The synthesis of 17 α-ethynylestradiol glucuronides by mammalian microsomes immobilised in hollow-fibres

Abstract

Uridine 5′-diphosphate glucuronosyl transferases (UGTs, E.C.2.4.1.17) catalyse the glucuronidation of xenobiotics. The oral contraceptive 17 α-ethynylestradiol is partly cleared by glucuronidation on the 3 and 17 positions. These glucuronides are not commercially available and were synthesised as standards for characterisation of the metabolic profile of the drug. (1) Rat and dog microsomal suspensions were investigated, and the rates of glucuronide formation were found to be <0.09 and 0.49 nmol min −1 mg −1 protein, respectively. Therefore, dog microsomes were selected and immobilised in hollow-fibre membrane bioreactors. (2) Two bioreactors (15-ml capacity) were run at 20°C and fed with a saturated solution (3.3 pmol/mg protein) of the sparingly soluble drug and co-factor UDPGA (0.15 mmol/mg protein) in recycle and single pass modes, at a flow rate of 20 ml/h. The recycle mode produced more glucuronide than the single pass up to 46 h (substrate to product peak area ratios of 1.6 and 5.9, respectively). The recycle mode was adopted and the bioreactors run continuously for 88 h. (3) A second batch using the same bioreactor was run for 120 h. The estimated conversion reduced from 50% on the first run to 25% on the second run. (4) A primary clean up was effected by adsorption on octadecyl silyl resin. Separation of the glucuronides from 17 α-ethynylestradiol was on a normal phase high pressure liquid chromatography (HPLC) system. After collection of the glucuronide fraction, the solvents were evaporated off providing 1 mg of the two glucuronides (recovery efficiency>90%).