Pre-Clinical Metabolism Studies with Fenretinide in Paediatric Cancer Nicola Ann Illingworth Thesis Submitted for the Degree of Doctor of Philosophy August 2011 Northern Institute for Cancer Research Faculty of Medical Sciences The Medical School Newcastle University Abstract Fenretinide (4-HPR) is a retinoic acid analogue used in clinical trials for the treatment of neuroblastoma and Ewing’s sarcoma. The work described involves investigations into factors that may impact on 4-HPR drug disposition. Metabolism of 4-HPR is of particular interest due to production of the active metabolite 4’-oxo 4-HPR and the clinical challenge of obtaining consistent 4-HPR plasma concentrations. The enzymes involved in 4-HPR metabolism were characterised and the impact of metabolism on efficacy in neuroblastoma and Ewing’s sarcoma cell lines assessed. In addition, the potential for 4-HPR to act as a substrate for common drug transporters was explored. 4-HPR was metabolised to 4’-oxo 4-HPR and 4’-OH 4-HPR primarily by CYPs 3A4, 3A5 and 2C8. Genetic variance in CYP2C8 affected oxidative metabolism, with much lower affinity for 2C8*4 (km of 59.8µM compared to 19.3µM for wild-type), and may be of clinical relevance. Both 4-HPR and 4’-oxo 4-HPR were glucuronidated. 4-HPR was glucuronidated by UGTs 1A1, 1A3 and 1A6, whilst 4’-oxo 4-HPR was glucuronidated by UGTs 1A1, 1A3, 1A8 and 1A9. However, very high Km values were observed (ranging from 389µM to 716µM for 4-HPR). Methylation of 4-HPR to the major metabolite 4-methoxyphenyl retinamide (4-MPR) was determined to be carried out by amine N-methyltransferases. Neuroblastoma and Ewing’s sarcoma cell lines metabolised 4-HPR to 4’-oxo 4-HPR, 4’-OH 4-HPR and 4-MPR. Although upregulation of CYP26A1 expression increased metabolism, inhibition of CYP26A1 had no effect on cell sensitivity. It is therefore unlikely that CYP26A1 expression will have a significant impact on 4-HPR efficacy. 4-HPR appears to be a substrate for the drug transporters MDR1, MRP2 and BCRP. However evidence for the role of these transporters is weak with no difference in 4-HPR sensitivity observed in cell lines over-expressing individual transporters in the presence or absence of specific transporter inhibitors. The major metabolites and metabolising enzymes of 4-HPR have been identified and characterised. This provides the potential to increase plasma concentrations of 4-HPR, and therefore optimise drug efficacy, through modulation of drug metabolism. i Acknowledgements I would like to thank my supervisors, Prof. Alan Boddy and Dr. Gareth Veal, for the opportunity to work on this project, as well as their invaluable support and guidance throughout my experimental work and write-up. I would also like to thank everyone at the NICR for their technical support, particularly the members of the pharmacology group for never tiring of answering ‘daft’ questions! I am also especially grateful to my family for their ceaseless encouragement, my friends for their continued belief in me, and my husband for his never-ending support. ii Table of Contents Chapter 1 Introduction ............................................................................ 1 1.1 Cancer .................................................................................................................. 1 1.2 Chemotherapy ..................................................................................................... 1 1.3 Retinoids .............................................................................................................. 3 1.3.1 Retinoid Clinical Use ........................................................................................ 4 1.4 Fenretinide (4-HPR)............................................................................................ 5 1.5 Neuroblastoma .................................................................................................... 7 1.5.1 Epidemiology .................................................................................................... 7 1.5.2 Diagnosis and Staging ....................................................................................... 8 1.5.3 Treatment .......................................................................................................... 9 1.6 Ewing’s Sarcoma ............................................................................................... 13 1.6.1 Epidemiology .................................................................................................. 13 1.6.2 Molecular and Cytogenetic Characteristics .................................................... 14 1.6.3 Diagnostic and Prognostic Factors .................................................................. 14 1.6.4 Treatment ........................................................................................................ 17 1.7 Xenobiotic Metabolism and Disposition ......................................................... 18 1.7.1 Human Liver and Intestine Microsomal Enzymes .......................................... 19 1.7.2 Cytochrome P450s (CYPs) ............................................................................. 20 1.7.3 Uridine 5'-Diphospho-glucuronosyltransferase Enzymes (UGTs) ................. 24 1.7.4 ATP-Binding Cassette (ABC) Transporters.................................................... 25 1.8 Retinoid Metabolism ......................................................................................... 28 1.8.1 4-HPR Metabolism ......................................................................................... 30 1.9 4-HPR Mechanism of Action............................................................................ 31 1.10 4-HPR Pre-Clinical Animal Studies ................................................................ 35 1.11 4-HPR Clinical Pharmacology ......................................................................... 37 1.12 Summary and Aims .......................................................................................... 40 Chapter 2 4-HPR Microsomal Metabolism ......................................... 42 iii 2.1 Introduction ....................................................................................................... 42 2.2 4-HPR Oxidative Metabolism .......................................................................... 43 2.2.1 Materials and methods .................................................................................... 43 2.2.2 Chemicals ........................................................................................................ 43 2.2.3 LC/MS analysis of 4-HPR and metabolites .................................................... 43 2.2.4 HPLC analysis of 4-HPR and metabolites ...................................................... 44 2.2.5 Incubation of 4-HPR with human liver microsomes (HLM), human intestinal microsomes (HIM) and CYPs ..................................................................................... 44 2.2.6 Inhibition of CYP metabolism ........................................................................ 45 2.2.7 Determination of kinetic parameters for 4’-OH 4-HPR and 4’-oxo 4-HPR formation ..................................................................................................................... 45 2.2.8 Transformation of E. coli cells to express CYP2C8 variants.......................... 45 2.2.8.1 Transformation of E. coli cells ...................................................................................... 46 2.2.8.2 Protein analysis .............................................................................................................. 47 2.2.8.3 Western Blot analysis .................................................................................................... 47 2.2.8.4 P450 CO difference spectral assay ................................................................................ 48 2.2.8.5 Cytochrome C reductase assay ...................................................................................... 48 2.2.9 Determination of kinetic parameters for 4’-OH 4-HPR and 4’-oxo 4-HPR formation by CYP2C8 variants ................................................................................... 48 2.3 4-HPR Methyltransferase Metabolism ........................................................... 49 2.3.1 Materials and methods .................................................................................... 49 2.3.2 Incubation of 4-HPR with HLM and S-adenosyl methionine (SAM) ............ 49 2.3.3 Determination of kinetic parameters for 4-MPR formation ............................ 49 2.3.4 Inhibition of methyltransferases ...................................................................... 49 2.4 Results – 4-HPR Oxidative Metabolism .......................................................... 51 2.4.1 Analytical assays ............................................................................................. 51 2.4.2 Incubation of 4-HPR with HLM ..................................................................... 51 2.4.3 Identification and characterization of 4-HPR metabolism by individual CYP isoforms ....................................................................................................................... 54 2.4.4 Transformation of E. coli cells to express CYP2C8 variants.......................... 57 2.4.5 Kinetic parameters for 4’-OH 4-HPR and 4’-oxo 4-HPR formation by CYP2C8 variants ......................................................................................................... 60 2.4.6