Characterization of the metabolism of fenretinide by human liver microsomes, cytochrome P450 enzymes and UDP-glucuronosyltransferases

Abstract

Background and purpose: Fenretinide (4-HPR) is a retinoic acid analogue, currently used in clinical trials in oncology. 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 in patients. Here, we assessed the enzymes involved in various 4-HPR metabolic pathways.

Experimental approach: Enzymes involved in 4-HPR metabolism were characterized using human liver microsomes (HLM), supersomes over-expressing individual human cytochrome P450s (CYPs), uridine 5'-diphospho-glucoronosyl transferases (UGTs) and CYP2C8 variants expressed in Escherichia coli. Samples were analysed by high-performance liquid chromatography and liquid chromatography/mass spectrometry assays and kinetic parameters for metabolite formation determined. Incubations were also carried out with inhibitors of CYPs and methylation enzymes.

Key results: HLM were found to predominantly produce 4'-oxo 4-HPR, with an additional polar metabolite, 4'-hydroxy 4-HPR (4'-OH 4-HPR), produced by individual CYPs. CYPs 2C8, 3A4 and 3A5 were found to metabolize 4-HPR, with metabolite formation prevented by inhibitors of CYP3A4 and CYP2C8. Differences in metabolism to 4'-OH 4-HPR were observed with 2C8 variants, CYP2C8*4 exhibited a significantly lower V(max) value compared with *1. Conversely, a significantly higher V(max) value for CYP2C8*4 versus *1 was observed in terms of 4'-oxo formation. In terms of 4-HPR glucuronidation, UGTs 1A1, 1A3 and 1A6 produced the 4-HPR glucuronide metabolite.

Conclusions and implications: The enzymes involved in 4-HPR metabolism have been characterized. The CYP2C8 isoform was found to have a significant effect on oxidative metabolism and may be of clinical relevance.

Figure 1

Representative chromatograms showing separation of fenretinide (4-HPR) and metabolites by LC/MS/MS (A) and reversed-phase high-performance liquid chromatography (B, C and D). Metabolites were generated following a 3-h incubation of 20 µM 4-HPR with 0.5 mg·mL⁻¹ human liver microsomes (HLM; A, B and C) or following a 3-h incubation of 200 µM 4-HPR with 0.5 mg·mL⁻¹ HLM (D). Generation of methoxy fenretinide (4-MPR) is shown in (C) in the presence of 0.2 mM S-adenosyl methionine (SAM).

Figure 2

Formation of 4′-OH 4-HPR and 4′-oxo fenretinide (4′-oxo 4-HPR) metabolites of 4-HPR by a panel of supersomes over-expressing individual human cytochrome P450s. Metabolite formation was determined by high-performance liquid chromatography analysis. Control supersomes were from cells transfected with an empty vector. Metabolites were generated by 3-h incubation of 50 µM 4-HPR with 1 mg·mL⁻¹ of each supersome for 3 h. Results are mean ± SD from three independent experiments. HLM, human liver microsomes.

Figure 3

Determination of kinetic parameters for the formation of (A) 4′-oxo fenretinide (4′-oxo 4-HPR) and (B) 4′-OH 4-HPR metabolites by a panel of supersomes over-expressing individual human CYPs. The major CYPs (0.5 mg mL⁻¹) found to metabolize 4-HPR (human cytochrome P450s 3A4, 3A5 and 2C8) were incubated with 0, 2.5, 5, 10, 15, 20, 50 and 100 µM 4-HPR for 3 h. Metabolite formation was determined by high-performance liquid chromatography analysis. Results are mean ± SD from three independent experiments.

Figure 4

Determination of kinetic parameters for the formation of (A) 4′-oxo fenretinide (4′-oxo 4-HPR) and (B) 4′-OH 4-HPR metabolites by CYP2C8 variants. E. coli membrane fractions (0.5 mg·mL⁻¹) co-expressing CYP2C8 variants and P450 reductase were incubated with 0, 2.5, 5, 10, 15 and 20 µM 4-HPR for 3 h. Metabolite formation was determined by high-performance liquid chromatography analysis. Results are mean ± SD from three independent experiments.

Figure 5

Formation of methoxy fenretinide (4-MPR) following a 3-h incubation of 0.5 mg·mL⁻¹ human liver microsomes (HLM) with 0–100 µM fenretinide (4-HPR) and 0.2 mM S-adenosyl methionine (SAM) (A) and following a 3 h incubation of 0.5 mg·mL⁻¹ HLM with 50 µM 4-HPR and 0.2 mM SAM in the presence of 0–5 mM imidazole (B).

Figure 6

Formation of glucuronide metabolites of fenretinide (4-HPR) by a panel of uridine 5′-diphospho-glucoronosyl transferases (UGT) enzymes, human intestinal microsomes (HIM) and human liver microsomes (HLM). Metabolite formation was determined by HPLC analysis. Metabolites were generated by 3-h incubation of 200 µM 4-HPR with 1 mg·mL⁻¹ of each UGT/microsome for 3 h. Results are mean ± SD from three independent experiments.

Figure 7

Determination of kinetic parameters for the formation of glucuronide metabolites of fenretinide (4-HPR) by a panel of uridine 5′-diphospho-glucoronosyl transferases (UGT) enzymes, human intestinal microsomes (HIM) and human liver microsomes (HLM); 1 mg·mL⁻¹ of the major UGTs found to metabolize 4-HPR (1A1, 1A3 and 1A6, as well as HIM and HLM) were incubated with 0, 5, 25, 50, 75, 100, 150, 200, 250, 300, 500, 1000 and 2000 µM 4-HPR for 3 h. Metabolite formation was determined by high-performance liquid chromatography analysis. Results are mean ± SD from three independent experiments.