Pre-clinical metabolism studies with Fenretinide in paediatric cancer

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.