under investigation, detection of the mdr1a versus the mdr1b isoform in rodents or study of wild-type or mutant p53, and if the latter, the particular p53 mutation (Bush and Li, 2002; Chen and Sikic, 2012).

With respect to ABCB1, the general consensus is that wild-type p53 represses ABCB1 promoter constructs and expression of the endogenous ABCB1 gene, while a number of p53 mutants activate the ABCB1 promoter. However some evidence is conflicting.

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Wild-type p53 is thought to transcriptionally repress the ABCB1 gene by binding to a site within the ABCB1 promoter (Johnson et al., 2001). Early supportive evidence includes wild-type p53-mediated repression of an ABCB1 promoter-driven construct in SW13 adrenocortical carcinoma, BHK (baby hamster kidney) and Saos-2 osteosarcoma cell lines (Chin et al., 1992; Strauss et al., 1995). Consistent with this, transfection with wild- type p53 repressed ABCB1 expression in the p53 null colon carcinoma Caco-2 cell line (Sampath et al., 2001). Similarly, transfection of trans-dominant negative p53 into a H35 rat hepatoma cell line impaired endogenous wild-type p53 function and enhanced mdr1a gene expression (Thottassery et al., 1997). In human astrocytes isolated from epileptic foci, loss of p53 function was associated with ABCB1 overexpression (Marroni et al., 2003), whilst in leukaemia and lymphoma cell lines, there was an inverse correlation between ABCB1 expression and wild-type p53 expression (Hirose and Kuroda, 1998). Therefore a range of experimental approaches support transcriptional repression of ABCB1.

However there are also multiple reports of no effect or positive modulatory effects of wild-type p53 on ABCB1. Lack of effect of wild-type p53 is supported by no correlation between expression of wild-type p53 and ABCB1 in colorectal tumour samples (de Kant et al., 1996). Positive modulatory effects are supported by transfection of wild-type p53 into a p53-negative H358 lung carcinoma cell line stimulating the transcription of an ABCB1 promoter-driven expression construct (Goldsmith et al., 1995). Furthermore, in a mouse in vivo paradigm, irradiation- or diethylnitrosamine-induced upregulation of mdr1b was observed in p53 wild-type animals but not p53 knockout animals consistent with stimulatory effects of p53 (Lecureur et al., 2001). In a p53-defective multidrug resistant human epidermoid carcinoma cell line the transfection of wild-type p53 increased ABCB1 expression, while in rat hepatoma cells, endogenous wild-type p53 was shown to mediate daunorubicin-induced upregulation of mdr1b expression (Li et al., 1997; Zhou and Kuo, 1998).

Mutant and wild-type p53 appear to differentially regulate ABCB1, a number of studies being consistent with mutant p53 stimulating ABCB1 expression. In SW13 adrenocortical carcinoma cells and NIH 3T3 fibroblast cells mutant p53 upregulates expression of an ABCB1 promoter-driven construct (Chin et al., 1992). In colorectal cancer specimens, a positive relationship has been identified between mutant p53 levels and ABCB1

119 expression, whilst similarly, in the p53 null colon carcinoma Caco-2 cell line, transfection with mutant p53 reportedly stimulates ABCB1 expression (de Kant et al., 1996; Oka et al., 1997; Sampath et al., 2001).

However there are also several examples of a lack of relationship between expression of mutant p53 and ABCB1. These include the absence of a correlation between mutant p53 and ABCB1 expression in specimens from patients with osteosarcoma, myelodysplastic syndrome and hepatocellular, breast, endometrial and cervical carcinoma (Akimoto et al., 2006; Preudhomme et al., 1993; Schneider et al., 1994; Serra et al., 1999). These particular reports suggest that p53 mutation is not a major determinant of ABCB1 regulation.

Similarly to ABCB1, most evidence supports involvement of wild-type p53 in transcriptional repression, and mutant p53 in enhanced expression of ABCG2, with some inconsistencies. For example, following genetic knockdown or survivin-induced downregulation of wild-type p53 expression in the MCF-7 breast cancer cell line ABCG2 expression was upregulated, consistent with p53-mediated repression of ABCG2 gene transcription (Wang et al., 2013; Wang et al., 2010). Both reports additionally implicate NF-κB as a mediator of the effects of p53 (Wang et al., 2013; Wang et al., 2010). However, in contrast, a positive modulatory effect of wild-type p53 has been reported. In isolated rat brain capillaries p53 was required for sulforaphane-induced increases in ABCB1 activity and expression and ABCG2 expression (Wang et al., 2014).

In parallel with their effects on ABCB1, different p53 mutations may confer heterogeneity in their modulatory effects on ABCG2. For example a head and neck cancer cell line with cytoplasmically sequestered mutant p53 exhibited ABCG2 upregulation relative to cells with nuclear mutant p53 (Tonigold et al., 2014). However the limited published evidence is consistent with a stimulatory effect of mutant p53, including in human colon carcinoma cell lines, where mutant p53 transcriptionally upregulated expression of ephrin-B2 and in turn ABCG2 (Alam et al., 2016).

Collectively, most of the evidence is consistent with a potential suppressive effect of CFM-stimulated p53-mediated signalling on ABCB1 and ABCG2 activity and/or expression in opposition to the likely net effects of glucocorticoid receptor and NF-κB signalling. However it is clear that the mutation status of p53 would influence this.

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6.11 Characterisation of the integrity of the in vitro blood-brain barrier model Two measures of PBEC monolayer integrity were employed in this study. Transendothelial electrical resistance (TEER) indicates resistance to ionic flux while the permeability coefficient of Lucifer yellow, Papp, reflects permeability of the paracellular pathway (Cantrill et al., 2012).

In the present work, mean TEER values were 534 ± 145 Ω.cm2 and 866 ± 122 Ω.cm2 on the penultimate and final day of culture respectively. The values are therefore intermediate between the 394 Ω.cm2 reported without puromycin treatment using primary rat astrocytes, and values of 1693 Ω.cm2 and 2147 Ω.cm2, reported with puromycin treatment using primary rat astrocytes or the CTX-TNA2 rat astrocyte line respectively, all originating from the host laboratory using PBECs in non-contact co- culture (Cantrill et al., 2012; Skinner et al., 2009). Puromycin pre-treatment was employed to eradicate contaminating cells, thereby helping improve monolayer integrity whilst the CTX-TNA2 cell line was employed as release of soluble astrocytic factors helps maintain the endothelial cell phenotype and produce highly restrictive cell monolayers (Cantrill et al., 2012). The higher TEER values reported by Cantrill et al may also be attributable to other methodological differences such as duration of culture on the Transwell® inserts.

TEER values in the current study align with those in PBECs in non-contact co-culture (680 Ω.cm2) or in contact co-culture with rat glial cells (1112 Ω.cm2) although the mixed glial population employed in that study differed in creating a triple co-culture system comprising endothelial cells, astrocytes and pericytes (Cohen-Kashi Malina et al., 2009).

The TEER values reported in the present study are similar to those reported for PBECs in non-contact co-culture with primary or immortalised C6 rat astrocytes (881 Ω.cm2 or 834 Ω.cm2 respectively) (Smith et al., 2007; Thomsen et al., 2015). The TEER values obtained in the current study are also consistent with those of PBECs in monoculture, reported as mean values of 344 Ω.cm2 or 789 Ω.cm2, or values up to 700 Ω.cm2 (Lelu et al., 2017; Patabendige et al., 2013; Thomsen et al., 2015). TEER values in the current study are also consistent with those of bovine brain endothelial cells in monoculture with ACM (mean 625 Ω.cm2 up to a maximum of 1500 Ω.cm2) in non-contact co-culture with primary astrocytes (up to 800 Ω.cm2), or in contact co-culture with rat astrocytes (mean 671 Ω.cm2) (Cecchelli et al., 1999; Gaillard et al., 2001; Rubin et al., 1991). 121

Notably, in the present work, a 1.6-fold increase in TEER in the final 24 h of culture following addition of switch medium was observed. This was consistent with 2.4-fold or 3.3-fold switch medium-evoked increases within the same cultures, or 3.5 to 5.4-fold differences in final TEER values between cultures employing the switch medium step relative to those omitting this step (Cantrill et al., 2012; Gaillard et al., 2001; Rubin et al., 1991; Smith et al., 2007). Increased TEER can be attributed to effects of serum depletion, hydrocortisone supplementation, and cAMP augmentation via a cell- permeable cAMP analogue and phosphodiesterase inhibitor, on the differentiation of BBB characteristics including enhanced barrier function of intercellular tight junction protein complexes (Cantrill et al., 2012; Gaillard et al., 2001; Patabendige et al., 2013; Rubin et al., 1991).

TEER values exceeding 200 Ω.cm2 have been considered suitable for quantifying the penetration of small drug-like molecules across the cell monolayers (Gaillard 2000, Liew 2016). Hence a threshold of 250 Ω.cm2 has previously been adopted for experimentation (Skinner et al., 2009). However higher TEER values reflect increased barrier tightness and molecular discrimination, and in vivo mean TEER values as high as 1462 Ω.cm2 and 1870 Ω.cm2 have been reported in neonatal rats, and in frogs (Butt et al., 1990; Crone and Olesen, 1982). Therefore, thresholds such as 500 Ω.cm2 or 600 Ω.cm2 have variously been employed (Muller et al., 2018; Patabendige et al., 2013).

Higher TEER values have been associated with lower Papp (Lelu et al., 2017). An exponential decay in Papp of PBEC monolayers occurs with increasing TEER values, permeability of Lucifer yellow stabilising at 2.14 x 10-6 at TEER values above 220 Ω.cm2, validating this as a threshold (Lelu et al., 2017). The present study only used PBEC monolayers with TEER values exceeding 500 Ω.cm2.

-7 - In the present work, the mean Papp value for PBEC monolayers was 10.5 x 10 ± 5.4 x 10

7 -1 cm.s . The Papp value is consistent with the Papp previously reported by the host laboratory for PBEC monolayer co-cultured with the CTX-TNA2 astrocyte line in similar conditions of 5.70 x 10-7 cm.s-1 (Cantrill et al., 2012). The 4.6-fold difference between the Papp of the empty insert and that of the PBEC-seeded insert is also consistent with the approximately 10-fold difference reported by other investigators, albeit in monoculture (Lelu et al., 2017).

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6.12 Toxicity-based indirect measurement of CFM penetration of the in vitro blood-brain barrier model In this study, the absorbance characteristics of the CFM-4.16 and CFM-4.17 were evaluated with a view to detecting these test compounds spectrophotometrically. This would have represented a direct means of quantifying the extent of any CFM penetration across PBEC monolayers. However, in the absence of significant absorption by these compounds at a defined wavelength, this approach was not possible. Should radiolabelled CFM compound analogues have been available, radioactivity could have been detected by scintillation counting. Ideally high performance liquid chromatography (HPLC) would have been employed, which is direct, accurate and sensitive, however the requirement for optimisation precluded this. Consequently an alternative indirect measure of CFM penetration was devised which involved assessing the cytotoxicity to SH-SY5Y cells of apical and basolateral samples from Transwell® inserts seeded with PBECs following a six hour incubation period with CFM compounds. Whilst this strategy is limited by complexities in the relationship between concentration and cell viability such that precise concentration values cannot be derived, and the approach also prevents direct comparisons of CFM-4.16 and CFM-4.17 penetration owing to different cytotoxic potencies, it remains suitable for indicative high throughput screening and was selected for use in this study.

6.13 Assessing penetration of CFM-4.16 and CFM-4.17 across the in vitro blood- brain barrier model In this study CFM-4.16 (1.25 μM), CFM-4.17 (5 μM) or vehicle were applied to the apical chamber of Transwell® inserts seeded with PBEC monolayers and apical and basolateral samples were collected six hours later. The major finding when evaluating the penetration of CFM-4.16 or CFM-4.17 across this in vitro model of the BBB was the lack of significant cytotoxicity of basolateral samples when applied to SH-SY5Y cells, despite the significant cytotoxicity of apical samples. This is consistent with limited or absent penetration of CFM-4.16 or CFM-4.17 across the PBEC monolayer such that cytotoxic concentrations were not attained basolaterally.

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To explain this lack of penetration, various features of the BBB and this particular model, and of the CFM compounds themselves can be highlighted. Brain endothelial cells are characterised by tight junctions (which predominantly determine the flux of ions and small hydrophilic molecules), a lack of fenestrae and minimal pinocytosis (Brightman and Reese, 1969; Fenstermacher et al., 1988; Reese and Karnovsky, 1967; Sedlakova et al., 1999). The two main potential routes of endothelial monolayer penetration are paracellular (interendothelial) and transcellular (through endothelial cells). The high TEER and low permeability of Lucifer yellow reported in this study are consistent with the tight junctions being intact, and are indicative of a restrictive paracellular pathway. Hence the transcellular route may be more likely to predominate and account for any penetration. Two of the major criteria for transcellular passive diffusion include a molecular mass below 400-500 Da and high lipid solubility stemming from the requirement to pass through transitory pores of finite size in the phospholipid bilayer (Fischer et al., 1998a; Marrink et al., 1996). Both CFM-4.16 and CFM-4.17, with molecular weights of 440.4 and 435.9 respectively, would be classed as small lipophilic molecules, albeit close to 500 Da upper threshold. However excess lipophilicity could theoretically have resulted in trapping of the compounds in the lipid membrane bilayer and consequent bioaccumulation, preventing them from reaching the basolateral compartment.

Other determinants of monolayer penetration include abundance of mitochondria, phase I and II enzymes and ABC transporters within brain endothelial cells, in turn potentially mediating metabolic degradation and active efflux (Cooray et al., 2002; Cordon-Cardo et al., 1989; Oldendorf et al., 1977; Shawahna et al., 2011). Should CFM- 4.16 or CFM-4.17 represent enzymatic substrates, metabolic conversion may have two outcomes responsible for lack of cytotoxicity of basolateral samples from CFM-treated Transwell® inserts. Pharmacological inactivation may produce non-cytotoxic or less cytotoxic metabolites, and modification of drug polarity via addition of a polar functional group or conjugation reactions may cause intracellular sequestration. Alternatively CFM-4.16 and CFM-4.17 may diffuse across the apical plasma membrane but undergo efflux by ABC transporters. For example lipophilicity and particularly amphiphilicity may increase the probability of ABCB1 substrate behaviour (Didziapetris et al., 2003). Potential substrate-like properties of CFM-4.16 were explored further.

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6.14 Evaluation of the substrate-like properties of CFM-4.16 and CFM-4.17 In order to assess whether CFM-4.16 might act as a substrate for ABCB1 or ABCG2 the permeation experiments were repeated in the presence of pre and coincubation with verapamil or Ko143. However there was no significant effect of either compound on indirectly measured CFM-4.16 penetration; basolateral samples from CFM-4.16-treated Transwell® inserts remained without cytotoxic effect when applied to SH-SY5Y cells. This suggested that CFM-4.16 was either not a substrate or was a very weak substrate for ABCB1 or ABCG2. Hence penetration of the BBB is probably not substantially limited by the activity of these transporters. Future experiments would involve repeating this work with CFM-4.17.

As a complementary indirect measure of substrate-like properties, the cytotoxicity of CFM-4.16 and CFM-4.17 to PBECs was evaluated in the presence and absence of verapamil or Ko143. It was expected that, had either compound acted as a substrate, inhibition of efflux would increase intracellular retention and hence toxicity. However CFM-4.16 and CFM-4.17 both remained without cytotoxic effect irrespective of verapamil or Ko143 coadministration. The concentrations employed might have been too low to see a cytotoxic effect, selection of concentrations at the threshold of toxicity may have been more appropriate. Alternative evidence to verify ABC transporter efflux would involve comparing permeability in both directions across the monolayer to detect polarisation of transport (Zhang et al., 2006). Also assessment of a range of CFM concentrations would permit detection of concentration-independent permeation reflecting a lack of saturation consistent with passive diffusion (Tian et al., 2009).

Together these data provide insufficient evidence to suggest that therapeutic coadministration of an ABCB1 or ABCG2 inhibitor alongside CFM-4.16 or CFM-4.17 would augment CNS delivery.

6.15 Clinical implications of limited penetration Given the usefulness of identifying a chemotherapeutic compound to treat CNS neuroblastoma metastases, a desired endpoint reflecting BBB penetration, was the attainment of cytotoxic concentrations in the basolateral compartment of the Transwell® apparatus. Poor bioavailability would theoretically result in variable,

125 restricted, unpredictable exposure. In particular, inadequate penetration reduces the probability that CFM-4.16 or CFM-4.17 would reach therapeutically effective CNS concentrations to ensure adequate target engagement. Ideally brain concentrations as least as high as free plasma levels would minimise the requirement for dose escalation and systemic toxicity. The lack of BBB penetration suggested by the results of the present study mean that, CFM-4.16 or CFM-4.17, in their current pharmaceutical form, administered alone, for example orally or intravenously, would not be expected to successfully treat CNS metastases, although peripheral manifestations of neuroblastoma may still be treatable.

Potential methods to overcome the inadequate penetration include, in vitro, increasing the concentration and duration of exposure, or clinically increasing the intensity and duration of the dosing schedule (Roda et al., 2014). Alternatively, the BBB may be bypassed via the intrathecal route of administration, involving direct injection into the cerebrospinal fluid. However this is limited by poor diffusion to the brain and invasiveness (Kaiser and McGee, 1975; Shapiro et al., 1975). Other options include transient therapeutic opening of the BBB, for example with agents that generate a hyperosmolar environment, and the ‘Trojan horse’ approach of coupling a therapeutic agent to a molecule which is recognised and transported via active uptake (Neuwelt and Dahlborg, 1987; Regina et al., 2008). Perhaps the most promising method of increasing penetration is that of delivery via nanocarriers such as liposomes (Chen et al., 2016a; Shaw et al., 2017). Analogous nano-lipid formulations of CFM-4.16 and the parent compound CFM-4 have been developed with a view to overcoming poor dissolution, gastrointestinal absorption and oral bioavailability and conferring protection from systemic metabolism (Cheriyan et al., 2018; Cheriyan et al., 2017; Cheriyan et al., 2016; Muthu et al., 2015). The bioavailability of orally administered CFM-4 or CFM-4.16 in rats was significantly increased in terms of AUC and Cmax, when formulated in a nano-lipid carrier compared with the free compound (Cheriyan et al., 2016; Muthu et al., 2015). Efficacy of the nano-lipid formulation of CFM-4 in suppressing the growth of xenografted tumours of breast or lung cancer cell origin also increased commensurately relative to the free compound (Muthu et al., 2015). Bioavailability of analogous formulations of CFM-4.16 is also implied by their capacity alone to inhibit breast cancer xenograft growth, or together with sorafenib to inhibit NSCLC xenograft growth (Cheriyan et al.,

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2018; Cheriyan et al., 2016). However it was reported that nanolipid formulations of CFM-4.16 increased efficacy against RCC xenografts versus the free compound when administered intravenously although not when administered orally (Cheriyan et al., 2017).

Although there are physiological differences between gastrointestinal absorption and BBB penetration, the above evidence cumulatively raises the possibility that nano-lipid formulations of CFM-4.16 and CFM-4.17 could improve the ability of these compounds to traverse biological membranes including those of PBEC monolayers. It would therefore be interesting to evaluate the penetration of such formulations across the Transwell® model. Nanolipid formulations may combine the demonstrable cytotoxicity of CFM-4.16 and CFM-4.17 towards neuroblastoma with the potential to access the CNS compartment where difficult to treat metastases are located.

6.16 Limitations and clinical extrapolation of the Transwell® model Some caveats apply to the extent to which data from the in vitro Transwell® model can be extrapolated to the in vivo and clinical contexts. This study employed a static system with a lack of fluid flow whereas, in vivo, a significant unstirred water later is not observed at the BBB (Patlak and Paulson, 1981). Sheer stress can tighten the barrier in vitro and stirring can increase the dynamic range, expressed as the ratio of transcellular marker permeability to paracellular marker permeability (Cucullo et al., 2013; Zhang et al., 2006).

A phenomenon that may occur in vivo is brain tissue binding (Hellinger et al., 2012). Lipophilic permeant compounds bind brain tissue to a greater extent than hydrophilic compounds (Heymans et al., 2018). This non-specific binding in turn is associated with slower equilibration of compounds across compartments; equilibration in vitro may therefore be faster (Summerfield et al., 2006). Binding may also compensate for ABC transporter efflux of passively permeant molecules increasing net permeability (Summerfield et al., 2006). Notably this study in particular did not rule out ABCC- mediated efflux of CFM-4.16 and CFM-4.17 and did not evaluate ABCB1 and ABCG2 substrate-like properties of CFM-4.17 in the Transwell® setup therefore this efflux phenomenon could still be relevant. Some investigators mimic brain tissue binding by

127 adding glial cells to the receiver compartment within permeability assays (Heymans et al., 2018). Similarly, in vivo, high affinity binding to serum albumin can reduce the free drug fraction and hence penetration (Fong, 2015). Finally, the ultimate aim of the permeation experiments is to evaluate the potential to treat CNS metastases. Clinically, brain tumours may disrupt and result in partial leakiness of the BBB however the barrier is thought to typically remain sufficiently intact to impair drug delivery albeit potentially with heterogeneous accessibility even within a single metastatic lesion (Lockman et al., 2010). This complexity is difficult to recapitulate reproducibly in an in vitro model.

6.17 Potential experimental refinements Other methods of observing post-experimental cellular integrity include staining with Toluidine blue or crystal violet and checking for acellular gaps (Hellinger et al., 2010; Hellinger et al., 2012; Lin et al., 2016; Patel et al., 2018). Observation of morphological changes such as diffusely distributed vimentin can also reflect subtoxic cellular stress (Roda et al., 2014). However, in the present work, lack of overt cytotoxicity of CFM-4.16 and CFM-4.17 to PBECs was established for up to 48 h exposure with the same concentrations employed for 6 h in the permeation experiments.

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7 Conclusion

7.1 Implications for the treatment of neuroblastoma CNS metastases Several characteristics of CFM-4.16 and CFM-4.17 were determined which are relevant to assessing their suitability for treatment of neuroblastoma CNS metastases. Both compounds potently inhibited the viability of SH-SY5Y cells at lower concentrations than required to effect cytotoxicity in PBECs, consistent with differential sensitivity of cancer and non-malignant cell types and therefore with potentially favourable therapeutic indices. The lack of effect of either compound on ABCB1 or ABCG2 activity in PBECs following both short and long-term incubation reduces the likelihood of any unintended transporter-mediated interactions with non-chemotherapeutic drugs which are ABCB1 or ABCG2 substrates. However, it also prevents the compounds from influencing both their own CNS penetration (should further experiments reveal substrate-like behaviour) and the CNS penetration of other chemotherapeutic substrates. CFM-4.17 inhibited ABCB1 transporter activity in SH-SY5Y cells therefore it may simultaneously effect cytotoxicity and increase intracellular accumulation of drugs which are ABCB1 substrates, however CFM-4.16 did not display inhibitory properties. Neither compound appeared to penetrate the Transwell® model of the BBB which indicates that an alternative nanolipid or nanomicellular formulation may be preferable therapeutically, and exploration of this both in vitro and in vivo would need to precede potential clinical development. ABCB1 and ABCG2 inhibition did not modulate the penetration of CFM- 4.16 across the same model indicating that CFM-4.16 may not be a substrate for these transporters therefore coadministration of other ABCB1 or ABCG2 inhibitors therapeutically would be unlikely to influence CNS bioavailability.

7.2 Future work Several additional experiments and techniques would complement and extend the present work. More extensive characterisation of SH-SY5Y and PBEC viability could be carried out in the presence of inhibitors of ABCB1 and ABCC, (or for PBECs additionally ABCG2) across a range of CFM-4.16 and CFM-4.17 concentrations in order to determine whether transporter inhibition can potentiate cytotoxicity at CFM concentrations close to the threshold for toxicity. Alternatively, a more direct means of monitoring ABC

129 transporter substrate-like behaviour of the CFM compounds, such as the membrane vesicular uptake assay using vesicles from drug-selected or stably transfected cell lines (Volk and Schneider, 2003), could be employed. Should it be possible to radiolabel or fluorescently label the CFM compounds without significantly affecting their properties, transport of CFM-4.16 or CFM-4.17 may be quantified by measuring intracellular accumulation via scintillation counting or fluorescence detection.

For completeness, and given the prognostic significance of ABCC in neuroblastoma, it would be instructive to evaluate the effects of long-term (24 h or 48 h) incubation with CFM-4.16 or CFM-4.17 on ABCC activity in SH-SY5Y cells. Ideally the effect of short (30 min) or long-term (24 h or 48 h) incubation with these compounds on ABCC activity in PBECs should be evaluated in order to investigate inhibitory or chronic regulatory actions of the CFMs in an additional transporter type. As discussed, the chosen time points for evaluation of long-term effects of CFM compounds on transporter activity were 24 h and 48 h. in the absence of effects at these times, it is unlikely that further extension of the incubation period to 72 h would result in different effects, however this could be explored to discount this possibility. If effects of long-term incubation with CFMs on ABCC transporter activity were detected, then an additional time-point would allow any time-dependency to be fully characterised.

It would be interesting to also determine whether the absence of changes in ABC transporter functional activity with long-term incubation is mirrored by a lack of change in transporter expression at the mRNA and protein levels for which RT-PCR and Western blot could be employed respectively. For example, transporter expression may be modulated with transporters remaining non-functional or with deficiencies in trafficking to the cell membrane.

The Transwell® experiments could be made more complex with the use of the triple culture approach employing pericytes to further replicate the cell types present in vivo. Microscopic visualisation of the PBEC monolayer before and after CFM-4.16 or CFM-4.17 treatment would have verified its structural integrity. In addition, it would be beneficial to develop a direct technique, such as HPLC, to quantify CFM compounds.

Future studies could further characterise the influence of potential ABC transporter substrate-like behaviour of CFM-4.16 and CFM-4.17 on BBB penetration. Firstly, the

130 effect of MK571-mediated inhibition of ABCC transporters on CFM-4.16 penetration could be evaluated. Analogous experiments with verapamil, Ko143 or MK571 could additionally be employed to determine the effect of ABCB1, ABCG2 or ABCC efflux on CFM-4.17 penetration. Use of combinations of ABCB1, ABCG2 and ABCC inhibitors may also enable any redundancy between transporters to be accounted for, if a CFM compound is a substrate for more than one transporter class.

The favourable apparent therapeutic index inferred from differential sensitivity of PBECs and SH-SY5Y to CFM-4.16 and CFM-4.17 could be further validated by demonstrating comparatively low cytotoxicity of these compounds to alternative non-cancer cells of different origins, including from outside the CNS. Experiments could also be extended to neuroblastoma cell lines other than SH-SY5Y to illustrate applicability to other neuroblastoma phenotypes. Analogous findings may be expected in view of the dose- dependent inhibition of viability reported in the literature with the parent compound CFM-4 in SK-N-AS, SK-N-DZ, SK-N-BE(2) and SK-N-SH neuroblastoma cell lines (Muthu et al., 2014).

131

Appendix: Assessment of the effects of CFM-4.16 or CFM-4.17 on ABCB1 or ABCG2 functional activity in PBECs following a washout period

In PBECs treated with 1.25 μM CFM-4.16, CFM-4.17 or vehicle for 24 h or 48 h followed by a 5 h washout period there was no significant difference in intracellular accumulation of the fluorescent probe calcein in cells exposed to CFM-4.16 or CFM-4.17 relative to those exposed to vehicle (Figure A1). Furthermore, there was no significant difference in calcein accumulation in PBECs treated with vehicle and verapamil compared to PBECs treated with both CFM-4.16 and verapamil or both CFM-4.17 and verapamil (Figure A1). These findings indicate that long-term treatment with CFM-4.16 or CFM-4.17 had no significant effect on ABCB1 activity both in the presence and absence of verapamil, irrespective of the inclusion of a washout period prior to carrying out the ABCB1 transporter functional assay.

132

Figure A1. Effect of long-term treatment with CFM-4.16 or CFM-4.17 followed by a washout period on ABCB1 activity in PBECs.

n .s . n .s . A n .s . B 1 5 0 0 1 5 0 0 n .s . n .s .

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PBECs were seeded (25,000 cells/well) into 96 well plates and incubated for 48 h. Cells were then pre-equilibrated with treatment medium (low-glucose, phenol red-free DMEM supplemented with 1% (v/v) FBS and 1% (v/v) glutamine) for 30 min. The medium was subsequently aspirated, and medium containing vehicle, CFM-4.16 or CFM-4.17 (1.25 µM) added. PBECs were incubated for A) 24 h or B) 48 h, washed with PBS, equilibrated for 5 h with assay medium (low-glucose, phenol red-free DMEM supplemented with 1% (v/v) glutamine), and pre-incubated for a further 30 min in medium with or without verapamil (final concentration 10 μM). Cell monolayers were then incubated for 30 min in assay medium containing calcein-AM (final concentration 0.5 μM) and vehicle, CFM-4.16, CFM-4.17 or verapamil as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of calcein was measured at an excitation wavelength of 484 nm and an emission wavelength of 530 nm. Data were analysed using ANOVA and Tukey’s post hoc test and are expressed in each panel as mean ± standard deviation of three replicate wells in a single experiment. RFU: relative fluorescence units. * p<0.05, ** p<0.01, **** p<0.0001, n.s. non-significant.

In PBECs treated with 1.25 μM CFM-4.16, CFM-4.17 or vehicle for 24 h or 48 h followed by a 5 h washout period there was no significant difference in intracellular accumulation of the fluorescent probe Hoechst 33342 in cells exposed to CFM-4.16 or CFM-4.17 relative to those exposed to vehicle (Figure A2). Additionally there was no significant difference in Hoechst accumulation in PBECs treated with vehicle and Ko143 compared to PBECs treated with CFM-4.16 and Ko143 or CFM-4.17 and Ko143 (Figure A2). These 133 findings indicate that long-term treatment with CFM-4.16 or CFM-4.17 had no significant effect on ABCG2 activity both in the presence and absence of Ko143, irrespective of the inclusion of a washout period prior to carrying out the ABCG2 transporter functional assay.

Figure A2. Effect of long-term treatment with CFM-4.16 or CFM-4.17 followed by a washout period on ABCG2 activity in PBECs

n .s . n .s . n .s . n .s . 3 0 0 A n .s . 3 0 0 B n .s .

n .s . * * * * n .s .

1 -

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

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PBECs were seeded (25,000 cells/well) into 96 well plates and incubated for 48 h. Cells were then pre-equilibrated with treatment medium (low-glucose, phenol red-free DMEM supplemented with 1% (v/v) FBS and 1% (v/v) glutamine) for 30 min. The medium was subsequently aspirated, and medium containing vehicle, CFM-4.16 or CFM-4.17 (1.25 µM) added. PBECs were incubated for A) 24 h or B) 48 h, washed with PBS, equilibrated for 5 h with assay medium (low-glucose, phenol red-free DMEM supplemented with 1% (v/v) glutamine), and pre-incubated for a further 30 min in medium with or without Ko143 (final concentration 0.5 μM). Cell monolayers were then incubated for 30 min in assay medium containing Hoechst 33342 (final concentration 1 μM) and vehicle, CFM-4.16, CFM-4.17 or Ko143 as appropriate. The medium was aspirated, cells washed with ice-cold PBS, and 160 μL ice cold PBS added to each well. Intracellular accumulation of Hoechst 33342 was measured at an excitation wavelength of 370 nm and an emission wavelength of 450 nm. Data were analysed using ANOVA and Tukey’s post hoc test and are expressed in each panel as mean ± standard deviation of three replicate wells in a single experiment. RFU: relative fluorescence units. ** p<0.01, *** p<0.001, **** p<0.0001, n.s. non-significant.

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